Carbonyl reductase mutant and application thereof in synthesis of chiral alcohol compound

Abstract

The invention discloses a carbonyl reductase, a mutant and an application of the carbonyl reductase in synthesis of a chiral alcohol compound. The carbonyl reductase mutant is obtained by performing single-site or multi-site mutation on the 96th site, the 145th site, the 202nd site or the 206th site of the amino acid sequence of the carbonyl reductase LbCR derived from lactobacillus brevis as shown in SEQ ID No.2. The activity and stability of the carbonyl reductase LbCR mutant are obviously improved, and the carbonyl reductase LbCR mutant has a wide substrate spectrum. Compared with a wild type, the mutant LbCRM4 has the advantages that the enzyme activity is improved by 86.3 times, the substrate conversion rate is greater than 99%, the e.e.p value of the product is always kept at 99.5% or above, the space time yield reaches 1288.9 g / L / d, and the half-life period at 40 DEG C is 101.4 h and is improved by 83.5 times. The LbCR mutant has an industrial application prospect.

Description

(I) Technical field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and specifically relates to a carbonyl reductase (LbCR) derived from Lactobacillus brevis and a mutant with improved activity thereof, a coding gene and a recombinant expression vector and a recombinant expression transformant carrying the gene, and the application of the recombinant transformant or its recombinant carbonyl reductase as a catalyst for asymmetric reduction to prepare a chiral alcohol compound, in particular, the application in the biocatalytic synthesis of a chiral intermediate of luliconazole drug (1S)-2-chloro-1-(2,4-dichlorophenyl)ethanol ((S)-TCPE). (II) Background technology

[0002] As an analog of lanoconazole, luliconazole has strong antibacterial effects and is safe and effective in treating tinea among many antifungal compounds. It has great potential in the antifungal treatment of the skin. The global market demand for antifungal treatment is huge, and it is crucial to improve the production technology level and production capacity of antifungal active compounds. (S)-TCPE is a key intermediate for the preparation of luliconazole. However, the process route for the chemical synthesis of (S)-TCPE is lengthy, and chiral auxiliary agents or expensive metal reagents are required to separate to obtain a single enantiomer with high optical purity. In addition, the process is seriously polluted and the product yield is low. Biocatalysis is a green and sustainable production method. The development of (S)-TCPE biocatalysis technology is in line with the development trend of green chemistry.

[0003] Carbonyl reductases (CRs) belong to the class of short-chain dehydrogenases / reduetases (SDRs), and usually exist in the form of dimers or tetramers. In the process of catalyzing the reduction of carbonyl compounds, carbonyl reductases require NAD(P)H as a hydrogen or electron transfer carrier. Under the action of the reductase, the carbonyl is attacked from the Ri or Se surface to generate the corresponding chiral alcohol, and NAD(P)H is oxidized to NAD(P) + Since carbonyl reductases from natural sources often have defects such as low catalytic efficiency and poor stability, the industrial application of wild-type (WT) carbonyl reductases is limited.

[0004] The key to the industrial application of enzymes is to use protein engineering technology to molecularly modify carbonyl reductase to obtain enzyme preparations with industrial properties. The present invention aims to solve the problems of low activity and poor stability of WT-LbCR, and successfully obtains a carbonyl reductase mutant with improved activity through protein engineering. In addition, the present invention optimizes the process parameters for the catalytic synthesis of (S)-TCPE, and effectively improves the production efficiency of its (S)-TCPE enzymatic synthesis. (III) Summary of the invention

[0005] The purpose of the present invention is to provide a carbonyl reductase LbCR mutant, a recombinant bacterium and its application in the synthesis of chiral alcohol compounds, especially the application in the asymmetric reduction of 2-chloro-1-(2,4-dichlorophenyl)ethanone (TCAP) to prepare the key chiral intermediate (S)-TCPE of luliconazole. Through protein engineering, a beneficial carbonyl reductase mutant with improved activity was successfully obtained. Further, a super mutant with high activity and high stability was obtained through combined mutation. Among them, the mutant LbCR-N96V-E145A-A202L-M206A (LbCR M4 ) has a significantly improved activity compared to WT-LbCR, and also has a significantly improved activity for different types of carbonyl compounds. In addition, the present invention optimizes the reaction process parameters and constructs LbCR M4 The catalytic synthesis of (S)-TCPE process solves the problems of lack of robust 2-chloro-1-(2,4-dichlorophenyl)ethanone asymmetric reductase, low activity of natural LbCR, and poor stability.

[0006] The technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides a carbonyl reductase LbCR mutant, wherein the carbonyl reductase LbCR mutant is obtained by subjecting the amino acid sequence of the carbonyl reductase LbCR from Levilactobacillus brevis shown in SEQ ID No. 2 to single-site or multi-site mutation at position 96, position 145, position 202 or position 206. The nucleotide sequence of the carbonyl reductase LbCR encoding gene of the present invention is shown in SEQ ID No. 1.

[0008] Furthermore, the carbonyl reductase LbCR mutant is preferably a mutant in which the amino acid sequence of SEQ ID No. 2 is mutated into one of the following: (1) asparagine at position 96 is mutated into valine (N96V); (2) glutamic acid at position 145 is mutated into alanine (E145A); (3) alanine at position 202 is mutated into leucine (A202L); (4) methionine at position 206 is mutated into alanine (M206A); (5) a mutant in which the single-site mutations in (1) to (4) are combined in pairs; (6) a mutant in which any three of the single-site mutations in (1) to (4) are combined; (7) asparagine at position 96 is mutated into valine, glutamic acid at position 145 is mutated into alanine, alanine at position 202 is mutated into leucine, and methionine at position 206 is mutated into alanine (N96V / E145A / A202L / M206A, the nucleotide sequence is as shown in SEQ ID The amino acid sequence is shown in SEQ ID No.3 and the amino acid sequence is shown in SEQ ID No.4).

[0009] The present invention also relates to the carbonyl reductase LbCR mutant encoding gene, recombinant expression vector and recombinant genetic engineering bacteria. The recombinant expression vector is preferably vector pET28a(+), and the recombinant genetic engineering bacteria is preferably host bacteria E.coliBL21(DE3).

[0010] In a second aspect, the present invention also relates to the application of the carbonyl reductase mutant in the asymmetric reduction of carbonyl compounds to prepare chiral alcohol compounds. The application method is: using wet bacteria obtained by induction culture of recombinant genetic engineering bacteria of carbonyl reductase mutant as a catalyst, carbonyl compounds as substrates, isopropanol as co-substrate and cosolvent, using pH4.0-10.0 buffer (pH 7.0, 100mM potassium phosphate buffer) as a reaction medium to form a reaction system, reacting at 20-60°C and 1000rpm, and after the reaction is completed, the reaction solution is separated and purified, and the reaction solution is extracted with ethyl acetate to obtain a chiral alcohol compound.

[0011] Furthermore, the carbonyl compound is one of the following: 2-chloro-1-(2,4-dichlorophenyl)ethanone (TCAP), methyl acetoacetate, ethyl acetoacetate, tert-butyl acetoacetate, methyl 3-oxopentanoate, ethyl pyruvate, ethyl benzoylformate, n-valeraldehyde, benzaldehyde, acetophenone, 2-chloroacetophenone, o-fluoroacetophenone, benzylacetone, 3-chloroacetophenone, 4-fluoroacetophenone, and 3,5-bis(trifluoromethyl)acetophenone.

[0012] Further, the prochiral ketone TCAP structure is as follows:

[0013]

[0014] Furthermore, in the reaction system, the substrate is added to a final concentration of 1 to 600 g / L (preferably 200 to 400 g / L); the volumetric concentration of isopropyl alcohol is 10 to 80% (V / V) (preferably 40% (V / V)); the amount of catalyst used is 10 to 50 g / L based on the weight of wet cells and 1 to 30 g DCW / L (preferably 10 g DCW / L) based on the dry weight of wet cells; when the catalyst is a purified enzyme, it is used in an amount of 10 to 100 mg / L (preferably 60 to 80 mg / L) based on the protein content.

[0015] Furthermore, when the carbonyl compound is TCAP, the carbonyl reductase mutant asymmetric reduces TCAP to prepare (S)-TCPE, and the conditions are preferably: temperature 45° C. and pH 8.0.

[0016] Further, the wet bacteria were prepared as follows: the engineered bacteria containing the carbonyl reductase LbCR mutant gene were inoculated into an LB liquid culture medium containing kanamycin at a final concentration of 50 μg / mL, and cultured at 37°C and 180-200 rpm for 10 h to obtain a seed solution; the seed solution was inoculated into a sterilized LB liquid culture medium containing kanamycin at a final concentration of 50 μg / mL at a volume concentration of 1% (V / V), and cultured at 37°C and 180-200 rpm until the OD 600 =0.6~0.8, add isopropyl thiogalactoside (IPTG) with a final concentration of 0.15mM into the culture medium, culture at 20℃ for 18h, and centrifuge at 4℃ and 8000rpm for 10min to obtain wet cells of carbonyl reductase LbCR mutant.

[0017] Furthermore, the LbCR mutant purified enzyme was prepared as follows: (1) Crude enzyme solution: The wet bacteria were resuspended in a pH 7.0, 100 mM PBS buffer at a volume of 50 g / L, and the bacterial suspension was ultrasonically disrupted on an ice-water mixture for 15 min. The ultrasonic disruption conditions were: power 250 W, disruption for 1 s, pause for 1 s, and the disruption mixture was taken to obtain a crude enzyme solution; (2) The crude enzyme solution was centrifuged at 8000 rpm and 4°C for 20 min, the precipitate was discarded, and the supernatant was collected. The supernatant was filtered through a 0.45 μm microfiltration membrane and then purified using a nickel affinity column (1.6×10 cm, Bio-Rad, USA). The purification procedure was as follows: ① Nickel-agarose gel affinity chromatography filler (2-3 mL) was added flatly to a pre-packed column of protein purification filler, and the column was equilibrated with 10-15 mL of 20 mM phosphate buffered saline (pH 7.0); ② The sample was loaded at a flow rate of 1.0 mL / min and eluted with 20 mM sodium phosphate buffer (pH 7.0) containing 0.5 M NaCl and 20 mM imidazole to remove unbound impurities; ③ Then, 1 column volume was eluted by gravity with 20 mM sodium phosphate buffer (pH 7.0) containing 0.5 M NaCl and 400 mM imidazole; all the eluate was collected and transferred to an ultrafiltration tube with a molecular weight cutoff of 30 kDa, and 20 mM, pH 7.0 potassium phosphate was used as the replacement buffer to remove imidazole, collect the retained protein, and obtain the purified enzyme of LbCR mutant.

[0018] The base sequences of the carbonyl reductase LbCR and the carbonyl reductase LbCR mutant of the present invention are both 756 bp in length, from the first base to the 756th base, the start codon is ATG, the stop codon is TAA, and 252 amino acids are encoded.

[0019] The carbonyl reductase LbCR mutants of the present invention were obtained by protein engineering, and finally four beneficial mutants N96V, E145A, A202L, and M206A were obtained. Through further iterative combination mutations, we obtained LbCRM4 The mutant plasmid obtained was transferred into E.coli BL21(DE3) competent cells by heat shock, the obtained strain was inoculated, transferred, induced, and the cells were recovered, and the resuspended bacterial solution was used to catalyze TCAP reduction and prepare (S)-TCPE. The mutant recombinant plasmid obtained was transferred into E.coli BL21(DE3) competent cells by heat shock, the obtained strain was inoculated, transferred, induced, and the cells were collected from the fermentation broth by solid-liquid separation.

[0020] The inoculation, transfer, induction culture and cell recovery of the carbonyl reductase mutant of the present invention can be any culture medium in the art that can grow the cells of the present invention, preferably LB culture medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, dissolved in water. There are no special restrictions on the culture method and culture conditions, which can be appropriately selected according to the common knowledge in the art based on factors such as the host type and the culture method.

[0021] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0022] The invention provides a novel carbonyl reductase LbCR mutant, which has significantly improved substrate conversion rate and stability.

[0023] (1) The activity of the carbonyl reductase LbCR mutant was significantly improved.

[0024] The beneficial mutants N96V, E145A, A202L, and M206A obtained in the present invention have increased their activities by 8.8, 2.7, 4.5, and 1.2 times, respectively, compared with the wild-type WT-LbCR. M4 The mutant has a specific activity that is 86.3 times higher than that of the wild-type WT-LbCR. The maximum feed amount can reach 400g / L TCAP. The product concentration gradually increases with time. The reaction can be completed within 7h. The substrate conversion rate is greater than 99%. The product ee p The value was always kept above 99.5%, and the space-time yield reached 1288.9 g / L / d. When the wild-type WT-LbCR in the control group catalyzed 200 g / L TCAP, the maximum yield was only 58 g / L (S)-TCPE, and the substrate conversion rate was low.

[0025] (2) The stability of the carbonyl reductase LbCR mutant was improved.

[0026] Compared with the wild-type WT-LbCR, the new mutants developed by the present invention have improved stability, among which LbCR M4The half-life at 40°C was 101.4 h, which was 83.5 times higher than that of the parent wild-type carbonyl reductase WT-LbCR. M4 It has more prospects for industrial application.

[0027] (3) The carbonyl reductase LbCR mutant has a broader substrate spectrum.

[0028] The carbonyl reductase LbCR mutant of the present invention has a wider substrate spectrum for carbonyl compounds, and its catalytic activity for most substrates in the substrate spectrum is significantly higher than that of WT-LbCR. (IV) Description of the drawings

[0029] Figure 1 Schematic diagram of the reaction of preparing (S)-TCPE by asymmetric reduction of TCAP catalyzed by carbonyl reductase LbCR and its mutants using isopropanol as a cosubstrate.

[0030] Figure 2 This is the gas chromatogram of the reaction liquid of LbCR asymmetric reduction of TCAP to synthesize (S)-TCPE.

[0031] Figure 3 The (S)-TCPE standard curve was drawn based on gas chromatography.

[0032] Figure 4 The gel electrophoresis of purified proteins of LbCR and its mutants; M: protein standard, lane 1 is WT-LbCR pure protein, lane 2 is LbCR M4 Pure protein.

[0033] Figure 5 Optimization curve of the optimal reaction temperature for WT-LbCR.

[0034] Figure 6 This is the optimal reaction pH optimization curve of WT-LbCR.

[0035] Figure 7 This is the optimal isopropanol concentration optimization curve for WT-LbCR.

[0036] Figure 8 Mutant LbCR M4 Optimization curve of optimal reaction temperature.

[0037] Fig. 9 Mutant LbCR M4 Optimization curve of optimal reaction pH.

[0038] Fig.10 Mutant LbCR M4 Optimization curve of optimal isopropanol concentration.

[0039] Fig.11This is the reaction time course curve of WT-LbCR using isopropanol as a cosubstrate to asymmetric reduction of TCAP to synthesize (S)-TCPE.

[0040] Fig.12 Mutant LbCR M4 Reaction time course curve of asymmetric reduction of TCAP to synthesize (S)-TCPE using isopropanol as cosubstrate.

[0041] Fig.13 For WT-LbCR and LbCR M4 Catalytic efficiency towards different carbonyl compound substrates. (V) Specific implementation methods

[0042] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0043] The LB liquid culture medium used in the present invention includes 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, dissolved in water. The LB solid culture medium is LB liquid culture medium with 18 g / L agar added thereto.

[0044] Example 1: Preparation of purified LbCR enzyme and determination of enzyme activity

[0045] (1) Wild-type carbonyl reductase genetically engineered bacteria: The carbonyl reductase gene sequence from Levilactobacillus brevis in GenBank (GenBank No. MCF7523072.1), i.e., the carbonyl reductase LbCR gene (nucleotide sequence as shown in SEQ ID No. 1, amino acid sequence as shown in SEQ ID No. 2) was artificially synthesized and ligated between the NdeI and XhoI restriction sites of the vector pET28a(+) to construct a recombinant expression vector, which was then transferred into the host bacteria E. coli BL21(DE3) to obtain the wild-type carbonyl reductase genetically engineered bacteria E. coli BL21(DE3) / pET28a(+)-lbcr.

[0046] SEQ ID No.2

[0047] MSNRLDGKVAIVTGGTLGIGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGI QRMKNKGLGASIINMSSIEGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDLPGAEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ.

[0048] (2) Wet bacteria

[0049] The wild-type carbonyl reductase genetically engineered bacteria E. coli BL21 (DE3) / pET28a (+) -lbcr constructed in step 1 was inoculated into an LB liquid culture medium containing a final concentration of 50 μg / mL kanamycin, and cultured at 37 ° C for 10 hours to obtain a seed solution; the seed solution was inoculated into a fresh LB liquid culture medium containing a final concentration of 50 μg / mL kanamycin at an inoculum concentration of 1% by volume, and cultured at 37 ° C, 180 rpm for 2 hours, and then 0.15 mM isopropyl β-D-thiogalactoside (Isopropylβ-D-thiogalactoside, IPTG) was added to the culture solution at a final concentration of 0.15 mM, and after culturing at 20 ° C for 18 hours, it was centrifuged at 4 ° C, 8000 rpm for 10 minutes to obtain wet bacteria.

[0050] (3) Enzyme purification

[0051] The wet bacterial cells of step (2) were dispersed in a pH 7.0, 100 mM potassium phosphate buffer at an amount of 50 g / L and resuspended. The bacterial suspension was ultrasonically disrupted on an ice-water mixture for 15 min. The ultrasonic disruption conditions were: power 250 W, disruption for 1 s, pause for 1 s, and the disruption mixture was taken to obtain a crude enzyme solution.

[0052] The crude enzyme solution was centrifuged at 8000 rpm and 4°C for 20 min, the precipitate was discarded, and the supernatant was collected. The supernatant was filtered through a 0.45 μm microfiltration membrane and the protein was purified using a nickel affinity column (1.6×10 cm, Bio-Rad, USA). The enzyme purification operation was as follows:

[0053] ① Add nickel-agarose gel affinity chromatography filler (2-3 mL) to the pre-packed column of protein purification filler, and use 10-15 mL of 20 mM phosphate buffer solution (pH 7.0) to balance the chromatography column; ② Load the sample at a flow rate of 1.0 mL / min, and elute with sodium phosphate buffer (20 mM, pH 7.0) containing 0.5 M NaCl and 20 mM imidazole to remove unbound impurities; ③ Then use sodium phosphate buffer (400 mM, pH 7.0) containing 0.5 M NaCl and 400 mM imidazole to gravity elute for 1 column volume; collect all the eluate and transfer it to an ultrafiltration tube with a molecular weight cutoff of 30 kDa, use 20 mM, pH 7.0 potassium phosphate buffer as the replacement buffer to remove imidazole, collect the retained protein, and obtain the LbCR purified enzyme, which is recorded as WT-LbCR.

[0054] (4) Enzyme activity assay

[0055] Enzyme activity is defined as: Under optimal conditions, the amount of enzyme required to generate 1 micromole (S)-TCPE per minute is defined as one enzyme activity unit, U. Specific enzyme activity is defined as the enzyme activity unit per milligram of enzyme protein, U / mg.

[0056] Enzyme activity detection conditions: 5mM NADPH, appropriate amount of purified enzyme (enzyme protein content of 70mg / L), 5g / L substrate TCAP, 1% DMSO, the substrate was first dissolved in DMSO and then added, pH 7.0, 100mM potassium phosphate buffer was added to a final volume of 500μL, reacted at 35°C, 1000rpm for 5min, the reaction solution was extracted with an equal volume of ethyl acetate, the extract was collected and microfiltered with a 0.22μm filter membrane, and the filtrate was detected by gas phase for the peak area of ​​(S)-TCPE ( Figure 2 ). According to the standard curve of (S)-TCPE, the concentration of (S)-TCPE in the filtrate was calculated, and the enzyme activity was calculated. The standard curve of (S)-TCPE is drawn with the (S)-TCPE peak area as the ordinate and the (S)-TCPE concentration as the abscissa, as shown in Figure 3 .

[0057] The substrate conversion rate, product ee value and yield were detected by Agilent (8860) high performance gas chromatograph: GC (CYCLOSIL-B, 30m×0.25mm, 0.25μm). The injector and detector were set at 250℃ and 250℃ respectively, the column oven temperature was set at 170℃ and maintained for 22min, the split ratio was 15:1, the injection volume was 2μL, and other conditions were the default detection conditions of the instrument.

[0058] Example 2: Selection of LbCR mutant sites and construction of single-site mutants

[0059] 1. Selection of LbCR mutant sites

[0060] The docking software YASARA was used to dock the substrate TCAP into the active pocket of WT-LbCR, with a preferred distance from the substrate. Among the amino acid residues in the range, these amino acids are most likely to affect the selectivity and activity of the enzymatic reaction. After excluding the conserved catalytic triad amino acids (N114, S143, Y156, K160), N96, E145, and M206 were selected as mutation sites. At the same time, a saturation mutation was performed on A202, a key site located in the substrate binding pocket.

[0061] 2. Site-directed saturation mutagenesis of N96

[0062] (1) Mutation of site 96: By designing the NNK codon, a saturation mutation was performed on site 96 of LbCR. The plasmid pET28a(+)-lbcr of the wild-type carbonyl reductase genetic engineering bacteria E. coli BL21(DE3) / pET28a(+)-lbcr of Example 1 was extracted, and PCR amplification was performed using the primers in Table 1. After the PCR product was enzymatically eliminated from the original template and cleaned up, it was transformed into E. coli BL21(DE3) competent cells, and the clones were inoculated into LB solid culture medium containing 50 μg / mL kanamycin and cultured at 37°C for 12 hours. Then, the clones were picked out and transferred to 10 mL of LB liquid culture medium containing 50 μg / mL kanamycin, and cultured at 37°C and 200 rpm for 10 hours. The culture solution was centrifuged at 8000 rpm for 10 minutes, and the precipitate was washed twice with 0.9% (w / V) physiological saline, and the wet bacteria were collected.

[0063] Table 1. Primer design for site-directed saturation mutagenesis of carbonyl reductase 96

[0064]

[0065] Note: In Table 1, NNK represents a degenerate codon, and MNN represents a degenerate codon paired with NNK.

[0066] PCR reaction system (25 μL): 0.5 μL forward primer (100 μM), 0.5 μL reverse primer (100 μM), 12.5 μL 2×Phanta buffer, 0.5 μL dNTP mixture (10 mM each), 0.5 μL plasmid template, 0.5 μL DNA polymerase and 10 μL ultrapure water. According to the Phanta Super-Fidelity DNA polymerase operation manual, the PCR program was set as follows: 95°C pre-denaturation for 5 min, then 30 cycles (95°C denaturation for 15 s, 55-65°C annealing for 15 s, 72°C extension for 10 s), 72°C final extension for 10 min, and 16°C incubation.

[0067] (2) Screening of mutants

[0068] Mutant screening reaction system: 50 g / L of each wet cell prepared in step (1), 5 g / L TCAP, 30% (V / V) isopropanol, the reaction medium is pH 7.0, 100 mM potassium phosphate buffer, forming a 1 mL reaction system, reacting at 35 ° C, 1000 rpm for 10 min, adding 1 mL of ethyl acetate to the reaction solution for extraction, and the extract is detected by gas chromatography in Example 1 and its stereoselectivity is calculated. The results are shown in Table 2. Three replicates are set for each mutant characterization. Through the activity detection of 19 mutants, the dominant mutant strain was screened as E. coli BL21 (DE3) / pET28a (+) -lbcr-N96V, which was stored in a -80 ° C refrigerator.

[0069] Table 2 Catalytic performance of beneficial mutants

[0070]

[0071] 3. Construction of LbCR site 145 mutant

[0072] The same method as step 2 was used, by designing NNK codons, and saturation mutation was performed on LbCR site 145 using primers in Table 3. The activity of 19 mutants was tested, and the results are shown in Table 4. The best mutant was screened and determined to be pET28a(+)-lbcr-E145A.

[0073] Table 3. Primer design for site-directed saturation mutagenesis of carbonyl reductase 145

[0074]

[0075] Note: In Table 3, NNK represents a degenerate codon, and MNN represents a degenerate codon paired with NNK.

[0076] Table 4. Catalytic performance of beneficial mutants

[0077]

[0078] Note: ND in Table 4 means not detected.

[0079] 4. Construction of LbCR site 202 mutant

[0080] The same method as step 2 was used, by designing NNK codons, and saturation mutation was performed on LbCR site 202 using primers in Table 5. The activity of 19 mutants was tested, and the results are shown in Table 6. The best mutant was screened and determined to be pET28a(+)-lbcr-A202L.

[0081] Table 5. Primer design for site-directed saturation mutagenesis of carbonyl reductase 202

[0082]

[0083] Note: In Table 5, NNK represents a degenerate codon, and MNN represents a degenerate codon paired with NNK.

[0084] Table 6. Catalytic performance of beneficial mutants

[0085]

[0086] Note: ND in Table 6 means not detected.

[0087] 5. Construction of LbCR site 206 mutant

[0088] The same method as step 2 was used, by designing NNK codons, and saturation mutation was performed on LbCR site 206 using primers in Table 7. The activity of 19 mutants was tested, and the results are shown in Table 8. The best mutant was screened and determined to be pET28a(+)-lbcr-M206A.

[0089] Table 7. Primer design for site-directed saturation mutagenesis of carbonyl reductase 206

[0090]

[0091] Note: In Table 5, NNK represents a degenerate codon, and MNN represents a degenerate codon paired with NNK.

[0092] Table 8. Catalytic performance of beneficial mutants

[0093]

[0094] Note: ND in Table 8 means not detected.

[0095] Example 3: Construction of combined mutants

[0096] The four single mutants obtained were combined for mutation to construct multiple mutants, as follows:

[0097] 1. Construction of the double mutant

[0098] Using the mutant LbCR-N96V plasmid constructed in Example 2 as a template, the E145A, A202L and M206A sites were subjected to site-directed mutagenesis using the primers in Table 9 to obtain the combined mutants LbCR-N96V / E145A, LbCR-N96V / A202L and LbCR-N96V / M206A, which were transformed into E. coli BL21 (DE3) to obtain strains E. coli BL21 (DE3) / pET28a (+) -lbcr-N96V / E145A, E. coli BL21 (DE3) / pET28a (+) -lbcr-N96V / A202L and E. coli BL21 (DE3) / pET28a (+) -lbcr-N96V / M206A.

[0099] Further, the mutant LbCR-E145A plasmid constructed in Example 2 was used as a template, and site-directed mutagenesis was performed using primers A202L and M206A in Table 9 to obtain mutants LbCR-E145A / A202L and LbCR-E145A / M206A, which were transformed into E. coli BL21 (DE3) to obtain strains BL21 (DE3) / pET28a (+) -lbcr-E145A / A202L and E. coli BL21 (DE3) / pET28a (+) -lbcr-E145A / M206A.

[0100] Finally, the mutant LbCR-A202L plasmid constructed in Example 2 was used as a template, and the M206A site was subjected to site-directed mutagenesis using the primers in Table 9 to obtain the mutant LbCR-A202L / M206A, which was transformed into E. coli BL21 (DE3) to obtain the strain E. coli BL21 (DE3) / pET28a (+) -lbcr-A202L / M206A.

[0101] 2. Construction of triple mutants

[0102] Using the above-mentioned double mutant LbCR-N96V / E145A plasmid as a template, the A202L and M206A sites were subjected to site-directed mutagenesis using the primers in Table 9 to obtain the triple mutants LbCR-N96V / E145A / A202L and LbCR-N96V / E145A / M206A, which were transformed into E. coli BL21 (DE3) to obtain mutant strains E. coli BL21 (DE3) / pET28a (+)-lbcr-N96V / E145A / A202L and E. coli BL21 (DE3) / pET28a (+)-lbcr-N96V / E145A / M206A.

[0103] Further, the double mutant LbCR-E145A / A202L plasmid was used as a template, and the M206A site was subjected to site-directed mutagenesis using the primers in Table 9 to obtain the triple mutant LbCR-E145A / A202L / M206A, which was transformed into E. coli BL21 (DE3) to obtain the mutant strain E. coli BL21 (DE3) / pET28a (+) -lbcr-E145A / A202L / M206A.

[0104] Finally, the double mutant LbCR-A202L / M206A plasmid was used as a template, and the primers in Table 9 were used to perform site-directed mutagenesis on the N96V site to obtain the triple mutant LbCR-N96V / A202L / M206A, which was transformed into E. coli BL21 (DE3) to obtain the mutant strain E. coli BL21 (DE3) / pET28a (+) -lbcr-N96V / A202L / M206A.

[0105] 3. Construction of quadruple mutants

[0106] Using the above mutant LbCR-N96V / E145A / A202L plasmid as a template, the M206A site was subjected to site-directed mutagenesis using the primers in Table 9 to obtain the quadruple mutant LbCR-N96V / E145A / A202L / M206A, which was transformed into E. coli BL21 (DE3) to obtain the mutant strain E. coli BL21 (DE3) / pET28a (+) -lbcr-N96V / E145A / A202L / M206A.

[0107] The activity of the combined mutants was tested using the method and conditions of Example 2. The results are shown in Table 10. The best combined mutant was determined to be a quadruple mutant, denoted as mutant LbCR M4 .

[0108] Table 9. Combined mutation primers

[0109]

[0110] Table 10. Catalytic performance of combined mutants

[0111]

[0112] Example 4: Inducible expression of mutants

[0113] The E. coli BL21 (DE3) / pET28a (+) -lbcr obtained in Example 3 was M4Inoculate into LB liquid medium containing kanamycin at a final concentration of 50 μg / mL, culture at 37°C for 10 h, inoculate into fresh LB liquid medium containing kanamycin at a final concentration of 50 μg / mL at a volume concentration of 1% (V / V), culture at 37°C and 180 rpm for 2 h, then add IPTG at a final concentration of 0.15 mM to the culture medium, culture at 20°C for 18 h, centrifuge at 4°C and 8000 rpm for 10 min, and wash the precipitate twice with 0.9% (w / V) saline to obtain LbCR M4 Wet bacterial cells. The cells obtained above contain the corresponding protein, which can be used to prepare pure protein enzyme solution and can also be used to catalyze the synthesis of (S)-TCPE.

[0114] Example 5: Purification of mutant enzyme protein

[0115] The wet cells prepared in Example 4 were resuspended in a pH 7.0, 100 mM potassium phosphate buffer at a dosage of 50 g / L, and ultrasonically disrupted for 15 min on an ice-water mixture. The ultrasonic disruption conditions were: power of 250 W, disruption for 1 s, and pause for 1 s. The disruption mixture was the crude enzyme solution. The crude enzyme solution was centrifuged at 8000 rpm and 4°C for 10 min, the precipitate was discarded, and the supernatant was collected. The supernatant was purified by the same method as in Example 1 to obtain the mutant LbCR. M4 The results of SDS-polyacrylamide gel electrophoresis of the enzyme protein are shown in Figure 4 .

[0116] Example 6: WT-LbCR and its mutant LbCR M4 Determination of kinetic parameters of enzyme-catalyzed reactions

[0117] WT-LbCR and its mutant LbCR M4 The purified enzyme was used as a catalyst to determine the kinetic parameters. Specifically, 5 mM NADPH and an appropriate amount of purified enzyme (based on protein content, WT-LbCR content was 0.5 mg, LbCR content was 0.5 mg) were added to 200 μL of potassium phosphate (100 mM, pH 7.0) buffer. M4 The content is 0.02 mg), the concentration range of substrate TCAP is 0-10 mM (0 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM), the substrate is first dissolved in DMSO and then added, and the final volume concentration of DMSO is 1%. The reaction was carried out at 30 ° C and 1000 rpm for 1-10 min (the reaction time of the mutant is 3 min, and the reaction time of WT-LbCR is 10 min). The conversion rate of the substrate TCAP or the yield of the product (S)-TCPE was determined by the method of Example 1, and the Michaelis-Menten equation in Origin 2024b software was used for fitting, and the calculation results were obtained based on WT-LbCR and its mutant LbCR.M4 Michaelis constant K for substrate M , turnover number k cat And the catalytic efficiency k cat / K M The results are shown in Table 11. Compared with the wild type, the turnover number and catalytic efficiency of the mutants were significantly increased. M4 The catalytic efficiency was increased 15-fold compared with that of wild-type WT-LbCR.

[0118] Table 11 Wild-type WT-LbCR and mutant LbCR M4 Kinetic parameters

[0119]

[0120] Example 7: WT-LbCR and its mutant LbCR M4 The optimal reaction pH, temperature, and isopropanol concentration

[0121] 1. Optimum reaction temperature

[0122] In 1 mL of pH 7.0, 100 mM potassium phosphate buffer, 50 g / L of WT-LbCR and LbCR prepared by the methods of Example 1 and Example 4 were added. M4 Wet bacterial cells, 20 g / L substrate TCAP, 30% (V / V) isopropanol, at 20-60°C (20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C), 1000 rpm reaction conditions (mutant LbCR M4 The reaction time was 10 min, and the reaction time for WT-LbCR was 30 min). The enzyme activity at different temperatures was detected according to the method in Example 1, and the WT-LbCR and its mutant LbCR were optimized. M4 The catalytic temperature is shown in Figure 5 , Figure 8 The results showed that within the investigated temperature range (20-60°C), the optimum catalytic temperature of WT-LbCR was 30°C, while that of mutant LbCR M4 The optimum catalytic temperature is 45°C.

[0123] 2. Optimal reaction pH

[0124] The optimal reaction pH of mutants was screened using pH 4.0-6.0 sodium acetate buffer, pH 6.0-8.0 potassium phosphate buffer, and pH 8.0-10.0 Tris-hydrochloric acid buffer, respectively.

[0125] Reaction system: 50 g / L of WT-LbCR and LbCR prepared by the methods of Example 1 and Example 4 were added to 1 mL of 100 mM buffer (pH 4.0-6.0 sodium acetate buffer; pH 6.0-8.0 potassium phosphate buffer; pH 8.0-10.0 Tris-HCl buffer) at different pH values ​​(pH 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0). M4 Wet bacterial cells, 20 g / L substrate TCAP, 30% (V / V) isopropanol, reacted at 35°C, 1000 rpm (mutant LbCR M4 The reaction time was 10 min for WT-LbCR and 30 min for WT-LbCR. The enzyme activity at different pH values ​​was detected by the method in Example 1, and the WT-LbCR and its mutant LbCR were optimized. M4 The catalytic reaction pH value is shown in Figure 6 , Fig. 9 In the investigated pH range (4.0-10.0), the optimal catalytic pH value of WT-LbCR was 7.0, while that of mutant LbCR M4 The optimum catalytic pH is 8.0.

[0126] 3. Optimal isopropanol concentration

[0127] In 10 mL of pH 7.0, 100 mM potassium phosphate buffer, 50 g / L of WT-LbCR and LbCR prepared by the methods of Example 1 and Example 4 were added. M4 Wet bacterial cells, 20 g / L substrate TCAP, different volume ratios (10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%) of isopropanol, reacted at 35°C, 1000 rpm (mutant LbCR M4 The reaction time was 5 min, and the reaction time for WT-LbCR was 20 min). The enzyme activity under different isopropanol concentrations was detected according to the method in Example 1, and the WT-LbCR and its mutant LbCR were optimized. M4 The concentration of co-substrate isopropanol is shown in Figure 7 , Fig.10 When the concentration of co-substrate isopropanol (10% to 80%, V / V) was investigated, the optimal isopropanol concentration for WT-LbCR was 20% (V / V), while the mutant LbCR M4 The optimal isopropanol concentration for the reaction is 40% (V / V).

[0128] Example 8: Synthesis of (S)-TCPE by asymmetric reduction of TCAP by WT-LbCR

[0129] First, the wet cells of WT-LbCR were resuspended with a pH 7.0, 100 mM potassium phosphate buffer. The amount of WT-LbCR cells added to the reaction system was 10 g DCW / L on a dry weight basis, the amount of substrate TCAP added was 200 g / L, 40% (V / V) isopropanol was added, and a reaction system of 30 mL was formed with a pH 7.0, 100 mM potassium phosphate buffer as the reaction medium. The reaction was carried out at 30° C., pH 7.0, and 1000 rpm for 0.5-8 h. Samples were taken every 1 h, and the concentration of (S)-TCPE was detected using the method of Example 1. The results are as follows: Fig.11 As shown, WT-LbCR could produce a maximum of 58 g / L TCAP and could not achieve complete conversion.

[0130] Example 9: Mutant LbCR M4 Synthesis of (S)-TCPE by Asymmetric Reduction of TCAP

[0131] First, the mutant LbCR M4 The wet cells were resuspended in 100 mM potassium phosphate buffer at pH 7.0. M4 The amount of bacterial cells added was 10 g DCW / L in terms of dry weight, the amount of substrate TCAP added was 200 g / L, 40% (V / V) isopropanol was added, and a 30 mL reaction system was formed with a pH 8.0, 100 mM Tris-hydrochloric acid buffer as the reaction medium. The reaction was carried out at 45°C, pH 8.0, and 1000 rpm for 0.5-8 h. Samples were taken every 1 h, and the concentration of (S)-TCPE was detected by the method of Example 1 to calculate the yield and space-time yield. Under the same conditions, the substrate addition concentration was changed to 300 g / L and 400 g / L. The results are as follows: Fig.12 shown.

[0132] The space-time yield calculation formula is: STY = m / (t×V)×100%, where m is the mass of the product generated (g); t is the reaction time (d); and V is the volume of the reaction solution (L). When the substrate concentration is 200 g / L, LbCR M4 It can catalyze the production of 193 g / L of product, and the substrate is completely reacted. Under the same conditions, when the substrate concentration is as high as 400 g / L, it can be completely reacted within 7 hours and produce 378 g / L of product. The results are shown in Table 12, and the space-time yield is as high as 1288.9 g / (L·d).

[0133] Table 12 LbCR M4 The yields of catalysis at different substrate concentrations

[0134]

[0135] Example 10: WT-LbCR and its mutant LbCR M4Catalytic substrate spectrum

[0136] In 1 mL of pH 7.0, 100 mM potassium phosphate buffer, add 10 g DCW / L LbCR M4 and WT-LbCR bacteria, 30% (V / V) isopropanol, a final concentration of 10mM substrate (1a, methyl acetoacetate; 2a, ethyl acetoacetate; 3a, tert-butyl acetoacetate; 4a, methyl 3-oxopentanoate; 5a, ethyl pyruvate; 6a, ethyl benzoylformate; 7a, n-valeraldehyde; 8a, benzaldehyde; 9a, acetophenone; 10a, 2-chloroacetophenone; 11a, o-fluoroacetophenone; 12a, benzylacetone; 13a, 3-chloroacetophenone; 14a, 4-fluoroacetophenone; 15a, 3,5-bis(trifluoromethyl)acetophenone; 16a, TCAP), the substrate was first dissolved in 1% (V / V) DMSO and then added to the reaction system, and reacted at 35°C and 1000rpm for 30min. The substrate conversion rate and stereoselectivity were tested using the method and conditions of Example 1, and each substrate was set up in parallel 3 times. The results are as follows Fig.13 As shown, mutant LbCR M4 The catalytic activity for most substrates in the substrate spectrum was significantly higher than that of WT-LbCR.

Claims

1. A carbonyl reductase mutant, characterized in that: The carbonyl reductase mutant is obtained by performing single-site or multi-site mutation on the 96th, 145th, 202nd or 206th position of the amino acid sequence of carbonyl reductase LbCR from Levilactobacillus brevis shown in SEQ ID No.

2.

2. The carbonyl reductase mutant according to claim 1, characterized in that The carbonyl reductase mutant is a mutant in which the amino acid sequence shown in SEQ ID No. 2 is mutated into one of the following: (1) asparagine at position 96 is mutated into valine; (2) glutamic acid at position 145 is mutated into alanine; (3) alanine at position 202 is mutated into leucine; (4) methionine at position 206 is mutated into alanine; (5) a mutant in which the single-site mutations in (1) to (4) are combined in pairs; (6) a mutant in which any three of the single-site mutations in (1) to (4) are combined; (7) asparagine at position 96 is mutated into valine, glutamic acid at position 145 is mutated into alanine, alanine at position 202 is mutated into leucine, and methionine at position 206 is mutated into alanine.

3. A recombinant genetically engineered bacterium containing the gene encoding the carbonyl reductase mutant according to claim 1.

4. Use of the carbonyl reductase mutant according to claim 1 in synthesizing chiral alcohol compounds by asymmetric reduction of carbonyl compounds.

5. The use according to claim 4, characterized in that The application method comprises the following steps: using wet bacteria obtained by induction culture of recombinant genetic engineering bacteria of a carbonyl reductase mutant as a catalyst, using a carbonyl compound as a substrate, using isopropanol as a co-substrate and a cosolvent, and using a potassium phosphate buffer with a pH of 4.0 to 10.0 as a reaction medium to form a reaction system, reacting at 20 to 60° C. and 1000 rpm, and after the reaction is completed, separating and purifying the reaction solution, and extracting the reaction solution with ethyl acetate to obtain a chiral alcohol compound.

6. The use according to claim 5, characterized in that The carbonyl compound is one of the following: 2-chloro-1-(2,4-dichlorophenyl)ethanone, methyl acetoacetate, ethyl acetoacetate, tert-butyl acetoacetate, methyl 3-oxopentanoate, ethyl pyruvate, ethyl benzoylformate, n-valeraldehyde, benzaldehyde, acetophenone, 2-chloroacetophenone, o-fluoroacetophenone, benzylacetone, 3-chloroacetophenone, 4-fluoroacetophenone, and 3,5-bis(trifluoromethyl)acetophenone.

7. The use according to claim 5, characterized in that In the reaction system, the substrate is added to a final concentration of 1-600 g / L; the isopropyl alcohol is added to a concentration of 10-80% by volume; the catalyst is used in an amount of 10-50 g / L based on the wet bacterial cell weight and 1-30 g DCW / L based on the wet bacterial cell dry weight.

8. The use according to claim 6, characterized in that When the carbonyl compound is 2-chloro-1-(2,4-dichlorophenyl)ethanone, the conditions for the carbonyl reductase mutant to asymmetric reduce the carbonyl compound to prepare (1S)-2-chloro-1-(2,4-dichlorophenyl)ethanol are: temperature 45°C and pH 8.

0.

9. The use according to claim 5, characterized in that The wet bacteria are prepared as follows: the engineered bacteria containing the carbonyl reductase mutant gene are inoculated into LB liquid culture medium containing kanamycin at a final concentration of 50 μg / mL, and cultured at 37° C. and 180-200 rpm for 10 hours to obtain seed liquid; The seed solution was inoculated into sterilized LB liquid medium containing a final concentration of 50 μg / mL kanamycin at a volume concentration of 1%, and cultured at 37°C and 180-200 rpm until the OD 600 =0.6-0.8, add isopropyl thiogalactoside with a final concentration of 0.15 mM to the culture medium, culture at 20°C for 18 h, and centrifuge at 4°C and 8000 rpm for 10 min to obtain wet cells of the carbonyl reductase mutant.

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