CN115261365B

CN115261365B - Tryptophan synthase mutant and application thereof

Info

Publication number: CN115261365B
Application number: CN202210714273.7A
Authority: CN
Inventors: 秦成; 任丽梅; 张礼; 汤燕; 靳杰
Original assignee: Nantong Zilang Biopharma Tech Co ltd
Current assignee: Nantong Zilang Biopharma Tech Co ltd
Priority date: 2022-06-22
Filing date: 2022-06-22
Publication date: 2024-01-30
Anticipated expiration: 2042-06-22
Also published as: CN115261365A

Abstract

The invention discloses a tryptophan synthase mutant and application thereof, wherein the invention uses genetic engineering means to connect the 51 st amino acid to the 75 th amino acid of alpha subunit of tryptophan synthase to the front of the C-terminal stop codon of beta subunit of tryptophan synthase by adopting a direct or short connecting sequence Linker indirect connection mode, thus obtaining the tryptophan synthase mutant; the tryptophan synthase mutant is used for industrially synthesizing the L-cysteine and the derivatives thereof, so that the production efficiency and the product yield can be obviously improved, and the TsMUT3 in the enzyme mutant has the capability of catalyzing the L-cysteine to be synthesized, which is 3 times higher than that of a wild type; in addition, the TsMUT3 has good stability at 50 ℃, is more prone to the synthesis reaction of L-cysteine with ammonium sulfide as a sulfur group donor, and the L-cysteine generation rate can be more than 98%. The invention is suitable for industrial production of L-cysteine and derivatives thereof.

Description

Tryptophan synthase mutant and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, and relates to a tryptophan synthase mutant, in particular to a tryptophan synthase mutant and application thereof.

Background

Coli tryptophan synthase (Ts) is a tetrameric bifunctional enzyme capable of catalyzing the synthesis of L-tryptophan from L-serine, whose tertiary structure comprises two subunits, α and β, arranged in a linear fashion with αββα, the active centers of each of the α and β subunit dimer units being linked to one another by a 25a ° long channel. Research shows that the catalytic mechanism of Ts is: the alpha subunit alone catalyzes the cleavage of indole-3-glycerophosphate to indole and glyceraldehyde-3-phosphate; the beta subunit catalyzes the synthesis of L-tryptophan from indole and L-serine under the action of pyridoxal phosphate (PLP). Further studies have found that Ts also has the ability to catalyze the production of L-cysteine from L-serine.

In recent years, L-cysteine and its derivatives have been widely used in pharmaceuticals, foods and cosmetics. In the pharmaceutical field, acetylcysteine is widely used in respiratory diseases; in the food field, L-cysteine and its derivatives are mainly used in baked products, including dough improvement, gluten-like promotion, flavor improvement, etc.; in addition, the cysteine product can be used as an antioxidant for whitening and removing freckle.

With the rapid increase of the demand of L-cysteine, the conventional method for producing L-cysteine by directly utilizing proteins such as hair, feather and the like to carry out acid hydrolysis has very limited yield while generating waste water and waste residues. At present, although development of bioengineering technology provides a more efficient and environment-friendly L-cysteine production method, there are still some problems, for example, german patent application published as DE19539952 discloses the direct synthesis of L-cysteine by glucose in recombinant E.coli cells, but a series of enzymes such as O-acetylserine sulfhydrylase and the like participate in the synthesis path, so that the reaction is seriously inhibited by feedback of intermediate products, final products and the like, coupled knock-in mutant enzymes insensitive to the products are required, and separation and purification are difficult. In the subsequent research, the production method of the L-cysteine based on the principle has the yield of about 19.2g/L at maximum and still has a larger lifting space. The Chinese patent with publication number of CN102517352B discloses a method for synthesizing L-cysteine by using NaHS as a sulfur donor and using escherichia coli tryptophan synthase, wherein the molar conversion rate of L-serine is 84.9-85.3%, and the method has the defect of overlong reaction time and takes more than 24 hours, so that the production efficiency is low although the yield of the L-cysteine is improved.

In order to meet the demands of the market on L-cysteine, a tryptophan synthase mutant with higher stability, better performance and shorter reaction time is required to be constructed, and the production efficiency can be improved, and the L-cysteine yield is further improved by popularizing the application of the tryptophan synthase mutant.

Disclosure of Invention

The invention aims to solve the technical problem of providing a tryptophan synthase mutant, and the enzyme mutant is constructed by a genetic engineering technology means so as to achieve the purposes of improving the stability and catalytic activity of tryptophan synthase and being capable of being used for high-efficiency synthesis of L-cysteine;

it is a further object of the present invention to provide the use of the tryptophan synthase mutants described above for the synthesis of L-cystine and derivatives thereof for the synthesis of L-cysteine, L-cystine and L-cysteine hydrochloride monohydrate.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

a tryptophan synthase mutant is obtained by connecting amino acid 51 to amino acid 75 of an alpha subunit of tryptophan synthase to a position before a C-terminal stop codon of a beta subunit of tryptophan synthase;

wherein the tryptophan synthase is derived from escherichia coli K-12;

tryptophan synthase (Ts) from Escherichia coli str.K-12 substre.MG1655, NCBI accession number NC_000913.3; wherein, the 51 st amino acid to 75 th amino acid sequence of the alpha subunit is shown as SEQ ID NO.2, and is:

Gly Ile Pro Phe Ser Asp Pro Leu Ala Asp Gly Pro Thr Ile Gln Asn Ala Thr Leu Arg Ala Phe Ala Ala Gly。

the connection mode is direct connection or indirect connection by adopting a short connection sequence Linker.

As a limitation of the present invention, the sequence of the short Linker sequence Linker is GG, GGG or GGA. Indirect ligation may increase the flexibility of tryptophan synthase mutants.

As another limitation of the invention, the nucleotide sequence of the tryptophan synthase mutant is shown as SEQ ID NO.1, and is:

ATGACAACATTACTTAACCCCTATTTTGGTGAGTTTGGCGGCATGTACGTGCCACAAATCCTGATGCCTGCTCTGCGCCAGCTGGAAGAAGCTTTTGTCAGTGCGCAAAAAGATCCTGAATTTCAGGCTCAGTTCAACGACCTGCTGAAAAACTATGCCGGGCGTCCAACCGCGCTGACCAAATGCCAGAACATTACAGCCGGGACGAACACCACGCTGTATCTCAAGCGTGAAGATTTGCTGCACGGCGGCGCGCATAAAACTAACCAGGTGCTGGGGCAGGCGTTGCTGGCGAAGCGGATGGGTAAAACCGAAATCATCGCCGAAACCGGTGCCGGTCAGCATGGCGTGGCGTCGGCCCTTGCCAGCGCCCTGCTCGGCCTGAAATGCCGTATTTATATGGGTGCCAAAGACGTTGAACGCCAGTCGCCTAACGTTTTTCGTATGCGCTTAATGGGTGCGGAAGTGATCCCGGTGCATAGCGGTTCCGCGACGCTGAAAGATGCCTGTAACGAGGCGCTGCGCGACTGGTCCGGTAGTTACGAAACCGCGCACTATATGCTGGGCACCGCAGCTGGCCCGCATCCTTATCCGACCATTGTGCGTGAGTTTCAGCGGATGATTGGCGAAGAAACCAAAGCGCAGATTCTGGAAAGAGAAGGTCGCCTGCCGGATGCCGTTATCGCCTGTGTTGGCGGCGGTTCGAATGCCATCGGCATGTTTGCTGATTTCATCAATGAAACCAACGTCGGCCTGATTGGTGTGGAGCCAGGTGGTCACGGTATCGAAACTGGCGAGCACGGCGCACCGCTAAAACATGGTCGCGTGGGTATCTATTTCGGTATGAAAGCGCCGATGATGCAAACCGAAGACGGGCAGATTGAAGAATCTTACTCCATCTCCGCCGGACTGGATTTCCCGTCTGTCGGCCCACAACACGCGTATCTTAACAGCACTGGACGCGCTGATTACGTGTCTATTACCGATGATGAAGCCCTTGAAGCCTTCAAAACGCTGTGCCTGCACGAAGGGATCATCCCGGCGCTGGAATCCTCCCACGCCCTGGCCCATGCGTTGAAAATGATGCGCGAAAACCCGGATAAAGAGCAGCTACTGGTGGTTAACCTTTCCGGTCGCGGCGATAAAGACATCTTCACCGTTCACGATATTTTGAAAGCACGAGGGGAAATCGGTATCCCCTTCTCCGACCCACTGGCGGATGGCCCGACGATTCAAAACGCCACTCTGCGCGCCTTTGCGGCAGGTTAA。

the sequence SEQ ID NO.1 is the nucleotide sequence of TsMUT3 below.

The sequence of the tryptophan synthase mutant can also comprise point mutation, addition of various labels before and after the sequence, conservative substitution at other positions, amino acid truncation and other mutations which do not influence the active center of the tryptophan synthase mutant.

As a further definition of the invention, the expression host of the tryptophan synthase mutant is a bacterium or fungus for gene expression; including hosts common in the art, such as E.coli BL21 (DE 3), E.coli BL21, E.coli M15, B.subtilis, yeast, aspergillus, streptomyces, and the like; the expression vector of the tryptophan synthase mutant removes the common pET vector of prokaryote, and also comprises vectors matched with each host system, wherein the integration mode of the vectors and the host not only comprises free existence in the host, but also comprises integration at specific positions of genome and integration at random positions; the expression pattern of the tryptophan synthase mutant is not limited to expression and secretory expression within the host.

The invention also provides an application of the tryptophan synthase mutant, and the tryptophan synthase mutant is adopted to catalyze L-serine to synthesize L-cysteine.

As the limitation of the invention, the catalytic reaction liquid comprises 53-125 g/L of L-serine, 0.1-1 g/L of pyridoxal phosphate and 8-10% of ammonium sulfide by mass concentration;

the dosage of the tryptophan synthase mutant is 5-35 g/L;

the catalytic temperature is 35-38 ℃, and the catalytic time is 2-8 h.

The invention also provides application of the tryptophan synthase mutant in preparation of L-cystine, and after L-serine is catalyzed by the tryptophan synthase mutant to synthesize L-cysteine, the L-cysteine is synthesized into L-cystine through oxidation reaction.

The invention also provides application of the tryptophan synthase mutant in preparation of L-cysteine hydrochloride monohydrate.

As a limitation of the present invention, after L-serine is catalyzed to synthesize L-cysteine by tryptophan synthase mutant, L-cysteine hydrochloride monohydrate is prepared by resin chromatography.

As a further limitation of the invention, after L-serine is catalyzed by tryptophan synthase mutant to synthesize L-cysteine, L-cysteine is synthesized into L-cystine through oxidation reaction, and then L-cysteine hydrochloride monohydrate is prepared by adopting an electrolytic reduction method.

By adopting the technical scheme, compared with the prior art, the invention has the following technical progress:

(1) the tryptophan synthase mutant is obtained by shortening the length of an alpha subunit to 25 amino acids through a PCR method after analyzing the crystal structure of the protein and then re-splicing the alpha subunit to a C-terminal stop codon of a beta subunit; the invention changes the primary structure of the tryptophan synthase in the wild type, and the tryptophan synthase mutant obtained by the gene editing is used for obviously improving the catalytic activity on the synthesis of L-cysteine on the basis of improving the tryptophan synthesis capacity of the tryptophan synthase, and the stability and substrate specificity of the tryptophan synthase mutant are suitable for industrialized production of L-cysteine and derivatives thereof;

(2) the capacity of the tryptophan synthase mutant TsMUT3 obtained by the invention for catalyzing the synthesis of L-cysteine is 3 times higher than that of a wild type; meanwhile, tsMUT3 has the characteristics of high efficiency, good stability after being heated to 50 ℃, and the like, in the synthesis reaction of L-cysteine with ammonium sulfide as a sulfur-based donor, the catalytic activity is 6 times that of a wild type, and the highest L-cysteine generation rate is more than 98%;

(3) the tryptophan synthase mutant obtained by the invention has wide application range, can be used for efficiently producing L-cysteine, L-cystine and L-cysteine hydrochloride monohydrate, and has the yield of more than 90 percent in the production process of the product, and the purity of the L-cysteine hydrochloride monohydrate is up to 99 percent;

the invention is suitable for industrial production of L-cysteine and derivatives thereof.

The invention will now be described in detail with reference to the accompanying drawings and specific examples.

Drawings

FIG. 1 is a schematic diagram showing the splicing process of tryptophan synthase mutants in example 1 of the invention.

Detailed Description

The invention is described in further detail below with reference to specific examples and figures. It should be understood that the described embodiments are only for explaining the present invention and do not limit the present invention.

Materials, reagents, and the like used in the examples of the present invention are commercially available unless otherwise specified. The experimental methods for which specific conditions are not specified in the examples are generally conducted under conventional conditions or under conditions recommended by the manufacturer.

EXAMPLE 1 construction and expression of Tryptophan synthase mutants

The embodiment comprises the following steps sequentially carried out:

construction of Tryptophan synthase mutants

Construction of Ts wild-type expression strains: based on the wild-type tryptophan synthase sequence (1316416-1318415) of Escherichia coli str.K-12 substre.MG1655, the single cut site was selected for ligation with EcoR1 in combination with the characteristics of E.coli expression vector pET-21a, and primers P1 and P2 as shown in Table 1 were PCR-constructed into pET-21a expression vector. And transforming the positive plasmid after verification into E.coli BL21 (DE 3) to obtain the Ts wild-type expression strain. Wherein, the PCR procedure is: 95 ℃ for 10min,1 cycle; 30s at 95 ℃, 15s at 55 ℃ and 1min at 72 ℃ for 30 cycles in total; 72℃for 5min,1 cycle.

TABLE 1 primer sequences

Primer name	Sequence numbering	Sequence(s)
			P1	SEQ ID NO.3	5’-GGGAATTCATGACAACATTACTTAACC-3’
P2	SEQ ID NO.4	5’-CCGGAATTCTTAACTGCGCGTCGCCGCTTTC-3’

Construction of Ts mutant expression strains: as shown in FIG. 1, the alpha subunit sequences in the wild type are randomly truncated to form 30 alpha subunit fragments with different lengths, the positions of the alpha subunits and the beta subunits in the wild type are taken as alpha 1, primers are respectively designed for each alpha subunit substrate segment, and each alpha subunit substrate segment is respectively spliced and combined to the C end of the wild type beta subunit to obtain 30 Ts mutant sequences. Different splicing and combining modes are selected according to the length of the alpha subunit fragment, and when the length of the alpha subunit fragment is greater than 100aa and does not comprise the alpha 1 position, the splicing and combining mode of the overlay PCR is adopted; when the length of the alpha subunit fragment is less than 100bp except the alpha 1 position, splicing and combining are carried out by adopting a primer annealing mode. And respectively constructing the Ts mutant expression strains by referring to a construction method of the Ts wild type expression strains, and marking the Ts mutant expression strains as TsMUT expression strains.

Culture and expression of Tryptophan synthase mutants

The Ts wild-type strain and TsMUT-expressing strain were placed in LB liquid medium containing ampicillin (resistance marker) at a concentration of 100mg/L, respectively, and shake-cultured at 37℃and 200 rpm. When the cell density OD600 is 0.7, adding IPTG with the final concentration of 0.2mM, inducing for 18 hours at 20 ℃, centrifuging for 10 minutes at 6000rpm, collecting cells, preparing into 80g/L concentration, and performing ultrasonic pyrolysis to obtain enzyme solution for performance detection so as to screen TsMUT with strong L-cysteine synthesis capability.

Example 2 Performance detection and screening of Tryptophan synthase mutants

In this example, the wild-type Ts and the TsMUT enzyme solution prepared in example 1 were subjected to measurement of synthesis activity of L-tryptophan and L-cysteine, detection of color reaction, measurement of stability, and substrate profile test, respectively, to select tryptophan synthase mutants having high synthesis ability of L-cysteine. The specific process is as follows:

(1) Determination of synthesis activity: preparing a 1mL reaction system with pH of 8.0 by using 900uL reaction solution and 100uL enzyme solution to be detected, wherein the reaction solution is prepared from 1mM indole, 80mM L-serine, 1mM PLP and 200mM PBK buffer solution; the reaction system is reacted for 10min at 37 ℃, and after dilution, the synthesis condition of the L-tryptophan is tested by utilizing HPLC; the HPLC detection conditions were as follows: the chromatographic column selects ODS-2.5,4.6 ×150mm; mobile phase a is 0.1% phosphoric acid solution at ph 2.0, mobile phase B is methanol, mobile phase a and B volume ratio = 4:1; the loading amount is 10uL; the flow rate is 0.8mL/min; the detection temperature is 30 ℃; the detection wavelength is 205nm; wherein, the enzyme activity is defined as: under the above reaction conditions, 1U of L-tryptophan is produced per minute. The L-cysteine synthesis activity was similarly measured, and the results are shown in Table 2.

(2) Color reaction detection: preparing a 1mL reaction system with the pH of 8.0 by using 900uL reaction solution and 100uL enzyme solution to be detected, wherein the concentration of each component in the reaction solution is NaHS 5g/L, L-serine 80Mm, PLP 1mM and PBK buffer solution 100mM; after dilution, the reaction was performed at 37℃for 10min, and the resultant was subjected to a color reaction test using ninhydrin, and compared with a standard curve, to obtain the amount of L-cysteine synthesized in percentage. Wherein, the enzyme activity is defined as: under the above reaction conditions, the amount of enzyme required for the production of 1um L-cysteine per minute was defined as 1U. L-tryptophan was similarly measured and the results are shown in Table 2.

(3) Stability determination: the enzyme solution was heated to 50℃for 10min, cooled to room temperature and then tested for stability by L-cysteine color reaction. The results are shown in Table 2.

Analysis of the above performance test results revealed that the TsMUT1 to TsMUT5 prepared in example 1 were strong in L-cysteine synthesis ability, and the primary structures of the TsMUT were shown in Table 2.

TABLE 2 Performance test results

Table 2 shows that the synthesis activity of L-tryptophan by TsMUT 1-TsMUT 5 is improved as compared with that of the wild type, and the phenomenon is consistent with the synthesis function of L-tryptophan contained in beta subunit. Wherein, tsMUT3 and TsMUT4 obviously enhance the synthesis capacity of L-cysteine, which is 4.07 times and 2.38 times of wild type respectively, which shows that the alpha subthread section selected by the 2 designs obviously enhances the synthesis capacity of L-cysteine of the enzyme. In stability analysis, compared with wild type, the stability of TsMUT3 is obviously enhanced, and more than 60% of catalytic activity can be reserved. The gene sequence of TsMUT3 is shown as SEQ ID NO. 1.

(4) Substrate spectrum test: to further investigate whether a change in the primary structure of Ts could bring about a more suitable sulfur-based donor, reference was made to the reaction system in (2) in which NaHS was replaced with (Na), respectively ₂ S and (NH) ₄ ) ₂ S two common sulfur-based donors, followed by (Na) ₂ S group sum (NH) ₄ ) ₂ S group two sets of color reaction, wherein the concentration of S element in each set of substrates is controlled to be the same, and calculated (Na) by taking L-cysteine combined by NaHS as 100 percent ₂ S group sum (NH) ₄ ) ₂ L-cysteine synthesis amount of group S. The results are shown in Table 3.

TABLE 3 results of substrate profiling

As is clear from Table 3, six enzymes, ts wild-type and TsMUT1 to TsMUT5, were contained in the substrate (Na) ₂ In S system, L-cysteine synthesis ability is inferior to that of system using NaHS as sulfur group donor. In particular, among TsMUT1 to TsMUT5, tsMUT1, tsMUT3, tsMUT4 pair (NH) ₄ ) ₂ S is a sulfur-based donor exhibiting a higher propensity, especially TsMUT3 as (NH) ₄ ) ₂ When S is a sulfur-based donor, the L-cysteine synthesis capacity is about 50% or more of that of the wild type Ts.

In summary, by analyzing the results of the synthesis activity assay, the color reaction assay, the stability assay, and the substrate spectrum assay of L-tryptophan and L-cysteine, tsMUT1 to TsMUT5 in all the tryptophan synthase mutants prepared in example 1 showed a combination of stronger synthesis ability of L-tryptophan and L-cysteine than that of the wild type, especially TsMUT3, and also showed efficient use (NH ₄ ) ₂ S is the ability of the sulfur donor.

Examples 3-8 Tryptophan synthase mutants methods for catalyzing L-cysteine Synthesis

Example 3 is a method for catalyzing L-cysteine synthesis by tryptophan synthase mutant, specifically, 1L system: taking a small amount of PBK buffer solution to completely dissolve L-serine 53g, and adding the TsMUT3 enzyme solution prepared in example 1, pyridoxal phosphate 1g and ammonium sulfide with the mass concentration of 16%, wherein the final concentration of TsMUT3 enzyme is 10g/L, the final concentration of ammonium sulfide is 8%, and the final concentration of PBK buffer solution is 200mM; stirring at 100rpm, monitoring and supplementing ammonium sulfide by using a pH electrode, maintaining the pH value at 8.0-9.5, reacting for 2 hours at 37 ℃, and detecting the color reaction of the reaction liquid obtained after the reaction is finished, wherein the result shows that the L-cysteine is successfully synthesized, the L-cysteine content is 60.70g/L, and the L-cysteine generation rate is more than 98%.

Examples 4 to 8 are methods for catalyzing L-cysteine synthesis using tryptophan synthase mutants, respectively, and the specific methods are substantially the same as example 3, except that the parameter settings are different, and the specific differences are shown in Table 4:

table 4 examples 4 to 8 parameter tables

The other parts of examples 4 to 8 are the same as those of example 3.

The reaction solutions obtained after the completion of the reactions in examples 3 to 8 were subjected to color reaction detection, and the results showed that all of the reactions were successful in synthesizing L-cysteine, and the L-cysteine content was 53.13 to 117.60g/L.

Comparative example Ts wild type and catalytic ability of TsMUT3 to different concentrations of reaction substrate

This comparative example was used to compare the catalytic ability of Ts wild-type and TsMUT3 to L-cysteine synthesis at different concentrations of L-serine and PLP as substrates. The catalytic reaction system of this comparative example was substantially the same as in example 3, except that the concentrations of L-serine and PLP were different as shown in Table 5, in which the Ts wild-type group used the Ts wild-type instead of TsMUT3. Under the condition that L-serine and PLP with different concentrations are detected by a color reaction, the L-cysteine production rates of a Ts wild type group and a TsMUT3 group are shown in the following table 5:

TABLE 5 substrate concentration set and L-cysteine production rate

The results show that the Ts wild type has a certain capacity of converting L-serine into L-cysteine, but has lower level. Compared with the wild type, the TsMUT3 has the yield of 84.31-98.35 percent and the highest L-cysteine yield of more than 98 percent when the L-serine concentration is 53-125 g/L, and has better catalytic capability.

EXAMPLE 9 Tryptophan synthase mutant for L-cystine production

In this example, the reaction solution obtained by the reaction of example 3 and having an L-cysteine content of 55g/L was used for the preparation of L-cystine. The specific method comprises the following steps: taking 2L of reaction solution, regulating the pH to 2.0, and standing at 100 ℃ for 10min to ensure that the system is free of H ₂ S gas is filtered by a 500nm ceramic membrane to remove solids such as denatured proteins. Adding 1.0% active carbon, decolorizing at 60deg.C for 30min, filtering to remove carbon, and recovering filtrate. Continuously introducing compressed air into the filtrate, and carrying out oxidation reaction on L-cysteine under the action of oxygen to generate L-cystine, continuously oxidizing for 24 hours, taking the supernatant to detect the concentration of the L-cysteine until the concentration of the L-cysteine is less than 0.1g/L, and considering that the L-cysteine is completely oxidized. And after the oxidation is finished, stirring for 2 hours continuously, precipitating and filtering in vacuum, washing the precipitate with pure water, filtering in vacuum to obtain a wet product, and drying to obtain the crude L-cystine.

The yield of this example was 98% as measured.

EXAMPLE 10 resin chromatography preparation of L-cysteine hydrochloride monohydrate

In this example, the reaction solution obtained by the reaction of example 3 and having an L-cysteine content of 55g/L was used for the preparation of L-cysteine hydrochloride monohydrate by resin chromatography.

The specific method comprises the following steps: taking 2L of reaction solution, regulating the pH to 2.0 by using 6mol/L hydrochloric acid solution, and standing at 100 ℃ for 10min to ensure that the system is free of H ₂ S gas is filtered by a 500nm ceramic membrane to remove solids such as denatured proteins. Adding 1.0% active carbon, decolorizing at 60deg.C for 30min, filtering to remove carbon, and recovering filtrate. The filtrate was adjusted to pH 5.0 using 6mol/L hydrochloric acid, the solids were filtered off, and the filtrate obtained was of defined molecular weight5000, ultrafiltering and concentrating, loading the filtrate into strong cation resin LX160 chromatographic column, eluting with 2mol/L hydrochloric acid, nanofiltration with membrane with defined molecular weight of 200, concentrating under reduced pressure, cooling at low temperature for crystallization, centrifuging, and drying to obtain L-cysteine hydrochloride monohydrate.

The yield of this example was 95% as measured.

EXAMPLE 11 electrolytic reduction of L-cysteine hydrochloride monohydrate

This example uses the L-cystine obtained in example 9 in an electrolytic reduction process to prepare L-cysteine hydrochloride monohydrate.

The specific method comprises the following steps: adding 100g of L-cystine and 6mol/L hydrochloric acid into an electrolytic reactor, stirring at room temperature for 1.5h to fully dissolve the L-cystine, filtering, and transferring the filtrate into a cathode region of an electrolytic tank; 6mol/L hydrochloric acid is added into the positive electrolytic tank region to keep the liquid level to be level with the cathode region. Energizing at 50deg.C, controlling current density to 5A/dm ² And (5) electrolyzing for 11h. 6mol/L hydrochloric acid is continuously added into the anode region in the electrolysis process, so that the original volume is maintained. And before the reaction reaches the end point, taking the cathode region electrolyte to detect the optical rotation every 15min until the optical rotation value of the cathode electrolyte is no longer increased, namely the reaction reaches the end point. After the electrolysis is finished, transferring the catholyte into a decoloring reactor, maintaining the temperature at 80 ℃, adding 1% of active carbon, decoloring for 30min, filtering, concentrating the filtrate under reduced pressure, cooling and crystallizing at low temperature, centrifugally separating and drying to obtain L-cysteine hydrochloride monohydrate.

The yield of the L-cysteine hydrochloride monohydrate obtained in the embodiment is more than 99% and the purity is more than 99%.

In other embodiments, the amount of L-cystine used is any number between 100 and 500g, resulting in an L-cysteine hydrochloride monohydrate having a purity of greater than 99%.

The results show that the invention successfully constructs the tryptophan synthase mutant which can be used for synthesizing the L-cysteine and the derivatives thereof, and obviously enhances the capability of catalyzing the synthesis of the Ts tryptophan. Furthermore, the ability of TsMUT3 in the enzyme mutant to catalyze L-cysteine synthesis is 3-fold greater than that of the wild type. Meanwhile, the TsMUT3 has the characteristics of good stability, high efficiency and the like, in the synthesis reaction of L-cysteine with ammonium sulfide as a sulfur-based donor, the catalytic activity is 6 times that of a wild type, and the highest L-cysteine generation rate is more than 98%.

It should be noted that the foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, but the present invention is described in detail with reference to the foregoing embodiment, and those skilled in the art may modify the technical solutions described in the foregoing embodiments or substitute some of the technical features thereof. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.

SEQUENCE LISTING

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ccggaattct taactgcgcg tcgccgcttt c 31

Claims

1. A tryptophan synthase mutant is characterized in that the 51 st amino acid to 75 th amino acid of an alpha subunit of tryptophan synthase are connected to the front of a C-terminal stop codon of a beta subunit of tryptophan synthase, and the nucleotide sequence of the tryptophan synthase mutant is shown as SEQ ID NO. 1.

2. The tryptophan synthase mutant according to claim 1, wherein the expression host of the tryptophan synthase mutant is a bacterium or fungus for gene expression.

3. Use of a tryptophan synthase mutant according to claim 1 or 2, wherein the tryptophan synthase mutant is used to catalyze the synthesis of L-cysteine from L-serine.

4. The use according to claim 3, characterized in that the catalytic reaction solution comprises 53-125 g/L-serine, 0.1-1 g/L pyridoxal phosphate and 8-10% ammonium sulphide by mass;

the dosage of the tryptophan synthase mutant is 5-35 g/L;

the catalysis temperature is 35-38 ℃, the catalysis time is 2-8 h, and the pH is 8.0-9.5.

5. The use of a tryptophan synthase mutant according to claim 1 or 2 for the preparation of L-cystine, wherein the L-cysteine is synthesized into L-cystine by oxidation after the L-serine is catalyzed by the tryptophan synthase mutant to synthesize L-cysteine.

6. Use of a tryptophan synthase mutant according to claim 1 or 2 in the preparation of L-cysteine hydrochloride monohydrate.

7. The use of a tryptophan synthase mutant according to claim 6, wherein after L-serine is catalyzed by the tryptophan synthase mutant to synthesize L-cysteine, the L-cysteine hydrochloride monohydrate is prepared by resin chromatography.

8. The use of a tryptophan synthase mutant according to claim 6, wherein after L-serine is catalyzed by the tryptophan synthase mutant to synthesize L-cysteine, L-cysteine is synthesized by oxidation and then L-cysteine hydrochloride monohydrate is prepared by electrolytic reduction.