阅读说明：本技术 一种无细胞自组装体系高效合成谷胱甘肽的方法 (Method for efficiently synthesizing glutathione by cell-free self-assembly system ) 是由 李志敏 张星 于 2019-03-07 设计创作，主要内容包括：本发明提供了一种无细胞自组装体系高效合成谷胱甘肽的方法,由重组细胞过量表达腺苷激酶、多聚磷酸激酶和谷胱甘肽双功能合成酶,通过多酶体系有序自组装形成超聚合分子,利用多聚磷酸盐,和少量腺苷,或AMP,或ADP,或ATP,循环催化生成ATP,并将甘氨酸、谷氨酸和半胱氨酸以及生成的ATP生成谷胱甘肽。本发明首次将无细胞自组装体系利用到体外合成谷胱甘肽中,该方法缩短了底物传递距离,提高了反应速率和催化效率,强化了蛋白的热稳定性。不仅实现了谷胱甘肽合成过程中ATP的高效循环再生,降低生产成本；而且操作简单、稳定性强；副反应少、生产速率较高,底物得率接近理论值。(The invention provides a method for efficiently synthesizing glutathione by a cell-free self-assembly system, which comprises the steps of over-expressing adenosine kinase, polyphosphate kinase and glutathione bifunctional synthetase by recombinant cells, forming a super-polymer molecule by orderly self-assembly of a multienzyme system, circularly catalyzing and generating ATP by utilizing polyphosphate and a small amount of adenosine, or AMP, or ADP, or ATP, and generating glutathione from glycine, glutamic acid, cysteine and the generated ATP. The invention utilizes the cell-free self-assembly system to synthesize glutathione in vitro for the first time, and the method shortens the transfer distance of the substrate, improves the reaction rate and the catalytic efficiency and strengthens the thermal stability of the protein. The ATP efficient cyclic regeneration in the glutathione synthesis process is realized, and the production cost is reduced; the operation is simple and the stability is strong; less side reaction, high production rate and high substrate yield.)

1. A method for efficiently synthesizing glutathione by a cell-free self-assembly system is characterized by comprising the following steps:

(1) culturing a recombinant cell containing a polyphosphate kinase and glutathione bifunctional synthetase gene with a combined joint to ensure that the polyphosphate kinase and the glutathione bifunctional synthetase containing the joint are over-expressed;

(2) harvesting the cells in the step (1) to obtain cell disruption solution, and carrying out intracellular self-assembly or extracellular self-assembly on the enzyme system;

(3) adding polyphosphate and one or more of AMP, ADP or ATP, catalyzing ATP by polyphosphate kinase in the self-assembly system through circulation of polyphosphate, AMP and/or ADP, and generating glutathione from glycine, glutamic acid, cysteine and synthesized ATP in vitro through the action of glutathione bifunctional synthetase in the self-assembly system.

2. The method for efficiently synthesizing glutathione by using the cell-free self-assembly system as claimed in claim 1, which comprises the following steps:

(1) culturing a recombinant cell containing a polyphosphate kinase, adenosine kinase and glutathione bifunctional synthetase gene with a combined joint to enable the polyphosphate kinase, adenosine kinase and glutathione bifunctional synthetase containing the joint to be over-expressed;

(2) harvesting the cells in the step (1) to obtain cell disruption solution, and carrying out intracellular self-assembly or extracellular self-assembly on the enzyme system;

(3) adding polyphosphate and adenosine and one or more of AMP, ADP or ATP, catalyzing ATP by polyphosphate kinase and adenosine kinase in the self-assembly system through circulation of polyphosphate, adenosine, AMP and/or ADP, and generating glutathione from glycine, glutamic acid, cysteine and synthesized ATP in vitro through the action of glutathione bifunctional synthetase in the self-assembly system.

3. The method for efficiently synthesizing glutathione by using a cell-free self-assembly system as claimed in claim 1 or 2, wherein the polyphosphate kinase may be PPK1 or PPK2 derived from one of the following microorganisms Thermusthermophilus, Thermosynechococcus elonga, Jhaorihela thermophila, Hydrogenophilaceae bacterium, Nocardia dokdannensis, Halofaxsulforosis, Pseudonocardia thermophile, and Roseophilum reptaeotiaceae.

4. The method for efficiently synthesizing glutathione by using the cell-free self-assembly system as claimed in claim 1 or 2, wherein the glutathione bifunctional enzyme is derived from one of the following microorganisms Streptococcus thermophilus, Streptococcus sanguinis, Streptococcus uberis, Streptococcus gordonii, Actinobacillus succinogenes, Actinobacillus pleuropneumoniae, Streptococcus sp.dd12 and Streptococcus equus.

5. The method for efficiently synthesizing glutathione by using the cell-free self-assembly system as claimed in claim 1 or 2, wherein the temperature for generating glutathione in the step (3) is 30-60 ℃.

6. The method for efficiently synthesizing glutathione by using a cell-free self-assembly system as claimed in claim 1 or 2, wherein the concentration of the polyphosphate in the step (3) is 1 to 50 mmol/L.

7. The method for efficiently synthesizing glutathione by using a cell-free self-assembly system as claimed in claim 1 or 2, wherein the polyphosphate in the step (3) comprises pyrophosphate, tripolyphosphate, tetrapolyphosphate, hexapolyphosphate or polyphosphate with higher polymerization degree.

Technical Field

The invention relates to a method for producing glutathione, in particular to an enzyme catalysis method for synthesizing glutathione in vitro by using a cell-free self-assembly system.

Background

Glutathione (GSH) is a sulfhydryl-containing compound with important physiological functions, and is a tripeptide composed of three amino acids, namely L-glutamic acid (L-Glu), L-cysteine (L-Cys) and glycine (Gly). GSH is widely present in normal cells, is involved in a series of reactions in the body, and is one of the main metabolic regulators in human cells. In the field of medicine, glutathione can be used for resisting radiation, tumors, cancers, oxygen poisoning, aging and the like. In the field of food, glutathione can be used for improving the quality and flavor of food, prolonging the shelf life and the like. The glutathione is also applied to industries such as cosmetics, sports nutrition, health care products and the like, and has wide industrial application prospect.

The microbial fermentation production of GSH is the most widely applied method, and many researches are carried out on the utilization of recombinant saccharomyces cerevisiae or recombinant escherichia coli, the optimization of a culture medium and a fermentation regulation and control strategy and the improvement of GSH yield. The bottleneck of the glutathione preparation by the fermentation method is low conversion efficiency, complex post-treatment and low product yield.

Patent No. CN200510122930.5 discloses "a method for promoting synthesis of glutathione by microbial enzyme method". It provides a technology for synthesizing glutathione by using a microbial cell enzyme method. The method takes cultured recombinant Escherichia coli E.coli WSH-KE1 cells as an enzyme source, reduces the permeability barrier of a cell outer membrane by directly adding a low-concentration organic solvent or surfactant into a reaction system, catalyzes L-Glu, L-Cys and Gly to synthesize glutathione in the presence of Adenosine Triphosphate (ATP), and the synthesis amount of the glutathione can reach 4.8g/L after reacting for 2 hours. The reaction process needs to add a large amount of expensive ATP, and the cost is high, so that the industrial production is not facilitated.

Patent No. CN201210201691.2 provides a "method for preparing glutathione by enzymatic method", which comprises performing two reactions of synthesizing GSH, i.e. a reaction for generating γ -glutamylcysteine and a reaction for generating GSH, in different reaction tanks, respectively, and separating the enzymes used in the reactions, i.e. γ -glutamylcysteine synthetase (GSH-I) and glutathione synthetase (GSH-II), after each reaction, so that the enzyme activities of both GSH I and GSH II enzymes are utilized to the maximum extent, and mutual inhibition between the two enzymatic reactions is reduced. The yield of the GSH in one circulation can reach 8 g/L. The method realizes the recycling of the GSH I and the GSHII, and uses a yeast ATP regeneration system which is suitable for the reaction for preparing the GSH, thereby reducing the production cost for preparing the GSH, but the method has more complex process and higher operation cost.

Patent No. CN201310538982.5 provides a method for producing glutathione by recombinant expression of glutathione synthetase and acetate kinase, using acetyl phosphate as donor to realize ATP cyclic regeneration, and catalyzing L-Glu, L-Cys and Gly to synthesize glutathione. The method realizes the cyclic regeneration of ATP, further reduces the cost of directly adding ATP, improves the regeneration efficiency of ATP compared with a yeast ATP regeneration system, but the acetyl phosphate is still expensive and unstable.

In the synthesis reaction of glutathione, 2 molecules of ATP need to be provided for each 1 molecule of glutathione produced, and ATP supply is one of the primary considerations for process selection. Meanwhile, how to maximize the utilization rate of the substrate, simplify the operation, reduce the cost and the like are all considered factors. The intracellular synthesis of glutathione by using a microbial enzyme system has the problems of high raw material cost, slow reaction rate caused by substrate transportation and mixing limitation, degradation of substrates and products and the like. Cell-free in vitro synthesis has the characteristics of high reaction rate, high substrate conversion rate, convenience for subsequent separation and the like, so that the research on an energy supply system which is low in cost and easy to operate for efficient synthesis of glutathione and the improvement on reaction rate and efficiency are needed in the field.

Disclosure of Invention

The invention aims to provide a method for efficiently synthesizing glutathione by a cell-free self-assembly system in order to improve the product synthesis efficiency and the substrate conversion rate and reduce the production cost of glutathione.

In order to achieve the above object, the present invention provides a method for efficiently synthesizing glutathione using a cell-free self-assembled system, comprising the steps of:

(1) culturing a recombinant cell containing a polyphosphate kinase and glutathione bifunctional synthetase gene with a combined joint to ensure that the polyphosphate kinase containing the joint and the glutathione bifunctional synthetase containing the joint are over-expressed;

(2) harvesting the cells in the step (1) to obtain cell disruption solution, and carrying out intracellular self-assembly or extracellular self-assembly on the enzyme system;

(3) adding polyphosphate and one or more of AMP, ADP or ATP, catalyzing ATP by polyphosphate kinase in the self-assembly system through circulation of polyphosphate, AMP and/or ADP, and generating glutathione from glycine, glutamic acid, cysteine and synthesized ATP in vitro through the action of glutathione bifunctional synthetase in the self-assembly system.

As a preferable scheme, the method for efficiently synthesizing the glutathione by the cell-free self-assembly system is characterized by comprising the following steps:

(1) culturing a recombinant cell containing a polyphosphate kinase, adenosine kinase and glutathione bifunctional synthetase gene with a combined joint to ensure that the polyphosphate kinase containing the joint, the adenosine kinase containing the joint and the glutathione bifunctional synthetase containing the joint are over-expressed;

(2) harvesting the cells in the step (1) to obtain cell disruption solution, and carrying out intracellular self-assembly or extracellular self-assembly on the enzyme system;

(3) adding polyphosphate and adenosine and one or more of AMP, ADP or ATP, catalyzing ATP by polyphosphate kinase and adenosine kinase in the self-assembly system through circulation of polyphosphate, adenosine, AMP and/or ADP, and generating glutathione from glycine, glutamic acid, cysteine and synthesized ATP in vitro through the action of glutathione bifunctional synthetase in the self-assembly system.

As a preferred embodiment, the polyphosphate kinase may be PPK1 or PPK2 derived from one of the microorganisms Thermus thermophilus, Thermoynechococcus elongates, Jhaorihela thermophila, Hydrogenophilaceae bacterium, Nocardia dokdannensis, Halofaxsulfornation, Pseudonocardia thermophile, Roseophilum reptoaenium.

As a preferred scheme, the glutathione bifunctional enzyme is derived from one of the following microorganisms Streptococcus thermophilus, Streptococcus sanguinis, Streptococcus uberis, Streptococcus gordonii, Actinobacillus succinogenes, Actinobacillus pleuropneumoniae, Streptococcus sp.DD12 and Streptococcus equus.

Preferably, the temperature for producing glutathione in step (3) is 30-60 ℃.

As a preferred embodiment, the concentration of the polyphosphate in the step (3) is 1 to 50 millimoles per liter.

As a preferable mode, the polyphosphate in the step (3) includes pyrophosphate, tripolyphosphate, tetrapolyphosphate, hexapolyphosphate or polyphosphate with higher polymerization degree.

The invention provides a technology for synthesizing glutathione in vitro in a cell-free high-efficiency manner, which comprises the screening and assembly of polyphosphate kinase and glutathione bifunctional synthetase, or the screening and assembly of polyphosphate kinase, adenosine kinase and glutathione bifunctional synthetase, wherein polyphosphate and one or more of a small amount of AMP, ADP or ATP are utilized by the polyphosphate kinase, or the polyphosphate and adenosine kinase utilize polyphosphate, a small amount of adenosine, and one or more of a small amount of AMP, ADP or ATP, ATP is generated by circulating catalysis, and glycine, glutamic acid, cysteine and the generated ATP are used for producing glutathione by the action of the bifunctional glutathione synthetase by utilizing an assembly system.

The polyphosphate kinase in the cell-free self-assembly system utilizes the polyphosphate and a small amount of AMP or ADP in a reaction system to synthesize ATP, glutathione is synthesized from three precursors by glutathione bifunctional synthetase, and the polyphosphate in the reaction system continuously provides phosphate groups required by ATP synthesis for the polyphosphate kinase (PPK) to achieve the cyclic regeneration of ATP.

The cell-free self-assembly system utilizes the interaction between biological molecules and the substrate-driven polymerization, orderly assembles according to the structure, and shortens the space distance of enzyme, thereby increasing the reaction catalysis efficiency and being beneficial to the thermodynamic equilibrium towards the final product. In such assemblies, a high concentration of one substrate can be rapidly converted to a product and transferred to an adjacent enzyme as a substrate, thereby avoiding the accumulation and diffusion of toxic, unstable intermediate compounds. In this system, the distance between adjacent enzymes is shortened to a nano-scale or shorter, and the interaction formed between the intermediate and the linker arm, like the substrate channel, ensures rapid transfer of the intermediate substrate, thereby maximizing the efficiency of the multi-enzyme catalytic reaction.

The construction of the recombinant bacterium comprises the following steps: and (3) over-expressing heterologous polyphosphate kinase (PPK), Adenosine Kinase (AK) or glutathione bifunctional enzyme (GshF) in escherichia coli to obtain the recombinant expression strain.

The glutathione bifunctional enzyme is derived from actinobacillus succinogenes (Actinobacillus succinogenes).

The adenosine kinase described in the present invention is derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae).

The polyphosphate kinase comprises crude enzyme liquid obtained by expression and crushing of recombinant bacteria; or purified enzyme.

The glutathione bifunctional synthetase comprises crude enzyme liquid obtained by expression and crushing of recombinant bacteria; or purified enzyme.

The adenosine kinase comprises crude enzyme liquid obtained by expression and crushing of recombinant bacteria; or purified enzyme.

The polyphosphate disclosed by the invention has a general formula of poly (P) n, and comprises pyrophosphate, tripolyphosphate, tetrapolyphosphate, hexapolyphosphate or polyphosphate with higher polymerization degree, wherein common potassium salt and sodium salt or phosphate compounds thereof are used directly.

The invention has the advantages that the cell-free self-assembly system is utilized to synthesize the glutathione in vitro for the first time, the method shortens the transfer distance of the substrate, improves the reaction rate and the catalytic efficiency, and strengthens the thermal stability of the protein. The ATP efficient cyclic regeneration in the glutathione synthesis process is realized, and the production cost is reduced; the operation is simple and the stability is strong; less side reaction, high production rate and high substrate yield.

Drawings

FIG. 1 concentration values of synthesized glutathione in example 1 of the present invention.

FIG. 2 concentration values of synthesized glutathione in example 2 of the present invention.

FIG. 3 concentration values of synthesized glutathione in example 3 of the present invention.

FIG. 4 concentration values of synthesized glutathione in example 4 of the present invention.

FIG. 5 example 5 Effect of PPKTE/SS enzyme ratio on GSH synthesis according to the invention.

FIG. 6 Effect of initial ADP concentration on GSH synthesis according to example 6 of the present invention.

Figure 7 effect of temperature on GSH synthesis of example 7 of the invention.

FIG. 8 example 8 of the invention the effect of polyP concentration on GSH synthesis.

FIG. 9 Effect of ADP concentration on the catalytic efficiency of MENRs or free systems according to example 9 of the present invention.

FIG. 10 stability analysis of the assemblies and free enzymes of example 10 of the present invention.

Detailed Description

Hereinafter, the technique of the present invention will be described in detail with reference to specific embodiments. It should be understood that the following detailed description is only for the purpose of assisting those skilled in the art in understanding the present invention, and is not intended to limit the present invention.

Preparation of glutathione-II bifunctional enzyme

a. The GshF-containing pET-28a vector is transformed into an Escherichia coli expression host BL21(DE3) by a calcium chloride method to obtain BL21(DE3)/pET28 a-GshF.

b. BL21(DE3)/pET28a-GshF was inoculated into LB medium (peptone 10g/L, yeast powder 5h/L, sodium chloride 10g/L), cultured overnight at 37 ℃ and then inoculated into fresh LB medium in an inoculum size of 1%, cultured at 37 ℃ to OD₆₀₀0.6-0.8, IPTG was added to a final concentration of 0.2mM, and induction was carried out at 18 ℃ for 18 hours.

c. The enzyme purification method comprises the following steps: and centrifuging and collecting the induced thallus, crushing the thallus under high pressure to obtain a crude enzyme solution, and centrifuging the crude enzyme solution to obtain a supernatant. The supernatant was purified by HisSep Ni-NTA Agarose affinity chromatography column. Eluting with 0-500mM imidazole concentration gradient, verifying purity by SDS-PAGE, and collecting eluate under 120mM imidazole to obtain pure enzyme solution. The quantification of the protein was determined by BCA kit (Tiangen Biotechnology Ltd.).

Preparation of adenosine kinase

The adenosine kinase gene derived from Saccharomyces cerevisiae was synthesized, inserted into plasmid pET28a and transformed into BL21(DE3) to give BL21(DE3)/pET28 a-AK.

A pure enzyme solution of adenosine kinase was prepared according to the same procedure as in method one.

Construction of polyphosphate kinase recombinant bacteria

The gene derived from Thermus elongatus polyphosphate kinase (TePPK) was synthesized, inserted into plasmid pET28a and transformed into BL21(DE3) to give BL21(DE3)/pET28 a-TePPK.

The gene of the JHAORHELLA thermophila polyphosphate kinase (JtPPK) is obtained by synthesis, inserted into a plasmid pET28a and transformed into BL21(DE3) to obtain BL21(DE3) -JtPPK.

Four, cell-free self-assembly

Equimolar amounts of PPK, AK and GshF containing the assembled linkers were mixed in a buffer of 20mM Tris-HCl (pH8.0) at 25 ℃ for 30min, centrifuged at 3000rpm for 10min, washed with 20mM Tris-HCl (pH8.0) and then resuspended slowly in 2.5mM Tris-HCl (pH 8.0).

Fifthly, in vitro synthesis of glutathione

Using the self-assembly system, Tris-HCl buffer system and Mg²⁺In the presence of the glutathione, ATP is recycled and regenerated from polyphosphoric acid and an initial small amount of ADP or ATP, and glutathione is synthesized using the ATP, glycine, glutamic acid and cysteine that are produced.