A METHOD FOR THE ENZYMATIC CONVERSION OF A PHENOL SUBSTRATE INTO A CORRESPONDING CATECHOL PRODUCT

Abstract

A method for the enzymatic conversion of a phenol substrate into a corresponding catechol product comprises the step of incubating the phenol substrate with a Ralstonia solanacearum tyrosinase enzyme, or a functional derivative thereof, in a reaction mixture, for a period of time sufficient to allow the enzyme convert at least some of the phenol substrate into the catechol product.

Claims

1. A method for the enzymatic conversion of a phenol substrate into a corresponding catechol product, in which the phenol substrate is selected from tyrosol and a 4-halophenol, the method comprising the steps of incubating the phenol substrate with a Ralstonia solanacearum tyrosinase enzyme, or a functional derivative thereof, in a reaction mixture, for a period of time sufficient to allow the enzyme convert at least some of the phenol substrate into the catechol product and in which the reaction mixture comprises ascorbic acid.

2. A method according to claim 1 in which the phenol substrate is a tyrosol and the catechol product is hydroxytyrosol.

3. A method according to claim 1 in which the phenol substrate is a halophenol and the catechol product is a halocatechol.

4. A method according to claim 1 in which the ascorbic acid is provided as a salt.

5. (canceled)

6. A method according to claim 1 in which the functional derivative of the Ralstonia solanacearum tyrosinase enzyme is an engineered variant of Ralstonia solanacearum tyrosinase enzyme that is capable of the 100% conversion of 175 mM tyrosol into hydroxytyrosol

7. A method according to claim 1 in which the functional derivative of Ralstonia solanacearum tyrosinase enzyme is an engineered enzyme selected from the group consisting of RVC10, RV145 and C10_N322S.

8. A method according to claim 1 in which the Ralstonia solanacearum tyrosinase enzyme, or functional derivative thereof, is provided as an extract from a bacteria that expresses the Ralstonia solanacearum tyrosinase enzyme, or functional derivative thereof.

9. A method according to claim 1 in which the Ralstonia solanacearum tyrosinase enzyme, or functional derivative thereof, is provided as a bacteria that expresses the Ralstonia solanacearum tyrosinase enzyme, or functional derivative thereof.

10. A method according to claim 1 in which the Ralstonia solanacearum tyrosinase enzyme, or functional derivative thereof, is provided as a purified enzyme.

11. A method according to claim 1 in which the Ralstonia solanacearum tyrosinase enzyme, or functional derivative thereof, is provided as a crude mixture in the form of a cell extract.

12. A method according to claim 1, in which the substrate concentration is at least 75 mM.

13. A method as claimed in claim 1 in which the catechol product is separated from impurities present in the reaction mixture by chilling the reaction mixture after the enzymatic conversion step.

14. A method as claimed in claim 1 in which the catechol product is separated from impurities present in the reaction mixture by refrigerating or freezing the reaction mixture after the enzymatic conversion step.

15. A method according to claim 2 in which the ascorbic acid is provided as a salt.

16. A method according to claim 3 in which the ascorbic acid is provided as a salt.

17. A method according to claim 4 in which the ascorbic acid is provided as a salt.

18. A method according to claim 2 in which the functional derivative of the Ralstonia solanacearum tyrosinase enzyme is an engineered variant of Ralstonia solanacearum tyrosinase enzyme that is capable of the 100% conversion of 175 mM tyrosol into hydroxytyrosol.

19. A method according to claim 3 in which the functional derivative of the Ralstonia solanacearum tyrosinase enzyme is an engineered variant of Ralstonia solanacearum tyrosinase enzyme that is capable of the 100% conversion of 175 mM tyrosol into hydroxytyrosol.

20. A method according to claim 4 in which the functional derivative of the Ralstonia solanacearum tyrosinase enzyme is an engineered variant of Ralstonia solanacearum tyrosinase enzyme that is capable of the 100% conversion of 175 mM tyrosol into hydroxytyrosol.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0021] FIG. 1: Hydroxylation of halophenols by the monophenolase activity and oxidation of halocatechols by the diphenolase activity of tyrosinase

[0022] FIG. 2: Biotransformation of 20 mM of tyrosol in shaking flasks using WT and RV145 enzymes (20 μg/mL) in the presence of 40 mM ascorbic acid sodium salt (TY: tyrosol, HT: Hydroxytyrosol).

[0023] FIG. 3: Biotransformation of tyrosol to hydroxytyrosol by engineered RV145 enzyme. a: 75 mM biotransformation of tyrosol using purified engineered RV145 enzyme in the presence of 150 mM ascorbic acid sodium salt. B: 100 mM biotransformation of tyrosol using cell free lysate of E. coli harbouring engineered RV145 enzyme in the presence of 200 mM ascorbic acid sodium salt

[0024] FIG. 4: 150 mM biotransformation of tyrosol using crude cell free lysate of E. coli harbouring engineered RV145 enzyme in the presence of 300 mM ascorbic acid sodium salt

[0025] FIG. 5a: Biotransformation of 10 mM of 4-fluorophenol in shaking flasks using WT and RV145 enzymes (20 μg/mL) in the presence of 20 mM ascorbic acid sodium salt. b Biotransformation of 10 mM of 4-iodophenol in shaking flasks using WT and RV145 enzymes (10 μg/mL) in the presence of 20 mM ascorbic acid sodium salt (4-IP: 4-iodocphenol, 4-IC: 4-iodocatechol, 4-FP: 4-fluorophenol, 4-FC: 4-fluorocatechol)

[0026] FIG. 6: 100 mM biotransformation of tyrosol using commercial mushroom tyrosinase in the presence of 200 mM ascorbic acid. The reaction yields only 30% product and stops once 30 mM product is formed showing its much poorer performance compared to R. solanacearum tyrosinase.

DETAILED DESCRIPTION OF THE INVENTION

[0027] Definitions:

[0028] “Phenol substrate” means an unsubstituted or substituted phenol, especially a 3-substituted or 4-substituted phenol. Examples of substituents include halogens or hydroxyalkyl substituents. Examples of hydroxyalkyl substituents include hydroxymethyl and hydroxyethyl substituents, especially 4-hydroxymethyl and 4-hydroxyethyl substituents. Typically, the 3-substituted or 4-substituted phenol is a 3-halophenol or 4-halophenol. Examples of halophenols include iodophenol, bromophenol, chlorophenol, and fluorophenol. Preferably, the phenol is tyrosol. Preferably, the phenol substrate is provided at a concentration up to their maximum solubility in aqueous environment. In one embodiment, the phenol substrate is selected from tyrosol, a 3-halophenol, and a 4-halophenol. In one embodiment, the phenol substrate is selected from tyrosol and a 4-halophenol.

[0029] “Corresponding catechol product” means a catechol that is produced by reacting a phenol substrate with a Ralstonia solanacearum tyrosinase enzyme. When the phenol substrate is a 4-halophenol (for example, 4-fluorophenol), the corresponding catechol product is the corresponding 4-fluorocatechol (4-fluorocatechol). Likewise, when the phenol substrate is 4-substituted hydroxyalkyl phenol (for example, tyrosol), the corresponding catechol product is the corresponding 4-substituted hydroxyalkyl catechol (hydroxytyrosol).

[0030] “Tyrosol” refers to 4-(2-hydroxyethyl)phenol.

[0031] “Hydroxytyrosol” means 4-(2-hydroxyethyl)-1,2-benzenediol.

[0032] “Ralstonia solanacearum tyrosinase enzyme” means a tyrosinease enzyme isolated from Ralstonia solanacearum. An example of such an enzyme is described in Molloy et al., Biotechnol. Bioeng. 2013, 110, pp 1849-1857. The amino acid and nucleic acid sequences for the wild-type enzyme are provided below:

TABLE-US-00001 Ralstonia solanacearum tyrosinase enzyme amino acid sequence (SEQ ID NO: 1) (NP_518458) MVVRRTVLKAIAGTSVATVFAGKLTGLSAVAADAAPLRVRRNLHGMKMDD PDLSAYREFVGIMKGKDQTQALSWLGFANQHGTLNGGYKYCPHGDWYFLP WHRGFVLMYERAVAALTGYKTFAMPYWNWTEDRLLPEAFTAKTYNGKTNP LYVPNRNELTGPYALTDAIVGQKEVMDKIYAETNFEVFGTSRSVDRSVRP PLVQNSLDPKWVPMGGGNQGILERTPHNTVHNNIGAFMPTAASPRDPVFM MHHGNIDRVWATWNALGRKNSTDPLWLGMKFPNNYIDPQGRYYTQGVSDL LSTEALGYRYDVMPRADNKVVNNARAEHLLALFKTGDSVKLADHIRLRSV LKGEHPVATAVEPLNSAVQFEAGTVTGALGADVGTGSTTEVVALIKNIRI PYNVISIRVFVNLPNANLDVPETDPHFVTSLSFLTHAAGHDHHALPSTMV NLTDTLKALNIRDDNFSINLVAVPQPGVAVESSGGVTPESIEVAVI Ralstonia solanacearum tyrosinase enzyme nucleic acid sequence (SEQ ID NO. 2) (gi|30407127) TCAAATGACGGCGACCTCGATCGATTCGGGCGTCACGCCGCCGCTGCTCT CCACGGCAACGCCGGGTTGGGGTACGGCCACCAGGTTGATCGAAAAGTTG TCGTCCCGGATGTTGAGCGCCTTCAGCGTGTCGGTCAGGTTCACCATGGT CGACGGCAGGGCATGGTGGTCGTGTCCCGCCGCATGCGTCAGGAAGCTGA GCGAGGTGACGAAGTGCGGGTCGGTTTCCGGCACATCGAGGTTGGCGTTC GGCAGGTTGACGAAGACCCGGATGCTGATCACGTTGTAGGGGATCCTGAT GTTCTTGATCAGGGCCACGACTTCGGTGGTACTGCCGGTACCAACATCGG CACCCAGGGCACCCGTCACGGTGCCGGCCTCGAACTGGACGGCGCTGTTG AGCGGTTCGACCGCCGTGGCAACCGGATGTTCCCCCTTCAGCACGCTGCG CAGCCGGATATGATCGGCCAGCTTGACGCTGTCGCCGGTCTTGAACAGGG CCAGCAGATGCTCGGCACGGGCGTTGTTCACCACCTTGTTGTCGGCGCGC GGCATGACGTCATAGCGGTAGCCCAGCGCCTCGGTGCTCAGCAGATCGCT CACGCCTTGCGTGTAGTACCGGCCCTGCGGATCGATGTAGTTGTTGGGGA ACTTCATGCCCAGCCACAGCGGGTCAGTCGAGTTCTTGCGGCCCAGCGCG TTCCAGGTGGCCCATACCCGGTCGATATTGCCGTGGTGCATCATGAACAC CGGGTCGCGCGGCGAGGCGGCGGTGGGCATGAAGGCGCCGATGTTGTTGT GGACGGTGTTGTGCGGCGTGCGCTCCAGGATGCCCTGGTTGCCGCCTCCC ATCGGCACCCATTTGGGGTCGAGGCTGTTCTGTACCAGCGGCGGCCGGAC CGAGCGGTCGACCGAACGGCTGGTGCCGAAGACTTCGAAGTTGGTTTCGG CATAGATCTTGTCCATGACCTCCTTCTGGCCGACGATGGCGTCGGTGAGC GCGTAGGGGCCGGTCAGCTCATTCCGGTTGGGCACGTAGAGCGGGTTCGT CTTGCCGTTGTAGGTCTTGGCGGTGAAGGCTTCGGGCAGCAGGCGGTCTT CGGTCCAGTTCCAGTACGGCATGGCGAAGGTCTTGTAGCCGGTGAGCGCG GCCACGGCGCGCTCGTACATCAGCACGAAGCCGCGGTGCCAGGGCAGGAA GTACCAGTCGCCGTGCGGGCAGTACTTGTAGCCGCCGTTGAGCGTACCGT GCTGGTTGGCAAAGCCGAGCCAGCTCAGCGCCTGCGTCTGGTCCTTGCCT TTCATGATGCCGACGAACTCGCGATAGGCCGACAGGTCCGGGTCGTCCAT CTTCATGCCATGCAGGTTGCGCCGCACGCGCAGCGGGGCGGCATCGGCCG CAACAGCGGAGAGGCCGGTCAGCTTGCCCGCGAATACCGTGGCGACACTT GTCCCGGCGATTGCCTTCAGCACCGTTCTACGCACGACCAT

[0033] “Functional derivative thereof” as applied to a Ralstonia solanacearum tyrosinase enzyme means an engineered variant of Ralstonia solanacearum tyrosinase enzyme that is typically capable of converting a phenol (i.e. tyrosol) into a corresponding catechol (i.e. hydroxytyrosol) at a concentration and rate that is significantly better than mushroom tyrosinease described in US2003180833 (D1). Examples of such engineered variants of Ralstonia solanacearum tyrosinase enzyme are described in Molloy et al (2013), including variants that are capable of converting tyrosol into hydroxytyrosol at a concentration and rate that is at least the equivalent of the wild-type Ralstonia solanacearum tyrosinase enzyme. In one embodiment, the engineered variant of Ralstonia solanacearum tyrosinase enzyme is capable of the 100% conversion of 150 mM tyrosol into hydroxytyrosol in the tyrosol biotransformation assay described below. In one embodiment, the engineered variant of Ralstonia solanacearum tyrosinase enzyme is capable of the 100% conversion of 175 mM tyrosol into hydroxytyrosol in the tyrosol biotransformation described below. Methods for generating and testing engineered variants of Ralstonia solanacearum tyrosinase enzyme will be apparent to the person skilled in the art, and are described in Molloy et al.

[0034] An “engineered variant” of the Ralstonia solanacearum tyrosinase enzyme protein shall be taken to mean enzymes having amino acid sequences which are substantially identical to wild-type Ralstonia solanacearum tyrosinase enzyme. Thus, for example, the term should be taken to include enzymes that are altered in respect of one or more amino acid residues. Preferably such alterations involve the insertion, addition, deletion and/or substitution of 5 or fewer amino acids, more preferably of 4 or fewer, even more preferably of 3 or fewer, most preferably of 1 or 2 amino acids only. Insertion, addition and substitution with natural and modified amino acids is envisaged. The engineered variant may have conservative amino acid changes, wherein the amino acid being introduced is similar structurally, chemically, or functionally to that being substituted. Generally, the variant will have at least 70% amino acid sequence homology, preferably at least 80% sequence homology, more preferably at least 90% sequence homology, and ideally at least 95%, 96%, 97%, 98% or 99% sequence homology with wild-type Ralstonia solanacearum tyrosinase enzyme. In this context, sequence homology comprises both sequence identity and similarity, i.e. a polypeptide sequence that shares 70% amino acid homology with wild-type Ralstonia solanacearum tyrosinase enzyme is one in which any 70% of aligned residues are either identical to, or conservative substitutions of, the corresponding residues in wild-type Ralstonia solanacearum tyrosinase enzyme. Specific variants included within the scope of the invention are the engineered variants described in (Molloy et al., 2013), especially (RVC10, RV145 and C10_N322S). In one embodiment, the engineered variant of Ralstonia solanacearum tyrosinase enzyme is capable of complete conversion of a tyrosol substrate to hydroxytyrosol at concentrations an order of magnitude higher than previously reported by any tyrosinase or other biocatalyst (e.g. 175 mM tyrosol)

[0035] The term “engineered variant” is also intended to include chemical derivatives of wild-type Ralstonia solanacearum tyrosinase enzyme, i.e. where one or more residues of the wild-type enzyme is chemically derivatized by reaction of a functional side group. Also included within the term variant are wild-type Ralstonia solanacearum tyrosinase enzymes in which naturally occurring amino acid residues are replaced with amino acid analogues. Examples of side chain modifications include modification of amino groups, such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH.sub.4; amidation with methylacetimidate; acetylation with acetic anhydride; carbamylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6, trinitrobenzene sulfonic acid (TNBS); alkylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxa-5′-phosphate followed by reduction with NABH.sub.4. The guanidino group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal. The carboxyl group may be modified by carbodiimide activation via o-acylisourea formation followed by subsequent derivatization, for example, to a corresponding amide. Sulfhydryl groups may be modified by methods, such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of mixed disulphides with other thiol compounds; reaction with maleimide; maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulfonic acid, phenylmercury chloride, 2-chloromercuric-4-nitrophenol and other mercurials; carbamylation with cyanate at alkaline pH. Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphonyl halides. Tryosine residues may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative. Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate. Examples of incorporating unnatural amino acids and derivatives during enzyme synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.

EXPERIMENTAL

[0036] Materials and Methods

[0037] Strains, Growth Conditions and Purification of Tyrosinase

[0038] Recombinant E. coli BL21 were used to express wild type (WT) tyrosinase gene from R. solanacearum and three engineered variants (RV145, RVC10, C10_N322S) of the same gene previously generated by Molloy et al (2013). All strains were maintained in Lysogeny broth (LB) with 50% glycerol at −80° C. In preparation for induction and tyrosinase purification, the E. coli strains stored at −80° C. were streaked on LB agar and incubated at 37° C. for 24 h. The primary inoculum was cultured in a rotary shaker (200 rpm, 37° C.) in 5 mL of LB overnight. Carbenicillin (50 μg/mL) was used as the antibiotic for the maintenance of the plasmid throughout the study. Tyrosinase production was induced and the enzyme was purified as described earlier (Molloy et al. 2013). Different fractions of the purified tyrosinase were analysed by 10% SDS-PAGE for confirmation of purity and molecular weight. The protein concentration was determined by bicinchoninic acid method (Smith et al. 1985) using bovine serum albumin as the standard.

[0039] Preparation of Substrates and other Chemicals

[0040] Various monophenol substrates such as 4-fluorophenol, 4-bromophenol, 4-chlorophenol, and 4-iodophenol were purchased from Sigma-Aldrich (Dublin, Ireland). Stock solutions (1 M) of the 4-bromophenol, and 4-iodophenol were prepared in 100% ethanol and subsequent working solutions were further diluted in 50 mM potassium phosphate buffer (pH 7). Similarly, stock solutions (500 mM) of 4-fluorophenol and 4-chlorophenol were prepared in deionized water and working stocks were prepared in 50 mM phosphate buffer. Tyrosol was purchased from TCI Europe (Belgium) and stocks were prepared in 50 mM phosphate buffer. Ascrobic acid and ascorbic acid sodium salt (AA) (used to reduce o-quinone to o-diphenol) was prepared in deionized water freshly before the biotransformation assay

[0041] Biotransformation of 4-halogenated Phenols and Tyrosol

[0042] Biotransformations of halophenols into corresponding halocatechols were carried out using purified enzymes (WT and engineered variants). Various concentrations (5, 10, and 20 mM) of halophenol biotransformations were carried out in 100 mL conical flasks with a working volume of 20 mL at 30° C. and 200 rpm in a shaker incubator. Biotransformation of tyrosol to hydroxytyrosol was carried out using purified enzymes, cell free lysate or whole cells. Various concentrations (75, 100, 150 and 175 mM) of tyrosol biotransformations were carried out in 500 mL baffled conical flasks with a working volume of 100 mL at 30° C. and 200-250 rpm in a shaker incubator. An enzyme concentration of 2 μg/mL per 1 mM of substrate was tested with a substrate to sodium ascorbate ratio of 1:2. Biotransformation of 100 mM tyrosol to hydroxytyrosol was also carried out using commercial mushroom tyrosinase (Sigma, Ireland) using the above specified conditions. Samples (450 μL) were withdrawn at various time points and immediately added to 50 μL of ice-cold 1N HCl. Samples were centrifuged (12,000 g), filtered using Whatman Mini-UniPrep™ syringeless filters (0.45μ Whatman Inc. NJ, USA), and a sample volume of 20 μL was used for all HPLC injections.

[0043] HPLC Analysis

[0044] Filtered samples were analyzed by HPLC using a 5 μm ACE 5 C18column (25 cm×4.6 mm ID; Apex Scientific, Ireland) and a Hewlett-Packard (Palo Alto, Calif., USA) HP1100 instrument equipped with an Agilent 1100 Series Diode Array Detector. The samples were isocratically eluted at 22° C. using a phosphoric acid (0.1%, v/v) and methanol mix at a flow rate of 1.0 mL/min. The ratio of phosphoric acid to methanol was 70:30 for 4-fluorophenol and 50:50 for 4-chloro-, 4-bromo-, and 4-iodophenol.