CN116769749A - Polyphosphate kinase and method for producing glutathione by coupling glutathione bifunctional enzyme - Google Patents
Polyphosphate kinase and method for producing glutathione by coupling glutathione bifunctional enzyme Download PDFInfo
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- CN116769749A CN116769749A CN202310738715.6A CN202310738715A CN116769749A CN 116769749 A CN116769749 A CN 116769749A CN 202310738715 A CN202310738715 A CN 202310738715A CN 116769749 A CN116769749 A CN 116769749A
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- glutathione
- polyphosphate kinase
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- mutant
- glycine
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- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 title claims abstract description 159
- 229960003180 glutathione Drugs 0.000 title claims abstract description 79
- 108010024636 Glutathione Proteins 0.000 title claims abstract description 77
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 58
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 58
- 108020000161 polyphosphate kinase Proteins 0.000 title claims abstract description 51
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 44
- 230000001588 bifunctional effect Effects 0.000 title claims abstract description 14
- 230000008878 coupling Effects 0.000 title claims abstract description 7
- 238000010168 coupling process Methods 0.000 title claims abstract description 7
- 238000005859 coupling reaction Methods 0.000 title claims abstract description 7
- 229920000388 Polyphosphate Polymers 0.000 claims abstract description 19
- 239000001205 polyphosphate Substances 0.000 claims abstract description 19
- 235000011176 polyphosphates Nutrition 0.000 claims abstract description 19
- 229920000037 Polyproline Polymers 0.000 claims abstract description 17
- 239000006166 lysate Substances 0.000 claims abstract description 14
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 36
- 238000006243 chemical reaction Methods 0.000 claims description 21
- 239000004471 Glycine Substances 0.000 claims description 18
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims description 16
- 241000894006 Bacteria Species 0.000 claims description 14
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 14
- 235000018417 cysteine Nutrition 0.000 claims description 14
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 12
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 12
- 239000004472 Lysine Substances 0.000 claims description 12
- 150000001413 amino acids Chemical group 0.000 claims description 12
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 11
- 239000007853 buffer solution Substances 0.000 claims description 11
- 235000013922 glutamic acid Nutrition 0.000 claims description 11
- 239000004220 glutamic acid Substances 0.000 claims description 11
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- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 claims description 8
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 7
- 239000003054 catalyst Substances 0.000 claims description 5
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- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 claims description 4
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 claims description 4
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- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 claims description 4
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- 101150094189 gshAB gene Proteins 0.000 abstract 1
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- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 34
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- XTWYTFMLZFPYCI-UHFFFAOYSA-N Adenosine diphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(O)=O)C(O)C1O XTWYTFMLZFPYCI-UHFFFAOYSA-N 0.000 description 7
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- 230000003197 catalytic effect Effects 0.000 description 4
- 235000021317 phosphate Nutrition 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 3
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- 238000000034 method Methods 0.000 description 3
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- 235000013878 L-cysteine Nutrition 0.000 description 2
- 208000037062 Polyps Diseases 0.000 description 2
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- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 101150040316 ppk2 gene Proteins 0.000 description 2
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- 241000203069 Archaea Species 0.000 description 1
- 101100409044 Chlorobaculum tepidum (strain ATCC 49652 / DSM 12025 / NBRC 103806 / TLS) ppk1 gene Proteins 0.000 description 1
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- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- XJLXINKUBYWONI-NNYOXOHSSA-O NADP(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-NNYOXOHSSA-O 0.000 description 1
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- MEFKEPWMEQBLKI-AIRLBKTGSA-N S-adenosyl-L-methioninate Chemical compound O[C@@H]1[C@H](O)[C@@H](C[S+](CC[C@H](N)C([O-])=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 MEFKEPWMEQBLKI-AIRLBKTGSA-N 0.000 description 1
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- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 description 1
- TTWYZDPBDWHJOR-IDIVVRGQSA-L adenosine triphosphate disodium Chemical compound [Na+].[Na+].C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O TTWYZDPBDWHJOR-IDIVVRGQSA-L 0.000 description 1
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- HXCHCVDVKSCDHU-LULTVBGHSA-N calicheamicin Chemical compound C1[C@H](OC)[C@@H](NCC)CO[C@H]1O[C@H]1[C@H](O[C@@H]2C\3=C(NC(=O)OC)C(=O)C[C@](C/3=C/CSSSC)(O)C#C\C=C/C#C2)O[C@H](C)[C@@H](NO[C@@H]2O[C@H](C)[C@@H](SC(=O)C=3C(=C(OC)C(O[C@H]4[C@@H]([C@H](OC)[C@@H](O)[C@H](C)O4)O)=C(I)C=3C)OC)[C@@H](O)C2)[C@@H]1O HXCHCVDVKSCDHU-LULTVBGHSA-N 0.000 description 1
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- 229960002743 glutamine Drugs 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
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- 229940111202 pepsin Drugs 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1229—Phosphotransferases with a phosphate group as acceptor (2.7.4)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
- C07K5/0215—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing natural amino acids, forming a peptide bond via their side chain functional group, e.g. epsilon-Lys, gamma-Glu
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- C12N15/09—Recombinant DNA-technology
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- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
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Abstract
The invention discloses a polyphosphate kinase and a method for producing glutathione by coupling glutathione bifunctional enzyme. The invention expands the double-substrate polyP of polyphosphate kinase through molecular docking and site-directed mutagenesis and rational design 6 And ADP channel cavity, and increasing specific enzyme activity of polyphosphate kinase, and screening to obtainThe polyphosphate kinase mutant is used for producing glutathione, gshAB and polyphosphate kinase are produced and accumulated through fermentation thalli, cells are broken after the thalli are collected to obtain lysate, the lysate has the advantage that endogenous ATP can start a regeneration system, the problems of cell barrier, complex path modification and the like in the glutathione intracellular production process are avoided, and a production path of the glutathione is quickly reconstructed in vitro. After the ATP regeneration system is coupled, the use of ATP can be reduced, and the efficient production of glutathione can be realized after the production system is optimized.
Description
Technical Field
The invention relates to a method for producing glutathione by polyphosphate kinase and glutathione bifunctional enzyme coupling, belonging to the technical field of enzyme engineering.
Background
Glutathione (GSH) is the most abundant non-protein thiol compound in all organisms, has the ability to antioxidant, antidote and immune booster, and is widely used in the medical, food and cosmetic industries. The starting strain for the fermentative production of glutathione is mainly Saccharomyces cerevisiae, and more recently, a glutathione bifunctional enzyme (GshAB) has been found in Streptococcus agalactiae, which can directly catalyze the precursors glutamate, cysteine and glycine to synthesize GSH, with the concomitant consumption of two molecules of ATP. The production of glutathione by fermentation may have problems of cell membrane barrier, substrate limitation, product transportation, etc., while whole cell catalysis requires the addition of a large amount of ATP, resulting in high cost.
Adenosine Triphosphate (ATP) is a high-energy phosphate compound necessary in organisms and provides energy for processes such as synthesis, transport, information transfer, etc. within living cells. In enzyme catalysis, ATP is often used as a cofactor for group transfer, participating in the production of high value products. The majority of biocatalytic reactions are phosphate transfer between ATP, ADP and AMP, e.g., ATP driven formation of amide bonds, dipeptides, glutamine, glutathione, S-adenosylmethionine, etc. Given the time and cost of the biological reaction, it seems not feasible to add large amounts of ATP, which requires us to seek sustainability and efficiency of ATP supply methods. Inorganic polyphosphates (polyps) are a linear polymer with several phosphates that have been found to act as stress and survival, energy, cell movement, biofilm formation and metal ion chelators in all living cells including archaea, bacteria and eukaryotes. In recent years, polyphosphate kinase (PPK) catalyzed reversible reactions of inorganic polyphosphates with ATP have been of interest. PPK can be divided into two families, PPK1 and PPK2, PPK1 being prone to synthesize polyP with the phosphate group at the ATP terminus as substrate, e.g. BlPPK from e.coli belongs to the class of typical PPK 1. PPK2 exhibits the opposite property and can catalyze polyP as a donor for adenosine phosphorylation. Currently, the bifunctional polyphosphate kinase PPK is widely focused, and the enzyme takes cheap and easily available phosphate (polyP) as a substrate, belongs to PPK 2-III, and can catalyze adenosine to generate adenosine diphosphate and adenosine triphosphate. How to apply polyphosphate kinase in glutathione production and improve the yield and conversion rate of glutathione is a problem to be solved urgently.
Disclosure of Invention
In order to solve the technical problems, the invention provides a polyphosphate kinase mutant and a method for producing glutathione by coupling glutathione bifunctional enzymes GshAB and polyphosphate kinase, wherein the GshAB and the polyphosphate kinase are produced and accumulated by fermentation thalli, and a lysate obtained by cell disruption after fermentation broth thalli is collected is used for producing glutathione, so that the ATP regeneration capacity of the polyphosphate kinase is improved, a production system is optimized, efficient production of the glutathione is realized, and the yield and conversion rate of the glutathione are improved.
The first object of the present invention is to provide a polyphosphate kinase mutant in which lysine at position 81 and/or lysine at position 103 of the polyphosphate kinase having the amino acid sequence shown in SEQ ID NO.4 is mutated.
Further, the polyphosphate kinase mutant is characterized in that lysine at position 81 of the polyphosphate kinase with an amino acid sequence shown in SEQ ID NO.4 is mutated into threonine or histidine.
Further, the polyphosphate kinase mutant is characterized in that the 103 rd lysine of the polyphosphate kinase with the amino acid sequence shown in SEQ ID NO.4 is mutated into aspartic acid, glycine or valine.
Further, the polyphosphate kinase mutant is characterized in that lysine at position 81 of the polyphosphate kinase with an amino acid sequence shown in SEQ ID NO.4 is mutated into threonine or histidine, and lysine at position 103 is mutated into aspartic acid, glycine or valine.
It is a second object of the present invention to provide a gene encoding the polyphosphate kinase mutant.
It is a third object of the present invention to provide an expression vector carrying the gene.
It is a fourth object of the present invention to provide a recombinant bacterium expressing the polyphosphate kinase mutant.
Furthermore, glutathione bifunctional enzymes are also expressed in the recombinant bacteria.
Further, the amino acid sequence of the glutathione bifunctional enzyme is shown as SEQ ID NO. 2.
Further, the recombinant bacteria take escherichia coli as a host.
Furthermore, the recombinant bacteria take escherichia coli E.coli BL21 (DE 3) as a host and pET28a (+) as an expression vector.
A fifth object of the present invention is to provide the use of said polyphosphate kinase mutant or recombinant bacteria expressing said polyphosphate kinase mutant in the production of glutathione.
Advancing oneThe application adopts polyphosphate kinase mutant and glutathione bifunctional enzyme as catalyst to contain 40-60 mM glutamic acid, 40-60 mM glycine, 40-60 mM cysteine, 4-6 mM ATP and 40-60 mM polyP 6 The buffer solution of (2) is a reaction system for catalyzing and producing glutathione.
Further, the application adopts the thallus lysate of the recombinant bacteria as a catalyst to contain 40-60 mM glutamic acid, 40-60 mM glycine, 40-60 mM cysteine, 4-6 mM ATP and 40-60 mM polyP 6 The buffer solution of (2) is a reaction system for catalyzing and producing glutathione.
Further, glutamic acid, glycine, cysteine and polyP 6 Added to the reaction system in portions.
Further, the batch addition is divided into 2 to 4 additions, one addition to the initial medium, and one addition at 1 to 3 hours later.
Further, in the cell lysate, the cell mass OD 600 10 to 20.
Further, the buffer solution is Tris-HCl buffer solution.
Further, the bacterial lysate is obtained by culturing the recombinant bacteria in an LB culture medium at 35-38 ℃ and performing induction culture by using IPTG to obtain a fermentation broth, and crushing the fermentation broth.
In the production of glutathione, enzyme is produced and accumulated through fermentation of thalli, cells are broken after the thalli are collected to obtain lysate, the lysate can provide endogenous ATP for producing glutathione by an ATP regeneration system, and meanwhile, the problems of cell barrier, complex path modification and the like in the intracellular production process of glutathione are avoided; after the ATP regeneration system is coupled, the use of ATP can be reduced, and the efficient production of glutathione can be realized after the production system is optimized.
The beneficial effects of the invention are as follows:
1. the invention expands the double-substrate polyP of polyphosphate kinase through molecular docking and site-directed mutagenesis and rational design 6 And ADP channel cavity, the specific enzyme activity of ChPPK before mutation is 605+ -2.1U/mgAfter mutation, the double mutant enzyme ChPPK K81H-K103V Exhibits the highest catalytic activity, and the specific enzyme activity is 1972+/-2.5U/mg.
2. The invention uses the polyphosphate kinase mutant obtained by screening to produce glutathione, produces and accumulates GshAB and polyphosphate kinase through fermentation thalli, and breaks cells after collecting thalli to obtain lysate, and the lysate has the advantage of an endogenous ATP (adenosine triphosphate) activatable regeneration system, avoids the problems of cell barrier, complex path modification and the like in the intracellular production process of glutathione, and rapidly reconstructs a production path of the glutathione in vitro. In EC01 system, sufficient ATP was added to produce 30.7+ -1.9 mM glutathione, while in EC03 system, chPPK was used K81H-K103V The ability of enzyme to regenerate ATP can enhance the sustainable supply of ATP, and 25.4+ -1.9 mM glutathione can be produced in 6 hours after 5mM ATP is added, and meanwhile, compared with the EC02 system before mutation, the glutathione yield is improved by 41.9%. After the buffer solution, the thallus quantity of the fermentation liquor and the feeding time of the system are optimized, the EC01 system can produce 47.9+/-1.3 mM glutathione, the EC03 system can produce 45.2+/-1.8 mM glutathione, and the conversion rate of the substrate L-cysteine reaches 90.4 percent.
3、ChPPK K81H-K103V The regeneration system of the catalyst realizes the unification of high yield, high conversion rate and high economic value of the enzyme-catalyzed production of the glutathione. After the efficient PPK mutant enzyme is obtained, chPPK is added in the biocatalysis reaction K81H-K103V The efficiency and the sustainability of ATP regeneration can be obviously improved, the catalytic efficiency and the substrate conversion rate can be improved after the system is optimized, and the cost for reaction can be saved.
Description of the drawings:
FIG. 1 shows the production yield and system optimization of glutathione production from double enzyme-coupled fermentation broth.
Detailed Description
The present invention will be further described with reference to specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the present invention and practice it.
The nucleotide sequence of glutathione bifunctional enzyme GshAB is shown as SEQ ID NO.1, and the amino acid sequence is shown as the sequenceAs shown in SEQ ID NO. 2; the nucleotide sequence of the polyphosphate kinase ChPPK is shown as SEQ ID NO.3, and the amino acid sequence is shown as SEQ ID NO. 4; mutant ChPPK K81H-K103V The nucleotide sequence of the plasmid pET-28a-ChPPK is shown in SEQ ID NO.5, the amino acid sequence is shown in SEQ ID NO.6, the nucleotide sequence of the plasmid pET-28a-GshAB is shown in SEQ ID NO.7, the nucleotide sequence of the plasmid pET-28a-GshAB is shown in SEQ ID NO.8, and the plasmid pET-28a-ChPPK is shown in the specification K81H-K103V The nucleotide sequence of (2) is shown in SEQ ID NO.9.
The media referred to in the examples below:
LB medium (1L): 10g NaCl, 10g tryptone, 5g yeast extract.
Example 1: construction of recombinant E.coli Strain BL21/pET-28a-GshAB, BL21/pET-28a-GshAB-ChPPK
The method comprises the following specific steps:
(1) Construction of overexpression plasmid pET-28a-ChPPK, pET-28a-GshAB
The polyphosphate kinase ChPPK (nucleotide sequence shown in SEQ ID NO. 3) and glutathione bifunctional enzyme GshAB (nucleotide sequence shown in SEQ ID NO. 1) are synthesized by Suzhou Jin Weizhi biotechnology limited company to obtain plasmids pET-28a-ChPPK (nucleotide sequence shown in SEQ ID NO. 7) and pET-28a-GshAB (nucleotide sequence shown in SEQ ID NO. 8).
(2) Preparation of E.coli BL21 competence
E.coli BL21 is streaked on an antibiotic-free LB plate, placed and cultured in a 37 ℃ incubator, colonies are picked up and inoculated in 10mL LB vials for culturing for 12 hours, 1% of inoculum size is transferred into 50mL LB culture medium bottles, and when the thallus concentration reaches 0.4-0.6, the preparation of the coliform competence is prepared. Pre-cooling the related reagents and instruments in advance, placing the solution A, the solution B, the 1.5mL EP tube and the 50mL centrifuge tube in the prepared kit on ice, and controlling the temperature of the centrifuge to 4 ℃. Packaging 50mL of the bacterial liquid in a sterile operation table, and centrifuging (8000 r.min -1 5 min), the supernatant was removed. 5mL of solution A was sucked for blowing suspension, and centrifuged (8000 r.min -1 5 min), the supernatant was removed. 5mL of solution B is sucked for blowing suspension, and the suspension is split into EP pipes which are pre-cooled in advance, and each pipe is filled with 100 mu L.
(3) Construction of recombinant BL21/pET-28a-GshAB, BL21/pET-28a-GshAB-ChPPK
Transforming pET-28a-ChPPK and pET-28a-GshAB plasmids obtained in the step (1) into competent cells of escherichia coli BL21 to obtain transformants, coating the transformants in an LB solid medium containing the calicheamicin with the concentration of 50 mug/mL, culturing for 12 hours at 37 ℃, picking positive colonies, designing a primer P1/P2 in the plasmid pET-28a, and carrying out colony PCR verification on single colonies by Taq DNA polymerase; positive single colonies with the target band size were inoculated into a vial containing LB liquid medium for 12h, and after the culture, plasmids were extracted and sent to the gold-only intelligent company for sequencing. And if the sequencing is correct, the E.coli BL21/pET-28a-ChPPK is successfully constructed. E.coli BL21/pET-28a-GshAB, named EC01, was constructed in the same manner.
The plasmid pET-28a-ChPPK is used as a template, a primer P3/P4 is amplified to obtain a ChPPK fragment, the plasmid pET-28a-GshAB is used as a template, a primer P5/P6 is amplified to obtain a linearization plasmid pET-28a-GshAB, the ChPPK fragment and the linearization plasmid pET-28a-GshAB are connected through homologous recombination enzymes, and the ChPPK fragment is transferred into E.coli BL21 (DE 3) competent cells to obtain a transformant. Likewise, BL21/pET-28a-GshAB-ChPPK constructed as described above was designated EC02.
P1：5’-cctgtggcgccggtgatgccggcc-3’；
P2：5’-atccggatatagttcctcctttca-3’；
P3：5’-atggcaaccgatttttctaagctg-3’；
P4：5’-ctggtggcagaaaagtcctccgat-3’；
P5：5’-ctgagttggctgctgccaccgct-3’；
P6：5’-ctgctaacaaagcccgaaaggaag-3’。
Example 2: site-directed mutagenesis and enzyme activity comparison
(1) Selection of mutation points
The three-dimensional structure of the ChPPK protein was predicted using the Swiss-model, and the optimal docking position was selected by docking analysis using the Schrodinger-glide docking substrate with the protein. After structural analysis of the ChPPK protein, dual-substrate polyP is found 6 And the K81 and K103 residues at the entrance of ADP channel may affect the bottomThe entry of the substance further influences the flexibility of the enzyme activity pocket, and presumably the rational design of the double-substrate channel cavity can be carried out, the channel of the substrate is enlarged, more substrates are allowed to enter the active center, and thus the enzyme activity is improved. Two residue positions K81 and K103 were selected, mutated using the Pymol software, and the distance between residues D77 and K81 and the distance between residues K81 and K103 were determined. Before mutation, the distance between the oxygen atom of residue D77 and the nitrogen atom of residue K81 isThe distance between the nitrogen atom of residue K81 and the nitrogen atom of residue K103 is +.>Mutation of residues K81 and K103 visualized by the Pymol software, residues D77 at a distance of +.>T81 and D103, G103 and V103 are each +.> Residue D77 is +.about.81 from H>The distances between H81 and D103, G103 and V103 are respectively-> Mutation of T81, H81 and D103, G103, V103 results in dual-substrate polyP 6 And the channel entrance of ADP became large, so K81T, K81H, K103D, K G and K103V were selected for subsequent mutation experiments.
(2) Site-directed mutagenesis
Site-directed mutagenesis of the K81 site of wild-type ChPPK was T, H, site-directed mutagenesis of the K103 site was V, G, D, site-directed mutagenesis was performed by designing primers containing specific mutated sequences, and site-directed mutagenesis was performed according to the primers of P7-P15 (P7/P8: K103V, P9/P10: K103G, P11/P12: K103D, P13/P14: K81H, P/P16: K81T). PCR conditions: pET28a-ChPPK is used as a DNA template, and the amplification conditions are as follows: pre-denaturation at 95 ℃,5min: denaturation at 95 ℃,30s, annealing at 58 ℃,30s, extension at 72 ℃,2.5min,30 cycles; finally, the extension is carried out for 5min at 72 ℃. The obtained recombinant plasmid was sent to Jin Weizhi (su zhou) for sequencing verification. The plasmid with correct sequence was constructed according to the construction method of example 1 (3) to obtain recombinant strain BL21/pET-28a-ChPPKmuts.
P7：5’-tgacttccttcgtggtgccatccaagatcgaactgtccca-3’；
P8：5’-tggcaccacgaaggaagtcaccttcacgccttgtg-3’；
P9：5’-tgacttccttcggcgtgccatccaagatcgaactgtccca-3’；
P10：5’-tggcacgccgaaggaagtcaccttcacgccttgtg-3’；
P11：5’-tgacttccttcgatgtgccatccaagatcgaactgtccca-3’；
P12：5’-tggcacatcgaaggaagtcaccttcacgccttgtg-3’；
P13：5’-atgcagccggccacgatggcaccgtgaagcacatc-3’；
P14：5’-caagcaatggatgcagccggccacgatggcaccgtgaa-3’；
P15：5’-atgcagccggcaccgatggcaccgtgaagcacatc-3’；
P16：5’-ccaagcaatggatgcagccggcaccgatggcacc-3’；
(3) Disruption and purification of proteins
Inoculating single colony of BL21/pET-28a-ChPPK and BL21/pET-28a-ChPPKmuts recombinant strain into 10mL of LB liquid medium, culturing at 37deg.C for 12 hr, inoculating 1% of the recombinant strain into 50mL of LB liquid medium, culturing to OD 600 About 0.8, IPTG was added and incubated at 16℃for 16h. Washing cells with PBS for three times, suspending the collected bacteria again with PBS, crushing cells with an ultrasonic crusher, crushing Escherichia coli for 1s, stopping for 3s, and crushing for 15min. Centrifugal 10000rpmAnd (5) taking supernatant, namely the pepsin glue, for 20 minutes. Purifying the crude enzyme liquid by a protein purification nickel column, and verifying the pure enzyme protein gel.
(4) Comparison of enzyme Activity
The reaction system (2 mL) for enzyme activity assay contained (final concentration) 100mM Tris-HCl (pH 8.0), 10mM glucose, 10mM NADP, 10mM ADP, 10mM polyP 6 5U/mL HK, 5U/mL G6PD and 50. Mu.L PPK enzyme. The reaction was carried out at 37℃for 30min, followed by 5min in boiling water to terminate the enzyme reaction, centrifugation at 12,000 rpm for 5min, and the absorbance was measured at 340nm from the supernatant. The enzyme activity (1U) of PPK is defined as the amount of enzyme required to produce 1. Mu. Mol ATP per minute.
Site-directed mutagenesis of two residues K81 and K103, respectively, was performed by Ni 2+ The wild-type WT and mutant enzyme were then reacted at 37℃for 30min, respectively, followed by purification by NTA column affinity chromatography, and their enzyme activities were determined. The specific enzyme activity of the wild type is 605+/-2.1U/mg, the specific enzyme activity of mutant enzyme K81H is improved most in site-directed mutation at site-specific mutation at site of 103 at site-specific mutation at site of 938+/-1.2U/mg, and the specific enzyme activity of K103V is improved most compared with the wild type at site-specific mutation at site of 1065+/-2.9U/mg, which also proves that double-substrate ADP and polyP are enlarged by rational transformation 6 Can effectively improve the catalytic activity of the ChPPK.
Thus, for the above single mutant enzyme with increased specific enzyme activity, we constructed 6 double mutant enzymes, and measured the specific enzyme activity of the double mutant enzymes, and expected to obtain mutant enzymes with more significant enzyme activity improvement. The double mutant enzyme K81H-K103V shows the highest catalytic activity, and the specific enzyme activity is 1972+/-2.5U/mg. The specific enzyme activities of all mutant enzymes are shown in Table 1.
TABLE 1
| Mutant enzymes | Specific enzyme activity (x 10, U/mg) |
| WT | 60.5±0.21 |
| K81T | 76.8±0.34 |
| K81H | 93.8±0.12 |
| K103D | 80.5±0.24 |
| K103G | 84.7±0.09 |
| K103V | 106.5±0.29 |
| K81T-K103D | 101.0±0.17 |
| K81T-K103G | 115.6±0.30 |
| K81T-K103V | 175.5±0.23 |
| K81H-K103D | 113.1±0.15 |
| K81H-K103G | 155.5±0.22 |
| K81H-K103V | 197.2±0.25 |
Example 3: production of glutathione from double-enzyme coupled fermentation broth
(1) Strain BL21/pET-28a-GshAB-ChPPK K81H-K103V Construction of (3)
With plasmid pET-28a-ChPPK K81H-K103V (the nucleotide sequence is shown as SEQ ID NO. 9) as a template, and the primer P3/P4 is used for amplifying to obtain the ChPPK K81H-K103V The fragment, the obtained gene fragment is connected with linearization plasmid pET-28a-GshAB (P5/P6 amplification) through homologous recombination enzyme after purification, and transferred into E.coli BL21 (DE 3) competent cells to obtain transformant. Similarly, E.coli BL21/pET-28a-GshAB-ChPPK was constructed as in example 1 K81H-K103V Designated EC03.
(2) Treatment of fermentation broths
Inoculating single colony of EC01, EC02 and EC03 into 10mL LB liquid vial, culturing at 37deg.C for 18h, inoculating into 100mL LB medium according to 1% (V/V) inoculum size, culturing at 37deg.C for 4h, OD 600 About 0.8, 0.2mM IPTG was added and incubated at 16℃for 18 hours to produce and accumulate the relevant enzymes. After collecting the cells of the fermentation broth, 800mL of PBS buffer (pH 8.0, 100 mM) was added to resuspend the cells (OD 600 10), the cells are crushed for 15min by a high-pressure refiner, and the obtained lysate is ready for use.
(3) Production of glutathione from double-enzyme coupled fermentation broth
EC01 production glutathione system: the PBS buffer contained 50mM glutamate, 50mM glycine, 50mM cysteine, 100mM ATP. EC02/EC03 production glutathione system: the PBS buffer contained 50mM glutamate, 50mM glycine, and 50mM,5mM ATP,polyP cysteine 6 50mM. The lysate of the above fermentation broth is added to the system. The pH was adjusted to 8.0 with NaOH and reacted at 37 ℃.
Upon addition of substrates glutamic acid, glycine, cysteine and sufficient ATP (100 mM) to the production system, after reaction at 37℃the sample was taken to determine glutathione production. As shown in FIG. 1A, 50mM substrate can be converted into 30.7+ -1.9 mM glutathione in 6h of EC01 production system, but because of the high price of ATP, it is not possible to add large amounts of ATP in actual production, therefore, we couple ChPPK with GshAB, besides the additional 5mM ATP, endogenous ATP in lysate can be further startedCatalytic reaction, EC02 production system 6h can produce 17.9+ -1.7 mM glutathione. In the mutant enzyme ChPPK K81H-K103V In the EC03 production system coupled with GshAB, 50mM substrate can be converted to generate 25.4+/-1.9 mM glutathione within 6 hours, 82.7% effect of the EC01 production system can be achieved, and the yield is improved by 41.9% compared with that of the coupling of unmutated ChPPK and GshAB. Mutant enzyme ChPPK K81H-K103V The addition of substrate ATP can be effectively reduced, and under the drive of the mutant enzyme, efficient ATP supply is provided for the production of glutathione, so that the reaction cost is greatly saved.
Example 4: optimization of glutathione production system by double-enzyme coupled fermentation broth
(1) Optimization of buffers
According to the system for producing glutathione in example 3, the buffer solution of the system is changed, and the system is respectively placed in PBS, tris-HCl and glycine-NaOH buffer solution to react at 37 ℃, and the sample is taken to measure the yield of the glutathione. As shown in FIG. 1B, the yields of glutathione in PBS, tris-HCl, glycine-NaOH buffer, and 27.6.+ -. 1.1mM and 24.3.+ -. 2.1mM, 26.8.+ -. 2.9mM, and 20.2.+ -. 2.5mM were 31.5.+ -. 1.4mM, 34.1.+ -. 2.7mM, and 27.6.+ -. 1.1mM, respectively, for EC01 at 6 hours. Thus, the yields of glutathione in the EC01 and EC03 systems were highest in Tris-HCl buffer, which was selected for subsequent experiments.
(2) Optimization of cell quantity
According to the glutathione production system of example 3, the amount of cells in the fermentation broth was changed, and OD was measured 600 5, 10, 15 and 20, crushing cells for 15min by a high-pressure homogenizer, adding the crushed lysate into the system, reacting at 37 ℃, sampling and measuring the yield of glutathione. As shown in FIG. 1C, OD 600 At 5, 10 and 15, the glutathione production in the EC01 and EC03 systems gradually increases, while the OD 600 At 20, glutathione production no longer increases, at OD 600 At 15, the highest glutathione yields of EC01 and EC03 were 39.9+ -1.5 mM, 30.4+ -1.4 mM, respectively, and OD was selected 600 15 was used for the subsequent experiments.
(3) Optimization of feed time
According to the system for producing glutathione in example 3, buffer Tris-HCl, cell mass OD was selected 600 15, changing the feeding time of the substrate, adding the substrate into the system at different time, reacting at 37 ℃, sampling and measuring the yield of glutathione. As shown in FIG. 1D, the amounts of substrate glutamic acid, glycine and cysteine in the initial medium were 16.6mM, respectively, and the substrates glutamic acid, glycine and cysteine (16.6 mM each substrate was added in total at about 50mM each substrate) were added at 2 hours and 4 hours, respectively, and the glutathione yield in the EC01 system was the highest at 47.9.+ -. 1.3mM at 6 hours; in the system for producing glutathione by EC03, under the condition of optimal buffer solution and bacterial body amount, the amounts of substrates of glutamic acid, glycine and cysteine in the initial culture medium are respectively 16.6mM, and the amounts of polyp are respectively 6 The substrates glutamic acid, glycine, cysteine (16.6 mM each substrate added and a total of about 50mM each substrate) and polyP were added at 2h and 4h, respectively, in amounts of 33.3mM each substrate 6 (33.3 mM each time), the glutathione yield in the EC03 system at 6 hours is 45.2+/-1.8 mM, the conversion rate of the substrate L-cysteine is 90.4%, and the glutathione yield (25.4+/-1.9 mM) is improved by 1.78 times compared with that of the non-optimized EC03 system; glutathione production did not continue to increase after 2h, 4h, 6h substrate addition, probably due to the gradual decrease in enzyme activity in the system over time, and the production efficiency decreased. The comparison of glutathione production is shown in Table 2 herein.
TABLE 2
The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.
Claims (10)
1. A polyphosphate kinase mutant is characterized in that the polyphosphate kinase mutant is obtained by mutating lysine at position 81 and/or lysine at position 103 of polyphosphate kinase with an amino acid sequence shown as SEQ ID NO. 4.
2. The polyphosphate kinase mutant of claim 1, wherein the polyphosphate kinase mutant is one of the following mutants:
mutating lysine at position 81 of polyphosphate kinase with amino acid sequence shown in SEQ ID NO.4 into threonine or histidine;
mutating lysine at 103 of polyphosphate kinase with amino acid sequence shown in SEQ ID NO.4 into aspartic acid, glycine or valine;
the 81 st lysine of polyphosphate kinase with the amino acid sequence shown in SEQ ID NO.4 is mutated into threonine or histidine, and the 103 rd lysine is mutated into aspartic acid, glycine or valine.
3. A gene encoding the polyphosphate kinase mutant according to any one of claims 1 to 2.
4. An expression vector carrying the gene of claim 3.
5. A recombinant bacterium expressing the polyphosphate kinase mutant of any one of claims 1 to 2.
6. The recombinant bacterium according to claim 5, wherein glutathione bifunctional enzyme is also expressed in the recombinant bacterium.
7. Use of a polyphosphate kinase mutant according to any one of claims 1 to 2 or a recombinant bacterium according to any one of claims 5 to 6 in the production of glutathione.
8. The use according to claim 7, characterized in thatThe application adopts the coupling of polyphosphate kinase mutant and glutathione bifunctional enzyme as catalyst to contain 40-60 mM glutamic acid, 40-60 mM glycine, 40-60 mM cysteine, 4-6 mM ATP and 40-60 mM polyP 6 The buffer solution of (2) is a reaction system for catalyzing and producing glutathione; or alternatively, the first and second heat exchangers may be,
the application adopts the thallus lysate of the recombinant bacterium as a catalyst, and comprises 40-60 mM glutamic acid, 40-60 mM glycine, 40-60 mM cysteine, 4-6 mM ATP and 40-60 mM polyP 6 The buffer solution of (2) is a reaction system for catalyzing and producing glutathione.
9. The use according to claim 8, characterized in that glutamic acid, glycine, cysteine and polyP 6 The batch addition is divided into 2 to 4 times, one part is added to the initial culture medium respectively, and one part is added at intervals of 1 to 3 hours.
10. The use according to claim 8, wherein the cell lysate contains an OD of the cell mass 600 10 to 20; the buffer solution is Tris-HCl buffer solution.
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| CN116083394A (en) | 2023-05-09 |
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