A transaminase mutant and its application in the production of sitagliptin intermediates

Abstract

The present invention discloses a mutant of a transaminase and its application in the production of a sitagliptin intermediate, wherein the mutant is an amino acid sequence mutated on the basis of the amino acid sequence of the transaminase as shown in SEQ ID NO: 2, and the mutation site is any single point or multiple point mutation in the 10th, 28th, and 271st positions. The activity of the mutated transaminase in methanol is greatly improved, and it can react in a methanol system. Methanol replaces DMSO, which can effectively solve the problem that the price of DMSO is high, it is basically impossible to recover it after use, and the production cost is very high. The present invention increases the activity of the enzyme in methanol by five times, and can be applied in industrialization.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and mainly relates to a mutant of transaminase and application thereof in the production of a sitagliptin intermediate. Background Art

[0002] Sitagliptin is one of a class of anti-hyperglycemic drugs that inhibit dipeptidyl peptidase-4 (DPP-4). It controls blood sugar levels by protecting endogenous incretins and enhancing their effects. Glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide-1 (GLP-1) are incretins released in response to dietary intake. Due to its highly effective glucose control effect, sitagliptin has been approved in many countries for the treatment of type II diabetes.

[0003] The main method for preparing sitagliptin by chemical method is asymmetric hydrogenation of enamine amide, and the reaction conditions required are heavy metal rhodium catalysis reaction under 250psi high pressure. The high pressure and heavy metals required in the chemical preparation process are not conducive to industrial green production. Therefore, the combined preparation method of chemistry and biological enzymes is gradually adopted by more and more manufacturers. Chinese patent CN102405281 discloses a transaminase and the application of the enzyme in the preparation of sitagliptin. The process uses DMSO as a substrate cosolvent to convert the substrate 4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazine-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-one (hereinafter referred to as: ketoamide substrate) into 2R-4-oxo-4-T3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazine-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine (hereinafter referred to as: product) under the joint action of isopropylamine (amino donor) and coenzyme pyridoxal phosphate (PLP). However, in actual production, the DMSO used in the process causes great damage to the enzyme and is difficult to recover, resulting in excessively high production costs, which is not conducive to large-scale industrial production.

[0004] Therefore, there is an urgent need in the art to develop a biocatalytic method that has high catalytic activity in a recyclable solvent and can reduce production costs. Specifically, there is a need to develop a method for preparing sitagliptin using an enzyme that efficiently catalyzes the conversion of substrates into products in a methanol solvent. Summary of the invention

[0005] The present invention aims at solving the problems that the current reaction system uses DMSO as solvent, which is expensive and cannot be recovered after use, and the transaminase has low activity in methanol. The transaminase is mutated to obtain a mutant capable of preparing sitagliptin in a methanol solvent.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] A transaminase mutant, wherein the mutant is an amino acid sequence mutated based on the amino acid sequence of the transaminase as shown in SEQ ID NO: 2, and the mutation site is any single point or multiple point mutation at position 10, position 28, and position 271.

[0008] The mutant is a mutant in which isoleucine is mutated to asparagine at position 10 of the amino acid sequence of the transaminase shown in SEQ ID NO:2, and is named as I10N mutant enzyme, and its amino acid sequence is shown in SEQ ID NO:3.

[0009] The mutant is a mutant in which leucine is mutated to glycine at position 28 of the amino acid sequence of the transaminase shown in SEQ ID NO:2, and is named L28G mutant enzyme, and its amino acid sequence is shown in SEQ ID NO:5.

[0010] The mutant is a mutant in which glutamic acid is mutated to aspartic acid at position 271 of the amino acid sequence of the transaminase shown in SEQ ID NO:2, and is named E271D mutant enzyme, and its amino acid sequence is shown in SEQ ID NO:7.

[0011] The mutant is a mutant in which isoleucine is mutated to asparagine at position 10, leucine is mutated to glycine at position 28, and glutamic acid is mutated to aspartic acid at position 271 of the amino acid sequence of the transaminase shown in SEQ ID NO:2, and is named I10N / L28G / E271D mutant enzyme, and its amino acid sequence is shown in SEQ ID NO:8.

[0012] An expressed gene, which encodes the transaminase mutant.

[0013] A recombinant plasmid is connected with the expression gene.

[0014] A recombinant cell comprising the above recombinant expression plasmid or the expression gene of the above transaminase mutant.

[0015] Construction of transaminase mutant expression vector:

[0016] The transaminase mutant gene sequence was amplified by PCR, and nucleotide electrophoresis was performed using 0.9% agarose gel. After the bands appeared clearly, the target band of about 1000 bp was cut with gel strips, and the target DNA fragment was recovered using a gel recovery kit (Takara Biotech). Then, the obtained DNA fragment and vector pET28b+ (Novagen) were double-digested with restriction endonucleases NdeI and XhoI according to the method described in the 2009 product catalog instructions of Takara Biotech (website: www.takara.com.cn).

[0017] The digested product was subjected to DNA electrophoresis at a voltage of 100 volts again, and the corresponding fragments and vectors of about 1000 bp and 5300 bp were recovered. Finally, the fragments and vectors were mixed in a ratio of 3:1, and 1.5 units of T4 ligase were added, and the connection was carried out at 16°C for 12 hours. After the ligation reaction, the ligation product was transformed into E. coli DH5-alpha competent cells, and positive clones were screened on LB plates containing kanamycin. After double digestion with NdeI and XhoI and DNA sequencing, positive clones containing a target DNA sequence of 1000 bp and their recombinant plasmids were obtained. The obtained recombinant plasmid was transformed into E. coli BL21 (DE3) by the calcium chloride transformation method, and the E. coli expression strain of the transaminase mutant was obtained.

[0018] A single microbial colony of the transaminase expression strain was inoculated into LB medium containing kanamycin and grown overnight with shaking. The overnight culture was inoculated into TB medium at an inoculum size of 5-15% and cultured with shaking. 600 When the pH is 0.7-0.8, 1 mM IPTG (isopropyl-β-D-thiogalactoside) is used to induce the expression of the ribokinase gene; after the culture is completed, the cells are collected by centrifugation and the supernatant is discarded, the cells are mechanically broken in a high-pressure homogenizer and centrifuged, and the supernatant is the obtained soluble transaminase mutant crude enzyme solution.

[0019] TB medium was: 12 g / L tryptone, 24 g / L yeast extract, 4 mL / L glycerol, 65 mM potassium phosphate pH 7.0, 1 mM magnesium sulfate, and 100 μg / mL kanamycin.

[0020] The transaminase mutant is used in preparing sitagliptin in a methanol solvent.

[0021] The method for preparing sitagliptin using the transaminase mutant in a methanol solvent comprises the following steps:

[0022] S1, weigh water, triethanolamine, isopropylamine, mix, adjust pH to 8.5, then add coenzyme pyridoxal phosphate PLP, enzyme solution, stir until completely mixed;

[0023] S2, methanol was added, stirring was continued, and the temperature was raised to 45 ℃, and then the pH was adjusted to 8.5 using 4M isopropylamine;

[0024] S3. Slowly add the dissolved ketoamide substrate to the reaction system for 2-5 hours. At the same time, evacuate the reaction system (vacuum degree 300 torr). Use 4 M isopropylamine to control pH 8.5 throughout the reaction.

[0025] The final concentration of the reaction system is substrate: water: methanol = 1: 5: 5, enzyme amount = substrate × 50%, reaction time is 24 hours, and temperature is 45°C.

[0026] Further preferably, the method for preparing sitagliptin using the transaminase mutant in a methanol solvent comprises the following steps:

[0027] S1, weigh 90mL of water, 1.94g of triethanolamine, 32.8g of 4M isopropylamine, mix well, adjust pH to 8.5, then add PLP0.134g, 10g of enzyme solution, stir well until completely mixed;

[0028] S2, add 10g of methanol, continue stirring, and raise the temperature to 45 ° C, then use 4M isopropylamine to adjust the pH to 8.5, and start adding ketoamide substrate;

[0029] S3. Slowly add the dissolved ketoamide substrate to the reaction system for about 3 hours, and complete the addition of all the substrates. At the same time, evacuate the reaction system (vacuum degree 300 torr), and use 4M isopropylamine to control pH 8.5 throughout the reaction.

[0030] Beneficial effects:

[0031] The present invention mutates the sequence, and the activity of the mutated transaminase in methanol is greatly improved, and it can react in a methanol system. Methanol replaces DMSO, which can effectively solve the problem of high production cost due to the high price of DMSO and the fact that it is basically impossible to recover it after use. The present invention increases the activity of the enzyme in methanol by five times and can be applied in industrialization. DETAILED DESCRIPTION

[0032] The technical scheme of the present invention is described in detail below in conjunction with specific embodiments. The DNA sequences involved in the present invention are synthesized using conventional methods, and the experimental conditions and operations not mentioned in the embodiments of the present invention are performed according to conventional methods in the art or conditions recommended by manufacturers.

[0033] 4-Oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-one (hereinafter referred to as ketoamide substrate);

[0034] 2R-4-Oxy-4-T3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazine-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine (hereinafter referred to as: product)

[0035] Example 1 Cloning of transaminase gene and construction of expression vector

[0036] Based on the amino acid sequence of the aminotransferase reported in the Chinese patent application CN102405281, a pair of primers TA-F: 5'-CATATGGCGTTCTCAGCGG-3' and TA-R: 5'-CTCGAGTTAGTACTGTACCG-3' were designed for polymerase chain reaction (PCR reaction). The reaction system included 18 μl of redistilled water, 2.5 μl of PCR buffer, 1 μl of magnesium chloride (25 mM concentration), 1.5 μl of dNTP (2.5 mM concentration), 0.5 μl of the above two primers, 20 ng of the above template, and 2 units of Taq enzyme (Shanghai Sangon Company).

[0037] PCR conditions: denaturation at 94°C for 10 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 1 minute, for a total of 30 cycles; and finally extension at 72°C for 10 minutes. The nucleotide sequence of the transaminase gene is shown in Sequence 1 of the sequence listing, and its amino acid sequence is shown in Sequence 2 of the sequence listing.

[0038] Example 2 Construction of transaminase expression vector

[0039] After the PCR amplification reaction, 0.9% agarose gel was used for nucleotide electrophoresis at a voltage of 100 volts. After the bands appeared clearly, the target band of about 1000 bp was cut with gel strips, and the target DNA fragment was recovered using a gel recovery kit (Takara Biotech). Then, according to the method described in the 2009 edition product catalog instructions of Takara Biotech (website: www.takara.com.cn), the obtained DNA fragment and vector pET28b+ (Novagen) were double-digested with restriction endonucleases NdeI and XhoI.

[0040] The digested products were subjected to DNA electrophoresis at 100 volts again to recover fragments and vectors of approximately 1000 bp and 5300 bp in size. Finally, the fragments and vectors were mixed in a ratio of 3:1, 1.5 units of T4 ligase were added, and the mixture was connected at 16°C for 12 hours.

[0041] After the ligation reaction, 10 μl of the ligation product was transformed into Escherichia coli DH5-alpha competent cells, and positive clones were screened on LB plates containing kanamycin. After double restriction digestion with NdeI and XhoI and DNA sequencing verification, positive clones containing a target DNA sequence of 1000 bp and their recombinant plasmids were obtained.

[0042] Example 3 Construction of genetically engineered bacteria expressing transaminase

[0043] The recombinant plasmid finally obtained in Example 1.2 was transformed into E. coli BL21 (DE3) (Novagen) using the calcium chloride transformation method (Hao Fuying et al., ed., "Molecular Biology Experimental Techniques", Peking University Press, 1998, pp. 12-15) to obtain a transaminase E. coli expression strain.

[0044] Example 4 Fermentation of transaminase bacteria and preparation of crude enzyme solution

[0045] A single microbial colony of the obtained transaminase expression strain was inoculated into 50 ml of LB medium (1% peptone, 0.5% yeast extract, 1% sodium chloride) containing 100 μg / mL kanamycin. The cells were grown overnight (at least 12 hours) in a 37°C incubator with shaking at 250 rpm. 250 ml of TB medium (12 g / L tryptone, 24 g / L yeast extract, 4 mL / L glycerol, 65 mM potassium phosphate pH 7.0, 1 mM magnesium sulfate, 100 μg / mL kanamycin) was placed in a 1-liter culture flask, and the above overnight culture was inoculated into it at an inoculum size of 8%, and then cultured at 37°C with shaking. When the OD of the culture reached 250, the culture was inoculated into 250 ml of TB medium (12 g / L tryptone, 24 g / L yeast extract, 4 mL / L glycerol, 65 mM potassium phosphate pH 7.0, 1 mM magnesium sulfate, 100 μg / mL kanamycin). 600 When the pH value was 0.7 to 0.8, the temperature was lowered to 28°C, and the expression of the ribokinase gene was induced with 1 mM IPTG (isopropyl-β-D-thiogalactoside), and the culture was continued for at least 12 hours.

[0046] After the culture was completed, the cells were collected and the supernatant was discarded by centrifuging at 4°C and 5000 rpm for 15 minutes. The cell pellet was resuspended in an equal volume of 4°C, 100 mM pH7.0 triethanolamine (chloride) buffer, and the cells were collected after centrifugation at 5000 rpm for 20 minutes. The washed cells were resuspended in 2 volumes of 4°C 100 mM pH7.0 triethanolamine (chloride) buffer, and mechanically disrupted twice at 12000 psi in a high-pressure homogenizer (ATS), and the temperature was kept at 4°C throughout the process. Cell debris was removed by centrifugation at 4°C and 10000 rpm in a refrigerated high-speed centrifuge for 40 minutes, and the supernatant was the obtained soluble transaminase crude enzyme solution.

[0047] Example 5 Site-directed mutagenesis at positions 10, 28, and 271

[0048] Primers were designed according to the amino acids to be mutated and the target amino acid sites, and mutation was performed using the Mega WHOP method (Arnold and Georgiou 2003). Primer pairs containing the mutation of position 10 to asparagine were designed: I10N-F: 5'-ACCCCTGAAAACGTTTACACCC-3'; I10N-R: 5'-CTCGAGTTAGTACTGTACCG-3'. The PCR system included: 60 pmol of each primer, 100 ng of the plasmid template obtained in Example 2, 18 μl of redistilled water, 2.5 μl of PCR buffer (KODPCR buffer), 1 μl of magnesium chloride (25 mM concentration), 1.5 μl of dNTP (2.5 mM concentration), and KOD plus 2 enzyme activity units. The PCR amplification conditions are: the first step is 95°C for 5 minutes, the second step is 94°C for 45 seconds, the third step is 55°C for 40 seconds, the fourth step is 72°C for 1 minute, and the second to fourth steps are repeated for a total of 25 cycles, and the sixth step is 72°C for 10 minutes. After the amplification, electrophoresis analysis was performed to obtain a band of about 1kb, and the target DNA fragment was recovered using a gel recovery kit (Takara Biotech). Then, the recovered band was used as a primer for PCR, with 60pmol of each primer, 100ng of the plasmid template obtained in Example 2, 18μl of redistilled water, 2.5μl of PCR buffer (KOD PCR buffer), 1μl of magnesium chloride (25mM concentration), 1.5μl of dNTP (2.5mM concentration), and KOD plus 2 enzyme activity units. The PCR amplification conditions are: the first step is 95°C for 5 minutes, the second step is 94°C for 45 seconds, the third step is 55°C for 40 seconds, the fourth step is 72°C for 6 minutes, and the second to fourth steps are repeated for a total of 25 cycles, and the sixth step is 72°C for 10 minutes. After the amplification, DpnI enzyme was added to the reaction system and treated at 37 degrees for 1 hour to eliminate the plasmid template. After the nucleic acid was purified, it was transformed into Escherichia coli BL21 (DE3) competent cells. After culturing overnight, the grown colonies were picked for sequencing verification. The amino acid sequence of the successfully mutated transformant mutated from isoleucine to asparagine at the tenth position, named I10N mutant enzyme, and its amino acid sequence is shown in sequence 3.

[0049] According to the same method as above, mutations were continued at positions 28 and 271. The primers used for the amino acid mutation at position 28 were sequence 4 (L28G-F: 5'-TACGAAGGGGACCCGGCTAACCC-3') and sequence 9 (5'-CTCGAGTTAGTACTGTACCG-3'), which mutated from leucine to glycine and was named L28G mutant enzyme, and its complete amino acid sequence was sequence 5. The primers used for the amino acid mutation at position 271 were sequence 6 (E271D-F: 5'-ATGGCGTTCTCAGCGGACAC-3') and sequence 10 (E271D-R: 5'-TCGTACAGATCAGCCGGGGT-3'), which mutated from glutamic acid to aspartic acid and was named E271D mutant enzyme, and its complete amino acid sequence was sequence 7.

[0050] The I10N mutant enzyme, L28G mutant enzyme, and E271D mutant enzyme were fermented to produce enzymes according to the method of Example 4 to obtain the corresponding crude enzyme liquid.

[0051] Example 6 Comparison of enzyme activities of transaminase and mutant enzyme in DMSO and methanol solvents

[0052] The crude enzyme solution was subjected to enzyme activity assay under DMSO / methanol solvent conditions as follows:

[0053] Equipment: 30mL stoppered test tube, beaker, measuring cylinder, pipette, electronic balance, volumetric flask, constant temperature shaking water bath, HPLC;

[0054] Reagents: PLP, isopropylamine, DMSO / methanol, Tris (tris(hydroxymethyl)aminomethane), hydrochloric acid, crude enzyme solution.

[0055] Solution preparation: a. Weigh 0.335 g of PLP, dissolve it in deionized water to make up to 100 mL, and set aside.

[0056] b. Preparation of 4M isopropylamine: Weigh 236 g of isopropylamine, dilute to 1 L, and set aside.

[0057] c. Preparation of 0.1MPH8.5 Tris-HCl buffer: weigh 12.1g Tris (tris(hydroxymethyl)aminomethane), dissolve it in 900mL deionized water, adjust the pH to 8.5 with concentrated hydrochloric acid, make up to 1000mL, and set aside.

[0058] d. Weigh 5 g of substrate and dissolve it in 10 mL DMSO / methanol for later use

[0059] e. Take 30mL DMSO / methanol, 10mL 4M isopropylamine (b), 10mL PLP solution (a), and 30mL Tris-HCl solution (c), mix well in a beaker, adjust the pH to 8.5 with concentrated hydrochloric acid, and set aside.

[0060] Operation steps: Pipette 8mL of solution e into a stoppered test tube, add 0.5mL of crude enzyme solution, add 0.5mL of solution c to rinse the residual enzyme on the test tube wall, and finally pipette 1mL of solution d. Place in a 45℃ water bath and shake at 250rpm for 20min. Take a sample and add a drop of hydrochloric acid to terminate the reaction. Detect the product concentration A (mg / mL) by HPLC.

[0061] Enzyme activity definition: The amount of enzyme required to convert 1 micromole of product in 1 minute at 45°C.

[0062] Enzyme activity calculation formula: Enzyme activity (U / mL enzyme) = (1000×(A×10) / 407) / (20×0.5)

[0063] Note: A: product concentration mg / mL

[0064] The activity of the transaminase in DMSO solvent was taken as a benchmark and recorded as 100%. The relative activities of the transaminase and the three mutant enzymes in DMSO and methanol solvents were measured. The results are shown in Table 1.

[0065] Table 1:

[0066]

[0067] Example 7 Superposition mutation of mutant enzyme and determination of enzyme activity and conversion rate

[0068] According to the method provided in Example 5, site-directed mutations were performed on positions 10, 28, and 271 of the transaminase and sequenced for verification. The transformant with successful mutation was named I10N / L28G / E271D mutant enzyme, and its amino acid sequence was sequence 8.

[0069] According to the method of Example 4, a crude enzyme solution was prepared for enzyme activity determination and conversion experiments under DMSO and methanol solvents. The conversion method was as follows: 38 mL of water, 1.94 g of triethanolamine, and 32.8 mL of isopropylamine (4M) were weighed in the reaction device, mixed, and the pH was adjusted to 8.5 with 12M hydrochloric acid or concentrated hydrochloric acid, and then 0.134 g of PLP and 40 g of enzyme solution were added, and stirred for 5-10 min until completely mixed; 44.4 mL of DMSO or methanol was added, stirring was continued, and the temperature was raised to 45°C, and then 4M isopropylamine was used to adjust the pH to 8.5, and the ketoamide substrate was started to be added. Substrate flow addition: The dissolved ketoamide substrate was slowly added to the reaction system for 3 hours. At the same time, the reaction system was evacuated (vacuum degree 300 torr), and 4M isopropylamine was used to control the pH to 8.5 (±0.1) throughout the reaction. The analysis method is a high performance liquid chromatography, and the conditions are: the chromatographic column is Agilent Eclipse XDB-C8 (4.6 X 150mm / 5 microns), the eluent is 10mM concentration of NH4Ac / MeCN, the flow rate is 1.5ml / min, and the column temperature is 40 degrees Celsius. Retention time: 1.7 minutes for ketoamide substrate and 1.4 minutes for sitagliptin, and the ketoamide substrate and product in the eluate are determined as the peak area at 210nm or 286nm.

[0070] The comparison of the activities of transaminase and I10N / L28G / E271D mutant enzyme in DMSO and methanol and the product formation rate within one hour are shown in Table 2.

[0071] Table 2:

[0072]

[0073] The above embodiments are only for illustrating the ideas and technical features of the present invention, and their purpose is to enable people familiar with the field to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications to a certain extent made according to the ideas of the present invention should be included in the protection scope of the present invention. Sequence Listing <110> Jiangsu Meike Biotechnology Co., Ltd. <120> A transaminase mutant and its application in the production of sitagliptin intermediates <160> 12 <170> SIPOSequenceListing 1.0 <210> 1 <211> 993 <212> DNA <213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 1 atggcgttct cagcggacac ccctgaaatc gtttacaccc acgacaccgg tctggactat 60 atcacctact ctgactacga actggacccg gctaacccgc tggctggtgg tgctgcttgg 120 atcgaaggtg ctttcgttcc gccgtctgaa gctcgtatct ctatcttcga ccagggtttt 180 tatacttctg acgctaccta caccaccttc cacgtttgga acggtaacgc tttccgtctg 240 ggggaccaca tcgaacgtct gttctctaat gcggaatcta ttcgtttgat cccgccgctg 300 acccaggacg aagttaaaga gatcgctctg gaactggttg ctaaaaccga actgcgtgaa 360 gcgatggtta ccgtttctat cacccgtggt tactcttcta ccccattcga gcgtgacatc 420 accaaacatc gtccgcaggt ttacatgagc gcttgcccgt accagtggat cgtaccgttt 480 gaccgccatc gtgacggtgt tcacctgatg gttgctcagt cagttcgtcg tacaccgcgt 540 agctctatcg acccgcaggt taaaaacttc cagtggggtg acctgatccg tgcaattcag 600 gaaacccacg accgtggttt cgagttgccg ctgctgctgg actgcgacaa cctgctggct 660 gaaggtccgg gtttcaacgt tgttgttatc aaagacggtg ttgttcgttc tccgggtcgt 720 gctgctctgc cgggtatcac ccgtaaaacc gttctggaaa tcgctgaatc tctgggtcac 780 gaagctatcc tggctgacat caccccggct gaactgtacg acgctgacga agttctgggt 840 tgctcaaccg gtggtggtgt ttggccgttc gtttctgttg acggtaactc tatctctgac 900 ggtgttccgg gtccggttac ccagtctatc atccgtcgtt actgggaact gaacgttgaa 960 ccttcttctc tgctgacccc ggtacagtac taa 993 <210> 2 <211> 330 <212> PRT <213> Artificial Sequence (2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 2 Met Ala Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr 1 5 10 15 Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn 20 25 30 Pro Leu Ala Gly Gly Ala Ala Trp Ile Glu Gly Ala Phe Val Pro Pro 35 40 45 Ser Glu Ala Arg Ile Ser Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp 50 55 60 Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu 65 70 75 80 Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu 85 90 95 Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu 100 105 110 Val Ala Lys Thr Glu Leu Arg Glu Ala Met Val Thr Val Ser Ile Thr 115 120 125 Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg 130 135 140 Pro Gln Val Tyr Met Ser Ala Cys Pro Tyr Gln Trp Ile Val Pro Phe 145 150 155 160 Asp Arg His Arg Asp Gly Val His Leu Met Val Ala Gln Ser Val Arg 165 170 175 Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp 180 185 190 Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu 195 200 205 Leu Pro Leu Leu Leu Asp Cys Asp Asn Leu Leu Ala Glu Gly Pro Gly 210 215 220 Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg 225 230 235 240 Ala Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu 245 250 255 Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu 260 265 270 Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp 275 280 285 Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly 290 295 300 Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu 305 310 315 320 Pro Ser Ser Leu Leu Thr Pro Val Gln Tyr 325 330 <210> 3 <211> 330 <212> PRT <213> Artificial Sequence (2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 3 Met Ala Phe Ser Ala Asp Thr Pro Glu Asn Val Tyr Thr His Asp Thr 1 5 10 15 Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn 20 25 30 Pro Leu Ala Gly Gly Ala Ala Trp Ile Glu Gly Ala Phe Val Pro Pro 35 40 45 Ser Glu Ala Arg Ile Ser Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp 50 55 60 Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu 65 70 75 80 Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu 85 90 95 Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu 100 105 110 Val Ala Lys Thr Glu Leu Arg Glu Ala Met Val Thr Val Ser Ile Thr 115 120 125 Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg 130 135 140 Pro Gln Val Tyr Met Ser Ala Cys Pro Tyr Gln Trp Ile Val Pro Phe 145 150 155 160 Asp Arg His Arg Asp Gly Val His Leu Met Val Ala Gln Ser Val Arg 165 170 175 Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp 180 185 190 Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu 195 200 205 Leu Pro Leu Leu Leu Asp Cys Asp Asn Leu Leu Ala Glu Gly Pro Gly 210 215 220 Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg 225 230 235 240 Ala Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu 245 250 255 Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu 260 265 270 Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp 275 280 285 Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly 290 295 300 Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu 305 310 315 320 Pro Ser Ser Leu Leu Thr Pro Val Gln Tyr 325 330 <210> 4 <211> 23 <212> DNA <213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 4 tacgaagggg acccggctaa ccc 23 <210> 5 <211> 330 <212> PRT <213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 5 Met Ala Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr 1 5 10 15 Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Gly Asp Pro Ala Asn 20 25 30 Pro Leu Ala Gly Gly Ala Ala Trp Ile Glu Gly Ala Phe Val Pro Pro 35 40 45 Ser Glu Ala Arg Ile Ser Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp 50 55 60 Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu 65 70 75 80 Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu 85 90 95 Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu 100 105 110 Val Ala Lys Thr Glu Leu Arg Glu Ala Met Val Thr Val Ser Ile Thr 115 120 125 Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg 130 135 140 Pro Gln Val Tyr Met Ser Ala Cys Pro Tyr Gln Trp Ile Val Pro Phe 145 150 155 160 Asp Arg His Arg Asp Gly Val His Leu Met Val Ala Gln Ser Val Arg 165 170 175 Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp 180 185 190 Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu 195 200 205 Leu Pro Leu Leu Leu Asp Cys Asp Asn Leu Leu Ala Glu Gly Pro Gly 210 215 220 Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg 225 230 235 240 Ala Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu 245 250 255 Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu 260 265 270 Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp 275 280 285 Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly 290 295 300 Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu 305 310 315 320 Pro Ser Ser Leu Leu Thr Pro Val Gln Tyr 325 330 <210> 6 <211> 20 <212> DNA <213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 6 atggcgttct cagcggacac 20 <210> 7 <211> 330 <212> PRT <213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 7 Met Ala Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr 1 5 10 15 Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn 20 25 30 Pro Leu Ala Gly Gly Ala Ala Trp Ile Glu Gly Ala Phe Val Pro Pro 35 40 45 Ser Glu Ala Arg Ile Ser Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp 50 55 60 Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu 65 70 75 80 Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu 85 90 95 Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu 100 105 110 Val Ala Lys Thr Glu Leu Arg Glu Ala Met Val Thr Val Ser Ile Thr 115 120 125 Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg 130 135 140 Pro Gln Val Tyr Met Ser Ala Cys Pro Tyr Gln Trp Ile Val Pro Phe 145 150 155 160 Asp Arg His Arg Asp Gly Val His Leu Met Val Ala Gln Ser Val Arg 165 170 175 Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp 180 185 190 Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu 195 200 205 Leu Pro Leu Leu Leu Asp Cys Asp Asn Leu Leu Ala Glu Gly Pro Gly 210 215 220 Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg 225 230 235 240 Ala Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu 245 250 255 Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Asp Leu 260 265 270 Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp 275 280 285 Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly 290 295 300 Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu 305 310 315 320 Pro Ser Ser Leu Leu Thr Pro Val Gln Tyr 325 330 <210> 8 <211> 330 <212> PRT <213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 8 Met Ala Phe Ser Ala Asp Thr Pro Glu Asn Val Tyr Thr His Asp Thr 1 5 10 15 Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Gly Asp Pro Ala Asn 20 25 30 Pro Leu Ala Gly Gly Ala Ala Trp Ile Glu Gly Ala Phe Val Pro Pro 35 40 45 Ser Glu Ala Arg Ile Ser Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp 50 55 60 Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu 65 70 75 80 Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu 85 90 95 Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu 100 105 110 Val Ala Lys Thr Glu Leu Arg Glu Ala Met Val Thr Val Ser Ile Thr 115 120 125 Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg 130 135 140 Pro Gln Val Tyr Met Ser Ala Cys Pro Tyr Gln Trp Ile Val Pro Phe 145 150 155 160 Asp Arg His Arg Asp Gly Val His Leu Met Val Ala Gln Ser Val Arg 165 170 175 Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp 180 185 190 Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu 195 200 205 Leu Pro Leu Leu Leu Asp Cys Asp Asn Leu Leu Ala Glu Gly Pro Gly 210 215 220 Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg 225 230 235 240 Ala Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu 245 250 255 Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Asp Leu 260 265 270 Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp 275 280 285 Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly 290 295 300 Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu 305 310 315 320 Pro Val Gln Tyr Free Mp3 Download 325 330 <210> 9 <211> 20 <212> DNA <213> Material(2 Lateral Ambystoma x Ambystoma jeffersonianum) <400> 9 ctcgagttag tactgtaccg <210> 10 <211> 20 <212> DNA <213> Material(2 Lateral Ambystoma x Ambystoma jeffersonianum) <400> 10 tcgtacagat cagccggggt <210> 11 <211> 19 <212> DNA <213> Material(2 Lateral Ambystoma x Ambystoma jeffersonianum) <400> 11 catatggcgt tctcagcgg <210> 12 <211> 20 <212> DNA <213> Material(2 Lateral Ambystoma x Ambystoma jeffersonianum) <400> 12 ctcgagttag tactgtaccg

Claims

1. A transaminase mutant, characterized in that: The mutant is a mutant in which isoleucine is mutated to asparagine at position 10 of the amino acid sequence of the transaminase as shown in SEQ ID NO:

2.

2. A transaminase mutant, characterized in that: The mutant is a mutant in which leucine is mutated to glycine at position 28 of the amino acid sequence of the transaminase as shown in SEQ ID NO:

2.

3. A transaminase mutant, characterized in that: The mutant is a mutant in which glutamic acid is mutated to aspartic acid at position 271 of the amino acid sequence of the transaminase as shown in SEQ ID NO:

2.

4. A transaminase mutant, characterized in that: The mutant is a mutant in which isoleucine is mutated to asparagine at position 10, leucine is mutated to glycine at position 28, and glutamic acid is mutated to aspartic acid at position 271 of the amino acid sequence of the transaminase as shown in SEQ ID NO:

2.

5. An expressed gene encoding the aminotransferase mutant according to any one of claims 1 to 4. A recombinant plasmid, wherein the expression gene according to claim 5 is connected to the recombinant plasmid.

7. Use of the transaminase mutant according to any one of claims 1 to 4 in the preparation of sitagliptin in methanol solvent.

8. A method for preparing sitagliptin in a methanol solvent using the aminotransferase mutant according to any one of claims 1 to 4, characterized in that: The steps include: S1, weigh water, triethanolamine, isopropylamine, mix, adjust pH to 8.5, then add coenzyme pyridoxal phosphate PLP, enzyme solution, stir until completely mixed; S2, methanol was added, stirring was continued, and the temperature was raised to 45 ℃, and then the pH was adjusted to 8.5 using 4M isopropylamine; S3. Slowly add the dissolved ketoamide substrate to the reaction system for 2-5 hours. Simultaneously, evacuate the reaction system and use 4 M isopropylamine to control the pH at 8.5 during the whole reaction.

9. The method according to claim 8, characterized in that The final concentration of the reaction system is substrate: water: methanol = 1: 5: 5, enzyme amount = substrate × 50%, reaction time is 24 hours, and temperature is 45°C.

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