Tryptophan synthetase trp B modified gene and preparation method of 6-fluoro-L-tryptophan

Abstract

The invention discloses a tryptophan synthetase trp B modified gene and a preparation method of 6-fluoro-L-tryptophan, belongs to the technical field of gene modification and amino acid synthesis, and provides a tryptophan synthetase trp B gene subjected to mutation modification, and the nucleotide sequence of the tryptophan synthetase trp B gene is shown as SEQ ID NO.1. After gene modification, substrate molecules can enter the channel more easily. After gene modification, a channel of an active center of tryptophan synthetase is more beneficial to entering of substrates 6-fluoroindole and L-serine, and the yield of 6-fluoro-L-tryptophan is increased. According to the method, a specific strain containing tryptophan synthetase is adopted, tryptophan synthetase can be efficiently expressed by culturing in an optimized culture medium, so that the 6-fluoro-L-tryptophan synthesized by an enzymic method has relatively high catalytic rate and conversion rate, the molar conversion rate of L-serine reaches 86.5% or above, the reaction conditions of the enzymic method synthesis are mild, the enzyme stereoselectivity is strong, the catalytic efficiency is high, and the method is suitable for industrial production. The method is suitable for industrial production.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic modification and amino acid synthesis, and specifically relates to a modified gene of tryptophan synthase trpB and a preparation method of 6-fluoro-L-tryptophan. Background Art

[0002] Due to their significant physiological properties, L-tryptophan derivatives are playing an increasingly important role in the pharmaceutical field. L-tryptophan derivatives can be used to synthesize a variety of polypeptide drugs. For example, the anticancer drugs Rebeccamycin and Diazonamide A contain the L-tryptophan derivative moiety. Substituting halogens or alkanes on the aromatic ring of tryptophan can synthesize novel natural product analogs, whose biological activities and bioavailabilities can vary significantly, making them common structural units in new drug research and development. Therefore, the study of the synthesis methods of unnatural L-tryptophan derivatives is of great significance.

[0003] The Chinese patent application document with the publication number CN113666861A discloses a preparation method of an unnatural L-tryptophan derivative. Using a substituted indole compound as a raw material, it undergoes a series of reactions including formylation, cyclization, ring opening, asymmetric hydrogenation, hydrolysis deprotection, and introduction of the Fmoc protecting group to obtain the unnatural L-tryptophan derivative, and the chemical synthesis method is relatively complex. There are literature reports on the method for preparing 4-bromo-L-tryptophan from 4-bromoindole, but the yield is only 9% (Organic Letters, 2014, 16, 10, 2622 - 2625).

[0004] In the industrial production of amino acids, a large amount of hair acid hydrolysis solution rich in L-serine that cannot be directly used in the food and pharmaceutical fields is generated. The direct separation of this mixed amino acid feed solution to prepare L-serine has high costs and low efficiency. Currently, there are few reports on the synthesis method of 6-fluoro-L-tryptophan. Summary of the Invention

[0005] The technical problem to be solved by the present invention is how to provide a preparation method of 6-fluoro-L-tryptophan to improve the catalytic rate and conversion rate of the reaction.

[0006] The present invention solves the above technical problems through the following technical means:

[0007] In the first aspect of the present invention, a mutated and modified gene of tryptophan synthase trpB is proposed, and its nucleotide sequence is as shown in SEQ ID NO.1.

[0008] The mutation modification specifically is: mutating ccc at the 7th position to gcc, cgg at the 100th position to ggg, and gaa at the 220th position to aga.

[0009] The second aspect of the present invention provides the application of the above-mentioned mutated and modified tryptophan synthase trpB gene in the preparation of 6-fluoro-L-tryptophan.

[0010] The third aspect of the present invention provides a recombinant construct, which contains the above-mentioned mutated and modified tryptophan synthase trpB gene.

[0011] Preferably, the vector used for the recombinant construct is a cloning vector or an expression vector for expressing the above-mentioned mutated and modified tryptophan synthase trpB gene.

[0012] Preferably, the recombinant construct is a recombinant plasmid.

[0013] The fourth aspect of the present invention provides a recombinant host cell, which contains the above-mentioned recombinant construct.

[0014] Preferably, the host cell includes any one of Escherichia coli ATCC11229, Bacillus subtilis ATCC21332, Pseudomonas stutzeri ATCC8482, and Bacillus cereus ATCC11778.

[0015] The fifth aspect of the present invention provides a method for preparing 6-fluoro-L-tryptophan, which includes the following steps:

[0016] Adding a transformation solution to the recombinant host cell and performing an enzymatic reaction to obtain 6-fluoro-L-tryptophan; the transformation solution includes a feed solution containing L-serine, pyridoxal phosphate, and 6-fluoroindole.

[0017] Preferably, the temperature of the enzymatic reaction is 25-45°C, the time is 10-20 h, and the pH is 6-11.

[0018] Preferably, after the reaction is completed, it further includes centrifuging, decolorizing, separating, washing, filtering by suction, and drying the reaction product.

[0019] The beneficial effects of the present invention are as follows:

[0020] 1. The present invention uses a specific strain with modified tryptophan synthase, and cultivates highly active tryptophan synthase in a preferred culture medium, so that the enzymatic synthesis of 6-fluoro-L-tryptophan has a high catalytic rate and conversion rate, and the molar conversion rate of L-serine reaches more than 95%; the enzymatic synthesis of 6-fluoro-L-tryptophan has mild reaction conditions, high catalytic efficiency, and is suitable for industrial production.

[0021] 2. During the genetic modification of tryptophan synthase gene, mutations were made at different sites. It was found that mutations at the 7th, 100th, and 220th positions promoted enzyme activity, while mutations at other sites did not. The reason is that the 7th, 100th, and 220th positions are channels of the catalytic active center, and after genetic modification, it is more conducive to the entry of substrate molecules into the channels. After genetic modification, the channels of the tryptophan synthase active center are more conducive to the entry of substrates 6-fluoroindole and L-serine, increasing the yield of 6-fluoro-L-tryptophan. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a physical picture of the culture of the tryptophan synthase strain in Example 1 of the present invention.

[0023] Figure 2 This is the infrared spectrum of 6-fluoro-L-tryptophan in Example 1 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0025] The test materials and reagents used in the following embodiments can be obtained from commercial channels without special instructions.

[0026] For those not specifying specific technologies or conditions in the embodiments, they can all be carried out according to the technologies or conditions described in the literature in the field or according to the product specifications. Without special instructions, the quantitative tests in the following embodiments are all set with more than three repeated experiments, and the results are averaged.

[0027] Example 1:

[0028] A method for preparing 6-fluoro-L-tryptophan, comprising the following steps:

[0029] (1) Site-directed mutagenesis of the tryptophan synthase trp B gene sequence, changing ccc at the 7th position to gcc, cgg at the 100th position to ggg, and gaa at the 220th position to aga. The tryptophan synthase trp B gene was synthesized by Sangon Biotech (Shanghai) Co., Ltd. The sequence of the mutated tryptophan synthase trp B gene is shown in SEQ ID NO.1. During the process of modifying the tryptophan synthase gene by the applicant's team, mutations were made at different sites. It was found that the mutations at the 7th, 100th, and 220th positions promoted enzyme activity, while mutations at other sites did not. The reason was analyzed that the 7th, 100th, and 220th positions were the channels of the catalytic active center, and after gene modification, it was more conducive to the entry of substrate molecules into the channels. After gene modification, the channels of the tryptophan synthase active center were more conducive to the entry of the substrates 6-fluoroindole and L-serine, increasing the yield of 6-fluoro-L-tryptophan.

[0030] Transfer the mutated trp B gene into Escherichia coli ATCC11229: ligate the modified trp B gene fragment into the vector pETDuet-1 to construct a recombinant plasmid. The ligation system is as follows: 10 μL of plasmid pETDuet-1, 2 μL of trpB, 2 μL of 10×T4 ligation Buffer, 1 μL of T4 DNA Ligase, and 5 μL of ddH 2 O, with a total volume of 20 μL. The ligation condition is: incubate at 16°C for more than 16 h. Transformation is carried out under the condition of heat shock at 42°C for 60 s. Take 50 μL of Escherichia coli ATCC11229, add 5 μL of the recombinant plasmid ligation product, react on ice for 30 min, plate (ampicillin resistance), and screen for bacteria.

[0031] Take 15 g of wet cells of Escherichia coli ATCC11229 containing the modified trp B gene (the physical objects of strain culture are as Figure 1 shown), a hair acid hydrolysis solution containing 21.00 g of L-serine, 27.02 g of 6-fluoroindole, 0.20 g / L of pyridoxal phosphate, and 0.005 g / L of petroleum ether in a 500 mL reaction solution, pH 8.0, and carry out an enzymatic reaction at 37°C for 12 h. After the reaction, the 6-fluoro-L-tryptophan in the conversion solution is 38.16 g, and the molar conversion rate to L-serine is 85.6%;

[0032] (2) Centrifuge the conversion solution at 4000 r / min for 10 min to remove the bacterial cells, heat, decolorize with activated carbon, filter by suction, adjust the pH of the filtrate to about 5.5, let it stand to precipitate, filter by vacuum suction, dry to obtain 35.61 g of crude 6-fluoro-L-tryptophan, wash with ethanol with a mass concentration of 95%, filter by vacuum suction, dry to obtain 33.6 g of fine 6-fluoro-L-tryptophan. The 6-fluoro-L-tryptophan was measured by infrared (the results are as Figure 2as shown in Figure B), which is consistent with the standard product ( Figure 2 A). Determined by an amino acid analyzer, the purity is 99.9%.

[0033] Example 2:

[0034] A method for preparing 6-fluoro-L-tryptophan, comprising the following steps:

[0035] (1) Site-directed mutation of the tryptophan synthase trp B gene sequence, and the specific steps are the same as those in Example 1.

[0036] Transfer the mutated trp B gene into Bacillus subtilis ATCC21332, and ligate the modified trp B gene fragment to the vector pETDuet-1 to construct a recombinant plasmid. The ligation system is as follows: plasmid pETDuet-1 10 μL, trpB 2 μL, 10×T4 ligation Buffer 2 μL, T4 DNA Ligase 1 μL, ddH 2 O 5 μL, with a total volume of 20 μL. The ligation condition is: incubate at 16 °C for more than 16 h. Transformation is carried out under the condition of heat shock at 42 °C for 60 s. Take 50 μL of Bacillus subtilis ATCC21332, add 5 μL of the recombinant plasmid ligation product, react on ice for 30 min, coat the plate (ampicillin resistance), and screen for bacteria.

[0037] Take 15 g of the wet cells of the modified trp B gene Bacillus subtilis ATCC21332. The reaction solution in 500 mL contains 11 g of L-serine, 13.5 g of 6-fluoroindole, 0.2 g / L of pyridoxal phosphate, and 0.5 g / L of benzene, pH 8.0. Carry out enzymatic reaction at 37 °C for 16 h. After the reaction, 18.23 g of 6-fluoro-L-tryptophan is obtained in the conversion solution, and the molar conversion rate to L-serine is 81.8%;

[0038] (2) Centrifuge the conversion solution at 3000 r / min for 20 min to remove the bacterial cells, heat, decolorize with activated carbon, filter by suction, adjust the pH of the filtrate to about 5.5, let it stand to precipitate, filter by vacuum suction, and dry to obtain 16.23 g of crude 6-fluoro-L-tryptophan. Wash it with ethanol with a mass concentration of 95%, filter by vacuum suction, and dry to obtain 15.56 g of fine 6-fluoro-L-tryptophan. Determined by an amino acid analyzer, the purity is 99.8%.

[0039] Example 3:

[0040] A method for preparing 6-fluoro-L-tryptophan, comprising the following steps:

[0041] (1) Site-directed mutation of the tryptophan synthase trp B gene sequence, and the specific steps are the same as those in Example 1.

[0042] The mutated trp B gene was transferred into Pseudomonas stutzeri ATCC8482. The modified trp B gene fragment was ligated into the vector pETDuet-1 to construct a recombinant plasmid. The ligation system was as follows: 10 μL of plasmid pETDuet-1, 2 μL of trpB, 2 μL of 10×T4 ligation Buffer, 1 μL of T4 DNA Ligase, and 5 μL of ddH 2 O, with a total volume of 20 μL. The ligation condition was: incubation at 16 °C for more than 16 h. Transformation was carried out under the condition of heat shock at 42 °C for 60 s. Take 50 μL of Pseudomonas stutzeri ATCC8482, add 5 μL of the recombinant plasmid ligation product, react on ice for 30 min, spread on plates (ampicillin resistance), and screen for bacteria.

[0043] Take 20 g of wet cells of Pseudomonas stutzeri ATCC8482, and in a 500 mL reaction solution containing 21 g of L-serine in hair acid hydrolysis solution, 27.02 g of 6-fluoroindole, 0.2 g / L of pyridoxal phosphate, and 0.01 g / L of methanol, with a pH of 8.0, carry out an enzymatic reaction at 37 °C for 15 h. After the reaction, 37.25 g of 6-fluoro-L-tryptophan was obtained in the conversion solution, and the molar conversion rate to L-serine was 83.6%;

[0044] (2) Centrifuge the conversion solution at 5000 r / min for 10 min to remove the bacterial cells, heat, decolorize with activated carbon, filter by suction, adjust the pH of the filtrate to about 5.5, let it stand to precipitate, filter by vacuum suction, dry to obtain 35.23 g of crude 6-fluoro-L-tryptophan, wash with ethanol with a mass concentration of 95%, filter by vacuum suction, dry to obtain 33.25 g of fine 6-fluoro-L-tryptophan. Determined by an amino acid analyzer, the purity was 99.9%.

[0045] Example 4:

[0046] A method for preparing 6-fluoro-L-tryptophan, comprising the following steps:

[0047] (1) Site-directed mutation of the tryptophan synthase trp B gene sequence, and the specific steps are the same as in Example 1.

[0048] The mutated trp B gene was transferred into Bacillus cereus ATCC11778. The modified trp B gene fragment was ligated into the vector pETDuet-1 to construct a recombinant plasmid. The ligation system was as follows: 10 μL of plasmid pETDuet-1, 2 μL of trpB2 μL, 2 μL of 10×T4 ligation Buffer, 1 μL of T4 DNA Ligase, and 5 μL of ddH 25 μL, with a total volume of 20 μL. The ligation conditions were: incubation at 16 °C for more than 16 h. Transformation was carried out under the condition of heat shock at 42 °C for 60 s. Take 50 μL of Bacillus cereus ATCC11778, add 5 μL of the recombinant plasmid ligation product, react on ice for 30 min, plate (ampicillin resistance), and screen for bacteria.

[0049] Take 20 g of wet cells of the modified Bacillus cereus ATCC11778 with the trp B gene. In a 500 mL reaction solution, there were 11 g of L-serine, 13.5 g of 6-fluoroindole, 0.2 g / L of pyridoxal phosphate, and 0.5 g / L of dimethylformamide. The pH was 8.0, and the enzymatic reaction was carried out at 37 °C for 20 h. After the reaction, the 6-fluoro-L-tryptophan in the conversion solution was 18.14 g, and the molar conversion rate to L-serine was 81.3%.

[0050] (2) Centrifuge the conversion solution at 4000 r / min for 15 min to remove the bacterial cells, heat it, decolorize it with activated carbon, filter it by suction. Adjust the pH of the filtrate to about 5.5, let it stand to precipitate, filter it by vacuum suction, and dry it to obtain 17.45 g of crude 6-fluoro-L-tryptophan. Wash it with ethanol with a mass concentration of 95%, filter it by vacuum suction, and dry it to obtain 16.60 g of fine 6-fluoro-L-tryptophan. Determined by an amino acid analyzer, the purity was 99.9%.

[0051] Example 5:

[0052] A method for preparing 6-fluoro-L-tryptophan, comprising the following steps:

[0053] (1) Site-directed mutagenesis of the tryptophan synthase trp B gene sequence, and the specific steps were the same as in Example 1.

[0054] Transfer the mutated trp B gene into Escherichia coli ATCC11229. Connect the modified trp B gene fragment to the vector pETDuet-1 to construct a recombinant plasmid. The ligation system was as follows: plasmid pETDuet-1 10 μL, trpB 2 μL, 10×T4 ligation Buffer 2 μL, T4 DNA Ligase 1 μL, ddH 2 O 5 μL, with a total volume of 20 μL. The ligation conditions were: incubation at 16 °C for more than 16 h. Transformation was carried out under the condition of heat shock at 42 °C for 60 s. Take 50 μL of Escherichia coli ATCC11229, add 5 μL of the recombinant plasmid ligation product, react on ice for 30 min, plate (ampicillin resistance), and screen for bacteria.

[0055] Take 15 g of wet cells of Escherichia coli ATCC11229 containing the modified trp B gene, 500 mL of reaction solution containing 42 g of hair acid hydrolysis solution of L-serine, 54.0 g of 6-fluoroindole, 0.4 g / L of pyridoxal phosphate and 0.01 g / L of petroleum ether, pH 8.0, enzymatic reaction at 37 °C for 16 h. After the reaction, 73.11 g of 6-fluoro-L-tryptophan was obtained in the conversion solution, and the molar conversion rate to L-serine was 86.5%;

[0056] (2) Centrifuge the conversion solution at 3000 r / min for 20 min to remove the bacterial cells, heat, decolorize with activated carbon, filter by suction, adjust the pH of the filtrate to about 5.5, let it stand to precipitate, filter by vacuum suction, and dry to obtain 71.22 g of crude 6-fluoro-L-tryptophan. Wash it with ethanol with a mass concentration of 95%, filter by vacuum suction, and dry to obtain 69.59 g of fine 6-fluoro-L-tryptophan. Determined by an amino acid analyzer, the purity is 99.8%.

[0057] Example 6:

[0058] A method for preparing 6-fluoro-L-tryptophan, comprising the following steps:

[0059] (1) Site-directed mutagenesis of the tryptophan synthase trp B gene sequence, and the specific steps are the same as in Example 1.

[0060] Transfer the mutated trp B gene into Pseudomonas stutzeri ATCC8482. Connect the modified trp B gene fragment to the vector pETDuet-1 to construct a recombinant plasmid. The ligation system is as follows: 10 μL of plasmid pETDuet-1, 2 μL of trpB, 2 μL of 10×T4 ligation Buffer, 1 μL of T4 DNA Ligase, ddH 2 O 5 μL, with a total volume of 20 μL. The ligation condition is: incubate at 16 °C for more than 16 h. Transformation is carried out under the condition of heat shock at 42 °C for 60 s. Take 50 μL of Pseudomonas stutzeri ATCC8482, add 5 μL of the recombinant plasmid ligation product, react on ice for 30 min, coat the plate (ampicillin resistance), and screen for bacteria.

[0061] Take 20 g of wet cells of Pseudomonas stutzeri ATCC8482 containing the modified trp B gene, 500 mL of reaction solution containing 11 g of hair acid hydrolysis solution of L-serine, 13.5 g of 6-fluoroindole, 0.1 g / L of pyridoxal phosphate and 0.05 g / L of dimethylformamide, pH 8.0, enzymatic reaction at 37 °C for 16 h. After the reaction, 17.98 g of 6-fluoro-L-tryptophan was obtained in the conversion solution, and the molar conversion rate to L-serine was 80.26%;

[0062] (2) Centrifuge the conversion solution at 5000 r / min for 10 min to remove the bacterial cells, heat it, decolorize with activated carbon, filter by suction, adjust the pH of the filtrate to about 5.5, let it stand to precipitate, filter by vacuum suction, and dry to obtain 16.45 g of crude 6-fluoro-L-tryptophan. Wash it with ethanol with a mass concentration of 95%, filter by vacuum suction, and dry to obtain 15.43 g of high-quality 6-fluoro-L-tryptophan. Determined by an amino acid analyzer, the purity is 99.8%.

[0063] Comparative Example 1:

[0064] The difference between this comparative example and Example 1 is that the trp B gene was not modified. The amount of 6-fluoro-L-tryptophan synthesized by the tryptophan synthase in this comparative example was 4.05 g, and the molar conversion rate to L-serine was 9.10%.

[0065] Comparative Example 2:

[0066] The difference between this comparative example and Example 2 is that the trp B gene was not modified. The amount of 6-fluoro-L-tryptophan synthesized by the tryptophan synthase in this comparative example was 1.98 g, and the molar conversion rate to L-serine was 8.91%.

[0067] Comparative Example 3:

[0068] The difference between this comparative example and Example 3 is that the trp B gene was not modified. The amount of 6-fluoro-L-tryptophan synthesized by the tryptophan synthase in this comparative example was 3.83 g, and the molar conversion rate to L-serine was 8.61%.

[0069] Comparative Example 4:

[0070] The difference between this comparative example and Example 4 is that the trp B gene was not modified. The amount of 6-fluoro-L-tryptophan synthesized by the tryptophan synthase in this comparative example was 1.93 g, and the molar conversion rate to L-serine was 8.71%.

[0071] Comparative Example 5:

[0072] The difference between this comparative example and Example 5 is that the trp B gene was not modified. The amount of 6-fluoro-L-tryptophan synthesized by the tryptophan synthase in this comparative example was 7.40 g, and the molar conversion rate to L-serine was 8.30%.

[0073] Comparative Example 6:

[0074] The difference between this comparative example and Example 6 is that the trp B gene was not modified. The amount of 6-fluoro-L-tryptophan synthesized by the tryptophan synthase in this comparative example was 2.16 g, and the molar conversion rate to L-serine was 9.69%.

[0075] Comparative Example 7:

[0076] The difference between this comparative example and Example 1 lies in that the positions of site-directed mutations in the tryptophan synthase trp B gene sequence are different from those in Example 1. Specifically, gag at the 11th position is mutated to gaa, aag at the 99th position is mutated to acg, and ccg at the 224th position is mutated to caa.

[0077] The amount of 6-fluorol-L-tryptophan synthesized by the tryptophan synthase in this comparative example is 1.35 g, and the molar conversion rate to L-serine is 6.05%.

[0078] The specific gene sequence (SEQ ID NO.1) of the mutated tryptophan synthase trp B is as follows:

[0079] atgacaacat tacttaacgc ctattttggt gagtttggcg gcatgtacgt gccacaaatc

[0080] ctgatgcctg ctctgcgcca gctggaagaa gcttttgtca gtgcgcaaaa agatcctgaa

[0081] tttcaggctc agttcaacga cctgctgaaa aactatgccg ggcgtccaac cgcgctgacc

[0082] aaatgccaga acattacagc cgggacgaac accacgctgt atctcaagcg tgaagatttg

[0083] ctgcacggcg gcgcgcataa aactaaccag gtgctggggc aggcgttgct ggcgaagggg

[0084] atgggtaaaa ccgaaatcat cgccgaaacc ggtgccggtc agcatggcgt ggcgtcggcc

[0085] cttgccagcg ccctgctcgg cctgaaatgc cgtatttata tgggtgccaa agacgttgaa

[0086] cgccagtcgc ctaacgtttt tcgtatgcgc ttaatgggtg cggaagtgat cccggtgcat

[0087] agcggttccg cgacgctgaa agatgcctgt aacgaggcgc tgcgcgactg gtccggtagt

[0088] tacgaaaccg cgcactatat gctgggcacc gcagctggcc cgcatcctta tccgaccatt

[0089] gtgcgtgagt ttcagcggat gattggcgaa gaaaccaaag cgcagattct ggaaagaaga

[0090] ggtcgcctgc cggatgccgt tatcgcctgt gttggcggcg gttcgaatgc catcggcatg

[0091] tttgctgatt tcatcaatga aaccaacgtc ggcctgattg gtgtggagcc aggtggtcac

[0092] ggtatcgaaa ctggcgagca cggcgcaccg ctaaaacatg gtcgcgtggg tatctatttc

[0093] ggtatgaaag cgccgatgat gcaaaccgaa gacgggcaga ttgaagaatc ttactccatc

[0094] tccgccggac tggatttccc gtctgtcggc ccacaacacg cgtatcttaa cagcactgga

[0095] cgcgctgatt acgtgtctat taccgatgat gaagcccttg aagccttcaa aacgctgtgc

[0096] ctgcacgaag ggatcatccc ggcgctggaa tcctcccacg ccctggccca tgcgttgaaa

[0097] atgatgcgcg aaaacccgga taaagagcag ctactggtgg ttaacctttc cggtcgcggc

[0098] gataaagaca tcttcaccgt tcacgatatt ttgaaagcac gaggggaaat ctga

[0099] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A mutant tryptophan synthase trp B gene, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

1.

2. Use of the mutant tryptophan synthase trp B gene according to claim 1 in the preparation of 6-fluoro-L-tryptophan.

3. A recombinant construct, characterized in that The recombinant construct contains the mutated tryptophan synthase trp B gene according to claim 1.

4. The recombinant construct according to claim 3, characterized in that The vector used in the recombinant construct is a cloning vector or an expression vector for expressing the mutated tryptophan synthase trp B gene.

5. The recombinant construct according to claim 3, characterized in that The recombinant construct is a recombinant plasmid.

6. A recombinant host cell, characterized in that It contains the recombinant construct according to claim 5.

7. The recombinant host cell according to claim 6, characterized in that The host cell includes any one of Escherichia coli ATCC11229, Bacillus subtilis ATCC21332, Pseudomonas stutzeri ATCC8482, and Bacillus cereus ATCC11778.

8. A method for preparing 6-fluoro-L-tryptophan, characterized in that: The following steps are involved: A transformation liquid is added to the recombinant host cell to carry out an enzymatic reaction to obtain 6-fluoro-L-tryptophan; the transformation liquid comprises a feed liquid containing L-serine, pyridoxal phosphate and 6-fluoroindole.

9. The preparation method according to claim 8, characterized in that: The temperature of the enzymatic reaction is 25-45° C., the time is 10-20 hours, and the pH is 6-11.

10. The preparation method according to claim 8, characterized in that: After the reaction is completed, the reaction product is centrifuged, decolorized, separated, washed, filtered and dried.

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