A recombinant carbonyl reductase mutant and its application in catalyzing synthesis of chiral alcohol

Abstract

The present application provides a kind of recombinant carbonyl reductase mutant, by the amino acid sequence of the recombinant carbonyl reductase from Rhodosporidium toruloides ZJB2014212 it is obtained by mutation of aspartic acid in the 25th position into threonine.The recombinant carbonyl reductase mutant has high activity, high stereoselectivity compared with wild-type enzyme, further improves the catalytic efficiency of substrate precursor ketone.The recombinant carbonyl reductase mutant is applied to the synthesis of chiral alcohol by biological enzyme method, asymmetric catalytic reduction is carried out on substrate precursor ketone, compared with wild-type enzyme, the catalytic activity is improved when converting the above reaction, and the reaction process time is shortened.In addition, in the reaction system for catalytic preparation of chiral alcohol in the present application, a single enzyme double substrate coupling system is constructed, and isopropanol is selected as the auxiliary substrate of coenzyme regeneration system, which can effectively save production cost, and has important significance for the industrial application of biological enzyme method for catalytic preparation of chiral alcohol.

Description

Technical Field

[0001] This invention belongs to the field of biotransformation, specifically relating to a recombinant carbonyl reductase mutant and its application in the catalytic synthesis of chiral alcohols. Background Technology

[0002] Carbonyl reductases are a class of enzymes involved in reversible redox reactions. They utilize the coenzyme NADP(H) or NAD(H) as a hydrogen carrier to catalyze the reaction of aldehydes / ketones with alcohols, and are considered one of the most commonly used biocatalysts in the synthesis of chiral alcohols. Carbonyl reductases are widely distributed and commonly found in natural environments, such as various plants, bacteria, fungi, and animal tissues. They are mainly distributed in three superfamilies: short-chain dehydrogenases (SDRs), aldehyde-ketoreductases (AKRs), and medium-chain dehydrogenases (MDRs). Although they differ significantly in spatial structure, catalytic mechanism, and protein chain length, they all catalyze similar reactions. Short-chain dehydrogenases (SDRs) are a class of non-metal-dependent enzymes, typically with a single subunit containing around 250 amino acid residues. They exist as homopolymers, including monomers, dimers, and tetramers. Based on differences in coenzyme binding sites and catalytic mechanisms, SDRs are generally divided into five subfamilies, the most important being classical and extended SDRs. Although the sequence similarity among SDRs is only 15-30%, they possess highly conserved higher-order structures. Their catalytic tetramer is typically Asn(aspartic acid)-Ser(serine)-Tyr(tyrosine)-Lys(lysine). Medium-chain dehydrogenases (MDRs) are a class of zinc- or non-zinc-dependent enzymes, usually existing as homopolymers. A single subunit typically contains 350 amino acid residues, exhibiting higher sequence similarity (40%-90%) compared to short-chain dehydrogenases. Aldehyde-ketone reductases (AKRs) are a class of non-metal-dependent dehydrogenases, typically existing as monomers containing 320 amino acids. Although they differ significantly in three-dimensional structure from short-chain dehydrogenases, they are functionally similar. AKRs can catalyze the metabolism of a wide variety of substances, including aldehydes, aldehyde esters, alkyl groups, and polycyclic aromatic hydrocarbons, demonstrating their broad substrate spectrum.

[0003] The catalytic mechanism of the short-chain carbonyl reductase involved in this patent is mainly as follows: As a non-metal-dependent enzyme, it first combines with the reduced coenzyme NAD(P)H to form a holoenzyme. Then, after the substrate enters the catalytic active site pocket, the catalytic tetramer begins to function. Tyrosine and serine form hydrogen bonds with the carbonyl oxygen in the substrate, firmly fixing it in the pocket. Simultaneously, as the most conserved amino acid in the reductase family, tyrosine also provides hydrogen ions to the carbonyl oxygen for subsequent catalysis. Asparagine activates lysine, causing it to form hydrogen bonds with the coenzyme, also firmly stabilizing it in the pocket. The hydrogen ions provided by tyrosine attack the carbonyl oxygen on the substrate, while the hydrogen ions on the coenzyme attack the carbonyl carbon on the substrate. This combination further reduces the substrate carbonyl group to the corresponding chiral alcohol. The product is formed, and the oxidized coenzyme, which has already been oxidized, separates from the enzyme, thus ending the entire catalytic process.

[0004] (3R,5S)-6-chloro-3,5-dihydroxyhexanoate (tert-butyl(3R,5S)-6-chloro-3,5-dihydroxyhexanoate, (3R,5S)-CDHH) is a chiral side-chain intermediate of rosuvastatin, an HMG-CoA enzyme inhibitor, and can be synthesized via chemical and biocatalytic methods. Traditional chemical synthesis typically requires expensive noble metal catalysts, which, despite their high cost, cannot guarantee the construction of the chiral center. Currently, the commonly used chemical synthesis of (3R,5S)-CDHH requires sodium cyanide for ring-opening and the catalyst methoxydiethylborane (Et2BOMe). While chemical synthesis has many advantages, it also has some unavoidable drawbacks. Firstly, cyanide is highly toxic, and Et2BOMe is also a hazardous chemical, prone to spontaneous combustion and expensive. Therefore, the enzymatic method, with its advantages of mild reaction conditions and simple operation, has gradually gained popularity. The enzymatic method exhibits high stereoselectivity, avoids the introduction of highly toxic substances, and is environmentally friendly. Consequently, in recent years, enzymatic methods have gradually replaced chemical methods for the synthesis of (3R,5S)-CDHH. Wolber reported that an alcohol dehydrogenase cloned from the Lactobacillus kefi strain was expressed in recombinant Escherichia coli, enabling asymmetric reduction catalysis of the synthesis of (3R,5S)-CDHH from tert-butyl(S)-6-chloro-5–hydroxy-3-oxohexanoate ((S)-CHOH) at laboratory gram scales. The de value reached 99.5% after 24 hours of reaction, and the overall product yield was 72%. Pfruende reported an efficient whole-cell asymmetric catalytic transformation process using *Lactobacillus kefi* DSM20587 in a two-step asymmetric reduction catalysis to synthesize (3R,5S)-CDHH from (S)-CHOH. A whole cell of 50 g / L (cell dry weight) *Lactobacillus kefi* could catalyze the production of 47.5 mmol / L (3R,5S)-CDHH from 100 mmol / L (S)-CHOH, with a conversion rate (e) > 99%. Xia mutated carbonyl reductase from *Candida theae* at positions 126, 129, and 194, achieving a conversion rate of 96% at a substrate concentration of 1 mol / L (or 220 g / L), with a product ee value exceeding 99% (CN 116814572A).Ni utilizes the carbonyl reductase NaKRED derived from Novosphingobium aromaticivorans by mutating at positions 45, 141, and 195, enabling it to catalyze substrate concentrations exceeding 100 g / L with a stereoselectivity of up to 99.5%, exhibiting high enzyme activity and excellent catalytic efficiency (CN 116676285A).

[0005] Similarly, the chiral intermediate 6-cyano-(3R,5R)-dihydroxyhexanoate tert-butyl ester, a HMG-CoA enzyme inhibitor, can also be synthesized via biocatalysis. Luo's team successfully reduced (R)-6-cyano-5-hydroxy-3-carbonylhexanoate tert-butyl ester with a substrate concentration of 450 g / L using the aldehyde-ketone reductase KlAKR to obtain the stereospecific intermediate 6-cyano-(3R,5R)-dihydroxyhexanoate tert-butyl ester, achieving a de selectivity >99.5% and a space-time yield of 1.24 kg / (Ld). Wang, using a carbonyl reductase derived from Starmerella magnolia to catalyze the reduction of (R)-6-cyano-5-hydroxy-3-carbonylhexanoate tert-butyl ester, achieved an ee ≥98% and a conversion rate ≥95% (CN 116515780A).

[0006] Therefore, it is of great significance to study the biocatalytic process of (3R,5S)-CDHH or 6-cyano-(3R,5R)-dihydroxyhexanoate tert-butyl ester. It can not only provide a new route for the synthesis of (3R,5S)-CDHH or 6-cyano-(3R,5R)-dihydroxyhexanoate tert-butyl ester, but also provide enzyme sources with high stereoselectivity and high catalytic activity. Summary of the Invention

[0007] To further improve the production efficiency and purity of chiral alcohol (3R,5S)-CDHH or 6-cyano-(3R,5R)-dihydroxyhexanoate tert-butyl ester, an intermediate for statin drugs, this invention conducted in-depth research on the stereospecific recombinant carbonyl reductase SCR derived from Rhodosporidium toruloides ZJB2014212. A highly active and stereoselective recombinant carbonyl reductase mutant, SCR-Asp25Thr, and its encoding gene, as well as a recombinant vector and genetically engineered bacteria containing this mutant encoding gene, were obtained. When the recombinant carbonyl reductase mutant was applied to the enzymatic synthesis of chiral alcohol, the yield of (3R,5S)-CDHH was >92%, ee >99%, and the yield of 6-cyano-(3R,5R)-dihydroxyhexanoate tert-butyl ester reached 97.1%, ee >99%.

[0008] This invention provides a recombinant carbonyl reductase mutant, obtained by mutating the 25th amino acid position of the recombinant carbonyl reductase derived from Rhodosporidium toruloides ZJB2014212. The amino acid sequence of the recombinant carbonyl reductase derived from Rhodosporidium toruloides ZJB2014212 is shown in SEQ ID NO. 2.

[0009] Preferably, the mutation is a threonine mutation at position 25 aspartic acid.

[0010] The present invention also provides a gene encoding the recombinant carbonyl reductase mutant. Preferably, the acylase mutant is obtained by mutating the codon encoding aspartic acid at position 25 to a codon encoding threonine, based on the recombinant carbonyl reductase SCR shown in SEQ ID NO. 1. As is known in the art, an allelic variant is a polynucleotide substitution, which may be a substitution, deletion, or insertion of a polynucleotide, but does not substantially alter the function of the encoded peptide protein.

[0011] This invention also provides a recombinant vector for the encoding gene of the recombinant carbonyl reductase mutant. The recombinant vector comprises a polynucleotide operatively linked to a control sequence suitable for guiding expression in a host cell. Various vectors conventional in the art, such as plasmids, phages, or viral vectors, linked to the nucleotide sequence of the recombinant carbonyl reductase mutant of this invention, are all within the scope of this invention. Preferably, the recombinant vector uses plasmid pET-28b as the expression vector, and the encoding gene of the novel carbonyl reductase mutant is linked to plasmid pET-28b.

[0012] This invention also provides a genetically engineered bacterium encoding the recombinant carbonyl reductase mutant gene. The exogenous recombinant carbonyl reductase mutant gene is introduced into a host cell using genetic engineering techniques to construct and express the genetically engineered bacterium, thereby obtaining the novel carbonyl reductase mutant described in this invention. The host cell can be bacteria, fungi, plant cells, or animal cells, with *Escherichia coli* BL21(DE3) being a preferred expression host.

[0013] This invention also provides the application of the recombinant carbonyl reductase mutant, the recombinant vector, or the genetically engineered bacteria in the biocatalytic preparation of chiral alcohols. Preferably, the application involves using wet cells obtained from the fermentation culture of genetically engineered bacteria containing the encoding gene of the recombinant carbonyl reductase mutant, or enzyme extract obtained after ultrasonic disruption of wet cells, as a catalyst; precursor ketone as a substrate; isopropanol as an auxiliary substrate; and NAD(P)H as a coenzyme, preferably in a buffer solution at pH 6-8, to construct the reaction system. Chiral alcohols are obtained through an asymmetric reduction reaction at 25-35°C and 200-800 rpm. It is understood that when applying the recombinant carbonyl reductase mutant of this invention, there are several feasible forms to choose from. These include: use in whole-cell form of genetically engineered bacteria; use in unpurified crude enzyme form; and use in partially or completely purified enzyme form. Furthermore, the acylase mutant of the present invention can be made into an immobilized enzyme or a biocatalyst in the form of an immobilized cell using known enzyme immobilization technology, thereby further improving the stability and reusability of the enzyme and facilitating its application in industrial production and other fields.

[0014] Preferably, the precursor ketone is selected from tert-butyl (S)-6-chloro-5-hydroxy-3-carbonylhexanoate or tert-butyl 6-cyano-(5R)-3-carbonylhexanoate. The substrate (S)-6-chloro-5-hydroxy-3-carbonylhexanoate ((S)-CHOH) is asymmetricly reduced to (3R,5S)-CDHH under the catalysis of a recombinant carbonyl reductase mutant. The substrate tert-butyl 6-cyano-(5R)-3-carbonylhexanoate is asymmetricly reduced to tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate under the catalysis of a recombinant carbonyl reductase mutant.

[0015] Preferably, the amount of catalyst in the reaction system is 3-50 g / L buffer solution based on the weight of wet bacterial cells.

[0016] Preferably, the initial concentration of the precursor ketone in the reaction system is 200-500 g / L.

[0017] Preferably, the final volume concentration of isopropanol in the reaction system is 10%-70%, more preferably 20%-50%, and even more preferably 40%. (Reference) Figure 1 The regeneration system for the coenzyme NAD(P)H of recombinant carbonyl reductase and its mutant is preferably a single-enzyme, dual-substrate coupled system. Choosing isopropanol as the auxiliary substrate for the coenzyme regeneration system can effectively save production costs. Furthermore, the inventors optimized the isopropanol concentration in the coenzyme cycling regeneration system. The results showed that when the isopropanol volume concentration was 40%, the coenzyme cycle could basically meet the NADPH requirement of carbonyl reductase, and the ee of the catalytic reaction product was >99%.

[0018] The beneficial effects of this invention are as follows: The recombinant carbonyl reductase mutant provided by this invention exhibits higher activity and stereoselectivity compared to the wild-type enzyme, further improving the catalytic efficiency for substrate precursor ketones. When the recombinant carbonyl reductase mutant is applied to the enzymatic synthesis of chiral alcohols, the yield of product (3R,5S)-CDHH is >92% and ee>99% after 6 hours of catalysis at a substrate concentration of 400 g / L; the yield of product 6-cyano-(3R,5R)-dihydroxyhexanoate tert-butyl ester reaches 97.1% and ee>99% after 3 hours of catalysis at a substrate concentration of 200 g / L. Furthermore, this invention constructs a single-enzyme, dual-substrate coupled system in the reaction system for the catalytic preparation of chiral alcohols, selecting isopropanol as the auxiliary substrate for the coenzyme regeneration system, which can effectively save production costs and is of great significance for the industrial application of enzymatic catalytic preparation of chiral alcohols. Compared to chemical methods for preparing chiral alcohols, this invention utilizes asymmetric catalysis of recombinant carbonyl reductase mutants to reduce precursor ketones and synthesize chiral alcohols. This simplifies the chemical catalysis steps, provides milder reaction conditions, requires less equipment, reduces reaction costs, and is environmentally friendly. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the synthesis of chiral alcohol (3R,5S)-CDHH by asymmetric catalytic reduction of precursor ketone using a recombinant carbonyl reductase mutant. Detailed Implementation

[0020] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. Unless otherwise specified, the methods used in the embodiments of the present invention are conventional methods, and the reagents used are commercially available.

[0021] Liquid phase detection methods

[0022] High Performance Liquid Chromatography Instrument: Shimadzu LC-20AD System - SPD-20A Ultraviolet Detector

[0023] For conversion detection, an Agilent Zorbax SB-C8 column (150×4.6mm, 5μm) was used. The mobile phase consisted of an acetonitrile aqueous solution with a volume ratio of 3:7 (v / v). The flow rate was 1 mL / min, the detection wavelength was set to 210 nm, the injection volume was 10 μm, and the column temperature was set to 40℃. The retention times of (S)-CHOH and (3R,5S)-CDHH were 8.348 min and 5.534 min, respectively. For ee detection, a chiral OD-H column (250×4.6mm, 5μm) was used. The mobile phase was n-hexane:isopropanol = 85:15. The flow rate was 1 mL / min, and the detection wavelength was 215 nm. The retention times of (S)-CHOH, (3R,5S)-CDHH, and (3S,5S)-6-chlorodihydroxyhexanoate tert-butyl ester were 5.99 min, 5.09 min, and 4.93 min, respectively.

[0024] The chromatographic column used was a J&K Scientific C18 reversed-phase column (4.6 mm × 150 mm, 5 μm). The mobile phase consisted of an acetonitrile aqueous solution with a volume ratio of 1:3 (v / v). The flow rate was 1 mL / min, the detection wavelength was set to 210 nm, the injection volume was 10 μm, and the column temperature was set to 40 °C. The retention time of tert-butyl 6-cyano-(5R)-3-carbonylhexanoate was approximately 15.353 min, and the retention times of tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate were approximately 9.057 min (S-configuration) and 9.501 min (R-configuration).

[0025] Example 1: Obtaining a recombinant carbonyl reductase mutant

[0026] Using the recombinant strain E. coli BL21(DE3)pET28b-SCR containing the expression vector pET28b-SCR encoding the recombinant carbonyl reductase SCR as the starting strain, site-directed saturation mutagenesis and site-directed mutagenesis were performed to further improve the catalytic activity and substrate tolerance of carbonyl reductase to substrate precursor ketones.

[0027] The mutation primers are shown in Table 1, with the mutation sites underlined. Using plasmid DNA containing the SCR gene as a template, mutations were introduced via PCR. The PCR reaction program was as follows: 98℃ for 3 min; 98℃ for 10 s, 55℃ for 5 s, 72℃ for 6 min 30 s, repeated 26 times; followed by a 10 min extension at 72℃. The PCR products were inactivated by DpnI treatment at 37℃ for 3 h and then transformed into *E. coli* BL21(DE3) recipient bacteria. The transformed bacteria were plated on LB agar plates containing a final concentration of 50 mg / L kanamycin and incubated at 37℃ for 12 h. Single colonies were randomly selected for sequencing analysis to obtain different mutated amino acids at different mutation sites.

[0028] Table 1. Mutant Primers

[0029]

[0030] A series of different amino acid mutants were obtained through site-directed saturation mutagenesis and site-directed mutagenesis. Clones with correct sequencing results were inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance and cultured at 37°C and 180 rpm for 8 h. Then, 1% (v / v) inoculation was added into fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance and cultured at 37°C and 180 rpm until the bacterial OD600 reached 0.6-0.8. IPTG was added to a final concentration of 0.1 mM, and the culture was induced at 28°C for 12 h. After centrifugation at 4°C and 8000 rpm for 10 min, the supernatant was discarded, and the precipitate was collected to obtain recombinant *E. coli* wet cells containing the gene expressing the recombinant carbonyl reductase mutant. The transformation reaction was carried out in a 10 mL transformation flask with a substrate concentration of 100 g / L ((S)-CHOH), 4 mL of isopropanol, and 15 g / L of wet mutant cells. The reaction was carried out at 30 °C with magnetic stirring at 600 rpm for 20 min. The reaction solution was analyzed by HPLC to determine the relative enzyme activity of the mutants, thereby screening for mutants with relatively high catalytic activity. The results are shown in Table 2.

[0031] Table 2. Relative enzyme activity of recombinant carbonyl reductase mutants

[0032] SCR 100±1.43 SCR-Asp25Thr 115.93±1.33 SCR-Lys211Ile 108.46±1.21 SCR-Val181Ile 102.31±1.32

[0033] The results showed that the optimal mutant obtained by site-directed saturation mutagenesis and site-directed mutagenesis was SCR-Asp25Thr, which was obtained by mutating aspartic acid at position 25 of the recombinant carbonyl reductase as shown in SEQ ID NO.2.

[0034] Example 2: Preparation of wet cells of recombinant carbonyl reductase mutant

[0035] The recombinant *E. coli* BL21(DE3) / pET28b-SCR-Asp25Thr containing the gene expressing the recombinant carbonyl reductase mutant SCR-Asp25Thr obtained in Example 1 was inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance and cultured at 37°C and 180 rpm for 8 h. Then, it was inoculated at a 1% inoculum (v / v) into fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance and cultured at 37°C and 180 rpm until the bacterial OD600 reached 0.6-0.8. IPTG was added to a final concentration of 0.1 mM, and the culture was induced at 28°C for 12 h. After centrifugation at 4°C and 8000 rpm for 10 min, the supernatant was discarded, and the precipitate was collected to obtain the recombinant *E. coli* wet cells containing the gene expressing the recombinant carbonyl reductase mutant. These wet cells can be used directly as a biocatalyst or for protein purification. Wet cells of E. coli BL21(DE3) / pET28b-SCR containing the gene expressing recombinant carbonyl reductase were prepared using the same method.

[0036] Example 3: Isolation and purification of carbonyl reductase mutants

[0037] The wet bacterial cells obtained in Example 2 were washed three times with physiological saline. The wet bacterial cells were resuspended in 20 mL of 20 mM, pH 7.0 potassium phosphate buffer at a ratio of 1 g of wet bacterial cells. The cells were then sonicated under ice bath conditions (60 W, 1 s duration, 1 s interval, continuous sonication for 30 min) to obtain cell lysate. The cell lysate obtained after sonication was centrifuged at 8000 rpm and 4°C for 25 min. The supernatant obtained was the required crude enzyme solution, designated as Crude Enzyme Solution I.

[0038] Ammonium sulfate fractional salting out: Determining the optimal precipitation concentration. The most commonly used salts in salting out are (NH4)2SO4, Na2SO4, and MgSO4. (NH4)2SO4 is the most commonly used salt in the salting out step because of its high solubility, small temperature coefficient, good separation effect, generally does not denature proteins, and is inexpensive and readily available. Because proteins can lose their hydration layer under the influence of high concentrations of neutral salts, their charges are neutralized, disrupting the colloidal stability of the proteins and causing precipitation. Furthermore, protein salting out is a reversible process, and proteins can be reconstituted upon dilution with water or buffer solution. Therefore, as the ammonium sulfate saturation increases, the protein content in the supernatant gradually decreases; conversely, the amount of protein precipitated in the precipitate gradually increases. The method involves adding 5 mL of crude enzyme solution to eight small beakers numbered 1-8. Grinded ammonium sulfate was added in increments of 0.57 g, 0.88 g, 1.215 g, 1.565 g, 1.95 g, 2.36 g, 2.805 g, and 3.31 g, respectively, to achieve ammonium sulfate saturation of 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90% in the crude enzyme solution. The ammonium sulfate was added slowly and stirred on ice until dissolved. After stirring, the solution was allowed to stand at 4°C for 4 hours. The liquid in each beaker was centrifuged (8000 rpm, 25 min), and the supernatant was weighed to calculate its volume. The enzyme activity and protein content were then determined. By calculating the specific activity and protein precipitation rate of ammonium sulfate fractional salting-out, the two saturations with the highest specific activity and the largest protein precipitation rate can be identified. These are the optimal precipitation saturations for contaminating proteins and the target protein, respectively, thus removing a large amount of contaminating proteins. For ammonium sulfate fractional salting-out, after cell sonication, crude enzyme solution I is continuously stirred in an ice-water bath, and ammonium sulfate is slowly added until the final concentration reaches 50% saturation. The solution is then incubated at 4°C for 4 hours. After centrifugation at 8000 rpm for 25 minutes, the precipitate is discarded. Ammonium sulfate is added to the supernatant until the final concentration reaches 70% saturation, and the solution is incubated at 4°C for 4 hours. After another centrifugation at 8000 rpm for 25 minutes, the supernatant is discarded, and the precipitate is reconstituted with an appropriate amount of 20 mM, pH 7.0 potassium phosphate buffer. The solution is then dialyzed overnight at 4°C in 20 mM, pH 7.0 potassium phosphate buffer to concentrate the crude enzyme solution II.

[0039] DEAE Sepharose Fast Flow Anion Exchange Chromatography: Ion exchange chromatography is a method of separation that utilizes reversible ion exchange between ion-exchange groups coupled to the stationary phase and ionic compounds dissociated from the mobile phase. Anion exchange chromatography is used in the preliminary chromatographic purification of most oxidoreductases. The DEAE Sepharose Fast Flow anion exchange column (1.6 cm × 20 cm) was first equilibrated with 20 mM, pH 7.0 pB potassium phosphate buffer at a flow rate of 1.5 mL / min. After the baseline leveled out, the dialyzed crude enzyme solution II was loaded onto the column at a flow rate of 1.0 mL / min. After loading, elution was first performed with 20 mM, pH 7.0 potassium phosphate buffer at a flow rate of 1.5 mL / min. After the baseline leveled out, elution was performed with a 0–0.8 M NaCl gradient. The active fraction was collected and concentrated to obtain pure enzyme. The entire purification process was performed at 4°C. The protein content of the retentate was determined using the BCA kit method and then frozen at -80°C to obtain the recombinant carbonyl reductase SCR and its mutant SCR-Asp25Thr purified enzymes.

[0040] Example 4: Enzyme activity of recombinant carbonyl reductase and its mutants

[0041] The carbonyl reductase SCR and its mutant SCR-Asp25Thr purified enzyme obtained in Example 3 were used to catalyze the synthesis of (3R,5S)-CDHH from the substrate (S)-CHOH.

[0042] The enzyme activity catalytic system composition and catalytic conditions are as follows: Carbonyl reductase mutant pure enzymes (final concentrations SCR 2.5 μg / mL and SCR-Asp25Thr 2.5 μg / mL) diluted to the same concentrations with the same buffer solution were added to 5 mL of 100 mM, pH 7.0 phosphate buffer. The final concentrations of (S)-CHOH were 10 mM, and the final concentrations of coenzyme NAD(P)H were 2 mM. The mixture was preheated at 30℃ and 600 rpm for 3 min, and then reacted for 3 min. The reaction was terminated by adding 5 mL of 30% acetonitrile aqueous solution. After thorough mixing, samples were taken to detect enzyme activity. The results are shown in Table 3.

[0043] Table 3. Relative enzyme activities of recombinant carbonyl reductases and their mutants

[0044] SCR 100±1.12 99 SCR-Asp25Thr 116.36±1.26 99

[0045] Note: Enzyme activity unit (U) is defined as the amount of enzyme required to produce 1 μmol of product (3R,5S)-CDHH within 1 min at 30℃ and pH 7.0. The amount of product generated is determined by HPLC detection.

[0046] Example 5: Optimization of isopropanol concentration in recombinant carbonyl reductase coenzyme regeneration system

[0047] refer to Figure 1 The regeneration system of recombinant carbonyl reductase and its mutant coenzyme NAD(P)H is preferably a single-enzyme dual-substrate coupling system. Choosing isopropanol as the auxiliary substrate of the coenzyme regeneration system can effectively save production costs.

[0048] Using the wet E. coli BL21(DE3) / pET28b-SCR-Asp25Thr cells obtained by the method in Example 2 as a biocatalyst, (3R,5S)-CDHH was synthesized using (S)-CHOH as a substrate.

[0049] The conversion system consisted of 10 mL of potassium phosphate buffer (100 mM, pH 7.0), wet cells of the recombinant carbonyl reductase mutant mut-Asp25Thr (15 g / L buffer), a final substrate concentration of 100 g / L buffer, and final isopropanol concentrations of 10%, 20%, 30%, 40%, 50%, 60%, and 70%. The reaction was carried out at 30℃ and 600 rpm in a magnetically stirred water bath for 20 min. After the reaction, 1 mL of acetonitrile was added to terminate the reaction. The reaction solution was thoroughly mixed, and an appropriate amount of the reaction solution was taken for HPLC analysis to determine the conversion rate. The results are shown in Table 4.

[0050] Table 4. Optimization of Isopropanol Concentration

[0051] 10％ 37.22％ 20％ 51.45％ 30％ 52.33％ 40％ 66.72％ 50％ 48.79％ 60％ 38.16％ 70％ 33.29％

[0052] The results showed that when the volume concentration of isopropanol was 40%, the coenzyme cycle could basically meet the NADPH requirements of carbonyl reductase, and the product ee of the above catalytic reaction was >99%.

[0053] Application of recombinant carbonyl reductase mutants in the preparation of (3R,5S)-CDHH

[0054] Example 6:

[0055] Using the wet E. coli BL21(DE3) / pET28b-SCR-Asp25Thr cells containing the recombinant expression plasmid obtained in Example 2 as a biocatalyst, and (S)-CHOH as a substrate, a biotransformation reaction was carried out to prepare (3R,5S)-CDHH.

[0056] The composition and catalytic conditions of the 10 mL catalytic system are as follows: 6 mL of potassium phosphate buffer solution (pH 7.0) and 50 g / L wet cells of the recombinant carbonyl reductase mutant SCR-Asp25Thr were added to the 10 mL reaction system, along with 4 mL of isopropanol. The initial substrate concentration was 400 g / L. The reaction was carried out in a 30 °C water bath with a magnetic stirrer at 600 rpm. Samples were taken periodically (100 μL each time), diluted 50 times with 30% acetonitrile aqueous solution, and the conversion rate was determined by HPLC. The results showed that when the substrate concentration in the 10 mL catalytic system was 400 g / L, the yield of product (3R,5S)-CDHH reached 97.3% after 8 h of catalysis, with ee > 99%.

[0057] Example 7:

[0058] Using the wet E. coli BL21(DE3) / pET28b-SCR-Asp25Thr cells containing the recombinant expression plasmid obtained in Example 2 as a biocatalyst, and (S)-CHOH as a substrate, a biotransformation reaction was carried out to prepare (3R,5S)-CDHH.

[0059] The composition and catalytic conditions of the 100 mL catalytic system are as follows: 60 mL of potassium phosphate buffer solution (pH 7.0), 50 g / L wet cells of the recombinant carbonyl reductase mutant mut-Asp25Thr, and 40 mL of isopropanol were added to the 100 mL reaction system. The initial substrate concentration was 400 g / L. The reaction was carried out in a 30 °C water bath with a magnetic stirrer at 1200 rpm. Samples were taken periodically (100 μL each time), diluted 50 times with 30% acetonitrile aqueous solution, and the conversion rate was determined by HPLC. The results showed that when the substrate concentration in the 100 mL catalytic system was 400 g / L, the yield of product (3R,5S)-CDHH reached 96.4% after 6 h of catalysis, with ee > 99%.

[0060] Example 8:

[0061] Using the wet E. coli BL21(DE3) / pET28b-SCR-Asp25Thr cells containing the recombinant expression plasmid obtained in Example 2 as a biocatalyst, and (S)-CHOH as a substrate, a biotransformation reaction was carried out to prepare (3R,5S)-CDHH.

[0062] Compared to Example 6, the only difference was the initial substrate concentration, which was 500 g / L; all other parameters remained the same. The results showed that with a substrate concentration of 500 g / L in a 10 mL catalytic system, the yield of product (3R,5S)-CDHH reached 92.1% after 6 hours of catalysis, with ee > 99%.

[0063] Application of recombinant carbonyl reductase in the preparation of (3R,5S)-CDHH

[0064] Comparative Example 1:

[0065] Compared to Example 6, the difference lies in the use of E. coli BL21(DE3) / pET28b-SCR wet cells containing the recombinant expression plasmid obtained in Example 2 as a biocatalyst, and (S)-CHOH as the substrate, to carry out a biotransformation reaction to prepare (3R,5S)-CDHH. All other aspects are the same. The results show that when the substrate concentration in a 10 mL catalytic system is 400 g / L, the yield of (3R,5S)-CDHH reaches 94.1% after 6 h of catalysis, with ee > 99%.

[0066] Comparative Example 2:

[0067] Compared to Example 8, the difference lies in the use of E. coli BL21(DE3) / pET28b-SCR wet cells containing the recombinant expression plasmid obtained in Example 2 as a biocatalyst, and (S)-CHOH as the substrate, to carry out a biotransformation reaction to prepare (3R,5S)-CDHH. All other aspects are the same. The results show that when the substrate concentration in a 10 mL catalytic system is 500 g / L, the yield of (3R,5S)-CDHH reaches 89.4% after 6 h of catalysis, with ee > 99%.

[0068] Application of recombinant carbonyl reductase mutant in the preparation of tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate

[0069] Using the wet E. coli BL21(DE3) / pET28b-SCR-Asp25Thr bacterial cells containing the recombinant expression plasmid obtained in Example 2 as a biocatalyst, and tert-butyl 6-cyano-(5R)-3-carbonylhexanoate as a substrate, a biotransformation reaction was carried out to prepare tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate.

[0070] The catalytic system composition and catalytic conditions are as follows: 6 mL of potassium phosphate buffer solution (pH 7.0) and 50 g / L wet cells of the recombinant carbonyl reductase mutant SCR-Asp25Thr were added to a 10 mL reaction system, along with 4 mL of isopropanol. The initial substrate concentration was 200 g / L. The reaction was carried out in a 30°C water bath with a magnetic stirrer at 600 rpm. Samples were taken periodically (100 μL each time), diluted 50 times with 30% acetonitrile aqueous solution, and the conversion rate was determined by HPLC. The results showed that at a substrate concentration of 200 g / L, the yield of tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate reached 97.1% after 3 h of catalysis, with ee > 99%.

[0071] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope of the present invention.

Claims

1. A recombinant carbonyl reductase mutant, composed of... Rhodosporidium toruloides The amino acid sequence of the recombinant carbonyl reductase derived from ZJB2014212 is shown in SEQ ID NO.

2. The recombinant carbonyl reductase was obtained by mutating aspartic acid at position 25 to threonine.

2. The gene encoding the recombinant carbonyl reductase mutant of claim 1.

3. A recombinant vector containing the gene of the recombinant carbonyl reductase mutant as described in claim 2.

4. Genetically engineered bacteria containing the gene of the recombinant carbonyl reductase mutant as described in claim 2.

5. The application of the recombinant carbonyl reductase mutant of claim 1, the recombinant vector of claim 3, or the genetically engineered bacteria of claim 4 in the biocatalytic preparation of chiral alcohols, characterized in that, The application involves using wet bacterial cells obtained through fermentation culture of genetically engineered bacteria containing a recombinant carbonyl reductase mutant encoding gene, or enzyme solution extracted after ultrasonic disruption of wet bacterial cells, as a catalyst; a precursor ketone as a substrate; and isopropanol as an auxiliary substrate to form a reaction system, thereby obtaining a chiral alcohol through an asymmetric reduction reaction; the precursor ketone is selected from (…). S 6-chloro-5-hydroxy-3-carbonylhexanoate tert-butyl ester or 6-cyano-(5-) R 3-Carbonylhexanoate tert-butyl ester.

6. The application as described in claim 5, characterized in that, The initial and final concentrations of the precursor ketone in the reaction system are 200-500 g / L.

7. The application as described in claim 5, characterized in that, The final volume concentration of isopropanol in the reaction system is 10%-70%.

Images