NOVEL OLEATE HYDRATASES

Abstract

The present invention relates to a method of producing a 10-hydroxy fatty acid, wherein the method comprises contacting a sample comprising a (9Z) or (9E)-fatty acid with a polypeptide having the activity of an oleate hydratase (EC 4.2.1.53) encoded by a nucleic acid molecule, wherein the nucleic acid molecule is (a) a nucleic acid molecule encoding a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 1 or 7; (b) a nucleic acid molecule comprising or consisting of the nucleotide sequence of SEQ ID NO: 2 or 8; (c) a nucleic acid molecule comprising or consisting of a nucleic acid molecule encoding a polypeptide having the activity of an oleate hydratase the amino acid sequence of which is at least 91% identical to the amino acid sequence of SEQ ID NO: 1 or 7; (d) a nucleic acid molecule encoding a polypeptide having the activity of an oleate hydratase and comprising or consisting of a nucleotide sequence which is at least 91% identical to the nucleotide sequence of SEQ ID NO: 2 or 8; (e) a fragment of the nucleic acid molecule of any of (a) to (d) comprising at least 1341 nucleotides and encoding a polypeptide having the activity of an oleate hydratase; or (f) the nucleic acid sequence of any of (a) to (d) wherein T is U.

Claims

1. A nucleic acid molecule encoding a polypeptide having the activity of an oleate hydratase (EC 4.2.1.53), which nucleic acid molecule is (a) a nucleic acid molecule encoding a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 1 or 7; (b) a nucleic acid molecule comprising or consisting of the nucleotide sequence of SEQ ID NO: 2 or 8; (c) a nucleic acid molecule comprising or consisting of a nucleic acid molecule encoding a polypeptide having the activity of an oleate hydratase the amino acid sequence of which is at least 91% identical to the amino acid sequence of SEQ ID NO: 1 or 7; (d) a nucleic acid molecule encoding a polypeptide having the activity of an oleate hydratase and comprising or consisting of a nucleotide sequence which is at least 91% identical to the nucleotide sequence of SEQ ID NO: 2 or 8; (e) a fragment of the nucleic acid molecule of one any of (a) to (d) comprising at least 1200 nucleotides and encoding a polypeptide having the activity of an oleate hydratase; or (f) the nucleic acid sequence of any of (a) to (d) wherein T is U.

2. A polypeptide encoded by the nucleic acid molecule of claim 1.

3. A fusion protein comprising the polypeptide of claim 2.

4. A vector comprising the nucleic acid molecule of claim 1.

5. A host cell carrying the vector of claim 4.

6. A method of producing a polypeptide having the activity of an oleate hydratase (EC 4.2.1.53) comprising (a) culturing the host cell of claim 5, and (b) isolating the produced protein having the activity of an oleate hydratase.

7. A composition comprising the nucleic acid molecule of claim 1.

8. The composition of claim 7, which is a large-scale composition, a food composition, a cosmetic composition, a pharmaceutical composition, or a diagnostic composition.

9. A method of producing a 10-hydroxy fatty acid, wherein the method comprises contacting a sample comprising a (9Z) or (9E)-fatty acid with a polypeptide having the activity of an oleate hydratase (EC 4.2.1.53) encoded by a nucleic acid molecule, wherein the nucleic acid molecule is (a) a nucleic acid molecule encoding a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 1 or 7; (b) a nucleic acid molecule comprising or consisting of the nucleotide sequence of SEQ ID NO: 2 or 8; (c) a nucleic acid molecule comprising or consisting of a nucleic acid molecule encoding a polypeptide having the activity of an oleate hydratase the amino acid sequence of which is at least 91% identical to the amino acid sequence of SEQ ID NO: 1 or 7; (d) a nucleic acid molecule encoding a polypeptide having the activity of an oleate hydratase and comprising or consisting of a nucleotide sequence which is at least 91° A identical to the nucleotide sequence of SEQ ID NO: 2 or 8; (e) a fragment of the nucleic acid molecule of any of (a) to (d) comprising at least 1200 nucleotides and encoding a polypeptide having the activity of an oleate hydratase; or (f) the nucleic acid sequence of any of (a) to (d) wherein T is U.

10. The method of claim 9, further comprising the esterification of the 10-hydroxy fatty acid, thereby producing one or more esters of the 10-hydroxy fatty acid.

11. The method of claim 9 or 10, further comprising the isolation of the 10-hydroxy fatty acid.

12. The method of claim 9, wherein the step of contacting the sample comprising the (9Z) or (9E)-fatty acid with a polypeptide having the activity of an oleate hydratase (EC 4.2.1.53) is in the presence of flavin adenine dinucleotide (FAD) and/or reduced nicotinamide adenine dinucleotide (NADH).

13. Use of the polypeptide having the activity of an oleate hydratase (EC 4.2.1.53) as defined in claim 9 for the production of a 10-hydroxy fatty acid.

14. The use of claim 13, wherein the polypeptide having the activity of an oleate hydratase (EC 4.2.1.53) is used together with FAD and/or NADH.

15. The method of claim 9, wherein (c′) the nucleic acid molecule of (c) encodes a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 3, and/or (d′) the nucleic acid molecule of (d) comprises or consists of the nucleotide sequence of SEQ ID NO: 4.

16. A composition comprising the polypeptide of claim 2.

17. A composition comprising the fusion protein of claim 3.

18. A composition comprising the vector of claim 4.

19. A composition comprising the host cell of claim 5.

20. The method of claim 10, further comprising the isolation of the one or more esters of the 10-hydroxy fatty acid.

Description

THE FIGURES SHOW

[0131] FIG. 1: Hydratase reaction of oleic acid to 10-hydroxystearic acid catalyzed by fatty acid hydratase.

[0132] FIG. 2: Construction of chimeric fatty acid hydratases from Lysinibacillus fusiformis and Lactococcus lactis

[0133] FIG. 3: Conversion of oleic acid to 10-hydroxystearic acid by fatty acid hydratases from Lysinibacillus fusiformis and Lactococcus lactis

[0134] FIG. 4: (A) SDS-PAGE (Coomassie stain) of oleate hydratase from Lactococcus lactis purified by Ni-NTA affinity chromatography showing the apparent molecular weight of approx. 68 kDa. 10 μL of cell-free extract (CFE), 10 μL of flow through (FT), 10 μL of concentrated elution fraction (EF) 1-12 were loaded onto an SDS-PA gel for providing correct size of approximately 68 kDa and high purity of the eluted protein. (B) SDS-PAGE (Coomassie stain) of chimeric oleate hydratase from Lysinibacillus fusiformis and Lactococcus lactis and purified by Ni-NTA affinity chromatography showing the apparent molecular weight of approx. 68 kDa. 10 μL of cell-free extract (CFE), 10 μL of flow through (FT), 10 μL of concentrated elution fraction (EF) 1-11 were loaded onto an SDS-PA gel for providing correct size of approximately 68 kDa and high purity of the eluted protein. (B) SDS-PAGE (Coomassie stain) of chimeric oleate hydratase from Lactococcus lactis and Lysinibacillus fusiformis showing the apparent molecular weight of approx. 67 kDa. 10 μL of cell-free extract (CFE), 10 μL of insoluble fraction (IF).

[0135] FIG. 5: Conversion of oleic acid to 10-hydroxystearic acid by fatty acid hydratase from Lactococcus lactis regarding pH profile (A) and temperature (B). FIG. 6: Conversion of oleic acid to 10-hydroxystearic acid by chimeric fatty acid hydratases from Lysinibacillus fusiformis and Lactococcus lactis regarding pH profile (A) and temperature (B).

[0136] The following Examples illustrate the invention.

Example 1

Cloning, Expression and Purification of the Fatty Acid Hydratases of Lactococcus lactis and Lysinibacillus fusiformis

[0137] Genomic DNA of Lactococcus lactis and Lysinibacillus fusiformis were isolated and used for a PCR screening with degenerated primers. The genes encoding fatty acid hydratases (EC 4.2.1.53) of Lactococcus lactis and Lysinibacillus fusiformis, respectively, were cloned in a pET26 expression vector, with the addition of a methionine initiation codon and a 6-histidine tag added at the C-terminal end. Overlap extension PCR technique was used for the creation of the chimeric enzyme. The first step was a conventional PCR reaction, in which oligonucleotide primers were partially complementary at their 5′ ends to the respective adjacent fragment which was subsequently fused to create the chimera. The reverse primer of fragment 1 Lf (N-terminal sequence from Lysinibacillus fusiformis) was complementary at its 5′ end to the 5′ end of the forward primer of fragment 2LI (C-terminal sequence from Lactococcus lactis).

[0138] The second PCR step consisted in the fusion of the PCR fragments generated in the first step using the complementary extremities of the primers. In the third step the fusion product was amplified by PCR.

[0139] In yet another example, the chimeric enzyme comprises an N-terminal fragment of Lactoccous lactis (fragment 1L1) and a C-terminal fragment of Lysinibacillus fusiformis (fragment 2Lf) The fusion site of the fragments 1LI and 2Lf is at the same amino acid position as for the chimeric enzyme described above (fragment 1Lf-2L1).

[0140] Competent E. coli BL21 (DE3) cells (Novagen) were transformed with these vectors by heat shock. The recombinant E. coli cells for protein expression were cultivated in a 2,000-ml flask containing 200 ml of Luria—Bertani (LB) medium and 25 μg ml.sup.−1 of kanamycin at 37° C. with shaking at 200 rpm. When the optical density of the bacterial culture at 600 nm reached 0.6, isopropyl-11-D-thiogalactopyranoside was added to a final concentration of 0.1 mM to induce enzyme expression, and the culture was incubated with shaking at 200 rpm at 25° C. for 16 h. The cells were collected by centrifugation at 4° C., 10.000 rpm for 20 min and the pellets were frozen at −80° C.

[0141] Cell-free extracts of the wild type enzymes and the chimera from Lysinibacillus fusiformis and Lactococcus lactis were assayed in 100 mM citrate/phosphate buffer (pH7.0) containing 10 mM MgSO.sub.4 at 30° C. FAD (0.1 mM) and NADH (5 mM) were applied for the FAD-reducing conditions. Reactions were started by the addition of 150 mM oleic acid for 60 min. The conversion of oleic acid to 10-hydroxystearic acid was confirmed by HPLC analysis for both wild type and both chimeric enzymes (oriented either fragment 1Lf-2LI or fragment 1LI-2Lf).

Example 2

Cell Lysis And Protein Purification of Fatty Acid Hydratase from Lactococcus lactis and Chimeric Oleate Hydratase from Lysinibacillus fusiformis and Lactococcus lactis

[0142] For protein purification cell lysates were obtained by resuspension of the cell pellet in buffer A (50 mM piperazine-N,N′-bis-(2-ethanesulfonic acid) (PIPES) buffer (pH 6.5) containing 1 mM CaCl.sub.2, 300 mM NaCl and 15 mM imidazole). The resuspended cells were disrupted using Branson Ultrasonics.sup.TM Sonifier S-250 (Branson Ultrasonics™ Cooperation, Danbury, Con., USA) at duty cycle 50%, output control 5.5 for 1 min, six times on ice.

[0143] The cell debris was removed by centrifugation at 3,894×g for 30 min at 4° C., and the supernatant was filtered through a 0.45-μm filter. The filtrate was applied to a His-Trap HP chromatography column (Amersham Biosciences, Uppsala, Sweden) equilibrated with 50 mM piperazine-N,N′-bis-(2-ethanesulfonic acid) (PIPES) buffer (pH 6.5) containing 1 mM CaCl.sub.2, 300 mM NaCl, 15 mM imidazole. The column was equilibrated with 10 column volumes of buffer A, clear supernatant was loaded at 1 ml/min, column washed with 10 column volumes buffer A and protein eluted in buffer B (buffer A with 0.3 M imidazol). Fractions of 1 ml were collected. After elution 10 μl aliquots of peak fractions were tested by SDS PAGE. The active fractions were collected and immediately buffer exchanged via disposable PD-10 desalting columns (GE Healthcare, UK) according to the recommended protocol. The resultant solution was used as the purified enzyme. Proteins were quantified by the BCA method. SDS-PAGE analyses were conducted in parallel: a main band is clearly visible around 68 kDa, and the purity of the protein is estimated to over 80% (FIG. 4).

Example 3

Effects of pH and Temperature on Enzyme Activity of Fatty Acid Hydratase from Lactococcus lactis and Chimeric Oleate Hydratase from Lysinibacillus fusiformis and Lactococcus lactis

(A) Effects of pH

[0144] The reactions were performed in 100 mM citrate/phosphate buffer (pH levels ranging from 5.6-8.0) containing MgSO4; 8.9 mg/ml total protein of Lactococcus lactis or 9.3 mg/mL of total protein of chimeric oleate hydratase from Lysinibacillus fusiformis and Lactococcus lactis. FAD (0.1 mM) and NADH (5 mM) were applied for the FAD-reducing conditions. Reactions were started by the addition of 150 mM oleic acid at 30° C. for 40 min. Following the reactions, the solutions of fatty acids and hydroxy fatty acids were recovered by three consecutive extractions with 1.6 volume of dichloromethane. After solvent evaporation, the resultant sample was diluted in ethanol and analyzed by HPLC-MS. Analyses were performed on an Agilent 1100 HPLC instrument equipped with evaporative light scattering detector (ELSD) and a Luna® C18 (2) HPLC column (RP-18e 5 μm, 250×4.6 mm) maintained at 50° C. The elution system consisted of ddH.sub.2O with 0.1% formic acid (A) and methanol with 0.1% formic acid (B). The gradient was set as follows: 0 min (80% B); 15 min (100% B); 23.2 min (80% B) at a flow rate of 0.7 mL min.sup.−1.

[0145] Data represent the mean±standard deviation of three independent experiments (FIG. 5A and FIG. 6A).

[0146] The pH optimum of the oleate hydratase from Lactococcus lactis was in the range of 6.9 to 8.0 (FIG. 5A) whereas the pH profile of the chimeric oleate hydratase from Lysinibacillus fusiformis and Lactococcus lactis was shifted to a slightly lower pH optimum at 6.5 with decreasing activity at higher pH values (FIG. 6A).

(B) Effects of Temperature

[0147] The reactions were performed in 100 mM citrate/phosphate buffer (pH7.0) containing MgSO.sub.4;

[0148] 3.1 mg/ml total protein of Lactococcus lactis (a) or 5.6 mg/mL of total protein of chimeric oleate hydratase from Lysinibacillus fusiformis and Lactococcus lactis (b). FAD (0.1 mM) and NADH (5 mM) were applied for the FAD-reducing conditions. Reactions were started by the addition of 150 mM oleic acid for 20 min. At the relative activity of 100%, the specific enzyme activity of the oleate hydratase from Lactococcus lactis was 0.8 μmol min.sup.−1 mg.sup.−1 total protein and the specific enzyme activity of the chimeric oleate hydratase from Lysinibacillus fusiformis and Lactococcus lactis was 1.0 μmol min.sup.−1 mg.sup.−1 total protein.

[0149] The oleate hydratase from Lactococcus lactis has its highest enzyme activity at 15° C. Maximal enzyme activity of the oleate hydratase from Lysinibacillus fusiformis was observed at 35° C. (Kim, Bi-Na et al. (2012) Appl. Microbiol. Biotechnol. 95, 929-937).

[0150] The chimeric enzyme 1 Lf-2L1 showed a surprisingly broader and higher temperature optimum of enzyme activity at 20° C. -25° C. (FIG. 6 B) than the wildtype enzyme from Lactococcus lactis (FIG. 5B).

[0151] The activity of both chimeric enzymes at 30° C. was higher than the wildtype enzyme from Lactococcus lactis and lower than the wildtype enzyme from Lysinibacillus fusiformis (FIG. 3). The temperature profile of the constructed chimeric enzymes surprisingly changed compared to the wildtype. The recombination of oleate hydratase fragments from different microbial flora unexpectedly enables the development of enzymes for different application fields. The use of enzymes that remain active at low temperatures has a great potential for industrial biocatalysis in terms of energy savings by lowering the required temperature of a reaction without sacrificing enzyme activity. The temperature adaptation of the catalytic properties has made cold-adapted enzymes promising biocatalysts for industrial applications, and they are now used in the synthesis of heat-labile fine chemicals, as additives in food processing at low temperatures, and in detergents for cold-water laundry. Chimerization may also be applied when the thermal stability of cold-adapted enzymes, especially at critical temperatures at which the enzymes begin to unfold, have to be improved.

[0152] The results of examples 1 and 3 show that the enzyme characteristics were changed significantly by creating a chimeric oleate hydratase using fragments of two wildtype enzymes from different microbial species. The new features offered by chimerization significantly increase the biotechnological potential of this biocatalyst, expanding its field of application and provide energetic advantages in technical processes which can be performed at ambient temperatures using the enzyme of the invention.