CN117683760B

CN117683760B - Tryptophan synthase mutant and application thereof in preparation of cysteine and cystine

Info

Publication number: CN117683760B
Application number: CN202410154368.7A
Authority: CN
Inventors: 林振泉; 乔玮博; 马成伟; 刘云慧; 金祥
Original assignee: Beijing Liangwei Biotechnology Research Institute Co ltd
Current assignee: Beijing Liangwei Biotechnology Research Institute Co ltd
Priority date: 2024-02-04
Filing date: 2024-02-04
Publication date: 2024-04-19
Anticipated expiration: 2044-02-04
Also published as: CN117683760A

Abstract

The invention relates to tryptophan synthase mutants and application thereof in preparation of cysteine and cystine. The invention is based on tryptophan synthase Trps from escherichia coli, and mutants with higher enzyme activity are obtained through site-directed mutation screening. The in vitro enzyme catalysis of the mutant is utilized to prepare the cysteine and the cystine, so that the synthesis speed can be improved, the production time can be shortened, the yield of the cysteine and the cystine can be greatly improved, the cost can be effectively reduced, and the method has important significance for the large-scale production of the cysteine and the cystine.

Description

Tryptophan synthase mutant and application thereof in preparation of cysteine and cystine

Technical Field

The invention relates to a tryptophan synthase mutant and application thereof in cysteine production, belonging to the technical fields of genetic engineering and enzyme engineering.

Background

L-cysteine and L-cystine are sulfur-containing nonessential amino acids, which are one of the basic units of various proteins constituting living bodies, and are particularly high in human and animal hair and finger keratin. Because the sulfhydryl groups in cysteine are unstable and are very easily oxidized, two cysteine molecules can be linked by disulfide bonds to form a more stable cystine molecule. L-cysteine and L-cystine are widely used for processing and manufacturing medicines, foods, cosmetics, feeds and the like, and particularly can replace methionine with higher price to be used as a feed additive, so that the L-cysteine and L-cystine have wide market demands and prospects.

Currently, the industrial production of L-cystine mainly adopts hair hydrolysate extraction, chemical synthesis and biological methods. The hair hydrolysate extraction method and the chemical synthesis method have lower yield and more operation steps, and a large amount of waste which is difficult to treat is produced in the production process, so that the environment is polluted, and the environmental protection pressure is high. The biological rule has the advantages of strong specificity, simple production process, less side reaction and by-product, less pollution and the like, and has wider development potential.

Biological methods for preparing cysteine include in vivo microbial fermentation and in vitro enzymatic conversion. In recent years, various scientists have studied a great deal for synthesizing L-cysteine by a microbial fermentation method, but the method is limited by the complexity of a microbial intracellular metabolic network and the mutual influence and restriction among different metabolic pathways, and the yield of the L-cysteine produced by the fermentation method is still not ideal, the sulfur conversion rate is low, and the requirement of industrial production cannot be met. The enzyme catalytic conversion method has the advantages of strong specificity, simple reaction steps, mild conditions, environmental friendliness and the like, thereby becoming a new direction of research and attention of people.

The method for producing L-cysteine by using the enzyme catalytic conversion method comprises the steps of expressing a required catalytic enzyme through an engineering strain, then treating the engineering strain, and using the engineering strain as a catalyst to catalyze serine and hydrosulfide to react in vitro to generate cysteine. If the production of L-cystine is needed to be continued, then the reaction solution is subjected to oxidation treatment to oxidize the L-cysteine into L-cystine; and then the L-cystine with extremely low solubility in water and easy precipitation is utilized to obtain the L-cystine product with higher purity by filtering and separating from the reaction liquid.

Tryptophan synthase (Tryptophan synthase, trps) is a key enzyme in the tryptophan synthesis pathway in organisms, consisting of two subunits, the β -subunit (TrpB) and the α -subunit (TrpA), catalyzing the reaction of indole with serine to synthesize tryptophan. The research shows that the tryptophan synthase (EcTrps) from escherichia coli not only has the function of synthesizing tryptophan, but also can catalyze serine and sodium hydrosulfide to generate cysteine, which opens up a new idea for the enzyme catalysis production of cysteine. However, ecTrps's study focused on tryptophan synthesis and metabolic pathways that it participates in the cell and little attention was paid to its ability to synthesize cysteine. In the current process of producing cysteine by EcTrps enzyme catalysis, the yield and conversion rate of cysteine are still not high, so that the production cost cannot be further reduced. In recent years, the demand of L-cysteine in various industries is increasing, and the existing yield can not meet the demands of domestic and foreign markets. Therefore, the molecular modification of tryptophan synthase EcTrps is of great significance in improving the yield and conversion rate of cysteine and reducing the cost.

Disclosure of Invention

In order to improve the yield and substrate conversion rate in the process of producing L-cystine by an enzyme catalysis method and also to reduce the impurity content in the product, tryptophan synthase EcTrps derived from escherichia coli is mutated and screened to obtain a EcTrps mutant with high enzyme activity for catalyzing serine and sodium hydrosulfide to synthesize L-cysteine (the enzyme activity, reaction enzyme activity, enzyme function, catalytic function and the like mentioned in the invention are referred to as the activity or function for catalyzing serine and sodium hydrosulfide to synthesize L-cysteine or similar expressions with the same meaning, unless otherwise specified).

The invention aims to provide a novel tryptophan synthase mutant and application thereof in cysteine production.

In order to achieve the object of the present invention, the present invention provides a tryptophan synthase mutant obtained by mutating EcTrps derived from E.coli (ESCHERICHIA COLI) with an amino acid substitution, the mutant consisting of an α -subunit and a β -subunit, wherein the amino acid sequence of the α -subunit is as shown in SEQ ID NO:2 is shown in SEQ ID NO:1, and a mutation at a site selected from the group consisting of:

1) Mutation of amino acids 36-40 from QKDPE to AAAPA;

2) Mutation of amino acid 47 from D to W or R;

3) Mutation of amino acid 64 from N to W;

4) Mutation of amino acid 127 from G to R;

5) Mutation of amino acid 143 from S to W or R;

6) Mutation of amino acid 170 from C to A;

7) Mutation of amino acid 246 from N to A;

The present invention provides nucleic acid molecules encoding the tryptophan synthase mutants.

The present invention provides biological materials comprising the nucleic acid molecules, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, or engineered bacteria.

The invention provides a recombinant microorganism which is constructed by introducing a nucleic acid molecule encoding the tryptophan synthase mutant into escherichia coli through a plasmid or integrating the nucleic acid molecule into the escherichia coli chromosome through a genetic engineering means.

In one embodiment of the invention, the recombinant microorganism uses E.coli as a host cell in which a tryptophan synthase mutant is expressed.

The present invention provides the use of said tryptophan synthase mutant or said biological material or said recombinant microorganism in cysteine production.

The recombinant microorganism may be a host cell of E.coli in which the tryptophan synthase mutant is expressed.

The E.coli strain may be an engineering strain commonly used in the field of genetic engineering, and is preferably, for example, BL21 (DE 3), BW25113, W3110 (DE 3), M5 HiPer BLR (DE 3), BL21 (DE 3) DUOs, and E.coli BL21 (DE 3) is particularly preferred.

The invention provides a preparation method of L-cysteine, which is characterized in that the tryptophan synthase mutant, a nucleic acid molecule encoding the tryptophan synthase mutant or a biological material containing the nucleic acid molecule is used.

The preparation method of the L-cysteine takes serine and sodium hydrosulfide as substrates, the substrates are contacted with the mutant, and the L-cysteine is synthesized by enzyme catalysis.

In one embodiment of the present invention, recombinant E.coli cells expressing the tryptophan synthase mutant are cultured, and the mutant cell disruption solution obtained after disrupting the recombinant cells at high pressure is used as a reaction enzyme solution, and is added to the reaction solution for enzyme-catalyzed reaction.

In one embodiment of the present invention, the enzyme-catalyzed reaction system comprises: l-serine, sodium hydrosulfide, pyridoxal phosphate, disodium hydrogen phosphate, mutant cell disruption solution.

The enzyme catalytic reaction system is as follows: l-serine 0.5-2M, sodium hydrosulfide 0.5-2M, pyridoxal phosphate 0.1-1 mM, disodium hydrogen phosphate 20-100 mM, and mutant cell disruption solution corresponding to 10-50 g/L wet cell.

The mutant cell disruption solution corresponding to 10-50 g/L wet thalli means a cell disruption solution obtained by disrupting 10-50g of wet thalli expressing the mutant under high pressure in each liter of an enzyme catalytic reaction system.

The method comprises the following steps of: 30-45 ℃ and pH 7.5-8.5.

The invention provides a preparation method of L-cystine, which is used for oxidizing the L-cysteine prepared by the method to obtain L-serine.

The invention provides a preparation method of L-cysteine or L-cystine, which specifically comprises the following steps:

(1) Culturing recombinant E.coli cells expressing said tryptophan synthase mutant;

(2) Performing cell disruption treatment on the culture, wherein the obtained cell disruption product is a reaction enzyme solution containing the tryptophan synthase mutant;

(3) The reaction enzyme liquid is used for catalyzing serine and sodium hydrosulfide to react to synthesize L-cysteine;

if L-cystine is to be prepared, the preparation method further comprises the following steps:

(4) Oxidizing the L-cysteine to obtain a crude L-cystine product;

(5) And separating and purifying the crude L-cystine to obtain a finished L-cystine product.

In the preparation method, the L-serine in the step (3) is L-serine pure product or L-serine fermentation liquor obtained by fermenting serine producing bacteria.

In the preparation method, the oxidation in the step (4) is to introduce air into a reaction solution obtained by synthesizing the L-cysteine by enzyme catalysis, and then add FeCl ₂ for oxidation.

The serine producing strain is constructed by the construction method of serine high-producing strain ES-134/pSC-08.

The preparation method of the L-serine fermentation broth comprises the following steps: inoculating the constructed serine producing strain into serine fermentation medium for fermentation.

The serine concentration at the end of the fermentation was found to be about 36 g/L.

The fermentation medium is M9 medium, and comprises ：6.8 g/L Na₂HPO₄、3 g/L KH₂PO₄、1g/L NaCl、5 g/L (NH₄)₂SO₄、0.015 g/L CaCl₂·2H₂O、3g/L MgSO₄·7H₂O、0.07 g/L FeSO₄·7H₂O、0.11 g/L sodium citrate, 2g/L yeast extract, 8 g/L glucose, and microelements (7 mg/L CoCl₂·6H₂O、2.5 mg/L CuSO₄·5H₂O、25 mg/L H₃BO₃、16 mg/L MnCl₂·4H₂O、1.5 mg/L Na₂MoO₄·2H₂O、3 mg/L ZnSO₄·7H₂O).

The preparation method of the L-serine fermentation liquor further comprises the following steps:

(1) After the fermentation, the pH of the serine fermentation broth is adjusted, and the serine fermentation broth is heated to inactivate and denature the protein, so that the serine fermentation broth becomes insoluble protein.

(2) And (3) passing the serine fermentation liquor after heat treatment through a ceramic membrane, and removing thallus fragments and denatured proteins to obtain ceramic membrane clear liquid.

(3) And concentrating the clear ceramic membrane liquid by using a reverse osmosis membrane or a nanofiltration membrane to obtain L-serine concentrated fermentation liquor.

Each 1L of the reaction system contains: l-serine concentrate fermentation broth, sodium hydrosulfide, pyridoxal phosphate, disodium hydrogen phosphate, recombinant E.coli cell disruption solution of the tryptophan synthase mutant.

The mutant Trps ^{QKDPE36-40AAAPA}、Trps^N64W、Trps^G127R、Trps^S143R、Trps^N246A with high enzyme activity, especially mutant Trps ^{QKDPE36-40AAAPA}、Trps^N64W、Trps^G127R with the enzyme activity of 2.87 times, 2 times and 1.8 times of that of the wild tryptophan synthase are obtained by mutating and screening the wild tryptophan synthase, and the mutants all improve the production of cysteine. The invention greatly improves the output of the cysteine and the cystine, reduces the cost and has important significance for the large-scale high-yield production of the cysteine and the cystine.

Drawings

FIG. 1 is a comparison of the relative enzyme activities of E.coli tryptophan synthase and mutants thereof;

FIG. 2 shows the results of cysteine and cystine production using a crude recombinant E.coli enzyme solution expressing tryptophan synthase and mutants thereof.

FIG. 3 shows the results of cysteine production using a recombinant E.coli crude enzyme solution expressing tryptophan synthase and mutants thereof to catalyze serine fermentation broths.

Detailed Description

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or in accordance with the manufacturer's instructions.

EXAMPLE 1 construction of Tryptophan synthase EcTrps mutant

(1) Template construction

Two subunits of tryptophan synthase are simultaneously expressed by using an escherichia coli dual expression vector pETDuet-1 with dual promoters, a beta-subunit (TrpB) of tryptophan synthase Trps of escherichia coli is cloned to a multiple cloning site 1 of pETDuet-1, an alpha-subunit (TrpA) is cloned to a multiple cloning site 2 of pETDuet-1, and a vector pETDuet-1-Trps capable of simultaneously expressing TrpB and TrpA is obtained, and the TrpB and TrpA gene sequences are shown as SEQ ID NO. 3 and SEQ ID NO. 4.

(2) Construction of mutant recombinant strains

The vector pETDuet-1-Trps is used as a template, a mutation primer pair (shown in table 1) is used for carrying out corresponding site mutation on the coding gene of the beta-subunit (TrpB) of Trps, and PCR amplified products are transferred into E.coli DH5 alpha competent cells after being digested for 1 hour by Dpn I, and monoclonal is selected for sequencing identification. The plasmid containing the mutant gene was transferred into E.coli W3110 (DE 3) to obtain mutant recombinant strain W3110-Trps^D47W、W3110-Trps^D47R、W3110-Trps^N64W、W3110-Trps^G127R、W3110-Trps^S143R、W3110-Trps^S143W、W3110-Trps^C170A、W3110-Trps^N246A、W3110-Trps^{QKDPE36-40AAAPA}. while the unmutated vector pETDuet-1-Trps was transferred into E.coli W3110 (DE 3) to obtain recombinant strain W3110-Trps expressing wild-type Trps as a control. ( And (3) injection: the above mutation sites are numbered 36-40, 47, 64, 127, 143, 170, 246 referring to the amino acid sequence number of the β -subunit (TrpB) of Trps of E.coli )

TABLE 1 TrpsB Gene site-directed mutagenesis primer

Example 2 Tryptophan synthase mutant enzyme Activity test

(1) Recombinant protein induced expression

The mutant recombinant strain and the control recombinant strain constructed in example 1 were cultured in LB medium containing ampicillin at 37℃with shaking, when the bacterial liquid concentration reached OD ₆₀₀ to 1.0, IPTG was added to the final concentration of 0.4mM for induction expression, and after culturing at 30℃for 20 h, the bacterial cells were collected.

(2) Tryptophan synthase mutant enzyme activity assay

After the collected tryptophan synthase mutant strain is resuspended in water, the culture is subjected to cell disruption by a high-pressure homogenizer (800-1000 bar) to obtain a cell disruption solution as an enzyme solution, and enzyme activity measurement is performed. The enzyme activity reaction system comprises the following components: tris-HCL 100mM, L-serine 100mM, sodium hydrosulfide 110 mM, pyridoxal phosphate 0.4 mM and cell disruption solution, wherein the addition amount of the cell disruption solution is equal to the addition amount of 1g wet thalli per liter of reaction system; the reaction conditions were 40℃and pH 8.0. After the reaction was completed, the reaction was terminated with an equal volume of 100. Mu.L of 1M HCL. Samples were tested by HPLC and mutant enzyme activity was determined.

As shown in FIG. 1, compared with the wild type, the mutants Trps ^N64W、Trps^G127R、Trps^{QKDPE36-40AAAPA} have higher enzyme activities, which respectively reach 1.8-3 times that of the wild type Trps, and especially the enzyme activity of the mutant Trps ^{QKDPE36-40AAAPA} obtained by continuously mutating 36-40 amino acids of beta subunit is more nearly 3 times that of the wild type and far less than that of other single-site mutations.

Example 3 catalytic production of L-cysteine Using Tryptophan synthase mutants

The mutant strain W3110-Trps ^{QKDPE36-40AAAPA} with the highest catalytic activity and the control strain W3110-Trps were selected and inoculated into LB medium containing ampicillin, and cultured with shaking at 37 ℃. When the concentration of the bacterial liquid reaches OD ₆₀₀ to 1.0, IPTG with the final concentration of 0.4mM is added for induction expression, and the bacterial liquid is cultured at 30 ℃ for 20 h, so that a recombinant cell culture is obtained.

And respectively carrying out high-pressure disruption on the recombinant cell culture to obtain a cell disruption solution serving as a reaction enzyme solution, and adding the cell disruption solution into an enzyme catalysis reaction solution for producing L-cysteine. The reaction liquid comprises the following components: l-serine 0.95M, sodium hydrosulfide 1.1M, pyridoxal phosphate 0.4 mM, disodium hydrogen phosphate 50 mM, equivalent to 20 g/L wet cell disruption solution (i.e., a cell disruption solution obtained by disrupting 20g of the wet cell of the mutant strain or the control strain under high pressure per liter of the reaction system). The reaction conditions are as follows: the reaction was carried out at 40℃and the pH was maintained at 8.0 by adjusting with 2M H ₂SO₄. The formation of L-cysteine or L-cystine within 4 hours was examined by HPLC, and the remaining amount of substrate serine: 10 min,60 min,120 min,180 min,240 min reaction solutions were taken and examined, and the reaction was terminated with 1M hydrochloric acid.

Note that: since L-cysteine is unstable, a small amount of L-cysteine as a product is naturally oxidized to L-cystine in the reaction system during the above enzyme-catalyzed reaction, and 1L-cystine molecule is generated from two L-cysteine molecules by the oxidation reaction, the molar concentration of L-cystine to be measured needs to be doubled when the yield is calculated. The method comprises the following steps:

Yield = molar concentration of L-cysteine +2 x molar concentration of L-cystine

Serine conversion = (molar concentration of L-cysteine+2×molar concentration of L-cystine)/(initial molar concentration of L-serine×100%

As a result, as shown in FIG. 2, when the wild-type Trps was used for the catalytic reaction for 4 hours, the yield was 868: 868 mM (cysteine 781mM, cystine 43.5 mM), and the serine conversion was 91.4%. When the mutant Trps ^{QKDPE36-40AAAPA} is adopted, the reaction is carried out for 3 hours, the yield can reach 949.5 mM (878.5 mM of cysteine and 35.5mM of cystine), and the serine is basically completely converted into the cysteine, so that the conversion rate is as high as 99.9%.

The results show that compared with the wild Trps, the mutant tryptophan synthase Trps ^{QKDPE36-40AAAPA} is used for catalyzing the synthesis of L-cysteine, so that the reaction time is shortened by 25%, the production efficiency is greatly improved, the yield and the conversion rate are also greatly improved, and especially, the complete conversion of substrate serine is basically realized, and the production cost is effectively reduced.

EXAMPLE 4 preparation of L-cysteine Using Tryptophan synthase mutant to catalyze serine fermentation broths

Because of the high price of the pure serine, the fermentation broth of serine producing bacteria is used as a substrate instead of the pure serine to perform the enzyme-catalyzed synthesis of L-cysteine to further reduce the cost.

Preparation of serine fermentation liquor

The purpose of serine fermentation production is to obtain fermentation liquor containing serine with higher concentration through fermentation, which is used for the subsequent preparation of L-cysteine, so as to reduce the high production cost caused by adopting pure serine. Thus, any strain in the art capable of producing serine by fermentation can be used in this step.

For example, a serine producing strain can be constructed according to the construction method of serine high producing strain ES-134/pSC-08 disclosed in document Wang C, Wu J, Shi B, Shi J, Zhao Z. Improving L-serine formation byEscherichia coliby reduced uptake of produced L-serine. Microb Cell Fact. 2020 Mar 14;19(1):66. doi: 10.1186/s12934-020-01323-2. , and a serine fermentation broth can be prepared by fermentation. The specific method comprises the following steps: the constructed serine producing strain was inoculated into a serine fermentation medium and fermented at 37℃for 51 hours, and the serine concentration was found to be about 36 g/L at the end of the fermentation. The fermentation medium is consistent with the above document, and is M9 medium, and comprises ：6.8 g/L Na₂HPO₄、3 g/L KH₂PO₄、1g/L NaCl、5 g/L (NH₄)₂SO₄、0.015 g/L CaCl₂·2H₂O、3g/L MgSO₄·7H₂O、0.07 g/L FeSO4·7H2O、0.11 g/L sodium citrate, 2 g/L yeast extract, 8 g/L glucose, and microelements (7 mg/L CoCl₂·6H₂O、2.5 mg/L CuSO₄·5H₂O、25 mg/L H₃BO₃、16 mg/L MnCl₂·4H₂O、1.5 mg/L Na₂MoO₄·2H₂O、3 mg/L ZnSO₄·7H₂O).

(II) fermentation liquor treatment

(1) After the fermentation is finished, the pH value of the serine fermentation liquid is adjusted to 5, and the serine fermentation liquid is heated to 70-90 ℃ to inactivate and denature the protein, so that the protein is changed from soluble protein to insoluble protein.

(2) And (3) passing the serine fermentation liquor after heat treatment through a ceramic membrane with the pore size of 50nM, and removing thallus fragments and denatured proteins to obtain ceramic membrane clear liquid.

(3) And concentrating the clear ceramic membrane liquid by using a reverse osmosis membrane or a nanofiltration membrane with the molecular weight of less than 150 so that the content of L-serine reaches 120g/L, thereby obtaining L-serine concentrated fermentation liquor.

(III) production of L-cysteine

(1) And respectively taking 20g of wet thalli from cultures of the recombinant mutant strains W3110-Trps ^{QKDPE36-40AAAPA} and the control strains W3110-Trps for high-pressure crushing, wherein the obtained cell crushing liquid is the reaction enzyme liquid.

(2) In vitro enzyme catalytic reaction to prepare L-cysteine.

Each 1L of the reaction system contains: l-serine concentrated fermentation broth 833 mL (L-serine concentration 0.95M), sodium hydrosulfide 1.1. 1.1 mol, pyridoxal phosphate 0.4 mmol, disodium hydrogen phosphate 50 mmol, cell disruption solution obtained by high pressure disruption of 20g wet cells in step (1).

The reaction conditions are as follows: the reaction was carried out at 40℃and the pH was maintained at 8.0 by adjusting with 2M H ₂SO₄.

(3) The formation of L-cysteine or L-cystine and the residual amount of serine as substrate were measured by HPLC for 4 hours, and 10 min,60 min,120 min,180 min,240 min reaction mixtures were taken and quenched with 1: 1M hydrochloric acid.

Note that: since L-cysteine is unstable, a small amount of L-cysteine as a product is naturally oxidized to L-cystine in the reaction system during the above enzyme-catalyzed reaction, and 1L-cystine molecule is generated from two L-cysteine molecules by the oxidation reaction, the molar concentration of L-cystine measured needs to be doubled when the yield and conversion rate are calculated. The method comprises the following steps:

Yield = molar concentration of L-cysteine +2 x molar concentration of L-cystine

As a result, as shown in FIG. 3, after 4 hours of the reaction of wild-type Trps, the yield was 731 mM (651 mM cysteine, 40mM cystine), the serine conversion was only 76.9%, and about one quarter of serine was not converted to L-cysteine. The mutant Trps ^{QKDPE36-40AAAPA} is adopted for reaction for 4 hours, the yield can reach 897 mM (807 mM of cysteine and 45mM of cystine), the yield is 23% higher than that of wild type Trps, and the serine conversion rate reaches 94.4% and is far higher than that of the wild type.

In addition, when the reaction was carried out using L-serine as a substrate in comparison with the case of using L-serine as a substrate in example 3, the conversion rate of the wild-type Trps in the reaction for 4 hours was reduced from 91.4% to 76.9%, which suggests that the impurity components in the L-serine concentrated fermentation liquid have a large interference and influence on the enzyme activity of the wild-type Trps. When the mutant Trps ^{QKDPE36-40AAAPA} is adopted for reaction, the conversion rate is only slightly reduced, and basically complete conversion can be achieved after 4 hours of reaction, which shows that the enzyme activity of the mutant Trps ^{QKDPE36-40AAAPA} is less influenced by impurities in serine concentrated fermentation liquor, and the method is more suitable for industrial production.

EXAMPLE 5 preparation of L-cystine from L-cysteine

(1) Oxidation

In example 4, a reaction was catalyzed by using a tryptophan synthase mutant Trps ^{QKDPE36-40AAAPA}, after the completion of the reaction, the pH of the reaction system was adjusted to 7.0, air was introduced to the reaction system, the ventilation was 1.67: 1.67 vvm, 200mg/L FeCl ₂ was added after 30 minutes of ventilation, and the reaction system was oxidized for 6 hours to oxidize L-cysteine to L-cystine.

(2) Separation

The oxidation product obtained in the step (1) is filtered and separated by a buchner funnel to obtain reaction solution and cystine precipitate, the reaction solution and cystine precipitate are leached for 2 times by a dilute sulfuric acid solution (pH is approximately equal to 5) with the concentration of 5 mu M, and the precipitate is collected.

(3) Purification

Adding 6M sulfuric acid solution into the precipitate obtained in the step (2) until the cystine precipitate is completely dissolved, adding 0.5% active carbon for decoloring treatment for 30min, and filtering and separating supernatant and precipitate by a Buchner funnel. Regulating pH of the supernatant to 5.6 with 10M NaOH, filtering with Buchner funnel, separating supernatant and cystine precipitate, eluting with pure water for 2 times, drying and crystallizing the cystine precipitate to obtain white powder crystal, i.e. L-cystine product.

The optical characteristics and purity of the obtained L-cystine product are detected, and the light transmittance is 98%, the specific rotation is-215% and the purity is 99%.

The above results demonstrate that the use of tryptophan synthase mutant Trps ^{QKDPE36-40AAAPA} for the preparation of cystine can result in a high purity and high quality L-cystine product with excellent optical properties even if the substrate is a low cost serine concentrate broth containing a large amount of impurities without purification.

In conclusion, the tryptophan synthase mutant with high enzymatic activity is obtained by mutating the escherichia coli Trps, so that the yield and the conversion rate of cysteine or cystine are greatly improved, the activity is not easily affected by impurities, the production efficiency is effectively improved, the production cost is reduced, and the method has important significance on large-scale high-yield production of cysteine and cystine.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims

1. A tryptophan synthase mutant comprising an α -subunit and a β -subunit, wherein the amino acid sequence of the α -subunit is set forth in SEQ ID NO:2 is shown in SEQ ID NO:1, and carrying out mutation at the following sites based on the amino acid sequence shown in the specification: the QKDPE th position 36-40 is mutated to AAAPA.

2. A nucleic acid molecule encoding the mutant of claim 1.

3. A biological material comprising the nucleic acid molecule of claim 2, wherein the biological material is a recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector, or an engineering bacterium.

4. A recombinant microorganism, wherein the recombinant microorganism is constructed by introducing the nucleic acid molecule of claim 2 into E.coli by means of a plasmid or integrating it into E.coli chromosome by means of genetic engineering.

5. Use of the mutant according to claim 1 or the biological material according to claim 3 or the recombinant microorganism according to claim 4 for the production of L-cysteine or L-cystine.

6. A process for the preparation of L-cysteine or L-cystine, characterized in that the mutant according to claim 1 or the biological material according to claim 3 or the recombinant microorganism according to claim 4 is used.

7. The method for producing L-cysteine or L-cystine according to claim 6, wherein L-serine and sodium hydrosulfide are used as substrates.

8. The method for producing L-cysteine or L-cystine according to claim 7, comprising the steps of:

(1) Culturing a recombinant microorganism expressing the recombinant microorganism of claim 4;

(3) Adding the reaction enzyme solution into a catalytic reaction system to catalyze L-serine and sodium hydrosulfide to react and synthesize L-cysteine;

in preparing L-cystine, the preparation method further comprises the following steps:

(4) Oxidizing the L-cysteine to obtain a crude L-cystine product;

9. The process according to claim 8, wherein the catalytic reaction system of step (3) comprises L-serine, sodium hydrosulfide, pyridoxal phosphate, disodium hydrogen phosphate.

10. The process according to any one of claims 7 to 9, wherein the L-serine is pure L-serine or an L-serine fermentation broth obtained by fermentation of serine producing bacteria.