Transaminase mutant with improved enzymatic activity and application thereof

Abstract

The invention discloses a transaminase mutant with improved enzymatic activity and application of the transaminase mutant, and the transaminase mutant is formed by mutation of one or more sites by taking transaminase with an amino acid sequence as shown in SEQ ID NO.2 as a parent. Compared with parent transaminase, the oseltamivir phosphate has higher catalytic activity, the production efficiency can be improved, the production cost is reduced, and the oseltamivir phosphate has a good application prospect in industrial production of oseltamivir phosphate.

Description

Technical Field

[0001] This invention relates to the field of biosynthesis technology, and in particular to a transaminase mutant with enhanced enzyme activity and its applications. Background Technology

[0002] Influenza (flu) is an acute respiratory infectious disease caused by the influenza virus. It is characterized by its high infectivity, rapid spread, and wide reach, posing a serious threat to human health and public health security. Early treatments for influenza primarily used amantadine compounds, which exert their antiviral effect by blocking the M2 ion channel of the influenza virus, inhibiting the virus's uncoating process within host cells. However, with widespread clinical use, the problem of influenza virus resistance to these drugs has become increasingly prominent, leading to a significant decline in their clinical efficacy and severely limiting their application.

[0003] Studies have shown that influenza virus neuraminidase (NA) is a key enzyme in the viral life cycle; therefore, developing specific inhibitors targeting NA has become a key direction in the research and development of anti-influenza drugs. Oseltamivir is a synthetic NA inhibitor developed under this background, which can effectively inhibit the NA activity of influenza A and B viruses, and has become an important clinical anti-influenza drug.

[0004] Currently, the main synthetic routes for oseltamivir are divided into asymmetric synthetic routes and semi-synthetic routes. The semi-synthetic method, relying on natural raw materials, is still the most applicable to industrial production. However, the oseltamivir produced by this method cannot solve the global problem. Therefore, Gilead Sciences in the United States and Roche in Switzerland collaborated to disclose an industrial production method for oseltamivir using shikimic acid and quinic acid as raw materials, through reduction and the introduction of functional groups. However, this method has many problems, such as requiring the use of flammable and explosive trimethylphosphorus and corrosive trifluoromethanesulfonic acid, which are expensive and generate corrosive byproducts, making subsequent product separation difficult and affecting the yield of the final product. While Roche's subsequent new method improved the overall yield, it still suffers from the drawbacks of cumbersome operation, numerous byproducts, and environmental pollution.

[0005] The combination of enzymatic and chemical processes has also provided new ideas for the production of oseltamivir phosphate. For example, using ethyl benzoate as a substrate, fermentation with a specific E. coli strain, E. coli JM109, yields cyclohexadiene-cis-diol, which is then converted into oseltamivir phosphate through multiple chemical transformations. The applicant previously designed a novel synthetic route (application number 202511804724.6) using shikimic acid as a raw material. This route synthesizes oseltamivir through esterification-ketalization, oxidation, transaminase-catalyzed construction of a chiral center, and phosphorylation. Compared to the cumbersome steps in traditional synthesis, this route significantly simplifies the process and greatly improves production safety. However, this route is still in the optimization stage, and the transaminase activity used is insufficient to catalyze high concentrations of substrate, limiting its industrialization. Summary of the Invention

[0006] The purpose of this invention is to provide a transaminase mutant with enhanced enzyme activity and its application. Compared with the parental transaminase, it has higher catalytic activity, which can improve production efficiency and reduce production costs. It has good application prospects in the industrial production of oseltamivir phosphate.

[0007] The technical solution adopted by this invention to solve its technical problem is: A transaminase mutant with enhanced enzyme activity, derived by mutation at one or more sites using the transaminase with the amino acid sequence shown in SEQ ID NO.2 as the parent, wherein the transaminase mutant is selected from one of the following: Mutant TA-G32A: The G at position 32 is mutated to A; Mutant TA-K51M: K at position 51 is mutated to M; Mutant TA-G110L: The G at position 110 is mutated to L; The mutant TA-G32A-K51M has the following mutations: G at position 32 is mutated to A and K at position 51 is mutated to M. The mutant TA-G32A-G110L has the following mutations: G at position 32 is mutated to A, and G at position 110 is mutated to L. The mutant TA-K51M-G110L has a K mutation at position 51 that is replaced by M and a G mutation at position 110 that is replaced by L. The mutant TA-G32A-G110L-H154A has the following mutations: G at position 32 is mutated to A, G at position 110 is mutated to L, and H at position 154 is mutated to A. The mutant TA-G32A-G110L-H154A-E199I has the following mutations: G at position 32 is mutated to A, G at position 110 is mutated to L, H at position 154 is mutated to A, and E at position 199 is mutated to I. The mutant TA-G32A-G110L-H154A-E199I-L208H has the following amino acid sequences: G at position 32 is mutated to A, G at position 110 is mutated to L, H at position 154 is mutated to A, E at position 199 is mutated to I, and L at position 208 is mutated to H. The amino acid sequence is shown in SEQ ID NO.20.

[0008] The transaminase with the amino acid sequence shown in SEQ ID NO.2 is derived from Chromobacterium violaceum ( Chromobacteriumviolaceum ).

[0009] In a previous application (application number 2025118047246), the applicant designed a novel synthetic route using the natural compound shikimic acid as a raw material. Through esterification-ketalization, oxidation, transaminase catalysis, and other steps, different chiral centers were synthesized, followed by phosphorylation to obtain oseltamivir phosphate. This route avoids the sodium azide step required in traditional routes, enhancing production safety and industrialization potential. Furthermore, by utilizing two different transaminases to construct different chiral centers, the traditional steps of protecting and deprotecting groups are eliminated. The overall route is concise, uses fewer chemical reagents, and achieves higher yields, better meeting the demands of green, environmentally friendly, and low-cost industrialization. However, this route is still in the optimization stage. The inventors discovered that the transaminase activity used in Step 5 was insufficient to catalyze high concentrations of substrate, limiting its industrialization. Therefore, this invention molecularly modifies transaminase 1 used in Step 5 to enhance its catalytic activity. This invention utilizes directed evolution technology to modify its activity, resulting in a transaminase mutant with catalytic activity 1.3 to 6.4 times that of the parent TA.

[0010] Conservative substitutions, additions or deletions of one or more amino acids, amino-terminal truncation, and carboxyl-terminal truncation of other amino acid sites of the above-mentioned transaminases are also included within the scope of this invention.

[0011] The parent transaminase TA (transaminase 1 in application number 2025118047246) requires a cell concentration of 50 g / L to catalyze the reaction of 50 g / L OS-05. If the substrate concentration is increased to a higher level, the substrate OS-05 cannot catalyze the reaction completely. The large amount of residual substrate will not only reduce the yield, but also add an extra impurity removal step, thus increasing the production cost.

[0012] The series of mutant transaminases prepared in this application were verified by activity testing. Compared with the parental transaminase TA, their catalytic activities were all significantly improved, as detailed below: The catalytic activity of the mutant TA-G32A was increased to 1.5 times that of the parental transaminase TA; The catalytic activity of the mutant TA-K51M was increased to 1.3 times that of the parental transaminase TA; The catalytic activity of the mutant TA-G110L was increased to 1.7 times that of the parental transaminase TA; The catalytic activity of the mutant TA-G32A-K51M was increased to 1.6 times that of the parental transaminase TA; The catalytic activity of the mutant TA-G32A-G110L was increased to 2.4 times that of the parental transaminase TA; The catalytic activity of the mutant TA-K51M-G110L was increased to 1.9 times that of the parental transaminase TA; The catalytic activity of the mutant TA-G32A-G110L-H154A was increased to 3.8 times that of the parental transaminase TA; The catalytic activity of the mutant TA-G32A-G110L-H154A-E199I was increased to 5.2 times that of the parental transaminase TA; The catalytic activity of the mutant TA-G32A-G110L-H154A-E199I-L208H was increased to 6.4 times that of the parental transaminase TA.

[0013] A polynucleotide sequence encoding the transaminase mutant described above.

[0014] A recombinant vector comprising the aforementioned polynucleotide sequence. Preferably, the recombinant plasmid is expressed using a pET28a(+) series vector.

[0015] A host cell comprising the recombinant vector. The host cell can be any conventional host cell in the art, with *Escherichia coli* BL21 being a preferred host cell.

[0016] The application of the transaminase mutant as an enzyme catalyst in the preparation of oseltamivir phosphate.

[0017] A chemical-enzymatic coupling synthesis method for oseltamivir phosphate, using shikimic acid OS-01 as a substrate, sequentially undergoes S1 esterification, S2 ketalization, S3 selective ring opening, S4 TEMPO oxidation, S5 primary transaminase reaction, S6 acetylation, S7 secondary transaminase reaction, and S8 phosphorylation reaction to obtain the final product oseltamivir phosphate OS-09. S5 primary transaminase reaction uses the transaminase mutant described in claim 1 as an enzyme catalyst; .

[0018] The steps S1-S4 and S6-S8 of this invention are the same as those in the applicant's prior application (application number 2025118047246), with the core difference being the improvement to transaminase 1 in step S5.

[0019] The transaminase mutant was added in the form of pure enzyme, immobilized enzyme, crude enzyme solution, or wet cells of recombinant genetically engineered bacteria.

[0020] The reaction system for the S5 primary transaminase reaction consists of: The substrate OS-05 concentration is 40-200 g / L, the recombinant genetically engineered bacterial cells expressing the transaminase mutant are 30-60 g / L, pyridoxal phosphate is 0.5-2.0 mM / L, and isopropylamine is used, with the amount of isopropylamine being 1-2 times the molar amount of substrate OS-05. The reaction medium is triethanolamine buffer (concentration 20-100 mM) with a pH of 6-9. The wet bacterial cells contain 70-90% water by weight.

[0021] The reaction temperature for the S5 primary transaminase reaction is 20-40℃, and the reaction time is 2-24 hours.

[0022] The beneficial effects of this invention are: 1. This invention modifies the molecular structure of transaminase TA through semi-rational design, site-directed mutagenesis, and saturation mutagenesis, and obtains the optimal mutant enzyme TA-G32A-G110L-H154A-E199I-L208H (hereinafter referred to as TA-M5) with an enzyme activity increased by 6.4 times.

[0023] 2. In this invention, the parental transaminase cannot react completely with excessively high substrate concentrations, resulting in reduced product yield and increased subsequent processing costs. However, the mutant TA-G32A-G110L-H154A-E199I-L208H (TA-M5) can catalyze the reaction of 200 g / L substrate at a bacterial cell concentration of 50 g / L, demonstrating extremely high industrial value. Attached Figure Description

[0024] Figure 1 Catalytic activity of transaminase mutant TA-M5 at different pH values; Figure 2 Catalytic activity of transaminase mutant TA-M5 at different temperatures. Detailed Implementation

[0025] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0026] In this invention, unless otherwise specified, all raw materials and equipment used are commercially available or commonly used in the field. The methods described in the following embodiments are conventional methods in the field, unless otherwise specified.

[0027] LB liquid medium consists of 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl; LB solid medium is LB liquid medium with 20 g / L agar powder added; autoclave at 121°C for 20 min.

[0028] Example 1: Construction of recombinant engineered bacteria containing transaminase mutants This invention relates to the targeted modification of the amino acid sequence of parental transaminases. First, site-directed mutations are designed targeting amino acid residues at positions 31, 51, and 110. Using whole-plasmid PCR technology, with the recombinant vector pET28a / TA as a template (the nucleotide sequence of TA is shown in SEQ ID NO.1), single-point mutations are introduced into the TA amino acid sequence (shown in SEQ ID NO.2) to construct transaminase single mutants, specifically TA-G32A, TA-K51M, and TA-G110L. Based on this, using the recombinant plasmid pET28a-TA-G32A or pET28a-TA-K51M as mutation templates, a series of multi-site mutants of transaminases are gradually constructed by sequentially introducing site-directed mutations at single sites, namely TA-G32A-K51M and TA-G32A-G110L. , TA-K51M-G110L, TA-G32A-G110L-H154A, TA-G32A-G110L-H154A-E199I and TA-G32A-G110L-H154A-E199I-L208H.

[0029] SEQ ID NO.1: SEQ ID NO.2: MQKQRTTSQWRELDAAHHLHPFTDTASLNQAGARVMTRGEGVYLWDSEGNKIIDGMAGAWCVNVGYGRKDFAEAARRQMEELSFMHTADGITHPAVVELSSLLAEVTPAGFDRVFY TNSGSESVDTMIRMVRRYWDVQGKPEKKTLIGRWNGYHGSTIGGASLSGMKYMHEQGDLPIPGVAHIEQPWWYKHGKDMTPDEFGVVAARWLEEKILEIGADKVAAFVGEPIQGAGG AIVPPATYWPEIERICRKYDVLLVADEVICGFGRTGEWFGHQHFGFQPDLFTAAKGLSSGYLPIGAVSVGKRVAEGLIAGGDFNHGHTTSGHPVCAAVAHANVAALRDEGIVQRVKD DIGPYMQKRWRETFSRFEHVDDVRGVGMVAAFTLVKNKAKRELFPDFGEIGTLCRDIFFRNNLIMRICGDHIVAAPPLVMTRAEVDEMLAVAERCLEELEQSLKARGLAHHHHHH*.

[0030] To achieve the precise introduction of the above-mentioned mutation sites, this invention designed specific primers corresponding to the construction of each mutant. The specific nucleotide sequences of all primers are detailed in Table 1.

[0031] PCR reaction system: 1U of 2×Phanta Max Master Mix (Nanjing Novozymes), 1μL of forward / reverse primers (10pmol / μL), 1μL of template DNA (10 ng / μL), 1µL of dNTPs (10mM), and ddH2O added to 20μL.

[0032] PCR amplification conditions were: 98℃ for 5 min; (98℃ for 10 s, 60℃ for 5 s, 72℃ for 30 s) for 35 cycles; 72℃ for 5 min.

[0033] After the PCR product was positive by 0.9% agarose gel electrophoresis, 20 µL of PCR reaction solution was taken, and 1 µL of restriction enzyme Dpn I was added for digestion at 37 °C for 3 h to remove template plasmid DNA. The plasmid was then inactivated at 65 °C for 10 min.

[0034] Take 10 μL of PCR product and add it to 100 μL of competent cell suspension in an ice bath. Incubate on ice for 30 min, then immediately heat shock at 42℃ for 90 s. After incubating on ice for 2-5 min, add 1 mL of LB liquid medium for 1 h of recovery. Centrifuge at 4000 rpm for 1 min, discard some of the supernatant, resuspend the remaining bacterial culture, plate it, and incubate upside down at 37℃ for 12 h. Then, pick 3-4 clones and add them to LB liquid medium containing a final concentration of 50 μg / mL kanamycin. Incubate overnight at 37℃, then extract bacterial culture samples from each strain for gene sequencing verification. The sequencing results confirm the correctness of the mutation site and the integrity of the sequence, finally obtaining recombinant engineered bacteria containing the target transaminase mutant. E. coli BL21(DE3) / pET28a-TA; E.coli BL21(DE3) / pET28a-TA-G32A; E.coli BL21(DE3) / pET28a-TA-K51M; E.coli BL21(DE3) / pET28a-TA-G110L; E.coli BL21(DE3) / pET28a-TA-G32A-K51M; E.coli BL21(DE3) / pET28a-TA-G32A-G110L; E.coli BL21(DE3) / pET28a-TA-K51M-G110L; E.coli BL21(DE3) / pET28a-TA-G32A-G110L-H154A; E. coli BL21(DE3) / pET28a-TA-G32A-G110L-H154A-E199I; E. coli BL21(DE3) / pET28a-TA-G32A-G110L-H154A-E199I-L208H.

[0035] Table 1 Primer Design .

[0036] Example 2: Preparation of recombinant engineered bacteria containing transaminase mutants The genetically engineered bacteria were inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37℃ and 200 r / min for 10–12 h to obtain seed culture. The seed culture was then inoculated into fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin at a volume concentration of 1% and cultured at 37℃ and 200 r / min until the OD600 reached 0.6–0.8. IPTG was then added to the medium to a final concentration of 0.1 mM and induced for expression at 16℃ for 12 h. After centrifugation at 4℃ and 8000 r / min for 10 min, the supernatant was discarded, and the bacteria were washed twice with PB buffer (100 mM, pH 8.0) and centrifuged again. The collected bacteria were stored at -20℃ for further reaction.

[0037] Example 3: Assay of the activity of recombinant Escherichia coli with transaminase mutants In this embodiment, the catalytic activity of the parental transaminase TA and a series of mutants was measured to verify the effect of the mutants on improving the conversion efficiency of compound OS-05.

[0038] Taking a 20 mL reaction system as an example: 50 g / L OS-5, 50 g / L catalyst (using the wet bacterial cells prepared in Example 2 as the catalyst), a final concentration of 1 mM pyridoxal phosphate, 20 g / L isopropylamine, and 20 mL triethanolamine buffer (100 mM, pH 8.0). After reacting in a water bath at 35°C for 4 h, the conversion rate was measured to determine the activity of each mutant and TA.

[0039] The specific results are shown in Table 2. The control group was the parental transaminase TA (denoted as WT), whose conversion rate in the above reaction system was only 10.75%. The catalytic activities of the three single mutant transaminases constructed based on TA were all improved. Among them, the mutant TA-G110L showed the largest increase in catalytic activity, with its relative enzyme activity reaching 1.67 times that of TA. The double mutant transaminases constructed by combining the above mutation sites also showed a significant trend of improved catalytic activity. Among them, the mutant TA-G32A-G110L showed the largest increase in catalytic activity, with its relative enzyme activity reaching 2.44 times that of TA. Based on this, further mutation sites were added to construct mutants TA-G32A-G110L-H154A, TA-G32A-G110L-H154A-E199I, and TA-G32A-G110L-H154A-E199I-L208H. The conversion rates of these three mutants in the same reaction system reached 41.17%, 56.12%, and 68.91%, respectively, all showing a significant improvement compared to TA. Among them, the mutant TA-G32A-G110L-H154A-E199I-L208H (referred to as TA-M5) showed the most significant increase in catalytic activity, with its relative enzyme activity reaching 6.4 times that of the parental transaminase TA. Subsequent experiments will use TA-M5 as the experimental subject.

[0040] Table 2 Comparison of transaminase mutant activities .

[0041] Example 4: Optimization of pH for the reaction of transaminase mutant TA-M5 This embodiment measures the catalytic activity of transaminase TA-M5 under different pH conditions to verify the optimal reaction pH of the mutant.

[0042] Taking a 20 mL reaction system as an example: 50 g / L OS-5, 30 g / L catalyst (using the transaminase TA-M5 prepared in Example 2 as the catalyst), a final concentration of 1 mM pyridoxal phosphate, 20 g / L isopropylamine, and 20 mL of buffer solutions at different pH values ​​(100 mM, pH 5.0, 6.0, 7.0, 8.0, and 9.0). After reacting in a water bath at 35°C for 4 hours, the reaction conversion rate was measured to determine the activity of TA-M5 under each pH condition.

[0043] Specific results are as follows Figure 1As shown, the catalytic activity of the transaminase mutant TA-M5 exhibits a clear gradient characteristic with changes in the pH of the reaction system: within the pH range of 5.0-8.0, its catalytic activity gradually increases with increasing pH, reaching its maximum conversion rate at pH 8.0; when the pH of the reaction system is increased to 9.0, the catalytic activity of the transaminase mutant TA-M5 decreases sharply, presumably due to the excessively high pH environment leading to a reduction in its catalytic activity. Based on the above experimental results, pH 8.0 was determined to be the optimal reaction pH for the conversion of OS-05 catalyzed by the transaminase mutant TA-M5.

[0044] Example 5: Optimization of reaction temperature for transaminase mutant TA-M5 This embodiment measures the catalytic activity of transaminase TA-M5 under different temperature conditions to verify the optimal reaction temperature of the mutant.

[0045] Taking a 20 mL reaction system as an example: 50 g / L OS-5, 30 g / L catalyst (using the transaminase TA-M5 prepared in Example 2 as the catalyst), a final concentration of 1 mM pyridoxal phosphate, 20 g / L isopropylamine, and 20 mL triethanolamine buffer (100 mM, pH 8.0). The reaction conversion rate was measured after reacting in water baths at 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, and 50℃ for 4 h to determine the activity of TA-M5 under each temperature condition.

[0046] Specific results are as follows Figure 2 As shown, the catalytic activity of the transaminase mutant TA-M5 exhibits a clear gradient characteristic with increasing reaction temperature: within the temperature range of 20-35℃, its catalytic activity increases with increasing temperature, reaching its highest activity at 35℃, at which point the conversion rate is 34.24%; when the reaction temperature is further increased, the catalytic activity of the transaminase mutant TA-M5 begins to decrease, and when the temperature reaches 50℃, TA-M5 is almost inactivated. Based on the above experimental results, 35℃ was determined to be the optimal reaction temperature for the conversion of OS-05 catalyzed by the transaminase mutant TA-M5.

[0047] Example 6: Optimization of cell and substrate concentrations in the transaminase mutant TA-M5 catalyzed reaction This embodiment measures the conversion rate of transaminase TA-M5 under different bacterial and substrate concentrations to determine the optimal bacterial and substrate concentrations for the mutant.

[0048] Taking a 20 mL reaction system as an example: 40-200 g / L OS-5, 30-60 g / L catalyst (using the transaminase TA-M5 prepared in Example 2 as the catalyst), a final concentration of 1 mM pyridoxal phosphate, 20-80 g / L isopropylamine (twice the substrate equivalent), and 20 mL triethanolamine buffer (100 mM, pH 8.0). After reacting in a water bath at 35°C for 24 h, the reaction conversion rate was measured to determine the activity of TA-M5 under each condition.

[0049] The specific results are shown in Table 3. When the concentration of TA-M5 wet cell was controlled at 30 g / L, it could effectively catalyze substrates with concentrations of 40-120 g / L and achieve complete transformation. However, when the substrate concentration was increased to 160 g / L, the transformation efficiency at this cell concentration decreased significantly, with a transformation rate of only 90.28%, failing to achieve complete transformation. By increasing the cell concentration to 40 g / L, catalytic experiments showed that the substrate at 160 g / L could be completely transformed under this condition. Based on this optimization approach, the combination of substrate and cell concentration was further expanded. Finally, under the combination of "200 g / L substrate + 50 g / L wet cell", the TA-M5 mutant could still achieve complete transformation of the substrate. However, when the substrate concentration was increased to 220 g / L and the cell concentration was correspondingly increased to 60 g / L, the transformation efficiency declined, and complete transformation could not be achieved. Taking into account factors such as conversion efficiency, substrate loading, and cost control, the optimal reaction conditions were determined to be "200 g / L OS-05 and 50 g / L TA-M5 recombinant Escherichia coli wet cells".

[0050] Table 3. Optimization of transaminase mutant TA-M5 cell concentration and substrate concentration. Note: The conversion rate is the average of three parallel experiments. "≥99.0%" indicates that the complete conversion standard under the detection method of this embodiment has been met.

[0051] Example 7: Synthesis of Oseltamivir Intermediate from OS-05 Catalyzed by Transaminase Mutant TA-M5 Based on the optimal reaction conditions determined in the above-mentioned small-scale experiments, this embodiment uses recombinant Escherichia coli TA-M5 as a catalyst to carry out a 30 L scale-up reaction for the preparation of oseltamivir intermediates, verifying the industrial application potential of this mutant.

[0052] The reaction system (30L) consisted of 200g / L OS-5, 50g / L catalyst (using the transaminase TA-M5 prepared in Example 2 as the catalyst), 1 mM pyridoxal phosphate, 80g / L isopropylamine, and 30L triethanolamine buffer (100 mM, pH 8.0).

[0053] During the reaction, the pH of the system was maintained at 8.0 using conventional pH control methods, the reaction temperature was controlled at 35℃, and the reaction time was 24 h. Samples were taken for testing after the reaction was completed. The reaction was immediately terminated once the sampling confirmed complete conversion. The reaction solution was centrifuged at 12000 rpm for 10 min, and the supernatant was collected. Oseltamivir intermediate was extracted from the supernatant using conventional solvent extraction. The organic phases containing the target product obtained from multiple extractions were combined, followed by vacuum concentration to remove the extraction solvent and residual impurities, finally yielding 5325.7 g of the intermediate. The calculated yield of oseltamivir intermediate in this scaled-up reaction was 88.9%, indicating that the transaminase mutant TA-M5 has good catalytic stability and application value in industrial-scale production.

[0054] Example 8: A chemical-enzymatic coupling synthesis method for oseltamivir phosphate, using shikimic acid OS-01 as a substrate, sequentially undergoes S1 esterification, S2 ketalization, S3 selective ring opening, S4 TEMPO oxidation, S5 primary transaminase reaction, S6 acetylation, S7 secondary transaminase reaction, and S8 phosphorylation reaction to obtain the final product oseltamivir phosphate OS-09. The S5 primary transaminase reaction used the transaminase mutant prepared in Example 2 as the enzyme catalyst; .

[0055] The steps S1-S4 and S6-S8 of this invention are the same as those in the applicant's prior application (application number 2025118047246), and step S5 adopts one of the solutions in embodiments 4-7.

[0056] Example 9 Preparation of oseltamivir intermediate: 200 g / L OS-5, 50 g / L catalyst (using the transaminase TA-M5 prepared in Example 2 as the catalyst), 2 mM pyridoxal phosphate, isopropylamine at a substrate concentration of 2 equivalents as the amino donor, and 20 mL triethanolamine buffer (100 mM, pH 8.0). The reaction was carried out at 35 °C for 24 h.

[0057] Example 10 Preparation of oseltamivir intermediate: 40 g / L OS-5, 30 g / L catalyst (using the transaminase TA-M5 prepared in Example 2 as the catalyst), 0.5 mM pyridoxal phosphate to a final concentration, isopropylamine at a substrate concentration of 1 equivalent as the amino donor, and 20 mL triethanolamine buffer (100 mM, pH 8.0). The reaction was carried out at 35 °C for 24 h.

[0058] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications are possible without departing from the technical solutions described in the claims.

Claims

1. A transaminase mutant with enhanced enzyme activity, characterized in that, The transaminase mutant is generated by mutating one or more sites using the amino acid sequence shown in SEQ ID NO.2 as the parent. The transaminase mutant is selected from one of the following: Mutant TA-G32A: The G at position 32 is mutated to A; Mutant TA-K51M: K at position 51 is mutated to M; Mutant TA-G110L: The G at position 110 is mutated to L; The mutant TA-G32A-K51M has the following mutations: G at position 32 is mutated to A and K at position 51 is mutated to M. The mutant TA-G32A-G110L has the following mutations: G at position 32 is mutated to A, and G at position 110 is mutated to L. The mutant TA-K51M-G110L has a K mutation at position 51 that is replaced by M and a G mutation at position 110 that is replaced by L. The mutant TA-G32A-G110L-H154A has the following mutations: G at position 32 is mutated to A, G at position 110 is mutated to L, and H at position 154 is mutated to A. The mutant TA-G32A-G110L-H154A-E199I has the following mutations: G at position 32 is mutated to A, G at position 110 is mutated to L, H at position 154 is mutated to A, and E at position 199 is mutated to I. The mutant TA-G32A-G110L-H154A-E199I-L208H has the following amino acid sequences: G at position 32 is mutated to A, G at position 110 is mutated to L, H at position 154 is mutated to A, E at position 199 is mutated to I, and L at position 208 is mutated to H. The amino acid sequence is shown in SEQ ID NO.

20.

2. The transaminase mutant according to claim 1, characterized in that, The transaminase with the amino acid sequence shown in SEQ ID NO.2 is derived from Chromobacterium violaceum.

3. A polynucleotide sequence, characterized in that, It encodes the transaminase mutant as described in claim 1.

4. A recombinant vector, characterized in that, It contains the polynucleotide sequence as described in claim 3.

5. A host cell, characterized in that, It comprises the recombinant vector as described in claim 4.

6. The use of the transaminase mutant as described in claim 1 as an enzyme catalyst in the preparation of oseltamivir phosphate.

7. A chemical-enzymatic coupling synthesis method for oseltamivir phosphate, characterized in that, Using shikimic acid OS-01 as a substrate, the final product oseltamivir phosphate OS-09 was obtained by sequentially undergoing S1 esterification, S2 ketalization, S3 selective ring opening, S4 TEMPO oxidation, S5 primary transaminase reaction, S6 acetylation, S7 secondary transaminase reaction, and S8 phosphorylation reaction. S5 primary transaminase reaction uses the transaminase mutant described in claim 1 as an enzyme catalyst; 。 8. The chemical-enzymatic coupling synthesis method according to claim 7, characterized in that, The transaminase mutant was added in the form of pure enzyme, immobilized enzyme, crude enzyme solution, or wet cells of recombinant genetically engineered bacteria.

9. The chemical-enzymatic coupling synthesis method according to claim 7, characterized in that, The reaction system for the S5 primary transaminase reaction consists of: The concentration of substrate OS-05 is 40-200 g / L, the wet bacterial cells of recombinant genetically engineered bacteria expressing the transaminase mutant of claim 1 are 30-60 g / L, pyridoxal phosphate is 0.5-2.0 mM / L, and isopropylamine is used. The amount of isopropylamine is 1-2 times the molar amount of substrate OS-05. The reaction medium is triethanolamine buffer at pH 6-9.

10. The chemical-enzymatic coupling synthesis method according to claim 7, characterized in that, The reaction temperature for the S5 primary transaminase reaction is 20-40℃, and the reaction time is 2-24 hours.

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