A carbonyl reductase mutant and its application in synthesizing chiral alcohol compounds

Abstract

The present invention discloses a carbonyl reductase, a mutant, and its use in synthesizing chiral alcohol compounds. The carbonyl reductase mutant is obtained by subjecting the amino acid sequence of carbonyl reductase LbCR from Lactobacillus 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 activity and stability of the mutant carbonyl reductase LbCR of the present invention are significantly improved, and the mutant has a wider substrate spectrum. M4 The enzyme activity was increased by 86.3 times compared with the wild type, the substrate conversion rate was greater than 99%, and the product ee p The value remained above 99.5%, the space-time yield reached 1288.9 g / L / d, and the half-life at 40°C was 101.4 h, an increase of 83.5 times. The LbCR mutant has promising industrial applications.

Description

(1) Technical field

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

[0002] Luliconazole, an analog of lanoconazole, has strong antibacterial activity among many antifungal compounds and is safe and effective in treating tinea. It has great potential in the antifungal treatment of the skin. The global market demand for antifungal treatments is huge, and improving the production technology level and production capacity of antifungal active compounds is crucial. (S)-TCPE is a key intermediate in the preparation of luliconazole. However, the chemical synthesis of (S)-TCPE is a lengthy process route and requires the use of chiral auxiliaries or expensive metal reagents for separation to obtain a single enantiomer with high optical purity. In addition, the process is highly polluting and the product yield is low. Biocatalysis is a green and sustainable production method. The development of (S)-TCPE biocatalytic 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 as dimers or tetramers. In the process of catalyzing the reduction of carbonyl compounds, carbonyl reductases require NAD(P)H as a hydrogen or electron carrier. Under the action of the reductase, the carbonyl group 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 the molecular modification of carbonyl reductase using protein engineering techniques to obtain enzyme preparations with industrial properties. This study addresses the issues of low activity and poor stability of WT-LbCR by successfully obtaining a carbonyl reductase mutant with enhanced activity through protein engineering. Furthermore, the present invention optimizes the process parameters for the catalytic synthesis of (S)-TCPE, effectively improving the production efficiency of its enzymatic synthesis of (S)-TCPE. (3) Summary of the invention

[0005] The present invention aims to provide a carbonyl reductase LbCR mutant, recombinant bacteria and their 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 enhanced activity was successfully obtained. Furthermore, 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 significant improvement in activity compared to WT-LbCR, and also has a significant improvement in 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 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 in the present invention is:

[0007] In a first aspect, the present invention provides a carbonyl reductase (LbCR) mutant, obtained by subjecting the carbonyl reductase (LbCR) mutant to single-site or multi-site mutations at positions 96, 145, 202, or 206 of the amino acid sequence of carbonyl reductase (LbCR) from Levilactobacillus brevis as shown in SEQ ID No. 2. The nucleotide sequence of the gene encoding the carbonyl reductase (LbCR) 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 shown in 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 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, a recombinant expression vector and a recombinant genetic engineering bacterium. The recombinant expression vector is preferably pET28a(+), and the recombinant genetic engineering bacterium is preferably a host bacterium E. coli BL21(DE3).

[0010] In a second aspect, the present invention also relates to the use of the carbonyl reductase mutant in the asymmetric reduction of carbonyl compounds to prepare chiral alcohol compounds. The method of the application is as follows: wet bacteria obtained by induction culture of recombinant genetically engineered bacteria of the carbonyl reductase mutant are used as catalysts, carbonyl compounds are used as substrates, isopropanol is used as a co-substrate and cosolvent, a pH 4.0-10.0 buffer (pH 7.0, 100mM potassium phosphate buffer) is used as a reaction medium to form a reaction system, the reaction is carried out at 20-60°C and 1000rpm, 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, 3,5-bis(trifluoromethyl)acetophenone.

[0012] Furthermore, 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 the wet cells and 1 to 30 g DCW / L (preferably 10 g DCW / L) based on the dry weight of the 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] Furthermore, the wet bacteria were prepared as follows: the engineered bacteria containing the carbonyl reductase LbCR mutant gene were inoculated into an LB liquid 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 a seed solution; the seed solution was inoculated into a sterilized LB liquid 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 isopropylthiogalactoside (IPTG) to the culture medium at a final concentration of 0.15 mM, 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 LbCR mutant.

[0017] Furthermore, the LbCR mutant purified enzyme was prepared as follows: (1) Crude enzyme solution: wet bacteria were resuspended in a pH 7.0, 100 mM PBS buffer at a concentration 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 affinity chromatography filler (2–3 mL) was layered onto a prepacked column of protein purification filler and the column was equilibrated with 10–15 mL of 20 mM phosphate buffer (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 One column volume of the supernatant was then eluted by gravity with 20 mM sodium phosphate buffer (pH 7.0) containing 0.5 M NaCl and 400 mM imidazole; all the eluates were collected and transferred to an ultrafiltration tube with a molecular weight cutoff of 30 kDa and filtered through a 20 mM, pH 7.0 filter. 7.0 potassium phosphate was used as the replacement buffer to remove imidazole, and the retained protein was collected to obtain the purified enzyme of the 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 and the stop codon is TAA, encoding 252 amino acids.

[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 heat-shocked into E. coli BL21(DE3) competent cells, and the resulting strain was inoculated, transferred, induced, and the cells were recovered. The resuspended bacteria were used to catalyze the reduction of TCAP and prepare (S)-TCPE. The resulting mutant recombinant plasmid was heat-shocked into E. coli BL21(DE3) competent cells, and the resulting strain was inoculated, transferred, induced, and the cells were collected from the fermentation broth by solid-liquid separation.

[0020] The culture medium for inoculation, transfer, induction culture, and bacterial cell recovery of the carbonyl reductase mutant of the present invention can be any culture medium known in the art that can grow the bacterial cells of the present invention, preferably LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, dissolved in water. The culture method and conditions are not particularly limited and can be appropriately selected based on factors such as the host type and culture method, according to common knowledge in the art.

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

[0022] The invention provides a new 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 to the wild-type WT-LbCR. M4 The mutant has an 86.3-fold increase in specific activity compared to the wild-type WT-LbCR. The maximum feed amount can reach 400 g / L TCAP. The product concentration gradually increases with time. The reaction can be completed within 7 hours. The substrate conversion rate is greater than 99%, and the product ee p The value was always maintained 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, indicating a low substrate conversion rate.

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

[0026] Compared with the wild-type WT-LbCR, the new mutants developed in 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 wild-type carbonyl reductase WT-LbCR. M4 It has more prospects for industrial application.

[0027] (3) The carbonyl reductase LbCR mutant has a wider 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 accompanying 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 the 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] Figure 9 Mutant LbCR M4 Optimum reaction pH optimization curve.

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

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

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

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

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

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

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

[0045] (1) Wild-type carbonyl reductase genetically engineered bacteria: The carbonyl reductase gene sequence from Levilactobacillus brevis in GenBank (GenBank No. MCF7523072.1), namely 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 LB liquid 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 medium containing a final concentration of 50 μg / mL kanamycin at an inoculum concentration of 1% by volume, and cultured at 37 ° C and 180 rpm for 2 hours. Then, isopropyl β-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, the culture was centrifuged at 4 ° C and 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 a concentration 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 as follows: 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 then purified using a nickel affinity column (1.6 × 10 cm, Bio-Rad, USA). The enzyme purification procedure was as follows:

[0053] ① Nickel-agarose affinity chromatography filler (2-3 mL) was layered onto a prepacked column of protein purification filler, and the column was equilibrated with 10-15 mL of 20 mM phosphate buffer (pH 7.0). ② The sample was loaded at a flow rate of 1.0 mL / min and eluted with sodium phosphate buffer (20 mM, pH 7.0) containing 0.5 M NaCl and 20 mM imidazole to remove unbound impurities. ③ The column was then gravity eluted with sodium phosphate buffer (400 mM, pH 7.0) containing 0.5 M NaCl and 400 mM imidazole for one column volume. The entire eluate was collected and transferred to an ultrafiltration tube with a molecular weight cutoff of 30 kDa. The imidazole was removed using 20 mM potassium phosphate buffer, pH 7.0, as the exchange buffer. The retained protein was collected to obtain the purified LbCR enzyme, which was designated as WT-LbCR.

[0054] (4) Enzyme activity determination

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

[0056] Enzyme activity assay conditions: 5 mM NADPH, appropriate amount of purified enzyme (enzyme protein content 70 mg / L), 5 g / L substrate TCAP, 1% DMSO, the substrate was first dissolved in DMSO and then added, pH 7.0, 100 mM potassium phosphate buffer was added to a final volume of 500 μL, the reaction was carried out at 35 ° C, 1000 rpm for 5 minutes, the reaction solution was extracted with an equal volume of ethyl acetate, the extract was collected and microfiltered with a 0.22 μm filter membrane, and the filtrate was detected by gas chromatography 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 thus 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, and is shown in Figure 3 .

[0057] Substrate conversion, product ee values, and yields were determined using an Agilent (8860) high-performance gas chromatograph (CYCLOSIL-B, 30 m × 0.25 mm, 0.25 μm). The injector and detector were set at 250°C and 250°C, respectively. The column oven was set at 170°C for 22 min, the split ratio was 15:1, and the injection volume was 2 μL. Other instrument settings were the default.

[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 distance of 1.5 to the substrate being preferred. Amino acid residues within the range 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, saturation mutation was performed on the key site A202 located in the substrate binding pocket.

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

[0062] (1) Mutation at position 96: By designing an NNK codon, a saturation mutation was performed at position 96 of the LbCR. The plasmid pET28a(+)-lbcr of the wild-type carbonyl reductase gene engineering bacteria E. coli BL21(DE3) / pET28a(+)-lbcr of Example 1 was extracted and PCR amplified using the primers in Table 1. The PCR product was enzymatically eliminated from the original template and cleaned up, and then transformed into E. coli BL21(DE3) competent cells. 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, cultured at 37°C and 200 rpm for 10 hours, and the culture solution was centrifuged at 8000 rpm for 10 minutes. The precipitate was washed twice with 0.9% (w / v) saline, and the wet cells 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] The PCR reaction system (25 μL) consisted of 0.5 μL forward primer (100 μM), 0.5 μL reverse primer (100 μM), 12.5 μL 2× Phanta buffer, 0.5 μL dNTP mix (10 mM each), 0.5 μL plasmid template, 0.5 μL DNA polymerase, and 10 μL ultrapure water. The PCR program was set according to the Phanta Super-Fidelity DNA Polymerase instruction manual as follows: initial denaturation at 95°C for 5 min, followed by 30 cycles (denaturation at 95°C for 15 s, annealing at 55–65°C for 15 s, and extension at 72°C for 10 s), a final extension at 72°C for 10 min, and a 16°C incubation period.

[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, and a reaction medium of pH 7.0, 100 mM potassium phosphate buffer, to form a 1 mL reaction system. After reacting at 35°C and 1000 rpm for 10 min, the reaction solution was extracted with 1 mL of ethyl acetate. The extract was detected by gas chromatography in Example 1 and its stereoselectivity was calculated. The results are shown in Table 2. Three replicates were set for each mutant characterization. Through 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] Similar to step 2, saturation mutagenesis was performed at LbCR site 145 using the designed NNK codon and the primers listed in Table 3. The activity of 19 mutants was tested, and the results are shown in Table 4. The optimal mutant was identified as 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] Similar to step 2, by designing the NNK codon, saturation mutagenesis was performed on LbCR site 202 using the primers in Table 5. The activity of 19 mutants was tested, and the results are shown in Table 6. The optimal mutant was screened and identified as 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] Similar to step 2, by designing the NNK codon, saturation mutagenesis was performed on LbCR site 206 using the primers in Table 7. The activity of 19 mutants was tested, and the results are shown in Table 8. The optimal 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 and mutated to construct multiple mutants, as follows:

[0097] 1. Construction of double mutants

[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] Furthermore, 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 the mutant strains E. coli BL21 (DE3) / pET28a (+) -lbcr-N96V / E145A / A202L and E. coli BL21 (DE3) / pET28a (+) -lbcr-N96V / E145A / M206A.

[0103] Furthermore, the double mutant LbCR-E145A / A202L plasmid was used as a template, and the primers in Table 9 were used to perform site-directed mutagenesis on the M206A site to obtain the triple mutant LbCR-E145A / A202L / M206A, which was then 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 N96V site was subjected to site-directed mutagenesis using the primers in Table 9 to obtain the triple mutant LbCR-N96V / A202L / M206A, which was then 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 M4The cells were inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37°C for 10 h. The cells were then inoculated into fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin at a volume concentration of 1% (V / V) and cultured at 37°C and 180 rpm for 2 h. IPTG was then added to the culture medium at a final concentration of 0.15 mM. The cells were cultured at 20°C for 18 h, and then centrifuged at 4°C and 8000 rpm for 10 min. The precipitate was washed twice with 0.9% (w / V) saline to obtain LbCR. M4 Wet bacterial cells. The cells obtained above contain the corresponding protein and can be used to prepare pure protein enzyme solution or 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 in an ice-water mixture for 15 min. The ultrasonic disruption conditions were: 250 W power, 1 second disruption, and 1 second pause. 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 using 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 kinetic parameters were determined by using the purified enzyme as a catalyst. 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 concentration of the substrate TCAP was 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 was first dissolved in DMSO and then added, with a final DMSO concentration of 1%. The reaction was carried out at 30°C and 1000 rpm for 1-10 min (the reaction time for the mutant was 3 min, and the reaction time for WT-LbCR was 10 min). The conversion rate of the substrate TCAP or the yield of the product (S)-TCPE was determined using the method of Example 1. The Michaelis-Menten equation in Origin 2024b software was used for fitting, and the conversion rate of the WT-LbCR and its mutant LbCR was calculated.M4 Michaelis constant K for substrate M , turnover number k cat and 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. Among them, the quadruple mutant LbCR M4 The catalytic efficiency was increased 15-fold compared with the 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 the optimal catalytic temperature of WT-LbCR was 30℃ within the investigated temperature range (20~60℃), while that of mutant LbCR was M4 The optimum catalytic temperature is 45℃.

[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, at 35 ° C, 1000 rpm conditions for the reaction (mutant LbCR M4 The reaction time was 10 min, and the reaction time for WT-LbCR was 30 min). The enzyme activity at different pH was detected using 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 、 Figure 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 was M4 The optimum catalytic pH value is 8.0.

[0126] 3. Optimal isopropyl alcohol 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 were reacted at 35 ° C and 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 、 Figure 10 When the concentration of co-substrate isopropanol was investigated (10% to 80%, V / V), 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: Asymmetric reduction of TCAP by WT-LbCR to synthesize (S)-TCPE

[0129] First, the wet cells of WT-LbCR were resuspended in a 100 mM potassium phosphate buffer at pH 7.0. The amount of WT-LbCR cells added to the reaction system was 10 g DCW / L on a dry weight basis, the amount of TCAP substrate added was 200 g / L, 40% (V / V) isopropanol was added, and a 30 mL reaction system was formed with a 100 mM potassium phosphate buffer at pH 7.0 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 shown in FIG. Figure 11 As shown, WT-LbCR could produce a maximum of 58 g / L of 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. LbCR M4 The amount of bacterial cells added 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 30 mL reaction system was prepared using a pH 8.0, 100 mM Tris-HCl 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 determined using the method of Example 1 to calculate the yield and space-time yield. Under the same conditions, the substrate concentration was changed to 300 g / L and 400 g / L. The results are as follows: Figure 12 shown.

[0132] The space-time yield is calculated as follows: 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 The reaction catalyzed the formation of 193 g / L of product, demonstrating complete substrate reaction. Under the same conditions, when the substrate concentration reached 400 g / L, the reaction was complete within 7 hours, yielding 378 g / L of product. The results are shown in Table 12, yielding a space-time yield of 1288.9 g / (L·d).

[0133] Table 12 LbCR M4 Catalytic yields 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, 10 g DCW / L LbCR was added. M4 and WT-LbCR bacteria, 30% (V / V) isopropanol, a final concentration of 10 mM 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-chloropropiophenone; 14a, 4-fluoroacetophenone; 15a, 3,5-bis(trifluoromethyl)acetophenone; 16a, TCAP), the substrate was first dissolved in 1% (V / V) DMSO and added to the reaction system, and the reaction was carried out at 35°C and 1000 rpm for 30 min. 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 Figure 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 mutating the amino acid sequence shown in SEQ ID No. 2 to the following sequence: asparagine at position 96 is mutated to valine, glutamic acid at position 145 is mutated to alanine, alanine at position 202 is mutated to leucine, and methionine at position 206 is mutated to alanine.

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

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

4. The use according to claim 3, characterized in that The application method comprises the following steps: using wet bacteria obtained by induction culture of recombinant genetically engineered bacteria of a carbonyl reductase mutant as a catalyst, using a carbonyl compound as a substrate, using isopropanol as a cosubstrate 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, carrying out the reaction at 20 to 60° C. and 1000 rpm, and separating and purifying the reaction solution after the reaction is completed, and extracting the reaction solution with ethyl acetate to obtain a chiral alcohol compound.

5. The use according to claim 4, 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.

6. The use according to claim 4, characterized in that In the reaction system, the substrate is added to a final concentration of 1-600 g / L; the volumetric concentration of isopropyl alcohol is 10-80%; the amount of catalyst used is 10-50 g / L based on the wet cell weight and 1-30 g DCW / L based on the dry weight of the wet cells.

7. The use according to claim 5, characterized in that When the carbonyl compound is 2-chloro-1-(2,4-dichlorophenyl)ethanone, the carbonyl reductase mutant asymmetric reduces the carbonyl compound to prepare (1 S The conditions for the reaction of )-2-chloro-1-(2,4-dichlorophenyl)ethanol are: temperature 45°C and pH 8.

0.

8. The use according to claim 5, characterized in that The wet bacteria were prepared as follows: the engineered bacteria containing the carbonyl reductase mutant gene were inoculated into LB liquid culture medium containing kanamycin at a final concentration of 50 μg / mL, and the mixture was incubated at 37 o C. Cultivate at 180-200 rpm for 10 h to obtain seed solution; The seed solution was inoculated into sterilized LB liquid medium containing kanamycin at a final concentration of 50 μg / mL at a volume concentration of 1%. o C. Cultivate at 180-200 rpm until OD 600 = 0.6~0.8, add isopropylthiogalactoside to the culture medium at a final concentration of 0.15 mM, 20 o After culturing for 18 h at 4 o C. Centrifuge at 8000 rpm for 10 min to obtain wet cells of the carbonyl reductase mutant.

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