Carbonyl reductase mutant, preparation method and use thereof, and preparation method of ethyl (R)-6-hydroxy-8-chlorooctanoate

Abstract

The present invention provides a carbonyl reductase mutant, preparation method and use thereof, and a preparation method of ethyl (R)-6-hydroxy-8-chlorooctanoate. The carbonyl reductase mutant is a carbonyl reductase with amino acid mutation; the carbonyl reductase comprises an amino acid sequence as set forth in SEQ ID NO: 2; the amino acid mutation includes E101V, F214R or E101V/F214R. In the present invention, by introducing mutations on the basis of the original carbonyl reductase sequence, the enzyme activity is improved, the stereoselectivity is improved, and ethyl (R)-6-hydroxy-8-chlorooctanoate can be obtained with high yield and high purity under relatively mild conditions, which reduces the production cost and is suitable for industrial production.

Claims

1.-11. (canceled)

12. A carbonyl reductase mutant comprising an amino acid mutation; wherein the carbonyl reductase mutant comprises the amino acid sequence of SEQ ID NO: 2, and the amino acid mutation comprises E101V, F214R or both E101V and F214R.

13. The carbonyl reductase mutant according to claim 12, which is derived from a carbonyl reductase derived from Pseudohyphozyma bogoriensis.

14. The carbonyl reductase mutant of claim 12, which is expressed by a genetically engineered bacterial strain.

15. The carbonyl reductase mutant of claim 14, wherein the genetically engineered bacterial strain comprises any one of Escherichia coli, Pichia pastoris or Bacillus subtilis.

16. A nucleotide sequence encoding the carbonyl reductase mutant according to claim 12.

17. The nucleotide sequence according to claim 16, which comprises SEQ ID NO: 1, SEQ ID NO: 8, or SEQ ID NO: 10.

18. An expression vector comprising at least one copy of the nucleotide sequence according to claim 16.

19. A carbonyl reductase mutant transformant comprising the nucleotide sequence of claim 16.

20. A carbonyl reductase mutant transformant comprising the expression vector of claim 18.

21. A method for preparing a carbonyl reductase mutant comprising an amino acid mutation; wherein the carbonyl reductase mutant comprises the amino acid sequence of SEQ ID NO: 2, and the amino acid mutation comprises E101V, F214R or both E101V and F214R; the method comprising: constructing an expression vector according to claim 18, and transforming the expression vector into a recipient cell to generate a carbonyl reductase mutant transformant; culturing the carbonyl reductase mutant transformant in a culture medium and collecting the culture medium; and obtaining the carbonyl reductase mutant from the culture medium.

22. The method of claim 21, wherein the expression vector comprises a pET series expression vector.

23. The method of claim 22, wherein the pET series expression vector comprises a pET-28a expression vector.

24. A method for catalyzing a carbonyl reduction reaction, comprising: contacting a substrate with a carbonyl reductase mutant according to claim 12.

25. The method of claim 24, wherein the substrate comprises ethyl 6-oxo-8-chlorooctanoate.

26. A method for preparing ethyl (R)-6-hydroxy-8-chlorooctanoate, comprising: mixing a carbonyl reductase mutant according to claim 12 with a reaction solution containing ethyl 6-oxo-8-chlorooctanoate, and reacting to obtain ethyl (R)-6-hydroxyl-8-chlorooctanoate.

27. The method according to claim 26, wherein: the ethyl 6-oxo-8-chlorooctanoate has a mass percent concentration of 4% to 30%; the carbonyl reductase mutant is used in an amount of 5% to 30% by the weight of ethyl 6-oxo-8-chlorooctanoate; the reaction solution further comprises glucose dehydrogenase, a coenzyme and glucose; the glucose dehydrogenase in the reaction system is used in an amount of 3% to 10% by the weight of ethyl 6-oxo-8-chlorooctanoate; the coenzyme is NADP.sup.+; the coenzyme is used in an amount of 1/10,000 to 5/10,000 of the weight of ethyl 6-oxo-8-chlorooctanoate; the glucose is used in an amount 0.9-2 times the weight of ethyl 6-oxo-8-chlorooctanoate; the reaction is carried out in a solvent of Tris-HCl buffer, phosphate buffer, triethanolamine hydrochloride buffer, sodium acetate buffer or Tris-phosphate buffer; the reaction is carried out at pH 6.0 to 7.5; the reaction comprises a cosolvent comprising any one or a combination of at least two of ethanol, propanol, isopropanol, DMF, DMSO, polyethylene glycol or Tween 80; the cosolvent in the reaction is used in a volume percentage of 5% to 10% by the volume of the solvent; the reaction is carried out at a temperature of 20? C. to 35? C.; and the reaction is carried for a time of 4 to 24 hours.

28. A method for manufacturing (R)-?-lipoic acid, comprising the following steps: (1) preparing ethyl (R)-6-hydroxy-8-chlorooctanoate by using the carbonyl reductase mutant according to claim 12, and (2) preparing (R)-?-lipoic acid from the ethyl (R)-6-hydroxy-8-chlorooctanoate.

29. A method for manufacturing (R)-?-lipoic acid, comprising the following steps: (1) preparing ethyl (R)-6-hydroxy-8-chlorooctanoate by using the carbonyl reductase mutant transformant according to claim 19, and (2) preparing (R)-?-lipoic acid from the ethyl (R)-6-hydroxy-8-chlorooctanoate.

30. A method for manufacturing (R)-?-lipoic acid, including the following steps: (1) preparing ethyl (R)-6-hydroxy-8-chlorooctanoate via the method according to claim 25, and (2) preparing (R)-?-lipoic acid from the ethyl (R)-6-hydroxy-8-chlorooctanoate.

31. A method for manufacturing (R)-?-lipoic acid, including the following steps: (1) preparing ethyl (R)-6-hydroxy-8-chlorooctanoate via the method according to claim 26, and (2) preparing (R)-?-lipoic acid from the ethyl (R)-6-hydroxy-8-chlorooctanoate.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0059] FIG. 1 shows a schematic diagram of the reaction for preparing ethyl (R)-6-hydroxyl-8-chlorooctanote in the present invention.

SPECIFIC MODELS FOR CARRYING OUT THE INVENTION

[0060] The technical solutions of the present invention will be further described below through specific models. It should be clear to those skilled in the art that the examples are only for helping to understand the present invention, and should not be regarded as specific limitations on the present invention.

Example 1: Construction of Carbonyl Reductase Mutant

[0061] Using an original carbonyl reductase nucleotide sequence (as set forth in SEQ ID NO: 2) of Pseudohyphozyma bogoriensis genome (GenBank: JALBUQ010000056.1) as a template, NdeI and XhoI (purchased from New England Biolabs, and operated according to the instructions) were used for the recombination into pET28a vector. The recombined vector was transformed into trans 5a competent cells (purchased from TransGen Biotech Co., Ltd.), spread on an LB agar plate containing 50 ?g/mL kana resistance and cultured overnight at 37? C. to obtain the recombinant strain PB-CR0 with the original carbonyl reductase plasmid.

[0062] The PB-CR0 monoclone was picked and placed into an LB liquid medium containing 50 ?g/mL kana resistance, and cultured at 37? C. and 200 rpm for 8 hours; and after culture, the recombinant plasmid with original carbonyl reductase was extracted by plasmid extraction kit. The mutation primers (SEQ ID NO: 3-6) were used to perform single-point mutation amplification PCR, respectively, and Takara Primestar max high-fidelity polymerase (purchased from Takara Company) was used for amplification, in which the PCR program comprised: 98? C. pre-denaturation for 3 minutes, 30 amplification cycles (98? C. 10s, 55? C. 5s, 72? C. 70s), 72? C. 10 min. The amplification product was digested with DpnI at 37? C. for 1 h to remove the template; the mutant plasmid was transformed into BL21(DE3) competent cells, spread on an LB agar plate containing 50 ?g/mL kana resistance and cultured overnight at 37? C. to obtain recombinant strains PB-CR1 (E101V) and PB-CR2 (F214R) with carbonyl reductase single-point mutation plasmids.

[0063] The monoclones of PB-CR1 and PB-CR2 were separately picked and placed into an LB liquid medium containing 50 ?g/mL kana resistance, and cultured at 37? C. and 200 rpm for 8 hours, after the culture, the bacterial solutions were subjected to PCR identification and sequencing verification, and the strain PB-CR1 with the correct sequencing result was continued to undergo other different point mutations to obtain a multi-point mutation recombinant strain PB-CR3 (E101V/F214R), and each of the recombinant strains PB-CR1/2/3 was used for fermentation expression.

[0064] A single colony of each recombinant strain was picked, inoculated into 5 mL of an LB liquid medium containing 50 ?g/mL kana resistance, and cultured on a shaker at 200 rpm and 37? C. for 8 hours. The seed liquid was taken at an inoculum amount of 2% and transferred into 50 mL of a TB liquid medium containing 50 ?g/mL kana resistance, cultured on a shaker at 200 rpm and 37? C. for 2 hours, until the OD600 reached above 0.6, then 0.1 mmol/L IPTG was added, cooled down to 30? C. and continuously cultured for 16 hours; after centrifugation at 4? C. and 10,000 rpm for 15 minutes, the bacterial cells were collected, added with 15 mL of 50 mmol/L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer and resuspended, the bacterial cells were disrupted by an ultrasonic breaker, and centrifuged at 4? C. and 10,000 rpm for 15 minutes to leave a supernatant as carbonyl reductase enzyme solution.

Example 2: Preparation of Ethyl (R)-6-Hydroxy-8-Chlorooctanoate Under Catalysis of Carbonyl Reductase

[0065] In four 100 mL centrifuge tubes, 5 g of ethyl 6-oxo-8-chlorooctanoate, 2.5 mL of ethanol solution, 50 mL of Tris-HCl buffer solution (pH7.0, 100 mM) and 5 g of glucose were added, respectively, and then added with 0.5 g of different carbonyl reductase enzyme solutions, 0.2 g of GDH enzyme solution, 2.5 mg of NADP.sup.+ dry powder to different tubes for reaction, the reaction system was maintained at a pH around 7.0, and shaken at 25? C. and 200 rpm for 8 hours, and samples were taken to measure conversion rate and ee value. The results were shown in Table 1.

[0066] Detection of conversion rate by gas chromatography: [0067] Detection instrument: Shimadzu gas chromatograph with hydrogen flame ionization detector; [0068] Chromatographic column: CP-Chirasil Dex CB (25 m*0.32 mm*0.25 ?m); [0069] Column flow rate of chromatographic column: 2 mL/min; column temperature of chromatographic column: using an initial temperature of 140? C., keeping for 2 min, increasing the temperature to 200? C. at 4? C./min, keeping for 5 min; injection volume: 1 ?L; detector temperature: 270? C.; Sample port temperature: 270? C.; split ratio: 50:1.

[0070] Detection of chirality: [0071] Product purification: 200 ?L of the reaction solution was taken, and extracted with 1000 ?L of ethyl acetate, the organic layer was dried over magnesium sulfate, and used to detect ee value. [0072] Instrument: Shimadzu gas chromatograph with hydrogen flame ionization detector; [0073] Chromatographic column: CYCLOSIL-B (30 m*0.25 mm*0.25 ?m); [0074] Column flow rate: 1 mL/min; [0075] Chromatographic column temperature: using an initial temperature of 160? C., keeping for 2 min, increasing the temperature to 190? C. at 1? C./min, keeping for 2 min, increasing the temperature to 220? C. at 20? C./min, keeping for 2 min; [0076] Injection volume; 0.2 ?L; [0077] Detector temperature: 270? C.; [0078] Injection port temperature: 270? C.; [0079] Split ratio: 50:1; [0080] ee value calculation formula: ee %=([R]-[S]/[R]+[S])*100%.

TABLE-US-00006 TABLE 1 Conversion ee Name Source species rate value Carbonyl reductase mutant Pseudohyphozyma 90.62% 99.84% (E101V/F214R) bogoriensis Carbonyl reductase mutant 67.34% 99.23% (E101V) Carbonyl reductase mutant 54.23% 95.16% (F214R) Original carbonyl reductase 26.95% 87.58%

[0081] It could be seen from Table 1 that the carbonyl reductase mutant derived from Pseudohyphozyma bogoriensis had a significantly improved enzymatic activity compared with the original carbonyl reductase, which could significantly increase the conversion rate of the product, improve the stereoselectivity, and increase the ee value of the product. Especially, the mutant E101V/F214R showed conversion rate of 90.62%, and ee value of 99.84%.

Example 3: Preparation of Ethyl (R)-6-Hydroxyl-8-Chlorooctanoate Under Catalysis of Carbonyl Reductase

[0082] In four 100 mL centrifuge tubes, 15 g of ethyl 6-oxo-8-chlorooctanoate, 2.5 mL of DMSO solution, 50 mL of phosphate buffer solution (pH7.2, 100 mM) and 20 g of glucose were added, respectively, and then added with 0.8 g of different carbonyl reductase enzyme solutions, 0.8 g of GDH enzyme solution, 5 mg of NADP.sup.+ dry powder to different tubes for reaction, the reaction system was maintained at a pH around 7.2, and shaken at 30? C. and 200 rpm for 8 hours, and samples were taken to measure conversion rate and ee value. The results were shown in Table 2.

TABLE-US-00007 TABLE 2 Conversion ee Name Source species rate value Carbonyl reductase mutant Pseudohyphozyma 87.63% 99.54% (E101V/F214R) bogoriensis Carbonyl reductase mutant 60.18% 99.17% (E101V) Carbonyl reductase mutant 48.65% 95.33% (F214R) Original carbonyl reductase 21.79% 86.77%

Example 4: Preparation of Ethyl (R)-6-Hydroxy-8-Chlorooctanoate Catalyzed by Carbonyl Reductase

[0083] In four 100 mL centrifuge tubes, 2 g of ethyl 6-oxo-8-chlorooctanoate, 2.5 mL of Tween 90 solution, 50 mL of sodium acetate buffer solution (pH6.0, 100 mM) and 2.5 g of glucose were added, respectively, and then added with 0.2 g of different carbonyl reductase enzyme solutions, 0.15 g of GDH enzyme solution, 1.0 mg of NADP.sup.+ dry powder to different tubes for reaction, the reaction system was maintained at a pH around 6.0, and shaken at 20? C. and 200 rpm for 8 hours, and samples were taken to measure conversion rate and ee value. The results were shown in Table 3.

TABLE-US-00008 TABLE 3 Conversion ee Name Source species rate value Carbonyl reductase mutant Pseudohyphozyma 75.32% 99.62% (E101V/F214R) bogoriensis Carbonyl reductase mutant 57.32% 99.19% (E101V) Carbonyl reductase mutant 44.66% 95.14% (F214R) Original carbonyl reductase 16.25% 84.68%

Example 5: Preparation of Ethyl (R)-6-Hydroxyl-8-Chlorooctanoate Catalyzed by Carbonyl Reductase

[0084] In four 100 mL centrifuge tubes, 5 g of ethyl 6-oxo-8-chlorooctanoate, 2.5 mL of polyethylene glycol solution, 50 mL of triethanolamine hydrochloride buffer solution (pH7.0, 100 mM) and 7.5 g of glucose were added, respectively, and then added with 0.5 g of different carbonyl reductase enzyme solutions, 0.2 g of GDH enzyme solution, 1.5 mg of NADP.sup.+ dry powder to different tubes for reaction, the reaction system was maintained at a pH around 7.0, and shaken at 20? C. and 200 rpm for 8 hours, and samples were taken to measure conversion rate and ee value. The results were shown in Table 4.

TABLE-US-00009 TABLE 4 Conversion ee Name Source species rate value Carbonyl reductase mutant Pseudohyphozyma 88.89% 99.64% (E101V/F214R) bogoriensis Carbonyl reductase mutant 62.54% 99.17% (E101V) Carbonyl reductase mutant 50.28% 94.87% (F214R) Original carbonyl reductase 25.33% 85.95%

Example 6: Effects of Different Temperatures on Reduction Reaction

[0085] In 100 mL three-necked flasks, 5 g of crude ethyl 6-oxo-8-chlorooctanoate, 2.5 mL of ethanol solution, 50 mL of Tris-HCl buffer solution (pH7.0, 100 mM) and 5 g of glucose were added in order, then stirred, and added to the flasks with 0.5 g of carbonyl reductase mutant enzyme solution (derived from carbonyl reductase mutant E101V/F214R), 0.2 g of GDH enzyme solution, 2.5 mg of NADP.sup.+ dry powder for reaction, in which the reaction temperature was controlled at 20? C., 30? C., 35? C., and 40? C., respectively, and the pH of the reaction system was maintained at around 7.0. After 4 hours of reaction, the corresponding conversion rates were 59.26%, 97.88%, 86.72%, 0.25%, respectively; after 7.5 hours of reaction, the corresponding conversion rates were 78.00%, 100%, 0.45%, respectively, and the ee values of the products were 99.24%, 99.35% %, 99.34%, 99.07%, respectively. It could be seen that the carbonyl reductase mutant of the present invention could realize the preparation of ethyl (R)-6-hydroxy-8-chlorooctanoate under relatively mild conditions.

Example 7: Effects of Different pH Values on Reduction Reaction

[0086] In 100 mL three-neck flasks, 5 g of crude ethyl 6-oxo-8-chlorooctanoate, 2.5 mL of ethanol solution, 50 mL of Tris-HCl buffer solution (pH7.0, 100 mM) and 5 g of glucose were added in order, then stirred, and added to the flasks with 0.5 g of carbonyl reductase mutant enzyme solution (derived from carbonyl reductase mutant E101V/F214R), 0.2 g of GDH enzyme solution, 2.5 mg of NADP.sup.+ dry powder for reaction, the reaction temperature was controlled at 30? C., and the pH of the reaction system was maintained at 6.0, 7.0, and 8.0, respectively. After 4 hours of reaction, the corresponding conversion rates were 72.75%, 97.88%, and 1.04%, respectively; after 7.5 hours of reaction, the corresponding conversion rates were 97.08%, 100%, and 2.18%, respectively, and the ee values of the products were 99.18%, 99.25%, and 99.07%, respectively.

Example 8: Effects of Different Amounts of Carbonyl Reductase on Reduction Reaction

[0087] In 100 mL three-neck flasks, 5 g of crude ethyl 6-oxo-8-chlorooctanoate, 2.5 mL of ethanol solution, 50 mL of Tris-HCl buffer solution (pH7.0, 100 mM) and 5 g of glucose were added in order, then stirred, and added to the flasks with carbonyl reductase enzyme solution (derived from carbonyl reductase mutant E101V/F214R), 0.2 g of GDH enzyme solution, 2.5 mg of NADP.sup.+ dry powder for reaction, the reaction temperature was controlled at 30? C., the reaction system pH was maintained at 7.0, in which the amounts of carbonyl reductase enzyme solution were controlled at 35%, 30%, 20%, 10%, 5%, and 3% of the substrate, respectively. After 4 hours of reaction, the corresponding conversion rates were 98.72%, 99.01%, 98.12%, 91.50%, 51.65%, 31.25%, respectively; after 7.5 hours of reaction, the corresponding conversion rates were 99.16%, 99.89%, 99.92%, 99.87%, 81.42%, 45.07%, respectively, the corresponding product yields were 89.77%, 90.02%, 86.31%, 85.52%, 80.27%, 44.15%, respectively; and the ee values of the products were 99.45%, 99.53%, 99.60%, 99.45%, 99.25%, 99.03%, respectively.

[0088] The applicant declares that the present invention illustrates the process method of the present invention through the above examples, but the present invention is not limited to the above process steps, that is, it does not mean that the present invention must rely on the above process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of the selected raw materials in the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.