A ketone reductase gene, ketone reductase, mutants and applications

Abstract

This invention belongs to the field of fermentation genetic engineering technology, specifically relating to a ketone reductase gene, ketone reductase, mutant, and application. The ketone reductase is obtained by mutation based on the amino acid sequence SEQ ID NO.2, with the mutation sites being 79, 116, 132, 144, and / or 225. This invention further improves enzyme activity and enables it to tolerate high concentrations of tert-butanol, which is beneficial for subsequent cell transformation.

Description

Technical Field

[0001] This invention belongs to the field of fermentation genetic engineering technology, specifically relating to a ketone reductase gene, ketone reductase, mutant, and application. Background Technology

[0002] DHEA (dehydroepiandrosterone) is a steroidal drug secreted by the reticular layer of the adrenal cortex. Studies have found that DHEA levels peak at age 25, and by age 80, they are only about 10% of their peak. Therefore, the decline in DHEA levels is believed to be closely related to a range of age-related diseases. Further research has revealed that DHEA has anti-inflammatory, antidepressant, anti-anxiety, anticonvulsant, and memory-enhancing effects.

[0003] Besides its inherent pharmacological activity, DHEA has been found to be an important precursor compound in the synthesis of other steroid drugs, such as 7-OH-DHEA and 11-OH-DHEA, in the steroid synthesis industry. Therefore, research into various processes for DHEA synthesis has led to a shift in chemical synthesis methods due to increased environmental concerns and the growing consumption of medical resources, resulting in the development of new process routes. Biotechnology has thus attracted widespread attention. Biotechnology is divided into bio-fermentation and enzymatic catalysis. Bio-fermentation involves the fermentation of strains to produce active proteins that catalyze the formation of products from substrates. Generally, wild-type or recombinant strains are used, but the overall fermentation time is relatively long, and sometimes it can cause the decomposition of the substrate. Enzymatic catalysis also involves the fermentation of strains to produce active proteins; the fermentation and catalysis steps can be separated. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a ketone reductase gene, ketone reductase, mutant and application, which further improves enzyme activity and can withstand high concentrations of tert-butanol, which is beneficial for subsequent cell transformation.

[0005] This invention provides a ketone reductase, which is obtained by mutation based on the amino acid sequence SEQ ID NO.2, wherein the mutation sites are 79, 116, 132, 144 and / or 225.

[0006] Preferably, the 79th position is mutated to R or V, or the 116th position is mutated to S or T, or the 132nd position is mutated to A, or the 144th position is mutated to D, or the 225th position is mutated to L or S.

[0007] Preferably, the 79th position is mutated to R and the 116th position is mutated to S; or the 132nd position is mutated to A and the 144th position is mutated to D; or the 79th position is mutated to R and the 225th position is mutated to L.

[0008] Preferably, the 132nd position is mutated to A, and the 225th position is mutated to L.

[0009] Preferably, the 132nd bit is mutated to A, the 225th bit is mutated to L, and the 144th bit is mutated to D; or the 132nd bit is mutated to A, the 225th bit is mutated to L, and the 116th bit is mutated to S.

[0010] This invention provides a ketone reductase gene for encoding the aforementioned ketone reductase.

[0011] The present invention provides a ketone reduction mutant containing the ketone reductase gene or a plasmid containing the ketone reductase gene.

[0012] The ketone-reducing mutant is preferably *Escherichia coli*. Escherichia coli .

[0013] This invention provides an application of the ketone reductase described above, wherein the ketone reductase is used to catalyze a substrate to convert the substrate into DHEA, wherein the substrate is AD or an AD derivative, such as 5-AD or 4-AD esters.

[0014] Preferably, the substrate, glucose, tert-butanol, buffer solution, ketone reductase and glucose dehydrogenase are mixed, an electron acceptor is added, an extraction solvent is added after the reaction, and the mixture is centrifuged to obtain the product.

[0015] Preferably, the electron acceptor is NAD. + The extractant is dichloromethane, and the pH value is controlled at 6-7 during the reaction.

[0016] The beneficial effects of this invention are that it constructs a recombinant ketone reductase derived from Nocardia, and performs saturation and combination mutations on the ketone reductase selection site to obtain a recombinant strain with high enzyme activity. Compared to the original ketone reductase, the ketone reductase of this invention exhibits significantly increased enzyme activity, reaching 1.26-31.25 times. Furthermore, the ketone reductase still shows good activity at high concentrations of tert-butanol, achieving a conversion rate of 99%, which is beneficial for subsequent transformation using fermentation cells and further reduces production costs. Detailed Implementation

[0017] Example 1

[0018] strain construction

[0019] The nucleotide sequence of the primitive ketone reductase is SEQ ID NO.1, and the protein sequence is SEQ ID NO.2.

[0020] The sequence of SEQ ID NO.1 is:

[0021] 1 atggcacggc tcgacggcaa ggtcgcgatc atcaccggag cggcgcaggg catgggagcg

[0022] 61 gccacggccc ggctgttcgt cgccgaaggc gcccgcgtgc tgctcgggga cgtgctcgac

[0023] 121 gagaaggggc gcgcgctcgc ggaggaactc ggcgacgccg cgatcttcac cccgctcgac

[0024] 181 gtgagcagcg agtccgcgtg gaaggcggcc accgcgctgg ccgtcgacca cttcggcggc

[0025] 241 ctcgacatcc tcgtcaacaa cgccggcgtg atgcactggg cgccgatcga ggagctcgat

[0026] 301 gtcgcccgga ccgaacgcct gctcgacgtc aacgtcctcg gaaatctgct gggcgccaag

[0027] 361 tccgtcgtcc cgatcatgaa gaaggccggg cggggcgtga tcgtcaacat ctcctcggtc

[0028] 421 gacgggctgc gcggcgtcaa cggcctggcc gcgtacaccg cgagcaagtg ggcggtgcgc

[0029] 481 ggtctcacca aggcactcgc gtacgagctg gggccgtcgg gcatccgcgt gtgctcggtg

[0030] 541 catccgggcg gtgtggacac cacgctcggc aatccgggcg gtctgatcgg cgacgacctg

[0031] 601 cagagcaagt acgtgggtgt gccgctgcag cgcatcggcg agtccgagga catcgcccgg

[0032] 661 gccacgctgt tcgtcgcgag cgacgaggcg tcgtacatct cgggcgcgga actggcagtg

[0033] 721 gacggcggct ggtccgccgg cacctactac ccgggcctgc cgggcacgcc gccggcgctg

[0034] 781 atgccgaact ga.

[0035] The sequence of SEQ ID NO.2 is:

[0036] 1 marldgkvai itgaaqgmga atarlfvaeg arvllgdvld ekgralaeel gdaaiftpld

[0037] 61 vssesawkaa talavdhfgg ldilvnnagv mhwapieeld varterlldv nvlgnllgak

[0038] 121 svvpimkkag rgvivnissv dglrgvngla aytaskwavr gltkalayel gpsgirvcsv

[0039] 181 hpggvdttlg npggligddl qskyvgvplq rigesediar atlfvasdea syisgaelav

[0040] 241 dggwsagtyy pglpgtppal mpn.

[0041] 1. The sequence SEQ ID NO.1 was sent to Shanghai Qingke Biotechnology for whole-gene synthesis and constructed into the pET-28a plasmid expression vector to obtain the recombinant plasmid PET28a-ADH1.

[0042] 2. Introduce the recombinant plasmid into 100 μL using the thermal shock method. Escherichia coli BL21(DE3) competent cells were plated onto LB solid medium containing cannabinoids and incubated overnight at 37°C (approximately 16 hours). Larger single clones were selected and colony PCR (polymerase chain reaction) was performed using universal primers (T7, T7ter) to screen for positive clones, thereby obtaining the engineered bacteria (PET28a-ADH1-BL21).

[0043] 3. Inoculate the engineered bacterial strain into LB medium at a rate of 0.1% and incubate at 37°C for 12-16 hours to activate the strain. Then, inoculate the activated bacterial solution into fresh LB medium (containing 50 μg / ml kanamycin) at a rate of 2% and incubate at 37°C for 4 hours. Add IPTG (isopropyl-β-D-thiogalactoside) inducer to a final concentration of 0.01 mM and induce at 30°C for 16 hours. Collect the bacterial cells by centrifugation at 4°C. The induced recombinant engineered bacteria are obtained. Detect the enzyme activity of the recombinant engineered bacteria using an enzyme activity assay.

[0044] Example 2

[0045] Using ADH1 (amino acid sequence as shown in SEQ ID NO.2) as a template, a search was conducted in the PDB database. Using 1VWH and 1VWE, which showed high similarity to the target protein in the PDB database, as templates, homology modeling was performed using DS software. After obtaining the simulated conformation, the substrate molecule was docked, and site selection was performed. Sites within a 10 Å range centered on the substrate and substrate channel sites were analyzed. The structures were analyzed, and approximately 20 sites were selected for the establishment of a mutation library. Saturation mutations were performed on each site. Degenerate codon NNK was used to design mutation primers, and whole-plasmid PCR amplification was performed using the PET28a-ADH1 plasmid as a template. Reagents were purchased from Takara, and the PCR system was prepared according to its instructions. The PCR program was 98℃ for 3 min; 98℃ for 15 s; 55℃ for 30 s; 72℃ for 3 min, 30 cycles, followed by incubation at 16℃. The PCR products obtained in the above steps were verified by agarose gel electrophoresis, and then digested with restriction endonuclease DpnI at 37℃ for 2 h. Transfer digestion products to E. coli BL21(DE3) competent cells were plated on plates containing 10 μg / mL kanamycin and incubated at 37°C for about 12 hours. Single colonies grew, yielding a 3β-ketoreductase mutant library.

[0046] The monoclonal colonies obtained after culturing in step 2 above were picked and placed in LB liquid medium containing 4 mL of ampicillin (100 mg / L) (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl), and cultured overnight at 37°C and 200 rpm to obtain the culture solution.

[0047] The culture medium was inoculated into fermentation medium (LB liquid medium) at a rate of 1% (v / v) and cultured at 37°C and 200 rpm on a shaker until the OD600 reached 0.6-0.8. IPTG was then added to a final concentration of 0.1 mM, and the culture was induced at 25°C and 200 rpm for 8-12 hours. The bacterial cells were collected by centrifugation at 6000 g, and then subjected to high-pressure disruption to obtain a crude ketone reductase enzyme solution for subsequent enzyme activity assays.

[0048] Beneficial mutation sites for enhancing enzyme activity were identified through enzyme activity assays: 79, 116, 132, 144, and 225. The enzyme activity assay method was as follows: Experimental groups were performed in triplicate, with an additional control group. 260 μL of Tris-HCl (0.05 M, pH 7.0) was added to the wells of the microplate, followed by 5 μL of 5AD stock solution (0.5% DMSO solution). The experimental groups received 20 μL of diluted enzyme solution, while the control group received 20 μL of water. All samples were shaken to mix thoroughly and incubated at 30°C for 10 min. Immediately after incubation, 15 μL of 2 g / L NADH stock solution was added, and the absorbance was measured at 340 nm.

[0049] Enzyme activity is defined as the amount of enzyme required to catalyze the conversion of 1 μM substrate into product per minute under optimal conditions. One unit of enzyme activity (U) is defined as the amount of enzyme required to catalyze the conversion of 1 μM substrate into product per minute.

[0050] Based on the results of saturation mutagenesis, a combination mutant with a single mutation site was constructed. The resulting mutants were picked and cultured in test tubes containing 4 mL of LB medium, and the activity of the expressed protein was detected.

[0051] The results are shown in Table 1.

[0052] Table 1. Relative enzyme activities of each mutant

[0053]

[0054] As shown in Table 1, mutants 12 and 14 had the highest activity, which was 26 and 31 times higher than that of the wild type, respectively.

[0055] The sequence of SEQ ID NO.3 is:

[0056] 1 marldgkvai itgaaqgmga atarlfvaeg arvllgdvld ekgralaeel gdaaiftpld

[0057] 61 vssesawkaa talavdhfrg ldilvnnagv mhwapieeld varterlldv nvlgnllgak

[0058] 121 svvpimkkag rgvivnissv dglrgvngla aytaskwavr gltkalayel gpsgirvcsv

[0059] 181 hpggvdttlg npggligddl qskyvgvplq rigesediar atlfvasdea syisgaelav

[0060] 241 dggwsagtyy pglpgtppal mpn。

[0061] SEQ ID NO.4

[0062] 1 marldgkvai itgaaqgmga atarlfvaeg arvllgdvld ekgralaeel gdaaiftpld

[0063] 61 vssesawkaa talavdhfvg ldilvnnagv mhwapieeld varterlldv nvlgnllgak

[0064] 121 svvpimkkag rgvivnissv dglrgvngla aytaskwavr gltkalayel gpsgirvcsv

[0065] 181 hpggvdttlg npggligddl qskyvgvplq rigesediar atlfvasdea syisgaelav

[0066] 241 dggwsagtyy pglpgtppal mpn。

[0067] SEQ ID NO.5

[0068] 1 marldgkvai itgaaqgmga atarlfvaeg arvllgdvld ekgralaeel gdaaiftpld

[0069] 61 vssesawkaa talavdhfgg ldilvnnagv mhwapieeld varterlldv nvlgnslgak

[0070] 121 svvpimkkag rgvivnissv dglrgvngla aytaskwavr gltkalayel gpsgirvcsv

[0071] 181 hpggvdttlg npggligddl qskyvgvplq rigesediar atlfvasdea syisgaelav

[0072] 241 dggwsagtyy pglpgtppal mpn。

[0073] SEQ ID NO. 6

[0074] 1 marldgkvai itgaaqgmga atarlfvaeg arvllgdvld ekgralaeel gdaaiftpld

[0075] 61 vssesawkaa talavdhfgg ldilvnnagv mhwapieeld varterlldv nvlgntlgak

[0076] 121 svvpimkkag rgvivnissv dglrgvngla aytaskwavr gltkalayel gpsgirvcsv

[0077] 181 hpggvdttlg npggligddl qskyvgvplq rigesediar atlfvasdea syisgaelav

[0078] 241 dggwsagtyy pglpgtppal mpn。

[0079] SEQ ID NO.7

[0080] 1 marldgkvai itgaaqgmga atarlfvaeg arvllgdvld ekgralaeel gdaaiftpld

[0081] 61 vssesawkaa talavdhfgg ldilvnnagv mhwapieeld varterlldv nvlgnllgak

[0082] 121 svvpimkkag ravivnissv dglrgvngla aytaskwavr gltkalayel gpsgirvcsv

[0083] 181 hpggvdttlg npggligddl qskyvgvplq rigesediar atlfvasdea syisgaelav

[0084] 241 dggwsagtyy pglpgtppal mpn。

[0085] SEQ ID NO.8

[0086] 1 marldgkvai itgaaqgmga atarlfvaeg arvllgdvld ekgralaeel gdaaiftpld

[0087] 61 vssesawkaa talavdhfgg ldilvnnagv mhwapieeld varterlldv nvlgnllgak

[0088] 121 svvpimkkag rgvivnissv dgldgvngla aytaskwavr gltkalayel gpsgirvcsv

[0089] 181 hpggvdttlg npggligddl qskyvgvplq rigesediar atlfvasdea syisgaelav

[0090] 241 dggwsagtyy pglpgtppal mpn。

[0091] SEQ ID NO. 9

[0092] 1 marldgkvai itgaaqgmga atarlfvaeg arvllgdvld ekgralaeel gdaaiftpld

[0093] 61 vssesawkaa talavdhfgg ldilvnnagv mhwapieeld varterlldv nvlgnllgak

[0094] 121 svvpimkkag rgvivnissv dglrgvngla aytaskwavr gltkalayel gpsgirvcsv

[0095] 181 hpggvdttlg npggligddl qskyvgvplq rigesediar atlflasdea syisgaelav

[0096] 241 dggwsagtyy pglpgtppal mpn。

[0097] SEQ ID NO.10

[0098] 1 marldgkvai itgaaqgmga atarlfvaeg arvllgdvld ekgralaeel gdaaiftpld

[0099] 61 vssesawkaa talavdhfgg ldilvnnagv mhwapieeld varterlldv nvlgnllgak

[0100] 121 svvpimkkag rgvivnissv dglrgvngla aytaskwavr gltkalayel gpsgirvcsv

[0101] 181 hpggvdttlg npggligddl qskyvgvplq rigesediar atlfsasdea syisgaelav

[0102] 241 dggwsagtyy pglpgtppal mpn。

[0103] The sequence of SEQ ID NO.11 is:

[0104] 1 marldgkvai itgaaqgmga atarlfvaeg arvllgdvld ekgralaeel gdaaiftpld

[0105] 61 vssesawkaa talavdhfrg ldilvnnagv mhwapieeld varterlldv nvlgnslgak

[0106] 121 svvpimkkag rgvivnissv dglrgvngla aytaskwavr gltkalayel gpsgirvcsv

[0107] 181 hpggvdttlg npggligddl qskyvgvplq rigesediar atlfvasdea syisgaelav <​​​​​​1 marldgkvai itgaaqgmga atarlfvaeg arvllgdvld ekgralaeel gdaaiftpld

[0111] 61 vssesawkaa talavdhfgg ldilvnnagv mhwapieeld varterlldv nvlgnllgak

[0112] 121 svvpimkkag ravivnissv dgldgvngla aytaskwavr gltkalayel gpsgirvcsv

[0113] 181 hpggvdttlg npggligddl qskyvgvplq rigesediar atlfvasdea syisgaelav

[0114] 241 dggwsagtyy pglpgtppal mpn。

[0115] SEQ ID NO. 13

[0116] 1 marldgkvai itgaaqgmga atarlfvaeg arvllgdvld ekgralaeel gdaaiftpld

[0117] 61 vssesawkaa talavdhfrg ldilvnnagv mhwapieeld varterlldv nvlgnllgak

[0118] 121 svvpimkkag rgvivnissv dglrgvngla aytaskwavr gltkalayel gpsgirvcsv

[0119] 181 hpggvdttlg npggligddl qskyvgvplq rigesediar atlflasdea syisgaelav

[0120] 241 dggwsagtyy pglpgtppal mpn。

[0121] SEQ ID NO. 14

[0122] 1 marldgkvai itgaaqgmga atarlfvaeg arvllgdvld ekgralaeel gdaaiftpld

[0123] 61 vssesawkaa talavdhfgg ldilvnnagv mhwapieeld varterlldv nvlgnllgak

[0124] 121 svvpimkkag ravivnissv dglrgvngla aytaskwavr gltkalayel gpsgirvcsv

[0125] 181 hpggvdttlg npggligddl qskyvgvplq rigesediar atlflasdea syisgaelav

[0126] 241 dggwsagtyy pglpgtppal mpn。

[0127] SEQ ID NO.15

[0128] 1 marldgkvai itgaaqgmga atarlfvaeg arvllgdvld ekgralaeel gdaaiftpld

[0129] 61 vssesawkaa talavdhfgg ldilvnnagv mhwapieeld varterlldv nvlgnllgak

[0130] 121 svvpimkkag ravivnissv dgldgvngla aytaskwavr gltkalayel gpsgirvcsv

[0131] 181 hpggvdttlg npggligddl qskyvgvplq rigesediar atlflasdea syisgaelav

[0132] 241 dggwsagtyy pglpgtppal mpn。

[0133] SEQ ID NO. 16

[0134] 1 marldgkvai itgaaqgmga atarlfvaeg arvllgdvld ekgralaeel gdaaiftpld

[0135] 61 vssesawkaa talavdhfgg ldilvnnagv mhwapieeld varterlldv nvlgnslgak

[0136] 121 svvpimkkag ravivnissv dglrgvngla aytaskwavr gltkalayel gpsgirvcsv

[0137] 181 hpggvdttlg npggligddl qskyvgvplq rigesediar atlflasdea syisgaelav

[0138] 241 dggwsagtyy pglpgtppal mpn.

[0139] Example 3

[0140] Preparation of ketone-reducing mutant enzyme solution:

[0141] Prepare 100 mL of LB liquid medium as seed culture medium and autoclave at 121 °C for 20 min. After cooling, use an inoculation loop to pick a single colony of mutant 14 and inoculate it into the medium. Incubate overnight at 37 °C and 200 rpm to prepare the seed culture. Prepare 1 L of fermentation medium using LB liquid medium. Inoculate 20 mL of the overnight cultured seed culture into the fermentation medium and incubate at 37 °C and 200 rpm until the O.D600 reaches 0.6-0.8. Add 0.01 mM IPTG to induce expression for 12 hours. Collect the cells by centrifugation at 8000 rpm at 4 °C. Weigh a portion of the cells, resuspend them in pure water, adjust the pH to 7.0 with phosphate, prepare a 300 g / L bacterial solution, and add a small amount of nuclease. Disrupt the cells using an autoclave and store at -20 °C. Freeze the remaining portion at -20 °C to obtain the ketone-reducing mutant enzyme solution.

[0142] Example 4

[0143] Weigh 10g of 5-AD and 10g of glucose monohydrate into a conversion flask, add 45ml of pure water and 30ml of tert-butanol, stir to disperse evenly, then add 10ml of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (pH 7.0, 0.5M), maintain the temperature at 30℃ and continue stirring until homogeneous. Add 5ml of the ketone reduction mutant enzyme solution from Example 3 with a concentration of 300g / L, and 1ml of glucose dehydrogenase; finally, add 0.02g of NAD.+ Initiate the reaction. During the reaction, maintain the pH of the system at 6.8-7.0 using 10% sodium hydroxide solution. Monitor the substrate residue using TLC during the reaction. Take 0.1 ml of the reaction sample, add 0.4 ml of dichloromethane as the extraction solvent, shake thoroughly, centrifuge for 20 s, and use a capillary pipette to collect the supernatant, spotting it onto a silica gel thin-layer plate. Develop with ethyl acetate:petroleum ether (1:1) as the developing solvent. Observe the substrate spots, stain with phosphomolybdic acid, and bake at 60℃ for further color development. The conversion rate is 99% after 1 hour of transformation.

[0144] Example 5

[0145] Weigh 10g of 5-AD and 10g of glucose monohydrate into a conversion flask, add 48ml of pure water and 30ml of tert-butanol, stir to disperse evenly, then add 10ml of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (pH 7.0, 0.5M), maintain the temperature at 30℃ and continue stirring until homogeneous. Add 2.5ml of the ketone reduction mutant enzyme solution from Example 3 (concentration 300g / L) and 1ml of glucose dehydrogenase; finally add 0.02g of NAD. + Initiate the reaction. During the reaction, the pH of the system was controlled at 6.8-7.0 using 10% sodium hydroxide solution. The substrate residue was monitored by TLC during the reaction. 0.1 ml of the reaction sample was taken, and 0.4 ml of dichloromethane was added as the extraction solvent. After thorough shaking, the mixture was centrifuged for 20 seconds. The supernatant was aspirated using a capillary pipette and spotted onto a silica gel thin-layer plate. The developing solvent was ethyl acetate:petroleum ether = 1:1. UV staining was performed, and the substrate was observed and stained with phosphomolybdic acid. The plate was then baked at 60°C for further color development. The conversion rate was 99% after 2 hours of transformation.

[0146] Example 6

[0147] Weigh 10g of 5-AD and 10g of glucose monohydrate into a conversion flask, add 49ml of purified water and 30ml of tert-butanol, stir to disperse evenly, then add 10ml of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (pH 6.0, 0.5M), maintain the temperature at 30℃ and continue stirring until homogeneous. Add 1ml of the ketone reduction mutant enzyme solution from Example 3 (concentration 300g / L) and 1ml of glucose dehydrogenase; finally, add 0.02g of NAD. + Initiate the reaction. During the reaction, the pH of the system was controlled at 6.4-6.5 using 10% sodium hydroxide solution. The substrate residue was monitored by TLC during the reaction. 0.1 ml of the reaction sample was taken, and 0.4 ml of dichloromethane was added as the extraction solvent. After thorough shaking, the mixture was centrifuged for 20 seconds. The supernatant was aspirated using a capillary pipette and spotted onto a silica gel thin-layer plate. The developing solvent was ethyl acetate:petroleum ether = 1:1. UV staining was performed, and the substrate was observed and stained with phosphomolybdic acid. The plate was then baked at 60°C for further color development. The conversion rate was 99% after 1 hour of transformation.

[0148] Example 7

[0149] Weigh 10g of 4-AD ester and 10g of glucose monohydrate into a conversion flask. Add 30ml of pure water and 40ml of tert-butanol, and stir to disperse evenly. Then add 10ml of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (pH 7.0, 0.5M), and continue stirring at 30℃ until homogeneous. Add 3ml of lipohydrolase solution (300g / L), 3ml of ketoreductase mutant solution from Example 3, and 1ml of glucose dehydrogenase. Finally, add 0.02g of NAD. + Initiate the reaction. During the reaction, the pH of the system was controlled at 6.5-6.8 using 10% sodium hydroxide solution. The substrate residue was monitored by TLC during the reaction. 0.1 ml of the reaction sample was taken, and 0.4 ml of dichloromethane was added as the extraction solvent. After thorough shaking, the mixture was centrifuged for 20 seconds. The supernatant was aspirated using a capillary pipette and spotted onto a silica gel thin-layer plate. The developing solvent was ethyl acetate:petroleum ether = 1:1. UV staining was performed, and the substrate was observed after phosphomolybdic acid staining. The plate was then baked at 60℃ for further color development. The conversion rate was 99% after 4 hours of transformation.

[0150] Example 8

[0151] Weigh 10g of 4-AD ester and 10g of glucose monohydrate into a conversion flask. Add 20ml of purified water and 50mL of tert-butanol, and stir to disperse evenly. Then add 10mL of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (pH 7.0, 0.5M), and continue stirring at 30℃ until homogeneous. Add 3ml of enzyme solution with a concentration of 300g / L, 3ml of ketoreductase mutant enzyme solution from Example 3, and 1ml of glucose dehydrogenase; finally, add 0.02g of NAD. + Initiate the reaction. During the reaction, the pH of the system was controlled at 6.5-6.8 using 10% sodium hydroxide solution. The substrate residue was monitored by TLC during the reaction. 0.1 ml of the reaction sample was taken, and 0.4 ml of dichloromethane was added as the extraction solvent. After thorough shaking, the mixture was centrifuged for 20 seconds. The supernatant was aspirated using a capillary pipette and spotted onto a silica gel thin-layer plate. The developing solvent was ethyl acetate:petroleum ether = 1:1. UV staining was performed, and the substrate was observed after phosphomolybdic acid staining. The plate was then baked at 60℃ for further color development. The conversion rate was 99% after 4 hours of transformation.

[0152] The above experiments show that the constructed recombinant strain has an enzyme activity as high as 12685 U / L. The combined catalysis of 4AD esterification by ketone reductase and glucose dehydrogenase meets industrial standards. It also exhibits high conversion rates even at high concentrations of tert-butanol, demonstrating tolerance to high concentrations of tert-butanol. Subsequent transformation using fermentation cells eliminates concerns about enzyme release during the transformation system.

[0153] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of protection of this application is limited to these examples; within the framework of this application, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of different aspects of one or more embodiments of this application as described above, which are not provided in detail for the sake of brevity.

[0154] One or more embodiments in this application are intended to cover all such substitutions, modifications, and variations that fall within the broad scope of this application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of one or more embodiments in this application should be included within the protection scope of this application.

Claims

1. A ketone reductase, characterized in that, The ketone reductase is obtained by mutation based on the amino acid sequence SEQ ID NO.2, and the mutation is any one of the following: The mutation site is position 132, where position 132 is mutated to A; The mutation at position 132 is A, and the mutation at position 225 is L; The mutation at position 132 is A, the mutation at position 225 is L, and the mutation at position 144 is D; Alternatively, the 132nd position may mutate to A, the 225th position to L, and the 116th position to S.

2. A ketone reductase gene, characterized in that, Used to encode the ketone reductase as described in claim 1.

3. A ketone-reducing mutant, characterized in that, The ketone reductase mutant contains the ketone reductase gene as described in claim 2, or a plasmid containing the ketone reductase gene as described in claim 2.

4. An application of the ketone reductase as described in claim 1, characterized in that, The ketone reductase is used to catalyze the conversion of a substrate into DHEA, wherein the substrate is a 5-AD or 4-AD ester.

5. The application as described in claim 4, characterized in that, The substrate, glucose, tert-butanol, buffer solution, ketone reductase and glucose dehydrogenase were mixed, an electron acceptor was added, an extraction solvent was added after the reaction, and the mixture was centrifuged to obtain the product.

6. The application as described in claim 5, characterized in that, The electron acceptor is NAD. + The extractant is dichloromethane, and the pH value is controlled at 6-7 during the reaction.

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