Transaminase mutants, methods and use thereof in the preparation of a key chiral intermediate for rotigotine

Abstract

The application belongs to the technical field of biology and pharmaceutical chemistry, and discloses a transaminase mutant, a method and application of the transaminase mutant in preparation of a key chiral intermediate of rotigotine, and a preparation method of the key intermediate of rotigotine is a biological conversion method with the transaminase mutant as a main enzyme, an amino acid sequence of the transaminase mutant is an amino acid sequence mutated from an amino acid sequence of a wild-type transaminase shown in SEQ ID NO. 1, the amino acid sequence of the transaminase mutant has a mutation site in the mutated amino acid sequence, and has more than 90% homology with the mutated amino acid sequence. When 5-methoxy-2-naphtalone (a compound of formula III) is catalyzed to prepare a key chiral intermediate (S)-5-methoxy-1,2,3,4-tetrahydronaphthalen-2-amine (a compound of formula II) of rotigotine through an enzymatic reaction method, the chiral purity of the product is greater than 99.9%, and the yield of the biological catalysis step is greater than 95%.

Description

Technical Field

[0001] This invention belongs to the fields of biochemistry and pharmaceutical chemistry, and in particular to a transaminase mutant, a method, and its application in the preparation of a key chiral intermediate of rotigotine. Background Technology

[0002] Neurological diseases such as Parkinson's disease (PD) are becoming increasingly globalized. In 2019, the number of PD patients worldwide reached 8.5 million, while my country alone had 2.94 million PD patients. It is projected that by 2030, the number of Parkinson's disease patients in my country will approach 5 million (China Chronic Disease Prevention and Control. 2022, 30: 649-654). The global market size for Parkinson's disease medications reached US$4.589 billion in 2020 and is projected to increase to US$6.025 billion by 2026. Among the marketed anti-Parkinson's disease drugs, rotigotine (Formula I) holds a 22.13% market share, making it the best-selling Parkinson's disease treatment. Rotigotine is a dopamine receptor agonist developed by Schwarz Pharma in Germany for the treatment of Parkinson's disease and restless legs syndrome. Rotigotine is marketed as a transdermal patch under the brand name Neupro®.

[0003]

[0004] Formula I Formula II Formula III

[0005] (S)-5-methoxy-1,2,3,4-tetrahydronaphthyl-2-amine (Formula II) is one of the key intermediates in the production of rotigotine, and it is currently mostly prepared by chemical synthesis. Its preparation methods can be divided into two categories ( Figure 1 The first type is the resolution method, which first synthesizes racemic 5-methoxy-1,2,3,4-tetrahydronaphthyl-2-amine, and then resolves it with different resolving reagents to obtain (S)-5-methoxy-1,2,3,4-tetrahydronaphthyl-2-amine. Figure 1 For example, Chinese patent publications CN103058985A and CN113234057A report the preparation of a racemic mixture of 5-methoxy-1,2,3,4-tetrahydronaphthyl-2-amine from 5-methoxy-2-tetrahydronaphthone, followed by several resolutions with different resolving agents to obtain (S)-5-methoxy-1,2,3,4-tetrahydronaphthyl-2-amine.

[0006] The second type involves using different chiral auxiliaries, chiral catalysts, or chiral compound raw materials to prepare intermediates with the target configuration predominant. Further resolution yields (S)-5-methoxy-1,2,3,4-tetrahydronaphthyl-2-amine with high chiral purity. Figure 1Chinese patent publications CN114249663A, CN102731326A, CN114276258A, and US patent publication US2014046095A1 report the use of R-α-phenylethylamine or L-phenylglycine as chiral auxiliaries to react with 5-methoxy-2-tetrahydronaphthone, followed by borohydride reduction and palladium-catalyzed hydrogenation to obtain a product with (S)-5-methoxy-1,2,3,4-tetrahydronaphthyl-2-amine as the dominant configuration. Further resolution yields a product with high chiral purity. For example, Kawada T et al. (J. Org. Chem. 2022, 87: 8458-8468) and Chen T et al. (Angew. Chem. Int. Ed. 2023, 62: e202303488) reported the preparation of a product with (S)-5-methoxy-1,2,3,4-tetrahydronaphthyl-2-amine as the dominant configuration using a chiral metal catalyst. Chinese patent publication CN113969303A reports the reduction of 5-methoxy-2-tetrahydronaphthone to a chiral alcohol using piriformin reductase, followed by configuration inversion to obtain (S)-5-methoxy-1,2,3,4-tetrahydronaphthyl-2-amine. However, this method requires two steps, and the second step carries the risk of racemization. The use of ammonium hydroxide and high temperatures during the reaction also compromises the safety of the process.

[0007] The biggest drawback of the first type of method is that enantiomers are treated as waste chemicals, which is not cost-effective. Furthermore, the separation process generates a significant amount of waste, posing environmental pollution problems and thus making it unsuitable for industrial application. The second type of method involves complex preparation and difficulty in obtaining chiral auxiliaries and catalysts, resulting in higher costs. The chiral purity of the products also fails to meet quality standards, necessitating further separation. Therefore, this type of method also suffers from cost-ineffectiveness. Both methods share the common problem of requiring multiple reaction steps and often involving catalytic hydrogenation, leading to poor safety and economic efficiency.

[0008] To address the problems encountered in the preparation of (S)-5-methoxy-1,2,3,4-tetrahydronaphthyl-2-amine, this invention utilizes 5-methoxy-2-tetrahydronaphthone, a transaminase mutant of high stability and chiral purity, to obtain (S)-5-methoxy-1,2,3,4-tetrahydronaphthyl-2-amine with a chiral purity exceeding 99.9% in 95% yield. Enzyme fermentation, extraction, and biocatalysis are all carried out in an aqueous phase at 20–40°C, making the process safer. Summary of the Invention

[0009] The purpose of this invention is to overcome the shortcomings of the prior art and provide a transaminase mutant, a method for preparing the key chiral intermediate (S)-5-methoxy-1,2,3,4-tetrahydronaphthyl-2-amine of rotigotine using 5-methoxy-2-naphthone catalyzed by the transaminase mutant, and its application in the preparation of the key chiral intermediate of rotigotine.

[0010] The technical solution adopted by this invention to solve its technical problem is:

[0011] A transaminase mutant, wherein the amino acid sequence of the transaminase mutant is a mutated amino acid sequence of the wild-type transaminase shown in SEQ ID NO.1, and the amino acid sequence of the transaminase mutant has a mutation site in the mutated amino acid sequence and has more than 90% homology with the mutated amino acid sequence.

[0012] Further, the mutated amino acid sequence has at least one of the following mutation sites: amino acid at position 10 is mutated from W to D or K; amino acid at position 16 is mutated from W to D or K; amino acid at position 20 is mutated from Y to K; amino acid at position 25 is mutated from F to A, V, or W; amino acid at position 30 is mutated from E to G, A, or S; amino acid at position 32 is mutated from Q to G, A, or S; amino acid at position 34 is mutated from V to G, A, or S; amino acid at position 46 is mutated from M to G, A, or S; amino acid at position 52 is mutated from L to G, A, or S; amino acid at position 56 is mutated from F to A, V, or W; amino acid at position 59 is mutated from L to A, F, or W; amino acid at position 60 is mutated from Y to A; amino acid at position 120 is mutated from T to V; amino acid at position 148 is mutated from Y to W; amino acid at position 151 is mutated from Y to K ...50 is mutated from Y to K; amino acid at position 16 is mutated from W to D or K; amino acid at position 20 is mutated from Y to K; amino acid at position 25 is mutated from F to A, V, or W; amino acid at position 16 is mutated from F to W; amino acid at position 151 is mutated from E to G, A, or S; amino acid at position 32 is mutated from Q to G, A, or S; amino acid at position 34 is mutated from V to G, A, or S; amino acid The amino acid at position 160 mutates from W to Y or S, the amino acid at position 164 mutates from Y to A, V, or W, the amino acid at position 197 mutates from M to D or E, the amino acid at position 225 mutates from N to D or K, the amino acid at position 242 mutates from A to V or F, the amino acid at position 303 mutates from S to F or Y, the amino acid at position 328 mutates from V to A or F, the amino acid at position 329 mutates from S to F or V, the amino acid at position 330 mutates from T to A or F, the amino acid at position 404 mutates from L to K or R, the amino acid at position 410 mutates from H to A, the amino acid at position 420 mutates from Q to A or S, the amino acid at position 423 mutates from M to A or S, the amino acid at position 424 mutates from K to A or S, and the amino acid at position 427 mutates from L to A or S.

[0013] Further, the amino acid sequences of the transaminase mutants are shown in SEQ ID NO. 2 to SEQ ID NO. 33. Preferably, the amino acid sequences of the highly active transaminase mutants with high chiral purity and good stability obtained by the present invention are shown in SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 13, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 19, SEQ ID NO 21, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 28, SEQ ID NO 29, and SEQ ID NO 32. More preferably, SEQ ID NO 2, as a transaminase mutant, exhibits good solubility and high expression level during high-density fermentation of *E. coli* to prepare enzyme mutants, and demonstrates high activity and good stability during the enzymatic reaction, resulting in a product with high chiral purity.

[0014] The application of the transaminase mutant described above in the catalytic preparation of the chiral intermediate (S)-5-methoxy-1,2,3,4-tetrahydronaphthyl-2-amine from 5-methoxy-2-naphthone.

[0015] A recombinant expression vector containing the coding gene of the transaminase mutant as described above.

[0016] Furthermore, the recombinant expression vector is a pET series vector plasmid.

[0017] A genetically engineered bacterium for producing transaminase mutants as described above, the genetically engineered bacterium comprising the recombinant expression vector as described in claim 5, wherein the host cell of the genetically engineered bacterium is Escherichia coli.

[0018] The application of the transaminase mutant gene as described above, the recombinant expression vector as described above, and the genetically engineered bacteria as described above in the preparation of the transaminase mutant as described above.

[0019] A method for preparing transaminase mutants includes the following steps:

[0020] Culture genetically engineered bacteria to obtain transaminase mutants; wherein, the genetically engineered bacteria is the genetically engineered bacteria as described in claim 6, and the transaminase mutant is the transaminase mutant as described above.

[0021] A method for preparing a key chiral intermediate of rotigotine, wherein the method uses a transaminase mutant as described above to catalyze the preparation of the chiral intermediate (S)-5-methoxy-1,2,3,4-tetrahydronaphthyl-2-amine of rotigotine.

[0022] Furthermore, when the transaminase mutant catalyzes the synthesis of (S)-5-methoxy-1,2,3,4-tetrahydronaphthyl-2-amine, its amino donor is α-phenylethylamine, isopropylamine, or L-alanine. Preferably, L-alanine is the most suitable amino donor.

[0023] Furthermore, in order to improve the product yield, L-alanine dehydrogenase and formate dehydrogenase are needed to regenerate the byproduct pyruvate into L-alanine.

[0024] Furthermore, the specific steps are as follows:

[0025] (1) Preparation of the chiral intermediate (S)-5-methoxy-1,2,3,4-tetrahydronaphthyl-2-amine of rotigotine

[0026] The following solutions were prepared: crude enzyme solution of transaminase mutant with a final concentration of 400 ml / L, crude enzyme solution of alanine dehydrogenase with a final concentration of 160 ml / L, crude enzyme solution of formate dehydrogenase with a final concentration of 160 ml / L, pH 7.5, phosphate buffer with a final concentration of 100 mM, dithiothreitol with a final concentration of 2 mM, 0.4 g / L PLP, 0.1 g / L nicotinamide adenine dinucleotide, L-alanine with a final concentration of 40 g / L, and ammonium formate with a final concentration of 28 g / L. After dissolving in water, the pH was adjusted to 7.5–8.5. Under nitrogen protection, DMSO solution of 5-methoxy-2-naphthylone with a final concentration of 40 g / L was added to the reaction system. The reaction was carried out at a constant temperature for 7 h to obtain the reaction system after 7 h of reaction.

[0027] (2) Extraction of (S)-5-methoxy-1,2,3,4-tetrahydronaphthyl-2-amine

[0028] The pH of the reaction system was adjusted to 2-3 after 7 hours of reaction with 6M hydrochloric acid, and then centrifuged at 6000g for 30 minutes to remove enzyme proteins. The supernatant was extracted once with an equal volume of ethyl acetate, and the aqueous phase was collected. An equal volume of ethyl acetate was added again, and the pH was adjusted to 9 with 5M sodium hydroxide solution under stirring. The aqueous phase was then separated. The aqueous phase was extracted again with 0.5 volume of ethyl acetate and separated. The two ethyl acetate phases were combined, decolorized with 1% activated carbon, and dehydrated with 2% anhydrous sodium sulfate. The supernatant was collected by filtration, concentrated to dryness under reduced pressure, dissolved in 5 times its weight of ethanol, and hydrogen chloride gas was passed through the ethanol at 5-10℃ to obtain white crystals, yielding the key chiral intermediate of rotigotine. The molar yield of its purification and refining process was 75-85%, the product purity was >99.5%, and the chiral purity was >99.9%.

[0029] The advantages and positive effects of this invention are as follows:

[0030] 1. In this invention, when the key chiral intermediate (S)-5-methoxy-1,2,3,4-tetrahydronaphthyl-2-amine (compound II) of rotigotine is prepared by catalytic reaction of 5-methoxy-2-naphthone (compound III), the product has a chiral purity of >99.9% and a yield of >95%.

[0031] 2. In the process of preparing the key chiral intermediate (S)-5-methoxy-1,2,3,4-tetrahydronaphthyl-2-amine from 5-methoxy-2-naphthone, the present invention uses L-alanine as the ammonia source in the enzymatic reaction system and provides an L-alanine regeneration pathway instead of using α-phenylethylamine as the ammonia source, which makes the extraction simpler and the product yield higher.

[0032] 3. In the enzymatic reaction system of this invention, the reaction temperature is 20–40°C, the pH is 6.5–8.5, the reaction pressure is atmospheric pressure, and the reaction is a single-step process without the need for resolution. Compared to the high temperature, high pressure, organic solvents, and precious metal catalysts required for organic synthesis, this system offers advantages such as shorter steps, lower cost, higher product quality, no need for heavy metal catalysts, and lower waste production.

[0033] 4. Compared with existing methods, the process of this invention is safer and simpler to operate, increases the utilization rate of raw materials, reduces the generation of by-products and isomers, reduces waste, lowers synthesis costs, and is more suitable for the needs of large-scale industrial production.

[0034] 5. The preparation method of the key intermediate (S)-5-methoxy-1,2,3,4-tetrahydronaphthyl-2-amine of rotigotine in this invention is a biotransformation method using a transaminase mutant as the main enzyme. The amino acid sequence of the transaminase mutant is the mutated amino acid sequence shown in SEQ ID NO.1. The amino acid sequence of the transaminase mutant has the mutation site in the mutated amino acid sequence and has more than 90% homology with the mutated amino acid sequence. When the transaminase mutant catalyzes the preparation of the chiral intermediate (S)-5-methoxy-1,2,3,4-tetrahydronaphthyl-2-amine and pyruvate of rotigotine from 5-methoxy-2-naphthylone, the yield can reach more than 95%, and the chiral purity can reach more than 99.9%. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of a preparation method for 1(S)-5-methoxy-1,2,3,4-tetrahydronaphthalene-2-amine in the prior art;

[0036] Figure 2 This is a technical route diagram for producing the chiral intermediate of rotigotine in this invention;

[0037] Figure 3The image shows the purity test results of (S)-5-methoxy-1,2,3,4-tetrahydronaphthalene-2-amine in Example 7 of this invention.

[0038] Figure 4 This is the NMR analysis result of (S)-5-methoxy-1,2,3,4-tetrahydronaphthyl-2-amine in Example 7 of this invention;

[0039] Figure 5 The graph shows the chiral purity test results of (S)-5-methoxy-1,2,3,4-tetrahydronaphthalene-2-amine in Example 7 of this invention. Detailed Implementation

[0040] The present invention will be further described below with reference to the embodiments. The following embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0041] The various experimental operations involved in the specific embodiments are all conventional techniques in the field. For parts not specifically annotated in this document, those skilled in the art can refer to various commonly used reference books, scientific and technological documents or related instructions and manuals prior to the filing date of this invention to carry out the operations.

[0042] In view of the shortcomings of existing methods for synthesizing key chiral intermediates of rotigotine, this invention provides a method for preparing key chiral intermediates of rotigotine. This method uses a transaminase mutant to catalyze the reduction of the compound represented by Formula III, achieving targeted and efficient preparation of high-purity chiral intermediates of rotigotine (Formula II). This method is safe, environmentally friendly, and low-cost, and the produced rotigotine intermediate has excellent optical purity.

[0043] Transaminases are enzyme proteins that use pyridoxal 5-phosphate as a coenzyme. They are widely distributed in various organisms in nature and play an important role in amino transfer during nitrogen metabolism. Transaminases catalyze the transfer of amino groups from amino donors (amino acids or simple amines) to prochiral acceptor ketones, yielding chiral amines and byproduct ketones or α-keto acids. However, these transaminases face some difficulties in industrial applications, such as substrate product inhibition, narrow substrate applicability, and poor product chiral purity. Based on the problems of low catalytic activity and poor product chiral purity of existing wild-type transaminases, this invention genetically engineered wild-type transaminases and obtained transaminase mutants with high catalytic activity and high product chiral purity for the substrates shown in formula (III) through high-throughput enzyme activity screening. Furthermore, a biocatalytic system and process suitable for this transaminase mutant were developed.

[0044] On the one hand, a transaminase mutant, the amino acid sequence of which is the amino acid sequence shown in SEQ ID NO.1 mutated; wherein the mutated amino acid sequence has at least one of the following mutation sites: position 10, position 16, position 20, position 25, position 30, position 32, position 34, position 46, position 52, position 56, position 59, position 60, position 120, position 148, position 151, position 160, position 164, position 197, position 225, position 242, position 303, position 328, position 329, position 330, position 404, position 410, position 420, position 423, position 424, and position 427. Specifically: the 10th amino acid changes from W to D or K; the 16th amino acid changes from W to D or K; the 20th amino acid changes from Y to K; the 25th amino acid changes from F to A, V, or W; the 30th amino acid changes from E to G, A, or S; the 32nd amino acid changes from Q to G, A, or S; the 34th amino acid changes from V to G, A, or S; the 46th amino acid changes from M to G, A, or S; the 52nd amino acid changes from L to G, A, or S; the 56th amino acid changes from F to A, V, or W; the 59th amino acid changes from L to A, F, or W; the 60th amino acid changes from Y to A; the 120th amino acid changes from T to V; the 148th amino acid changes from Y to W; and the 151st amino acid changes from W to Y or S. The amino acid at position 160 is mutated from R to F or Y; the amino acid at position 164 is mutated from Y to A, V, or W; the amino acid at position 197 is mutated from M to D or E; the amino acid at position 225 is mutated from N to D or K; the amino acid at position 242 is mutated from A to V or F; the amino acid at position 303 is mutated from S to F or Y; the amino acid at position 328 is mutated from V to A or F; the amino acid at position 329 is mutated from S to F or V; the amino acid at position 330 is mutated from T to A or F; the amino acid at position 404 is mutated from L to K or R; the amino acid at position 410 is mutated from H to A; the amino acid at position 420 is mutated from Q to A or S; the amino acid at position 423 is mutated from M to A or S; the amino acid at position 424 is mutated from K to A or S; and the amino acid at position 427 is mutated from L to A or S.

[0045] The amino acid sequence of the transaminase mutant contains the mutation site in the mutated amino acid sequence and has more than 90% homology with the mutated amino acid sequence. The yield and chiral purity of the product synthesized using the transaminase mutant of the present invention are shown in Table 1.

[0046] Table 1 Results of transaminase and mutant catalytic reactions

[0047]

[0048]

[0049]

[0050] For enzyme activity: - indicates no product peak detected; + indicates substrate conversion rate less than 10%; ++ indicates substrate conversion rate 20%; +++ indicates substrate conversion rate 30%.

[0051] For enzyme stability: After storing the enzyme in phosphate buffer at 40℃ and pH 8.0 for 24 hours, its activity was measured. + indicates enzyme activity remaining below 20%; ++ indicates enzyme activity remaining 20%-40%; +++ indicates enzyme activity remaining 40%-60%; ++++ indicates enzyme activity remaining 60%-80%; +++++ indicates enzyme activity remaining >80%.

[0052] Regarding product chiral purity: - indicates that the product quantity is too small to be detected.

[0053] In Table 1, each letter is a single-letter abbreviation for amino acids, which is well known to those skilled in the art.

[0054] The mutation shown in SEQ ID NO. 3 (M46A+L52A+F56A+M197E+Q420A) is transformed into a combination of mutations in SEQ ID NO. 2, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 23, and SEQ ID NO. 26. After optimizing the codons according to E. coli, the gene is synthesized and ligated to the NdeI and Xho I sites of pET24a. After transformation into E. coli, E. coli genetically engineered bacteria expressing the mutant shown in SEQ ID NO. 3 can be obtained.

[0055] The amino acid sequence shown in SEQ ID NO.1 is the amino acid sequence of the wild-type transaminase, derived from Bacillus megaterium (UNIPROT ID: A0A0Q9UXH6). Specifically, "wild-type" refers to the form found in nature. For example, naturally occurring or wild-type polypeptide or polynucleotide sequences are sequences present in organisms, which can be isolated from natural sources and have not been artificially manipulated or intentionally modified. However, enzymes obtained after expressing these genes generally exhibit low catalytic activity for certain substrates, poor chiral alcohol content, and poor thermal stability.

[0056] Mutating positions 10, 16, 20, 25, 30, 32, 34, 46, 52, 56, 59, 60, 120, 148, 151, 160, 164, 197, 225, 242, 303, 328, 329, 330, 404, 410, 420, 423, 424, and 427 of the wild-type enzyme alters the enzyme's activity, stability, and the chiral purity of the product. Combining the more favorable mutations yields transaminase mutants with superior performance. Preferably, the amino acid sequences of the transaminase mutants obtained by the present invention, which exhibit high activity, high chiral purity, and good stability, are shown in SEQ ID NO2, SEQ ID NO3, SEQ ID NO4, SEQ ID NO13, SEQ ID NO16, SEQ ID NO17, SEQ ID NO19, SEQ ID NO21, SEQ ID NO24, SEQ ID NO25, SEQ ID NO28, SEQ ID NO29, and SEQ ID NO32. More preferably, SEQ ID NO2, as a transaminase mutant, exhibits good solubility and high expression level during high-density fermentation of *E. coli* to prepare enzyme mutants. It also demonstrates high activity and good stability during the enzymatic reaction, resulting in a product with high chiral purity.

[0057] In another aspect, the present invention provides a recombinant expression vector, which can be constructed by linking the nucleotide sequence containing the transaminase mutant gene described in the present invention to various prokaryotic or eukaryotic expression vectors using conventional methods in the art. Examples include prokaryotic expression vectors such as pGEX, pMAL, and pET series, as well as eukaryotic expression vectors, more preferably selected from the pET series. The vector plasmid used in the present invention is pET-24a.

[0058] The present invention also provides a genetically engineered bacterium for producing the transaminase mutant, wherein the genetically engineered bacterium contains the transaminase mutant gene or the recombinant expression vector of the present invention; the host cell of the above-mentioned genetically engineered bacterium is preferably Escherichia coli BL21(DE3).

[0059] In another aspect, the present invention provides a method for preparing the above-mentioned transaminase mutant, comprising fermenting and culturing the genetically engineered bacteria, and collecting and preparing recombinant transaminase mutants.

[0060] The above method includes the step of industrially preparing the transaminase mutant under certain fermentation conditions in a production tank; the preferred fermentation conditions in the production tank are: DO ≥ 30% and air flow rate 1:1~2vvm.

[0061] Furthermore, the transaminase mutant of the present invention can be used to convert the compound shown in formula (III) into a chiral amine compound shown in formula (II). When the transaminase mutant of the present invention catalyzes the synthesis of the chiral amine compound shown in formula (II), the amino donor can be isopropylamine, L-alanine, racemic α-phenylethylamine, or S-α-phenylethylamine (Table 1). Preferably, L-alanine is the most suitable amino donor. When the transaminase mutant of the present invention uses L-alanine as the amino donor to catalyze the synthesis of the chiral amine compound shown in formula (II), the yield is low. To improve the product yield, various schemes can be used to assist in improving the product yield, such as alanine dehydrogenase and formate dehydrogenase, or pyruvate dehydrogenase and formate dehydrogenase, or alanine dehydrogenase and glucose dehydrogenase, or pyruvate dehydrogenase and glucose dehydrogenase. The present invention uses alanine dehydrogenase and formate dehydrogenase to improve the product yield (…). Figure 2 Those skilled in the art can obtain the sequences of alanine dehydrogenase and formate dehydrogenase from publicly available databases, journals, and patents in the field, and link their genes to expression vectors for expression in Escherichia coli.

[0062] Specifically, the relevant preparation and testing methods are as follows:

[0063] Example 1: Establishment of Wild-Type Transaminase Genetically Engineered Bacteria

[0064] Based on the amino acid sequence of the transaminase derived from Bacillus megaterium (UNIPROT ID: A0A0Q9UXH6) (i.e., SEQ ID NO.1) recorded in Uniprot, after codon optimization, the complete gene fragment was artificially synthesized and then... Nde I and Xho The gene was inserted into the pET-24a vector using an I endonuclease, and the ligated plasmid was then transferred into Escherichia coli BL21(DE3) to establish a transaminase-engineered bacterium.

[0065] Example 2: Obtaining the transaminase mutant gene

[0066] The three-dimensional structure of the transaminase shown in SEQ ID NO.1 has been disclosed, and its PDB ID is 5G0A. The three-dimensional structure of this enzyme was analyzed, and substrate-protein binding simulations were performed using Docking, as shown in Formula III. Finally, Pymol analysis was used to select amino acids that are likely related to substrate binding and coenzyme (pyridoxal 5-phosphate) binding as mutant amino acids.

[0067] In addition to the rational design described above, this application also utilizes the error-prone PCR random mutagenesis method to engineer transaminases for protein modification. Error-prone PCR amplifies the target gene using DNA polymerase by adjusting reaction conditions (including increasing magnesium ion concentration, adding manganese ions, changing the concentration of the four dNTPs in the system, or using low-fidelity DNA polymerase, etc.) to alter the mutation frequency during amplification, thereby randomly introducing mutations into the target gene at a certain frequency to obtain random mutants of the protein molecule.

[0068] This embodiment uses a lower fidelity Taq polymerase, while utilizing Mn 2+ Alternative natural cofactor Mg 2+ To increase the probability of error, the system is designed as follows:

[0069] The 50μL PCR system is as follows:

[0070] 5×PCR Buffer 10 μL;

[0071] dATP / GTP / CTP / TTP (2.5mmol / L) 1 μL each;

[0072] MgCl2 (5 mmol / L) 1 μL;

[0073] MnCl2 (5 mmol / L) 1 μL;

[0074] 1 μL of transaminase template gene;

[0075] Taq DNA polymerase (5 U / μL) 0.5 μL;

[0076] 1 μL each of upstream and downstream primers;

[0077] Add sterile double-distilled water to a final volume of 50 μL.

[0078] Wherein: the transaminase template gene was constructed by PCR amplification of the transaminase gene according to the method in Example 1 and insertion of the gene into the pET-24a plasmid; the primer design was based on the upstream and downstream sequences of the target gene in the recombinant plasmid constructed in Example 1 (primer 1: TCAGCTTCCTTTCGGGCTTTG (SEQ ID NO.34), primer 2: CACTATAGGGGAATTGTGAGC (SEQ ID NO.35)).

[0079] The PCR reaction conditions were as follows: 95℃ pre-denaturation for 2.5 min; 94℃ denaturation for 15 s, 48~53℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; 72℃ extension for 10 min, and cooling to 4℃.

[0080] The obtained PCR amplification product was ligated into the pET-24a vector and transformed into Escherichia coli BL21(DE3) to construct a transaminase gene mutant library.

[0081] Using Escherichia coli BL21(DE3) as the host and pET-24a plasmid as the vector, an extended transaminase mutant was expressed. The amino acid sequence of the transaminase mutant is the amino acid sequence after mutation at the corresponding sites of the amino acid sequence shown in SEQ ID NO.1; wherein the mutated amino acid sequence has at least one mutation site in the amino acid sequence shown in SEQ ID NO.1: position 10, position 16, position 20, position 25, position 30, position 32, position 34, position 46, position 52, position 56, position 59, position 60, position 120, position 148, position 151, position 160, position 164, position 197, position 225, position 242, position 303, position 328, position 329, position 330, position 404, position 410, position 420, position 423, position 424, and position 427. The amino acid sequence of the transaminase mutant has the mutation site in the mutated amino acid sequence and has more than 90% homology with the mutated amino acid sequence.

[0082] Furthermore, by using the enzyme activity detection method described in Example 5 for high-throughput screening, a variety of mutant strains with high activity, good stability, and high product chirality can be obtained, and their amino acid sequences are shown in SEQ ID NO.2-33.

[0083] Example 3: Small-scale production of wild-type transaminase and transaminase mutants in shake flasks

[0084] E. coli containing the recombinant plasmids constructed in Examples 1 and 2, or E. coli carrying alanine dehydrogenase or formate dehydrogenase, were inoculated into 100 mL of LB medium containing kanamycin (50 μg / mL) (peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, pH 7.2). The culture was incubated in a shaker at 37°C and 210 rpm for 16 hours. Then, the culture was transferred at a 1:100 volume ratio to 100 mL of LB medium containing kanamycin (50 μg / mL) and incubated under the same conditions with shaking. The absorbance (OD) of the bacterial culture at 600 nm was measured periodically. 600 This is used to monitor bacterial cell growth density. When the OD of the culture... 600When the concentration of the enzyme is 0.6~0.8, add isopropyl β-D-thiogalactoside (IPTG) to a final concentration of 0.8 mM to induce the expression of the target gene, and culture overnight (≥16 hours). Centrifuge the culture at 10000 rpm for 10 min at 4°C, discard the supernatant, and resuspend the cell pellet in pre-cooled 50 mM Tris-HCl buffer (pH 8.5) at 200 g / L. Sonicate the cells under the following conditions: Φ3 amplitude transformer, 3 s on, 6 s off, 10 min for disruption. Centrifuge again at 13000 rpm for 30 min at 4°C, collect the supernatant (crude enzyme solution), and store at -20°C.

[0085] Example 4: Fermentation production of transaminase, alanine dehydrogenase and formate dehydrogenase

[0086] Fermentation protocol: *E. coli* containing the recombinant plasmid carrying the mutant gene described in SEQ ID No. 2 constructed in Examples 1 and 2, or *E. coli* carrying alanine dehydrogenase or formate dehydrogenase, were inoculated as single colonies in 400 mL LB medium (containing 50 μg / mL kanamycin) and cultured overnight (≥16 hours) at 37°C with shaking at 210 rpm. Fermentation was then carried out in a 30 L fermenter: 2% of the seed culture was inoculated into 15 L of fermentation medium (the fermentation medium consisted of: 5.0 g / L peptone, 7.5 g / L yeast extract, 8.5 g / L K₂HPO₄·3H₂O, 5 g / L NaCl, 1.5 g / L (NH₄)₂SO₄, 11 g / L glucose, 2.5 g / L MgSO₄·7H₂O, 0.11 g / L FeSO₄·7H₂O, and 1 mL / L trace element stock solution; wherein the trace element stock solution included: 8.4 g / L MgSO₄·7H₂O, 0.11 g / L FeSO₄·7H₂O, and 1 mL / L trace element stock solution; wherein the trace element stock solution included: 8.4 g / L MgSO₄·7H₂O, 0.11 g / L FeSO₄·7H₂O, and 1 mL / L trace element stock solution). The fermentation broth was prepared with 2 g / L EDTA, 2.5 g / L CoCl₂·6H₂O, 2.19 g / L CuSO₄·5H₂O, 15 g / L MnCl₂·4H₂O, 3 g / L H₃BO₃, 2.5 g / L Na₂MoO₄·2H₂O, 17 g / L ZnSO₄·7H₂O, and a final concentration of 80.0 mmol / L HCl. The pH of the fermentation broth was maintained at 7.0–7.2 by adding ammonia. The tank temperature was 37°C, the stirring speed was 300–900 rpm, the dissolved oxygen was controlled at approximately 30%, and the air flow rate was 1:1–2 vvm. After 8 hours of cultivation, IPTG (final concentration 0.8 mmol / L) was added to induce transaminase expression, the tank temperature was adjusted to 22°C, and fermentation continued for 12–16 hours. During fermentation, a feed solution (300 g / L glucose, 150 g / L yeast extract, pH 7.2) was added to maintain the growth of the culture. After fermentation, the culture was homogenized and broken 2-4 times using a high-pressure homogenizer at 800-1000 bar. Polyethyleneimine (2 g / L) and diatomaceous earth (150 g / L) were added, and the mixture was stirred for 30 minutes. After flocculation and sedimentation, the mixture was filtered through a filter cloth lined with diatomaceous earth. The filtered enzyme solution was concentrated using an ultrafiltration membrane to prepare crude transaminase, crude alanine dehydrogenase, and crude formate dehydrogenase solutions, which were then stored at -20℃.

[0087] Example 5: Determination of the activity and stability of transaminases and their mutants

[0088] The transaminase activity assay system is as follows:

[0089] The final concentrations of each compound in the reaction system were as follows: 30 g / L of the compound shown in Formula III, 100 ml / L of DMSO, 100 g / L of L-alanine, 20 mM / L of dithiothreitol, 300 ml / L of crude transaminase or its mutant (prepared in Example 3), with water as the solvent, DMSO as the co-solvent, and 5 g / L of PLP (pyridoxal phosphate, hereinafter the same) to adjust the pH to 7.0-8.5. The reaction was carried out at 25 °C for 2 h. The results are shown in Table 1. As can be seen from Table 1, multiple mutants can improve the chiral purity of the enzyme-catalyzed product. Specifically, mutations at positions W16, F25, E30, Q32, V34, M46, L52, F56, Y164, M197, L404, M423, K424, and L427 to specific amino acids can increase the chiral purity of the product to over 99%. The multi-point mutant shown in SEQ ID NO 3 can also achieve a chiral purity of 99.72% for the product obtained by enzyme catalysis. In particular, the product obtained by catalysis of F56A (as shown in SEQ ID NO 2) can achieve a chiral purity of over 99.9%.

[0090] Enzyme stability assay:

[0091] The enzyme was added to phosphate buffer at pH 8.5 and incubated at 40°C for 24 h. Residual enzyme activity was then detected using the enzyme activity assay described above. Stability results are shown in Table 1. Table 1 shows that amino acid mutations at positions F25, E30, M46, L52, F56, L59, T120, Y148, R160, M197, N225, A242, V328, Q420, and M423 significantly improved enzyme stability at 40°C. In particular, mutations at positions M46, L52, F56, M197, V328, and Q420, as well as the multi-point mutant (SEQ ID NO 3), improved both enzyme stability and catalytic activity. Mutations at positions L52, F56, M197, and M423, as well as multi-point mutants (SEQ ID NO 3), can improve enzyme stability and the chiral purity of the product obtained through enzyme catalysis. In particular, mutations at positions L52A (SEQ ID NO 18), F56A (SEQ ID NO 2), and M197E (SEQ ID NO 23), as well as multi-point mutants (SEQ ID NO 3), can simultaneously improve enzyme stability, enzyme catalytic activity, and product chiral purity.

[0092] Example 6: Preparation of the chiral intermediate (S)-5-methoxy-1,2,3,4-tetrahydronaphthyl-2-amine of rotigotine

[0093] The crude enzyme solution of the transaminase mutant obtained in Example 4 (final concentration 400 ml / L), crude enzyme solution of alanine dehydrogenase (final concentration 160 ml / L), crude enzyme solution of formate dehydrogenase (final concentration 160 ml / L) (the transaminase-catalyzed reaction is a reversible reaction, especially the reaction with L-alanine as the ammonia source, the yield is extremely low. Therefore, alanine dehydrogenase and formate dehydrogenase are added to the reaction system to improve the product yield), phosphate buffer (pH 7.5, final concentration 100 mM), and dithiothreitol (final concentration) were added. Dissolve 2 mM / L nicotinamide adenine dinucleotide (NAD) (final concentration 0.1 g / L), L-alanine (final concentration 40 g / L), and ammonium formate (final concentration 28 g / L) in water (unless otherwise specified in this invention). Adjust the pH to 7.5–8.5. Under nitrogen protection, add a DMSO solution of 5-methoxy-2-naphthone (final concentration 40 g / L) to the reaction system and react at a constant temperature for 7 hours. The yield of the biocatalytic step can reach over 95%.

[0094] Example 7 Extraction of (S)-5-methoxy-1,2,3,4-tetrahydronaphthyl-2-amine

[0095] The pH of the reaction system in Example 6 (7 hours of isothermal reaction in Example 6, 2 hours for enzyme activity detection, 7 hours for product production) was adjusted to 2-3 after 7 hours of isothermal reaction with 6M hydrochloric acid. The system was then centrifuged at 6000g for 30 minutes to remove enzyme proteins. The supernatant was extracted once with an equal volume of ethyl acetate, and the aqueous phase was collected. An equal volume of ethyl acetate was added again, and the pH was adjusted to 9 with 5M sodium hydroxide solution under stirring. The aqueous phase was then separated. The aqueous phase was extracted again with 0.5 volume of ethyl acetate and separated. The two ethyl acetate phases were combined, decolorized with 1% activated carbon, and dehydrated with 2% (w / v, mass percentage) anhydrous sodium sulfate. The supernatant was collected by filtration, concentrated to dryness under reduced pressure, dissolved in 5 times its weight of ethanol, and then purified by passing hydrogen chloride gas through the ethanol at 5-10°C to obtain white crystals. The molar yield of the purification and refining process was 75-85%, and the product purity was >99.5% (results are shown in Figure 1). Figure 3 (As shown in Table 2), chiral purity > 99.9% ( Figure 5 The product's structure was confirmed to be correct by nuclear magnetic resonance imaging (NMR). Figure 4 ).

[0096] Table 2 Peak Area Detection Table

[0097]

[0098] Example 8 Analysis Method

[0099] Enzyme-catalyzed reaction process detection: The reaction system (0.1 mL) was diluted with 1.9 mL of methanol, filtered through a 0.45 μm filter membrane, and the content of substrate and product was detected by HPLC. The HPLC detection method is as follows: Column: Agilent ZORBAX Eclipse XDB-C18 (250 mm × 4.6 mm, 5 μm); Mobile phase (A): 0.1% H3PO4 aqueous solution; Mobile phase (B): acetonitrile; Flow rate: 1 mL / min; Column temperature: 30℃; Injection volume: 10 μL; Detection wavelength: 230 nm; Mobile phase gradient: t = 0–4.5 min, 77% (A), 23% (B); t = 4.5–8 min, 15% (A) and 85% (B); t = 8.5–11.5 min, 5% (A) + 95% (B); t = 11.5–16 min, 77% (A) + 23% (B).

[0100] Product purity testing: The product was dissolved in acetonitrile-water at a concentration of 1 g / L in a 1:1 volume ratio, filtered through a 0.45-micron filter membrane, and the purity of the product was determined by HPLC. The HPLC detection method is as follows: Column: Agilent ZORBAX Eclipse XDB-C18 (250 mm × 4.6 mm, 5 μm); Mobile phase (A): 0.01 mol / L H3PO4 aqueous solution; Mobile phase (B): acetonitrile; Flow rate: 1 mL / min; Column temperature: 30℃; Injection volume: 10 μL; Detection wavelength: 230 nm; Mobile phase gradient: t = 0–5 min, 90% (A), 10% (B); t = 5–45 min, 15% (A) and 85% (B); t = 45–50 min, 15% (A) + 85% (B); t = 50.1–60 min, 90% (A) + 10% (B).

[0101] Product chiral purity test: Column: CHIRALPAK IC (250 mm × 4.6 mm); Mobile phase: 95% n-hexane, 5% isopropanol, 0.1% diethylamine; Flow rate: 1 mL / min; Detection wavelength: 230 nm; Column temperature: 40 ℃; Injection volume: 10 μL.

[0102] As shown in Example 5, the various transaminase mutants involved in this invention can effectively improve the enzyme's stability at 40°C, the chiral purity of the enzyme-catalyzed product, and the enzyme's catalytic activity. In particular, the transaminase described in SEQ ID NO 2 exhibits significantly improved catalytic activity, stability, and product chiral purity compared to the wild-type enzyme. Its product chiral purity can reach over 99.9%, exceeding data from existing literature and patents. The enzyme catalytic system composed of L-alanine dehydrogenase and formate dehydrogenase can further increase the molar yield of the biocatalytic process to over 95%, and the molar yield after purification can still reach 75-85%.

[0103] The relevant sequences used in this invention are as follows:

[0104] SEQ ID NO.1 (Wild type)

[0105] MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYQPVPIESTEGDYLIMPDGTRLLDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFV STGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITE VSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVE QAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTQIIMKKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0106] SEQ ID NO.2 (A mutation occurs at position 56 of the amino acid sequence of the wild-type enzyme)

[0107] MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYQPVPIESTEGDYLIMPDGTRLLDFANQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTQIIMKKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0108] SEQ ID NO.3 (Mutations occurred at positions 46, 52, 56, 197, and 420 of the amino acid sequence of the wild-type enzyme)

[0109] MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYQPVPIESTEGDYLIAPDGTRALDFANQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNEFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTAIIMKKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0110] SEQ ID NO.4 (Mutation at the 56th position of the amino acid sequence of the wild-type enzyme)

[0111] MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYQPVPIESTEGDYLIMPDGTRLLDFWNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTQIIMKKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0112] SEQ ID NO.5 (Mutation at the 16th position of the amino acid sequence of the wild-type enzyme)

[0113] MSLTVQKINWEQVKEDDRKYLMRTFSTQNEYQPVPIESTEGDYLIMPDGTRLLDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTQIIMKKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0114] SEQ ID NO.6 (Mutation at the 25th position of the amino acid sequence of the wild-type enzyme)

[0115] MSLTVQKINWEQVKEWDRKYLMRTWSTQNEYQPVPIESTEGDYLIMPDGTRLLDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTQIIMKKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0116] SEQ ID NO.7 (Mutation at position 30 of the amino acid sequence of the wild-type enzyme)

[0117] MSLTVQKINWEQVKEWDRKYLMRTFSTQNGYQPVPIESTEGDYLIMPDGTRLLDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTQIIMKKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0118] SEQ ID NO.8 (Mutation at position 30 of the amino acid sequence of the wild-type enzyme)

[0119] MSLTVQKINWEQVKEWDRKYLMRTFSTQNAYQPVPIESTEGDYLIMPDGTRLLDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTQIIMKKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0120] SEQ ID NO.9 (Mutation at position 32 of the amino acid sequence of the wild-type enzyme)

[0121] MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYGPVPIESTEGDYLIMPDGTRLLDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTQIIMKKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0122] SEQ ID NO.10 (Mutation at position 32 of the amino acid sequence of the wild-type enzyme)

[0123] MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYAPVPIESTEGDYLIMPDGTRLLDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTQIIMKKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0124] SEQ ID NO.11 (Mutation at position 32 of the amino acid sequence of the wild-type enzyme)

[0125] MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYSPVPIESTEGDYLIMPDGTRLLDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTQIIMKKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0126] SEQ ID NO.12 (Mutation at position 34 of the amino acid sequence of the wild-type enzyme)

[0127] MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYQPGPIESTEGDYLIMPDGTRLLDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTQIIMKKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0128] SEQ ID NO.13 (Mutation at the 34th position of the amino acid sequence of the wild-type enzyme)

[0129] MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYQPAPIESTEGDYLIMPDGTRLLDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTQIIMKKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0130] SEQ ID NO.14 (Mutation at position 34 of the amino acid sequence of the wild-type enzyme)

[0131] MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYQPSPIESTEGDYLIMPDGTRLLDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTQIIMKKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0132] SEQ ID NO.15 (Mutation at position 46 of the amino acid sequence of the wild-type enzyme)

[0133] MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYQPVPIESTEGDYLIGPDGTRLLDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTQIIMKKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0134] SEQ ID NO.16 (mutation at the 46th position of the amino acid sequence of the wild-type enzyme)

[0135] MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYQPVPIESTEGDYLIAPDGTRLLDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTQIIMKKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0136] SEQ ID NO.17 (Mutation at position 46 of the amino acid sequence of the wild-type enzyme)

[0137] MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYQPVPIESTEGDYLISPDGTRLLDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTQIIMKKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0138] SEQ ID NO.18 (Mutation at position 52 of the amino acid sequence of the wild-type enzyme)

[0139] MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYQPVPIESTEGDYLIMPDGTRALDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTQIIMKKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0140] SEQ ID NO.19 (Mutation at the 52nd position of the amino acid sequence of the wild-type enzyme)

[0141] MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYQPVPIESTEGDYLIMPDGTRSLDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTQIIMKKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0142] SEQ ID NO.20 (Mutation at the 164th position of the amino acid sequence of the wild-type enzyme)

[0143] MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYQPVPIESTEGDYLIMPDGTRLLDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSARSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTQIIMKKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0144] SEQ ID NO.21 (Mutation at position 164 of the amino acid sequence of the wild-type enzyme)

[0145] MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYQPVPIESTEGDYLIMPDGTRLLDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSVRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTQIIMKKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0146] SEQ ID NO.22 (mutation at the 197th position of the amino acid sequence of the wild-type enzyme)

[0147] MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYQPVPIESTEGDYLIMPDGTRLLDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNDFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTQIIMKKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0148] SEQ ID NO.23 (Mutation at the 197th position of the amino acid sequence of the wild-type enzyme)

[0149] MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYQPVPIESTEGDYLIMPDGTRLLDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNEFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTQIIMKKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0150] SEQ ID NO.24 (Mutation at the 404th position of the amino acid sequence of the wild-type enzyme)

[0151] MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYQPVPIESTEGDYLIMPDGTRLLDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKKDRNFTHGMNPNQIPTQIIMKKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0152] SEQ ID NO.25 (Mutation at position 404 of the amino acid sequence of the wild-type enzyme)

[0153] MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYQPVPIESTEGDYLIMPDGTRLLDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKRDRNFTHGMNPNQIPTQIIMKKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0154] SEQ ID NO.26 (Mutation at the 420th position of the amino acid sequence of the wild-type enzyme)

[0155] MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYQPVPIESTEGDYLIMPDGTRLLDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTAIIMKKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0156] SEQ ID NO.27 (Mutation at the 420th position of the amino acid sequence of the wild-type enzyme)

[0157] MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYQPVPIESTEGDYLIMPDGTRLLDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTSIIMKKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0158] SEQ ID NO.28 (Mutation at position 423 of the amino acid sequence of the wild-type enzyme)

[0159] MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYQPVPIESTEGDYLIMPDGTRLLDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTQIIAKKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0160] SEQ ID NO.29 (mutation at amino acid position 423 of the amino acid sequence of the wild-type enzyme)

[0161] MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYQPVPIESTEGDYLIMPDGTRLLDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTQIISKKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0162] SEQ ID NO.30 (Mutation at amino acid position 424 of the amino acid sequence of the wild-type enzyme)

[0163] MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYQVPPIESTEGDYLIMPDGTRLLDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNNLLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGAGSAMPPYEYIPQIRKMTKELGVLINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTGMNPNQIPTQIIMAKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0164] SEQ ID NO.31 (Mutation at amino acid position 424 of the wild-type enzyme) MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYQPVPIESTEGDYLIMPDGTRLLDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNLLKDENGELLSVKYTRRMIENY GPEQVAAVITEVSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMME ENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTQIIMSKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0165] SEQ ID NO.32 (A mutation occurred at position 427 of the amino acid sequence of the wild-type enzyme)

[0166] MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYQPVPIESTEGDYLIMPDGTRLLDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTQIIMKKAAEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0167] SEQ ID NO.33 (mutation at amino acid position 427 of the amino acid sequence of the wild-type enzyme)

[0168] MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYQPVPIESTEGDYLIMPDGTRLLDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFV STGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITE VSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVE QAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTQIIMKKASEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0169] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. The application of a transaminase mutant in the catalytic preparation of the chiral intermediate (S)-5-methoxy-1,2,3,4-tetrahydronaphthyl-2-amine from 5-methoxy-2-naphthone, wherein the amino acid sequence of the transaminase mutant is shown in SEQ ID NO.3; in the catalytic synthesis of (S)-5-methoxy-1,2,3,4-tetrahydronaphthyl-2-amine, the amino donor of the transaminase mutant is L-alanine, and the chiral purity of the product is >99.72%, and the yield is >95%.

2. The application according to claim 1, characterized in that: The method for preparing transaminase mutants includes the following steps: A genetically engineered bacterium was cultured to obtain a transaminase mutant; wherein the genetically engineered bacterium contained a recombinant expression vector, and the host cell of the genetically engineered bacterium was Escherichia coli; the recombinant expression vector contained the encoding gene of the transaminase mutant as shown in SEQ ID NO.

3.

3. A method for preparing a key chiral intermediate of rotigotine, characterized in that: The method uses the transaminase mutant as described in claim 1 or 2 to catalyze the preparation of the chiral intermediate (S)-5-methoxy-1,2,3,4-tetrahydronaphthyl-2-amine of rotigotine using 5-methoxy-2-naphthylone. The amino acid sequence of the transaminase mutant is shown in SEQ ID NO.3; When the transaminase mutant catalyzes the synthesis of (S)-5-methoxy-1,2,3,4-tetrahydronaphthyl-2-amine, its amino donor is L-alanine.

4. The preparation method according to claim 3, characterized in that: The specific steps are as follows: (1) Preparation of the chiral intermediate (S)-5-methoxy-1,2,3,4-tetrahydronaphthyl-2-amine of rotigotine The following solutions were prepared: crude enzyme solution of transaminase mutant with a final concentration of 400 ml / L, crude enzyme solution of alanine dehydrogenase with a final concentration of 160 ml / L, crude enzyme solution of formate dehydrogenase with a final concentration of 160 ml / L, pH 7.5, phosphate buffer with a final concentration of 100 mM, dithiothreitol with a final concentration of 2 mM, 0.4 g / L PLP, 0.1 g / L nicotinamide adenine dinucleotide, L-alanine with a final concentration of 40 g / L, and ammonium formate with a final concentration of 28 g / L. After dissolving in water, the pH was adjusted to 7.5–8.

5. Under nitrogen protection, DMSO solution of 5-methoxy-2-naphthylone with a final concentration of 40 g / L was added to the reaction system. The reaction was carried out at a constant temperature for 7 h to obtain the reaction system after 7 h of reaction. (2) Extraction of (S)-5-methoxy-1,2,3,4-tetrahydronaphthyl-2-amine The pH of the reaction system was adjusted to 2-3 after 7 hours of reaction with 6M hydrochloric acid, and then centrifuged at 6000g for 30 minutes to remove enzyme proteins. The supernatant was extracted once with an equal volume of ethyl acetate, and the aqueous phase was collected. An equal volume of ethyl acetate was added again, and the pH was adjusted to 9 with 5M sodium hydroxide solution under stirring. The aqueous phase was then separated. The aqueous phase was extracted again with 0.5 volume of ethyl acetate and separated. The two ethyl acetate phases were combined, decolorized with 1% activated carbon, and dehydrated with 2% anhydrous sodium sulfate. The supernatant was collected by filtration, concentrated to dryness under reduced pressure, dissolved in 5 times its weight of ethanol, and hydrogen chloride gas was passed through the ethanol at 5-10℃ to obtain white crystals, yielding the key chiral intermediate of rotigotine. The molar yield of its purification and refining process was 75-85%, the product purity was >99.5%, and the chiral purity was >99.72%.

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