CN110628736B

CN110628736B - Tryptophan 2,3 dioxygenase mutant and preparation method and application thereof

Info

Publication number: CN110628736B
Application number: CN201910955751.1A
Authority: CN
Inventors: 王喜庆; 魏研欣; 蒋胜胜
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2019-10-09
Filing date: 2019-10-09
Publication date: 2021-05-11
Anticipated expiration: 2039-10-09
Also published as: CN110628736A

Abstract

The invention discloses a tryptophan 2,3 dioxygenase mutant and a preparation method and application thereof, belonging to the field of protein engineering. The tryptophan 2,3 dioxygenase mutant is obtained by mutating the amino acid sequence of the tryptophan 2,3 dioxygenase at the 51 st position and the 127 th position. The biocatalyst constructed by the invention can synthesize 3 a-hydroxy-hexahydropyrrole [2,3-b ] indole-2-carboxylic acid (HPICs) and derivatives thereof in one step by using tryptophan, 5-chlorotryptophan and 6-chlorotryptophan without protecting groups as substrates. The novel enzyme converts tryptophan dioxygenase into monooxygenase, generates HPICs in a quantity far larger than that of wild-type tryptophan 2,3 dioxygenase, and has excellent diastereoselectivity; the reaction and the purification process are carried out in an environment-friendly aqueous solution at room temperature, the reaction system is simple, the reaction condition is mild, and the energy consumption is low.

Description

Tryptophan 2,3 dioxygenase mutant and preparation method and application thereof

Technical Field

The invention relates to a tryptophan 2,3 dioxygenase mutant and a preparation method and application thereof, belonging to the field of protein engineering.

Background

Three-membered ring 3 a-hydroxy-hexahydropyrrole [2,3-b ] indole-2-carboxylic acid and derivatives (HPICs for short) are common molecular frameworks in many alkaloids and polypeptides, and have strong structural diversity and biological activity. For example, kapakahine C is cytotoxic to P388 mouse leukemia cells; most natural products in the NWG family (e.g., NWG01) have greater antibacterial activity against gram-positive bacteria than against gram-negative bacteria. In addition to antibacterial activity, himastatin and chlorphostin inhibit the growth of several cancer cell lines. Thus, natural products containing structural units of HPICs are a popular target for synthetic and pharmacological research. HPIC can also be oxidized to intermediates useful in the synthesis of pyrrolobenzazine, for example: paeciloxazine, CJ12662, and CJ 12663. In addition, monocyclic peptides having the backbone of HPICs can be converted enzymatically into bicyclic peptides with improved biological activity. Thus, in addition to its presence in a wide variety of biologically active molecules, HPICs are also important synthetic intermediates.

The construction of HPICs from tryptophan derivatives has been the focus of research in the field of organic chemical synthesis. Earlier, Danishefsky could prepare diastereomerically selective HPICs in 3-4 steps using free Trp as a substrate, which provided a powerful strategy for the synthesis of complex natural products. The cyclization reaction initiated by cations utilizes electrophiles such as N-phenylselenophthalimide (N-PSP) or N-bromosuccinimide (NBS) to activate indole ring, and then utilizes m-chloroperbenzoic acid (mCPBA) or AgNO to activate indole ring₃The aqueous solution is oxidized to denitrify, and oxygen atoms are substituted for activated heterocyclic atoms. Unlike the stepwise approach described above, oxidation of DMDO can directly convert Trp derivatives to HPICs, but diastereoselectivity depends on the carboxyl and amine protecting groups. Without bulky protecting groups like trityl (Tr) and tert-butyl (tBu), the resulting HPIC is a mixture of 1:1 diastereomers. Recently, free radical initiated cyclization has also been used to step-wise prepare HPIC from protected Trp derivatives. Despite significant advances, multiple protecting groups are required in the preparation of HPIC, which is not an environmentally friendly synthetic approach. To address this problem, Nakagawa researchers have attempted to oxidize unprotected tryptophan, including Me, by a two-step/one-pot process using photosensitization₂S reduces the generated tricyclic peroxide by cracking. Although the reaction process is simple, the stereoselectivity of this method is poor. In addition, unstable peroxide intermediates are susceptible to rapid decomposition to N' -formyl kynurenines (NFKs).

The research in 2011 finds that tryptophan 2,3 dioxygenase xcTDO can catalyze tryptophan to generate HPIC through a one-step reaction, which provides a new idea for generating HPIC, but the problem that the yield of HPIC is extremely low because NFK is mainly generated through the reaction is solved, and the application prospect of synthesizing HPICs by the enzyme is limited.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects of the prior art and provide a tryptophan 2,3 dioxygenase mutant and a preparation method and application thereof, wherein the amount of HPICs generated by the prepared tryptophan 2,3 dioxygenase mutant is far greater than that generated by wild-type tryptophan 2,3 dioxygenase, and the mutant has excellent diastereoselectivity.

In order to solve the technical problem, the invention provides a tryptophan 2,3 dioxygenase mutant which comprises an amino acid sequence shown as SEQ ID NO. 1.

The invention also provides a nucleotide for coding the tryptophan 2,3 dioxygenase mutant, wherein the nucleotide sequence is shown as SEQ ID NO. 2.

The invention also provides a carrier containing the nucleotide of the tryptophan 2,3 dioxygenase mutant.

The invention also provides cells expressing the tryptophan 2,3 dioxygenase mutant.

The invention also provides a method for improving the catalytic efficiency of tryptophan 2,3 dioxygenase, which is characterized in that the 51 st amino acid residue phenylalanine of tryptophan 2,3 dioxygenase is replaced by methionine, and the 127 th amino acid residue glutamine is replaced by tyrosine.

The invention also provides a preparation method of the tryptophan 2,3 dioxygenase mutant, which comprises the following steps:

(1) construction of a plasmid expressing wild-type tryptophan 2,3 dioxygenase: the nucleotide sequence of SEQ ID NO 3 is recombined on an expression vector through enzyme digestion;

(2) constructing a saturated mutation library through three rounds of fixed-point saturated mutation;

(3) transforming the plasmids expressing the tryptophan 2,3 dioxygenase and the mutant constructed in the steps (1) and (2) into escherichia coli;

(4) expression of tryptophan 2,3 dioxygenase TDO and mutants: after overnight culture of the strain obtained in the step (3) in an LB liquid culture medium, adding the strain into a TB liquid culture medium for propagation, adding a heme precursor delta-aminolevulinic acid and IPTG for induction expression at the late logarithmic phase, and centrifuging to collect the strain;

(5) preparation of cell lysates containing tryptophan 2,3 dioxygenase TDO and mutants: resuspending the thallus obtained in the step (4) by using a buffer solution, cracking cells by ultrasonic disruption, and centrifuging to obtain a supernatant;

(6) preparation of cell lysates containing tryptophan 2,3 dioxygenase TDO and mutants: resuspending the thallus obtained in the step (4) by using a buffer solution, cracking cells by ultrasonic disruption, and centrifuging to obtain a supernatant;

(7) purification of tryptophan 2,3 dioxygenase TDO and mutants: adding the supernatant obtained in the step (5) into a nickel ion affinity column which is pretreated in advance, enabling the target protein with the His label to be combined with the nickel column, then removing the hybrid protein at a lower imidazole concentration, and finally eluting the target protein by using a buffer solution containing high-concentration imidazole; dialyzing the eluted target protein to remove imidazole, concentrating the protein, and storing at-80 deg.C.

The preparation method of the tryptophan 2,3 dioxygenase mutant comprises the following specific steps:

(1) construction of expression vector for tryptophan 2,3 dioxygenase TDO gene: the whole gene synthesized xcTDO fragment is treated by restriction endonucleaseNdeIAndXhoIafter double digestion according to the instructions, ligation was performed using T4 ligaseNdeIAndXhoIon the expression vector pET22b (+) after double enzyme digestion; transforming escherichia coli DH5 alpha competent cells with the ligation product; picking the successfully transformed monoclonal colony from a solid LB culture medium plate containing 100 mu g/ml ampicillin, and culturing the colony in an LB liquid culture medium containing ampicillin at the same concentration at 37 ℃ and the rotation speed of a shaking table of 220rpm overnight; extracting recombinant plasmids from the cultured bacterial liquid by using a plasmid miniprep kit, and sending the extracted plasmids to sequencing for identification;

(2) construction of tryptophan 2,3 dioxygenase mutant plasmid: using the plasmid with successful sequencing as a PCR template, constructing a mutant plasmid by a Quickchange method, and sending the mutant plasmid to sequencing for identification;

(3) preparation of tryptophan 2,3 dioxygenase TDO and mutants: transforming the recombinant plasmid with successful sequencing into escherichia coli BL21 (DE 3), picking the positive clone into 5 ml LB liquid culture medium containing 100 mug/ml ampicillin for overnight culture, adding the bacterial liquid into 500 ml TB liquid culture medium containing 100 mug/ml kanamycin at the ratio of 1:100, and performing amplification at 37 ℃ and 220rpm of shaker rotation speed; to be OD₆₀₀Approximately 0.6, the final concentration of heme precursor, delta-aminolevulinic acid, was added at 0.5mM and IPTG was added at 0.5mM for induction; centrifuging at a rotating speed of 5000 rpm for 10 min to collect the bacteria after the temperature is 22 ℃ and the rotating speed of a shaking table is 220rpm and the rotating speed is 20 h; discarding the supernatant culture medium, then resuspending the thallus with 85 ml buffer A, cracking the cells by ultrasonic disruption, and centrifuging at 4 ℃ and a centrifugal speed of 10000 rpm for 45 min to obtain a supernatant cell lysate; adding the cell lysate into a nickel ion affinity column of 10 ml pre-treated by buffer A in advance, and combining the target protein with the His tag with the nickel column; subsequently, the heteroproteins were removed by buffer C containing 20 mM, 85 mM imidazole, respectively, and the protein of interest was eluted from the column by buffer B; the purity of the target protein was identified by SDS-PAGE; dialyzing the eluted target protein with buffer D4 ℃ overnight to remove imidazole; after centrifugal concentration, storing at-80 ℃; wherein buffer A is a buffer of 0.1M KPi (potassium phosphate), 0.3M KCl, 10% glycerol, pH 7.8; buffer B was 0.1M KPi, 0.3M KCl, 300 mM imidazole, 10% glycerol, pH7.8 buffer; buffer C was 20 mM or 85 mM imidazole, 0.1M KPi, 0.3M KCl, 10% glycerol, pH7.8 buffer. Buffer D was 50 mM Tris, 10% glycerol, pH8.0 Buffer.

The specific method of the three rounds of site-specific saturation mutagenesis comprises the following steps:

(1) the xcTDO residues of the first round of site-directed saturation mutagenesis were: y24, Y27, L28, F51, H55, Y113, R117, and L120. They are located in the α B (F51 and H55) and α D (Y113, R117 and L120) helices and amino terminal residues from other monomers (Y24, Y27 and L28) and are all located to one side of the active center.

(2) Constructing a second round of saturated mutation library, and constructing double mutant plasmids by taking the mutant plasmids with higher activity than the wild type plasmids obtained by the first round of mutation screening as templates, wherein the targets of the round of screening are residues G121, P122, S123, S124, G125, Q127, G253, T254 and G255 which are positioned on the other side of the active site. These residues are located in the loop between the alpha D-alpha E (residues 121-126 and 127) and alpha J-alpha K (residue 253-255) helices. Different conformations of these two loops are observed in the xcTDO crystal structure, showing dynamic interactions with the substrate.

(3) Third round of mutation screening the most active mutant plasmids from the first or second round of screening were used as templates, and the target residues for this round of screening were those associated with heme binding (W102, L105, F126, Y131, R132, W236, H240, V244, I248, G256, F262, and L263).

The invention also provides a method for producing 3 a-hydroxy-hexahydropyrrole [2,3-b ] indole-2-carboxylic acid and derivatives thereof, which takes tryptophan, 5-chlorotryptophan and 6-chlorotryptophan as substrates, adds catalase, methylene blue, ascorbic acid and reaction buffer solution, and carries out catalytic reaction by using the tryptophan 2,3 dioxygenase mutant as the catalyst in claim 1.

Preferably, the reaction system has a pH of 6.0, a reaction temperature of 25 ℃ and a reaction time of 24 hours. After the reaction is finished, adding 50% acetonitrile to precipitate protein for 30 min, centrifuging for 20 min, taking supernatant, and freeze-drying.

The invention also provides application of the tryptophan 2,3 dioxygenase mutant in preparing products containing 3 a-hydroxy hexahydropyrrole [2,3-b ] indole-2-carboxylic acid and derivatives thereof.

The invention achieves the following beneficial effects:

(1) the constructed biocatalyst can utilize tryptophan, 5-chlorotryptophan and 6-chlorotryptophan without protecting groups as substrates to synthesize HPICs in one step;

(2) the invention is from Xanthomonas campestris (A)Xanthomonas campestris) Replacement of the amino acid residue phenylalanine of tryptophan 2,3 dioxygenase at position 51 with methionineAnd replacing the amino acid residue glutamine at the 127 th position with the tyrosine to form the tryptophan 2,3 dioxygenase mutant. The novel enzyme tryptophan 2,3 dioxygenase mutant converts tryptophan dioxygenase into monooxygenase, and the amount of HPICs generated by the novel enzyme tryptophan 2,3 dioxygenase mutant is far greater than that generated by wild-type tryptophan 2,3 dioxygenase (yield of wild-type tryptophan 2,3 dioxygenase HPICs)<1%) has excellent diastereoselectivity;

(3) the reaction and the purification process are carried out in an environment-friendly aqueous solution at room temperature, the reaction system is simple, the reaction condition is mild, and the energy consumption is low.

Detailed Description

The invention is further described below. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.

Unless otherwise specified, all compounds and reagents below were purchased from Sigma-Aldrich, EMD Millipore, Tokyo Chemical Industry, Biotechnology (Shanghai) Ltd, with a purity of > 99%.

Example 1 construction of Gene expression vector for xcTDO

The whole gene synthesized xcTDO fragment (sequence shown in SEQ ID NO:3, synthesized by Jinzhi Biometrics, Inc., Suzhou) was subjected to restriction enzymeNdeIAndXhoI(New England Biolabs) following the protocol, ligated to the DNA using T4 ligase (New England Biolabs)NdeIAndXhoIthe double-digested expression vector pET22b (+). Coli DH 5. alpha. competent cells (Tiangen Biochemical technology Co., Ltd.) were transformed with the ligation product. The successfully transformed monoclonal colonies were picked from solid LB medium plates containing 100. mu.g/ml ampicillin and cultured overnight in LB liquid medium containing the same concentration of ampicillin at 37 ℃ with a shaker speed of 220 rpm. Recombinant plasmids were extracted from the cultured bacterial solution using a plasmid miniprep kit (Tiangen Biochemical technology Co., Ltd.) according to the instructions, and the extracted plasmids were subjected to sequencing and identification (Suzhou Jinwei Zhi Biol., Ltd.).

Example 2 creation of mutant pools

Three rounds of site-directed saturation mutagenesis were used.

The xcTDO residues of the first round of site-directed saturation mutagenesis were: y24, Y27, L28, F51, H55, Y113, R117, and L120. They are located in the α B (F51 and H55) and α D (Y113, R117 and L120) helices and amino terminal residues from other monomers (Y24, Y27 and L28) and are all located to one side of the active center. In the screening process, the expression plasmid synthesized by the whole gene (the nucleotide sequence shown by the code SEQ ID NO:3 is cloned on the plasmid) is used as a template, a primer is designed around each mutation site, and a single mutation library is constructed. Screening revealed that most of the mutants in the F51X pool exhibited significantly higher HPIC production than wild-type xcTDO, while little or no HPIC production was detected with mutants from other pools.

(2) A second round of saturated mutation library was constructed, using the highly active mutant plasmid in library F51X as template, and this round of screening was targeted at residues G121, P122, S123, S124, G125, Q127, G253, T254 and G255, all of which were on the other side of the active site. These residues are located in the loop between the α D- α E (residue 121-127) and α J- α K (residue 253-255) helices. Different conformations of these two loops are observed in the xcTDO crystal structure, showing dynamic interactions with the substrate. Screening found that xcTDO-51M/Q127Y activity was most prominent, showing significantly increased overall reactivity (>99% conversion) and moderately improved HPIC/NFK ratio of the product.

(3) A third round of saturation mutation library was constructed using the plasmid xcTDO-51M/Q127Y as a template, and the target residues for this round of screening were those involved in heme binding (W102, L105, F126, Y131, R132, W236, H240, V244, I248, G256, F262, and L263). No more excellent mutants were found by the screening.

Example 3 activation and inducible expression of wild-type xcTDO and mutant pools

The recombinant plasmid was transformed into E.coli BL21 (DE 3) (Tiangen Biochemical technology Co., Ltd.), and a single clone was picked from the overnight-cultured plate to 96-depth plates containing 300. mu.l of LB liquid medium containing ampicillin at a final concentration of 100. mu.g/mlIn a well plate, the cells were incubated overnight at 37 ℃ with a shaker rotation speed of 250 rpm. The next day, 15. mu.l of the culture broth was aspirated from the overnight-cultured 96-deep-well plate, and added to a fresh 1.5 ml 96-deep-well plate containing ampicillin TB broth at a final concentration of 100. mu.g/ml, at 37 ℃ and a shaker rotation speed of 250 rpm, until OD was reached₆₀₀Approximately 0.6, the final concentration of heme precursor, delta-aminolevulinic acid, was added at 0.5mM and IPTG was added at 0.5mM for induction. After 20 hours at 22 ℃ with a shaker speed of 250 rpm, the mixture was centrifuged at 5000 rpm for 10 min and the supernatant was discarded.

Example 4 Activity screening of mutant libraries

The cells were suspended in 375. mu.l buffer E (50 mM Tris, pH 8.0), lysozyme was added to a final concentration of 1.0 mg/mL, deoxyribonuclease I was added to the cells at 0.1 mg/mL, after ultrasonication, the cells were centrifuged at 5000 rpm at 4 ℃ for 30 min, 337.5. mu.l of the supernatant was transferred to a new 96-well plate, and 142. mu.l of buffer F (50 mM Tris, 5mM Trp, pH 8.0), 20. mu.l of 500 mM ascorbic acid and 0.5. mu.l of 10 mM methylene blue were added in this order. 25 ℃, 120 rpm, 5 h. Adding 500 μ l acetonitrile to precipitate protein for 30 min, centrifuging at 5000 rpm for 20 min, collecting supernatant, and lyophilizing. After lyophilization 500. mu.l ddH was used₂Redissolving, filtering and detecting by HPLC, and taking wild enzyme as a control. HPLC detection conditions: c18 column (5 μm, 2.1X 150 mm); the mobile phase was acetonitrile/water (0.1% formic acid); the flow rate is 0.3 mL/min; the detection wavelength is 295 nm; the column temperature was 27 ℃. HPLC method: 100% water for 10 min, 100% water to 0% water for 15 min, 100% acetonitrile for 3 min.

Example 5 expression and Activity verification of mutants

(1) Expression of the mutant: after selecting the mutant clone into 5 ml LB liquid culture medium containing 100 mug/ml ampicillin for overnight culture, adding the bacterial liquid into 500 ml TB liquid culture medium containing 100 mug/ml kanamycin in a ratio of 1:100, and performing expansion culture at 37 ℃ and 220rpm of shaking table rotation speed; to be OD₆₀₀Approximately 0.6, the final concentration of heme precursor, delta-aminolevulinic acid, was added at 0.5mM and IPTG was added at 0.5mM for induction; centrifuging at a rotating speed of 5000 rpm for 10 min to collect the bacteria after the temperature is 22 ℃ and the rotating speed of a shaking table is 220rpm and the rotating speed is 20 h; after discarding the supernatant medium, the cells were washed with 85 ml of buffer AResuspending, cracking cells by ultrasonic disruption, centrifuging at 4 ℃ and 10000 rpm for 45 min to obtain supernatant cell lysate; adding the cell lysate into a nickel ion affinity column of 10 ml pre-treated by buffer A in advance, and combining the target protein with the His tag with the nickel column; subsequently, the heteroproteins were removed by buffer C containing 20 mM, 85 mM imidazole, respectively, and the protein of interest was eluted from the column by buffer B; the purity of the target protein was determined by SDS-PAGE and was generally > 98% pure. Dialyzing the eluted target protein with buffer D4 ℃ overnight to remove imidazole; after centrifugal concentration at 5000 rpm, preserving at-80 ℃; wherein buffer A is buffer solution with 0.1M KPi, 0.3M KCl, 10% glycerol and pH7.8; buffer B was 0.1M KPi, 0.3M KCl, 300 mM imidazole, 10% glycerol, pH7.8 buffer; buffer C was 20 mM or 85 mM imidazole, 0.1M KPi, 0.3M KCl, 10% glycerol, pH7.8 buffer. Buffer D was 50 mM Tris, 10% glycerol, pH8.0 Buffer.

(2) And (3) activity verification: reaction system: reaction buffer (Bis-Tris, pH 6.0), tryptophan 2,3 dioxygenase mutant (1. mu.M), tryptophan (5 mM), catalase (10. mu.g/mL), methylene blue (10. mu.M), ascorbic acid (20 mM), pH 6.0, 25 ℃, 120 rpm, and reaction for 24 hours were added to the reaction vessel, respectively. After the reaction is finished, 50% acetonitrile is added to precipitate protein, centrifugation is carried out, and supernatant is taken and freeze-dried. Freeze-drying the sample with ddH₂Redissolving in water, filtering, detecting by HPLC, and using wild enzyme as control.

HPLC detection conditions: c18 column (5 μm, 2.1X 150 mm); the mobile phase was acetonitrile/water (0.1% formic acid); the flow rate is 0.3 mL/min; the column temperature is 27 ℃; HPIC yield was measured at 295 nm; NFK production was measured at 321 nm and Trp residual at 280 nm. HPLC method: 100% water 36 min, 100% acetonitrile 13 min.

HPLC detection shows that the F51M/Q127Y mutant is most suitable for industrial production, the TON is 3312, the conversion rate is more than 99%, and the ratio of the HPIC/NFK product is 2.0: 1.

example 6: large Scale preparation of HPIC, 5Cl-HPIC, 6Cl-HPIC

Reaction system: reaction buffer (Bis-Tris, pH 6.0), tryptophan 2,3 dioxygenase mutant xcTDO-F51M/Q127Y, substrate, catalase (10. mu.g/mL), methylene blue (10. mu.M), ascorbic acid (20 mM), pH 6.0, 25 ℃, 120 rpm, and reaction for 24 hours were added to the reaction vessel, respectively. After the reaction is finished, 50% acetonitrile is added to precipitate protein, the mixture is centrifuged at 1000 rpm for 20 min, and the supernatant is taken out and freeze-dried. A small amount of water was redissolved, loaded on an anion exchanger (DE 52) previously equilibrated with water, and NFK and HPIC were eluted separately with water containing 0.1% glacial acetic acid. Samples were lyophilized to a powder, weighed, and stored at-20 ℃. The yield of HPIC was 132 mg (60%), 5Cl-HPIC was 66 mg (52%), and 6Cl-HPIC was 39.5 mg (31%).

Substrate: trp (1.0 mmol, 204 mg), 5Cl-Trp (0.5 mmol, 119 mg); the amounts of the enzyme xcTDO-F51M/Q127Y were 0.03 mol%, 0.1 mol%, and 0.05 mol%, respectively.

The foregoing illustrates the general principles, principal features, and advantages of the invention. The method for synthesizing HPICs in one step by using tryptophan, 5-chlorotryptophan and 6-chlorotryptophan without protecting groups as substrates by the biocatalyst constructed by the invention has the advantages that: 1) the novel enzyme converts tryptophan 2,3 dioxygenase into monooxygenase, and the yield of HPICs generated by the novel enzyme is far greater than that of wild type (the yield of HPICs is less than 1 percent), and the novel enzyme has excellent diastereoselectivity; 2) the reaction and the purification process are carried out in an environment-friendly aqueous solution at room temperature, the reaction system is simple, the reaction condition is mild, and the energy consumption is low. Therefore, the method has good application prospect in the aspect of preparing HPICs.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.

Sequence listing

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Met Pro Val Asp Lys Asn Leu Arg Asp Leu Glu Pro Gly Ile His Thr

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Asp Leu Glu Gly Arg Leu Thr Tyr Gly Gly Tyr Leu Arg Leu Asp Gln

20 25 30

Leu Leu Ser Ala Gln Gln Pro Leu Ser Glu Pro Ala His His Asp Glu

35 40 45

Met Leu Met Ile Ile Gln His Gln Thr Ser Glu Leu Trp Leu Lys Leu

50 55 60

Leu Ala His Glu Leu Arg Ala Ala Ile Val His Leu Gln Arg Asp Glu

65 70 75 80

Val Trp Gln Cys Arg Lys Val Leu Ala Arg Ser Lys Gln Val Leu Arg

85 90 95

Gln Leu Thr Glu Gln Trp Ser Val Leu Glu Thr Leu Thr Pro Ser Glu

100 105 110

Tyr Met Gly Phe Arg Asp Val Leu Gly Pro Ser Ser Gly Phe Tyr Ser

115 120 125

Leu Gln Tyr Arg Tyr Ile Glu Phe Leu Leu Gly Asn Lys Asn Pro Gln

130 135 140

Met Leu Gln Val Phe Ala Tyr Asp Pro Ala Gly Gln Ala Arg Leu Arg

145 150 155 160

Glu Val Leu Glu Ala Pro Ser Leu Tyr Glu Glu Phe Leu Arg Tyr Leu

165 170 175

Ala Arg Phe Gly His Ala Ile Pro Gln Gln Tyr Gln Ala Arg Asp Trp

180 185 190

Thr Ala Ala His Val Ala Asp Asp Thr Leu Arg Pro Val Phe Glu Arg

195 200 205

Ile Tyr Glu Asn Thr Asp Arg Tyr Trp Arg Glu Tyr Ser Leu Cys Glu

210 215 220

Asp Leu Val Asp Val Glu Thr Gln Phe Gln Leu Trp Arg Phe Arg His

225 230 235 240

Met Arg Thr Val Met Arg Val Ile Gly Phe Lys Arg Gly Thr Gly Gly

245 250 255

Ser Ser Gly Val Gly Phe Leu Gln Gln Ala Leu Ala Leu Thr Phe Phe

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Pro Glu Leu Phe Asp Val Arg Thr Ser Val Gly Val Asp Asn Arg Pro

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aagaatccgc agatgctgca ggtgttcgcc tatgaccctg ccggccaggc acggctgcgc 480

gaagtactgg aagccccgag cctgtacgag gaattcctgc gctacctggc gcgcttcggg 540

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tcgctgtgcg aagacctggt ggatgtggaa acccagttcc agctgtggcg cttccggcac 720

atgcgcacgg tgatgcgggt gatcggcttc aaacgcggca ccggcggctc cagtggcgtg 780

gggttcctgc agcaggccct ggcactgacc ttcttcccgg agctgttcga cgtgcgtacg 840

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tccgagccgg cgcaccacga cgaaatgctg ttcatcatcc agcaccagac ctccgagctg 180

tggctgaaac tgctggccca cgagctgcgc gcggccatcg tgcacctgca gcgcgacgag 240

gtctggcaat gccgcaaggt gctggcgcgc agcaagcagg tgctgcgcca gctgaccgag 300

caatggtcgg tgctggaaac gctgaccccc agcgagtaca tgggctttcg cgatgtgctc 360

ggcccctcat cgggcttcca gtcgctgcaa taccgctaca tcgagttcct gctgggcaac 420

aagaatccgc agatgctgca ggtgttcgcc tatgaccctg ccggccaggc acggctgcgc 480

gaagtactgg aagccccgag cctgtacgag gaattcctgc gctacctggc gcgcttcggg 540

catgcgattc cgcagcaata ccaggcccgc gactggaccg ctgcgcacgt ggccgacgac 600

acgctgcggc cggtgttcga gcgcatctac gaaaataccg accgctactg gcgcgaatat 660

tcgctgtgcg aagacctggt ggatgtggaa acccagttcc agctgtggcg cttccggcac 720

atgcgcacgg tgatgcgggt gatcggcttc aaacgcggca ccggcggctc cagtggcgtg 780

gggttcctgc agcaggccct ggcactgacc ttcttcccgg agctgttcga cgtgcgtacg 840

tccgtaggcg tggacaaccg accgccgcag gggagtgcgg acgctgggaa gcgctga 897

Claims

1. A tryptophan 2,3 dioxygenase mutant is characterized in that the amino acid sequence of the mutant is shown as SEQ ID NO. 1.

2. Nucleotide sequence encoding a tryptophan 2,3 dioxygenase mutant as claimed in claim 1, characterized in that the nucleotide sequence is as shown in SEQ ID NO 2.

3. A vector comprising the nucleotide sequence of a tryptophan 2,3 dioxygenase mutant as claimed in claim 2.

4. A cell expressing the tryptophan 2,3 dioxygenase mutant according to claim 1, comprising the vector according to claim 3.

5. The method for preparing a tryptophan 2,3 dioxygenase mutant according to claim 1, wherein the method comprises the following steps:

substituting the 51 st amino acid residue of tryptophan 2,3 dioxygenase with phenylalanine and substituting the 127 th amino acid residue of glutamine with tyrosine to obtain a tryptophan 2,3 dioxygenase mutant as claimed in claim 1;

transforming the constructed plasmid expressing the tryptophan 2,3 dioxygenase mutant into escherichia coli;

and (3) expressing and purifying the tryptophan 2,3 dioxygenase mutant.

6. A process for producing 3 a-hydroxypyrrolo [2,3-b ] indole-2-carboxylic acid, which comprises using tryptophan, 5-chlorotryptophan and 6-chlorotryptophan as substrates, adding catalase, methylene blue, ascorbic acid and reaction buffer, and carrying out catalytic reaction using the tryptophan 2,3 dioxygenase mutant of claim 1 as a catalyst.

7. The process for producing 3 a-hydroxypyrrolo [2,3-b ] indole-2-carboxylic acid according to claim 6, wherein the reaction system has a pH of 6.0, a reaction temperature of 25 ℃ and a reaction time of 24 hours.

8. Use of the tryptophan 2,3 dioxygenase mutant according to claim 1 for the preparation of a product containing 3 a-hydroxyhexahydropyrrolo [2,3-b ] indole-2-carboxylic acid.