CN117904066A

CN117904066A - Recombinant carbonyl reductase mutant and application thereof in catalytic synthesis of chiral alcohol

Info

Publication number: CN117904066A
Application number: CN202410092889.4A
Authority: CN
Inventors: 柳志强; 方韦湄; 张晓健; 李向阳; 郑裕国
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2024-01-23
Filing date: 2024-01-23
Publication date: 2024-04-19

Abstract

The invention provides a recombinant carbonyl reductase mutant, which is obtained by mutating aspartic acid at the 25 th position of an amino acid sequence of Rhodosporidium toruloides ZJB 2014212-derived recombinant carbonyl reductase into threonine. The recombinant carbonyl reductase mutant has high activity and high stereoselectivity compared with wild enzyme, and further improves the catalysis efficiency of substrate precursor ketone. The recombinant carbonyl reductase mutant is applied to the synthesis of chiral alcohol by a biological enzyme method, and asymmetric catalytic reduction is carried out on substrate precursor ketone, so that compared with the wild type enzyme, the catalytic activity is improved during the reaction, and the time consumption of the reaction process is shortened. In addition, in the reaction system for preparing chiral alcohol by catalysis, a single-enzyme double-substrate coupling system is constructed, isopropanol is selected as an auxiliary substrate of a coenzyme regeneration system, so that the production cost can be effectively saved, and the method has important significance for industrial application of preparing chiral alcohol by catalysis of a biological enzyme method.

Description

Recombinant carbonyl reductase mutant and application thereof in catalytic synthesis of chiral alcohol

Technical Field

The invention belongs to the field of bioconversion, and particularly relates to a recombinant carbonyl reductase mutant and application thereof in catalytic synthesis of chiral alcohol.

Background

Carbonyl reductases are a class of enzymes that undergo reversible redox reactions, with the co-enzyme NADP (H) or NAD (H) being the hydrogen mediator involved in catalyzing aldehydes/ketones with alcohols, and are considered to be one of the most commonly used biocatalysts in the synthesis of chiral alcohols. Carbonyl reductases are widely available and are commonly found in natural environments such as various plant, bacterial, fungal and animal tissues. Carbonyl reductase is mainly distributed in 3 superfamilies of short-chain dehydrogenase (short-chain Dehydrogenase, SDRs), aldehyde-ketone reductase (aldo-ketoreductases, AKRs), medium-chain dehydrogenase (medium-chain dehydrogenases, MDRs), which all catalyze similar reactions, although they differ greatly in steric structure, catalytic mechanism, protein chain length, etc. Among them, short-chain dehydrogenases, SDRs are a non-metal dependent enzyme type, usually a single subunit contains about 250 amino acid residues, and have a multimeric protein such as a monomer, a dimer or a tetramer, and SDRs are usually divided into 5 subfamilies according to the difference of coenzyme binding sites and catalytic mechanisms, and most importantly, classical and extended SDRs. The sequence similarity between SDRs, although only 15-30%, is highly conserved, higher-order structure, with the catalytic quadruplet typically being Asn (aspartic acid) -Ser (serine) -Tyr (tyrosine) -Lys (lysine). The MDRs of medium-chain dehydrogenase are zinc or non-zinc dependent enzymes, usually exist in the form of homologous polymers, and the single subunit usually contains 350 amino acid residues, and has higher sequence consistency of 40% -90% compared with the short-chain dehydrogenase. Aldehyde ketoreductase AKRs is a non-metal dependent type of dehydrogenase that typically exists in the form of a single polymer containing 320 amino acids. Although they differ greatly in three-dimensional structure from short-chain dehydrogenases, they are functionally similar. AKRs is capable of catalyzing the metabolism of various substances such as furfural, aldehyde esters, alkyl groups, polycyclic aromatic hydrocarbons and the like, so that the substrate spectrum is very wide.

The catalytic mechanism of the short-chain carbonyl reductase related to the patent is mainly as follows: as a non-metal dependent enzyme, it is firstly combined with reduced coenzyme NAD (P) H to form holoenzyme, then after the substrate enters a catalytic active center pocket, the catalytic tetrad starts to act, tyrosine and serine form hydrogen bonds with carbonyl oxygen in the substrate to firmly fix the substrate in the pocket, meanwhile, as the most conserved amino acid of reductase family, tyrosine is also responsible for providing hydrogen ions for carbonyl oxygen for subsequent catalysis, and asparagine activates lysine to form hydrogen bonds with the coenzyme to firmly stabilize the substrate in the pocket. The hydrogen ions provided by the tyrosine attack the carbonyl oxygen on the substrate, while the hydrogen ions on the coenzyme attack the carbonyl carbon on the substrate, and the two phases further reduce the carbonyl of the substrate to the corresponding chiral alcohol, so that the oxidized coenzyme which is released and oxidized is also separated from the enzyme, and the whole catalytic process is ended.

The (3R, 5S) -6-chloro-3,5-dihydroxyhexanoate (tert-butyl (3R, 5S) -6-chloro-3,5-dihydroxyhexanoate, and (3R, 5S) -CDHH) is a chiral side chain intermediate of the HMG-CoA enzyme inhibitor rosuvastatin medicine, and can be synthesized by a chemical method and a biocatalysis method. Traditional chemistry generally requires expensive noble metal catalysts, which, despite the high cost, still do not guarantee the construction of chiral centers. At present, sodium cyanide is required to carry out ring opening reaction for synthesizing (3R, 5S) -CDHH by a chemical method which is commonly adopted at home and abroad, and a catalyst diethyl methoxyborane (Methoxydiethylborane, et ₂ BOMe) is also required. Chemical synthesis, while having many advantages, has some unavoidable drawbacks, primarily cyanide is a highly toxic species, and Et ₂ BOMe is also a hazardous chemical that is prone to spontaneous combustion and is expensive. Therefore, the biological enzyme method with the advantages of mild reaction conditions, simple operation and the like gradually goes into the field of view of the public, has high stereoselectivity, avoids the introduction of extremely toxic substances and is environment-friendly, so that the enzyme method gradually replaces a chemical method in recent years to synthesize (3R, 5S) -CDHH. Wolber reports that alcohol dehydrogenase cloned by Lactobacillus kefi strain is expressed in recombinant escherichia coli, and can asymmetrically reduce and catalyze gram-scale (S) -6-chloro-5-hydroxy-3-carbonyl hexanoic acid tert-butyl ester (tert-butyl (S) -6-chloro-5-hydroxy-3-oxohexanoate, (S) -CHOH) to synthesize (3R, 5S) -CDHH, d.e. value reaches 99.5% after 24 hours of reaction, and total yield of product reaction reaches 72%. Pfruende reports an effective whole-cell biological asymmetric catalytic conversion process, and (3R, 5S) -CDHH is synthesized by utilizing a two-step asymmetric reduction catalysis method of lactobacillus caucasians (Lactobacillus kefi DSM 20587), and 50g/L (dry cell weight) of lactobacillus caucasians can catalyze 100mmol/L (S) -CHOH to generate 47.5mmol/L (3R, 5S) -CDHH and d.e. >99%. Xia was mutated at positions 126, 129 and 194 using a carbonyl reductase from Candida theai (CANDIDA THEAE) to achieve 96% conversion at substrate concentrations up to 1mol/L (or 220 g/L) and a product e.e. value higher than 99% (CN 116814572A). Ni makes mutation at 45, 141 and 195 by utilizing carbonyl reductase NaKRED from Novosphingobium aromaticivorans, so that the concentration of the catalytic substrate can reach more than 100g/L, and the catalyst has stereoselectivity as high as 99.5%, high enzyme activity and good catalytic efficiency (CN 116676285A).

Likewise, the chiral intermediate 6-cyano- (3R, 5R) -dihydroxyhexanoate of the HMG-CoA enzyme inhibitor atorvastatin can be synthesized by a biocatalysis method. The Luo team successfully obtained the key intermediate, i.e., the tertiary butyl 6-cyano- (3R, 5R) -dihydroxyhexanoate with specific stereostructure by using aldehyde ketone reductase KlAKR and efficiently reducing the tertiary butyl (R) -6-cyano-5-hydroxy-3-carbonyl hexanoate with the concentration of 450g/L substrate, and the d.e. selectivity is more than 99.5 percent, and the space-time yield reaches 1.24 kg/(L.d). Wang reduced (R) -6-cyano-5-hydroxy-3-carbonyl hexanoic acid tert-butyl ester with carbonyl reductase from STARMERELLA MAGNOLIA, e.e. 98% or more, conversion 95% or more (CN 116515780A).

Therefore, the research on the biocatalysis process of the (3R, 5S) -CDHH or the 6-cyano- (3R, 5R) -dihydroxyhexanoate has important significance, not only can provide a new route for the synthesis of the (3R, 5S) -CDHH or the 6-cyano- (3R, 5R) -dihydroxyhexanoate, but also can provide an enzyme source with high stereoselectivity and high catalytic activity for the (3R, 5S) -CDHH or the 6-cyano- (3R, 5R) -dihydroxyhexanoate.

Disclosure of Invention

In order to further improve the production efficiency and the product purity of statin intermediate chiral alcohol (3R, 5S) -CDHH or 6-cyano- (3R, 5R) -dihydroxyhexanoate, the invention carries out intensive research on Rhodosporidium toruloides ZJB 2014212-derived stereospecific recombinant carbonyl reductase SCR, and obtains a recombinant carbonyl reductase mutant SCR-Asp25Thr with high activity and high stereoselectivity, and a coding gene thereof, a recombinant vector containing the mutant coding gene and genetic engineering bacteria. The recombinant carbonyl reductase mutant is applied to the biological enzyme method for synthesizing chiral alcohol, the yield of the product (3R, 5S) -CDHH is more than 92%, e.e. is more than 99%, and the yield of the product 6-cyano- (3R, 5R) -dihydroxyhexanoate tert-butyl ester reaches 97.1%, e.e. is more than 99%.

The invention provides a recombinant carbonyl reductase mutant, which is obtained by carrying out mutation on the 25 th site of an amino acid sequence of Rhodosporidium toruloides ZJB 2014212-derived recombinant carbonyl reductase. The amino acid sequence of the Rhodosporidium toruloides ZJB 2014212-derived recombinant carbonyl reductase is shown as SEQ ID NO. 2.

Preferably, the mutation is an aspartic acid mutation threonine at position 25.

The invention also provides a gene encoding the recombinant carbonyl reductase mutant. Preferably, the acylase mutant is obtained by mutating a codon encoding aspartic acid at position 25 into a codon encoding threonine on the basis of a recombinant carbonyl reductase SCR having a base sequence shown in SEQ ID NO. 1. As known in the art, an allelic variant is an alternative form of a polynucleotide, which may be a substitution, deletion, or insertion of a polynucleotide, without substantially altering the function of the peptide protein it encodes.

The invention also provides a recombinant vector of the recombinant carbonyl reductase mutant coding gene. The recombinant vector comprises a polynucleotide operably linked to control sequences suitable for directing expression in a host cell. Various vectors conventional in the art, such as various plasmids, phage or viral vectors, etc., are linked to the recombinant carbonyl reductase mutant nucleotide sequences of the present invention and are intended to fall within the scope of the present invention. The recombinant vector preferably uses a plasmid pET-28b as an expression vector, and the coding gene of the novel carbonyl reductase mutant is connected to the plasmid pET-28b.

The invention also provides a genetic engineering bacterium of the recombinant carbonyl reductase mutant coding gene. Introducing exogenous recombinant carbonyl reductase mutant coding genes into host cells through a genetic engineering technology to construct genetically engineered bacteria and express the genetically engineered bacteria so as to obtain the novel carbonyl reductase mutant; wherein the host cell may be a bacterial, fungal, plant cell or animal cell, preferably E.coli ESCHERICHIA COLI BL (DE 3) as an expression host.

The invention also provides application of the recombinant carbonyl reductase mutant, the recombinant vector or the genetically engineered bacterium in preparing chiral alcohol by biological catalysis. Preferably, the application is: the method comprises the steps of using wet thalli or enzyme liquid extracted after ultrasonic disruption of the wet thalli obtained by fermenting and culturing genetic engineering bacteria containing recombinant carbonyl reductase mutant encoding genes as a catalyst, using precursor ketone as a substrate, using isopropanol as an auxiliary substrate, using NAD (P) H as a coenzyme, preferably forming a reaction system in a buffer solution with pH value of 6-8, and obtaining chiral alcohol through asymmetric reduction reaction under the conditions of 25-35 ℃ and 200-800 rpm. It will be appreciated that there are a number of possible format choices when reference is made to the use of the recombinant carbonyl reductase mutants of the present invention. This includes the following forms: the whole cell form of the genetically engineered bacterium is used, the crude enzyme form without purification is used, and the partially or completely purified enzyme form is used. In addition, the acylase mutant of the invention can be prepared into immobilized enzyme or immobilized cell form biocatalyst by utilizing the known enzyme immobilization technology, thereby further improving the stability and reusability of the enzyme and being beneficial to the application thereof in the fields of industrial production and the like.

Preferably, the precursor ketone is selected from (S) -tert-butyl 6-chloro-5-hydroxy-3-carbonyl hexanoate or 6-cyano- (5R) -3-carbonyl hexanoate. The substrate (S) -tert-butyl 6-chloro-5-hydroxy-3-carbonyl hexanoate ((S) -CHOH) is asymmetrically reduced to (3R, 5S) -CDHH under the catalysis of recombinant carbonyl reductase mutant. Substrate tert-butyl 6-cyano- (5R) -3-carbonyl hexanoate asymmetrically reduces tert-butyl 6-cyano- (3R, 5R) -dihydroxyhexanoate under the catalysis of recombinant carbonyl reductase mutant

Preferably, the catalyst is used in the reaction system in an amount of 3-50g/L buffer based on the weight of wet bacteria.

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

Preferably, the final concentration of isopropanol volume in the reaction system is 10% -70%, preferably 20% -50%, more preferably 40%. Referring to FIG. 1, the coenzyme NAD (P) H regeneration system of the recombinant carbonyl reductase and the mutant thereof is preferably a single-enzyme double-substrate coupling system, and the isopropanol is selected as an auxiliary substrate of the coenzyme regeneration system, so that the production cost can be effectively saved. Further, the inventor team optimizes the concentration of isopropanol in the coenzyme cycle regeneration system, and the result shows that when the volume concentration of isopropanol is 40%, the coenzyme cycle can basically meet the requirement of carbonyl reductase on NADPH, and the catalytic reaction product e.e. >99%.

The invention has the beneficial effects that: compared with wild enzyme, the recombinant carbonyl reductase mutant provided by the invention has high activity and high stereoselectivity, and further improves the catalytic efficiency on substrate precursor ketone. The recombinant carbonyl reductase mutant is applied to the synthesis of chiral alcohol by a biological enzyme method, and the yield of the product (3R, 5S) -CDHH after catalysis for 6 hours is more than 92% and e.e. is more than 99% when the substrate concentration is 400 g/L; the yield of the product tert-butyl 6-cyano- (3R, 5R) -dihydroxyhexanoate after 3h catalysis reached 97.1%, e.e. >99% at a substrate concentration of 200 g/L. In addition, in the reaction system for preparing chiral alcohol by catalysis, a single-enzyme double-substrate coupling system is constructed, isopropanol is selected as an auxiliary substrate of a coenzyme regeneration system, so that the production cost can be effectively saved, and the method has important significance for industrial application of preparing chiral alcohol by catalysis of a biological enzyme method. Compared with the chemical method for preparing chiral alcohol, the method utilizes the recombinant carbonyl reductase mutant to asymmetrically catalyze and reduce the precursor ketone to synthesize the chiral alcohol, simplifies the chemical catalysis step, has milder reaction conditions, has low requirements on equipment, reduces the reaction cost and is environment-friendly.

Drawings

FIG. 1 is a schematic diagram of the synthesis of chiral alcohols (3R, 5S) -CDHH using asymmetric catalytic reduction of precursor ketones with recombinant carbonyl reductase mutants according to the invention.

Detailed Description

The following specific examples are presented to illustrate the present invention, and those skilled in the art will readily appreciate the additional advantages and capabilities of the present invention as disclosed herein. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention. It should be noted that the following embodiments and features in the embodiments may be combined with each other without conflict. The methods used in the examples of the present invention are conventional methods, and the reagents used are commercially available.

Liquid phase detection method

High performance liquid chromatography instrument: shimadzu LC-20AD system-SPD-20A ultraviolet detector

The conversion was measured using a chromatographic column: agilent Zorbax SB-C8 column (150X4.6mm, 5 μm), mobile phase composed of acetonitrile water solution with volume ratio of 3:7 (v/v), flow rate of 1mL/min, detection wavelength of 210nm, sample injection amount of 10 μm, column temperature box of 40 deg.C, (S) -CHOH, (3R, 5S) -CDHH retention time of 8.348min and 5.534min respectively; when detecting e.e.: chiral column OD-H column (250×4.6mm,5 μm) was used, mobile phase: n-hexane: isopropanol=85: the retention times of the tert-butyl (S) -CHOH, (3R, 5S) -CDHH, (3S, 5S) -6-chlorodihydroxyhexanoate were 5.99min,5.09min and 4.93min, respectively, at a flow rate of 1mL/min at a detection wavelength of 215 nm.

The chromatographic column J & K SCIENTIFIC C18 reversed phase column (4.6mm.times.150mm, 5 um) was used, the mobile phase consisted of acetonitrile aqueous solution with volume ratio of 1:3 (v/v), the flow rate was 1mL/min, the detection wavelength was set to 210nm, the sample injection amount was 10 μm, the column oven was set to 40 ℃, the retention time of tert-butyl 6-cyano- (5R) -3-carbonyl hexanoate was about 15.353min, the retention time of tert-butyl 6-cyano- (3R, 5R) -dihydroxyhexanoate was about 9.057min (S-configuration) and 9.501min (R-configuration).

Example 1: obtaining recombinant carbonyl reductase mutants

Recombinant bacteria E.coli BL21 (DE 3) pET28b-SCR containing recombinant carbonyl reductase SCR coding gene expression vector pET28b-SCR is used as an initial strain, and site-directed saturation mutation and site-directed mutation are respectively carried out so as to further improve the catalytic activity and substrate tolerance of carbonyl reductase to substrate precursor ketone.

The mutant primers are shown in Table 1, with the mutation sites underlined. The mutation was introduced by PCR using plasmid DNA containing the SCR gene as a template, and the PCR reaction procedure was as follows: 98 ℃ for 3min; 26 cycles are repeated at 98℃for 10s,55℃for 5s and 72℃for 6min for 30 s; the extension was continued for 10min at 72 ℃. The PCR product was treated with DpnI at 37℃for 3 hours, inactivated and transformed into E.coli BL21 (DE 3) recipient bacteria, which were plated on LB solid plates containing a final concentration of 50mg/L of kana resistance, and incubated at 37℃for 12 hours. Single colony is randomly picked for sequencing analysis, and different mutant amino acids at different mutation points are obtained.

TABLE 1 mutant primers

A series of different amino acid mutants were obtained by site-directed saturation mutagenesis. The correct clone of sequencing analysis is inoculated into LB liquid medium containing 50 mug/mL kanamycin resistance of final concentration, cultured for 8 hours at 37 ℃, inoculated into fresh LB liquid medium containing 50 mug/mL kanamycin resistance of final concentration at 1 percent of inoculum size (v/v), cultured until the cell OD600 reaches 0.6-0.8 at 37 ℃ and 180rpm, added with IPTG of final concentration of 0.1mM, induced and cultured for 12 hours at 28 ℃, centrifuged for 10 minutes at 4 ℃ and 8000rpm, the supernatant is discarded, and the precipitate is collected, thus obtaining the recombinant escherichia coli wet cell containing recombinant carbonyl reductase mutant gene. The conversion reaction was carried out in a 10mL conversion bottle with a substrate ((S) -CHOH) concentration of 100g/L, isopropyl alcohol of 4mL, wet cell of the mutant of 15g/L, magnetic stirring at 30℃and 600rpm for 20min, and the reaction solution was analyzed by HPLC to determine the relative enzyme activity of the mutant and screen mutants having relatively high catalytic activity, and the results are shown in Table 2.

TABLE 2 relative enzyme activities of recombinant carbonyl reductase mutants

Carbonyl reductase type	Relative enzyme Activity (%)
		SCR	100±1.43
SCR-Asp25Thr	115.93±1.33
		SCR-Lys211Ile	108.46±1.21
SCR-Val181Ile	102.31±1.32

The result shows that the optimal mutant obtained by the site-directed saturation mutation and the site-directed mutation method is SCR-Asp25Thr, namely, the optimal mutant is obtained by mutation of threonine by aspartic acid mutation at the 25th site of recombinant carbonyl reductase with the amino acid sequence shown as SEQ ID NO. 2.

Example 2: preparation of recombinant carbonyl reductase mutant wet thalli

The recombinant E.coli BL21 (DE 3)/pET 28b-SCR-Asp25Thr gene containing recombinant carbonyl reductase mutant SCR-Asp25Thr gene obtained in example 1 was inoculated into LB liquid medium containing kanamycin resistance at a final concentration of 50. Mu.g/mL, cultured at 37℃and 180rpm for 8 hours, then inoculated into fresh LB liquid medium containing kanamycin resistance at a final concentration of 50. Mu.g/mL at 1% inoculum size (v/v), cultured at 37℃and 180rpm until the cell OD600 reached 0.6-0.8, IPTG at a final concentration of 0.1mM was added, after 12 hours of induction culture at 28℃and at 4℃and 8000rpm, the supernatant was discarded, and the precipitate was collected to obtain recombinant E.coli wet cell containing recombinant carbonyl reductase mutant gene. The wet cell can be directly used as a biocatalyst or used for protein purification. E.coli BL21 (DE 3)/pET 28b-SCR wet cells containing the gene expressing recombinant carbonyl reductase were prepared in the same manner.

Example 3: isolation and purification of carbonyl reductase mutants

The wet cells obtained in example 2 were washed three times with physiological saline, and resuspended in 1g of wet cells in 20mL of 20mM potassium phosphate buffer pH7.0, and sonicated (60W, for 1s, intermittently for 1s, continuously for 30 min) under ice bath conditions to obtain a cell disruption solution. And centrifuging the cell disruption solution obtained after ultrasonic disruption at 8000rpm and at 4 ℃ for 25min to obtain a supernatant which is the required crude enzyme solution, and determining the supernatant as the crude enzyme solution I.

Ammonium sulfate fractional salting out: the most common salt used in salting-out is (NH₄)₂SO₄、Na₂SO₄、MgSO₄.(NH₄)₂SO₄, which is a salt most commonly used in salting-out step, because of its high solubility and small temperature coefficient, and its good separation effect, generally does not denature proteins, and is available at low cost. The protein can be dehydrated under the influence of high-concentration neutral salt, so that the charge is neutralized, the colloid stability of the protein is destroyed and separated out, and meanwhile, the salting-out effect of the protein is a reversible process and can be redissolved when diluted by water or buffer solution. Thus, as the saturation of ammonium sulfate increases, the protein content in the supernatant gradually decreases; conversely, the amount of protein precipitated in the precipitate increases gradually. The method is to add 5mL of crude enzyme solution in 8 small beakers numbered 1-8. 0.57g, 0.88g, 1.215g, 1.565g, 1.95g, 2.36g, 2.805g and 3.31g of ground ammonium sulfate are added respectively, so that the saturation of the ammonium sulfate in the crude enzyme solution reaches 20%, 30%, 40%, 50%, 60%, 70%, 80% and 90% respectively. Ammonium sulfate is slowly added, stirred on ice to dissolve, and kept stand for 4 hours in a refrigerator at 4 ℃ after stirring. The liquid in the beaker was taken out and centrifuged (8000 rpm,25 min), and the supernatant was weighed to calculate the supernatant volume and the enzyme activity and protein content were measured. The two saturations with the highest specific activity and the highest protein precipitation rate can be found out through calculation to obtain an ammonium sulfate fractional salting-out specific activity chart and an ammonium sulfate fractional salting-out protein precipitation rate chart, namely the optimal precipitation saturation of the hybrid protein and the optimal precipitation saturation of the target protein, so that a large amount of hybrid protein can be removed; ammonium sulfate is graded and salted out, the crude enzyme solution I is continuously stirred in an ice-water bath after cells are broken by ultrasonic, ammonium sulfate is slowly added to reach the final concentration of 50 percent of saturation degree, and the mixture is kept stand for 4 hours in a refrigerator at the temperature of 4 ℃. Centrifugation was performed at 8000rpm for 25min, the precipitate was discarded, and the supernatant was further added with ammonium sulfate to a final concentration of 70% saturation, and allowed to stand in a refrigerator at 4℃for 4 hours. Again, centrifugation was performed at 8000rpm for 25min, the supernatant was discarded, and the pellet was reconstituted with an appropriate amount of 20mM potassium phosphate buffer, pH 7.0. The crude enzyme solution II was obtained by dialysis against potassium phosphate buffer at pH 7.0 at 20mM overnight at 4 ℃.

DEAE Sepharose Fast Flow anion exchange chromatography: ion exchange chromatography is a method of separation using reversible ion exchange that occurs between stationary phase coupled ion exchange groups and mobile phase dissociated ionic compounds. Anion exchange chromatography is used in the primary chromatographic purification of most oxidoreductases. DEAE Sepharose Fast Flow anion exchange columns (1.6cm.times.20 cm) were first equilibrated with 20mM, pH 7.0, pB potassium phosphate buffer at a flow rate of 1.5mL/min, and after the baseline was leveled off, the dialyzed crude enzyme solution II was loaded onto the column at a loading flow rate of 1.0mL/min. After loading, the sample is eluted with 20mM potassium phosphate buffer with pH 7.0 at a flow rate of 1.5mL/min, and after the baseline is leveled, the sample is eluted with a gradient of 0-0.8M NaCl. Collecting active part, concentrating to obtain pure enzyme. The whole purification process was operated at 4 ℃. And (3) determining the protein content of the trapped fluid by adopting a BCA kit method, and freezing the trapped fluid in a refrigerator at the temperature of minus 80 ℃ to obtain recombinant carbonyl reductase SCR and mutant SCR-Asp25Thr pure enzyme thereof.

Example 4: recombinant carbonyl reductase and mutant enzyme activity thereof

The carbonyl reductase SCR and its mutant SCR-Asp25Thr pure enzyme obtained in example 3 were used to catalyze the substrate (S) -CHOH to synthesize (3R, 5S) -CDHH.

The composition of the enzyme activity catalytic system and the catalytic conditions are as follows: to 5mL of 100mM phosphate buffer solution at pH 7.0, carbonyl reductase mutant pure enzyme diluted to the same concentration with the same buffer solution (final concentration of SCR 2.5. Mu.g/mL, SCR-Asp25Thr 2.5. Mu.g/mL), (S) -CHOH final concentration of 10mM, coenzyme NAD (P) H final concentration of 2mM, pre-heated at 30℃for 3min at 600rpm, and reacted for 3min. The reaction was terminated by adding 5mL of 30% acetonitrile aqueous solution by volume, and after mixing uniformly, the sample was taken to detect the enzyme activity, and the results are shown in Table 3.

TABLE 3 recombinant carbonyl reductase and its mutant relative enzyme activities

Carbonyl reductase type	Relative enzyme Activity (%)	Ee value (%)
			SCR	100±1.12	99
SCR-Asp25Thr	116.36±1.26	99

Note that: the enzyme activity unit (U) is defined as: the amount of enzyme required to produce 1. Mu. Mol of product (3R, 5S) -CDHH in 1min at 30℃and pH 7.0 was defined as 1U. The amount of product formed was determined by HPLC detection.

Example 5: optimization of isopropanol concentration in recombinant carbonyl reductase coenzyme regeneration system

Referring to FIG. 1, the coenzyme NAD (P) H regeneration system of the recombinant carbonyl reductase and the mutant thereof is preferably a single-enzyme double-substrate coupling system, and the isopropanol is selected as an auxiliary substrate of the coenzyme regeneration system, so that the production cost can be effectively saved.

The wet cell E.coli BL21 (DE 3)/pET 28b-SCR-Asp25Thr was obtained as in example 2 and used as biocatalyst to synthesize (3R, 5S) -CDHH using (S) -CHOH as substrate.

Conversion system composition and catalytic conditions: 10mL of potassium phosphate buffer (100 mM, pH 7.0), 10%, 20%, 30%, 40%, 50%, 60% and 70% of the final concentration of the recombinant carbonyl reductase mutant mut-Asp25Thr wet cell (15 g/L buffer) in 100g/L buffer and isopropyl alcohol. The reaction was carried out in a magnetic stirring water bath at 30℃and 600rpm for 20min. After the completion of the reaction, the reaction was terminated by adding 1mL of acetonitrile, the reaction mixture was thoroughly mixed, and an appropriate amount of the reaction mixture was measured for conversion by HPLC, and the results are shown in Table 4.

TABLE 4 optimization of isopropyl alcohol concentration

Final volume concentration of isopropanol	Conversion rate
		10％	37.22％
20％	51.45％
		30％	52.33％
40％	66.72％
		50％	48.79％
60％	38.16％
		70％	33.29％

The results show that at an isopropanol volume concentration of 40%, the coenzyme cycle has been substantially able to meet the NADPH requirement of the carbonyl reductase, catalyzing the reaction product e.e. >99% above.

Application of recombinant carbonyl reductase mutant in preparation of (3R, 5S) -CDHH

Example 6:

The E.coli BL21 (DE 3)/pET 28b-SCR-Asp25Thr wet cell containing the expression recombinant plasmid obtained in example 2 is used as a biocatalyst, and (S) -CHOH is used as a substrate to carry out bioconversion reaction to prepare (3R, 5S) -CDHH.

The composition of the 10mL catalyst system and the catalyst conditions are as follows: in a 10mL reaction system, 6mL of potassium phosphate buffer solution (pH 7.0) is added, recombinant carbonyl reductase mutant SCR-Asp25Thr wet thalli (the dosage is 50g/L buffer solution) is added, 4mL of isopropanol is added, the initial substrate final concentration is 400g/L, a water bath is carried out at 30 ℃, the magnetic stirring device is at 600rpm, the reaction is sampled at regular time, the sampling volume is 100 mu L, 50 times of dilution is carried out by using 30% acetonitrile water solution by volume concentration, and the conversion rate is determined by HPLC analysis. The results showed that the yield of the product (3R, 5S) -CDHH after 8h of catalysis reached 97.3%, e.e. >99% at a substrate concentration of 400g/L in 10mL of catalytic system.

Example 7:

100ML of the catalytic system consists of the following components and catalytic conditions: in a 100mL reaction system, 60mL of a potassium phosphate buffer solution (pH 7.0) was added, a recombinant carbonyl reductase mutant mut-Asp25Thr wet cell (50 g/L buffer solution was used), 40mL of isopropyl alcohol was added, the initial substrate final concentration was 400g/L, a water bath was carried out at 30℃and the magnetic stirring apparatus was set at 1200rpm, the reaction was sampled at regular time, the sampling volume was 100. Mu.L, and 50-fold dilution was carried out with a 30% acetonitrile aqueous solution by volume concentration, and the conversion was determined by HPLC analysis. The results show that the yield of the product (3R, 5S) -CDHH after 6h of catalysis reaches 96.4%, e.e. >99% at a substrate concentration of 400g/L in 100mL of the catalytic system.

Example 8:

The difference compared to example 6 is that the initial substrate concentration is 500g/L, the remainder being identical. The results showed that the yield of the product (3R, 5S) -CDHH reached 92.1%, e.e. >99% after 6h of catalysis at a substrate concentration of 500g/L in 10mL of the catalytic system.

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

Comparative example 1:

Compared with example 6, the difference is that the E.coli BL21 (DE 3)/pET 28b-SCR wet cell containing the expression recombinant plasmid obtained in example 2 is used as a biocatalyst, and (S) -CHOH is used as a substrate for biological conversion reaction to prepare (3R, 5S) -CDHH, and the rest are the same. The results showed that the yield of the product (3R, 5S) -CDHH reached 94.1%, e.e. >99% after 6h of catalysis at a substrate concentration of 400g/L in 10mL of the catalytic system.

Comparative example 2:

Compared with example 8, the difference is that the E.coli BL21 (DE 3)/pET 28b-SCR wet cell containing the expression recombinant plasmid obtained in example 2 is used as a biocatalyst, and (S) -CHOH is used as a substrate for biological conversion reaction to prepare (3R, 5S) -CDHH, and the rest are the same. The results show that the yield of the product (3R, 5S) -CDHH after 6h of catalysis reaches 89.4%, e.e. >99% at a substrate concentration of 500g/L in 10mL of the catalytic system.

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

The E.coli BL21 (DE 3)/pET 28b-SCR-Asp25Thr wet cell containing the expression recombinant plasmid obtained in example 2 is used as a biocatalyst, and 6-cyano- (5R) -3-carbonyl tert-butyl caproate is used as a substrate for biological conversion reaction to prepare 6-cyano- (3R, 5R) -dihydroxyhexanoate tert-butyl ester.

The catalytic system comprises the following components and catalytic conditions: in a 10mL reaction system, 6mL of potassium phosphate buffer solution (pH 7.0) is added, recombinant carbonyl reductase mutant SCR-Asp25Thr wet thalli (the dosage is 50g/L buffer solution) is added, 4mL of isopropanol is added, the initial substrate final concentration is 200g/L, a water bath is carried out at 30 ℃, the magnetic stirring device is at 600rpm, the reaction is sampled at regular time, the sampling volume is 100 mu L, 50 times of dilution is carried out by using 30% acetonitrile water solution by volume concentration, and the conversion rate is determined by HPLC analysis. The results show that the yield of the product tert-butyl 6-cyano- (3R, 5R) -dihydroxyhexanoate reaches 97.1%, e.e. >99% after 3h of catalysis at a substrate concentration of 200 g/L.

The above examples are merely illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solution of the present invention should fall within the protection scope of the present invention without departing from the design spirit of the present invention.

Claims

1. A recombinant carbonyl reductase mutant is obtained by mutating the 25 th position of an amino acid sequence of Rhodosporidium toruloides ZJB 2014212-derived recombinant carbonyl reductase.

2. The recombinant carbonyl reductase mutant of claim 1, wherein the mutation is an aspartic acid mutant threonine at position 25.

3. A gene encoding the recombinant carbonyl reductase mutant of claim 1 or 2.

4. A recombinant vector comprising the gene encoding the recombinant carbonyl reductase mutant of claim 3.

5. A genetically engineered bacterium comprising a gene encoding the recombinant carbonyl reductase mutant of claim 3.

6. The use of the recombinant carbonyl reductase mutant of claim 1 or 2, the recombinant vector of claim 4, or the genetically engineered bacterium of claim 5 in biocatalysis for preparing chiral alcohols.

7. The application of claim 6, wherein the application is: the method comprises the steps of using wet thalli or enzyme liquid extracted after ultrasonic crushing of wet thalli obtained by fermenting and culturing genetic engineering bacteria containing recombinant carbonyl reductase mutant encoding genes as a catalyst, using precursor ketone as a substrate, using isopropanol as an auxiliary substrate to form a reaction system, and obtaining chiral alcohol through asymmetric reduction reaction.

8. Use according to claim 8, wherein the precursor ketone is selected from (S) -tert-butyl 6-chloro-5-hydroxy-3-carbonyl hexanoate or tert-butyl 6-cyano- (5R) -3-carbonyl hexanoate.

9. The use according to claim 8, wherein the initial final concentration of precursor ketone in the reaction system is 200-500g/L.

10. The use according to claim 8, wherein the final concentration of isopropanol volume in the reaction system is between 10% and 70%.