Multi-domain enzymes having cutinase activity, compositions comprising same and uses thereof

Abstract

An isolated chimeric recombinant protein has cutinase activity. The protein includes a cutinase catalytic domain and a polymer binding domain operably linked by a proline/threonine-rich linker domain. The proline/threonine-rich linker domain includes at least 50% proline or threonine residues over a stretch of 15 to 55 consecutive amino acids.

Claims

1. An isolated chimeric recombinant protein having cutinase activity, said protein comprising a cutinase catalytic domain and a polymer binding domain operably linked by a proline/threonine-rich linker domain, wherein the proline/threonine-rich linker domain comprises at least 85% proline or threonine residues over a stretch of at least 17 consecutive amino acids, or comprises an amino acid sequence at least 85% identical to SEP ID NO: 3.

2. The isolated chimeric recombinant protein of claim 1, wherein the proline/threonine-rich linker domain comprises at least 90% proline or threonine residues over a stretch of at least 17 consecutive amino acids, or comprises an amino acid sequence at least 90% identical to SEQ ID NO: 3.

3. The isolated chimeric recombinant protein of claim 1, wherein at least two of the cutinase catalytic domain, the polymer binding domain, and the proline/threonine-rich linker domain originate from native enzymes of different species.

4. The isolated chimeric recombinant protein of claim 3, wherein the polymer binding domain and the proline/threonine-rich linker domain are selected from a multi-domain cutinase from the species Kineococcus radiotolerans, Cellulomonas flavigena, Cellulomonas bogoriensis, and Cellulomonas cellasea.

5. The isolated chimeric recombinant protein of claim 3, wherein the polymer binding domain is from a poly(3-hydroxybutyrate) (PHB) depolymerase of the species Paucimonas lemoignei, Thermobifida sp. BCC23166, Comamonas testosterone, Thermobifida fusca, or Nocardiopsis dassonvillei.

6. The isolated chimeric recombinant protein of claim 1, which is recombinantly produced fused to a heterologous carrier protein or heterologous tag that enables secretion of said recombinant protein, and/or facilitates purification or detection of said recombinant protein.

7. The isolated chimeric recombinant protein of claim 6, wherein said carrier protein is YebF.

8. A composition comprising: (i) an isolated recombinant protein having cutinase activity, said protein comprising a cutinase catalytic domain and a polymer binding domain operably linked by a proline/threonine-rich linker domain, wherein the proline/threonine-rich linker domain comprises at least 85% proline or threonine residues over a stretch of at least 17 consecutive amino acids, or comprises an amino acid sequence at least 85% identical to SEQ ID NO: 3; and (ii) a bleaching agent and/or an organic solvent, wherein said recombinant protein retains said cutinase activity when present in said composition, and wherein the composition is: a cleaning product, a degreaser, a disinfectant, a bleaching product, a product for controlling biofilm, or any combination thereof; feed or a feed additive; a product for inactivating mycotoxin; a product for bioscouring or other treatment of fabrics or textiles; a product for catalyzing trans-esterification reactions; a product for vegetable oil extraction; or a product for enhancing oil recovery.

9. The composition of claim 8 comprising a bleaching agent which is: hydrogen peroxide, a peroxide other than hydrogen peroxide, a non-peroxide oxidizing agent, ozone, sodium percarbonate, sodium perborate, and any combination thereof.

10. The composition of claim 8 comprising a peroxide.

11. The composition of claim 8 comprising a bleaching agent which is: sodium dithionite, sodium dithionite, sulfur dioxide, a sulfite, a bisulfite, a sodium borohydride, or any combination thereof.

12. The composition of claim 8 comprising an organic solvent which is: methanol, 2-propanol, or an alcohol other than methanol or 2-propanol, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), or acetone.

13. The composition of claim 8 having a pH between 7.5 and 9.5.

14. The composition of claim 8, further comprising: a detergent, a surfactant, a buffer, a chelator, or any combination thereof.

15. The composition of claim 8, further comprising a chelator which is EDTA or GLDA.

16. The composition of claim 8, further comprising a polyol and/or boric acid as a stabilizer.

17. The composition of claim 8, further comprising an additional recombinant enzyme which is: a lipase, an esterase, a pectate lyase, a pectinase, a cutinase, a cellulase, a hemicellulose, an amylase, or any combination thereof.

18. The composition of claim 8, wherein the proline/threonine-rich linker domain comprises at least 90% proline or threonine residues over a stretch of at least 17 consecutive amino acids, or comprises an amino acid sequence at least 90% identical to SEQ ID NO: 3.

19. The composition of claim 8, wherein at least two of the cutinase catalytic domain, the polymer binding domain, and the proline/threonine-rich linker domain originate from native enzymes of different species.

20. The composition of claim 8, wherein: (a) the proline/threonine-rich linker domain is selected from a multi-domain cutinase from the species Kineococcus radiotolerans, Cellulomonas flavigena, Cellulomonas bogoriensis, and Cellulomonas cellasea; or (b) the polymer binding domain is selected from a multi-domain cutinase from the species Kineococcus radiotolerans, Cellulomonas flavigena, Cellulomonas bogoriensis, and Cellulomonas cellasea; or from a poly(3-hydroxybutyrate) (PHB) depolymerase of the species Paucimonas lemoignei, Thermobifida sp. BCC23166, Comamonas testosterone, Thermobifida fusca, and Nocardiopsis dassonvillei.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In the appended drawings:

(2) FIG. 1 is a schematic representation of the domain organization of KrCUT and sequence characteristics of some conserved regions. SP, signal peptide; TIP-rich, threonine-praline rich linker; KrCUT206 (containing 206 amino acids) is a truncated version of KrCUT lacking the polymer-binding domain. KrCUT189 (containing 189 amino acids) is a truncated version of KrCUT206 further lacking the T/P-rich linker. Inset (a): cutinase motifs containing the predicted catalytic triads (S114-D184-H198) and conserved cysteines (C103, C180, C187) according to Wang et al., 2010. The sequences shown in panel (a) are represented in the Sequence Listing as follows:

(3) Kr SEQ ID NO: 49 and SEQ ID NO: 50;

(4) Cf SEQ ID NO: 51 and SEQ ID NO: 52;

(5) Fs SEQ ID NO: 53 and SEQ ID NO: 54;

(6) Pi SEQ ID NO: 55 and SEQ ID NO: 56;

(7) Rh SEQ ID NO: 57 and SEQ ID NO:58;

(8) Mg SEQ ID NO: 59 and SEQ ID NO: 60;

(9) Mf SEQ ID NO: 61 and SEQ ID NO: 62.

(10) Inset (b): Homology to substrate-binding domains of PH8 depolymerases. The sequences shown in panel (b) are represented in the Sequence Listing as follows:

(11) Kr SEQ ID NO: 63;

(12) Cf SEQ ID NO: 64;

(13) PI SEQ ID NO: 65;

(14) Ts SEQ ID NO: 66;

(15) Ct SEQ ID NO: 67;

(16) Tf SEQ ID NO: 68;

(17) Nd SEQ ID NO: 69.

(18) Abbreviations of bacterial source: Cf, Cellulomonas flavigena DSM 20109 (ZP_04367703); Ct, Comamonas testosteroni (BAA22882); Fs, Fusarium solani (P00590); Kr, Kineococcus radiotolerans cutinase (YP_001363838); Mf, Monilinia fructicola (AAZ95012); Mg, Magnaporthe grisae (CAA43717); Nd, Nocardiopsis dassonvillei subsp PHB depolymerase (ZP_04334644); Pi, Phytophthora infestans (AAY43367); PI, Paucimonas lemoignei (AAB48166); Rh, Rhodococcus (jostii) RHA1 (YP_700622). Tf, Thermobifida fusca YX PHB depolymerase (YP_288143, re-annotated as WP_011290529.1); and Ts, Thermobifida sp. BCC23166 PHB depolymerase (ACF17837).

(19) FIG. 2 shows purification of recombinant K. radiotolerans cutinase and truncated derivatives. Proteins were analyzed by 10% SDS PAGE, and silver stained. The molecular weight markers are indicated alongside; (a) lane 1, fraction from SP-Sepharose showing YebF-KrCUT fusion protein (39 kDa) and mature KrCUT (31 kDa); lanes 2 and 3 from HiLoad Superdex 75 showing the separated protein bands; (b) lane 4, YebF-KrCUT189 (33 kDa) from HiLoad Superdex 75.

(20) FIG. 3 shows substrate specificity of full-length KrCUT and KrCUT189. Activity (U/mg) towards selected p-nitrophenol esters are compared.

(21) FIG. 4 shows pH optima curves for activity of KrCUT and KrCUT189.

(22) FIG. 5 shows thermostability curves for KrCUT. Activity of the enzyme after incubation at the defined temperatures and for the selected time periods was measured.

(23) FIG. 6 shows the enzymatic activity of protein purification elution fractions corresponding to KrCUT (fractions C1-C10) and YebF-KrCUT (fractions D9-D11).

(24) FIG. 7 shows GC analysis of cutin monomers released by KrCUT. The numbers on the peaks correspond to the retention times of the released products as described in the text.

(25) FIG. 8 shows degradation of synthetic polyesters by YebF-KrCUT and KrCUT189. Enzyme concentration is 2.5 M, unless otherwise stated. (A) Degradation of PCL films (.square-solid., YebF-KrCUT; .box-tangle-solidup., YebF-KrCUT, 1.25 M; and , KrCUT189), and PCL pellets (.circle-solid., YebF-KrCUT; and , KrCUT189). The curve of PCL film degradation by 5 M YebF-KrCUT approximated that of 2.5 M enzyme concentration (not shown). (B) Degradation of PLA (.circle-solid., YebF-KrCUT; and , KrCUT189) and polypropylene adipate (.square-solid., YebF-KrCUT; and , KrCUT189).

(26) FIG. 9 shows adsorption isotherm of cutinase and PHB granules at 25 C. Purified KrCUT of concentrations ranging from 0.16 to 3.2 mg/mL were mixed with 25 mg PHB in 50 mM sodium phosphate (pH 8.0) in a total volume of 1 mL and mixed by gently shaking for 3 h. The PHB polymer was removed by centrifugation and the concentration of protein in the supernatant was determined by the BCA method. The concentration of bound protein at a particular concentration of cutinase was calculated as the difference between a control without added PHB and free cutinase after incubation with PHB.

(27) FIG. 10 shows amounts of pectin released from natural hemp fibres by pectate lyase (PL) or in combination with cutinase (Cut) at different concentrations. The shaded bars indicate the two different incubation periods (1.5 h and 3 h).

(28) FIGS. 11 (A) and (B) show light microscopic analysis of hemp fibre obtained by enzymatic retting. (A) pectate lyase treatment, fibers are embedded into pectin and wax material; (B) pectate lyase and cutinase treatment, fiber, F cell in bundles. (C) and (D) represents images from scanning electron microscopy (SEM): (C) pectate lyase; (D) pectate lyase and cutinase.

(29) FIG. 12 shows the long term stability of recombinant KrCUT in stabilisation buffer.

(30) FIG. 13 shows examples of purified recombinant proteins separated by SDS-PAGE. Panel (A) shows the enzyme KrCUT, while panel (B) shows the variant AmKr alone or fused to the heterologous carrier protein, YebF (YebFAmKr).

(31) FIG. 14 shows CD spectroscopy measurements in order to monitor the unfolding/refolding of various recombinant proteins: (A) KrCUT; (B) AmKr; (C); CfCUT; and (D) Cf-TP.

(32) FIG. 15 shows a multiple sequence alignment (A) and a phylogenic tree (B) of native multi-domain cutinases from Kineococcus radiotolerans, Cellulomonas bogoriensis, Cellulomonas cellasea, and Cellulomonas flavigena.

SEQUENCE LISTING

(33) This application contains a Sequence Listing in computer readable form entitled Sequence_Listing.txt, created Jan. 28, 2016 having a size of 84 kb. The computer readable form is incorporated herein by reference.

(34) TABLE-US-00001 SEQ ID NO: 1 ssKrCUT amino acid sequence SEQ ID NO: 2 KrCUT189 amino acid sequence (cutinase catalytic domain of KrCUT) SEQ ID NO: 3 T/P-rich linker of KrCUT amino acid sequence SEQ ID NO: 4 Polymer binding domain of KrCUT amino acid sequence SEQ ID NO: 5 DNA encoding KrCUT optimized for expression in E. coli SEQ ID NO: 6 KrCUT amino acid sequence, lacking residues 1-33 of ssKrCUT SEQ ID NO: 7 DNA encoding YebF SEQ ID NO: 8 YebF amino acid sequence SEQ ID NO: 9 DNA encoding YebF-KrCUT SEQ ID NO: YebF-KrCUT amino acid 10 sequence SEQ ID NO: DNA encoding YebF-KrCUT189 11 SEQ ID NO: YebF-KrCUT189 amino acid 12 sequence SEQ ID NO: ssCfCUT: cutinase from Cf amino 13 acid sequence SEQ ID NO: ssCbCUT1: cutinase from Cb 14 amino acid sequence SEQ ID NO: ssCcCUT: cutinase from Cc 15 amino acid sequence SEQ ID NO: ssAmCUT: cutinase from Am 16 ATCC 39116 amino acid sequence SEQ ID NO: ssFrCUT: cutinase from Fr amino 17 acid sequence SEQ ID NO: ssAcCUT: cutinase from Ac 431 18 amino acid sequence SEQ ID NO: ssCaCUT: cutinase from Ca 19 amino acid sequence SEQ ID NO: ssRhCUT: cutinase from Rh 20 amino acid sequence SEQ ID NO: CfKr fusion protein amino acid 21 sequence SEQ ID NO: DNA encoding CfKr fusion 22 protein SEQ ID NO: KrCf fusion protein amino acid 23 sequence SEQ ID NO: DNA encoding KrCf fusion 24 protein SEQ ID NO: ssCbCUT2: cutinase from Cb 25 69B4 (KGM14008.1) amino acid sequence SEQ ID NO: AmCUT: cutinase from Am 26 ATCC 39116 amino acid sequence SEQ ID NO: Am-Kr: variant amino acid 27 sequence SEQ ID NO: CfCUT: cutinase from Cf 28 amino acid sequence SEQ ID NO: Cf-TP: variant amino acid 29 sequence SEQ ID NO: cutinase catalytic domain of 30 CfCUT amino acid sequence SEQ ID NO: T/P-rich linker domain of 31 CfCUT amino acid sequence SEQ ID NO: polymer binding domain of 32 CfCUT amino acid sequence SEQ ID NO: cutinase catalytic domain of 33 CbCUT1 (KGM14009.1) amino acid sequence SEQ ID NO: linker of CbCUT1 34 (KGM14009.1) amino acid sequence SEQ ID NO: polymer binding domain of 35 CbCUT1 (KGM14009.1) amino acid sequence SEQ ID NO: cutinase catalytic domain of 36 CbCUT2 (KGM14008.1) amino acid sequence SEQ ID NO: T/P-rich linker of CbCUT2 37 (KGM14008.1) amino acid sequence SEQ ID NO: polymer binding domain of 38 CbCUT2 (KGM14008.1) amino acid sequence SEQ ID NO: cutinase catalytic domain of 39 CcCUT (DSM 20118) amino acid sequence SEQ ID NO: T/P-rich linker of CcCUT 40 (DSM 20118) amino acid sequence SEQ ID NO: polymer binding domain of 41 CcCUT (DSM 20118) amino acid sequence SEQ ID NO: cutinase catalytic domain of 42 FrCUT (BMG5.12) amino acid sequence SEQ ID NO: cutinase catalytic domain of 43 AcCUT (431) amino acid sequence SEQ ID NO: cutinase catalytic domain of 44 CaCUT (DSM 44712) amino acid sequence SEQ ID NO: cutinase catalytic domain of 45 RhCUT amino acid sequence SEQ ID NO: CbCUT2: cutinase from Cb 46 (KGM14008.1) amino acid sequence SEQ ID NO: CcCUT: cutinase from 47 CcCUT (DSM 20118) amino acid sequence SEQ ID NO: CbCUT1: cutinase from 48 CbCUT1 (KGM14009.1) amino acid sequence ss = native N-terminal signal sequence Kr = Kineococcus radiotolerans Cf = Cellulomonas flavigena Cb = Cellulomonas bogoriensis Cc = Cellulomonas cellasea Am = Amycolatopsis sp. Fr = Frankia sp. BMG5.12 Ac = Actinoplanes missouriensis Ca = Cryptosporangium arvum Rh = Rhodococcus TP = threonine/proline-rich linker domain from Kr

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

(35) Recombinant Proteins

(36) In one aspect, the present description relates to an isolated multi-domain recombinant protein having cutinase activity. The multi-domain recombinant protein may comprise: (a) a cutinase catalytic domain; (b) a proline/threonine-rich linker domain positioned C-terminal of domain (a); and (c) a polymer binding domain positioned C-terminal of domain (b).

(37) As used herein, the expression cutinase catalytic domain refers to a polypeptide domain that has the ability to hydrolyze cutin (and/or another polymer substrate of interest hydrolyzable by the cutinase catalytic domain) in a modular fashion (i.e., when present within a larger recombinant polypeptide of the present invention).

(38) In some embodiments, the cutinase catalytic domain may be: (1) a cutinase catalytic domain having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity of SEQ ID NOs: 2, 30, 33, 36, 39, or 42-45; (2) a cutinase catalytic domain comprising the amino acid sequence of SEQ ID NOs: 2, 30, 33, 36, 39, or 42-45; or (3) both (1) and (2).

(39) In some embodiments, the cutinase catalytic domain may share at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the cutinase catalytic domain of any one of SEQ ID NOs: 2 and 13-21.

(40) As used herein, the expression threonine/proline-rich linker domain or proline/threonine-rich linker domain (T/P-rich linker domain) refers to a polypeptide domain that shares functional and/or structural similarity to T/P-rich linker domains found in native multi-domain cutinases, such as those described herein from Kineococcus radiotolerans (e.g., accession ABS05574.1), Cellulomonas flavigena (e.g., accession ADG75999.1), Cellulomonas bogoriensis (e.g., accessions KGM14008.1), and Cellulomonas cellasea (e.g., accession KGM03336.1).

(41) In some embodiments, the T/P-rich linker domain may be a threonine/proline-rich linker domain having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 3, 31, 34, 37, or 40. In some embodiments, the T/P-rich linker domain may be a T/P-rich linker domain comprising the amino acid sequence of SEQ ID NO: 3, 31, 34, 37, or 40.

(42) In some embodiments, the T/P-rich linker domain may be a polypeptide domain which has at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, or at least 65% of its residues being a proline or a threonine, over a stretch of 15 to 55 consecutive amino acids.

(43) In some embodiments, the linker domain may comprise an amino acid sequence sharing at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to: SEQ ID NO: 3; residues 220-247 of SEQ ID NO: 13; residues 226-236 of SEQ ID NO: 14; or residues 228-276 of SEQ ID NO: 15.

(44) As used herein, the expression polymer binding domain refers to a protein domain that may have the ability to bind in a modular fashion (i.e., when present within a larger recombinant polypeptide of the present description) to a given polymer of interest (e.g., a polyester hydrolyzable by a cutinase catalytic domain of the present description).

(45) In some embodiments, the polymer binding domain may have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 4, 32, 35, 38, or 41. In some embodiments, the polymer binding domain may comprise the amino acid sequence of SEQ ID NO: 4, 32, 35, 38, or 41.

(46) In some embodiments, the polymer binding domain of a polypeptide of the present description may bind to poly(hydroxybutyrate), triacin, tributyrin, trimiristin, tripalmitin, polyvinyl acetate (PVA), polyethylene terephthalate (PET), or polytrimethylene terephthalate (PTT). In some embodiments, the polymer binding domain may comprise an amino acid sequence sharing at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the polymer binding domain of any one of SEQ ID NOs: 4 and 13-15. In some embodiments, the polymer binding domain comprises the amino acid sequence of any one of SEQ ID NOs: 4 and 13-15.

(47) In some embodiments, an isolated recombinant protein of the present description may be a chimeric recombinant protein, wherein at least one of domains (a), (b) and (c) is from a cutinase belonging to a different species. Native multi-domain cutinases can be found in species such as Kineococcus radiotolerans (e.g., accession ABS05574.1), Cellulomonas flavigena (e.g., accession ADG75999.1), Cellulomonas bogoriensis (e.g., accessions KGM14008.1 and KGM14009.1), or Cellulomonas cellasea (e.g., accession KGM03336.1).

(48) In some embodiments, the above mentioned protein further comprises a polymer binding domain operably linked to the cutinase catalytic domain. As used herein, the expression operably linked refers to the covalent linkage of two or more modular protein domains (e.g., a cutinase catalytic domain and a polymer binding domain) such that each of the modular protein domains at least substantially retains its enzymatic activity. Two or more protein domains that cooperate synergistically when covalently linked is also within the meaning of operably linked as used herein.

(49) In some embodiments, the above mentioned cutinase domain and the above mentioned polymer binding domain are operably linked by a linker domain. In some embodiments, the linker domain is 5 to 60, 5 to 50, 5 to 40, 5 to 30, 5 to 20, 10 to 60, 10 to 50, or 10 to 40 amino acids in length. In some embodiments, the linker domain is a proline/threonine-rich linker domain. As used herein, the expression proline/threonine-rich refers to a polypeptide domain which has at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, or at least 65% of its residues being a proline or a threonine. In some embodiments, the linker domain comprises an amino acid sequence sharing at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to: SEQ ID NO: 3; residues 220-247 of SEQ ID NO: 13; residues 226-236 of SEQ ID NO: 14; or residues 228-276 of SEQ ID NO: 15.

(50) In another aspect, the present invention relates to isolated recombinant proteins sharing a minimum threshold of amino acid sequence identity with any one of the polypeptides as described herein. In specific embodiments, the present invention relates to isolated polypeptides having at least 60%, 65%, 70%, 71%, 72, 73%, 74%, 75%, 76%, 77%, 78%, 79, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to any one of the above-mentioned polypeptides. Other specific percentage units that have not been specifically recited here for brevity are nevertheless considered within the scope of the present invention.

(51) In some embodiments, the isolated recombinant protein of the present description may have improved stability to a bleaching agent, as compared to a native cutinase enzyme that comprises the corresponding cutinase catalytic domain. As used herein, a bleaching agent refers to an agent that lightens or whitens a substrate through chemical reaction. Bleaching reactions usually involve oxidative or reductive processes that degrade color. As used in the present context, increased stability refers to an enzyme that generally retains a higher relative level of enzymatic activity, or exhibits a lower relative loss of enzymatic activity, after being exposed to increasing concentrations of a given bleaching agent, as compared to a suitable control enzyme (e.g., native enzyme).

(52) In some embodiments, the bleaching agent may be an oxidizing agent or a reducing agent. The oxidizing agent may include hydrogen peroxide, a peroxide other than hydrogen peroxide, a non-peroxide oxidizing agent, ozone, sodium percarbonate, sodium perborate, or any combination thereof. The reducing agent may include sodium dithionite, sodium dithionite, sulfur dioxide, a sulfite, a bisulfite, a sodium borohydride, or any combination thereof.

(53) In some embodiments, the isolated recombinant protein of the present description retains activity in the presence of a chelator (e.g., EDTA or GLDA). In some embodiments, the isolated recombinant protein of the present description retains activity in the presence of EDTA at a concentration of between 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9%, and 10% w/v). In some embodiments, the isolated recombinant protein of the present description retains activity in the presence of GLDA at a concentration between 0.5%, 1%, 2%, 3%, or 4%, and 5% w/v).

(54) In some embodiments, the isolated recombinant protein of the present description may have improved stability to an organic solvent (e.g., methanol, 2-propanol, or another alcohol, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), acetone, or any combination thereof), as compared to a native cutinase enzyme that comprises the corresponding cutinase catalytic domain. As used in the present context, increased stability refers to an enzyme that generally retains a higher relative level of enzymatic activity, or exhibits a lower relative loss of enzymatic activity, after being exposed to increasing concentrations of a given organic solvent, as compared to a suitable control enzyme (e.g., native enzyme).

(55) In some embodiments, the isolated recombinant protein of the present description may exhibit a broader temperature activity range, as compared to a native cutinase enzyme that comprises the corresponding cutinase catalytic domain. Such properties may increase the versatility of an enzyme for industrial applications. As used in the present context, broader temperature activity range refers to an enzyme that generally retains a higher relative level of enzymatic activity, or exhibits a lower relative loss of enzymatic activity, when incubated across a broader range of reaction temperatures, as compared to a suitable control enzyme (e.g., native enzyme). This is in contrast to an enzyme that, for example, shows a maximal level of activity at a given optimal temperature, but exhibits a relatively sharp decrease in activity when the reaction temperature is progressively shifted away from that optimal temperature.

(56) It will be appreciated that this disclosure embraces the degeneracy of codon usage as would be understood by one of ordinary skill in the art and as illustrated in the table below.

(57) TABLE-US-00002 Codon Degeneracies Amino Codons Ala/A GCT, GCC, GCA, GCG Arg/R CGT, CGC, CGA, CGG, Asn/N AAT, AAC Asp/D GAT, GAC Cys/C TGT, TGC Gln/Q CAA, CAG Glu/E GAA, GAG Gly/G GGT, GGC, GGA, GGG His/H CAT, CAC Ile/I ATT, ATC, ATA Leu/L TTA, TTG, CTT, CTC, CTA, Lys/K AAA, AAG Met/M ATG Phe/F TTT, TTC Pro/P CCT, CCC, CCA, CCG Ser/S TCT, TCC, TCA, TCG, AGT, Thr/T ACT, ACC, ACA, ACG Trp/W TGG Tyr/Y TAT, TAC Val/V GTT, GTC, GTA, GTG START ATG STOP TAG, TGA, TAA

(58) Conservative substitutions: Furthermore, it will be understood by one skilled in the art that conservative substitutions may be made in the amino acid sequence of a polypeptide without disrupting the structure or function of the polypeptide. Conservative substitutions are accomplished by the skilled artisan by substituting amino acids with similar hydrophobicity, polarity, and R-chain length for one another. Additionally, by comparing aligned sequences of homologous proteins from different species, conservative substitutions may be identified by locating amino acid residues that have been mutated between species without altering the basic functions of the encoded proteins. The table below provides an exemplary list of conservative substitutions.

(59) TABLE-US-00003 Conservative Substitutions Type of Amino Acid Substitutable Amino Acids Hydrophilic Ala, Pro, Gly, Glu, Asp, Gln, Asn, Ser Sulphydryl Cys Aliphatic Val, Ile, Leu, Met Basic Lys, Arg, His Aromatic Phe, Tyr, Trp

(60) In another embodiment, the present invention relates to functional variants of the recombinant proteins as described herein. Functional variants refers to recombinant proteins differing from their parent recombinant protein by one or more amino acid substitutions, deletions, and/or insertions, yet which at least retain or provide higher biological activity as compared to their parent recombinant protein. Functional variants include functional fragments (i.e., biologically active fragments) of any one of the recombinant polypeptides of e present invention. Such fragments include fewer amino acids than the full length protein from which they are derived, but exhibit at least one biological activity of the corresponding full-length protein.

(61) In another aspect, the present invention includes fusion proteins comprising a polypeptide of the present invention or a functional variant thereof, which is operatively linked to one or more unrelated polypeptide (e.g., heterologous amino acid sequences). Unrelated polypeptides or heterologous polypeptides or heterologous sequences refer to polypeptides or sequences which are usually not present close to or fused to one of the polypeptides of the present invention. Such unrelated polypeptides or heterologous polypeptides having amino acid sequences corresponding to proteins which are not substantially homologous to the polypeptide sequences disclosed herein. Such unrelated polypeptides can be derived from the same or a different organism. In one embodiment, a fusion protein of the present invention comprises at least two biologically active portions or domains of polypeptide sequences disclosed herein.

(62) In another embodiment, a recombinant protein of the present invention can be fused to a protein which enables or facilitates recombinant protein purification and/or detection. For example, a polypeptide of the present invention can be fused to a protein such as glutathione S-transferase (GST), and the resulting fusion protein can then be purified/detected through the high affinity of GST for glutathione. In another embodiment, a recombinant protein of the present invention can be fused or otherwise operably linked to a carrier protein (e.g., YebF) which enables its extracellular secretion. Such a linkage can be made such the carrier protein is cleavable upon secretion of the recombinant protein of the present invention.

(63) Recovery and Purification

(64) In another aspect, polypeptides of the present invention may be present alone (e.g., in an isolated or purified form), within a composition including a carrier (e.g., an enzymatic composition for carrying out an industrial process), or in an appropriate host. In one embodiment, polypeptides of the present invention can be recovered and purified from cell cultures (e.g., recombinant cell cultures) by methods known in the art. In another embodiment, high performance liquid chromatography (HPLC) can be employed for the purification.

(65) In another aspect, polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, fungal, plant, insect, animal and mammalian cells. Depending on the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.

(66) Polynucleotides

(67) In some aspects, the present description relates to polynucleotides encoding the recombinant proteins described herein. In some embodiments, the recombinant protein encoded by said polynucleotide may lack an N terminal signal sequence often found on native cutinases. The recombinant truncation of such native N terminal signal sequences may increase the enzymatic activity, and thus the utility of the recombinant enzyme.

(68) In some embodiments, the polynucleotides of the present description may encode a recombinant protein described herein which is fused to a heterologous carrier protein (e.g., YebF) and/or a heterologous tag (e.g., a purification or detection tag). Such fusions may not only enable the proper expression and/or secretion of the recombination protein from a host production cell, but also may facilitate the autolysis of recombination proteins over time, which may result in truncated enzyme having higher activity and/or increased structural stability.

(69) In some embodiments, the polynucleotides described here may be fused to a heterologous promoter to improve their expression in a host production cell.

(70) In some aspects, the present invention relates to a polynucleotide encoding a recombinant protein as described herein. In some embodiments, the polynucleotide may be RNA or DNA (e.g., cDNA).

(71) In another aspect, the present invention relates to isolated polynucleotides sharing a minimum threshold of nucleic acid sequence identity with any one of the polynucleotides as described herein. In specific embodiments, the present invention relates to isolated polynucleotides having at least 60%, 65%, 70%, 71%, 72, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleic acid sequence identity to any one of the polynucleotides as described herein. Other specific percentage units that have not been specifically recited here for brevity are nevertheless considered within the scope of the present invention. Polynucleotides having the aforementioned thresholds of nucleic acid sequence identity can be created by introducing one or more nucleotide substitutions, additions or deletions into the coding nucleotide sequences of the present invention such that one or more amino acid substitutions, deletions or insertions are introduced into the encoded polypeptide. Such mutations may be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.

(72) In some aspects, the present invention relates to a vector comprising a polynucleotide as described herein operably linked to a heterologous promoter. As used herein in the context of polynucleotides, the term heterologous refers to the combination of two or more polynucleotide molecules when such a combination is not normally found in nature. For example, the two molecules may be derived from different species and/or the two molecules may be derived from different genes, e.g. different genes from the same species or the same genes from different species. A promoter is thus heterologous with respect to an operably linked transcribable polynucleotide molecule if such a combination is not normally found in nature, i.e., that transcribable polynucleotide molecule is not naturally occurring operably linked in combination with that promoter molecule. In some embodiments, the promoter may be a constitutively active promoter, or may be an inducible promoter. In some embodiments, the promoter may be a viral promoter, bacterial cell promoter, a yeast cell promoter, a fungal cell promoter, an insect cell promoter, an animal cell promoter, or a mammalian cell promoter. A heterologous promoter may be a synthetic promoter that differs from an endogenous promoter by the addition, deletion or substitution of one or more nucleotides.

(73) Vectors

(74) Another aspect of the invention pertains to vectors (e.g., expression vectors), containing a polynucleotide encoding a polypeptide of the present invention. As used herein, the term vector includes a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked.

(75) Cells

(76) In another aspect, the present description features cells, e.g., transformed host cells or recombinant host cells that contain a polynucleotide or vector described herein. A transformed cell or recombinant cell is a cell into which (or into an ancestor of which) has been introduced a polynucleotide or vector of the present description by means of recombinant DNA techniques. Both prokaryotic and eukaryotic cells may be included, e.g., a bacterial cell, a yeast cell, a non-yeast fungal cell (e.g., a filamentous fungus), an insect cell, a plant cell, an animal cell, or a mammalian cell. In some embodiments, the cells may be from E. coli, Bacillus subtilis, or Streptomyces spp.

(77) Expression of Transgenic Proteins

(78) Several experiments were conducted trying to express KrCUT as well as other recombinant proteins in E. coli. Various vectors were tested with the codon-optimized gene of krCUT including and excluding the enzyme's natural signal sequence to express the protein intracellular. Extensive growth and induction conditions were tested but all experiments yielded very low or no expression of the protein.

(79) However, it has presently been found that expression of the KrCUT protein could be achieved by the fusion of KrCUT to the YebF protein. This strategy yielded reproducible protein expression and secretion.

(80) The active proteins (confirmed by N-terminal sequencing) have an insertion of two amino acids (leucine and glutamine) that have been introduced as linkers between YebF and the natural occurring KrCUT and KrCUT206 sequence, respectively. In the mature expressed protein, those two amino acids are not cleaved off through the autocatalytic action of the enzyme but remain on the protein's N-terminus.

(81) Monitoring the protein after production reveals a cleavage of the N-terminal fused YebF protein which also results in an increase in enzyme activity (about 5 times). At the same time this N-terminal protein cleavage appears to be a slow process which is not related to protease activity but rather to autocatalytic protein hydrolysis as it occurs also in a purified protein solution.

(82) Protein fractions after purification on SP-Sepharose contain KrCUT or other recombinant enzymes described herein with and without intact YebF-N-terminal fusion, which can be separated using size exclusion chromatography. Over time, the proteins undergo a change in the protein patterns by completely losing the fusion protein YebF. After storage for several weeks at 4 C. The only protein band left is KrCUT (or other recombinant proteins lacking the YebF carrier protein) showing a single protein band which consists now exclusively of the truncated recombinant protein.

(83) Uses & Compositions

(84) In some aspects, the present description relates to a composition comprising (i) the isolated recombinant protein as defined herein; and (ii) an additive. As used herein, an additive refers to an agent that, when added to a composition of the present description, increases performance, stability, and/or shelf-life, as compared to a composition comprising the recombinant protein alone.

(85) In some aspects, the present description relates to a composition comprising (i) the isolated recombinant protein as defined herein; and (ii) a bleaching agent and/or an organic solvent, wherein said recombinant protein retains said cutinase activity when present in said composition.

(86) In some embodiments, the composition may be a ready to use formulation wherein the bleaching agent may be present at a concentration of at least 100 ppm, 150 ppm, 200 ppm, 250 ppm, 300 ppm, 350 ppm, 400 ppm, 450 ppm, 500 ppm, 550 ppm, 600 ppm, 650 ppm, 700 ppm, 750 ppm, 800 ppm, 850 ppm, 900 ppm, 950 ppm, 1000 ppm, 1500 ppm, 2000 ppm, 2500 ppm, 3000 ppm, 3500 ppm, 4000 ppm, 4500 ppm, 5000 ppm, 5500 ppm, 6000 ppm, 6500 ppm, or 7000 ppm. In some embodiments, the bleaching agent may be present at a concentration of between 100 ppm, 150 ppm, 200 ppm, 250 ppm, 300 ppm, 350 ppm, 400 ppm, 450 ppm, 500 ppm, 550 ppm, 600 ppm, 650 ppm, 700 ppm, 750 ppm, 800 ppm, 850 ppm, 900 ppm, 950 ppm, or 1000 ppm, and 1500 ppm, 2000 ppm, 2500 ppm, 3000 ppm, 3500 ppm, 4000 ppm, 4500 ppm, 5000 ppm, 5500 ppm, 6000 ppm, 6500 ppm, or 7000 ppm.

(87) In some embodiments, the composition may be a dilutable concentrate wherein the bleaching agent may be present at a concentration of at least 3%, 4%, 5%, or 6% v/v.

(88) In some embodiments, the bleaching agent may be an oxidizing agent or a reducing agent. The oxidizing agent may include hydrogen peroxide, a peroxide other than hydrogen peroxide, a non-peroxide oxidizing agent, ozone, sodium percarbonate, sodium perborate, or any combination thereof. The reducing agent may include sodium dithionite, sodium dithionite, sulfur dioxide, a sulfite, a bisulfite, a sodium borohydride, or any combination thereof.

(89) In some embodiments, the organic solvent may be: methanol, 2-propanol, or another alcohol, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), or acetone. In some embodiments, the organic solvent may be present at a concentration of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% v/v. In some embodiments, the organic solvent may be present at a concentration of between 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or 25%, and 30%, 35%, 40%, 45%, or 50% v/v. In some embodiments, the organic solvent is DMSO and is present at a maximum concentration of 50% v/v. In some embodiments, the organic solvent is acetone and is present at a maximum concentration of 30% v/v.

(90) In some embodiments, the compositions of the present description may comprise one or more an additional recombinant enzyme(s). Examples of such additional recombinant enzymes may include: a lipase, an esterase, a pectate lyase, a pectinase, a cutinase, a cellulase, a hemicellulose, an amylase, or any combination thereof. In some embodiments, the composition does not comprise a protease that degrades or inactivates a recombinant protein as defined herein. In some embodiments, the composition comprises a protease and a stabilizer (e.g., a polyol and/or boric acid).

(91) In some embodiments, the compositions of the present description may comprise one or more additives such as a detergent; a surfactant; a buffer; a chelator (e.g., EDTA or GLDA); a stabilizer; or any combination thereof. In some embodiments, the composition comprises EDTA (ethylenediaminetetraacetic acid) at a concentration of between 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9%, and 10% w/v. In some embodiments, the composition comprises GLDA (L-Glutamic acid N,N-Diacetic acid Tetra sodium) at a concentration between 0.5%, 1%, 2%, 3%, or 4%, and 5% w/v). In some embodiments, the composition comprises GLDA at a concentration of at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% w/v). In some embodiments, the composition comprises GLDA at a concentration of at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% w/v).

(92) In some embodiments, the compositions may comprise an additive which is a stabilization buffer comprising one or more of sodium gluconate (e.g., 1-5% v/v), sodium carbonate (e.g., 1-5% v/v), sodium citrate (e.g., 1-5% v/v), and propylene glycol (e.g., 1-5% v/v). In some embodiments, the compositions may comprise a stabilization buffer comprising one or more of a polyol such as propylene glycol and glycerol, boric acid and boron derivatives, and sodium formate. In some embodiments, the composition comprises a polyol and/or boric acid. In some embodiments, the compositions of the present description may be: a cleaning product, a degreaser, a disinfectant, a bleaching product, or any combination thereof; or feed or a feed additive.

(93) In some embodiments, the present description relates to the use of the isolated recombinant protein as defined herein, or the composition as defined herein, for: (i) cleaning, degreasing, disinfecting, bleaching, biofilm control, or any combination thereof; (ii) for inactivating mycotoxin (e.g., ochratoxin and/or aflatoxin); (iii) bioscouring or other treatment of fabrics or textiles; (iv) catalyzing trans-esterification reactions; (v) vegetable oil extraction; or (vi) enhanced oil recovery.

(94) In some embodiments, the present description relates to the isolated recombinant protein as defined herein, or the composition as defined herein, for use in: (i) cleaning, degreasing, disinfecting, bleaching, biofilm control, or any combination thereof; (ii) for inactivating mycotoxin; (iii) bioscouring or other treatment of fabrics or textiles; (iv) catalyzing trans-esterification reactions; (v) vegetable oil extraction; or (vi) enhanced oil recovery.

(95) In some embodiments, the present description relates to the use of the isolated recombinant protein as defined herein, or the composition as defined herein, for the manufacture of an industrial product for: (i) cleaning, degreasing, disinfecting, bleaching, biofilm control, or any combination thereof; (ii) for inactivating mycotoxin; (iii) bioscouring or other treatment of fabrics or textiles; (iv) catalyzing trans-esterification reactions; (v) vegetable oil extraction; or (vi) enhanced oil recovery.

(96) In some embodiments, the present description relates to a kit comprising a recombinant protein as defined herein and a suitable container. In some embodiments, the kit may be for one or more of the above mentioned uses.

(97) In some aspects, the present invention relates to the use of a recombinant protein as described herein for inactivating mycotoxin.

(98) In some aspects, polypeptides of the present invention may be useful for mycotoxin detoxification (mycotoxin inactivation). Advantageously, and unlike some other fungal cutinases that may have been used for mycotoxin detoxification, polypeptides of the present invention may be useful for inactivating more than one mycotoxin. In some embodiments, polypeptides of the present invention may be useful for inactivating Ochratoxin. In some embodiments, polypeptides of the present invention may be useful for inactivating Aflatoxin. In some embodiments, polypeptides of the present invention may be useful for inactivating both Ochratoxin and Aflatoxin (e.g., from the fungi strains Aspergillus flavus and/or Aspergillus niger).

(99) In some aspects, the present invention relates to a kit for inactivating mycotoxins, said kit comprising a recombinant protein of the present invention as defined herein; and a suitable container.

(100) In some aspects, the present invention relates to the use of a recombinant protein as described herein for degrading a polymer. In some embodiments the polymer is a homopolymer or a heteropolymer. In some embodiments, the polymer is a polyester. In some embodiments, the polymer is: cutin; polycaprolactone (PCL); polylactic acid (PLA); poly(1,3-propylen adipate (PPA); poly(hydroxybutyrate); triacin; tributyrin; trimiristin; tripalmitin; polyvinyl acetate (PVA); polyethylene terephthalate (PET); or polytrimethylene terephthalate (PTT).

(101) In other embodiments, polypeptides of the present invention may be employed with one or more other enzymes (e.g., pectate lyase) for the degradation/hydrolysis of a polymer of interest.

(102) Applications of the recombinant proteins of the present invention in industries such as food, laundry/detergent, textiles, recycling, and/or polymer manufacturing are also envisioned. In some aspects, the present invention may relate to the use of a recombinant protein as described herein in cleaners/degreasers (industrial and/or domestic); animal feed; the treatment of surface in the pulp and paper industries; and/or in the degradation/control of biofilm.

(103) The present invention is illustrated in further details by the following non-limiting examples.

Example 1

Material and Methods

(104) 1.1 Reagents and Chemicals.

(105) p-Nitrophenyl (pNP)-esters were purchased from Fluka (Steinheim, Germany) or from Sigma-Aldrich (USA). Lactic acid, 6-hydroxyhexanoic acid, adipic acid, polycaprolactone (PCL; M.sub.n=42500; M.sub.w=65000), polylactic acid (PLA; M.sub.n=30000; M.sub.w=60000), poly(1,3-propylen adipate) (PPA; M.sub.n=42500; M.sub.w=4800), and poly(R)-3-hydroxybutyrate (PHB) were purchased from Sigma-Aldrich, Fluka or Alfa Aesar (Ward Hill, USA). Restriction endonucleases were from New England Biolabs (Pickering, ON, Canada), T4 DNA ligase was from Roche (Mannheim, Germany) and Titanium DNA polymerase from Clontech (Takara Bio company, USA).

(106) 1.2 Protein Expression (Small Scale).

(107) A single colony of E. coli JM109 cells harboring either YebF-KrCUT or YebF-KrCUT189 was grown at 30 C. on a rotary shaker at 200 rpm in 10 mL Terrific Broth (TB) medium supplemented with 100 g/ml ampicillin. The overnight culture was used to inoculate 1 L fresh TB medium, then grown to OD.sub.600 0.4-0.5 under the same culture conditions and induced with 0.1 mM IPTG for 16 h. The cells were collected by centrifugation at 6000 rpm for 30 min (Beckman centrifuge, Model J2-21M) and the supernatant was used as a source of cutinase. Protein concentration was determined using the bicinchoninic acid (BCA) method (Smith et al., 1985).

(108) 1.3 Fermentation in DASGIP System (Large Scale Protein Expression).

(109) A single colony of E. coli JM109 cells harboring either YebF-KrCUT or YebF-KrCUT189 were cultured in LB-media containing ampicillin (0.1 g/L) and incubated at 30 C. in a rotary shaker at 200 rpm overnight. The preculture (5% v/v of the total fermentation volume) was added to TB media (1 L) in a 2 L fermenter (DASGIP, Germany). The temperature was set to 30 C. and the pH was controlled at 7.0 by the addition of concentrated ammonium hydroxide. The cell broth was aerated at 1.0 wm and stirred at 400-900 rpm to maintain 30% dissolved oxygen. If necessary, the airflow was supplemented with oxygen. At an OD.sub.600 of 9 (7 h), cutinase expression was induced by the addition of IPTG (final concentration 1 mM); the temperature was set to 25 C., and glucose (33% w/w aqueous solution) was fed at an initial rate of 3 mL/h and gradually increased to 6 mL/h. The cells were harvested after 16 h and centrifugation of the cell broth (6000 rpm, 4 C., 20 min) yielded 0.82 to 0.87 L of supernatant that was used to purify the corresponding cutinase.

(110) 1.4 Protein Purification.

(111) All purification procedures were performed at 4 C. on an KTAexplorer 100 Air chromatography system (GE Healthcare). Culture supernatant containing active enzyme was concentrated and dialyzed against sodium phosphate buffer (20 mM, pH 7.0) by cross flow filtration using the QuixStand system (GE Healthcare) equipped with a hollow fibre cartridge (3 kDa cut-off). The enzyme solution was loaded on a SP-Sepharose FF column (XK 50/12) previously equilibrated with 20 mM sodium phosphate buffer (pH 6.0). The flow rate was 4.0 mL/min. The column was washed with the same buffer until no protein could be detected in the flow through, and the enzyme subsequently eluted with a linear gradient of 0-0.6 M NaCl. Active fractions were pooled and concentrated by ultrafiltration (membrane exclusion size 10 kDa, in a 100 mL stirring cell (Amicon, USA) and then applied to a HiLoad Superdex 75 prep grade (16/60) column previously equilibrated with 20 mM sodium phosphate buffer (pH 7.0) containing 150 mM NaCl. Protein was eluted with the same buffer at a flow rate of 1.5 mL/min and collected in 2 mL fractions. The protein profile was monitored by its absorbance at 280 nm.

(112) 1.5 Protein Sequencing.

(113) Purified enzymes were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (BioRAD, USA). N-terminal sequencing was carried out by the Sheldon Biotechnology Centre (McGill University, Montreal, Canada). Phenylthiohydantoin amino acids were analyzed by HPLC using a reversed-phase column.

(114) 1.6 Esterase Assay.

(115) Esterase activity was determined by monitoring the formation of pNP at 410 nm from various pNP-esters using a Beckman UV spectrophotometer (model DU 640) (21). Substrates were dissolved in isopropanol and were added to 50 mM sodium phosphate buffer pH 8.0 to give 10% (v/v) total isopropanol concentration in a 1 mL reaction volume. pNP-butyrate was used at a final concentration of 1.2 mM. For pNP-caprylate, myristate and palmitate, the final concentration ranged from 0.1 to 0.5 mM. The reaction (1 mL) was started by adding an appropriate amount of enzyme. One enzyme unit (U) is defined as the amount of enzyme that produced 1 mol of product (pNP) per min; specific activity is expressed as U/mg protein. A molar extinction coefficient of 15 mM.sup.1 cm.sup.1 for pNP is used for calculating enzyme activity.

(116) 1.7 Determination of Cutinolytic Activity.

(117) Apple (Golden Delicious) cutin was used as substrate. The outer layer of the fruit was peeled by hand and cutin preparation carried out as described by Gerard et al. (1993) with minor modifications. Discs of apple peels were incubated at 50 C. in oxalate buffer [(oxalic acid (4 g/L)/ammonium oxalate (16 g/L)] for 16 h and then collected and washed several times with deionized water. The sample was treated with 0.5% Novozymes Aspergillus niger pectinase (Sigma P2736) in 100 mM phosphate citrate buffer pH 5.8 for 16 h. The cutin sheets were washed with ethanol, followed by several washes with chloroform, and then incubated in chloroform at 45 C. for 16 h, and finally dried to constant weight. The cutin was ground in a Cyclone sample Mill (UDY Corporation, Fort Collings, Colo., USA).

(118) A typical reaction mixture (2 mL) contained 20 mg cutin and an appropriate amount of cutinase in 25 mM sodium phosphate buffer, pH 8.0. The mixture was incubated in a water bath at 37 C. with gentle shaking for 16 h. Assays were conducted in duplicate. At the end of the reaction, the remaining cutin was removed by centrifugation (10 min, 5000 rpm). The resulting solution was acidified with hydrochloric acid and cutin monomers extracted with chloroform/methanol, according to Bligh and Dyer (1959). The organic phase was removed by evaporation under an air stream and the dried residue dissolved in 1 mL chloroform/methanol (85:15) and used for further derivatization. Trimethylsilylation of cutin monomers was performed with BSTFA [N, O-bis(trimethylsilyl) trifluoroactamide] and TMCS (trimethylchlorosilane) provided by the supplier in the ratio 99:1 (Supelco, Bellefonte, USA) at 70 C. for 3 h, and the composition of the mixture determined by GC-MS.

(119) 1.8 Analysis of Cutin Monomers by GC-MS.

(120) Cutin monomers were identified by GC-MS on an Agilent 6890 gas chromatography system coupled to a 5973 quadrupole mass spectrometer. 1 L was injected under splitless condition on a 50 m0.20 mm0.33 m DB-5MS capillary column (Agilent). The oven temperature program was 70 C. (for 1.39 min) to 136 C. at 64 C./min and to 290 C. at 2.5 C./min (for 55 min). Helium was used as carrier gas. The temperature at the injection port was 250 C. The detector was run in El mode at 70 eV. Masses were scanned between 50 and 650 amu.

(121) Components were identified by comparison of the MS spectra of their trimethylsilyl (TMSi) derivatives, as methyl ester TMSi ether or as TMSi ester TMSi ether, with published spectra (Kallio et al., 2006; Philipps et al., 2002).

(122) 1.9 HPLC-Analysis of Monomers after Polymer Degradation.

(123) HPLC analysis was carried out on a Waters system consisting of a model 600 pump and 717 Plus autosampler, equipped with a refractive index detector (Waters, model 2414) or PDA detector (model 2996). Organic acids were analyzed with a Transgenomic ICSep ICION-300 (300 mm7.8 mm OD) column using 0.01N H.sub.2SO.sub.4 as mobile phase at a flow rate of 0.4 mL/min at 35 C. Retention times are as follows: lactic acid, 23.2 min; 6-hydroyhexanoic acid, 45.2 min; and adipic acid, 33.4 min.

(124) 1.10 Determination of Polymer Degradation by Size Exclusion Chromatography.

(125) Size exclusion chromatography (SEC) was performed using a multi-detection system from Viscotek (Houston, Tex.) consisting of a Model 302 Triple Detector Platform, including a refractive index detector, a four capillary viscometer and a 2 angles laser light scattering detector, and a GPCmax Integrated pump, autosampler, and degasser. Molar masses of the samples were determined with universal calibration with the refractive index detector and the viscosity detector. The universal calibration curve was based on polystyrene standards [Polymer Laboratories (Amherst, Mass.) and Aldrich (Oakville, ON)] using the molar masses determined by the manufacturer and the intrinsic viscosities measured by the apparatus. The software OmniSEC software from Viscotek was used for data collection and calibration. Separation in THF was performed by injecting 100 L of 1.5-2 mg/mL solutions of standards or samples into thermostatically controlled SuperRes columns [35 C.; PAS-102, PAS102.5, PAS-103L, each 300 mm8 mm; PolyAnalytik (London, ON)]. The flow rate was 1 mL/min.

(126) 1.11 Degradation of Polyesters.

(127) An appropriate amount of cutinase was added to 20 mg of the polymer to be analyzed (PCL, PLA, PHB, and PPA) in 50 mM phosphate buffer, pH 8. The reaction mixture (2 mL) was shaken at 37 C. at 175 rpm on an orbital shaker. Samples were taken after 1, 3, 6, and 24 h and aliquots of 50 L were analyzed by HPLC; the remaining mixtures were frozen and subsequently lyophilised. The resulting solid (40-50 mg) was dissolved in THF, filtered, diluted (1.5-2 mg/mL), and analyzed by SEC.

(128) Alternatively, a thin film was prepared from 1 g of commercial available PCL pellets and used for degradation. The polymer was dissolved in dichloromethane in a 1 L beaker. The solvent was allowed to evaporate overnight and the resulting film dried to constant weight under reduced pressure. Prior to enzymatic treatment the film was cut into pieces (20 mg) of approximately 1.5 cm1.5 cm and thickness of approximately 0.1 mm. The reaction mixture was monitored by HPLC and SEC as described above.

(129) 1.12 Action of KrCUT on Plant Material.

(130) Hemp stems (50 cm, 25 g) were treated enzymatically with KrCUT (100 U/g) together with a cloned pectate lyase (20 U/g) described by Xiao et al. (2008). The reaction was carried out in 50 mM Tris-HCl, pH 8.0 supplemented with 0.5 mM CaCl.sub.2 (required for pectate lyase). The samples were incubated at 37 C. with gentle shaking at 100 rpm for 16 h. The stems were removed from the enzymatic solution, peeled off by hand, and the resulting fibre washed with hot water to inactivate the enzymes. After removal of the outer layer debris by rinsing with cold water, the fibres were air-dried. A sample of the hemp stem treated only with pectate lyase was prepared for comparison. These experiments were conducted in duplicate. The dried fibres were spread onto a microscope slide and observed by light microscope. A scanning electron microscopy (SEM) analysis was also conducted using a Hitachi S-4700 at a voltage of 2 kV to observe the fibre surface and also fiber bundle.

(131) 1.13 Effect of KrCUT in Removing Pectin from Hemp Fibre.

(132) Hemp fibres (German variety, 150 mg) were treated with either pectate lyase (PL, 10 U) or PL combined with various amounts of KrCUT (40, 60 and 100 U). Control experiments containing no enzyme or 100 U of KrCUT were conducted for comparison. After incubation at 37 C., aliquots of the reaction mixture were taken at 1 h 30 min and 3 h, and analyzed for pectin degradation products using the thio-barbituric acid (TBA) method (52) which detects released unsaturated compounds spectrophotometrically at 550 nm.

(133) 1.14 Adsorption Assays.

(134) Adsorption assays were carried out following procedures of Kasuya et al. (1999). Purified KrCUT and KrCUT189 at concentrations ranging from 0.16 to 3.2 mg/mL were added to a suspension of 25 mg PHB in 50 mM sodium phosphate (pH 8.0) in a total volume 1 mL. The mixture was incubated at room temperature with gentle shaking for at least 3 h. The PHB polymer was removed by centrifugation and the concentration of protein in the supernatant was determined by the BCA method. The concentration of bound protein at a particular concentration of cutinase was calculated as the difference between a control without added PHB and free cutinase after incubation with PHB. An isotherm of [Bound](mg/mg PHB) vs [Free] (mM) was generated and binding parameters were determined by non-linear regression using the Langmuir equation:

(135) $E_{\mathrm{ad}}=E_{\max }\left(\frac{K\left[E\right]e}{1+K\left[E\right]e}\right)$
where [E]=E.sub.ad+[E]e, is the concentration of the protein added; E.sub.max is the maximum amount of protein adsorbed on the polyester granules, and K is the adsorption equilibrium constant of the protein.

Example 2

Sequence and Phylogenetic Analyses of Cutinase from K. radiotolerans

(136) The 885-bp Krad_4111 locus tag of the K. radiotolerans genome (YP_001363838.1; Copeland, 2007) was selected as the result of a BLAST search using the amino acid sequence of FsCUT as a query sequence. A polypeptide having 294 amino acids corresponding to the full length sequence of a cutinase from K. radiotolerans was identified. A bioinformatic analysis of the 294-amino acid sequence of full length KrCUT (hereinafter referred to as ssKrCUT; SEQ ID NO: 1) revealed a modular domain organization consisting of a 33-amino-acid signal peptide (amino acids 1-33 of SEQ ID NO: 1), a catalytic domain (amino acids 34-222 of SEQ ID NO: 1; SEQ ID NO: 2), and unlike other characterized cutinases, the presence of a C-terminal extension made up of a Pro/Thr-rich linker (amino acids 223-239 of SEQ ID NO: 1; SEQ ID NO: 3), and a designated polymer-binding domain (amino acids 240-294 of SEQ ID NO: 1; SEQ ID NO: 4) (FIG. 1).

Example 3

Cloning of Cutinase-Encoding Gene and Variants

(137) A synthetic gene encoding full-length cutinase from K. radiotolerans identified in Example 2 but lacking the 33-amino acid endogenous N-terminal signal sequence was constructed after codon optimization by the reduction of G+C content from 77.2% to 59% for its expression in E. coli. The synthetic gene having the nucleotide sequence set forth in SEQ ID NO: 5 was cloned in a recombinant pUC57 plasmid provided by GenScript Corporation (Piscataway, N.J. USA). The specific primers, P1: 5-GAGCTGCAGGCGACTTGCAGTGACGTC-3 and P2 5-GCAGAAGCTTTCAAC AAGCAGTTACCAAG-3, were then designed to amplify the mature portion of the KrCUT-encoding gene (amino acids 34-294 of SEQ ID NO: 1; SEQ ID NO: 6; herein referred to as KrCUT); P1 and P3: 5-TGTAAGCTTTCATGTGGTAGGA ACCGGCGT-3 were used to amplify a truncated version KrCUT206 (amino acids 34-222 of SEQ ID NO: 1; SEQ ID NO: 2; herein referred to as KrCUT189) lacking the C-terminal 72 amino acids, which includes the T/P-rich linker as well as the polymer-binding domain. The indicated primer sets were designed to incorporate a 5 PstI site and 3 HindIII site (underlined) for directional cloning in the IPTG-inducible vector pKK223-3. The PCR parameters were: 94 C. 3 min, 30 cycles of 94 C. for 45 s, Tm-3 C. for 30 s, 68 C. for 1 min, followed by a final extension at 68 C. for 10 min.

(138) To express KrCUT and KrCUT189 in a secreted form, the E. coli extracellular carrier protein YebF (Zhang et al., 2006; SEQ ID NO: 8) was used to construct fusion proteins. The gene encoding YebF (366-bp minus stop codon but including the signal peptide; SEQ ID NO: 7) was amplified by oligonucleotides P4: 5-CGAGGAATTCATGGAGAAAAACATGAAAAAAAG-3 and P5: 5-GAACTGCAGACGCCGCTGATATTCCGC-3, containing the EcoRI and PstI restriction sites (underlined). The PCR fragment was gel purified and cloned upstream of the cutinase gene in pKK223-2 to produce pKK223-3-YebF-KrCUT and pKK223-3-YebF-KrCUT189. These recombinant plasmids were transformed into E. coli JM109 cells by conventional techniques (Sambrook et al., 1989). The cloned genes were verified by DNA sequencing using a Big Dye DNA sequencing kit (Applied Biosystems) and an automated DNA sequencer (Model 377, ABI Prism). The nucleotide sequence encoding the YebF-KrCUT fusion protein is set forth in SEQ ID NO: 9 and the corresponding amino acid sequence of YebF-KrCUT is set forth in SEQ ID NO: 10. The nucleotide sequence encoding the YebF-KrCUT189 fusion protein is set forth in SEQ ID NO: 11 and the corresponding amino acid sequence of YebF-KrCUT189 is set forth in SEQ ID NO: 12.

Example 4

Expression and Purification of YebF-KrCUT and YebF-KrCUT189 Fusion Proteins

(139) The YebF-KrCUT and YebF-KrCUT189 fusion proteins were expressed from E. coli as described in Example 1, sections 1.2 and 1.3.

(140) Cutinase activity, measured using pNP-butyrate as substrate as described in Example 1, section 1.6, was detected in E. coli [pKK233-2-YebF-KrCUT and pKK233-2-YebF-KrCUT189] culture 16 h after IPTG induction with the maximum activity found by 20 h (not shown).

(141) Production of the 39-kDa YebF-KrCUT (FIG. 2A, lane 1) fusion protein was confirmed by SDS-PAGE analysis), consistent with the summed masses of the 10-kDa YebF carrier protein and the 29-kDa mature KrCUT. FIG. 2A, lane 1, resulted from one column purification step using the SP-Sepharose FF as described in Example 1, section 1.4. Production of the YebF-KrCUT189 fusion protein was also confirmed by SDS-PAGE analysis.

(142) The YebF-KrCUT fusion protein was further purified as described in Example 1, section 1.4 to electrophoretic homogeneity in two steps involving cation exchange and size exclusion chromatography. Proteins bound on the SP-Sepharose eluted in two active peaks: peak 1 (minor) at a lower ionic strength, and peak 2 (major) at higher ionic strength (not shown). The height of peak 1 eluting from the SP-Sepharose column increased over time while peak 2 decreased in height, indicating some unknown processing of peak 1 protein species under storage conditions (not shown). This was confirmed by SDS-PAGE analysis of fractions from the major peak 2, which showed two protein bands of M.sub.r 39-kDa and 31-kDa (for YebF-KrCUT). These two species could be separated by an additional SEC step (FIG. 2, lanes 2 and 3). In FIG. 2A, lanes 2 and 3, the two bands from lane 1 were separated by loading the fraction of lane 1 on a HiLoad Superdex 75 prep grade (16/60) column.

(143) The YebF-KrCUT189 fusion protein, which also eluted in two active peaks, was purified to electrophoretic homogeneity the same way as for YebF-KrCUT. FIG. 2B, lane 4 represents YebF-KrCUT189 from HiLoad Superdex 75 prep grade (16/60) column, the fraction was collected after a first column using SP-Sepharose FF.

(144) N-terminal amino acid sequencing performed as described in Example 1, section 1.5 helped to establish the identity of each of the protein bands with respect to YebF-KrCUT and YebF-KrCUT189. The procedure is described with respect to YebF-KrCUT in the following paragraph, but a similar approach was used to establish the identity of each of the protein bands for YebF-KrCUT189.

(145) The sequence ANNETSKSVT derived from the 39 kDa protein band corresponds to the first 10 amino acids of the mature portion of YebF, confirming previous result that the carrier protein is cleaved immediately after the 21-amino acid secretory leader sequence (Zhang et al., 2006). The N-terminal amino acid sequence (AT[A]SDVDVVF) of the 31-kDa species is virtually identical to the first ten amino acids of the mature cutinase. The discrepancy of Ala in the third position versus Cys in the DNA-predicted sequence is due to the fact that Cys without modification is not detectable by Edman degradation.

(146) The above analysis confirmed that when the YebF-KrCUT and YebF-KrCUT189 proteins were purified immediately after fermentation, they exclusively yielded the intact YebF-KrCUT and YebF-KrCUT189 fusion proteins, respectively. Upon storage for several weeks at 4 C., the intact fusion proteins underwent processing to produce a lower molecular weight species corresponding to the cleavage of the YebF portion, resulting in the polypeptides KrCUT and KrCUT189.

(147) The presence of YebF appeared to greatly decrease the activity of the fusion proteins. For example, the processed KrCUT exhibited some three-time higher specific activity compared to that of YebF-KrCUT (162 versus 47 U/mg with pNP-palmitate as substrate). The results are shown in FIG. 6, in which fractions C1-C10 correspond to the first peak (KrCUT) and fractions D9-D11 correspond to the second peak (YebF-KrCUT).

Example 5

Substrate Range

(148) The KrCUT189 protein was purified in a similar way as YebF-KrCUT as described above (FIG. 2B) and its activity towards a number of p-NP esters was compared to those of KrCUT (FIG. 3). With KrCUT, the highest activity was obtained with pNP butyrate showing a 8-fold higher activity over pNP-palmitate which is in accordance with the activity profile of other cutinases (Kim et al., 2003). The KrCUT189 variant generally gave lower activity with various substrates compared to the full length protein. The highest activity of KrCUT189 was towards pNP-myristate, which was surprisingly higher than that of KrCUT. In both cases, there is little activity towards pNB-acetate.

Example 6

pH Optimum and Thermostability

(149) Both KrCUT and KrCUT189 showed a pH optimum of 8 (FIG. 4). Universal Buffer solutions were prepared according to Britton and Robinson (Surinenaite et al., 2002), having the selected pH value from 5-11. Activity of KrCUT and KrCUT189 were measured at the indicated pH, using p-nitrophenyl butyrate as substrate as described in Example 1, section 1.6). Curves were obtained by plotting values of activity against pH.

(150) Enzyme stability was studied over the temperature range of 40-60 C. using purified KrCUT. Aliquots were withdrawn at defined times (10, 30 and 80 min), chilled on ice, and assayed for activity against p-NP butyrate at room temperature. KrCUT appeared thermostable up to 45 C. At 50 C. and above there was virtually no activity left after 10 min of heat treatment (FIG. 5).

Example 7

Potential Inhibitors of Cutinase Activity

(151) KrCUT cutinase activity was assayed as described in Example 1, section 1.6 in the presence of various chemicals using p-NP palmitate as substrate under standard assay conditions. The zinc ion dramatically inhibited cutinase activity with a complete loss of activity at 1 mM and over 90% at 0.1 mM. Mg.sup.2+ showed only 19% enzyme inhibition at 10 mM; and 50% for Fe.sup.2+ at 1 mM. Cu.sup.2+ at 1.0 mM had little effect. The enzyme was not affected by the metal-chelating agent EDTA even at 10 mM. The anionic surfactant SDS was a strong inhibitor of cutinase even at 0.01% retaining only 28% activity. 0.1% SDS resulted in a complete loss of activity. Organic solvents, e.g. alcohols exhibited a strong inhibiting effect on cutinase in concentration above 15-20%. In particular, the 2-propanol used to dissolve the substrate and that is present in the standard assay at 10% (v/v), strongly reduces enzyme activity when it exceeds 15%. Dimethylsulfoxide (at 30%) and dimethylformide (at 5%) inhibited cutinase activity by about 70%. Interestingly, para-chloromercuribenzoate at concentrations 0.01 to 1 mM had no discernible effect on KrCUT implying that there is no free sulfhydryl group among the six cysteines found in KrCUT.

Example 8

Hydrolysis of Cutin

(152) To measure cutinase activity, assays of cutin hydrolysis were performed as described in Example 1, section 1.7 at two different enzyme concentrations. Characteristic cutin components, C.sub.16 and C.sub.18 hydroxyl fatty acids, were identified by GC-MS analysis. One representative mass spectrum of the enzymatic reaction is shown in FIG. 7. 18-Hydroxy-octadeca-9,12-dienoic acid, 10,16-dihydroxyhexadecanoic acid and 9,10,18-trihydroxyoctadecanoic acid, with retention times of 55.13, 55.92 and 64.20, respectively, were found to be the major products of hydrolysis (Table 1).

(153) TABLE-US-00004 TABLE 1 Momoneric products released from apple cutin by cloned K. radiotolerans cutinase. Retention 20 Units 200 Units time Cutin hydrolysis products Area (%) Area (%) 55.13 18-hydroxy-octadeca-9,12- 18.0 6.3 2.22 0.13 dienoic acid 55.24 9,16-dihydroxyhexadecanoic n.d 3.81 0.62 acid 55.92 10,16-dihydroxyhexadecanoic 39.25 2.5 27.74 0.41 acid 63.37 9,10,18-trihydroxyoctadec- n.d 13.25 0.40 10,12-dienoic acid 64.20 9,10,18-trihydroxyoctadecanoic n.d 25.55 0.10 acid 66.19 9,10,18-trihydroxyoctadec-9 n.d 6.25 0.40 enoic acid Hydrolysis was carried out at two different units of activity of cutinase. Not detected (n.d)

(154) Additional components of C16 and C18 fatty acids were identified as 9,16-dihydroxyhexadecanoic acid (55.24 min), 9,10,18-trihydroxyoctadec-10,12-dienoic acid (63.37 min), and 9,10,18-trihydroxyoctadec-9 enoic acid (66.19 min).

Example 9

Degradation of Synthetic Polyesters by KrCUT and KrCut189

(155) An initial plate clearing assay indicated that a lawn of E. coli JM109 cells harboring YebF-KrCUT was capable of producing a large clearing zone with PCL as substrate (not shown). To quantify this further, pellets of polycaprolactone (PCL) as well as films of PCL were incubated with KrCUT at various enzyme concentrations (1.25 M, 2.5 M, and 5 M representing 4.7, 9.5 and 19 enzyme Units) at 37 C. The degradation of the polymer was followed over time by analyzing aliquots of the reaction mixture by HPLC for the detection of the released monomer (6-hydroxyhexanoic acid) and characterization of the remaining polymer by SEC after lypholization of the sample. KrCUT was able to release nearly 90% of the acidic monomers with enzyme concentration of either 2.5 or 5 M in 6 hours. Even with a very low concentration of the enzyme (1.25 M) thin films of PCL were nearly completely degraded after 24 hours (FIG. 8A).

(156) SEC analysis confirmed the results obtained by HPLC. Comparison of the peak area corresponding to that of the polymer showed a gradual decrease over time with complete disappearance of the polymer in experiments conducted with 2.5-5 M after 24 hours. This correlated well with the increase in acid detected (Table 2).

(157) TABLE-US-00005 TABLE 2 Evidence of PCL degradation: comparison of SEC and HPLC analysis M.sub.n M.sub.w time (h) PCL (SEC) (Daltons) (Daltons) acid (HPLC) 0 100% 44000 63300 0% 1 61% 53800 64400 28% 3 27% 55000 66600 60% 6 12% 58200 68100 85% 24 0.3% 69500 74000 100%

(158) A dramatic increase in M.sub.n and M.sub.w from 44000 and 63300 to 69500 and 74000 emphasized the preference of KrCUT for low molecular polymers. The degradation of PCL pellets proceeded more slowly with 18% of the maximum yield of 6-hydroxy hexanoic acid were detected in the reaction mixture after incubation with 2.5 M of protein for 24 hours (FIG. 8A). The decrease in depolymerization is presumably due to the limited surface area of the PCL pellets compared to the films. Nearly identical results were obtained when PCL pellets and PCL films were incubated with KrCUT189 (2.5 M) (FIG. 8A).

(159) Both KrCUT and KrCUT189 (2.5 M; 2 Units) only degraded approximately 5% of PLA (FIG. 8B). However, in the case of poly(1,3-propylene adipate), a polymer consisting of alternating diacid and diol subunits, 12% and 17% of adipic acid were found in the reaction mixture containing KrCUT and KrCUT189, respectively, after 24 hours (FIG. 8B). All in all, it appears that both enzymes exhibited a preference for longer chain monomers such as 6-hydroxy hexanoic acid and adipic acid (both C.sub.6 unit in polymer) compared to lactic acid (C.sub.2 unit).

Example 10

Polymer-Binding Domain of KrCUT

(160) Different polymers (cellulose, PLA, PCL, and PHB) were tested to evaluate the ability of KrCUT and KrCUT189 to bind these materials. Binding assays for PCL and PLA could not be performed since both polymers were degraded by the enzymes. KrCUT did not bind to cellulose, indicating that the binding domain of the cutinase is distinct from a cellulose binding domain (CBD). On the other hand, KrCUT binds efficiently to PHB but did not degrade. The KrCUT189 variant showed no binding to PHB, indicating that essentiality of the C-terminal extension for this activity.

(161) The kinetics of KrCUT adsorption to PHB is shown in FIG. 9. The curve shows the relation between the adsorbed cutinase and the equilibrium concentration of the enzyme, with the adsorption to the PHB surface expressed by the Langmuir adsorption equation. As a result, the maximum amount of cutinase absorbed on the PHB granules (Emax) was determined to be 23.680.03 g/mg, and the adsorption equilibrium constant (K) was calculated to be 0.120.03 ml/mg PHB.

Example 11

Effect of KrCUT on Hemp Fibre

(162) The quantity of pectin released from natural hemp fibre by KrCUT in conjunction with the action of a thermostable pectate lyase was assessed as described in Example 1, section 1.13. The thermostable pectate lyase alone released a certain amount of pectin as expected of the pectinolytic activity (Xiao et al., 2008). With an increasing amount of added cutinase, a higher percentage of pectin degradation products were detected when the quantity of added pectate lyase was fixed. When KrCUT was incubated with hemp fibre at the highest concentration in the absence of pectate lyase, negligible amounts of pectin from the fibre material were detected as compared to a control reaction without any added enzyme (FIG. 10).

(163) The effect of KrCUT on the hemp fibre integrity was visualized using light microscopy and SEM. Light microscopic images revealed that treatment of fibres by both cutinase and pectate lyase appeared to produce more separated fibre bundles, whereas pectate lyase alone showed more fibres embedded in pectin and wax material (FIG. 11). The SEM pictures showed that cutinase treatment gave more separated fibres having a cleaner surface, compared to those of pectate lyase treated samples.

Example 12

Effect of KrCUT on Inactivation of Mycotoxin

(164) Fungi strains Aspergillus flavus and Aspergillus niger were cultivated and used for Aflatoxin and Ochratoxin isolation, respectively.

(165) Reactions were performed in 2 mL volumes in Eppendorf tubes comprising the mycotoxin to be analyzed and 0.3 mg/mL KrCUT enzyme. Ochratoxin A was added at a concentration of 200 ppb. The samples for Aflatoxin contain the B1 and B2 types of the mycotoxin in the concentration of 600 ppb and 9 ppb, respectively. In control reactions, the enzyme volume was replaced by an equivalent amount of water. The reactions were incubated at 37 C., samples were withdrawn at time periods 0, 1 h, 2 h, followed by the addition of equal volume of methanol 80% (v/v) for extraction. Reactions were stored, if needed until chromatographic analysis.

(166) Analysis: samples were centrifugated and the supernatant analyzed for Ochratoxin A and Aflatoxin by LC-MS/MS as described by Rudrabhatla et al., 2007. Results are shown in Tables 3 and 4.

(167) TABLE-US-00006 TABLE 3 Effect of KrCUT on Ochratoxin Incubation time (h) Residual Ochratoxin % 0 100 1 56.63 2 0.68

(168) TABLE-US-00007 TABLE 4 Effect of KrCUT on Aflatoxin mixture Residual Incubation time (h) Residual Aflatoxin B1 (%) Aflatoxin B2 (%) 0 100 100 3 48 48

(169) The results in Tables 3 and 4 show that the KrCUT is able to act on both aflatoxin and ochratoxin, in contrast to most other cutinases that can act only on one type of mycotoxin.

Example 13

Effect of KrCUT on Inactivation of Mycotoxin

(170) Fungi strains Aspergillus flavus and Aspergillus niger were cultivated and used for Aflatoxin and Ochratoxin isolation, respectively.

(171) Reactions were performed in 2 mL volumes in Eppendorf tubes comprising the mycotoxin to be analyzed and 0.3 mg/mL enzyme. The samples for Aflatoxin contain the B1 type of the mycotoxin in the concentration of 1000 ppb. In control reactions, the enzyme volume was replaced by an equivalent amount of water. The reactions were incubated at 37 C., samples were withdrawn at time periods 0, 1 h, 2 h, 4 h and 24 h followed by the addition of equal volume of methanol 80% (v/v) for extraction. Reactions were stored, if needed until chromatographic analysis.

(172) Analysis: samples were centrifugated and the supernatant analysed for Ochratoxin A and Aflatoxin by LC-MS/MS as described by Rudrabhatla et al., 2007. Results are shown in Table 5.

(173) TABLE-US-00008 TABLE 5 Effect of KrCUT on Aflatoxin Incubation time (h) Residual Aflatoxin B1 (%) 0 100 1 80 2 61.5 4 49 24 20

(174) The results in Table 5 show that KrCUT is able to reduce amount of 1000 ppb Aflatoxin B1 to 200 ppb within 24 h.

Example 14

Construction of Recombinant Fusion Polypeptides Having a Cutinase Catalytic Domain Operably Linked to a Polymer-Binding Domain

(175) Recombinant fusion polypeptides having a cutinase catalytic domain operably linked to a polymer-binding domain may be constructed using basic recombinant DNA cloning techniques, making use of suitable domains (cutinase catalytic domains, linker domains, and polymer binding-domains) from different cutinases. Examples of different cutinases and the domains therein are shown in Table 6.

(176) TABLE-US-00009 TABLE6 Examplesofdifferentcutinasesandthedomainstherein Cutinaseaminoacidsequence(signal sequencesareinlowercaseletters; Gene/ cutinasecatalyticdomainsareinbold; SEQ proteinname/ linkersareitalicized;polymer ID organism bindingdomainsareunderlined) NO: >GI:502883354 mqvtptrrtlagyvaavaalasvttlatgasaAPACPDVEL 13 WP_013118330 VFARGTGEAAGLGIVGRPLERALAAELPGRTVVATAVDYAA cutinase SSSQASAGPGSGDMVAKVRSRAAACPGTQFVLGGYSQGATV [Cellulomonas TDLALGIRTGVTAGTALPEDLAARVAAVVVYGNPLGLTRRT flavigena] IAQAAPAFATRTVEYCNAGDPVCEPGGGRFTAHITYATNGT VLEGARFAAARVTAPAPTPTPGPTGTPAPVPSAEPTPAPGD TCVTASSLQHVRDGRAYPLWMRTYARGSGDPLGVLSSRTVV SLQADGTDTWRKVAAC >gi|697974256| msitpggrkvaaalittaaltstaaiatpataaapvagpva 14 gb|KGM14009.1| tsTGDCPDVHVVFARGTGEPRGLGIVGRPFVSDLGDALPTM cutinase TVTSYAVNYSANASQTSAGPGAGDMTSHVTSMAARCPGTQF [Cellulomonas VLGGYSQGATVTSIAVGARSTSIRSRVLPANLEPRVAAVVV bogoriensis FGNPLGLTRRTIASEAPAYAAKSRDYCNRSDTVCGGRGDAR 69B4= DSM GGHLAYVSNGSVADGAAFAASLVGVAPGDPASCVTARAVDH 16987] VEADRAFRSVLRAYARGSRDPLGRLTSSDLVSLQRTGQDSW SVVPAC cutinase mstspvrrtltaallaaglaatsvalspaasatslpaaapl 25 [Cellulomonas sAGSCPDVQVVFARGTGERAGLGIIGRPFARALADELPGMT bogoriensis VTSHAVDYAAAASQRSAGPGATAMTDHVTAMAARCPGTQFV 69B4= DSM LGGYSQGATVTSIALGIRAGTTTGRAIPDELSDRVAAVVVF 16987]; GNPLGMRGQTIASASRTYADRAKDYCNSGDSICGRQPSTGR accession GTHTGYATNGSTTDGARFAAGLVTANPPEAAPPTAPPTTPP KGM14008.1 TTPAPPSDQVTCVTAQVSEHVEARRAIRGLTRAYARGTLED IGRLRSTEEVSLRRSGTFSWTPTASC >gi|697886498| mhqltppaapsrpraarrprrllaaalalaatsgalstlaa 15 gb|KGM03336.1| ptavaAPACADVEVLFARGTGEAPGLGVLGTPFVRSVTSAL hypothetical SDRTVTSYAVNYAAESSQRSAGPGATDLTNHLTATAAACPG protein TRFVLGGYSQGATVVDLALGIRTGTTTGTAIPAALEPRVAA Q760_05675 IVVFGNPLGISGRTIATASPTYAARARDFCATGDPVCGGGS [Cellulomonas SFAAHLAYRTNGDVTAGADFAAGLARATTVPVPTPTVPGTP cellaseaDSM TPSPTAPGTPTPVPTTSPTPAPSPTAPGAACVRASTRAHVE 20118] AGRAERRHGIAYATGSGDRIGWVSSFVRVSVQQTADGWERV LSC >gi|521090090|ref| mvhnrlrkltllacavvglagtatvgtasgatsaaACSDLE 16 WP_020420995.1| IVFARGSGEAPGLGITGTPLVSDVKSALSSASVSSYAVDYA cutinase ASYDQTSAGPGATDMSNHIKSTAAACPDTKFAIGGYSQGAS [Amycolatopsis VTDIAIGIRTYLGTGQTIPTELAPRVVAVIAFGNPLGLYGQ sp.ATCC39116] TIKTASPTYGPKSLEFCNRGDNVCGGTGTGPGYGHLSYARD GSVDQAAAFIAKQYNAS >gi|648619273:1- mrlarswkqravavfltavagtlgagitaspvsAATCSDVD 17 219cutinase VVFARGSGELPGLGITGTPFVNSVKQGLTGKTVSSYAVNYA [Frankiasp. ADIAQTSDGAGATDMTRHVRSVAASCPNTKFVLGGYSQGAS BMG5.12] VTDISIGIRTFLGSGETIPTELAPRVAAVVVFGNPLALFGQ KITTASPLYGPKAKEFCNLGDPVCAGGFNVFAHLTYGFDGS TANGASFAVSKVRA >gi|383779171|ref| mnkrrkrnvatvgicaavvaagavitpqafagtlfggratt 18 YP_005463737.1| aAGDCSDVELVFARGTGEPQGLGIVGRPLARELAAALPDLA putative VGSFAVVYAAAGNQRSAGPGATNMSRHITEVAGECPDTRFV cutinase IGGYSQGASVTDIAIGIRGAGTAGEAIPERLADRVAAVVVF [Actinoplanes GNPLGLQRRTIAGSSAVFGPKAKEFCNTGDPVCGGGGNFAA missouriensis HLAYPRNGSVQQAAAFAASKIAG 431] >gi|589395439| msylltvrrvvtavaltatgltgiafaaspaTAACSDVQVV 19 gb|EXG80196.1| FARGSTEAPGLGILGRPLVSAVQQQLPGLTVDSYAVDYAAN Cutinase VSQTSAGPGATDMSDHITEVAARCPDTEFVIGGYSQGASVT [Cryptosporangium DIAIGIRTTLGRGGTIPENLAPRIKAVTVFGNPLRLSRQTI arvumDSM NSASQLYGRKAIDICATGDPVCGNGANAAAHLRYAFDGSVT 44712] RAAQFAANLVRTT >gi|653334177|ref| mkrrliaysiaalaisatavalptgvasAAPCSDVDVSFAR 20 WP_027494901.1| GTGELPGLGITGTPFVNSVKSQLSDRSVSTYAVNYAADFTQ MULTISPECIES: ASAGPGSRDLVAHLNSVAASCPSTKFVIGGYSQGATVVTNA cutinase VGLRTPSSFTGAVIPAAIADRIEAVVVFGNPFGLTGRKIET [Rhodococcus] ASSTYGSRTNSFCNFGDPVCQIGGFNTFAHLTYGTNGSTTQ GASFAAAQVRS

(177) Further examples of constructions of fusion proteins are shown below.

(178) 14.1 Fusion Protein CfKr

(179) Amino acid sequences of this construct contain the catalytic domain of cutinase from Cellulomonas flavigena (CfCUT) fused to the C-terminal region (T/P-rich linker and polymer binding domain) from Kinecoccus radiotolerans cutinase. The amino acid sequence of this fusion protein is set forth in SEQ ID NO: 21, in which residues 1-187 correspond to the cutinase catalytic domain from CfCUT, residues 188-206 correspond to the T/P-rich linker of KrCUT, and residues 207-262 correspond to the polymer binding domain of KrCUT. To ensure extracellular secretion of this fusion protein, the N-terminus thereof can be fused to the C-terminus of YebF (SEQ ID NO: 8) to give YebF-CfKr encoded by the nucleotide sequence of SEQ ID NO: 22.

(180) 14.2 Fusion Protein KrCf

(181) Amino acid sequences of this construct contain the cutinase catalytic domain of KrCUT (SEQ ID NO: 2) fused to the T/P-rich linker of KrCUT (SEQ ID NO: 3), which in turn is fused to polymer binding domain of CfCUT. The amino acid sequence of this fusion protein is set forth in SEQ ID NO: 23, in which residues 1-189 correspond to the cutinase catalytic domain from KrCUT, residues 190-206 correspond to the T/P-rich linker of KrCUT, and residues 207-262 correspond to the polymer binding domain of CfCUT. To ensure extracellular secretion of this fusion protein, the N-terminus thereof can be fused to the C-terminus of YebF (SEQ ID NO: 8) to give YebF-KrCf encoded by the nucleotide sequence of SEQ ID NO: 24.

Example 15

Long Term Stability of KrCUT in Culture Medium

(182) Stability over time and/or with temperature represents one of the main desirable properties of an enzyme, for their utilization as ingredients in cleaning product formulation. Most enzymes tend to lose activity with the time.

(183) The KrCUT was routinely produced in the form of secreted fusion protein, YebF-KrCUT in culture medium. Culture medium having KrCUT activity was stored at 4 C. without any additives. Activity was determined periodically by using p-nitrophenyl laurate as substrate, and reported in Table 7.

(184) TABLE-US-00010 TABLE 7 Storage stability of KrCUT in culture medium at 4 C. Months 0 3 4 17 19 Activity 3.0 0.94 25.67 0.94 17.13 0.70 16.66 2.47 21.94 (U/mL) 1.83

(185) At room temperature, KrCUT in culture medium without additives retains full activity for a period of 4 months.

Example 16

Stability of KrCUT in Stabilisation Buffer

(186) For stability testing, culture medium showing KrCUT activity, and containing 100 mg total protein was used. Stabilisation buffer composed of sodium gluconate 1-5%, sodium carbonate 1-5%, sodium citrate 1-5% and propylene glycol 1-5% was prepared; 50 mL enzyme solution was added to 200 mL of buffer and stored at room temperature, after mixing. Samples were taken at defined time periods and the enzymatic activity was measured using p-nitrophenyl dodecanoate as substrate.

(187) Activity loss could be observed only after 4 months, and up to 65% of the initial activity was present after 9 months at room temperature.

(188) Data are reported in FIG. 12.

(189) For most of enzymes, more complex mixtures with different ingredients at higher concentrations are used for stabilisation, namely polyol such as propylene glycol and glycerol, boric acid and boron derivatives and sodium formate.

(190) In some test cleaning product formulations, the KrCUT enzyme was combined with a mix of proteases from Bacillus. The efficacy of the cleaning product was reduced, demonstrating the contribution of the KrCUT to the overall efficacy of the cleaning product. However, the loss of KrCUT activity could be avoided or substantially reduced by the additional of stabilizers to the formulation (e.g., polyols and/or boric acid).

Example 17

Stability of KrCUT in the Presence of Metal Ions

(191) Enzymes such as lipases, amylases, and proteases are widely used in cleaning product formulations, and these types of enzymes are generally sensitive to the presence or absence of metal ions. For example, lipases generally require calcium for optimal activity. Amylases and proteases also generally require metal ions for optimal activity.

(192) For improved performance, chelating agents (namely EDTA) are often added to cleaning products. These chelating agents sequester metal ions and thus may negatively impact enzyme activity (e.g, of lipases, amylases, and proteases).

(193) Interestingly, activity of KrCUT is not affected by EDTA or other chelating agents often found in cleaning products. More specifically, 10% w/v of EDTA does not inhibit KrCUT activity (data not shown), and the enzyme retains 100% activity in the presence of GLDA (L-Glutamic acid N,N-Diacetic acid Tetra sodium), at a concentration of 5% w/v.

(194) More particularly, stability in the presence of hydrogen peroxide makes KrCUT an interesting candidate for cleaning products with disinfection properties.

Example 18

Construction and Expression of Recombinant Enzymes Having Cutinase Activity

(195) The recombinant enzymes listed in Table 8 were constructed, expressed and purified for further characterization as generally described in Examples 3 and 4.

(196) TABLE-US-00011 TABLE8 Recombinantenzymeshavingcutinaseactivity Aminoacidsequence(YebFcarrier proteinsareinlowercaseletters; SEQID cutinasecatalyticdomainsarein NO: bold;linkersareitalicized; (without Enzyme Organism(s) polymer-bindingdomainsareunderlined) YebF) YebF- Kineococcus meknmkkrgaflglllvsacasvfaannetsks 6 KrCUT radiotolerans vtfpkcedldaagiaasvkrdyqqnrvarwadd qkivgqadpvawvslqdiqgkddkwsvpltvrg ksadihyqvsvdckagmaeyqrrATCSDVDVVF ARGTGETPGLGVVGGPFVRSLTGELSDRTVTSH AVDYAASSSQASAGPGATAMSAHVREVAAACPS TRFVLGGYSQGATVTDIALGIRTGTTTGTPVPA ELAGRVAAVVVFGNPLGLSGRTIATASSTYGPK SKDYCNSSDSVCGSAPKTGTGGHLSYASNGSTT DGARFAAGLVRAAGTPTTPTPTPTPTPVPTTCV RDSTRDHVAADRAVSLYGRAYARGSRDSLGATS SYNVVSLQQVEGGWRLVTAC YebF- Amycolatopsis meknmkkrgaflgillvsacasvfaannetsks 26 AmCUT sp.ATCC39116 vtfpkcedldaagiaasvkrdyqqnrvarwadd qkivgqadpvawvslqdiqgkddkwsvpltvrg ksadihyqvsvdckagmaeyqrrACSDLEIVFA RGSGEAPGLGITGTPLVSDVKSALSSASVSSYA VDYAASYDQTSAGPGATDMSNHIKSTAAACPDT KFAIGGYSQGASVTDIAIGIRTYLGTGQTIPTE LAPRVVAVIAFGNPLGLYGQTIKTASPTYGPKS LEFCNRGDNVCGGTGTGPGYGHLSYARDGSVDQ AAAFIAKQYNAS YebF- Amycolatopsis meknmkkrgaflglllvsacasvfaannetsks 27 Am-Kr sp.ATCC39116; vtfpkcedldaagiaasvkrdyqqnrvarwadd Kineococcus qkivgqadpvawvslqdiqgkddkwsvpltvrg radiotolerans ksadihyqvsvdckagmaeyqrrACSDLEIVFA RGSGEAPGLGITGTPLVSDVKSALSSASVSSYA VDYAASYDQTSAGPGATDMSNHIKSTAAACPDT KFAIGGYSQGASVTDIAIGIRTYLGTGQTIPTE LAPRVVAVIAFGNPLGLYGQTIKTASPTYGPKS LEFCNRGDNVCGGTGTGPGYGHLSYARDGSVDQ AAAFIAKQYNASAGTPTTPTPTPTPTPVPTTCV RDSTRDHVAADRAVSLYGRAYARGSRDSLGATS SYNVVSLQQVEGGWRLVTAC YebF- Cellulomonas meknmkkrgaflglllvsacasvfaannetsks 28 CfCUT flavigena vtfpkcedldaagiaasvkrdyqqnrvarwadd qkivgqadpvawvslqdiqgkddkwsvpltvrg ksadihyqvsvdckagmaeyqrrAPACPDVELV FARGTGEAAGLGIVGRPLERALAAELPGRTVVA TAVDYAASSSQASAGPGSGDMVAKVRSRAAACP GTQFVLGGYSQGATVTDLALGIRTGVTAGTALP EDLAARVAAVVVYGNPLGLTRRTIAQAAPAFAT RTVEYCNAGDPVCEPGGGRFTAHITYATNGTVL EGARFAAARVTAPAPTPTPGPTGTPAPVPSAEP TPAPGDTCVTASSLQHVRDGRAYPLWMRTYARG SGDPLGVLSSRTVVSLQADGTDTWRKVAAC YebF- Cellulomonas meknmkkrgaflglllvsacasvfaannetsks 29 Cf-TP flavigena; vtfpkcedldaagiaasvkrdyqqnrvarwadd Kineococcus qkivgqadpvawvslqdiqgkddkwsvpltvrg radiotolerans ksadihyqvsvdckagmaeyqrrAPACPDVELV FARGTGEAAGLGIVGRPLERALAAELPGRTVVA TAVDYAASSSQASAGPGSGDMVAKVRSRAAACP GTQFVLGGYSQGATVTDLALGIRTGVTAGTALP EDLAARVAAVVVYGNPLGLTRRTIAQAAPAFAT RTVEYCNAGDPVCEPGGGRFTAHITYATNGTVL EGARFAAARVTAAGTPTTPTPTPTPTPVPTTCV TASSLQHVRDGRAYPLWMRTYARGSGDPLGVLS SRTVVSLQADGTDTWRKVAAC

(197) The constructed and expressed enzymes include the predicted mature wild-type cutinases from Kineococcus radiotolerans (KrCUT), Amycolatopsis sp. ATCC 39116 (AmCUT, which lacks a linker domain and a polymer binding domain), and Cellulomonas flavigena (CfCUT), as well as the chimeric variants YebFAmKr (having AmCUT fused to the T/P-rich linker domain and the polymer binding domain of KrCUT), and YebF-Cf-TP (having the cutinase catalytic domain of CfCUT, the T/P-rich linker domain of KrCUT, and the polymer binding domain of CfCUT). The domain structures of each of the recombinant enzymes listed in Table 8 are summarized in Table 9 (following autolytic cleavage of the respective YebF carrier proteins).

(198) TABLE-US-00012 TABLE 9 Summary of domain structure of recombinant enzymes having cutinase activity Enzyme Cutinase catalytic Linker Polymer binding name domain from: domain from: domain from: KrCUT Kineococcus Kineococcus Kineococcus radiotolerans radiotolerans radiotolerans AmCUT Amycolatopsis sp. ATCC n/a n/a 39116 Am-Kr Amycolatopsis sp. ATCC Kineococcus Kineococcus 39116 radiotolerans radiotolerans Cf Cellulomonas flavigena Cellulomonas Cellulomonas flavigena flavigena Cf-TP Cellulomonas flavigena Kineococcus Cellulomonas radiotolerans flavigena
Constructions

(199) To ensure expression of the recombinant enzymes, synthetic genes encoding fusion proteins consisting of the carrier protein YebF (357 bp) and the corresponding full-length cutinases lacking the native N-terminal signal sequences were constructed.

(200) The optimized sequences for reduction of G+C content were provided by Biomatik USA. The nucleotide sequences were cloned into the NcoI and HindIII sites of the pTrc2Bd vector, a derivative of the plasmid pTrcHis2B, obtained by deletion of the His Tag. The recombinant plasmids were transformed in the -galactosidase-positive and lactose inducible E. coli MG1655 strain.

(201) The synthetic gene of Amycolatopsis sp. ATCC 39116, cutinase (AmCUT) corresponds to 564 bp, after optimization the G+C content was reduced from 71% to 55%.

(202) The recombinant enzyme variant AmKr was constructed as a 786 bp polynucleotide consisting of 561 bp AmCUT and 225 bp of the C-terminal portion of KrCUT. Codon optimization led to G+C content reduction from 68% to 56%.

(203) The synthetic gene encoding CfCUT corresponded to an 812 bp nucleotide sequence, in which G+C content was reduced from 78% to 59% after codon optimization.

(204) The polynucleotide encoding the variant CfTP represented a DNA fragment of 789 bp.

(205) Production at Small Scale

(206) Expression of the recombinant enzymes were performed by growing the E. coli MG1655 harboring the recombinant plasmids pTrc3Bd. Colonies of the cells on LB agar plates were selected and used to prepare pre-cultures consisting of 5 mL TB media supplemented with ampicillin (50 g/mL). The culture was incubated at 37 C., on a rotary shaker at 200 rpm, for 16 h. The overnight culture was used to inoculate 1 L of fresh TB media. Recombinant enzyme production was induced by addition of 10 g/L lactose, at optical density (OD) 0.5, cells were allow to grow for 24 h, collected by centrifugation at 4000 rpm, 20 min (Eppendorf Centrifuge 5810R). The supernatant represents the source of cutinase for activity measurements.

(207) Production of Cutinase Variants in Bioreactor

(208) Fermentation was performed in a 30-Liter computer-controlled, in-situ sterilizable bioreactor (INFOR HT Techfors), with a working volume of 20 L. The bioreactor was filled with 18 L of TB culture medium and sterilized at 121 C. for 20 min. A regulation system was used to control the temperature at 30 C. throughout the experiment. A pH of 7.0 was maintained constant by using acidic H.sub.2SO.sub.4 and basic NaOH regulation.

(209) The stirring speed was first set at 400 rpm and then linked to dissolved oxygen (DO) concentration at 25% saturation. The airflow velocity was set to 1 wm. The bioreactor was inoculated with 5% volume of culture under aseptic conditions, using pre-culture. First pre-culture was performed in 5 mL TB media, supplemented by ampicillin (50 g/mL), and incubated at 37 C., 16 h, using bacteria colonies from LB agar plate. This culture was used to inoculate the second pre-culture, in a 1 L flask, TB medium, incubation under the same conditions.

(210) Expression of recombinant enzymes was initiated at OD600 of 7, by adding lactose at a concentration of 10 g/L. The cultivation was allowed to continue for 24 h, after induction, fermentation liquid was collected by centrifugation, 8000 rpm for 30 min (Beckman centrifuge, Model Avanti J26 XPI).

(211) The following crude culture supernatants were obtained: KrCUT (8 L); 1.6 U/mL (pNP-dodecanoate); AmCUT (8 L); 1.2 U/mL (pNP-dodecanoate); AmKr (16 L); 0.5 U/mL (pNP-dodecanoate); CfCUT (16 L); 0.5 U/mL (pNP-dodecanoate); and Cf-TP (8 L); 0.6 U/mL (pNP-dodecanoate).
Purification

(212) In a first step, all 5 enzymes were concentrated to about 0.5 L using a 10 kD cut-off holo fiber membrane. During this step, the enzyme containing solutions became much darker, probably due to medium ingredients that remained loosely bound to proteins contained in the solution and thus did not pass the 10 kD barrier.

(213) After concentration, each enzyme solution was subjected to dynamic dialysis on a SPECTRA/POR 500-016 HP-75 dialysis module, 10 kD, 6500 cm.sup.2. In this step, the individual protein concentration was kept constant, but salt and medium components were removed gradually. The enzyme-containing solution was dialyzed against 20 mM sodium phosphate, pH 6.0 until the conductivity of the solution was lower than 2 mS/cm.sup.2.

(214) Theoretical isoelectric points and the molecular masses of the enzymes were calculated using their putative amino acid sequences. The results and properties of each of the enzyme variants are shown in Table 10.

(215) TABLE-US-00013 TABLE 10 Properties of recombinant enzymes expressed 1 Absorbance Calc. 1 Molar Unit [280 nm] Absorbance Predicted Length MW microgram = extinction corr. [280 nm] Isoelectric charge Enzyme (aa) (kD) (pmol) coefficient to (mg/mL) of 1 mg/mL point at pH 7 YebF- 393 39.5 25.31 37920 1.04 0.96 8.81 6.13 KrCUT KrCUT 261 26.1 38.37 16650 1.57 0.64 8.59 3.07 YebF- 309 32.3 30.98 34550 0.93 1.07 6.98 0.03 AmCUT AmCUT 187 18.8 53.11 13280 1.42 0.71 5.20 3.10 YebF- 383 40 25.0 44320 0.90 1.11 8.04 2.04 Am-Kr Am-Kr 261 26.6 37.65 23050 1.15 0.87 6.32 1.03 YebF- 393 40.6 24.60 42320 0.96 1.04 8.69 4.97 CfCUT CfCUT 271 27.2 36.77 21060 1.29 0.77 8.29 1.90 YebF- 384 39.9 25.06 42330 0.94 1.06 8.94 6.97 Cf-TP Cf-TP 262 26.5 37.80 21060 1.26 0.80 8.84 3.90

(216) Based on their calculated molecular weights, their isoelectric points, and other properties, the enzymes YebF-KrCUT and KrCUT, YebF-CfCUT and CfCUT, as well as YebF-Cf-TP and Cf-TP, were all predicted to bind to a cation exchanger (Capto-S at a pH of 6.0 and a conductivity below 2 mS/cm.sup.2). The enzyme YebF-AmCUT was predicted to weakly bind to the cation exchanger, but not the enzyme AmCUT. The enzyme Yeb-FAmKr was predicted to bind to the cation exchanger, whereas the enzyme AmKr was considered to bind minimally.

(217) Despite the generally positive binding assessment of the above enzymes (with exception of AmCUT) to a cation exchanger (Capto-S and SP-Sepharose XL were tested) under the selected conditions (pH 6.0, 2 mS/cm.sup.2), only KrCUT bound completely to the resin. CfCUT did not bind at all and appeared completely in the flow through, whereas active enzyme for AmCUT, AmKr, and Cf-TP were found in both the column flow through and in bound fractions.

(218) Bound fractions of each enzyme (with the exclusion of CfCUT) were concentrated using stirring cell with a cut-off membrane of 10 kD, and subjected to size exclusion chromatography using a Hi-Load Superdex pg 16/60 column, which also allowed the assessment of the native molecular mass of the enzymes based on their retention time.

(219) For KrCUT separated on SP-Sepharose XL, two active fractions (A7/A8, and A11-B1; chromatogram on shown) were collected. Both fractions showed similar activity but, due to the higher yield, fraction A11-B1 was selected for further studies and purified by size exclusion chromatography.

(220) For AmCUT separated on Capto-S, only a very small amount of active enzyme bound to the column and was subsequently concentrated and subjected to size exclusion chromatography (chromatogram not shown). In parallel, the flow through containing most of the active Am enzyme was collected, concentrated, dialysed against 20 mM Na-phosphate, pH 7.0, and applied to a DEAE-Sepharose FF 16/10. Under the chosen conditions, active enzyme did not bind to the column and appeared in the flow through that was collected, concentrated and subjected to size exclusion chromatography. Even though AmCUT did not bind to DEAE-Sepharose, most of the contaminating proteins did and thus this step resulted in an almost pure enzyme. Active almost pure AmCUT was thus only found in fraction X1 (flow through). All bound factions did not contain enzyme activity.

(221) For AmKr separated on Capto-S, in contrast to AmCUT, AmKr did bind to the cation exchanger in much larger quantity, although active enzyme was also found in the flow through. Fractions B3-B9 were active and were pooled (chromatograph not shown), concentrated and subjected to size exclusion chromatography as final purification step.

(222) For CfCUT separated on Capto-S, CfCUT did not bind to the cation exchanger at all. Thus, a similar purification approach was chosen as for AmCUT cutinase. The active flow-through of CfCUT was collected, concentrated and diafiltrated against 20 mM sodium phosphate pH 7.0, and the enzyme was loaded to a DEAE-Sepharose FF column (16/10). The flow through contained the active enzyme and was collected, concentrated and subjected to size exclusion chromatography on Superdex 75 g. Active CfCUT was exclusively found in fraction X1 (chromatograph not shown). None of the bound fractions contained active enzyme.

(223) For Cf-TP separated on Capto-S, in contrast to CfCUT, parts of Cf-TP did bind to the cation exchanger, although active enzyme was also found in the flow through. Fractions B3-B7 (chromatograph not shown) were active and were pooled, concentrated and subjected to size exclusion chromatography as final purification step.

(224) In summary, using different techniques for enzyme purification, pure enzyme preparations were obtained and tested for their activity towards a standard esterase substrate, pNP-palmitate. Enzymatic activities of the purified variants are summarized below:

(225) TABLE-US-00014 KrCUT 197 3 U/mg AmCUT 244 18 U/mg Am-Kr 174 11 U/mg CfCUT 26 1 U/mg Cf-TP 30 2 U/mg

(226) The active fraction of KrCUT (Superdex 75) was shown to have a native molar mass of about 28 kD (deducted from its retention time, using a standard curve), showing a single band in SDS-PAGE at about 27 kD (FIG. 13A), which corresponds well with the theoretical molar mass for full length Kr (26 kD, after YebF cleavage).

(227) AmCUT and AmKr behaved unusual in size exclusion chromatography, which made determining their native molar masses impossible, as the retention time of both enzymes did not correspond to their predicted molar mass. Instead, both enzymes were retained on the column, suggesting some interaction of the enzymes with the column matrix. SDS-PAGE of AmKr (FIG. 13B) revealed two distinct species with molar masses of about 40 kD (which is in agreement with the predicted molar mass for YebFAmKr) and about 22 kD which is slightly below the predicted molar mass of full length AmKr (YebF removed, 26 kDa).

Example 19

Circular Dichroism Spectroscopy and Estimation of Melting Temperature (Tm)

(228) The enzymes purified in Example 18 were characterized to determine their melting temperatures.

(229) 19.1 Methods

(230) CD spectra were recorded using a Jasco J-815 spectrometer operating with the Spectra Manager software. Temperature was controlled using a Jasco PFD-452S peltier unit. Previously purified protein solutions were adjusted to 0.15 mg/mL (if quantities allowed for) and CD spectra were recorded between 190 and 250 nm using a Quartz cuvette (ID=0.1 cm). Blanks containing buffer only were prepared and used as baseline. Temperature dependent protein unfolding was monitored at 222 nm with thermo profiles ranging from 20 up to 90 C. (2 C. min-1). Samples were kept for 5 min at the respective maximum temperature and protein refolding was monitored using the same conditions as above reversing the thermo profiles. Thermodynamic parameters (Tm, H, S, G) for the folding/unfolding process, if applicable, were calculated using the Spectra Manager software.

(231) 19.2 Results

(232) Variable temperature measurement using CD spectroscopy (monitored at 222 nm) shows the unfolding of KrCUT with increased temperature (FIG. 14A). The melting temperature (Tm) was estimated to be 50.7 C.0.1. KrCUT also showed refolding to some extent (FIG. 14A, upper curve, going from high to low temperature) with a midpoint transition temperature of 42.7 C.0.2.

(233) The Tm of AmKr was estimated to be 55.0 C.0.1 (FIG. 14B). In contrast to KrCUT, AmKr did not show any refolding.

(234) The Tm of CfCUT was estimated to be 65.2 C.0.1 (FIG. 14C). CfCUT did not show any refolding.

(235) The CD signal of Cf-TP was a slightly low, probably due to lower protein concentration. The Tm of Cf-TP was estimated to be 56.6 C.0.1 (FIG. 14D). Cf-TP showed some refolding, but to a much lower extend when compared to KrCUT.

(236) The CD signal of AmCUT was too weak to allow for the estimation of a Tm for this enzyme.

(237) All enzymes showed the typical spectrum of alpha-helix dominant secondary structure, as expected being hydrolases.

Example 20

Enzyme Substrate Profiles

(238) The enzymes purified in Example 18 were characterized to determine their substrate profiles on common esterase substrates.

(239) 20.1 Methods: Esterase Assays

(240) Esterase activity was determined by monitoring the formation of para-nitrophenol (pNP) at 410 nm from various pNP-esters using a Beckman UV spectrophotometer (model DU 640). Substrates were completely dissolved in 2-propanol (8 mM) and were then added to 50 mM Tris/HCl buffer pH 8.0 to give a final substrate concentration of 0.8 mM. The reaction (1 mL) was started by adding an appropriate amount of enzyme. One enzyme unit (U) was defined as the amount of enzyme that produced 1 mol of product (pNP) per min; specific activity was expressed as U/mg protein. A molar extinction coefficient of 15 mM.sup.1 cm.sup.1 was used for the calculation.

(241) 20.2 Results

(242) TABLE-US-00015 TABLE 11 Substrate profiles of enzymes tested Substrates KrCUT AmCUT Am-Kr CfCUT Cf-TP pNP-acetate (C2) 2 8 9 1 6 5 pNP-butyrate (C4) 26 2 20 2 75 8 93 8 80 3 pNP-caprylate (C8) 25 3 76 2 100* 11 2 33 1 pNP-decanoate (C10) 25 3 80 2 72 2 40 5 66 8 pNP-myristate (C14) 100* 100* 46 8 47 8 40 4 pNP-palmitate (C16) 98 6 78 2 49 6 100* 100* *Relative activities in % for the respective cutinase, highest activity towards a specific substrate set to 100%

(243) As can be seen in Table 11, the highest enzymatic activity for KrCUT and AmCUT was achieved with pNP-myristate, whereas the CfCUT and Cf-TP variants showed the highest activity towards pNP-palmitate. In contrast, AmKr seemed to prefer the lower chain substrates with highest activity towards pNP-caprylate. pNP-acetate seems to be a relatively poor substrate for all variants. KrCUT, AmCUT and AmKr showed preference for the longer carbon chain substrates, whereas the CfCUT and Cf-TP substrate profile seemed to be more inhomogeneous, with preference for both pNP-butyrate and pNP-palmitate.

Example 21

Determination of Enzyme Optimal Temperatures and Thermostability

(244) The enzymes purified in Example 18 were characterized to determine their optimum temperatures.

(245) 21.1 Methods: Temperature Optimum

(246) For estimation of the temperature optimum, buffer containing pNP-palmitate (see esterase assay in Example 20.1) was pre-incubated to reach the desired temperature, enzyme was then added, and the formation of pNP was assayed. For this experiment, Tris/HCl buffer was replaced with sodium phosphate buffer (50 mM, pH 8.0) to avoid a pH shift at the different temperatures.

(247) 21.2 Results: Temperature Optimum

(248) TABLE-US-00016 TABLE 12 Temperature dependence of the different enzymes tested Temperature ( C.) KrCUT AmCUT Am-Kr CfCUT Cf-TP 20 83 5 92 11 50 2 69 2 54 5 25 84 4 100* 61 2 100* 84 9 30 83 4 67 3 69 2 63 7 95 5 35 84 4 27 4 78 1 45 5 100* 40 89 6 15 2 87 2 38 8 89 6 45 100* 100* 1 72 7 50 90 4 81 10 37 6 55 38 3 37 5 9 1 60 15 5 9 1 *Relative activities in % for the respective cutinase, highest activity at a specific temperature set to 100%

(249) As shown in the results above, the multi-domain enzyme KrCUT showed enzymatic activity at a broader range of temperatures than single-domain AmCUT and fellow multi-domain CfCUT (despite CfCUT having an higher estimated melting temperature than KrCUT, see Example 19.2). Interestingly, fusing the T/P-rich linker domain and the polymer binding domains from KrCUT to the cutinase catalytic domain of AmCUT, resulted in a chimeric enzyme (AmKr) having higher enzymatic activity at broader range of temperatures than AmCUT. Furthermore, replacing only the T/P-rich linker domain of CfCUT with the corresponding domain from KrCUT was sufficient to produce a chimeric enzyme (Cf-TP) having enzymatic activity at broader range of temperatures than CfCUT.

(250) 21.3 Methods: Thermostability

(251) Enzyme thermostability was studied over a temperature range of 50-95 C. using the purified enzyme preparations. Aliquots were incubated at the desired temperature for 5, 10, 30, 60, and 120 minutes, chilled on ice, and assayed for remaining activity towards p-NP-palmitate (pNPP) at room temperature.

(252) 21.4 Results: Thermostability

(253) TABLE-US-00017 TABLE 13 Thermostability of the cutinase variants is shown in the following table and graphs. Time (min) 50 C. 55 C. 60 C. 65 C. 70 C. KrCUT* 5 61 5 45 4 34 4 19 2 10 31 4 26 4 12 2 30 12 2 4 60 6 120 AmCUT* 5 46 6 21 4 3 10 18 3 10 2 30 5 60 120 Am-Kr* 5 96 3 82 17 19 2 12 1 10 85 8 64 3 15 2 30 75 15 56 8 5 1 60 79 12 42 8 120 80 9 30 3 cfCUT* 5 109 3 72 7 70 6 65 5 40 5 10 118 3 75 10 75 7 64 4 37 6 30 129 9 77 9 56 10 55 1 33 3 60 120 6 65 10 40 4 30 5 26 3 120 125 20 58 2 28 4 25 4 15 1 Cf-TP* 5 68 8 42 4 35 3 31 3 10 43 3 30 42 3 31 1 30 3 25 3 60 22 4 15 2 120 21 3 15 3 9 2 *Relative activities in % for the respective cutinase, activity set to 100% at time 0.

(254) The results above show that single-domain enzyme AmCUT seemed to be very unstable at higher temperatures, whereas all the multi-domain enzymes (particularly the CfCUT) showed higher thermostability. The letter variant was also shown to have the highest melting temperature, however, the optimal temperature does not seem to reflect the enzyme's higher thermostability.

Example 22

Enzymatic Activity in the Presence of Organic Solvents

(255) The enzymes purified in Example 18 were characterized to determine their stability and activity in presence of diverse organic solvents.

(256) 22.1 Methods

(257) Enzyme activity in various organic solvents was studied over a concentration range of 1-30% at room temperature. pNP-palmitate was completely dissolved in 2-propanol (8 mM) and then added to 50 mM Tris/HCl buffer pH 8.0 containing up to 30% organic solvent to give a final substrate concentration of 0.8 mM. The reaction was started by addition of enzyme and the formation of pNP was assayed as described above in Example 20.1.

(258) 22.2 Results

(259) TABLE-US-00018 TABLE 14 Enzymatic activity in the presence of organic solvents Solvent addition to Solvent Relative enzymatic activity (%) assay conc. (v/v) KrCUT AmCUT Am-Kr CfCUT Cf-TP Buffer only 100* 100* 100* 100* 100* Methanol 10% 91 12 116 4 87 10 115 11 121 14 20% 8 16 2 60 13 62 3 94 1 2-Propanol 5% 55 11 60 2 70 10 70 3 93 1 10% 8 9 2 23 2 17 8 92 2 Dimethyl sulfoxide 10% 106 4 142 12 114 2 105 11 150 14 (DMSO) 20% 14 2 53 2 90 8 107 1 146 23 30% 2 7 2 60 5 57 15 129 18 Dimethylformamide 5% 55 11 149 7 120 11 101 17 134 10 (DMF) Acetone 1% 71 2 170 2 118 1 92 9 103 12 5% 47 7 126 5 90 14 95 1 112 10 10% 26 7 74 7 52 5 109 6 *Relative activities in % for the respective cutinase, activity with buffer only as negative control set to 100%

(260) The results above show that the chimeric enzymes AmKr and Cf-TP impressively exhibited increased tolerance to higher concentrations of most of the organic solvents tested. More particularly, the chimeric AmKr enzymes exhibited higher relative activity in the presence of methanol (20% v/v), 2-propanol (10% v/v), dimethyl sulfoxide (DMSO; 20% or 30% v/v), and acetone (10% v/v), as compared to its corresponding wild-type enzymes (KrCUT and AmCUT). Similarly, the chimeric Cf-TP enzyme exhibited higher relative activity in the presence of all the organic solvents tested, as compared to its corresponding wild-type enzymes (KrCUT and CfCUT).

Example 23

pH Dependence of Enzymes

(261) The enzymes purified in Example 18 were characterized to determine their stability and activity at different pHs.

(262) 23.1 Methods: pH Optimum

(263) The influence of different pH values (from 5.5 to 11) on the activities of the purified enzymes was examined in three buffer systems (sodium acetate, sodium phosphate, and Tris-HCl) at room temperature using pNP-palmitate (see esterase assay in Example 20.1) as a substrate at a final substrate concentration of 0.8 mM.

(264) 23.2 Results

(265) TABLE-US-00019 TABLE 15 pH dependence of each of the enzymes Relative activity (%) pH KrCUT AmCUT Am-Kr CfCUT Cf-TP 6.5 22 1 35 6 23 2 13 2 12 1 7.0 53 2 56 6 46 3 40 4 22 1 7.5 72 4 56 2 54 3 65 5 36 1 8.0 87 6 38 8 84 2 62 2 65 7 8.5 100* 69 4 88 9 85 6 88 8 9.0 96 4 94 4 91 5 93 9 95 3 9.5 76 7 100* 100* 100* 100* 10 37 5 73 2 47 4 31 1 35 4 10.5 60 2 10 1 *Relative activities in % for the respective cutinase, highest activity at a specific pH set to 100%, sodium phosphate buffer (6.5-7.5), Tris/HCl (8.0-10.5)

Example 24

Stability of Enzymes Towards Oxidizing Agent

(266) The enzymes purified in Example 18 were characterized to determine their stability and activity after being exposed to an oxidizing agent (i.e., hydrogen peroxide).

(267) 24.1 Methods: Stability in the Presence of Hydrogen Peroxide

(268) Purified cutinases were incubated at room temperature in the presence of 0, 30, 100, 300, 1500, and 3000 ppm of hydrogen peroxide. Residual enzyme activity was assayed (see Example 20.1) after 1, 2, 3, and 7 days of incubation.

(269) 24.1 Results

(270) TABLE-US-00020 TABLE 16 Enzyme stability following exposure to hydrogen peroxide Relative activity (%) H.sub.2O.sub.2 (ppm) Day 0 Day 1 Day 2 Day 3 Day 7 KrCUT* 30 100 9 90 6 75 3 75 8 76 2 100 104 6 91 6 88 6 76 2 66 2 300 102 4 87 2 74 1 63 2 49 4 1,500 102 10 80 4 67 4 44 2 11 1 3,000 98 9 60 2 33 1 20 4 AmCUT* 30 94 6 100 94 6 300 85 12 1,500 65 6 3,000 44 10 Am-Kr* 30 92 3 90 15 82 8 71 4 63 5 100 101 3 97 5 83 2 73 5 69 4 300 100 9 88 5 83 2 72 10 61 4 1,500 91 8 97 9 75 15 73 3 60 6 3,000 86 6 68 9 63 4 67 2 55 3 CfCUT* 30 101 22 89 8 82 8 76 6 72 7 100 92 5 114 15 118 15 92 11 94 7 300 93 12 106 20 118 12 102 14 106 13 1,500 126 1 106 12 109 2 95 4 54 16 3,000 128 18 115 2 99 23 60 6 35 1 Cf-TP* 30 97 1 89 4 78 3 86 2 65 7 100 98 6 91 3 89 7 94 7 74 6 300 95 6 88 3 85 12 98 3 77 5 1,500 95 9 89 3 88 6 87 4 68 7 3,000 80 2 74 5 77 2 78 5 57 5 *Relative activities in % for the respective cutinase, activity with buffer only as negative control set to 100% at day 0.

(271) As shown above, all of the multi-domain enzymes that were tested (i.e., KrCUT, Kr-Am, CfCUT, and Cf-TP) retained some level of activity after 1 or more days exposure to hydrogen peroxide, whereas the single-domain enzyme AmCUT did not. Furthermore, chimeric enzymes (Kr-Am and Cf-TP) exhibited increased resistance to hydrogen peroxide, as compared to their corresponding wild-type multi-domain enzymes (KrCUT and CfCUT). For example, after exposure to 3000 ppm hydrogen peroxide for 7 days, no activity was detected for the wild-type KrCUT enzyme. In contrast, the chimeric enzyme AmKr (containing the cutinase catalytic domain of AmCUT on the backbone of KrCUT), retained 55% of its activity under the same conditions. Moreover, the relative activity of CfCUT was reduced from 128% at day 0 to 35% at day 7 of exposure to 3000 ppm hydrogen peroxide. In contrast, the relative activity of the chimeric enzyme Cf-TP (containing the T/P-rich linker domain of KrCUT on the backbone of CfCUT) went from 80% to 57% under the same conditions.

Example 25

Summary of Biochemical Properties of the Enzymes

(272) A summary of some of the results shown in Examples 19-24 is provided below.

(273) TABLE-US-00021 TABLE 17 Summary of biochemical properties of the tested enzymes Mol. Mol. Temp. Mass Mass opt. Spec. Act. Tm Enzyme (native).sup.1 (SDS) ( C.) pH opt. (U/mg).sup.2 ( C.).sup.3 KrCUT 28,000 27,000 45 8.5 197 3 51 1 AmCUT n.a..sup.4 20,000 25 9.5 244 18 n.d..sup.5 Am-Kr n.a..sup.5 22,000 45 9.5 174 11 55 1 CfCUT.sup.6 53,000 27,000 25 9.5 26 1 65 1 Cf-TP.sup.7 42,000 25,000 35 9.5 30 2 57 1 .sup.1Estimated by retention time of the active fraction on Superdex 75 pg using a standard curve .sup.2Towards pNP-palmitate (standard assay) .sup.3Melting temperature of the enzyme, estimated using CD-spectroscopy .sup.4Not applicable, Am and Am-Kr showed some interaction with the resin and thus a delayed retention time .sup.5Not determined, Am concentration was too low to give a good signal in CD spectroscopy .sup.6Cf shows a higher molecular mass native than in SDS-PAGE, which may be due to dimer formation .sup.7Cf-TP showed more than one band in SDS-PAGE: 25,000 and 28,000 Da.

Example 26

Assessment of Ochratoxin A Degradation Ability by the Enzymes

(274) The enzymes purified in Example 18 were tested for their ability to degrade the mycotoxin Ochratoxin A. In general, the mycotoxin was incubated with different concentrations of the enzyme, and the remaining mycotoxin concentration, as well the respective degradation products, were monitored by LC-MS over time (where possible/applicable).

(275) 26.1 Methods: Mycotoxin Testing

(276) Reactions were performed in 1 mL volumes in glass HPLC vials comprising the mycotoxin to be analyzed and the enzyme to be tested. For Ochratoxin A (Petromyces albertensis, 01877 Sigma), 50 L of a 100 ppm stock solution in methanol (final concentration of 5 ppm) was added to 910 L of distilled water and 40 L of the enzyme to be tested. In control reactions, the volume of enzyme was substituted with distilled water. The reactions were incubated in an Eppendorf thermomixer at 37 C. with shaking at 750 rpm. 100 L samples were withdrawn at 1, 4 and 24 h for chromatographic analysis.

(277) Mycotoxin analysis was carried out using a Shimadzu LCMS-2020 equipped with a UV/vis detector. Chromatographic separation of 10 L injected sample was carried out at 30 C. on a Kinetex 2.6 m XB-C18 column 100 (502.1 mm) using a binary gradient. For Ochratoxin A, eluent A consisted of water and eluent B consisted of acetonitrile and the UV detection wavelength was 360 nm. Gradient elution at a flow rate of 0.4 mL/min was performed as follows: 0-2 min, 10% B; 2.01-10.0 min, 10 to 80% B; 10.01-12.0 min, 80% B; 12.01-15 min, 10% B. The electrospray interface (ESI) was operated simultaneously in positive and negative mode, scanning from m/z 50-1,000.

(278) 26.2 Results

(279) The degradation of Ochratoxin A was monitored by HPLC-UV at 360 nm and LCMS. Possible degradation products that would arise from either hydrolysis of the amide bond or hydrolysis of the lactone moiety are shown below.

(280) Chemical Structures of Ochratoxin A and Hypothetical Degradation Products

(281) ##STR00001##

(282) The five enzymes tested were screened for their ability to degrade 5 ppm (5,000 ppb) Ochratoxin A at 37 C. The results are summarized in Table 18.

(283) TABLE-US-00022 TABLE 18 Results of Enzyme testing for Ochratoxin A degradation Residual ochratoxin A Ochratoxin A (%).sup.2 Degradation.sup.3 per Enzyme Activity (U).sup.1 1 h 4 h 24 h U and h (mol) KrCUT 3.272 63 17 7 0.02 Am-Kr 0.072 6 0 0 3 AmCUT 0.048 59 20 6 2 CfCUT 0.056 11 2 0 3 Cf-TP 0.002 5 0 0 98 .sup.1Activity used in assay towards p-nitrophenyl palmitate (standard assay) .sup.2Relative to Ochratoxin A incubated without enzyme .sup.3Describes the potency of 1 U of enzyme (pNP standard test) to degrade a specific amount of ochratoxin per hour of incubation at 37 C.

(284) Degradation of Ochratoxin A was observed with all enzymes, even after 1 h incubation. The chimeric enzymes AM-Kr and Cf-TP degraded 94-95% of the Ochratoxin A after 1 hours, and 100% after 4 hours. Cf-TP showed the highest potency in terms of the amount of Ochratoxin A that 1 U the enzyme (pNP standard test) can degrade per hour of incubation at 37 C.

Example 27

Alignment and Phylogenic Analyses of Multi-Domain Cutinase Enzymes

(285) A database search for other multi-domain enzymes using KrCUT (SEQ ID NO: 6) as query sequence revealed five hits, as shown in Table 19.

(286) TABLE-US-00023 TABLE 19 Native cutinase orthologs having multi-domain organization Enzyme Query name Organism coverage Identity Accession SEQ ID NO: ssKrCUT Kineococcus radiotolerans 100% 100% ABS05574.1 1 SRS30216 = ATCC BAA-149 ssCbCUT2 Cellulomonas bogoriensis 95% 60% KGM14008.1 25 69B4 = DSM 16987 ssCcCUT Cellulomonas cellasea DSM 98% 56% KGM03336.1 15 20118 ssCbCUT1 Cellulomonas bogoriensis 99% 55% KGM14009.1 14 69B4 = DSM 16987 ssCfCUT Cellulomonas flavigena DSM 99% 56% ADG75999.1 13 20109

(287) A multiple sequence alignment was performed for the enzymes listed in Table 19, after omitting their predicted N-terminal signal sequences, as shown in Table 20. The results of the alignment are shown in FIG. 15A, and a phylogenic tree is shown in FIG. 15B.

(288) TABLE-US-00024 TABLE20 KrCUTorthologswithouttheirpredicted N-terminalsignalsequences Aminoacidsequence(cutinasecatalytic Enzyme domainsareinbold;linkersareitalicized; SEQID name polymerbindingdomainsareunderlined) NO: KrCUT ATCSDVDVVFARGTGETPGLGVVGGPFVRSLTGELSDRTVTSHA 6 VDYAASSSQASAGPGATAMSAHVREVAAACPSTRFVLGGYSQGA TVTDIALGIRTGTTTGTPVPAELAGRVAAVVVFGNPLGLSGRTI ATASSTYGPKSKDYCNSSDSVCGSAPKTGTGGHLSYASNGSTTD GARFAAGLVRAAGTPTTPTPTPTPTPVPTTCVRDSTRDHVAADR AVSLYGRAYARGSRDSLGATSSYNVVSLQQVEGGWRLVTAC CbCUT2 AGSCPDVQVVFARGTGERAGLGIIGRPFARALADELPGMTVTSH 46 AVDYAAAASQRSAGPGATAMTDHVTAMAARCPGTQFVLGGYSQG ATVTSIALGIRAGTTTGRAIPDELSDRVAAVVVFGNPLGMRGQT IASASRTYADRAKDYCNSGDSICGRQPSTGRGTHTGYATNGSTT DGARFAAGLVTANPPEAAPPTAPPTTPPTTPAPPSDQVTCVTAQ VSEHVEARRAIRGLTRAYARGTLEDIGRLRSTEEVSLRRSGTFS WTPTASC CcCUT APACADVEVLFARGTGEAPGLGVLGTPFVRSVTSALSDRTVTSY 47 AVNYAAESSQRSAGPGATDLTNHLTATAAACPGTRFVLGGYSQG ATVVDLALGIRTGTTTGTAIPAALEPRVAAIVVFGNPLGISGRT IATASPTYAARARDFCATGDPVCGGGSSFAAHLAYRTNGDVTAG ADFAAGLARATTVPVPTPTVPGTPTPSPTAPGTPTPVPTTSPTP APSPTAPGAACVRASTRAHVEAGRAERRHGIAYATGSGDRIGWV SSFVRVSVQQTADGWERVLSC CbCUT1 TGDCPDVHVVFARGTGEPRGLGIVGRPFVSDLGDALPTMTVTSY 48 AVNYSANASQTSAGPGAGDMTSHVTSMAARCPGTQFVLGGYSQG ATVTSIAVGARSTSIRSRVLPANLEPRVAAVVVFGNPLGLTRRT IASEAPAYAAKSRDYCNRSDTVCGGRGDARGGHLAYVSNGSVAD GAAFAASLVGVAPGDPASCVTARAVDHVEADRAFRSVLRAYARG SRDPLGRLTSSDLVSLQRTGQDSWSVVPAC CfCUT APACPDVELVFARGTGEAAGLGIVGRPLERALAAELPGRTVVAT 28 AVDYAASSSQASAGPGSGDMVAKVRSRAAACPGTQFVLGGYSQG ATVTDLALGIRTGVTAGTALPEDLAARVAAVVVYGNPLGLTRRT IAQAAPAFATRTVEYCNAGDPVCEPGGGRFTAHITYATNGTVLE GARFAAARVTAPAPTPTPGPTGTPAPVPSAEPTPAPGDTCVTAS SLQHVRDGRAYPLWMRTYARGSGDPLGVLSSRTVVSLQADGTDT WRKVAAC

(289) The threonine/proline contents of the T/P-rich linker domains of four of the enzymes listed above are shown in Table 21.

(290) TABLE-US-00025 TABLE21 Threonine/prolinecontentofselectedmulti-domaincutinases # of Enzyme Aminoacidsequence SEQID TorP Linker T/P name ofT/P-richlinker NO: residues length content KrCUT TPTTPTPTPTPTPVPTT 3 16 17 94.1% CbCUT2 NPPEAAPPTAPPTTPPTTPAPP 37 16 22 72.7% CcCUT GLARATTVPVPTPTVPGTPTPSP 40 17 28 60.7% TAPGTPTPVPTTSPTPAPSPTAP GAA CfCUT PAPTPTPGPTGTPAPVPSAEPTP 31 29 49 59% APGDT

(291) The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.

(292) The scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.

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