A glutathione bifunctional synthetase mutant S722A with improved enzyme activity and its application

Abstract

The present invention relates to the field of genetic engineering technology, and in particular to a mutant of Escherichia coli glutathione bifunctional synthetase protein, genetically engineered bacteria and applications. The present invention firstly heterologously expresses the glutathione bifunctional synthetase derived from thermophilic streptococcus, constructs a recombinant Escherichia coli, transforms the glutathione bifunctional synthetase protein, performs site-directed mutation on it, and improves the enzyme activity. The present invention successfully expresses the glutathione bifunctional synthetase in Escherichia coli, and further improves the enzyme activity by 48.93%, which is conducive to further improving the yield of glutathione. Through whole-cell catalysis, the glutathione yield of the mutant S722A reaches 20.10mmol / L, and both the yield and conversion rate are further improved.

Description

Technical Field

[0001] The invention relates to a glutathione bifunctional synthetase mutant S722A with improved enzyme activity and application thereof, and belongs to the field of genetic engineering and fermentation engineering. Background Art

[0002] Glutathione (glutathione, r-glutamyl cysteingl+glycine, GSH) is a non-coding tripeptide thiol, which is formed by peptide bond condensation of three amino acids, L-glutamic acid, L-cysteine ​​and glycine. It contains γ-amide bonds and sulfhydryl groups and exists in most prokaryotes and eukaryotes at a concentration of 0.2-10mM. The main functions of GSH are detoxification, anti-oxidation, and maintaining intracellular redox balance. It is widely used in the fields of medicine, food and cosmetics.

[0003] The biosynthesis of GSH is mainly catalyzed by two ATP-dependent enzymes, γ-glutamylcysteine ​​synthetase and glutathione synthetase, of which the first enzyme is the rate-limiting enzyme and is feedback inhibited by glutathione. In 2005, glutathione bifunctional synthetase (GshF) was first discovered in Streptococcus agalactiae, which can synthesize GSH in the presence of ATP and Mg. 2+ or Mn 2+ In the presence of , the above two steps of GSH synthesis are catalyzed simultaneously.

[0004] Since the discovery of GSH, the production of GSH has mainly been directly extracted from animals and plants, and later developed into chemical synthesis, enzymatic and microbial fermentation production. Currently, the most widely used are enzymatic and microbial fermentation methods. At present, although the high-yield bacteria modified by genetic engineering have initially achieved industrial production of GSH, there are still problems such as intracellular inhibition of the product and high separation and purification costs. The advantage of enzymatic production of GSH is that the in vitro system simplifies the production process, effectively eliminates feedback inhibition, and enables the production of GSH to reach a higher level. Among them, whole-cell catalysis has the following advantages over free enzyme or immobilized enzyme catalysis: no enzyme separation and purification is required, reducing investment; the enzyme recovery rate is high and the stability is good; it does not increase the burden of downstream purification, etc. The complete cell structure can increase the number of cell reuses, thereby improving the intensity of industrial production.

[0005] The key to the current enzymatic synthesis of GSH is to screen and obtain a high-performance GSH synthase enzyme system. Therefore, the present invention improves the enzyme activity of GshF through site-directed mutagenesis to increase the yield of GSH. Summary of the invention

[0006] Purpose of the invention: Currently, the enzymatic production of glutathione has the problems of low catalytic efficiency and poor stability of the synthase. In order to overcome the defects of the prior art, the present invention provides a method for improving the activity of glutathione bifunctional synthetase, that is, a glutathione bifunctional synthetase mutant S722A with improved enzyme activity is proposed, the activity of glutathione bifunctional synthetase is improved, and the yield of GSH synthesized by the enzymatic method is increased, which has a general significance for promoting the synthesis of glutathione.

[0007] The first object of the present invention is to provide a glutathione bifunctional synthetase mutant, the amino acid sequence of the mutant is shown in SEQ ID NO.3.

[0008] Furthermore, the mutant is a mutant of the parent thermophilic Streptococcus GshF with an amino acid sequence as shown in SEQ ID NO.1, with the amino acid at position 722 mutated. Furthermore, the gene encoding the parent thermophilic Streptococcus GshF, in one embodiment of the present invention, has a nucleotide sequence as shown in SEQ ID NO.2.

[0009] Furthermore, in one embodiment of the present invention, the gene gshF (Gene ID: GU138096) of Streptococcus thermophilus GshF is synthesized by GeneWeichi Biotechnology Co., Ltd. and codon-optimized for Escherichia coli.

[0010] Furthermore, in one embodiment of the present invention, the mutant is a mutant in which the serine at position 722 is mutated into alanine.

[0011] The second object of the present invention is to provide a gene encoding the glutathione bifunctional synthetase mutant, the nucleotide sequence of which is shown in SEQ ID NO.4.

[0012] The third object of the present invention is to provide a vector or a cell carrying the gene.

[0013] The fourth object of the present invention is to provide an expression plasmid, which contains the gene mentioned above and uses pET-28a(+) as a vector.

[0014] The fifth object of the present invention is to provide a genetically engineered bacterium that expresses the glutathione bifunctional synthetase mutant.

[0015] Furthermore, in one embodiment of the present invention, the genetically engineered bacteria uses Escherichia coli BL21 (DE3) as a host.

[0016] The sixth object of the present invention is to provide a method for producing glutathione by fermentation using genetically engineered bacteria expressing the mutant.

[0017] Furthermore, the method, in one embodiment of the present invention, comprises: using microbial cells expressing the above-mentioned glutathione bifunctional synthetase mutant to transform a substrate to produce glutathione, wherein the substrate is a combination of L-glutamate, L-cysteine ​​and glycine.

[0018] Further, the method, in one embodiment of the present invention, comprises: culturing the microbial cells in a culture system until OD 600 The pH value is 0.6-0.8, the inducer is added to induce at 25°C for 10-12 hours, and the bacteria are collected; the bacteria are added to the reaction system containing the substrate and reacted for 6-12 hours.

[0019] Further, in one embodiment of the present invention, the substrate concentration in the reaction system is 20-40 mM, the strain OD 600 =30±0.5, react at pH 8±1, 37~45℃.

[0020] Further, the method, in one embodiment of the present invention, is specifically as follows: inoculate the recombinant monoclonal into 10 mL LB liquid medium containing kanamycin, culture at 37°C 220 rpm for 10 hours, then transfer 1% of the culture solution to a new 50 mL LB liquid medium containing kanamycin, culture for about 2-3 hours to OD = 0.6-0.8, add the filter-sterilized inducer IPTG to a final concentration of 0.2 mM, then culture at 25°C 220 rpm for 10 hours, and collect the bacteria by centrifugation. Glutathione is synthesized by whole cell catalysis.

[0021] The seventh object of the present invention is to provide glutathione obtained by the present invention.

[0022] The eighth object of the present invention is to provide the application of the glutathione obtained by the present invention in the fields of food, cosmetics, and drug preparation.

[0023] Beneficial effects of the present invention:

[0024] (1) Site-directed mutagenesis of glutathione synthetase 722 resulted in a mutant S722A with improved enzyme activity, whose relative enzyme activity was increased by 48.93% compared with that of the parent, indicating that the enzyme activity of the mutant was greatly improved;

[0025] (2) Through whole-cell catalysis, the glutathione production of S722A was 20.10 mmol / L, and that of WT was 16.57 mmol / L, an increase of 21.46%, which was significantly improved compared with the parent. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1: Construction and expression of recombinant Escherichia coli BL21(DE3) / pET28a-gshFst, wherein A is the electrophoresis diagram of gshF gene PCR amplification, B is the colony PCR verification diagram, and C is the SDS-PAGE diagram of glutathione bifunctional synthetase, wherein M: 180kDa protein marker, 1: protein in the ultrasonic precipitate of BL21(DE3) / pET28a, 2: protein in the ultrasonic precipitate of BL21(DE3) / pET28a-gshFst, 3: protein in the ultrasonic precipitate of BL21(DE3) / pET28a-gshFss, 4: protein in the supernatant of BL21(DE3) / pET28a, 5: protein in the supernatant of BL21(DE3) / pET28a-gshFst, 6: protein in the supernatant of BL21(DE3) / pET28a-gshFss.

[0027] Figure 2 : Construction and expression of site-directed mutation strains, wherein A is the mutant colony PCR verification diagram: 1-4 is S27G, 5-8 is E130G, 9-12 is E622D, 13-16 is F719V, 17-20 is S722A, 21-24 is Y724H, B is the SDS-PAGE diagram of the mutant: M: 180kDa protein marker, 1: BL21 / pET28a, 2: BL21 / pET28a-gshFst(WT), 3: S27G, 4: E130G, 5: E622D, 6: F719V, 7: S722A, 8: Y724H.

[0028] Figure 3 : Relative enzyme activities of different site-directed mutant strains.

[0029] Figure 4 :S722A and WT whole cells catalyze the synthesis of glutathione. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0031] Example

[0032] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the specific material ratios, process conditions and results described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0033] The determination method or culture method involved in the following examples is as follows:

[0034] Glutathione production determination method:

[0035] The yield of GSH was detected by HPLC. The sample was filtered with a 0.22 μm aqueous mixed cellulose ester microporous filter membrane. The HPLC used a C18 column, the mobile phase was 95% potassium dihydrogen phosphate solution containing 0.01 mol / L sodium heptane sulfonate and 0.05 mol / L, and 5% methanol, the flow rate was 0.6 mL / min, the injection volume was 10 μL, the wavelength of the UV detector was set to 210 nm, and the column temperature was 30 ° C. The curve formed by the concentration and peak area of ​​GSH was used as the standard curve, and the GSH concentration gradients were 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L and 1 g / L.

[0036] Biomass determination method (UV-visible photometer): dilute the samples at each sampling point to an appropriate multiple to OD 600 The value is 0.2-0.8, measure 200μL, and measure the absorbance at a wavelength of 600nm.

[0037] Culture medium: LB culture medium (g / L): 10% sodium chloride, 10% tryptone, 5% yeast extract, solid culture medium plus 2% agar powder. Fill 50 mL of the liquid in each 250 mL conical flask and sterilize at 121°C for 20 min.

[0038] E. coli transformation method (plasmid): (1) Add 10 μL of ligation product to each tube of competent cells and place on ice for 30 min; (2) Heat shock in a 42°C water bath for 90 s and place in an ice bath for 2 min; (3) Add 800 μL of 37°C LB medium (liquid) to each tube and incubate at 37°C on a shaker at 220 rpm for 1 h; (4) Centrifuge at 8000 rpm for 1 min, discard the supernatant, resuspend the cells and spread them on a Kan-resistant LB plate, and culture in a 37°C incubator for generally 10-12 h.

[0039] Example 1: Construction and expression of GshF in E. coli

[0040] 1. The glutathione bifunctional synthetase gene from Streptococcus thermophilus was synthesized by GENEWIZ (Suzhou Jinweizhi Biotechnology Co., Ltd.) and codon optimized for Escherichia coli. The nucleotide sequence of the gene is shown in SEQ ID NO: 2, and the amino acid sequence is shown in SEQ ID NO: 1. The synthesized gene is located on the pUC57-Kan standard vector.

[0041] 2. Using the vector as a template, amplify the target fragment using the primers in Table 1 (such as Figure 1A), connect to plasmid pET28a by homologous recombination to construct recombinant plasmid pET28a-gshFst; transform the recombinant plasmid pET28a-gshFst into Escherichia coli BL21 (DE3), pick transformants for colony PCR verification and sequencing, and verify that they are correct (such as Figure 1 B), the recombinant strain BL21(DE3) / pET28a-gshFst was successfully constructed.

[0042] Table 1 Primers used in this study

[0043]

[0044] Example 2: Construction and expression of mutants

[0045] The site-directed mutation site was introduced by the inverse PCR method, and the recombinant plasmid pET28a-gshFst obtained in Example 1 was used as a template for single-point mutation. The basic process is to first design mutation primers (primers are shown in Table 2), use S27G-F / S27G-R, E130G-F / E130G-R, E622D-F / E622D-R, F719V-F / F719V-R, S722A-F / S722A-R and Y724H-F / Y724H-R as primers, introduce mutation sites on the primers, perform inverse PCR, then use Dpn1 enzyme to recognize the methylation site and digest the template with enzyme digestion, transform the PCR product treated with Dpn1 enzyme, and finally perform bacterial selection sequencing verification (such as Figure 2 A), if the verification is correct, the plasmid is used for future use.

[0046] The mutants with correct sequencing were selected for fermentation experiments. The culture conditions were as follows: the genetically engineered bacteria seeds cultured at 37° C. and 220 rpm for 10 h were transferred to the fermentation medium at a 1% inoculation rate and cultured at 25° C. and 220 rpm for 10 h.

[0047] Take 2mL of fermentation liquid and centrifuge it. After resuspending and washing, resuspend the bacteria in 1mL of Tris-HCl buffer and break them with an ultrasonic disruptor. The conditions for breaking are: voltage 350V, electric shock time 3s, rest time 7s, and ultrasonic 18 times. After breaking, centrifuge the bacterial liquid at 4℃ and 12000r / min for 10min, and collect the supernatant as the crude enzyme liquid. Figure 2 As shown in B, SDS-PAGE results showed that the mutant was successfully expressed.

[0048] GSH synthesis reaction system: 0.1mol / L Tris-HCl (pH8.0), 40mmol / L Glu, 20mmol / L Cys, 40mmol / L Gly, 20mmol / L MgCl2, 20mmol / L ATP, add 100μL crude enzyme solution to each 1mL reaction system, react in a water bath at 37℃ for 20min. Take out 500μL and mix with an equal volume of 10% TCA (stop the reaction), then place on ice for 30min, centrifuge at 12000r / min for 10min, collect the supernatant and measure the GSH concentration.

[0049] The enzyme activity unit (U) is defined as the amount of GshF enzyme required to produce 1 μmol of glutathione per minute, measured by volume specific enzyme activity (U / mL). Figure 3 As shown, the enzyme activity of S722A was increased by 48.93% compared with the original strain WT, while the enzyme activities of other mutant strains were lower than those of the original strain WT.

[0050] Table 2 Primers used in this study

[0051]

[0052]

[0053] Note: Lowercase letters represent mutated bases

[0054] Example 3: Synthesis of glutathione by catalytic activity of recombinant Escherichia coli and mutant whole cells

[0055] The recombinant monoclonal clone was inoculated into 10 mL LB liquid culture medium containing kanamycin, and cultured at 37°C 220 rpm for 10 h. Then 1% of the culture solution was transferred to a new 50 mL LB liquid culture medium containing kanamycin and cultured for about 2 h until OD = 0.6-0.8. Filter-sterilized inducer IPTG was added to a final concentration of 0.2 mM, and then cultured at 25°C 220 rpm for 10 h. The bacteria were collected by centrifugation.

[0056] The collected bacteria were used to synthesize glutathione through whole-cell catalysis. The reaction system: the bacteria controlled OD 600 =30. Reaction solution: Tris-HCl Buffer 200mmol / L pH 8, L-Glu 40mmol / L, L-Cys 20mmol / L, Gly40mmol / L, MgCl2 20mmol / L, ATP 20mmol / L, 220r / min, 37℃ reaction, sampling at 0h, 2h, 4h, 6h, 8h, and detection of GSH production. Thaw the bacteria stored at -20℃ at room temperature, and repeat this three times. The results are shown in Figure 4As shown in the figure, at 2h, the yield of S722A was 20.10mmol / L, and that of WT was 16.57mmol / L, an increase of 21.46%. The molar conversion rate of Cys based on S722A was 100.5%, and that of Cys based on WT was 82.85%. The yield was higher than the theoretical value, mainly because the frozen-thawed cells contained GSH produced by the cells themselves.

[0057] In summary, the present invention heterologously expresses the glutathione bifunctional synthetase from thermophilic Streptococcus, constructs recombinant Escherichia coli, transforms the glutathione bifunctional synthetase protein, performs site-directed mutagenesis on it, and improves the enzyme activity. Through whole-cell catalysis, the yield of mutant S722A reaches 20.10mmol / L. The present invention successfully constructs an exogenous expression pathway for glutathione, further improves the enzyme activity, saves raw materials and costs, improves economic benefits, and lays a foundation for industrial production.

[0058] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A glutathione bifunctional synthetase mutant, characterized in that: The amino acid sequence of the mutant is shown in SEQ ID NO.

3.

2. A glutathione bifunctional synthetase mutant according to claim 1, characterized in that: The mutant is based on the glutathione bifunctional synthetase with an amino acid sequence as shown in SEQ ID NO.1, and the serine at position 722 is mutated into alanine.

3. A gene encoding the mutant according to claim 1 or 2, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

4.

4. A vector or cell carrying the gene according to claim 3.

5. An expression plasmid, characterized in that: The expression plasmid comprises the gene as claimed in claim 3, with pET-28a(+) as the vector.

6. A genetically engineered bacterium expressing the glutathione bifunctional synthetase mutant according to claim 1 or 2, characterized in that: Escherichia coli BL21 (DE3) was used as the host.

7. A method for producing glutathione, characterized in that Glutathione is produced by converting a substrate using a microbial cell expressing the glutathione bifunctional synthetase mutant according to claim 1 or 2, wherein the substrate is a combination of L-glutamate, L-cysteine ​​and glycine.

8. The method according to claim 7, characterized in that The microbial cells are cultured in a culture system until OD 600 The pH value is 0.6-0.8, the inducer is added to induce at 25°C for 10-12 hours, and the bacteria are collected; the bacteria are added to the reaction system containing the substrate and reacted for 6-12 hours.

9. The method according to claim 8, characterized in that In the reaction system, the concentration of the substrate L-glutamic acid is 20-40 mM, the concentration of the substrate L-cysteine ​​is 20-40 mM, the concentration of the substrate glycine is 20-40 mM, and the strain OD 600 =30±0.5, react at pH 8±1, 37~45℃.

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