CN115247158A - Glycerol phosphate oxidase mutant and screening method, preparation method and application thereof - Google Patents

Glycerol phosphate oxidase mutant and screening method, preparation method and application thereof Download PDF

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CN115247158A
CN115247158A CN202110807042.6A CN202110807042A CN115247158A CN 115247158 A CN115247158 A CN 115247158A CN 202110807042 A CN202110807042 A CN 202110807042A CN 115247158 A CN115247158 A CN 115247158A
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宫安
罗漫杰
钱坤
鄢文琪
陈敏娟
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Abstract

The invention discloses a glycerol phosphate oxidase mutant and a screening method, a preparation method and application thereof. The invention constructs a mutation library of wild glycerophosphate oxidase (G3 PO), performs single-point mutation and combined mutation at more than 50 sites, respectively constructs recombinant expression strains, performs induced expression, analyzes the vitality and thermal stability of the obtained mutation product, and screens 20 kinds of G3PO mutants with obviously improved thermal stability in single-point mutation modes, including but not limited to four single-point mutants of D102N, T90V, A363S and F555E, and also screens 40 kinds of better combined mutants containing the single-point mutation. The invention also discloses a fusion protein, a nucleic acid construct, a recombinant vector, an engineering bacterium of the G3PO mutant and a preparation method thereof. The G3PO mutant screened by the invention has great application potential in the aspect of serum triglyceride content detection.

Description

Glycerol phosphate oxidase mutant and screening method, preparation method and application thereof
Technical Field
The invention relates to the technical field of biology, in particular to a glycerophosphate oxidase mutant and a screening method, a preparation method and application thereof.
Background
Glycerophosphate oxidase (G3 PO, EC 1.1.3.21) is one of the key enzymes for determining triglyceride content by coupled enzyme method, and is widely applied to clinical judgment of heart disease and hyperlipidemia with its special high specificity and high sensitivity.
The study of glycerophosphate oxidase began in the thirties of the twentieth century, and the main study object was a protein derived from Streptococcus faecalis. The current commercial glycerophosphate oxidase is usually obtained from multi-step separation and purification products, and the major production countries comprise Japan, united states, UK and the like. The research on the glycerophosphate oxidase is relatively late in China, related reports are not particularly many, and most of the glycerophosphate oxidase for clinical diagnosis needs to be imported. Meanwhile, with the increasing improvement of the living standard of people, the working pressure is increased day by day, and the idea of paying attention to health is gradually taken as one of the standards for measuring the quality of life, so that the requirement of China on the glycerol phosphate oxidase is on an increasing trend.
The current research on glycerophosphate oxidase has mainly focused on sources such as Pediococcus acililacticii, aerococcus viridans, streptococcus faecalis and Enterococcus SP. The patent document CN110938607A reports that glycerophosphate oxidase derived from Pediococcus acidilactici is subjected to random mutation to improve the thermal stability [1] . A method for preparing phosphoglycerol oxidase by inducing culture medium Streptococcus is reported by Wangteng, etc. and uses the G3PO gene from Streptococcus faecalis to make recombination expression and uses polyethylene to make the recombination expressionObtaining G3PO target protein by two-step chromatography purification and two-aqueous phase extraction of diol-ammonium sulfate [2] . Screening Enterococcus SP. Source glycerophosphate oxidase from 23 strains in Cabernet Sauvignon of Sichuan university, and performing multi-step purification of ammonium sulfate fractional precipitation, DADE-Sepharose FF ion exchange chromatography, phenyl Sepharose CL-4B hydrophobic chromatography, chemical Sepharose FF affinity chromatography and Sephacryls-200HR treatment, wherein the operation is complicated [3] . In addition, with the increasing quality of life of people, health problems are more and more emphasized, and the detection of the content of serum triglyceride is more and more concerned. The reagent for detecting the content of the serum triglyceride relates to an important enzyme, namely glycerophosphate oxidase, so that the enzyme has better market prospect.
Serum triglyceride level measurements have been essentially completely converted from previous chemical methods to current enzymatic methods. The method for detecting triglyceride by the enzyme method is quick and convenient, has high accuracy, can be applied to a biochemical analyzer, and can also be used for large-batch detection. The detection principle of the enzyme detection is as follows: hydrolyzing a sample to be detected under the action of Lipoprotein esterase (lipoproteinase) to generate Glycerol (Glycerol) and Fatty acid (Fatty acid), generating Glycerol-3-phosphate (Glycerol-3-P) by the Glycerol and ATP under the action of Glycerol kinase (Glycerol kinase), and generating Dihydroxyacetone phosphate (Dihydroxyacetone-P) and hydrogen peroxide (H) by the Glycerol-3-phosphate under the action of Glycerol phosphate oxidase (L-alpha-glycerophosphooxidase) 2 O 2 ) The hydrogen peroxide can generate a chromogenic substance quinonimine (Quinoneimine dye) under the action of 4-Aminoantipyrine (4-AA), 2-hydroxy-3-m-toluidine sodium propanesulfonate (TOOS) and horse radish Peroxidase (Peroxidase), and the content of triglyceride can be reflected according to the absorption change of the quinonimine under visible light, so that the detection purpose is achieved. The specific reaction principle is as follows:
Figure BDA0003166986700000021
Figure BDA0003166986700000022
Figure BDA0003166986700000023
Figure BDA0003166986700000024
most of the glycerol phosphate oxidase is obtained by fermentation culture of natural microorganisms, the yield is low, the purification difficulty is high, the research and development and popularization of TG reagent detection by an enzyme method are limited, and meanwhile, the stability of the glycerol phosphate oxidase can greatly influence the detection accuracy of the reagent.
At present, the stability of the wild type of the glycerol phosphate oxidase (G3 PO) from the aerococcus viridis is deficient, and a glycerol phosphate oxidase mutant with improved stability needs to be developed, so that the application stability of the glycerol phosphate oxidase mutant is improved.
[1] Zhoushuang, zhoushanhua, zhangyusheng, wanyi, shuaishui, zhang Yong, glycerol-3-phosphate oxidase with good thermal stability and its application in reagent kit, CN 110938607A.
[2] Wang, menyangming, a method for preparing phosphoglycerol oxidase by using induction medium streptococcus, CN101070529A.
[3] A recombinant glycerophosphate oxidase expression vector and its construction method, CN 110184289A.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, one of the technical problems to be solved by the present invention is to develop a glycerophosphate oxidase (G3 PO) mutant with improved stability, thereby improving the application stability.
The invention provides a glycerol phosphate oxidase mutant (G3 PO mutant) which is subjected to site-directed mutagenesis based on the amino acid sequence of a wild-type glycerol phosphate oxidase, wherein the amino acid sequence of the wild-type glycerol phosphate oxidase (wild-type G3 PO) is shown as SEQ ID No. 4, and the mutagenesis mode is selected from the following modes: D102N, T90V, A363S, F555E, F6L, G29P, G399K, G399E, A376K, G399P, H92Q, A341T, E204D, V209N, S218T, L131V, V198A, T221S, H227R, V228R, E392K, T370S, I179S, S501A, K113G, Q474F, V492T, S82A, Q464N and combinations comprising the foregoing.
In some preferred embodiments of the present invention, the combinatorial mutation mode is selected from the group consisting of at least two of D102N, T90V, a363S, F555E, F6L, G29P, G399K, G399E, a376K, G399P, H92Q, a341T, E204D, V209N, S218T, L131V, V198A, T221S, H227R, V228R, E392K, T370S, I179S, S501A, K113G, Q474F, V492T, S82A, Q464N.
In some preferred embodiments of the invention, the mutation pattern is selected from: D102N, T90V, A363S, F555E and a combination of the foregoing mutations.
In some preferred embodiments of the present invention, the combination mutation is selected from any one of the following combinations:
L4F + E574K, D102N + T90V + E574K, T370S + F555E + E574K, and the like;
D102N+T90V+A363S+Q464N+F555E+E574K，
T90V+A363S+F555E+I179S+V198A，
D102N+T90V+Q464N+V492T+F555E+E574K，
D102N+T90V+A363S+F555E+F6L，
D102N+T90V+A376K+G399K+S501A+F555E，D102N+T90V+E574K，
D102N+T90V+A341T+T370S+Q474F+E574K，D102N+T90V+A363S+F555E，
F6L + G29P + A341T + A363S + F555E + E574K and the like;
D102N+T90V+G399K+S501A+Q464N，T90V+L131V+A341T+Q464N，
T90V+F555E+A376K+G399E+Q474F，K113G+I179S+S218T+Q474F+V492T，
V198A+E204D+Q464N+S501A，T90V+A363S+Q464N，
T90V+A363S+Q474F+F555E，S82A+A363S+A341T+T370S+Q464N，
S82A + Q464N, D102N + T90V + Q464N, and so on.
The second aspect of the invention provides a fusion protein of a glycerol phosphate oxidase mutant, wherein the fusion protein of the glycerol phosphate oxidase mutant is connected with a fusion tag at the N end and/or the C end of the glycerol phosphate oxidase mutant in the first aspect.
In some preferred embodiments of the invention, the fusion tag comprises an affinity tag.
In some preferred embodiments of the invention, the affinity tag is selected from the group consisting of: his-tag, GST, flag-tag, MBP, and combinations thereof.
In a third aspect, the present invention provides a nucleic acid construct having a nucleotide sequence selected from any one of:
(i) A nucleotide sequence encoding a glycerophosphate oxidase mutant according to the first aspect or a fusion protein according to the second aspect; and
(ii) (ii) a nucleotide sequence complementary to the nucleotide sequence in (i).
The nucleic acid construct may be DNA, mRNA, or a combination thereof.
In a fourth aspect, the invention provides a recombinant vector comprising a nucleic acid construct according to the third aspect.
In some preferred embodiments of the invention, the recombinant vector is an expression plasmid comprising the nucleic acid construct of the third aspect.
In some preferred embodiments of the invention, the type of the recombinant vector is selected from the group consisting of: pET28a, pET 30a, pANY1, pQE30, pG-KJE8, pGRO7, pKJE7, pG-Tf2, pTf16, pUC18, pUC19 and pGEX.
In the fifth aspect, the invention provides an engineered bacterium, the genome of which is integrated with the nucleic acid construct described in the third aspect, or the engineered bacterium contains the recombinant vector described in the fourth aspect.
In some preferred embodiments of the invention, the engineered bacteria are derived from escherichia coli.
In a sixth aspect, the invention provides a genetically engineered cell having integrated into its genome the nucleic acid construct of the third aspect, or comprising the recombinant vector of the fourth aspect.
In some preferred embodiments of the invention, the genetically engineered cell is derived from Escherichia coli.
The seventh aspect of the invention provides a method for screening a glycerophosphate oxidase mutant from a mutation library, which comprises the following steps:
(1) Constructing a mutation library of the wild type G3PO as shown in SEQ ID No. 4, wherein mutation sites comprise: 4.6, 29, 55, 78, 82, 90, 92, 100, 102, 113, 119, 123, 127, 131, 147, 179, 196, 198, 204, 209, 218, 221, 227, 228, 261, 269, 341, 363, 370, 376, 392, 399, 452, 460, 463, 464, 466, 470, 474, 492, 501, 531, 547, 555, 560, 572, 574, and 584, to give a library of mutations consisting of G3PO mutants; the mutation mode comprises a single-point mutation mode and a combined mutation mode;
(2) Obtaining the coding sequence of each G3PO mutant in the mutation library by taking the gene of the wild G3PO as a template;
(3) Respectively constructing a recombinant vector (such as the recombinant vector described in the fourth aspect) for each G3PO mutant in the mutation library, transforming the recombinant vector into a host bacterium to obtain a recombinant expression strain, inducing the recombinant expression strain, and expressing the G3PO mutant or the fusion protein thereof (corresponding to the G3PO mutant described in the first aspect or the fusion protein described in the second aspect) to obtain the mutation library; when the coding sequence of the G3PO mutant in the recombinant vector is connected with a fusion tag (such as an affinity tag), expressing the fusion protein of the G3PO mutant of the mutation library;
(4) And (3) analyzing the protein specific activity and stability of the G3PO mutant or the fusion protein thereof, and screening to obtain the G3PO mutant of the first aspect or the fusion protein of the second aspect.
The eighth aspect of the invention provides a preparation method of a glycerol phosphate oxidase mutant, which comprises the following steps:
(1) Cloning the nucleic acid construct of the third aspect into an expression vector to obtain a recombinant vector containing the coding sequence of the G3PO mutant or the fusion protein thereof;
(2) Transforming the recombinant vector into host bacteria to obtain a recombinant expression strain containing the coding sequence of the G3PO mutant or the fusion protein thereof;
(3) Inducing the recombinant expression strain to express the G3PO mutant or the fusion protein thereof (corresponding to the G3PO mutant of the first aspect or the fusion protein of the second aspect) under the appropriate expression condition.
In some preferred embodiments of the invention, the recombinant expression strain is escherichia coli, and in this case, the suitable expression conditions include: 0.1mM isopropyl-. Beta. -D-thiogalactoside (IPTG), 0.1wt% of an induction precursor, at 25 ℃ for 16 hours. The inducible precursor substance may be Flavin Adenine Dinucleotide (FAD), a riboflavin analog, or a combination thereof.
The ninth aspect of the invention provides the glycerophosphate oxidase mutant of the first aspect, the fusion protein of the second aspect, the nucleic acid construct of the third aspect, the recombinant vector of the fourth aspect, the engineered bacterium of the fifth aspect, and the genetically engineered cell of the sixth aspect for use in detecting serum triglyceride content.
The technical scheme disclosed by the invention has the beneficial effects that:
1. the invention constructs a stable mutation library of wild type glycerophosphate oxidase, clones G3PO mutant genes to an expression vector, then transforms the genes to host bacteria to construct corresponding single-site mutant strains, after induced expression, carries out stability analysis on the expression products, screens and obtains 20 kinds of G3PO mutants with obviously improved thermal stability (including but not limited to four single-site mutants of D102N, T90V, A363S and F555E), screens and obtains 40 kinds of better combined mutants containing the single-site mutations (such as six-site combined mutations of D102N, T90V, A363S, Q464N, F555E and E574K), and can be used for catalyzing glycerol-3-phosphoric acid reaction to generate dihydroxyacetone phosphate.
2. The invention discloses more than 20G 3PO single-point mutants with thermal stability improved by at least 40%, and the quantitative analysis of the activity is based on the specific activity of protein or the residual activity percentage of 20min after incubation at 46 ℃. Wherein, the protein specific activity of the F6L, G29P and G399K mutants is slightly improved compared with that of wild G3PO, and the thermal stability is improved by 0.5 to 1.6 times; the specific protein activities of the D102N, T90V, A363S and F555E mutants can reach more than 90% of that of wild G3PO, and the thermal stability is improved by more than 2 times; the specific protein activities of G399E, A376K, G399P, H92Q, A341T, E204D and V209N mutants can reach more than 90 percent of that of wild type G3PO, and the heat stability is improved by 0.8 to 2.4 times; compared with wild type G3PO, the heat stability of the S501A, K113G, Q474F, V492T, S82A and Q464N mutants is improved by 1.5-3.1 times, and the specific activity of the protein reaches 21-60% of that of the wild type G3 PO.
3. By comprehensively considering the activity and stability of the G3PO mutant, the invention obtains the G3PO mutant with obviously improved comprehensive performance in a single-point mutation mode, including the G3PO mutants in D102N, T90V, A363S and F555E single-point mutation modes.
4. The invention also discloses a combined mutant which is obviously improved in stability on the basis of keeping a certain activity and is combined in the following single-point mutation mode: L4F, E574K, D102N, T90V, E574K, T370S, F555E, A363S, V492T, G399P, E204D, A376A, G29P, etc.
5. In addition, the invention specifically discloses more than 40G 3PO mutants with the thermal stability improved by at least over 60 percent in a combined mutation mode, and the quantitative analysis of the activity is based on the specific activity of the protein or the percentage of the residual activity after incubation for 20min at 48 ℃. The specific activity of the L4F + E574K, D102N + T90V + E574K and T370S + F555E + E574K proteins is improved by more than 10%, and the stability is improved by more than 80%; mutants such as D102N + T90V + A363S + Q464N + F555E + E574K, T90V + A363S + F555E + I179S + V198A, D102N + T90V + Q464N + V492T + F555E + E574K, D102N + T90V + A363S + F555E + F6L, D102N + T90V + A376K + G399K + S501A + F555E, D102N + T90V + E574K, D102N + T90V + A341T + T370S + Q474F + E574K, D102N + T90V + A363S + F555E, F6L + G29P + A341T + A363S + F555E + E574K and the like have the stability improved by more than 400% and the protein activity is basically maintained without being reduced by the ratio; D102N + T90V + G399K + S501A + Q464N, T90V + L131V + A341T + Q464N, T90V + F555E + A376K + G399E + Q474F, K113G + I179S + S218T + Q474F + V492T, V198A + E204D + Q464N + S501A, T90V + A363S + Q464N, T90V + A363S + Q474F + F555E, S82A + A363S + A341T + T370S + Q464N, S82A + Q464N, D102N + T90V + Q464N and the like mutants have the stability improved by more than 400 percent, and the protein specific activity is reduced to 30 to 80 percent of the wild type.
6. Comprehensively considering the activity and stability of the G3PO mutant, the combined mutant with extremely remarkable comprehensive performance improvement is obtained, and comprises a D102N + T90V + A363S + Q464N + F555E + E574K G3PO six-point combined mutant, the specific activity of the protein is improved to 109.3% of that of wild type G3PO, and the half-life period at 48 ℃ is improved to 822.9% of that of the wild type G3PO (namely, the half-life period is improved by more than 7 times).
7. The invention also provides a method for preparing and purifying the glycerophosphate oxidase recombined and heterogeneously expressed in escherichia coli cells and a mutant thereof in batches, and a G3PO mutant with better thermal stability, in particular a G3PO mutant with good thermal stability and high enzyme activity, is screened by combining a stability mutation library.
The advantages enable the glycerophosphate oxidase mutant of the invention to have great application potential in the aspect of serum triglyceride content detection.
The conception, the specific technical solutions and the technical effects produced by the present invention will be further described in the following with reference to the accompanying drawings so as to fully understand the objects, the features and the effects of the present invention.
Drawings
Other features, objects and advantages of the invention will be made apparent by reading the following detailed description of non-limiting embodiments with reference to the accompanying drawings in which:
FIG. 1 shows a comparison of properties of wild-type G3PO and G3PO mutants, including an analytical comparison of protein specific activity (U/mg), half-life (min) at 46 ℃, percent residual activity after incubation for 20min at 46 ℃, the comparison data being shown in a normalized manner with respect to wild-type G3 PO; wherein WT represents a wild type;
FIG. 2 shows a comparison of properties of wild-type G3PO and G3PO combination mutants, including an analytical comparison of protein specific activity (U/mg), half-life (min) at 48 ℃, and percent residual activity after incubation for 20min at 48 ℃, the comparison data being shown in a normalized manner based on wild-type G3 PO; wherein WT represents a wild type;
FIG. 3 shows SDS-PAGE of samples from purification of recombinantly expressed G3PO mutants, exemplified by T90V; wherein M is Marker; lanes 1 to 9 correspond to the following samples, respectively: lane 1, break supernatant; lane 2, pellet after disruption; lane 3, flow through; lane 4, edulcoration; lane 5, 200mM elution; lane 6, 400mM elution; lane 7, dialysate (5 and 6 mixed); lane 8, 500mM column 1; lane 9, 500mM column 2.
Detailed Description
The following detailed description of the present invention will be provided in conjunction with the accompanying drawings.
The technical contents of the preferred embodiments and preferred embodiments of the present invention will be more clearly understood and appreciated by referring to the drawings attached to the specification. The invention may be embodied in many different forms of embodiments and examples, and the scope of the invention is not limited to only the embodiments and examples described herein.
G3PO: glycerophosphate oxidase, EC 1.1.3.21.
G3PO mutants: glycerol phosphate oxidase mutant.
TABLE 1 abbreviations, chinese and English full names of partial terms
Figure BDA0003166986700000071
TABLE 2 Chinese and foreign translation control of partial terms
Figure BDA0003166986700000072
Figure BDA0003166986700000081
In the present invention, the G3PO mutant, i.e., the glycerol phosphate oxidase mutant, is a mutant obtained by genetic engineering techniques based on wild-type G3PO, and refers to a mutant obtained by amino acid substitution, without any particular limitation.
In the present invention, the fusion protein of the G3PO mutant refers to a protein in which a fusion tag is further linked to the N-terminus and/or C-terminus of the G3PO mutant and which still exhibits the G3PO activity. From the aspect of label size, the fusion protein can be a peptide label and also can be a protein label. Functionally, the fusion tag can be at least one of an affinity tag (useful for protein purification), a fluorescent tag (useful for fluorescent labeling), and the like.
In the present invention, the words "preferred", "particularly", "specific" and the like merely indicate preferred or more specific embodiments or examples, and it should be understood that the scope of the present invention is not limited thereto.
In the present invention, "and/or" means any one or any combination of the listed items.
In the present invention, "any combination", and "any combination" of the listed related items are intended to mean any suitable combination for successfully implementing the present invention. The number of related items constituting the combination is at least 2.
In the present invention, "combinatorial mutation" refers to a mutation mode in which at least 2 sites are mutated. The mutants corresponding to the "combinatorial mutations" are designated as "combinatorial mutants". Examples of the combination mutation mode include two-point, three-point, four-point, five-point, six-point, and the like. The combination of mutation patterns may be represented by "+" indicating a combination of mutation patterns of the relevant mutation sites. For example, "L4F + E574K" represents a two-point mutation in which the L → F mutation at position 4 and the E → K mutation at position 574 are combined.
The percent concentrations referred to in the present invention, unless otherwise specified or stated, represent the final concentrations in the relevant system.
In the present invention, wt% means mass percentage concentration.
The technical features mentioned above and below can be combined in any suitable way as long as there is no contradiction, and the combined technical solution can be implemented smoothly and can solve the technical problems of the present invention. The technical problem of the invention can be solved and the corresponding technical effect can be realized in any suitable way.
In a first aspect, the present invention provides a mutant glycerophosphate oxidase, which is site-directed mutated based on the amino acid sequence of a wild-type glycerophosphate oxidase, wherein the amino acid sequence of the wild-type glycerophosphate oxidase is shown in SEQ ID No. 4, and in some preferred embodiments of the present invention, the mutation mode is selected from: D102N, T90V, A363S, F555E, F6L, G29P, G399K, G399E, A376K, G399P, H92Q, A341T, E204D, V209N, S218T, L131V, V198A, T221S, H227R, V228R, E392K, T370S, I179S, S501A, K113G, Q474F, V492T, S82A, Q464N and combinations comprising the foregoing mutations.
The wild-type glycerophosphate oxidase is derived from Aerococcus viridans.
In some preferred embodiments of the present invention, the combinatorial mutation is selected from the group consisting of at least two mutations selected from D102N, T90V, a363S, F555E, F6L, G29P, G399K, G399E, a376K, G399P, H92Q, a341T, E204D, V209N, S218T, L131V, V198A, T221S, H227R, V228R, E392K, T370S, I179S, S501A, K113G, Q474F, V492T, S82A, and Q464N.
In some preferred embodiments of the invention, the mutation pattern is selected from: D102N, T90V, A363S, F555E and a combination of the foregoing mutations.
In some preferred embodiments of the present invention, the combination mutation is selected from any one of the following combinations:
L4F + E574K, D102N + T90V + E574K, T370S + F555E + E574K and the like;
D102N+T90V+A363S+Q464N+F555E+E574K，
T90V+A363S+F555E+I179S+V198A，
D102N+T90V+Q464N+V492T+F555E+E574K，
D102N+T90V+A363S+F555E+F6L，
D102N+T90V+A376K+G399K+S501A+F555E，D102N+T90V+E574K，
D102N+T90V+A341T+T370S+Q474F+E574K，D102N+T90V+A363S+F555E，
F6L + G29P + A341T + A363S + F555E + E574K and the like;
D102N+T90V+G399K+S501A+Q464N，T90V+L131V+A341T+Q464N，
T90V+F555E+A376K+G399E+Q474F，K113G+I179S+S218T+Q474F+V492T，
V198A+E204D+Q464N+S501A，T90V+A363S+Q464N，
T90V+A363S+Q474F+F555E，S82A+A363S+A341T+T370S+Q464N，
S82A + Q464N, D102N + T90V + Q464N, and the like.
The amino acid sequence of the mutant corresponding to each mutation mode of G3PO can be directly deduced according to the amino acid sequence of wild type G3PO (SEQ ID No.: 4) in combination with the corresponding mutation mode. For example, the amino acid sequence of the D102N mutant is shown as SEQ ID No.:1, the amino acid sequence of the T90V mutant is shown as SEQ ID No.:2, and the amino acid sequence of the D102N + T90V + A363S + Q464N + F555E + E574K six-point combination mutant is shown as SEQ ID No.: 3.
In a preferred embodiment of the invention, the protein specific activity (U/mg), half-life (min) at 46 ℃ and 20min after incubation at 46 ℃ as a percentage of the residual activity of the D102N mutant are 97.35%, 343.92% and 383.41% of wild-type G3PO, respectively.
In some preferred embodiments of the invention, the protein specific activities (U/mg) of the T90V, A363S and F555E mutants are 92.29-93.25% of the wild-type G3PO, the half-life (min) at 46 ℃ is 271.96-321.69% of the wild-type G3PO, and the residual activity percentage after 20min incubation at 46 ℃ is 343.78-378.80% of the wild-type G3 PO.
In some preferred embodiments of the invention, the protein specific activity (U/mg) of the F6L, G29P and G399K mutants is 101.45-104.82% of wild-type G3PO, the half-life (min) at 46 ℃ is 116.40-182.01% of wild-type G3PO, and the residual activity percentage after 20min incubation at 46 ℃ is 153.46-256.22% of wild-type G3 PO.
In some preferred embodiments of the invention, the protein specific activity (U/mg) of the G399E, a376K, G399P, H92Q, a341T, E204D and V209N mutants is 90.60% to 99.76% of the wild-type G3PO, the half-life (min) at 46 ℃ is 145.50% to 210.58% of the wild-type G3PO, and the percentage residual activity after 20min incubation at 46 ℃ is 144.24% to 258.99% of the wild-type G3 PO.
In some preferred embodiments of the invention, the protein specific activity (U/mg) of the S218T, L131V, V198A, T221S, H227R, V228R, E392K, T370S and I179S mutants is 68.67-88.67% of wild-type G3PO, the half-life (min) at 46 ℃ is 124.34-291.53% of wild-type G3PO, and the percentage of residual activity after 20min incubation at 46 ℃ is 183.87-337.33% of wild-type G3 PO.
In some preferred embodiments of the invention, the protein specific activity (U/mg) of the S501A, K113G, Q474F, V492T, S82A and Q464N mutants is 21.20-59.76% of wild-type G3PO, the half-life (min) at 46 ℃ is 242.3-451.85% of wild-type G3PO, and the residual activity percentage after 20min incubation at 46 ℃ is 247.93-413.82% of wild-type G3 PO.
In some preferred embodiments of the invention, the protein specific activity of some combination mutants (e.g., L4F + E574K, D102N + T90V + E574K and T370S + F555E + E574K) is improved by more than 10% and the stability is improved by more than 80% compared to wild-type G3 PO.
In some preferred embodiments of the invention, the stability of some of the combined mutants (e.g., D102N + T90V + a363S + Q464N + F555E + E574K, T90V + a363S + F555E + I179S + V198A, D102N + T90V + Q464N + V492T + F555E + E574K, D102N + T90V + a363S + F555E + F6L, D102N + T90V + a376K + G399K + S501A + F555E, D102N + T90V + E574K, D102N + T90V + a341T + T370S + Q474F + E574K, D102N + T90V + a363S + F555E, F6L + G29P + a341T + a363S + F555E + E) is increased by more than 400% compared to the wild-type G3PO while maintaining the protein at least 90% less than the activity of the wild-type G3PO protein.
In some preferred embodiments of the invention, the stability of some combinatorial mutants (e.g., D102N + T90V + G399K + S501A + Q464N, T90V + L131V + a341T + Q464N, T90V + F555E + a376K + G399E + Q474F, K113G + I179S + S218T + Q474F + V492T, V198A + E204D + Q464N + S501A, T90V + a363S + Q464N, T90V + a363S + Q474F + F555E, S82A + a363S + a341T + T370S + Q464N, S82A + Q464N, D102N + T90V + Q464N, etc.) is improved by more than 400% compared to the wild-type G3PO, and the protein specific activity is 30% -80% of the wild type.
In some preferred embodiments of the invention, the protein specific activity of the combination mutants (e.g., G29P + a341T, T370S + F555E + E574K, F6L + G29P + a341T + a363S + F555E + E574K, L4F + E574K, D102N + T90V + a363S + Q464N + F555E + E574K, T90V + a363S + E574K, and D102N + T90V + Q464N + V492T + F555E + E574K) is improved to 106.0% to 127.6% and the thermal stability is improved by more than 1.8 times compared to the wild-type G3 PO.
In some preferred embodiments of the invention, the half-life of the combination mutants (e.g., D102N + T90V + a363S + Q464N + F555E + E574K, T90V + a363S + E574K, D102N + T90V + Q464N + V492T + F555E + E574K, D102N + T90V + a341T + T370S + Q474F + E574K, a363S + T370S + E574K, F6L + G29P + a341T + a363S + F555E + E574K) at 48 ℃ is increased to 386.7% to 822.9% compared to the wild type and the protein activity is more than 100% compared to the wild type G3 PO; some combinatorial mutants (such as D102N + T90V + a376K + G399K + S501A + F555E, T90V + a363S + F555E + I179S + V198A, T90V + F555E + G399P + Q464N + S501A + E574K, D102N + T90V + a363S + F555E, D102N + F6L + G399K + S501A) have half-lives at 48 ℃ that are increased to 422.7% -695.2% compared to the wild type, and protein specific activities are 90% -100% of the wild type; some combined mutants (such as T90V + F555E + A376K + G399E + Q474F, T90V + T221S + G399E + I179S, D102N + H92Q + H227R + K113G, E574K + Q464N, D102N + T90V + V492T, G29P + I179S + V198A + V228R + A363S) have half-life period increased to 343.4% -580.7% at 48 ℃ compared with wild type, and protein specific activity is 80% -90% of wild type; the half life of some combined mutants (such as D102N + T90V + G399K + S501A + Q464N, T90V + A363S + Q474F + F555E, D102N + T90V + Q464N) at 48 ℃ is improved to 434.9% -627.7% compared with the wild type, and the specific activity of the protein is 70% -80% of that of the wild type; the half-life of some combined mutants (such as S82A + A363S + A341T + T370S + Q464N, T90V + Q474F, D102N + V492T) at 48 ℃ is improved to 374.7% -518.1% compared with the wild type, and the protein specific activity is 60% -70%; the half-life of some combined mutants (such as T90V + L131V + A341T + Q464N, T90V + A363S + Q464N, D102N + Q464N, S501A + F555E, A363S + Q464N) at 48 ℃ is improved to 386.7% -562.7% compared with the wild type, and the specific activity of the protein is 50% -60% of that of the wild type; some combination mutants (such as K113G + I179S + S218T + Q474F + V492T, S82A + Q464N) have half-lives at 48 ℃ increased to 434.9.7% -467.5% compared to the wild type, but protein specific activities were less than 50% of the wild type.
In some preferred embodiments of the present invention, the stability of some single-point or combination mutants such as D102N, T90V, etc. is improved significantly, while the activity is not affected much, and the specific activity of the protein can be kept substantially unchanged or changed to a smaller extent.
In some preferred embodiments of the invention, the protein specific activity, half-life at 48 ℃ and residual activity after 20min incubation of some combination mutants are all greatly improved relative to wild-type G3 PO. For example, the protein specific activity of the D102N + T90V + A363S + Q464N + F555E + E574K six-point combined mutant is increased to 109.3% compared with the wild type, the half-life period at 48 ℃ is increased to 822.9% of the wild type, and the residual activity after incubation for 20min is also greatly increased compared with the wild type.
It should be noted that the effect of the reduced specific activity of the protein on the application of the G3PO mutant can be overcome or alleviated by increasing the enzyme dosage, and therefore, even if the specific activity of the protein is reduced to some extent under the condition of significantly improved stability, the invention is also within the protection scope of the present invention. For example, the acceptable degree of change in specific activity of the protein is 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 100% or more of the wild-type G3 PO. The term "above" as used herein includes the present numbers. In some preferred embodiments of the invention, the protein specific activity of the preferred G3PO mutant is at least 50% of that of the wild type.
In a second aspect, the present invention provides a fusion protein of a glycerol phosphate oxidase mutant (G3 PO mutant), which is a fusion protein of the G3PO mutant of the first aspect. In some preferred embodiments of the invention, the fusion protein comprises a fusion tag attached to the N-terminus and/or C-terminus of the mutant glycerophosphate oxidase of the first aspect.
In some preferred embodiments of the invention, the fusion tag comprises at least one of an affinity tag, a fluorescent tag, and the like.
In some preferred embodiments of the invention, the affinity tag is selected from the group consisting of: his-tag, GST, flag-tag, MBP, and combinations thereof, and the selection of affinity tags can also be referenced to Protein Expression and Purification,41 (2005) 98-105, including but not limited to resin types and elution reagents.
In a third aspect, the present invention provides a nucleic acid construct having a nucleotide sequence selected from any one of:
(i) A nucleotide sequence encoding a glycerophosphate oxidase mutant of the first aspect (G3 PO mutant) or a fusion protein of the second aspect; and
(ii) (ii) a nucleotide sequence complementary to the nucleotide sequence described in (i).
The nucleic acid construct may be DNA, mRNA, or a combination thereof.
A nucleic acid construct encoding the G3PO mutant can be obtained using the gene of wild-type G3PO as a template.
The coding sequence for the G3PO mutant of the first aspect, and the coding sequence for the fusion protein of the second aspect, are within the scope of the nucleic acid construct of the third aspect of the invention.
In a fourth aspect, the invention provides a recombinant vector comprising a nucleic acid construct according to the third aspect.
In some preferred embodiments of the invention, the nucleic acid construct of the third aspect is cloned into an expression vector to obtain a recombinant vector comprising the coding sequence of the G3PO mutant or fusion protein thereof.
In some preferred embodiments of the invention, the nucleic acid construct encoding the G3PO mutant is cloned into an expression vector to provide a recombinant vector containing the coding sequence of the G3PO mutant.
In some preferred embodiments of the invention, an affinity tag is also added to the 5 'end or 3' end of the G3PO mutant gene on a plasmid containing the coding sequence of the G3PO mutant. The affinity tag can be one of His-tag, GST, flag-tag, MBP and the like or a combination thereof.
In the present invention, various vectors known in the art, such as commercially available vectors, including plasmids, cosmids, and the like, can be used. In producing the G3PO mutant of the present invention, the coding sequence of the G3PO mutant can be operably linked to expression regulatory sequences to form an expression vector for the G3PO mutant. The expression control sequences include, but are not limited to, transcription enhancing elements, translation enhancing elements, and the like.
In some preferred embodiments of the invention, the recombinant vector is an expression plasmid comprising the nucleic acid construct of the third aspect.
In some preferred embodiments of the invention, the type of the recombinant vector is selected from the group consisting of: pET28a, pET 30a, pANY1, pQE30, pG-KJE8, pGRO7, pKJE7, pG-Tf2, pTf16, pUC18, pUC19 and pGEX, as referred to in the relevant literature Biotechnol Lett (2008) 30; appl Environ Microb (2002) 68 (1): 263-270, available from the Biovector Chinese plasmid vector strain Gene Collection. These are known plasmid vectors in the art and can be known, understood and used by those of ordinary skill in the art.
In the fifth aspect, the invention provides an engineered bacterium, the genome of which is integrated with the nucleic acid construct described in the third aspect, or the engineered bacterium contains the recombinant vector described in the fourth aspect.
The recombinant vector of the fourth aspect may be used in free form or integrated into the genome.
In a sixth aspect, the invention provides a genetically engineered cell having integrated into its genome the nucleic acid construct of the third aspect, or comprising the recombinant vector of the fourth aspect. In some preferred embodiments of the invention, the genetically engineered cell is derived from Escherichia coli.
The recombinant vector of the fourth aspect may be used in free form or integrated into the genome.
In some preferred embodiments of the invention, the engineered bacteria are derived from prokaryotes.
In some preferred embodiments of the invention, the engineered bacterium is derived from Escherichia coli.
In some preferred embodiments of the present invention, the engineered bacterium is derived from escherichia coli BL21 (DE 3).
In some preferred embodiments of the present invention, the recombinant vector of the fourth aspect is transformed into a host bacterium, and the resulting recombinant expression strain is capable of expressing and synthesizing the G3PO mutant of the first aspect or the fusion protein of the second aspect.
In some preferred embodiments of the present invention, a recombinant expression strain capable of expressing the gene of the G3PO mutant can be obtained by transforming a host bacterium with a recombinant vector containing the coding sequence of the G3PO mutant. The recombinant expression strain contains a nucleic acid construct for coding the G3PO mutant and the recombinant vector.
The seventh aspect of the invention provides a method for screening a glycerophosphate oxidase mutant from a mutation library, which comprises the following steps:
(1) Constructing a mutation library of wild type G3PO as shown in SEQ ID No. 4, designing more than 50 mutants based on rationality and semi-rationality on the basis of the wild type, wherein mutation sites comprise: 4.6, 29, 55, 78, 82, 90, 92, 100, 102, 113, 119, 123, 127, 131, 147, 179, 196, 198, 204, 209, 218, 221, 227, 228, 261, 269, 341, 363, 370, 376, 392, 399, 452, 460, 463, 464, 466, 470, 474, 492, 501, 531, 547, 555, 560, 572, 574, and 584, to give a library of mutations consisting of G3PO mutants; the mutation mode comprises a single-point mutation mode and a combined mutation mode;
(2) Obtaining the coding sequence of each G3PO mutant in the mutation library by taking the gene of wild G3PO as a template;
(3) Respectively constructing a recombinant vector for each G3PO mutant in the mutation library, transforming the recombinant vector into host bacteria to obtain a recombinant expression strain, inducing the recombinant expression strain, and expressing to obtain the G3PO mutant of the mutation library or a fusion protein thereof (corresponding to the G3PO mutant of the first aspect or the fusion protein of the second aspect); when the coding sequence of the G3PO mutant in the recombinant vector is connected with a fusion tag (such as an affinity tag), expressing the fusion protein of the G3PO mutant of the mutation library;
(4) And (3) analyzing the protein specific activity and stability of the G3PO mutant or the fusion protein thereof, and screening to obtain the G3PO mutant of the first aspect or the fusion protein of the second aspect.
The eighth aspect of the invention provides a preparation method of a glycerol phosphate oxidase mutant (G3 PO mutant), which comprises the following steps:
(1) Cloning the nucleic acid construct of the third aspect into an expression vector to obtain a recombinant vector containing the coding sequence of the G3PO mutant or the fusion protein thereof;
(2) Transforming the recombinant vector into host bacteria to obtain a recombinant expression strain containing the coding sequence of the G3PO mutant or the fusion protein thereof;
(3) Inducing the recombinant expression strain to express under a suitable expression condition to obtain the G3PO mutant or the fusion protein thereof (corresponding to the G3PO mutant of the first aspect or the fusion protein of the second aspect). In some preferred embodiments of the present invention, the preparation method of G3PO mutant further comprises a separation and purification step after the step (3) to prepare a purified G3PO mutant. The separation and purification can be performed by methods known in the art for separating and purifying proteins from host bacteria, including but not limited to centrifugation, column chromatography, membrane filtration, ultrafiltration, and the like.
The preparation method can be used for batch preparation and purification.
In some preferred embodiments of the present invention, the host bacterium is escherichia coli, and in this case, the recombinant expression strain obtained in step (2) is a recombinant expression strain of escherichia coli.
In some preferred embodiments of the invention, suitable expression conditions for recombinant expression strains of E.coli include: 0.1mM IPTG, 0.1% mass concentration of an induction precursor substance, and inducing at 25 ℃ for 16 hours. Further, in some preferred embodiments of the present invention, the inducible precursor substance may be FAD, riboflavin analogs, or combinations thereof. In some preferred embodiments of the invention, examples of the riboflavin analogs include, but are not limited to: riboflavin sodium phosphate, riboflavin potassium phosphate, riboflavin iodine salt, and the like.
In some preferred embodiments of the invention, an affinity tag may be added to the 5 'end or 3' end of the G3PO mutant gene on a plasmid containing the coding sequence of the G3PO mutant. The affinity tag can be one of His-tag, GST, flag-tag, MBP and the like or a combination thereof. After the connection is correct, the recombinant expression strain (which is the engineering bacterium of the fifth aspect) is obtained by transforming into a host bacterium, and the G3PO mutant of the second aspect can be obtained by expression. The expression product can be purified by affinity chromatography column using affinity tag. In some preferred embodiments of the invention, the G3PO mutant can be purified to a purity of greater than 90% using simple affinity chromatography.
The principle of activity detection of the glycerol phosphate oxidase mutant product is as follows:
Figure BDA0003166986700000151
Figure BDA0003166986700000152
the amount of quinoneimine produced by the reaction can be detected by a spectrophotometer at a wavelength of 555 nm.
The enzyme activity of the glycerophosphate oxidase and the mutant thereof is defined as follows: the unit enzyme activity is defined as the amount of enzyme required to consume 1. Mu. Mol of glycerol-3-phosphate per minute under certain conditions.
The ninth aspect of the present invention provides the use of the mutant glycerophosphate oxidase of the first aspect, the fusion protein of the second aspect, the nucleic acid construct of the third aspect, the recombinant vector of the fourth aspect, the engineered bacterium of the fifth aspect, and the genetically engineered cell of the sixth aspect in the detection of serum triglyceride content.
In the present invention, the serum triglyceride content can be detected by a detection method known to those skilled in the art, for example, the detection method disclosed in patent document CN 107991477A can be referred to.
The following provides a detailed description of embodiments of the invention. The embodiments of the present invention are implemented on the premise of the technical solution of the present invention, and detailed embodiments and specific operation procedures are given, but the scope of the present invention is not limited to the following embodiments. Experimental methods (such as PCR amplification, chemical conversion method, etc.) without specifying specific conditions in the following examples are preferably performed according to the methods and conditions of the detailed description; the molecular cloning is then generally carried out according to conventional methods and conditions, for example Sambrook et al: methods and conditions as described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.
Example 1: glycerol phosphate oxidase stability mutation library construction
The molecular directed evolution is to create a large number of mutant homologous gene libraries by using modern molecular biology methods to artificially simulate the natural evolution mechanism, and to create mutant proteins or other biomolecules which do not exist in the nature or have obviously changed certain characteristics by using a sensitive directed screening strategy. Directed molecular evolution has been widely applied to molecular engineering of proteins and is considered to be the most efficient method for improving properties or regulatory sequences of entirely new proteins. In this example, based on the protein structure and bioinformatics related information of Aerococcus viridans glycerophosphate oxidase, a site-directed saturation mutagenesis library was constructed by applying the Consenssus Concept, and the sites of these mutations are shown in Table 3 and correspond to a series of glycerophosphate oxidase mutants (G3 PO mutants), wherein WT represents the wild type. And respectively designing primers (table 3) according to the sites, determining the coding sequence of each G3PO mutant by taking wild type G3PO as a template, carrying out PCR amplification, transferring the obtained mutated PCR product into an expression system, constructing to obtain a recombinant expression strain, and carrying out sequencing verification to obtain a mutant strain with correct sequencing verification. The mutant strain is also a recombinant expression strain of the G3PO mutant.
TABLE 3 primer sequences for library construction of stability mutants
Figure BDA0003166986700000153
Figure BDA0003166986700000161
Figure BDA0003166986700000171
Figure BDA0003166986700000181
TABLE 4 Single-Point mutant viability and stability test data for G3PO in stability mutation library, and normalization data based on data for wild-type G3PO
Figure BDA0003166986700000182
Figure BDA0003166986700000191
Figure BDA0003166986700000201
Example 2: construction of recombinant expression strains of Glycerol phosphate oxidase (including wild-type and mutant)
Cloning the glycerol phosphate oxidase gene to an expression vector to construct a recombinant vector and a recombinant expression strain.
Wild-type G3PO: based on literature research and sequence analysis, the Aerococcus viridans strain contains a wild-type gene sequence of glycerol-3-phosphate oxidase (G3 PO), and a primer is designed by taking the strain genome as a template to perform PCR amplification to obtain a target gene sequence. Constructing an expression plasmid by adopting an enzyme digestion connection method, adopting a BamH I and Xho I double-enzyme digestion PCR product, connecting a purified fragment with a plasmid pET28a subjected to BamH I and Xho I double-enzyme digestion, and converting the purified fragment into E.coli BL21 (DE 3) by adopting a chemical conversion method, wherein the N end of the constructed plasmid is provided with 6 His, and kanamycin resistance screening positive clone. A plasmid extraction kit sold in the market is adopted to extract a positively cloned plasmid, a DNA fragment of about 1900bp is obtained through double enzyme digestion identification of BamH I and Xho I, and the gene sequence is identified to be correct through sequencing, namely the construction is successful. Carrying out induction expression and glycerol phosphate oxidase production under the conditions of 25 ℃,0.1mM IPTG and 0.1% mass concentration of riboflavin phosphate, and carrying out activity detection and SDS-Page expression verification.
G3PO mutants: by the same method, the coding sequences of the G3PO mutants of the mutation library determined in example 1 are independently cloned to expression vectors, and recombinant expression strains are constructed after the recombinant vectors are constructed, so that the recombinant expression strains of the G3PO mutants can be obtained respectively.
Example 3: expression, purification and SDS-PAGE validation of Glycerol phosphate oxidase (including wild type and mutant)
Glycerol phosphate oxidase was used as the target protein, including wild-type G3PO and the G3PO mutant in the mutation library of example 1.
3.1. Expression of Glycerol phosphate oxidase
Taking the recombinant expression cell bacterial liquid of the recombinant expression strain constructed in the example 2, inoculating the recombinant expression cell bacterial liquid into a test tube containing 10mL of LB culture medium in an inoculation amount of 0.1%, carrying out overnight culture at 37 ℃, inoculating the seed liquid after the overnight culture into a culture medium containing 100mL of LB cone flask in a ratio of 1. Resuspending the collected thalli according to the mass volume ratio of the strains to a resuspension buffer solution (50 mM Tris-HCl, pH 8.0,0.1M NaCl) to 1. Centrifuging the liquid after ultrasonic crushing to obtain supernatant, namely the enzyme liquid of the target protein obtained by expression.
3.2. Purification of Glycerol phosphate oxidase
Filtering the enzyme solution obtained after the ultrasonic disruption in the step 3.1 by using a 0.22-micron filter membrane for later use, preparing a 2mL NTA affinity chromatography packed column, balancing by using 10 times of column volume of balance buffer solution (50 mM Tris-HCl, pH 8.0 and 0.1M NaCl), then loading the enzyme solution after passing through the membrane, washing the column by using 5 times of column volume of impurity removal buffer solution (10 mM imidazole, 50mM Tris-HCl, pH 8.7 and 400mM NaCl), eluting the target protein by using 5 times of column volume of 200mM imidazole eluent, then washing the column by using 5 times of column volume of 500mM imidazole buffer solution, and collecting the process sample purified in each step to obtain the eluent containing the target protein, namely the imidazole eluent, which is convenient for subsequent analysis.
3.3. SDS-PAGE testing of Glycerol phosphate oxidase
The wild-type glycerophosphate oxidase consists of 611 amino acids, the amino acid sequence is shown in SEQ ID No. 4, and the Molecular Weight (Molecular Weight) is about 66kDa. The 200mM imidazole eluate of the target protein of step 3.2 above was dialyzed twice against 50mM Tris-HCl at 4 ℃ to remove NaCl and imidazole from buffer. Then, the samples obtained in the above purification process and the dialyzed enzyme solution were subjected to 12-% SDS-PAGE gel running test, disrupted and resuspended in 10mL of a buffer solution corresponding to 1g of the cells, and the supernatant and the pellet were diluted 10-fold respectively and then subjected to sampling. FIG. 3 shows the results of SDS-PAGE of T90V, one of the G3PO mutants, in which lanes 1 to 9 correspond to the following: lane 1, break supernatant; lane 2, pellet after disruption; lane 3, flow through; lane 4, impurity removal; lane 5, 200mM elution; lane 6, 400mM elution; lane 7, dialysate (5 and 6 mixed); lane 8, 500mM column 1; lane 9, 500mM column 2. As can be seen from FIG. 3, there is a clear target protein band at 66kDa, indicating that the G3PO mutant was successfully expressed by heterologous recombination in E.coli.
Example 4: activity and half-life assays for Glycerol phosphate oxidase (wild-type and mutant)
Reaction solution: 50mM Tris-HCl buffer pH 8.0,0.1M disodium glycerophosphate, 75U/mL POD,7.5mM TOOS,7.5mM 4-Aminoantipyrine.
Enzyme dilution of the samples: the enzyme solution to be tested was released to 0.15-0.35U/mL with 20mM Tris-HCl buffer, pH 7.5, BSA at a mass concentration of 0.2% by mass for detection.
Half-life detection: diluting enzyme solution after wild type and mutant purification with enzyme diluent to 1mg/ml protein concentration, placing single point mutation in 46 deg.C water bath for different time, taking out, placing on 4 deg.C or ice, placing combined mutant in 48 deg.C water bath for different time, taking out, and placing on 4 deg.C or ice; the specific activity of the residual protein after the water bath for different time is detected according to the following activity detection methods respectively, and the residual activity after the water bath for 20min is calculated.
The enzyme activity determination method comprises the following steps: taking 1mL of reaction solution, incubating the reaction solution in a spectrophotometer at 37 ℃ for 2min, adding 0.02mL of enzyme to dilute the sample, uniformly blowing by a pipette, and measuring the OD change value within 1min at the wavelength of 555 nm. The enzyme activity calculation formula is as follows:
Figure BDA0003166986700000211
Weight activity(U/mg)＝Volume activity×1/C
wherein each symbol or number is represented as follows,
volume activity: volume specific activity (U/mL)
Weight activity: specific mass activity (U/mg)
Δ A: change in absorbance
1.02: total volume of reaction (mL);
0.02: enzyme solution volume (mL);
1: reaction time (min);
1/2: 1/2mol of quinone imine dye is generated by 1mol of hydrogen peroxide;
df: dilution times;
c: enzyme concentration (mg/mL);
39.2: under standard reaction conditions, the chromophore has a millimolar absorption coefficient (cm) at 555nm 2 /μmol)。
Example 5: stability site-directed saturation mutagenesis library screening
The sequence-verified mutant strain was expressed, purified and verified according to the respective procedures described in example 3. The eluted enzyme solution after dialysis was diluted with 50mM Tris-HCl buffer solution having pH 8.0 to a protein concentration of 1mg/mL, and the enzyme solution having a concentration of less than 1mg/mL was concentrated and diluted in an ultrafiltration tube. The following three parameters of each mutant were detected according to the viability detection method, respectively: specific activity of protein, half-life period at 46 ℃, and residual activity after 20min incubation at 46 ℃. The test results of the wild type G3PO (control group) and each G3PO mutant are shown in table 1, and the data of each G3PO mutant is normalized for comparison based on the value of the wild type G3PO in table 4, and the comparison result of the normalized data is also shown in fig. 1; wherein WT represents a wild type.
The protein specific activity (U/mg), half-life (min) at 46 ℃ and residual activity percentage of 20min after incubation at 46 ℃ of the D102N mutant are respectively 97.35%, 343.92% and 383.41% of wild G3 PO. The thermal stability is improved by more than 2 times.
The specific protein activities (U/mg) of the T90V, A363S and F555E mutants are 92.29-93.25% of wild G3PO, the half-life period (min) at 46 ℃ is 271.96-321.69% of the wild G3PO, and the residual activity percentage after 20min incubation at 46 ℃ is 343.78-378.80% of the wild G3 PO. The thermal stability is improved by more than 2 times.
The protein specific activity (U/mg) of F6L, G29P and G399K mutants is 101.45-104.82% of wild type G3PO, the half-life (min) at 46 ℃ is 116.40-182.01% of wild type G3PO, and the residual activity percentage after 20min incubation at 46 ℃ is 153.46-256.22% of wild type G3 PO. The specific activity and the thermal stability of the protein of the three mutants are improved relative to the wild type, the specific activity of the protein is slightly improved, and the thermal stability is obviously improved.
The protein specific activity (U/mg) of G399E, A376K, G399P, H92Q, A341T, E204D and V209N mutants is 90.60-99.76% of wild type G3PO, the half-life (min) at 46 ℃ is 145.50-210.58% of wild type G3PO, and the residual activity percentage after 20min incubation at 46 ℃ is 144.24-258.99% of wild type G3 PO. Under the condition of obviously improving the heat stability, the specific activity of the protein is only slightly reduced.
The protein specific activity (U/mg) of the S218T, L131V, V198A, T221S, H227R, V228R, E392K, T370S and I179S mutants is 68.67-88.67% of wild type G3PO, the half-life (min) at 46 ℃ is 124.34-291.53% of wild type G3PO, and the residual activity percentage after 20min incubation at 46 ℃ is 183.87-337.33% of wild type G3 PO. The heat stability is obviously improved, but the specific activity of the protein is reduced.
The protein specific activity (U/mg) of the S501A, K113G, Q474F, V492T, S82A and Q464N mutants is 21.20-59.76% of wild type G3PO, the half-life (min) at 46 ℃ is 242.3-451.85% of the wild type G3PO, and the residual activity percentage after 20min incubation at 46 ℃ is 247.93-413.82% of the wild type G3 PO. Although the increase in thermostability was very significant, the specific activity of the protein decreased relatively much.
Wherein the protein specific activity of the D102N, T90V, A363S and F555E mutants reaches more than 90% of that of wild type G3PO, the half-life period is increased from 18.9min to 51.4-65 min of the wild type G3PO at the temperature of 46 ℃, and the residual activity of the D102N, T90V, A363S and F555E mutants is increased from 21.7% to 74.6% -83.2% after incubation for 20 min. Among them, the relatively preferred mutant is D102N.
Example 6: combinatorial mutation library construction and screening
According to the results of single point mutation in example 5, it can be seen that mutants D102N, T90V, a363S, F555E, F6L, G29P, G399K, G399E, a376K, G399P, H92Q, a341T, E204D, V209N, S218T, L131V, V198A, T221S, H227R, V228R, E392K, T370S, I179S, S501A, K113G, Q474F, V492T, S82A, Q464N, etc. are improved in viability or half-life compared to wild-type G3PO, so a series of mutants as shown in table 5 below were designed, and after gene synthesis directly after construction of a combinatorial mutant library, these mutants were characterized according to the expression, purification and detection methods in example 2, example 3 and example 4.
Wild type WT was used as a control to raise the half-life detection temperature of the detection mutant to 48 deg.C, and the mutants in different combinations were observed to further increase the thermal stability of G3PO, and the data of each combination mutant of G3PO was normalized for comparison, and the comparison results of the normalized data are shown in Table 5 and FIG. 2.
From the experimental results, the protein ratio activity of the G29P + A341T, T370S + F555E + E574K, F6L + G29P + A341T + A363S + F555E + E574K, L4F + E574K, D102N + T90V + E574K, D102N + T90V + A363S + Q464N + F555E + E574K, T90V + A363S + E574K and D102N + T90V + Q464N + V492T + F555E + E574K combined mutants is improved compared with that of wild type WT, and reaches 106.0% -127.6%, and the thermal stability is improved by more than 1.8 times.
It can also be known from the experimental results that T90V + A363S + Q464N, T90V + A363S + E574K, D102N + F6L + G399K + S501A, A363S + T370S + E574K, T90V + A363S + Q474F + F555E, E574K + Q464N, D102N + Q464N, D102N + T90V + A363S + F555E + F6L, T90V + Q474F, D102N + T90V + Q464N, D102N + V492T, D102N + T90V + G399K + S501A + Q464N, D102N + T90V + V492T, T90V + F555E + A376K + G399E + Q474F, S501A + F555E, D102N + T90V + E574K, D102N + T90V + A363S + F555E, S82A + Q464N, S82A + A363S + A341T + T370S + Q464N, K113G + I179S + S218T + Q474F + V492T, D102N + T90V + A376K + G399K + S501A + F555E, T90V + L131V + A341T + Q464N, D102N + H92Q + H227R + K113G, A363S + Q464N, D102N + T90V + A341T + T370S + Q474F + E574K, V198A + E204D + Q464N + S501A, T90V + F555E + G399P + Q464N + S501A + E574K, T90V + A363S + F555E + I179S + V198A, F6L + G29P + A341T + A363S + F555E + E574K, the half life of the G29P + I179S + V198A + V228R + A363S, D102N + T90V + A363S + Q464N + F555E + E574K, T90V + T221S + G399E + I179S, and D102N + T90V + Q464N + V492T + F555E + E574K combined mutant at 48 ℃ is improved to 374.7% -822.9% compared with the wild type, and the specific activity of the protein is 38.2% -109.3% of that of the wild type. Some mutants have more improved stability but relatively reduced activity, for example, the site Q464N has very obvious improved stability but relatively reduced activity; E574K obviously improves the enzyme activity, but the stability is sharply reduced; the D102N, T90V and other mutants have obvious stability improvement, do not have obvious disadvantages on the activity, and the specific activity of the protein can be basically kept unchanged or has small change amplitude.
As can be seen from the results of table 4, table 5, fig. 1 and fig. 2, the combinatorial mutations including preferred single point mutations, particularly those combined with preferred single point mutations, have a better effect on improving stability.
The following combinatorial mutants based on the above single point mutations do not list all possible permutations, but can summarize some sites that are of paramount importance for stability and protein specific activity, in combination with the data shown in Table 5. In the combination mutation modes illustrated in table 5 and fig. 2, the most preferable example is the six-point combination mutant D102N + T90V + a363S + Q464N + F555E + E574K, the protein specific activity is increased to 109.3% of the wild-type G3PO, the half-life at 48 ℃ is increased to 822.9% of the wild-type G3PO, and the stability is greatly improved.
TABLE 5 Activity of some combinatorial mutants in the stability mutation library and their half-life at 48 ℃
Figure BDA0003166986700000241
Figure BDA0003166986700000251
Figure BDA0003166986700000261
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the prior art according to the concepts of the present invention should be within the scope of protection determined by the claims.
Sequence listing
<110> Han Hai New enzyme Biotechnology Limited
<120> glycerophosphate oxidase mutant and screening method, preparation method and application thereof
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Lys Glu Asn Gln Tyr Asp Leu Phe Ile Ile Gly Gly Gly Ile Thr Gly
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Ala Gly Val Ala Ile Gln Ala Ala Ala Ser Gly Leu Lys Thr Ala Leu
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Val Asp Met Gln Asp Phe Ser Glu Gly Thr Ser Ser Arg Ser Thr Lys
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Leu Val His Gly Gly Ile Arg Tyr Leu Lys Asn Phe Asp Leu Glu Val
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Val Ser Asp Thr Val Thr Glu Arg Ala Thr Val His Asn Ile Ala Pro
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His Ile Pro Gln Pro Asn Pro Met Leu Met Pro Leu Tyr Asp Glu Pro
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Lys Val Thr Phe Asn Pro Leu Arg Leu Gln Ile Ala Met Asp Ile Tyr
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Asp Ser Leu Ala Gly Val Lys Asp Ser Gln Tyr Ala Asn Glu Met Leu
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Ser Lys Asp Glu Val Leu Ser Arg Gln Pro Asp Leu Met Ala Glu Gly
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Leu Ile Gly Gly Gly Lys Tyr Leu Asp Phe Asn Asn Asn Asp Ser Arg
165 170 175
Leu Val Ile Glu Asn Ile Lys Gln Ala Asn Asp Asp Gly Ala Asp Leu
180 185 190
Leu Ser His Ala Lys Val Val Gly Phe Glu Tyr Glu Asn Asp Lys Ile
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Val Ala Val Lys Val Glu Asp Leu Leu Ser Gly Glu Thr Phe Thr Val
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Lys Ser His Val Val Ile Asn Thr Thr Gly Pro Trp Ser Asp Thr Ile
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Arg Gln Leu Asp Gly Ser Asp Lys Lys Pro Ala Gln Met Arg Pro Thr
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Lys Gly Val His Phe Val Val Asp Lys Ser Lys Leu Thr Val Ser Gln
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Pro Ile Tyr Phe Asp Thr Gly Glu Gln Asp Gly Arg Met Val Phe Val
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Leu Pro Arg Glu Asn Lys Thr Tyr Phe Gly Thr Thr Asp Thr Asp Tyr
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Thr Gly Asp Phe Glu His Pro Thr Val Thr Gln Glu Asp Val Asp Tyr
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Leu Leu Arg Val Val Asn His Arg Phe Pro Asn Ala Asn Leu Ser Ile
325 330 335
Asn Asp Ile Glu Ala Ser Trp Ala Gly Leu Arg Pro Leu Ile Asp Ser
340 345 350
Asn Asn Ala Ser Asp Tyr Asn Gly Gly Asp Ala Gly Arg Leu Ser Glu
355 360 365
Arg Thr Phe Asp Glu Leu Val Ala Leu Phe Asp Asp Tyr Ser Lys Asp
370 375 380
Lys Val Glu Arg Ser Thr Val Glu Asp Lys Leu Gln Asp Leu Gly Ser
385 390 395 400
Asn Thr Ser Glu Arg Gly Asp Gly Ser Pro Ser Ser Val Ser Arg Gly
405 410 415
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Lys Ile Thr Asp Tyr Arg Lys Met Ala Lys Gly Ala Met Glu Arg Ile
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Ile Pro Val Val Thr Asp Ile Thr Gly Lys Ser Tyr Glu Leu Val Gln
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Ser Ser Thr Tyr Pro Ile Ser Gly Gly Gln Phe Asp Pro Asn Ser Tyr
465 470 475 480
Glu Thr Ala Met Glu Lys Phe Ala Asn Val Gly Val Ala Arg Gly Leu
485 490 495
Thr Tyr Gly Gln Ser Leu Asn Leu Ala Lys Leu Tyr Gly Ser Asn Met
500 505 510
Asn Arg Val Ile Ser Tyr Leu Pro Val Ala Lys Glu Tyr Ala Ala Lys
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530 535 540
Glu Glu Gly Val Tyr Thr Pro Leu Asp Phe Phe Ala Arg Arg Thr Thr
545 550 555 560
Phe Met Leu Phe Gln His Asp Lys Met Leu Ala Val Lys Glu Ala Val
565 570 575
Ser Gln Thr Ile Val Asp Tyr Phe Gly Leu Asp Gln Ala Thr Ala Asp
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Gln Gln Lys Thr Ala Leu Asp Glu Glu Ile Ala Lys Ala Glu Leu Gln
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Tyr Leu Lys
610
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Met Ser Lys Leu Ser Phe Lys Tyr Arg Lys Glu Thr Val Glu Gln Leu
1 5 10 15
Lys Glu Asn Gln Tyr Asp Leu Phe Ile Ile Gly Gly Gly Ile Thr Gly
20 25 30
Ala Gly Val Ala Ile Gln Ala Ala Ala Ser Gly Leu Lys Thr Ala Leu
35 40 45
Val Asp Met Gln Asp Phe Ser Glu Gly Thr Ser Ser Arg Ser Thr Lys
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Leu Val His Gly Gly Ile Arg Tyr Leu Lys Asn Phe Asp Leu Glu Val
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Val Ser Asp Thr Val Thr Glu Arg Ala Val Val His Asn Ile Ala Pro
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His Ile Pro Gln Pro Asp Pro Met Leu Met Pro Leu Tyr Asp Glu Pro
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Lys Val Thr Phe Asn Pro Leu Arg Leu Gln Ile Ala Met Asp Ile Tyr
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Asp Ser Leu Ala Gly Val Lys Asp Ser Gln Tyr Ala Asn Glu Met Leu
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Ser Lys Asp Glu Val Leu Ser Arg Gln Pro Asp Leu Met Ala Glu Gly
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Leu Ile Gly Gly Gly Lys Tyr Leu Asp Phe Asn Asn Asn Asp Ser Arg
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Leu Val Ile Glu Asn Ile Lys Gln Ala Asn Asp Asp Gly Ala Asp Leu
180 185 190
Leu Ser His Ala Lys Val Val Gly Phe Glu Tyr Glu Asn Asp Lys Ile
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Val Ala Val Lys Val Glu Asp Leu Leu Ser Gly Glu Thr Phe Thr Val
210 215 220
Lys Ser His Val Val Ile Asn Thr Thr Gly Pro Trp Ser Asp Thr Ile
225 230 235 240
Arg Gln Leu Asp Gly Ser Asp Lys Lys Pro Ala Gln Met Arg Pro Thr
245 250 255
Lys Gly Val His Phe Val Val Asp Lys Ser Lys Leu Thr Val Ser Gln
260 265 270
Pro Ile Tyr Phe Asp Thr Gly Glu Gln Asp Gly Arg Met Val Phe Val
275 280 285
Leu Pro Arg Glu Asn Lys Thr Tyr Phe Gly Thr Thr Asp Thr Asp Tyr
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Thr Gly Asp Phe Glu His Pro Thr Val Thr Gln Glu Asp Val Asp Tyr
305 310 315 320
Leu Leu Arg Val Val Asn His Arg Phe Pro Asn Ala Asn Leu Ser Ile
325 330 335
Asn Asp Ile Glu Ala Ser Trp Ala Gly Leu Arg Pro Leu Ile Asp Ser
340 345 350
Asn Asn Ala Ser Asp Tyr Asn Gly Gly Asp Ala Gly Arg Leu Ser Glu
355 360 365
Arg Thr Phe Asp Glu Leu Val Ala Leu Phe Asp Asp Tyr Ser Lys Asp
370 375 380
Lys Val Glu Arg Ser Thr Val Glu Asp Lys Leu Gln Asp Leu Gly Ser
385 390 395 400
Asn Thr Ser Glu Arg Gly Asp Gly Ser Pro Ser Ser Val Ser Arg Gly
405 410 415
Ser Asp Leu Ser Val Ala Pro Ser Gly Leu Phe Thr Leu Ala Gly Gly
420 425 430
Lys Ile Thr Asp Tyr Arg Lys Met Ala Lys Gly Ala Met Glu Arg Ile
435 440 445
Ile Pro Val Val Thr Asp Ile Thr Gly Lys Ser Tyr Glu Leu Val Gln
450 455 460
Ser Ser Thr Tyr Pro Ile Ser Gly Gly Gln Phe Asp Pro Asn Ser Tyr
465 470 475 480
Glu Thr Ala Met Glu Lys Phe Ala Asn Val Gly Val Ala Arg Gly Leu
485 490 495
Thr Tyr Gly Gln Ser Leu Asn Leu Ala Lys Leu Tyr Gly Ser Asn Met
500 505 510
Asn Arg Val Ile Ser Tyr Leu Pro Val Ala Lys Glu Tyr Ala Ala Lys
515 520 525
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530 535 540
Glu Glu Gly Val Tyr Thr Pro Leu Asp Phe Phe Ala Arg Arg Thr Thr
545 550 555 560
Phe Met Leu Phe Gln His Asp Lys Met Leu Ala Val Lys Glu Ala Val
565 570 575
Ser Gln Thr Ile Val Asp Tyr Phe Gly Leu Asp Gln Ala Thr Ala Asp
580 585 590
Gln Gln Lys Thr Ala Leu Asp Glu Glu Ile Ala Lys Ala Glu Leu Gln
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Tyr Leu Lys
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Met Ser Lys Leu Ser Phe Lys Tyr Arg Lys Glu Thr Val Glu Gln Leu
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Val Asp Met Gln Asp Phe Ser Glu Gly Thr Ser Ser Arg Ser Thr Lys
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Val Ser Asp Thr Val Thr Glu Arg Ala Val Val His Asn Ile Ala Pro
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Asp Ser Leu Ala Gly Val Lys Asp Ser Gln Tyr Ala Asn Glu Met Leu
130 135 140
Ser Lys Asp Glu Val Leu Ser Arg Gln Pro Asp Leu Met Ala Glu Gly
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Leu Ile Gly Gly Gly Lys Tyr Leu Asp Phe Asn Asn Asn Asp Ser Arg
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Leu Ser His Ala Lys Val Val Gly Phe Glu Tyr Glu Asn Asp Lys Ile
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Val Ala Val Lys Val Glu Asp Leu Leu Ser Gly Glu Thr Phe Thr Val
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Lys Ser His Val Val Ile Asn Thr Thr Gly Pro Trp Ser Asp Thr Ile
225 230 235 240
Arg Gln Leu Asp Gly Ser Asp Lys Lys Pro Ala Gln Met Arg Pro Thr
245 250 255
Lys Gly Val His Phe Val Val Asp Lys Ser Lys Leu Thr Val Ser Gln
260 265 270
Pro Ile Tyr Phe Asp Thr Gly Glu Gln Asp Gly Arg Met Val Phe Val
275 280 285
Leu Pro Arg Glu Asn Lys Thr Tyr Phe Gly Thr Thr Asp Thr Asp Tyr
290 295 300
Thr Gly Asp Phe Glu His Pro Thr Val Thr Gln Glu Asp Val Asp Tyr
305 310 315 320
Leu Leu Arg Val Val Asn His Arg Phe Pro Asn Ala Asn Leu Ser Ile
325 330 335
Asn Asp Ile Glu Ala Ser Trp Ala Gly Leu Arg Pro Leu Ile Asp Ser
340 345 350
Asn Asn Ala Ser Asp Tyr Asn Gly Gly Asp Ser Gly Arg Leu Ser Glu
355 360 365
Arg Thr Phe Asp Glu Leu Val Ala Leu Phe Asp Asp Tyr Ser Lys Asp
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Lys Val Glu Arg Ser Thr Val Glu Asp Lys Leu Gln Asp Leu Gly Ser
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Asn Thr Ser Glu Arg Gly Asp Gly Ser Pro Ser Ser Val Ser Arg Gly
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Ser Asp Leu Ser Val Ala Pro Ser Gly Leu Phe Thr Leu Ala Gly Gly
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Lys Ile Thr Asp Tyr Arg Lys Met Ala Lys Gly Ala Met Glu Arg Ile
435 440 445
Ile Pro Val Val Thr Asp Ile Thr Gly Lys Ser Tyr Glu Leu Val Asn
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Ser Ser Thr Tyr Pro Ile Ser Gly Gly Gln Phe Asp Pro Asn Ser Tyr
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Glu Thr Ala Met Glu Lys Phe Ala Asn Val Gly Val Ala Arg Gly Leu
485 490 495
Thr Tyr Gly Gln Ser Leu Asn Leu Ala Lys Leu Tyr Gly Ser Asn Met
500 505 510
Asn Arg Val Ile Ser Tyr Leu Pro Val Ala Lys Glu Tyr Ala Ala Lys
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Tyr Asp Tyr Pro Val Asp Ile Ala Val Ser Leu Ile Tyr Ala Leu Glu
530 535 540
Glu Glu Gly Val Tyr Thr Pro Leu Asp Phe Glu Ala Arg Arg Thr Thr
545 550 555 560
Phe Met Leu Phe Gln His Asp Lys Met Leu Ala Val Lys Asp Ala Val
565 570 575
Ser Gln Thr Ile Val Asp Tyr Phe Gly Leu Asp Gln Ala Thr Ala Asp
580 585 590
Gln Gln Lys Thr Ala Leu Asp Glu Glu Ile Ala Lys Ala Glu Leu Gln
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Tyr Leu Lys
610
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<213> Aerococcus viridans Wide-type G3PO
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Met Ser Lys Leu Ser Phe Lys Tyr Arg Lys Glu Thr Val Glu Gln Leu
1 5 10 15
Lys Glu Asn Gln Tyr Asp Leu Phe Ile Ile Gly Gly Gly Ile Thr Gly
20 25 30
Ala Gly Val Ala Ile Gln Ala Ala Ala Ser Gly Leu Lys Thr Ala Leu
35 40 45
Val Asp Met Gln Asp Phe Ser Glu Gly Thr Ser Ser Arg Ser Thr Lys
50 55 60
Leu Val His Gly Gly Ile Arg Tyr Leu Lys Asn Phe Asp Leu Glu Val
65 70 75 80
Val Ser Asp Thr Val Thr Glu Arg Ala Thr Val His Asn Ile Ala Pro
85 90 95
His Ile Pro Gln Pro Asp Pro Met Leu Met Pro Leu Tyr Asp Glu Pro
100 105 110
Lys Val Thr Phe Asn Pro Leu Arg Leu Gln Ile Ala Met Asp Ile Tyr
115 120 125
Asp Ser Leu Ala Gly Val Lys Asp Ser Gln Tyr Ala Asn Glu Met Leu
130 135 140
Ser Lys Asp Glu Val Leu Ser Arg Gln Pro Asp Leu Met Ala Glu Gly
145 150 155 160
Leu Ile Gly Gly Gly Lys Tyr Leu Asp Phe Asn Asn Asn Asp Ser Arg
165 170 175
Leu Val Ile Glu Asn Ile Lys Gln Ala Asn Asp Asp Gly Ala Asp Leu
180 185 190
Leu Ser His Ala Lys Val Val Gly Phe Glu Tyr Glu Asn Asp Lys Ile
195 200 205
Val Ala Val Lys Val Glu Asp Leu Leu Ser Gly Glu Thr Phe Thr Val
210 215 220
Lys Ser His Val Val Ile Asn Thr Thr Gly Pro Trp Ser Asp Thr Ile
225 230 235 240
Arg Gln Leu Asp Gly Ser Asp Lys Lys Pro Ala Gln Met Arg Pro Thr
245 250 255
Lys Gly Val His Phe Val Val Asp Lys Ser Lys Leu Thr Val Ser Gln
260 265 270
Pro Ile Tyr Phe Asp Thr Gly Glu Gln Asp Gly Arg Met Val Phe Val
275 280 285
Leu Pro Arg Glu Asn Lys Thr Tyr Phe Gly Thr Thr Asp Thr Asp Tyr
290 295 300
Thr Gly Asp Phe Glu His Pro Thr Val Thr Gln Glu Asp Val Asp Tyr
305 310 315 320
Leu Leu Arg Val Val Asn His Arg Phe Pro Asn Ala Asn Leu Ser Ile
325 330 335
Asn Asp Ile Glu Ala Ser Trp Ala Gly Leu Arg Pro Leu Ile Asp Ser
340 345 350
Asn Asn Ala Ser Asp Tyr Asn Gly Gly Asp Ala Gly Arg Leu Ser Glu
355 360 365
Arg Thr Phe Asp Glu Leu Val Ala Leu Phe Asp Asp Tyr Ser Lys Asp
370 375 380
Lys Val Glu Arg Ser Thr Val Glu Asp Lys Leu Gln Asp Leu Gly Ser
385 390 395 400
Asn Thr Ser Glu Arg Gly Asp Gly Ser Pro Ser Ser Val Ser Arg Gly
405 410 415
Ser Asp Leu Ser Val Ala Pro Ser Gly Leu Phe Thr Leu Ala Gly Gly
420 425 430
Lys Ile Thr Asp Tyr Arg Lys Met Ala Lys Gly Ala Met Glu Arg Ile
435 440 445
Ile Pro Val Val Thr Asp Ile Thr Gly Lys Ser Tyr Glu Leu Val Gln
450 455 460
Ser Ser Thr Tyr Pro Ile Ser Gly Gly Gln Phe Asp Pro Asn Ser Tyr
465 470 475 480
Glu Thr Ala Met Glu Lys Phe Ala Asn Val Gly Val Ala Arg Gly Leu
485 490 495
Thr Tyr Gly Gln Ser Leu Asn Leu Ala Lys Leu Tyr Gly Ser Asn Met
500 505 510
Asn Arg Val Ile Ser Tyr Leu Pro Val Ala Lys Glu Tyr Ala Ala Lys
515 520 525
Tyr Asp Tyr Pro Val Asp Ile Ala Val Ser Leu Ile Tyr Ala Leu Glu
530 535 540
Glu Glu Gly Val Tyr Thr Pro Leu Asp Phe Phe Ala Arg Arg Thr Thr
545 550 555 560
Phe Met Leu Phe Gln His Asp Lys Met Leu Ala Val Lys Glu Ala Val
565 570 575
Ser Gln Thr Ile Val Asp Tyr Phe Gly Leu Asp Gln Ala Thr Ala Asp
580 585 590
Gln Gln Lys Thr Ala Leu Asp Glu Glu Ile Ala Lys Ala Glu Leu Gln
595 600 605
Tyr Leu Lys
610

Claims (10)

1. The mutant glycerol phosphate oxidase is characterized in that site-directed mutagenesis is carried out on the mutant glycerol phosphate oxidase based on the amino acid sequence of wild glycerol phosphate oxidase, the amino acid sequence of the wild glycerol phosphate oxidase is shown as SEQ ID No. 4, and the mutagenesis mode is selected from the following modes: D102N, T90V, A363S, F555E, F6L, G29P, G399K, G399E, A376K, G399P, H92Q, A341T, E204D, V209N, S218T, L131V, V198A, T221S, H227R, V228R, E392K, T370S, I179S, S501A, K113G, Q474F, V492T, S82A, Q464N and combinations comprising the foregoing.
2. The glycerol phosphate oxidase mutant according to claim 1, wherein the combinatorial mutation pattern is selected from the group consisting of at least two of D102N, T90V, A363S, F555E, F6L, G29P, G399K, G399E, A376K, G399P, H92Q, A341T, E204D, V209N, S218T, L131V, V198A, T221S, H227R, V228R, E392K, T370S, I179S, S501A, K113G, Q474F, V492T, S82A, and Q464N; or
The mutation mode is selected from: D102N, T90V, A363S, F555E and a combination mutation mode comprising the mutation modes;
preferably, the combination mutation mode is selected from any one of the following combination mutation modes:
L4F+E574K，D102N+T90V+E574K，T370S+F555E+E574K；
D102N+T90V+A363S+Q464N+F555E+E574K，
T90V+A363S+F555E+I179S+V198A，
D102N+T90V+Q464N+V492T+F555E+E574K，
D102N+T90V+A363S+F555E+F6L，
D102N+T90V+A376K+G399K+S501A+F555E，D102N+T90V+E574K，
D102N+T90V+A341T+T370S+Q474F+E574K，D102N+T90V+A363S+F555E，
F6L+G29P+A341T+A363S+F555E+E574K；
D102N+T90V+G399K+S501A+Q464N，T90V+L131V+A341T+Q464N，
T90V+F555E+A376K+G399E+Q474F，K113G+I179S+S218T+Q474F+V492T，
V198A+E204D+Q464N+S501A，T90V+A363S+Q464N，
T90V+A363S+Q474F+F555E，S82A+A363S+A341T+T370S+Q464N，
S82A+Q464N，D102N+T90V+Q464N。
3. a fusion protein of a mutant glycerophosphate oxidase, wherein the fusion protein of a mutant glycerophosphate oxidase is characterized in that a fusion tag is attached to the N-terminus and/or the C-terminus of the mutant glycerophosphate oxidase of any one of claims 1-2;
preferably, the fusion tag comprises an affinity tag;
more preferably, the affinity tag is selected from the group consisting of: his-tag, GST, flag-tag, MBP, and combinations thereof.
4. A nucleic acid construct having a nucleotide sequence selected from any one of the group consisting of:
(i) A nucleotide sequence encoding the glycerophosphate oxidase mutant of any of claims 1-2 or the fusion protein of claim 3; and
(ii) (ii) a nucleotide sequence complementary to the nucleotide sequence described in (i);
preferably, the nucleic acid construct is DNA, mRNA, or a combination thereof.
5. A recombinant vector comprising the nucleic acid construct of claim 4;
preferably, the type of the recombinant vector is selected from the group consisting of: pET28a, pET 30a, pANY1, pQE30, pG-KJE8, pGRO7, pKJE7, pG-Tf2, pTf16, pUC18, pUC19 and pGEX.
6. An engineered bacterium having a genome into which the nucleic acid construct of claim 4 is integrated, or comprising the recombinant vector of claim 5;
preferably, the engineering bacteria are derived from escherichia coli.
7. A genetically engineered cell having integrated into its genome the nucleic acid construct of claim 4, or comprising the recombinant vector of claim 5;
preferably, the genetically engineered cell is derived from escherichia coli.
8. A method for screening glycerol phosphate oxidase mutants from a mutation library is characterized by comprising the following steps:
(1) Constructing a mutation library of the wild type G3PO as shown in SEQ ID No. 4, wherein the mutation sites comprise: 4.6, 29, 55, 78, 82, 90, 92, 100, 102, 113, 119, 123, 127, 131, 147, 179, 196, 198, 204, 209, 218, 221, 227, 228, 261, 269, 341, 363, 370, 376, 392, 399, 452, 460, 463, 464, 466, 470, 474, 492, 501, 531, 547, 555, 560, 572, 574, and 584, to give a library of mutations consisting of G3PO mutants; wherein the mutation mode comprises a single point mutation mode and a combined mutation mode;
(2) Obtaining the coding sequence of each G3PO mutant in the mutation library by taking the gene of wild G3PO as a template;
(3) Respectively constructing a recombinant vector for each G3PO mutant in the mutation library, transforming the recombinant vector into host bacteria to obtain a recombinant expression strain, inducing the recombinant expression strain, and expressing to obtain the G3PO mutant of the mutation library or a fusion protein thereof; when the coding sequence of the G3PO mutant in the recombinant vector is connected with a fusion tag, expressing to obtain a fusion protein of the G3PO mutant of the mutation library;
(4) Analyzing the stability and activity of the G3PO mutant or the fusion protein thereof, and screening to obtain the G3PO mutant of any one of claims 1-2 or the fusion protein of claim 3.
9. A preparation method of a glycerophosphate oxidase mutant is characterized by comprising the following steps:
(1) Cloning the nucleic acid construct of claim 4 into an expression vector to obtain a recombinant vector comprising the coding sequence of the glycerol phosphate oxidase mutant or a fusion protein thereof;
(2) Transforming the recombinant vector into host bacteria to obtain a recombinant expression strain containing the coding sequence of the glycerophosphate oxidase mutant or the fusion protein thereof;
(3) Inducing the recombinant expression strain to express under a proper expression condition to obtain the glycerophosphate oxidase mutant or the fusion protein thereof;
preferably, the recombinant expression strain is escherichia coli;
more preferably, the suitable expression conditions include: 0.1mM isopropyl-beta-D-thiogalactoside, 0.1wt% of an induction precursor substance, and inducing for 16 hours at 25 ℃; wherein the inducing precursor substance is flavin adenine dinucleotide, riboflavin, a riboflavin analog, or a combination thereof.
10. Use of the glycerol phosphate oxidase mutant according to any of claims 1-2, the fusion protein according to claim 3, the nucleic acid construct according to claim 4, the recombinant vector according to claim 5, the engineered bacterium according to claim 6, and the genetically engineered cell according to claim 7 for detecting serum triglyceride levels.
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