Carbonyl reductase bs cr mutant, engineering bacteria and application of synthetic crizotinib chiral intermediate

Abstract

The application discloses a carbonyl reductase BsCR mutant, an engineering bacterium and application of the carbonyl reductase BsCR mutant in synthesis of a chiral intermediate of crizotinib. M2 The specific activity of the mutant is 87.7 times that of the wild-type BsCR, and the highest substrate feeding amount can reach 100 g / L 2,6-dichloro-3-fluoroacetophenone (CFA); the product concentration gradually increases with the prolongation of the reaction time; most of the substrate can be converted (the substrate conversion rate is 92.06%) within 6 h; the e.e. value of the product is always kept above 99.5%; and the space-time yield is 368.24 g / L / d.

Description

(I)TECHNICAL FIELD

[0001] The application belongs to the field of biopharmaceuticals, and relates to a carbonyl reductase BsCR mutant, an engineering bacterium and application of the carbonyl reductase BsCR mutant in catalytic synthesis of a chiral crizotinib intermediate. (II)BACKGROUND

[0002] Crizotinib is the first generation of anaplastic lymphoma kinase (ALK) inhibitor, which can effectively improve the survival time of patients with ALK-positive non-small cell lung cancer (NSCLC). (S)-2,6-dichloro-3-fluorophenethyl alcohol ((S)-CFL) is a key chiral intermediate for synthesizing crizotinib, and the synthesis of (S)-CFL is usually achieved by asymmetric reduction of 2,6-dichloro-3-fluorophenylacetone (CFA) by using a chemical method. However, the chemical asymmetric reduction process usually has harsh reaction conditions, uses a metal catalyst with high cost, and the product has a risk of metal contamination. Biocatalysis has the advantages of mild reaction conditions, high catalytic efficiency and good stereoselectivity, and is more and more applied to the pharmaceutical industry. The development of the technology for preparing (S)-CFL by biologically asymmetric reduction of CFA conforms to the development direction of green chemistry.

[0003] Carbonyl reductase (CR) belongs to the NAD(P)H-dependent oxidoreductase superfamily, and can asymmetrically reduce ketones, aldehydes and keto acids (esters) to generate alcohol compounds. Wild-type (WT) carbonyl reductases in nature often exhibit low activity, low selectivity and low stability for artificially synthesized unnatural compounds, and it is difficult to meet the technical requirements of industrial application. Therefore, the structure of the natural enzyme needs to be modified by using enzyme engineering technology to strengthen its industrial properties so as to be applied to large-scale production.

[0004] The application obtains a carbonyl reductase mutant with industrial properties by modifying the carbonyl reductase, and constructs a (S)-CFL biomanufacturing technology with high space-time yield. (III)SUMMARY

[0005] The application aims to provide a carbonyl reductase BsCR mutant derived from B.subtilis, a recombinant plasmid, a recombinant genetically engineered bacterium and application of the carbonyl reductase BsCR mutant in asymmetric reduction of CFA to synthesize a key chiral intermediate (S)-CFL of crizotinib. By enzyme engineering modification, a carbonyl reductase beneficial mutant with significantly improved activity is obtained. Further, a super mutant with high activity and high stability is obtained by combined mutation, wherein the mutant BsCR-I178L / T179L (BsCR M2) is significantly more active than WT-BsCR. The present invention effectively solves the problems of lack of robust S-selective CFA reduction enzyme elements, low activity of natural BsCR, and poor stability. In addition, the present invention further optimizes the process parameters of the biocatalytic reaction and constructs the preferred BsCR M2 The process of synthesizing (S)-CFL by catalytic enantioselective reduction of CFA effectively improves the space-time yield of (S)-CFL biomanufacturing.

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

[0007] In a first aspect, the present invention provides a carbonyl reductase (BsCR) mutant, obtained by subjecting the BsCR mutant to single-site mutation or multi-site combined mutation at position 130, position 178, position 179, or position 182 of the amino acid sequence of the BsCR carbonyl reductase from B. subtilis as shown in SEQ ID No. 2. The nucleotide sequence of the gene encoding the BsCR carbonyl reductase of the present invention is shown in SEQ ID No. 1.

[0008] Furthermore, it is preferred that the carbonyl reductase BsCR mutant is a mutant in which the amino acid sequence shown in SEQ ID No. 2 is mutated into one of the following: (1) glutamine at position 130 is mutated into leucine, Q130L; (2) isoleucine at position 178 is mutated into leucine, I178L; (3) threonine at position 179 is mutated into leucine, T179L; (4) lysine at position 182 is mutated into glutamic acid, K182E; (5) a double mutation obtained by combining any two of the single point mutations in (1) to (4); (6) a triple mutation obtained by combining any three of the single point mutations in (1) to (4).

[0009] Furthermore, more preferably, the carbonyl reductase BsCR mutant is one in which the amino acid sequence shown in SEQ ID No. 2 is mutated to one of the following: (1) the threonine at position 179 is mutated to leucine, T179L; (2) the isoleucine at position 178 is mutated to leucine, and the threonine at position 179 is mutated to leucine (I178L / T179L).

[0010] The present invention also relates to a gene encoding the carbonyl reductase BsCR mutant, a recombinant expression vector, and a recombinant genetically engineered bacterium. The recombinant expression vector is preferably pET28a(+), and the recombinant genetically engineered bacterium is preferably a host bacterium, E. coli BL21(DE3).

[0011] In a second aspect, the present invention also relates to the use of the carbonyl reductase BsCR mutant in the asymmetric reduction of CFA to prepare the key chiral intermediate (S)-CFL of crizotinib. The application method is as follows: the wet bacteria obtained by induction culture of the carbonyl reductase BsCR mutant strain are used as a catalyst, CFA is used as a substrate, glucose is used as a co-substrate, and a pH 4.0-9.0 buffer (preferably pH 6.0, 100mM sodium phosphate buffer (PB)) is used as a reaction medium to form a reaction system, and the reaction is carried out at 30-80°C (preferably 60°C) and 800-1000rpm. After the reaction is completed, the reaction solution is separated and purified, and the reaction solution is extracted with ethyl acetate to obtain the (S)-CFL compound.

[0012] Furthermore, the prochiral CFA structure is shown below:

[0013]

[0014] Furthermore, in the reaction system, the substrate is added to a final concentration of 1 to 100 g / L; the amount of glucose added is 43.5 to 217.4 g / L, and the molar ratio of glucose to substrate is 1 to 5:1 (preferably 3:1); the amount of catalyst used is 10 to 50 g / L based on the weight of the wet cells and 2 to 10 g DCW / L (preferably 10 g DCW / L) based on the dry weight of the wet cells.

[0015] Furthermore, dimethyl sulfoxide is added as a cosolvent in the reaction system, and the volume concentration of dimethyl sulfoxide is 10-80%, preferably 50%.

[0016] Furthermore, the wet cell was prepared as follows: the genetically engineered bacteria carrying the carbonyl reductase BsCR mutant gene were inoculated into an LB liquid medium containing a final concentration of 50 μg / mL kanamycin, and cultured at 37°C for 10 h 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 volume concentration of 1% (V / V), and cultured at 37°C and 180 rpm until the OD 600 =0.6~0.8, and then add isopropylβ-D-thiogalactoside (IPTG) to the culture medium with a final concentration of 0.15mM. After culturing at 20℃ for 16h, centrifuge at 4℃ and 8000rpm for 10min to obtain wet cells.

[0017] Furthermore, the BsCR mutant purified enzyme was prepared as follows: (1) cell lysis solution: wet bacteria were resuspended in 100 mM sodium phosphate buffer containing 300 mM sodium chloride at pH 7.0 according to 50 g WCW (wet bacteria) / L buffer, 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 cell lysis solution; (2) the cell lysate was centrifuged at 8000 rpm and 4°C for 10 min, the precipitate was discarded, and the supernatant was the crude enzyme solution; the crude enzyme solution was microfiltered through a 0.22 μm filter membrane and then protein was purified by affinity chromatography. The specific method was as follows: nickel-agarose gel affinity chromatography filler was added to the chromatography column, and after sufficient sedimentation, pH 7.0 containing 300 mM sodium chloride was used. The column was equilibrated with a pH 7.0, 20 mM sodium phosphate buffer; the sample was loaded; 5 column volumes of pH 7.0, 20 mM sodium phosphate buffer containing 300 mM sodium chloride and 50 mM imidazole were used to elute the column to remove unbound proteins; 5 column volumes of pH 7.0, 20 mM sodium phosphate buffer containing 300 mM sodium chloride and 100 mM imidazole were used to elute the column to remove unbound proteins; the eluate was then eluted with a pH 7.0, 20 mM sodium phosphate buffer containing 300 mM sodium chloride and 100 mM imidazole to collect the eluate containing the target enzyme protein; the eluate was transferred to an ultrafiltration centrifuge tube with a molecular weight cutoff of 30 kDa, the eluate in the ultrafiltration centrifuge tube was concentrated to 3% of the original volume at 4°C and 3000 rpm, 5 column volumes of pH 7.0, 20 mM sodium phosphate buffer were added, and the column was further concentrated to 3% of the original volume. This was repeated three times to remove the imidazole in the enzyme solution. The resulting concentrated solution was the pure enzyme solution.

[0018] The base sequences of the carbonyl reductase BsCR and the carbonyl reductase BsCR mutant of the present invention are both 858 bp in length, from the first base to the 858th base, with the start codon being ATG and the stop codon being TAA, encoding 285 amino acids.

[0019] The carbonyl reductase BsCR mutants of the present invention were obtained by protein engineering, and finally four beneficial mutants Q130L, T179L, I178L, and K182E were obtained. Through further iterative combination mutations, the optimal mutant BsCR was obtained. M2, the obtained mutant plasmid is transferred into E. coli BL21 (DE3) competent cells by heat shock to obtain recombinant engineered bacteria. The obtained strain is inoculated, transferred, induced cultured, and the bacteria are recovered, and the resuspended bacteria liquid is used to catalyze CFA reduction to prepare (S)-CFL. The inoculation, transfer, induction culture, and bacteria recovery of the carbonyl reductase BsCR mutant of the present invention, the culture medium preparation can be any culture medium in the art that can grow the bacteria of the present invention, preferably LB 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. The culture method and conditions can be appropriately selected according to the basic knowledge in the art based on factors such as the host type and culture method.

[0020] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0021] (1) The activity of the carbonyl reductase BsCR mutant was significantly improved.

[0022] The mutant obtained by the present invention has significantly higher enzyme activity than WT-BsCR, wherein BsCR M2 The mutant enzyme activity was 87.7 times higher than that of WT-BsCR. The maximum substrate dosage was 100 g / L CFA. The product concentration increased gradually with time. Most of the substrate could be converted within 6 h (substrate conversion rate was 92.06%). The product ee p The value was always maintained above 99.5%, and the space-time yield was 368.24 g / L / d.

[0023] (2) The stability of the carbonyl reductase BsCR mutant was improved.

[0024] Compared with the wild-type BsCR, the mutants constructed in the present invention have improved stability, among which BsCR M2 At 45℃ half-life (t 1 / 2 ) was 52.1h, which was 2.9h higher than that of the parent WT-BsCR. M2 It shows good industrial application prospects. (IV) Description of the accompanying drawings

[0025] Figure 1 Schematic diagram of the reaction in which glucose is used as a cosubstrate and carbonyl reductase BsCR and its mutants catalyze the asymmetric reduction of CFA to prepare (S)-CFL.

[0026] Figure 2 Gas chromatograms of CFA, (S)-CFL, and (R)-CFL standard analyses.

[0027] Figure 3Standard curve for (S)-CFL detection established for gas chromatography.

[0028] Figure 4 The SDS-PAGE gel electrophoresis images of WT-BsCR and its mutants. M, protein standard; lane 1, E. coli BL21 (DE3) / pET28a (+) (negative control); lane 2, WT-BsCR crude enzyme solution; lane 3, BsCR M1 Crude enzyme solution; Lane 4, BsCR M2 Crude enzyme solution; Lane 5, WT-BsCR purified enzyme; Lane 6, BsCR M1 Purified enzyme; lane 7, BsCR M2 Purify the enzyme.

[0029] Figure 5 The effect of temperature on BsCR M2 Effect of enzyme activity.

[0030] Figure 6 Effect of pH on BsCR M2 Effect of enzyme activity.

[0031] Figure 7 Effect of glucose addition on BsCR M2 Effect of enzyme activity.

[0032] Figure 8 The effect of cosolvent type on BsCR M2 Effect of enzyme activity.

[0033] Figure 9 The amount of DMSO used as a cosolvent has an effect on BsCR M2 Effect of enzyme activity.

[0034] Figure 10 Mutant BsCR M2 Reaction time course curve of asymmetric reduction of CFA to synthesize (S)-CFL using glucose as cosubstrate. (V) Specific implementation methods

[0035] The present invention is further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto:

[0036] 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.

[0037] Example 1: Preparation of purified WT-BsCR enzyme

[0038] (1) Construction of wild-type BsCR genetically engineered bacteria

[0039] The gene sequence of B. subtilis carbonyl reductase from GenBank (GenBank No. NC_000964.3), i.e., WT-BsCR gene (nucleotide sequence shown in SEQ ID No. 1, amino acid sequence shown in SEQ ID No. 2), was artificially synthesized and ligated between the Nde I and Xho I restriction sites of the vector pET28a(+) to construct a recombinant expression vector. The recombinant expression vector was then transformed into the host bacterium E. coli BL21(DE3) to obtain the WT-BsCR genetically engineered bacterium E. coli BL21(DE3) / pET28a(+)-bscr.

[0040] SEQ ID No. 2

[0041] MANQKKKTLPPQHQNQQPGFEYLMDPRPVFDKPKKAKKLEGKTAIITGGDSGIGRAVSVLFAKEGANVVIVYLNEHQDAEETKQYVEKEGVKCLLIAGDVGDEAFCNDVVGQASQVFPSIDILVNNAAEQHVQPSIEKITSHQ LIRTFQTNIFSMFYLTKAVLPHLKKGSSIINTASITAYKGNKTLIDYSATKGAIVTFTRSLSQSLVQQGIRVNAVAPGPIWTPLIPASFAAKDVEVFGSDVPMERPGQPVEVAPSYLYLASDDSTYVTGQTIHVNGGTIVNG.

[0042] (2) Wet cell preparation

[0043] The wild-type carbonyl reductase genetically engineered bacteria E. coli BL21 (DE3) / pET28a (+) -bscr constructed in step (1) was inoculated into LB liquid medium containing kanamycin at a final concentration of 50 μg / mL, and cultured at 37 ° C for 10 h to obtain seed liquid; the seed liquid was inoculated into fresh LB liquid medium containing kanamycin at a volume concentration of 1% (V / V) and cultured at 37 ° C and 180 rpm until the OD 600 =0.6-0.8, and then add IPTG to the culture solution with a final concentration of 0.15mM. After culturing at 20°C for 16h, centrifuge at 4°C and 8000rpm for 10min to obtain wet cells.

[0044] (3) Enzyme purification

[0045] The wet bacterial cells of step (2) were dispersed in a pH 7.0, 20 mM sodium phosphate buffer at a volume of 50 g WCW / L buffer 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 1 s, pause 1 s, and obtain cell lysate for subsequent enzyme purification.

[0046] The enzyme purification operation is as follows: the cell lysate is centrifuged at 8000 rpm and 4°C for 10 min, the precipitate is discarded, and the supernatant is collected. The supernatant was microfiltered (filter membrane: 0.22 μm) to obtain a permeate, and the permeate was purified by affinity chromatography. The specific method was as follows: nickel-agarose gel affinity chromatography filler (3 mL) was spread evenly and added to the chromatography column. After sufficient sedimentation, 15 mL of pH 7.0, 20 mM sodium phosphate buffer solution containing 300 mM sodium chloride was used to equilibrate the chromatography column; sample was loaded; 5 column volumes of pH 7.0, 20 mM sodium phosphate buffer containing 50 mM imidazole and 300 mM sodium chloride were used to elute the unbound proteins; 15 mL of pH 7.0, 20 mM sodium phosphate buffer containing 100 mM imidazole and 300 mM sodium chloride were used to elute the protein for 5 column volumes, and the eluate containing the target enzyme protein was collected; the eluate was transferred to an ultrafiltration centrifuge tube with a molecular weight cutoff of 30 kDa, centrifuged at 4 ° C and 3000 rpm, the eluate was concentrated to 500 μL, and then 15 mL of pH 7.0, 20mM sodium phosphate buffer, continue to concentrate to 500μL, repeat three times to remove the imidazole in the enzyme solution, the obtained concentrate is the pure enzyme solution, gel electrophoresis shows Figure 4 .

[0047] (4) Enzyme activity determination

[0048] The enzyme activity unit (U) is defined as the amount of enzyme required to generate 1 micromole of (S)-CFL per minute under optimal conditions. The specific enzyme activity is defined as the number of activity units per milligram of enzyme protein, U / mg.

[0049] Enzyme activity assay conditions: 1 mM reduced nicotinamide mononucleotide (NMNH), appropriate amount of pure enzyme solution (enzyme protein mass 100 μg), 1 mM substrate CFA, 5% DMSO by volume. The substrate was first dissolved in DMSO and then added. The final volume was made up to 500 μL with pH 6.0, 100 mM sodium phosphate buffer. The reaction was incubated at 60°C, 1000 rpm for 30 min. Substrate consumption and product formation were measured using an Agilent 8860 high-performance gas chromatograph.

[0050] The substrate conversion, product yield, 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 both set at 250°C, the column oven temperature was set at 155°C and maintained for 15 minutes, the split ratio was 15:1 (V / V), the injection volume was 2μL, and other conditions were the default detection conditions. The gas phase detection spectra of CFA, (S)-CFL, and (R)-CFL standards are shown in Figure 2 The standard curve of (S)-CFL gas chromatography detection is shown in Figure 3 .

[0051] Example 2: Selection of WT-BsCR mutant sites and construction of single-site mutants

[0052] 1. Selection of WT-BsCR mutant sites

[0053] The docking software YASARA was used to dock the substrate CFA into the active pocket of WT-BsCR, and the distance from the substrate was selected to be The amino acid residues in the range are most likely to affect the selectivity and activity of the enzymatic reaction. After excluding the conserved catalytic triad amino acid residues (S177, Y190, K194), Q130, I178, T179, and K182 were selected as the mutation sites for investigation.

[0054] 2. Q130 saturation mutation

[0055] (1) Q130 saturation mutation process: The plasmid pET28a(+)-bscr of the wild-type carbonyl reductase genetically engineered bacteria E. coli BL21(DE3) / pET28a(+)-bscr of Example 1 was extracted, and PCR amplification was performed using the primers in Table 1 to mutate the glutamine at position 130 to 19 other amino acids. The PCR product was digested with Dpn I endonuclease and transformed into E. coli BL21(DE3) competent cells. The transformants were spread onto LB solid medium containing 50 μg / mL kanamycin and cultured at 37°C for 12 h. Then, single clones were picked and transferred to 10 mL of LB liquid medium containing 50 μg / mL kanamycin, cultured at 37°C and 200 rpm for 10 h, the bacterial culture was collected, the plasmid was extracted, and the mutation was verified by sequencing.

[0056] Table 1 Primer design for site-directed saturation mutagenesis of BsCR130

[0057]

[0058] Note: NDT stands for degenerate codon; AHN stands for the degenerate codon paired with NDT.

[0059] 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.

[0060] (2) Screening of mutants

[0061] Mutant screening reaction system: 50 g / L of each wet cell prepared in step (1), 10 g / L CFA, 80 g / L glucose, pH 7.0, 100 mM sodium phosphate buffer, and 10% (V / V) DMSO as a cosolvent were added to form a 10 mL reaction system. After reacting at 60°C and 1000 rpm for 4 hours, 1 mL of the reaction solution was removed and extracted with 1 mL of ethyl acetate. The ethyl acetate layer was centrifuged and microfiltered using a 0.22 μm filter membrane. The permeate was detected by gas chromatography according to Example 1 for product and substrate concentration, and the substrate conversion rate, enzyme specific activity, and stereoselectivity were calculated. The results are shown in Table 2. Three replicates were set for each mutant characterization. Through activity testing of 12 mutants, the best mutant was screened and determined to be E. coli BL21 (DE3) / pET28a (+) -bscr-Q130L, which was stored in a -80°C refrigerator.

[0062] Table 2 Catalytic performance of Q130 site mutants

[0063]

[0064] Note: ND means no enzyme activity was detected.

[0065] 3. T179 saturation mutation

[0066] (1) T179 site saturation mutation process: the plasmid pET28a(+)-bscr of the wild type carbonyl reductase gene engineering bacteria E. coli BL21(DE3) / pET28a(+)-bscr of Example 1 was extracted, and the 179 site threonine was mutated into other 19 kinds of amino acids by PCR amplification using the primers in Table 3. After the PCR product was digested by Dpn I endonuclease, it was transformed into E. coli BL21(DE3) competent cells, and the transformants were coated on LB solid medium containing 50 μg / mL kanamycin and cultured at 37°C for 12 h. Then single colonies were picked and transferred to 10 mL of LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C, 200 rpm for 10 h. The bacterial liquid was collected, the plasmid was extracted, and the mutation was verified by sequencing.

[0067] Table 3 BsCR 179 site saturation mutation primer design

[0068]

[0069] Note: NDT represents a degenerate codon; AHN represents a degenerate codon paired with NDT.

[0070] The PCR reaction system (25 μL) was 0.5 μL of forward primer (100 μM), 0.5 μL of reverse primer (100 μM), 12.5 μL of 2×Phanta buffer, 0.5 μL of dNTP mixture (10 mM each), 0.5 μL of plasmid template, 0.5 μL of DNA polymerase and 10 μL of ultrapure water. According to the operation manual of Phanta Super-Fidelity DNA polymerase, 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.

[0071] (2) Screening of mutants

[0072] Mutant screening reaction system: 50 g / L of each wet cell prepared in step (1), 10 g / L CFA, 80 g / L glucose, pH 7.0, 100 mM sodium phosphate buffer, and 10% (V / V) DMSO as a cosolvent were added to form a 10 mL reaction system. After reacting at 60°C and 1000 rpm for 4 hours, 1 mL of the reaction solution was removed and extracted with 1 mL of ethyl acetate. The ethyl acetate layer was centrifuged and microfiltered using a 0.22 μm filter membrane. The permeate was detected by gas chromatography according to Example 1 for product and substrate concentration, and the substrate conversion rate, enzyme specific activity, and stereoselectivity were calculated. The results are shown in Table 4. Three replicates were set for each mutant characterization. Through activity testing of 12 mutants, the best mutant was screened and determined to be E. coli BL21 (DE3) / pET28a (+) -bscr-T179L, which was stored in a -80°C refrigerator.

[0073] Table 4 Catalytic performance of T179 site mutants

[0074]

[0075] Note: ND means no enzyme activity was detected.

[0076] 4. Saturation mutation at position I178

[0077] (1) Saturation mutation process at position I178: The plasmid pET28a(+)-bscr of the wild-type carbonyl reductase genetically engineered bacteria E. coli BL21(DE3) / pET28a(+)-bscr of Example 1 was extracted, and PCR amplification was performed using the primers in Table 5 to mutate the isoleucine at position 178 to the other 19 amino acids. The PCR product was digested with Dpn I endonuclease and transformed into E. coli BL21(DE3) competent cells. The transformants were spread onto LB solid medium containing 50 μg / mL kanamycin and cultured at 37°C for 12 h. Then, single clones were picked and transferred to 10 mL of LB liquid medium containing 50 μg / mL kanamycin, cultured at 37°C and 200 rpm for 10 h, the bacterial culture was collected, the plasmid was extracted, and the mutation was verified by sequencing.

[0078] Table 5 Primer design for site-directed saturation mutagenesis of BsCR178

[0079]

[0080]

[0081] Note: NDT stands for degenerate codon; AHN stands for the degenerate codon paired with NDT.

[0082] (2) Screening of mutants

[0083] Mutant screening reaction system: 50 g / L of each wet cell prepared in step (1), 10 g / L CFA, 80 g / L glucose, pH 7.0, 100 mM sodium phosphate buffer, and 10% (V / V) DMSO as a cosolvent were added to form a 10 mL reaction system. After reacting at 60°C and 1000 rpm for 4 hours, 1 mL of the reaction solution was removed and extracted with 1 mL of ethyl acetate. The ethyl acetate layer was centrifuged and microfiltered using a 0.22 μm filter membrane. The permeate was detected by gas chromatography according to Example 1 for product and substrate concentration, and the substrate conversion rate, enzyme specific activity, and stereoselectivity were calculated. The results are shown in Table 6. Three replicates were set for each mutant characterization. Through activity testing of 12 mutants, the best mutant was screened and determined to be E. coli BL21 (DE3) / pET28a (+) -bscr-I178L, which was stored in a -80°C refrigerator.

[0084] Table 6 Catalytic performance of I178 site mutants

[0085]

[0086] Note: ND means no enzyme activity was detected.

[0087] 5. Saturation mutation at K182

[0088] (1) Saturation mutation process at K182: The plasmid pET28a(+)-bscr of the wild-type carbonyl reductase genetically engineered bacteria E. coli BL21(DE3) / pET28a(+)-bscr of Example 1 was extracted, and PCR amplification was performed using the primers in Table 7 to mutate the lysine at position 182 to the other 19 amino acids. The PCR product was digested with Dpn I endonuclease and transformed into E. coli BL21(DE3) competent cells. The transformants were spread onto LB solid medium containing 50 μg / mL kanamycin and cultured at 37°C for 12 h. Then, single clones were picked and transferred to 10 mL of LB liquid medium containing 50 μg / mL kanamycin, cultured at 37°C and 200 rpm for 10 h, the bacterial culture was collected, the plasmid was extracted, and the mutation was verified by sequencing.

[0089] Table 7 BsCR182 site-directed saturation mutagenesis primer design

[0090]

[0091] Note: NDT stands for degenerate codon; AHN stands for the degenerate codon paired with NDT.

[0092] (2) Screening of mutants

[0093] Mutant screening reaction system: 50 g / L of each wet cell prepared in step (1), 10 g / L CFA, 80 g / L glucose, pH 7.0, 100 mM sodium phosphate buffer, and 10% (V / V) DMSO as a cosolvent were added to form a 10 mL reaction system. After reacting at 60°C and 1000 rpm for 4 hours, 1 mL of the reaction solution was removed and extracted with 1 mL of ethyl acetate. The ethyl acetate layer was centrifuged and microfiltered using a 0.22 μm filter membrane. The permeate was detected by gas chromatography according to Example 1 for product and substrate concentration, and the substrate conversion rate, enzyme specific activity, and stereoselectivity were calculated. The results are shown in Table 8. Three replicates were set for each mutant characterization. Through activity testing of 12 mutants, the best mutant was screened and determined to be E. coli BL21 (DE3) / pET28a (+) -bscr-K182E, which was stored in a -80°C refrigerator.

[0094] Table 8 Catalytic performance of K182 site mutants

[0095]

[0096] Note: ND means no enzyme activity was detected.

[0097] Example 3: Construction of combined mutants

[0098] The plasmids of the engineering bacteria E. coli BL21 (DE3) / pET28a (+) -bscr-Q130L, E. coli BL21 (DE3) / pET28a (+) -bscr-I178L, E. coli BL21 (DE3) / pET28a (+) -bscr-T179L, and E. coli BL21 (DE3) / pET28a (+) -bscr-K182E of Example 2 were used as templates, and PCR amplification was performed using the primers in Table 9 and the method and conditions of Example 2 to construct the engineering bacteria E. coli BL21 (DE3) / pET28a (+) -bscr-Q130L / I178L, E. coli BL21 (DE3) / pET28a (+) -bscr-Q130L / T179L, and E. coli BL21(DE3) / pET28a(+)-bscr-Q130L / K182E, E.coli BL21(DE3) / pET28a(+)-bscr-I178L / T179L, E.coli BL21(DE3) / pET28a(+)-bscr-I178L / K182E, E.coli BL21(DE3) / pET28a(+)-bscr-T179L / K182E, E.coli BL21(DE3) / pET28a(+)-bscr-Q130L / I178L / T179L, E.coli BL21(DE3) / pET28a(+)-bscr-Q130L / I178L / K182E, E.coli BL21(DE3) / pET28a(+)-bscr-Q130L / T179L / K182E, E. coli BL21(DE3) / pET28a(+)-bscr-I178L / T179L / K182E, and E. coli BL21(DE3) / pET28a(+)-bscr-Q130L / I178L / T179L / K182E were stored at -80°C. Wet cells of each mutant were prepared using the method in Example 1.

[0099] Table 9 BsCR combination mutation primer design

[0100]

[0101] The substrate conversion rate and stereoselectivity were tested using the method and conditions of Example 2, and the optimal mutant was determined to be E. coli BL21 (DE3) / pET28a (+) -bscr-I178L / T179L, which was recorded as mutant BsCR M2; E. coli BL21(DE3) / pET28a(+)-bscr- / T179L is recorded as mutant BsCR M1 , stored in a -80℃ freezer.

[0102] Table 10 Effects of mutations on BsCR stereoselectivity and relative activity

[0103]

[0104]

[0105] Note: ND means no enzyme activity was detected.

[0106] Example 4: Inducible expression of mutants

[0107] The E. coli BL21 (DE3) / pET28a (+) -BsCR obtained in Example 3 was M1 and E. coli BL21(DE3) / pET28a(+)-BsCR M2 The 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 1.5% (V / V), cultured at 37°C and 180 rpm for 2 h, and IPTG was added to the culture medium at a final concentration of 0.15 mM. The cells were cultured at 20°C for 15 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 BsCR. M1 Wet bacterial cells and BsCR M2 Wet bacterial cells. The cells obtained above contain the corresponding proteins, which can be used for enzyme purification and catalytic synthesis of (S)-CFL.

[0108] Example 5: Purification of mutant enzyme protein and enzyme activity determination

[0109] Resuspend the cells in 300mM sodium chloride pH 7.0 and 100mM sodium phosphate buffers at a rate of 50g wet cells / L of buffer, then ultrasonically disrupt the cells for 15min on an ice-water mixture. The ultrasonic disruption conditions are: power 250W, disruption for 1s, and pause for 1s. The disrupted mixture is the cell lysate. The cell lysate is centrifuged at 8000rpm and 4°C for 10min, the precipitate is discarded, and the supernatant is collected as the crude enzyme solution. The supernatant is purified by the same method as in Example 1 to obtain mutant BsCR. M1 Pure enzyme solution and mutant BsCR M2 Pure enzyme solution, gel electrophoresis see Figure 4 WT-BsCR, BsCR M1and BsCR M2 The enzyme activity detection method is the same as in Example 1, and the enzyme activity detection data are shown in Table 11. M2 Compared with WT-BsCR, it increased by about 87.7 times.

[0110] Table 11 Stereoselectivity and specific activity of BsCR and its mutants

[0111]

[0112] Example 6: Determination of half-life of BsCR and mutants

[0113] (1) Determination of half-life

[0114] Half-life (t 1 / 2 ) Determination reaction system: WT-BsCR and BsCR obtained in Example 1 and Example 5 M1 and BsCR M2 The purified enzyme was diluted to a protein concentration of 1 μg / μL, and 200 μL was dispensed into each tube. The tube was kept warm in a 45°C water bath. One tube was taken out every 4 h, and the temperature was restored to room temperature. The residual enzyme activity after different incubation times was detected according to the enzyme activity detection system of Example 1.

[0115] (2) Fitting of half-life

[0116] Half-life (t 1 / 2 ) Calculated according to the thermal inactivation equation: A t =A0e -kd t In the thermal inactivation equation, k d is the inactivation kinetic constant, A0 and A t Represent the initial enzyme activity and the enzyme activity at time t respectively. According to the thermal inactivation equation, ln(A t / A0) is the ordinate, sampling time t is the abscissa, and Origin is used for drawing. The ordinate is ln(A t The t value when / A0) = -ln2 is the half-life t 1 / 2 The half-lives of WT-BsCR and mutants are shown in Table 12.

[0117] Table 12 Half-life of BsCR and mutants at 45°C

[0118]

[0119] Example 7: BsCR M2 Optimal reaction temperature, pH, glucose concentration, DMSO volume concentration

[0120] 1. Optimum reaction temperature

[0121] BsCR prepared by the method of Example 4 was added to 10 mL of pH 7.0, 100 mM PB buffer M2 Wet bacterial cells, 20 g / L of substrate CFA, 80 g / L of glucose, 10% (V / V) of DMSO, 30-80°C (30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C), 800 rpm for 30 min, and the enzyme activity at different temperatures was detected according to the method of Example 1 to optimize the catalytic temperature of BsCR M2 The relative enzyme activity was calculated with the highest enzyme activity as 100%, and the results are shown in Figure 5 The results show that the temperature optimization range is 30-80°C, and the optimal catalytic temperature of BsCR M2 is 60°C.

[0122] 2, Optimal reaction pH

[0123] The optimal reaction pH of the mutant was screened using pH 4.0-6.0 sodium acetate buffer, pH 6.0-8.0 sodium phosphate buffer, and pH 7.0-9.0 Tris-HCl buffer, respectively.

[0124] Reaction system: BsCR prepared by the method of Example 4 was added to 10 mL of different pH (pH 4.0, 5.0, 6.0, 7.0, 8.0, 9.0), 100 mM buffer M2 Wet bacterial cells, 20 g / L of substrate CFA, 80 g / L of glucose, 10% (V / V) of DMSO, 60°C, 800 rpm for 30 min, and the enzyme activity at different pH was detected according to the method of Example 1 to optimize the catalytic reaction pH of BsCR M2 The relative enzyme activity was calculated with the enzyme activity at pH 4.0 as 100%, and the results are shown in Figure 6 The pH optimization range is 4.0-9.0, and the optimal catalytic pH of BsCR M2 is 6.0.

[0125] 3, Optimal glucose concentration

[0126] BsCR prepared by the method of Example 4 was added to 10 mL of pH 7.0, 100 mM sodium phosphate buffer M2 Wet bacterial cells, 50 g / L of substrate CFA, glucose to substrate molar ratio (1:1, 2:1, 3:1, 4:1, 5:1), 10% (V / V) of DMSO, 60°C, 800 rpm for 30 min, and the enzyme activity at different temperatures was detected according to the method of Example 1 to optimize BsCR M2The relative enzyme activity was calculated with the enzyme activity at a ratio of 1:1 as 100%. The results are shown in Figure 7 The results showed that the investigation range of the cosubstrate glucose was 1 to 5 times the molar amount of the substrate, and the BsCR M2 The optimal glucose concentration was 3 times the molar amount of substrate.

[0127] 4. Reaction cosolvent

[0128] In 9 mL of pH 6.0, 100 mM sodium phosphate buffer, 50 g / L of BsCR prepared by the method of Example 4 was added. M2 Wet bacterial cells and 80 g / L glucose were added to different organic solvents (DMSO, DMF, acetonitrile, methanol, ethanol, isopropanol) at a final volume concentration of 10%. The substrate was first dissolved in the organic solvent and then added to the reaction system to a final substrate concentration of 20 g / L. The reaction was carried out at 60°C and 800 rpm for 30 min. The enzyme activity under different cosolvents was detected according to the method of Example 1. The enzyme activity without adding organic solvent was defined as 100%. The relative enzyme activity was calculated and the results are shown in Figure 8 , BsCR M2 The best co-solvent for the reaction is DMSO.

[0129] 5. Optimal DMSO concentration

[0130] In 10 mL of pH 7.0, 100 mM sodium phosphate buffer, 50 g / L of BsCR prepared by the method of Example 4 was added. M2 Wet bacterial cells, 80 g / L glucose, and different volume concentrations (V / V) of DMSO (10%, 20%, 30%, 40%, 50%, 60%, 70%, and 80%) were added to the reaction system. The substrate was first dissolved in an organic solvent and then added to the reaction system to a final concentration of 20 g / L. The reaction was carried out at 60°C and 800 rpm for 30 min. The enzyme activity at different temperatures was detected according to the method in Example 1 to optimize BsCR. M2 The relative enzyme activity was calculated with the enzyme activity of the control without adding organic solvent as 100%. The results are shown in Figure 9 The results showed that in the concentration range of 10%-80% (v / v) of cosolvent DMSO, BsCR M2 The optimal DMSO concentration for the reaction was 50% (V / V).

[0131] Example 8: BsCR M2 Synthesis of (S)-CFL by Asymmetric Reduction of CFA

[0132] First, the BsCR prepared according to the method of Example 4 M2 Wet bacterial cells were resuspended in 100 mM sodium phosphate buffer at pH 6.0. M2The amount of wet cells added was 10 g DCW / L on a dry weight basis. The substrate CFA was fed to a total feed amount of 50 g / L. Glucose was added at 3 times the molar amount of the substrate. A pH 6.0, 100 mM sodium phosphate buffer was used as the reaction medium. 50% (V / V) DMSO was added to form a 50 mL reaction system. The reaction was carried out at 60°C, pH 6.0, and 800 rpm for 5 to 8 hours. Samples were taken every hour, and the concentration of (S)-CFL was determined using the method of Example 1 to calculate the space-time yield. Under the same conditions, the substrate concentration was changed to 100 g / L. The results are shown in FIG. Figure 10 shown.

[0133] The calculation formula of space-time yield is: m is the mass of the product generated (g); t is the reaction time (d); V is the volume of the reaction solution (L). M2 It can catalyze the production of 47.26 g / L of product. Under the same conditions, when the substrate concentration is 100 g / L, the reaction endpoint can be reached within 6 hours, and 92.06 g / L of product is produced, with a space-time yield of 368.24 g / L / d.

[0134] Table 13 BsCR M2 Space-time yields at different substrate concentrations

[0135]

[0136] a, S / C represents the feed mass ratio of substrate to wet bacterial cell dry weight.

Claims

1. A carbonyl reductase Bs CR mutant, characterized in that The carbonyl reductase Bs The CR mutant is obtained by mutating the amino acid sequence shown in SEQ ID No. 2 to the following sequence: isoleucine at position 178 is mutated to leucine, and threonine at position 179 is mutated to leucine.

2. A method comprising the carbonyl reductase according to claim 1 Bs Recombinant genetically engineered bacteria containing the gene encoding the CR mutant.

3. A carbonyl reductase according to claim 1 Bs Application of CR mutant in the asymmetric reduction of 2,6-dichloro-3-fluoroacetophenone to prepare key chiral intermediates of crizotinib.

4. The use according to claim 3, characterized in that The application method is: using carbonyl reductase Bs The wet cells obtained by inducing culture of the recombinant genetically engineered bacteria of the CR mutant were used as a catalyst, 2,6-dichloro-3-fluoroacetophenone was used as a substrate, glucose was used as a co-substrate, and a pH 4.0-9.0 buffer was used as a reaction medium to form a reaction system. The reaction was carried out at 30-80°C and 800-1000 rpm. After the reaction was completed, the reaction solution was separated and purified, and the reaction solution was extracted with ethyl acetate to obtain ( S )-2,6-dichloro-3-fluorophenethyl alcohol.

5. The use according to claim 4, characterized in that In the reaction system, the substrate is added to a final concentration of 1-100 g / L; the molar ratio of glucose addition to substrate is 1-5:1; the amount of catalyst used is 10-50 g WCW / L based on the wet cell weight and 2-10 g DCW / L based on the dry weight of the wet cell.

6. The use according to claim 4, characterized in that Dimethyl sulfoxide is added as a cosolvent in the reaction system, and the volume concentration of dimethyl sulfoxide is 10-80%.

7. The use according to claim 4, characterized in that The reaction medium is a 100 mM sodium phosphate buffer at pH 6.

0.

8. The use according to claim 4, characterized in that The wet cells are prepared as follows: Bs The engineered bacteria expressing the CR mutant gene were inoculated into LB liquid medium containing kanamycin at a final concentration of 50 μg / mL and cultured at 37°C for 10 h to obtain seed solution; The seed solution was inoculated into 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 until the OD 600 = 0.6~0.8, and then add isopropyl to the culture medium at a final concentration of 0.15 mM β -D-thiogalactoside, cultured at 20°C for 16 h, and then centrifuged at 4°C and 8000 rpm for 10 min to obtain wet cells.

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