Carbonyl reductase mutants, their preparation methods, applications, and the preparation method of (R)-6-hydroxy-8-chlorooctanoate ethyl ester

Abstract

This invention provides a carbonyl reductase mutant, its preparation method, applications, and a method for preparing (R)-6-hydroxy-8-chlorooctanoate ethyl ester. The carbonyl reductase mutant is a carbonyl reductase with an amino acid mutation. The carbonyl reductase includes the amino acid sequence shown in SEQ ID NO:2. The type of amino acid mutation includes E101V, F214R, or E101V / F214R. This invention, by introducing a mutation into the original carbonyl reductase sequence, improves enzyme activity and stereoselectivity, enabling the high-yield and high-purity production of (R)-6-hydroxy-8-chlorooctanoate ethyl ester under relatively mild conditions, reducing production costs and making it suitable for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of protein modification technology, and relates to a carbonyl reductase mutant, its preparation method, application, and preparation method of (R)-6-hydroxy-8-chlorooctanoate ethyl ester. Background Technology

[0002] Ethyl 6-hydroxy-8-chlorooctanoate is an important organic intermediate, currently mainly used in the synthesis of certain drugs and pharmaceutical intermediates, particularly in the synthesis of α-lipoic acid. α-Lipoic acid possesses antioxidant properties, acting as a vitamin-like compound that eliminates free radicals that accelerate aging and cause disease. (S)-α-lipoic acid exhibits almost no oxidizing activity; therefore, optically pure (R)-α-lipoic acid has attracted significant attention. Ethyl (R)-6-hydroxy-8-chlorooctanoate, as a chiral precursor for the preparation of (R)-α-lipoic acid, is therefore crucial.

[0003] The main synthetic routes for ethyl 6-hydroxy-8-chlorooctanoate include the MPV reduction of ethyl 6-hydroxy-8-oxooctanoate (CN114149324A); reduction with sodium borohydride in methanol as solvent; hydrolysis of racemic lipoic acid intermediate ethyl 6-hydroxy-8-chlorooctanoate by lipase to prepare (R)-ethyl 6-hydroxy-8-chlorooctanoate; and resolution of racemic 6-hydroxy-8-chlorooctanoate by enzymatic acetylation reaction kinetics to obtain (R)-enantiomers. In these methods, the product obtained by sodium borohydride reduction is a racemic mixture, which, after subsequent resolution by lipase, theoretically has a maximum yield of only 50%, which is contrary to atom economics. Moreover, the reaction steps are numerous, and the amount of waste generated is significantly less advantageous.

[0004] Therefore, it is desirable in the art to develop a method for preparing ethyl (R)-6-hydroxy-8-chlorooctanoate in high yield under milder conditions. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a carbonyl reductase mutant, its preparation method, applications, and a method for preparing (R)-6-hydroxy-8-chlorooctanoate ethyl ester. This invention introduces a mutation into the original carbonyl reductase sequence, thereby increasing enzyme activity and stereoselectivity. It enables the high-yield and high-purity production of (R)-6-hydroxy-8-chlorooctanoate ethyl ester under relatively mild conditions, reducing production costs and making it suitable for industrial production.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a carbonyl reductase mutant, wherein the carbonyl reductase mutant is a carbonyl reductase that has undergone amino acid mutation;

[0008] The carbonyl reductase comprises the amino acid sequence shown in SEQ ID NO:2;

[0009] The types of amino acid mutations include E101V, F214R, or E101V / F214R.

[0010] SEQ ID NO:2:

[0011] MMSVSGFLGSATLVAFVSRGVHVRATVRSQSKADAWVAQYPEYKHLIEWCIVPDIAKEGAFIDAVKGVTRVVHTASPFHFNSTTPSDLLDPAIKGTLSILEAALTEPAIKTVVITSSDAAIRDHDKGWREGYTYTSADWNPFTMEQALAETNPHSIYVASKALAE RAAWKFMDDKHPSFTLTTICPVLILGPMLQPVSKLSDMNLSTTIVWDLFHKPTVPDTWVPMFVDVRDCALAHYEATFRPVAAGKRYLCAASEHYSDVDVAIALREAFPEEGHRIPTGGNRPTNHYGWDSKPAEEDLGIKWTPLAKCVEDCGKQLLEMEKAEKGSA.

[0012] In this invention, by introducing the mutation described above into carbonyl reductase, the activity and stereoselectivity of the enzyme are significantly improved, enabling the high yield and high purity of (R)-6-hydroxy-8-chlorooctanoate to be obtained under relatively mild conditions, thereby reducing production costs and making it suitable for industrial production.

[0013] Preferably, the coding sequence of the carbonyl reductase includes the nucleotide sequence shown in SEQ ID NO.1.

[0014] SEQ ID NO.1:

[0015] ATGATGAGCGTCTCCGGGTTCCTCGGATCCGCAACGCTGGTCGCGTTCGTCTCGCGAGGCGTGCATGTCAGGGCGACTGTGCGTTCCCAGTCTAAAGCAGATGCTTGGGTTGCTCAATACCCCGAGTACAAGCACCTTATCGAGTGGTGCATCGTTCCCGACATCGCGAAAGAGGGCGCGTTCATTGATGCCGTGAAAGGCGTTACGAGGGTCGTTCATACCGCCAGTCCTTTCCACTTCAACTCCACTACCCCATCCGACCTCCTCGATCCTGCCATAAAAGGAACTCTCAGTATTCTCGAAGCGGCTCTCACTGAGCCGGCGATCAAAACGGTCGTCATCACGTCTTCAGATGCTGCTATCCGTGATCACGATAAGGGCTGGAGAGAGGGCTATACCTACACTTCGGCCGATTGGAACCCGTTCACTATGGAACAGGCGTTGGCGGAGACTAACCCTCACTCAATCTATGTCGCATCCAAAGCGCTCGCCGAGCGCGCCGCCTGGAAATTCATGGACGACAAGCACCCCTCGTTCACGCTCACGACCATCTGCCCAGTCCTCATCCTCGGTCCCATGCTCCAACCCGTCTCCAAACTCTCCGACATGAACCTCAGCACCACGATCGTGTGGGACCTCTTCCACAAACCTACTGTGCCGGACACGTGGGTGCCTATGTTCGTCGACGTGAGAGATTGCGCTCTGGCGCACTACGAAGCGACGTTCCGTCCCGTCGCGGCGGGGAAACGCTACCTCTGCGCTGCCTCCGAACACTATTCCGACGTCGACGTCGCCATCGCGCTTCGTGAGGCTTTCCCTGAGGAGGGTCACAGGATCCCCACTGGAGGAAATAGGCCGACGAACCATTATGGGTGGGATTCGAAGCCAGCGGAGGAAGACCTCGGAATCAAGTGGACGCCGTTGGCCAAGTGTGTTGAGGATTGCGGGAAACAGTTGTTGGAGATGGAAAAGGCTGAGAAAGGCTCCGCATGA。

[0016] Preferably, the carbonyl reductase is derived from *Pseudohyphozyma bogoriensis*.

[0017] Preferably, the nucleotide sequence of the forward primer (E101V-F) of the E101V mutation type includes the sequence shown in SEQ ID NO:3, and the nucleotide sequence of the reverse primer (E101V-R) includes the sequence shown in SEQ ID NO:4.

[0018] SEQ ID NO:3:

[0019] AAGGAACTCTCAGTATTCTCGTAGCGGCTTCTCACTGAG.

[0020] SEQ ID NO:4:

[0021] TACGAGAATACTGAGAGTTCCTTTTATGGCAGGATCGA.

[0022] Preferably, the nucleotide sequence of the forward primer (F214R-F) of the F214R mutation type includes the sequence shown in SEQ ID NO:5, and the nucleotide sequence of the reverse primer (F214R-R) includes the sequence shown in SEQ ID NO:6.

[0023] SEQ ID NO:5:

[0024] CCACGATCGTGTGGGACCTCCCGCCACAAACCTACTGTG.

[0025] SEQ ID NO:6:

[0026] GCGGAGGTCCCACACGATCGTGGTGCTGAGGTTCATGT.

[0027] The mutated E101V mutant includes the amino acid sequence shown in SEQ ID NO:7, and the coding sequence of the E101V mutant includes the nucleotide sequence shown in SEQ ID NO:8.

[0028] The F214R mutant includes the amino acid sequence shown in SEQ ID NO:9, and the coding sequence of the F214R mutant includes the nucleotide sequence shown in SEQ ID NO:10.

[0029] The E101V / F214R mutant includes the amino acid sequence shown in SEQ ID NO:11, and the coding sequence of the E101V / F214R mutant includes the nucleotide sequence shown in SEQ ID NO:12.

[0030] SEQ ID NO:7:

[0031] MMSVSGFLGSATLVAFVSRGVHVRATVRSQSKADAWVAQYPEYKHLIEWCIVPDIAKEGAFIDAVKGVTRVVHTASPFHFNSTTPSDLLDPAIKGTLSILVAALTEPAIKTVVITSSDAAIRDHDKGWREGYTYTSADWNPFTMEQALAETNPHSIYVASKALAE RAAWKFMDDKHPSFTLTTICPVLILGPMLQPVSKLSDMNLSTTIVWDLFHKPTVPDTWVPMFVDVRDCALAHYEATFRPVAAGKRYLCAASEHYSDVDVAIALREAFPEEGHRIPTGGNRPTNHYGWDSKPAEEDLGIKWTPLAKCVEDCGKQLLEMEKAEKGSA.

[0032] SEQ ID NO:8:

[0033] ATGATGAGCGTCTCCGGGTTCCTCGGATCCGCAACGCTGGTCGCGTTCGTCTCGCGAGGCGTGCATGTCAGGGCGACTGTGCGTTCCCAGTCTAAAGCAGATGCTTGGGTTGCTCAATACCCCGAGTACAAGCACCTTATCGAGTGGTGCATCGTTCCCGACATCGCGAAAGAGGGCGCGTTCATTGATGCCGTGAAAGGCGTTACGAGGGTCGTTCATACCGCCAGTCCTTTCCACTTCAACTCCACTACCCCATCCGACCTCCTCGATCCTGCCATAAAAGGAACTCTCAGTATTCTCGTAGCGGCTCTCACTGAGCCGGCGATCAAAACGGTCGTCATCACGTCTTCAGATGCTGCTATCCGTGATCACGATAAGGGCTGGAGAGAGGGCTATACCTACACTTCGGCCGATTGGAACCCGTTCACTATGGAACAGGCGTTGGCGGAGACTAACCCTCACTCAATCTATGTCGCATCCAAAGCGCTCGCCGAGCGCGCCGCCTGGAAATTCATGGACGACAAGCACCCCTCGTTCACGCTCACGACCATCTGCCCAGTCCTCATCCTCGGTCCCATGCTCCAACCCGTCTCCAAACTCTCCGACATGAACCTCAGCACCACGATCGTGTGGGACCTCTTCCACAAACCTACTGTGCCGGACACGTGGGTGCCTATGTTCGTCGACGTGAGAGATTGCGCTCTGGCGCACTACGAAGCGACGTTCCGTCCCGTCGCGGCGGGGAAACGCTACCTCTGCGCTGCCTCCGAACACTATTCCGACGTCGACGTCGCCATCGCGCTTCGTGAGGCTTTCCCTGAGGAGGGTCACAGGATCCCCACTGGAGGAAATAGGCCGACGAACCATTATGGGTGGGATTCGAAGCCAGCGGAGGAAGACCTCGGAATCAAGTGGACGCCGTTGGCCAAGTGTGTTGAGGATTGCGGGAAACAGTTGTTGGAGATGGAAAAGGCTGAGAAAGGCTCCGCATGA。

[0034] SEQ ID NO:9:

[0035] MMSVSGFLGSATLVAFVSRGVHVRATVRSQSKADAWVAQYPEYKHLIEWCIVPDIAKEGAFIDAVKGVTRVVHTASPFHFNSTTPSDLLDPAIKGTLSILEAALTEPAIKTVVITSSDAAIRDHDKGWREGYTYTSADWNPFTMEQALAETNPHSIYVASKALAERAAWKFMDDKHPSFTLTTICPVLILGPMLQPVSKLSDMNLSTTIVWDLRHKPTVPDTWVPMFVDVRDCALAHYEATFRPVAAGKRYLCAASEHYSDVDVAIALREAFPEEGHRIPTGGNRPTNHYGWDSKPAEEDLGIKWTPLAKCVEDCGKQLLEMEKAEKGSA。

[0036] SEQ ID NO:10:

[0037] ATGATGAGCGTCTCCGGGTTCCTCGGATCCGCAACGCTGGTCGCGTTCGTCTCGCGAGGCGTGCATGTCAGGGCGACTGTGCGTTCCCAGTCTAAAGCAGATGCTTGGGTTGCTCAATACCCCGAGTACAAGCACCTTATCGAGTGGTGCATCGTTCCCGACATCGCGAAAGAGGGCGCGTTCATTGATGCCGTGAAAGGCGTTACGAGGGTCGTTCATACCGCCAGTCCTTTCCACTTCAACTCCACTACCCCATCCGACCTCCTCGATCCTGCCATAAAAGGAACTCTCAGTATTCTCGAAGCGGCTCTCACTGAGCCGGCGATCAAAACGGTCGTCATCACGTCTTCAGATGCTGCTATCCGTGATCACGATAAGGGCTGGAGAGAGGGCTATACCTACACTTCGGCCGATTGGAACCCGTTCACTATGGAACAGGCGTTGGCGGAGACTAACCCTCACTCAATCTATGTCGCATCCAAAGCGCTCGCCGAGCGCGCCGCCTGGAAATTCATGGACGACAAGCACCCCTCGTTCACGCTCACGACCATCTGCCCAGTCCTCATCCTCGGTCCCATGCTCCAACCCGTCTCCAAACTCTCCGACATGAACCTCAGCACCACGATCGTGTGGGACCTCCGCCACAAACCTACTGTGCCGGACACGTGGGTGCCTATGTTCGTCGACGTGAGAGATTGCGCTCTGGCGCACTACGAAGCGACGTTCCGTCCCGTCGCGGCGGGGAAACGCTACCTCTGCGCTGCCTCCGAACACTATTCCGACGTCGACGTCGCCATCGCGCTTCGTGAGGCTTTCCCTGAGGAGGGTCACAGGATCCCCACTGGAGGAAATAGGCCGACGAACCATTATGGGTGGGATTCGAAGCCAGCGGAGGAAGACCTCGGAATCAAGTGGACGCCGTTGGCCAAGTGTGTTGAGGATTGCGGGAAACAGTTGTTGGAGATGGAAAAGGCTGAGAAAGGCTCCGCATGA。

[0038] SEQ ID NO:11:

[0039] MMSVSGFLGSATLVAFVSRGVHVRATVRSQSKADAWVAQYPEYKHLIEWCIVPDIAKEGAFIDAVKGVTRVVHTASPFHFNSTTPSDLLDPAIKGTLSILVAALTEPAIKTVVITSSDAAIRDHDKGWREGYTYTSADWNPFTMEQALAETNPHSIYVASKALAERAAWKFMDDKHPSFTLTTICPVLILGPMLQPVSKLSDMNLSTTIVWDLRHKPTVPDTWVPMFVDVRDCALAHYEATFRPVAAGKRYLCAASEHYSDVDVAIALREAFPEEGHRIPTGGNRPTNHYGWDSKPAEEDLGIKWTPLAKCVEDCGKQLLEMEKAEKGSA。

[0040] SEQ ID NO:12:

[0041] ATGATGAGCGTCTCCGGGTTCCTCGGATCCGCAACGCTGGTCGCGTTCGTCTCGCGAGGCGTGCATGTCAGGGCGACTGTGCGTTCCCAGTCTAAAGCAGATGCTTGGGTTGCTCAATACCCCGAGTACAAGCACCTTATCGAGTGGTGCATCGTTCCCGACATCGCGAAAGAGGGCGCGTTCATTGATGCCGTGAAAGGCGTTACGAGGGTCGTTCATACCGCCAGTCCTTTCCACTTCAACTCCACTACCCCATCCGACCTCCTCGATCCTGCCATAAAAGGAACTCTCAGTATTCTCGTAGCGGCTCTCACTGAGCCGGCGATCAAAACGGTCGTCATCACGTCTTCAGATGCTGCTATCCGTGATCACGATAAGGGCTGGAGAGAGGGCTATACCTACACTTCGGCCGATTGGAACCCGTTCACTATGGAACAGGCGTTGGCGGAGACTAACCCTCACTCAATCTATGTCGCATCCAAAGCGCTCGCCGAGCGCGCCGCCTGGAAATTCATGGACGACAAGCACCCCTCGTTCACGCTCACGACCATCTGCCCAGTCCTCATCCTCGGTCCCATGCTCCAACCCGTCTCCAAACTCTCCGACATGAACCTCAGCACCACGATCGTGTGGGACCTCCGCCACAAACCTACTGTGCCGGACACGTGGGTGCCTATGTTCGTCGACGTGAGAGATTGCGCTCTGGCGCACTACGAAGCGACGTTCCGTCCCGTCGCGGCGGGGAAACGCTACCTCTGCGCTGCCTCCGAACACTATTCCGACGTCGACGTCGCCATCGCGCTTCGTGAGGCTTTCCCTGAGGAGGGTCACAGGATCCCCACTGGAGGAAATAGGCCGACGAACCATTATGGGTGGGATTCGAAGCCAGCGGAGGAAGACCTCGGAATCAAGTGGACGCCGTTGGCCAAGTGTGTTGAGGATTGCGGGAAACAGTTGTTGGAGATGGAAAAGGCTGAGAAAGGCTCCGCATGA。

[0042] In a second aspect, the present invention provides a nucleic acid molecule that encodes the carbonyl reductase mutant described in the first aspect.

[0043] Thirdly, the present invention provides an expression vector containing at least one copy of the nucleic acid molecule described in the second aspect.

[0044] Fourthly, the present invention provides a carbonyl reductase mutant transformant, wherein the carbonyl reductase mutant transformant is a genetically engineered strain expressing the carbonyl reductase mutant described in the first aspect.

[0045] Preferably, the carbonyl reductase mutant transformant contains the nucleic acid molecule described in the second aspect.

[0046] Preferably, the carbonyl reductase mutant transformant contains the expression vector described in the third aspect.

[0047] Preferably, the genetically engineered strain includes any one of Escherichia coli, Pichia pastoris, or Bacillus subtilis.

[0048] Fifthly, the present invention provides a method for preparing the carbonyl reductase mutant described in the first aspect, the method comprising:

[0049] An expression vector was constructed and transformed into recipient cells to create a carbonyl reductase mutant transformant.

[0050] The carbonyl reductase mutant transformant was cultured, and the culture was collected to obtain the carbonyl reductase mutant.

[0051] Preferably, the carrier is a pET series carrier, preferably pET-28a.

[0052] As a preferred technical solution, the method for preparing the carbonyl reductase mutant of the present invention specifically includes the following steps:

[0053] The nucleic acid molecule encoding the carbonyl reductase mutant was digested and ligated into the expression vector pET-28a(+), which was then transformed into host cells BL21(DE3); positive clones were screened to obtain carbonyl reductase mutant transformants.

[0054] The carbonyl reductase mutant transformant was activated in LB liquid medium and then transferred to LB liquid medium for cultivation. After fermentation, the cells were collected, and the cell bodies were broken to obtain the carbonyl reductase mutant enzyme solution.

[0055] In a sixth aspect, the present invention provides the application of the carbonyl reductase mutant described in the first aspect in catalyzing carbonyl reduction reactions;

[0056] Preferably, the substrate for the carbonyl reduction reaction is ethyl 6-oxo-8-chlorooctanoate.

[0057] In a seventh aspect, the present invention provides a method for preparing ethyl (R)-6-hydroxy-8-chlorooctanoate, the method comprising the following steps:

[0058] The carbonyl reductase mutant described in the first aspect is mixed with a reaction solution containing ethyl 6-oxo-8-chlorooctanoate to obtain (R)-6-hydroxy-8-chlorooctanoate ethyl ester.

[0059] Preferably, the mass percentage concentration of ethyl 6-oxo-8-chlorooctanoate in the reaction system is 4%-30%.

[0060] Preferably, the amount of the carbonyl reductase mutant is 5%-30% of the weight of ethyl 6-oxo-8-chlorooctanoate, for example 5%, 8%, 10%, 15%, 18%, 20%, 23%, 25%, 28% or 30%.

[0061] Preferably, the reaction solution further includes glucose dehydrogenase, coenzyme, and glucose;

[0062] Preferably, the amount of glucose dehydrogenase used in the reaction system is 3%-10% of the weight of ethyl 6-oxo-8-chlorooctanoate, for example, 3%, 5%, 8%, 9% or 10%.

[0063] Preferably, the coenzyme is NADP. + ;

[0064] Preferably, the amount of the coenzyme is from 0.01% to 0.05% of the weight of ethyl 6-oxo-8-chlorooctanoate, for example, 0.01%, 0.02%, 0.03%, 0.04%, or 0.05%.

[0065] Preferably, the amount of glucose used is 0.9 to 2 times the weight of ethyl 6-oxo-8-chlorooctanoate, for example, 0.9 times, 1 time, 1.3 times, 1.5 times, 1.8 times or 2 times.

[0066] Preferably, the solvent for the reaction is Tris-HCl buffer, phosphate buffer, triethanolamine hydrochloride buffer, sodium acetate buffer, or Tris-phosphate buffer, with Tris-HCl buffer being the most preferred.

[0067] Preferably, the reaction is carried out at pH 6.0-7.5 (e.g., 6.0, 6.2, 6.4, 6.8, 7.0, 7.2 or 7.5).

[0068] Preferably, the reaction is carried out in the presence of a co-solvent, which is preferably any one or a combination of at least two of ethanol, propanol, isopropanol, DMF, DMSO, polyethylene glycol, or Tween 80. Ethanol is preferred.

[0069] Preferably, the volume percentage of the co-solvent in the reaction system is 5%-10% of the solvent volume, for example, 5%, 6%, 7%, 8%, 9% or 10%.

[0070] Preferably, the reaction temperature is 20-35℃ (e.g., 20℃, 23℃, 25℃, 28℃, 30℃, 33℃ or 35℃), and the reaction time is 4-24h (e.g., 4h, 6h, 8h, 10h, 12h, 15h, 18h, 20h, 22h or 24h).

[0071] In an eighth aspect, the present invention provides the use of the carbonyl reductase mutant described in the first aspect or the carbonyl reductase mutant transformant described in the fourth aspect or the method described in the seventh aspect in the preparation of (R)-α-lipoic acid.

[0072] Compared with the prior art, the present invention has the following beneficial effects:

[0073] The carbonyl reductase mutant of the present invention introduces mutations based on the original carbonyl reductase sequence, thereby increasing enzyme activity and stereoselectivity. It can obtain (R)-6-hydroxy-8-chlorooctanoate ethyl ester in high yield and high purity under relatively mild conditions, reducing production costs and making it suitable for industrial production. Attached Figure Description

[0074] Figure 1 This is a schematic diagram illustrating the reaction principle for preparing (R)-6-hydroxy-8-chlorooctanoate ethyl ester in this invention. Detailed Implementation

[0075] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0076] Example 1 Construction of carbonyl reductase mutant

[0077] Using the original carbonyl reductase nucleotide sequence of the Pseudohyphozyma bogoriensis genome (GenBank: JALBUQ010000056.1) (as shown in SEQ ID NO:2) as a template, NdeⅠ and XhoⅠ (purchased from New England Biolabs, operated according to the manufacturer's instructions) were used for recombination into the pET28a vector. The recombinant vector was transformed into trans 5α competent cells (purchased from TransGen), plated on LB agar plates containing 50 μg / mL kanamycin, and cultured overnight at 37°C to obtain the recombinant strain PB-CR0 carrying the original carbonyl reductase plasmid.

[0078] Single PB-CR0 clones were picked and cultured in LB liquid medium containing 50 μg / mL kana resistance at 37°C and 200 rpm for 8 h. After culture, the recombinant plasmid containing the original carbonyl reductase was extracted using a plasmid extraction kit. Single-point mutation amplification PCR was then performed using the respective mutant primers (SEQ ID NO: 3-6) with Takara Primestar max high-fidelity polymerase (purchased from Takara). The PCR program was: 98°C pre-denaturation for 3 min, 30 amplification cycles (98°C 10 s, 55°C 5 s, 72°C 70 s), 72°C 10 min. The amplified products were digested with DpnI at 37°C for 1 h to remove the template. The mutant plasmid was transformed into BL21DE3 competent cells and plated on LB plates containing 50 μg / mL kana resistance, cultured overnight at 37°C, yielding recombinant strains PB-CR1 (E101V) and PB-CR2 (F214R) carrying the single-point mutant plasmid of carbonyl reductase.

[0079] Single clones of PB-CR1 and PB-CR2 were picked and cultured in LB liquid medium with 50 μg / mL kana resistance for 8 h at 37℃ and 200 rpm. After culture, the bacterial culture was identified by PCR and sequenced. The PB-CR1 strain with correct sequencing results was further subjected to other different point mutations to obtain the recombinant strain PB-CR3 (E101V / F214R) with multiple point mutations. Each recombinant strain PB-CR1 / 2 / 3 was used for fermentation expression.

[0080] Single colonies of each recombinant strain were picked and inoculated into 5 mL of LB liquid medium containing 50 μg / mL kana resistance. The culture was incubated at 37°C and 200 rpm for 8 h. Seed culture was transferred at a 2% inoculation rate to 50 mL of TB liquid medium containing 50 μg / mL kana resistance. The culture was incubated at 37°C and 200 rpm for 2 h until the OD600 reached above 0.6. 0.1 mmol / L IPTG was added, and the culture was cooled to 30°C and incubated for another 16 h. The cells were collected by centrifugation at 4°C and 10,000 rpm for 15 min. 15 mL of 50 mmol / L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer was added and the cells were resuspended. The cells were then disrupted using an ultrasonic homogenizer, centrifuged at 4°C and 10,000 rpm for 15 min, and the supernatant was collected as the carbonyl reductase enzyme solution.

[0081] Example 2: Preparation of (R)-6-hydroxy-8-chlorooctanoate ethyl catalyzed by carbonyl reductase

[0082] In four 100 mL centrifuge tubes, add 5 g of ethyl 6-oxo-8-chlorooctanoate, 2.5 mL of ethanol solution, 50 mL of Tris-HCl buffer solution (pH 7.0, 100 mM), and 5 g of glucose sequentially. Then, add 0.5 g of carbonyl reductase enzyme solution, 0.2 g of GDH enzyme solution, and 2.5 mg of NADP to each tube respectively. + The dry powder was reacted, and the pH of the reaction system was maintained at around 7.0. The mixture was shaken at 25°C and 200 rpm for 8 hours. Samples were taken to measure the conversion rate and ee value. The results are shown in Table 1.

[0083] Conversion rate gas chromatography detection:

[0084] Detection instrument: Shimadzu gas chromatograph with flame ionization detector;

[0085] Chromatographic column: CP-Chirasil Dex CB (25m*0.32mm*0.25um);

[0086] Column flow rate: 2 mL / min; Column temperature: initial temperature 140℃, hold for 2 min, increase to 200℃ at 4℃ / min, hold for 5 min; Injection volume: 1 μL; Detector temperature: 270℃; Injector temperature: 270℃; Split ratio: 50:1.

[0087] Chirality detection:

[0088] Product purification: Take 200 μL of reaction solution and extract with 1000 μL of ethyl acetate. The organic layer after drying with magnesium sulfate can be used to test the ee value.

[0089] Instrument: Shimadzu gas chromatograph with flame ionization detector;

[0090] Chromatographic column: CYCLOSIL-B (30m*0.25mm*0.25μm);

[0091] Column flow rate: 1 mL / min;

[0092] Column temperature: Initial temperature 160℃, hold for 2 min, increase to 190℃ at 1℃ / min, hold for 2 min, increase to 220℃ at 20℃ / min, hold for 2 min;

[0093] Injection volume: 0.2 μL;

[0094] Detector temperature: 270℃;

[0095] Inlet temperature: 270℃;

[0096] Flow split ratio: 50:1;

[0097] The formula for calculating the ee value is: ee%=([R]-[S] / [R]+[S])*100%.

[0098] Table 1

[0099]

[0100] As shown in Table 1, the carbonyl reductase mutants derived from Pseudohyphozyma bogoriensis showed significantly increased enzyme activity compared to the original carbonyl reductase, which significantly improved the conversion rate of the product, enhanced stereoselectivity, and increased the product ee value. In particular, the mutant E101V / F214R achieved a conversion rate of 90.62% and an ee value of 99.84%.

[0101] Example 3: Preparation of (R)-6-hydroxy-8-chlorooctanoate ethyl ester catalyzed by carbonyl reductase

[0102] In four 100 mL centrifuge tubes, add 15 g of ethyl 6-oxo-8-chlorooctanoate, 2.5 mL of DMSO solution, 50 mL of phosphate buffer (pH 7.2, 100 mM), and 20 g of glucose sequentially. Then, add 0.8 g of carbonyl reductase solution, 0.8 g of GDH enzyme solution, and 5 mg of NADP to each tube respectively. + The dry powder was used for the reaction, and the pH of the reaction system was maintained at around 7.2. The mixture was shaken on a shaker at 30℃ and 200 rpm for 8 hours. Samples were taken to measure the conversion rate and ee value. The results are shown in Table 2.

[0103] Table 2

[0104]

[0105] Example 4: Preparation of (R)-6-hydroxy-8-chlorooctanoate ethyl ester catalyzed by carbonyl reductase

[0106] In four 100 mL centrifuge tubes, add 2 g of ethyl 6-oxo-8-chlorooctanoate, 2.5 mL of Tween 80 solution, 50 mL of sodium acetate buffer (pH 6.0, 100 mM), and 2.5 g of glucose, respectively. Then, add 0.2 g of carbonyl reductase enzyme solution, 0.15 g of GDH enzyme solution, and 1.0 mg of NADP to each tube, respectively. + The dry powder was reacted, and the pH of the reaction system was maintained at 6.0. The mixture was shaken on a shaker at 200 rpm for 8 hours at 20°C. Samples were taken to measure the conversion rate and ee value. The results are shown in Table 3.

[0107] Table 3

[0108]

[0109]

[0110] Example 5: Preparation of (R)-6-hydroxy-8-chlorooctanoate ethyl catalyzed by carbonyl reductase

[0111] In four 100 mL centrifuge tubes, add 5 g of ethyl 6-oxo-8-chlorooctanoate, 2.5 mL of polyethylene glycol solution, 50 mL of triethanolamine hydrochloride buffer (pH 7.0, 100 mM), and 7.5 g of glucose, respectively. Then, add 0.5 g of carbonyl reductase enzyme solution, 0.2 g of GDH enzyme solution, and 1.5 mg of NADP to each tube, respectively. + The dry powder was reacted, and the pH of the reaction system was maintained at 7.0. The mixture was shaken on a shaker at 200 rpm for 8 hours at 20°C. Samples were taken to measure the conversion rate and ee value. The results are shown in Table 4.

[0112] Table 4

[0113]

[0114] Example 6: Effect of different temperatures on the reduction reaction

[0115] In a 100 mL three-necked flask, add 5 g of crude ethyl 6-oxo-8-chlorooctanoate, 2.5 mL of ethanol solution, 50 mL of Tris-HCl buffer solution (pH 7.0, 100 mM), and 5 g of glucose sequentially. Stir. Then add 0.5 g of carbonyl reductase mutant enzyme solution (from carbonyl reductase mutant E101V / F214R), 0.2 g of GDH enzyme solution, and 2.5 mg of NADP. +The dry powder was reacted at controlled temperatures of 20℃, 30℃, 35℃, and 40℃, with the pH of the reaction system maintained around 7.0. The conversion rates after 4 hours of reaction were 59.26%, 97.88%, 86.72%, and 0.25%, respectively; the conversion rates after 7.5 hours of reaction were 78.00%, 100%, and 0.45%, respectively, with product ee values ​​of 99.24%, 99.35%, 99.34%, and 99.07%, respectively. It can be seen that the carbonyl reductase mutant of this invention can achieve the preparation of (R)-6-hydroxy-8-chlorooctanoate ethyl ester under relatively mild conditions.

[0116] Example 7: Effect of different pH values ​​on the reduction reaction

[0117] In a 100 mL three-necked flask, add 5 g of crude ethyl 6-oxo-8-chlorooctanoate, 2.5 mL of ethanol solution, 50 mL of Tris-HCl buffer solution (pH 7.0, 100 mM), and 5 g of glucose sequentially. Stir. Then add 0.5 g of carbonyl reductase mutant enzyme solution (from carbonyl reductase mutant E101V / F214R), 0.2 g of GDH enzyme solution, and 2.5 mg of NADP. + The dry powder was reacted at a controlled temperature of 30℃, and the pH of the reaction system was maintained at 6.0, 7.0, and 8.0, respectively. The conversion rates after 4 hours of reaction were 72.75%, 97.88%, and 1.04%, respectively; the conversion rates after 7.5 hours of reaction were 97.08%, 100%, and 2.18%, respectively, and the ee values ​​of the products were 99.18%, 99.25%, and 99.07%, respectively.

[0118] Example 8: Effect of different amounts of carbonyl reductase on the reduction reaction

[0119] In a 100 mL three-necked flask, add 5 g of crude 6-oxo-8-chlorooctanoate, 2.5 mL of ethanol solution, 50 mL of Tris-HCl buffer solution (pH 7.0, 100 mM), and 5 g of glucose sequentially. Stirring is then initiated. Next, carbonyl reductase enzyme solution (from the carbonyl reductase mutant E101V / F214R), 0.2 g of GDH enzyme solution, and 2.5 mg of NADP are added to the flask. +The dry powder was used for the reaction, and the reaction temperature was controlled at 30℃. The pH of the reaction system was maintained at 7.0. The amount of carbonyl reductase enzyme solution was controlled at 35%, 30%, 20%, 10%, 5%, and 3% of the substrate, respectively. The conversion rates after 4 hours of reaction were 98.72%, 99.01%, 98.12%, 91.50%, 51.65%, and 31.25%, respectively; the conversion rates after 7.5 hours of reaction were 99.16%, 99.89%, 99.92%, 99.87%, 81.42%, and 45.07%, respectively, with corresponding product yields of 89.77%, 90.02%, 86.31%, 85.52%, 80.27%, and 44.15%, respectively; and product ee values ​​of 99.45%, 99.53%, 99.60%, 99.45%, 99.25%, and 99.03%, respectively.

[0120] The applicant declares that the present invention is illustrated by the above embodiments, 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 improvements to the present invention, equivalent substitutions of the raw materials used in the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A carbonyl reductase mutant, characterized in that, The carbonyl reductase mutant is a carbonyl reductase with amino acid mutation; The amino acid sequence of the carbonyl reductase is shown in SEQ ID NO: 2; The amino acid mutation is E101V, F214R or E101V / F214R.

2. The carbonyl reductase mutant according to claim 1, wherein The coding sequence of the carbonyl reductase comprises the nucleotide sequence shown in SEQ ID NO.

1.

3. The carbonyl reductase mutant according to claim 1, wherein The carbonyl reductase is derived from Pseudohyphozyma bogoriensis.

4. The carbonyl reductase mutant according to claim 1, wherein The nucleotide sequence of the forward primer of the E101V mutation type comprises the sequence shown in SEQ ID NO: 3, and the nucleotide sequence of the reverse primer comprises the sequence shown in SEQ ID NO:

4.

5. The carbonyl reductase mutant of claim 1, wherein The nucleotide sequence of the forward primer of the F214R mutation type comprises the sequence shown in SEQ ID NO: 5, and the nucleotide sequence of the reverse primer comprises the sequence shown in SEQ ID NO:

6.

6. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the carbonyl reductase mutant according to any one of claims 1-5.

7. An expression vector, characterized by, The expression vector contains at least one copy of the nucleic acid molecule according to claim 6.

8. A carbonyl reductase mutant transformant, characterized by, The carbonyl reductase mutant transformant is a genetically engineered strain expressing the carbonyl reductase mutant according to any one of claims 1-5.

9. The carbonyl reductase mutant transformant according to claim 8, characterized by, The carbonyl reductase mutant transformant contains the nucleic acid molecule according to claim 6.

10. The carbonyl reductase mutant transformant according to claim 8, characterized by, The carbonyl reductase mutant transformant contains the expression vector according to claim 7.

11. The carbonyl reductase mutant transformant according to claim 8, characterized by, The genetically engineered strain comprises any one of Escherichia coli, Pichia pastoris or Bacillus subtilis.

12. A method for producing the carbonyl reductase mutant according to any one of claims 1 to 5, characterized by, The preparation method comprises: constructing an expression vector and transforming it into a recipient cell to construct a carbonyl reductase mutant transformant; culturing the carbonyl reductase mutant transformant, collecting the culture and obtaining the carbonyl reductase mutant.

13. The method of claim 12, wherein, The vector is selected from the pET series vectors.

14. The method of claim 12, wherein, The vector is pET-28a.

15. The carbonyl reductase mutant according to any one of claims 1-5 for use in catalyzing a carbonyl reduction reaction. The substrate of the carbonyl reduction reaction is ethyl 6-oxo-8-chlorooctanoate.

16. A process for the preparation of (R)-6-hydroxy-8-chlorooctanoic acid ethyl ester, characterized in that, The method comprises the following steps: mixing the carbonyl reductase mutant according to any one of claims 1-5 and a reaction solution containing ethyl 6-oxo-8-chlorooctanoate, and reacting to obtain (R)-ethyl 6-hydroxy-8-chlorooctanoate.

17. The method of claim 16, wherein, The mass percentage concentration of ethyl 6-oxo-8-chlorooctanoate in the reaction system is 4%-30%.

18. The method of claim 16, wherein, The amount of the carbonyl reductase mutant is 5%-30% of the weight of ethyl 6-oxo-8-chlorooctanoate.

19. The method of claim 16, wherein, The reaction solution further comprises glucose dehydrogenase, coenzyme and glucose.

20. The method of claim 19, wherein, The amount of glucose dehydrogenase in the reaction system is 3%-10% of the weight of ethyl 6-oxo-8-chlorooctanoate.

21. The method of claim 19, wherein, The coenzyme is NADP + .

22. The method of claim 19, wherein, The amount of coenzyme is one ten-thousandth to five ten-thousandths of the weight of ethyl 6-oxo-8-chlorooctanoate.

23. The method of claim 19, wherein, The amount of glucose is 0.9-2 times the weight of ethyl 6-oxo-8-chlorooctanoate.

24. The method of claim 16, wherein, The solvent of the reaction is Tris-HCl buffer, phosphate buffer, triethanolamine hydrochloride buffer, sodium acetate buffer or Tris-phosphate buffer.

25. The method of claim 24, wherein, The solvent for the reaction is Tris-HCl buffer.

26. The method of claim 16, wherein, The reaction is carried out at pH 6.0-7.

5.

27. The method of claim 16, wherein, The reaction is carried out in the presence of a co-solvent.

28. The method of claim 27, wherein, The co-solvent is selected from any one or a combination of at least two of ethanol, propanol, isopropanol, DMF, DMSO, polyethylene glycol or Tween 80.

29. The method of claim 27, wherein, The co-solvent is ethanol.

30. The method of claim 27, wherein, The volume percentage of the co-solvent in the reaction system is 5%-10% of the volume of the solvent.

31. The method of claim 16, wherein, The temperature of the reaction is 20-35℃, and the reaction time is 4-24 h.

32. Use of the carbonyl reductase mutant of any one of claims 1-5 or the carbonyl reductase mutant transformant of any one of claims 8-11 or the method of any one of claims 12-14 in the preparation of (R)-alpha-lipoic acid, wherein the substrate of the reaction in the preparation of (R)-alpha-lipoic acid is ethyl 6-oxo-8-chlorooctanoate.

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