CN117384873A - 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 PDFInfo
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- C12Y101/03021—Glycerol-3-phosphate-oxidase (1.1.3.21)
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Abstract
The invention discloses a glycerophosphate oxidase mutant and a screening method, a preparation method and application thereof. The invention constructs a mutation library of wild type glycerophosphate oxidase (G3 PO), carries out single-point mutation and combined mutation at more than 50 sites, respectively constructs recombinant expression strains, carries out induced expression, carries out activity and thermal stability analysis on the obtained mutation products, screens G3PO mutants with more than 20 single-point mutation modes with obviously improved thermal stability, comprises but is not limited to four single-point mutants of T90V, A363S and F555E, and screens more than 40 better combined mutants containing the single-point mutation. The invention also discloses a fusion protein of the G3PO mutant, a nucleic acid construct, a recombinant vector, engineering bacteria and a preparation method thereof. The G3PO mutant screened by the method has great application potential in the aspect of detecting the content of serum triglyceride.
Description
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
Glycerol phosphate oxidase (G3 PO, EC 1.1.3.21) is one of the key enzymes for determining triglyceride content by a coupled enzyme method, and is widely used in clinical judgment of heart disease and hyperlipidemia at present with its unique high specificity and high sensitivity.
The study of glycerophosphate oxidase began in the thirty-second twentieth century, and the main subject was Streptococcus faecalis-derived protein. Currently available commercial glycerophosphate oxidase often comes from multi-step separation and purification products, and major production countries include japan, the united states, the united kingdom, and the like. The research of the glycerophosphate oxidase in China is started later, the related reports are not particularly large, and most of the glycerophosphate oxidase for clinical diagnosis still needs to be imported. Meanwhile, along with the increasing of the living standard of people, the working pressure is increased, and the concept of paying attention to health is gradually taken as one of the standards for measuring the quality of life, so that the demands of China on the glycerophosphate oxidase in China also show an increasing trend.
Current research on glycerophosphate oxidase has focused mainly on Pediococcus acidilactici, aerococcus viridans, streptococcus faecalis and Enterococcus SP. sources. Patent document CN110938607a reports a glycerophosphate oxidase derived from Pediococcus acidilactici, which has been subjected to random mutation to improve the thermal stability [1] . Wang Teng et al report a method for preparing phosphoglycerate oxidase by using induction medium streptococcus, wherein Streptococcus faecalis-derived G3PO gene is subjected to recombinant expression, and G3PO target protein is obtained by polyethylene glycol-ammonium sulfate double-aqueous phase extraction and two-step chromatographic purification [2] . The glycerol phosphate oxidase from Enterococcus SP. is screened from 23 strains by the university of Sichuan and the color nepheline, and is prepared by multi-step purification of ammonium sulfate fractional precipitation, DADE-Sepharose FF ion exchange chromatography, phenyl Sepharose CL-4B hydrophobic chromatography, chelating Sepharose FF affinity chromatography and Sephacryls-200HR treatment, and has complex operation [3] . In addition, with the increasing improvement of life quality of people, health problems are increasingly emphasized, and detection of serum triglyceride content is increasingly focused. The reagent for detecting the content of the serum triglyceride relates to an important enzyme, namely glycerophosphate oxidase, so that the enzyme has good market prospect.
Serum triglyceride content detection has been converted substantially completely to current enzymatic methods by previous chemical methods. The enzyme method for detecting the triglyceride is quick and convenient, has high accuracy, can be applied to a biochemical analyzer, and can also be used for mass detection. The detection principle of the enzymatic detection is as follows: the sample to be detected is hydrolyzed under the action of lipoprotein esterase (Lipoprotein lipase) to generate Glycerol (Glycerol) and Fatty acid (Fatty acid), the Glycerol and ATP are reacted under the action of Glycerol kinase (Glycerol kinase) to generate Glycerol-3-phosphate (Glycerol-3-P), and the Glycerol-3-phosphate is reacted under the action of Glycerol phosphate oxidase (L-alpha-glycerophosphate oxidase) to generate Dihydroxyacetone phosphate (Dihydroxyacetone-P) and hydrogen peroxide (H) 2 O 2 ) The hydrogen peroxide can be selected from 4-Aminoantipyrine (4-AA) and 2-hydroxy3-m-toluidine sodium propane sulfonate (TOOS) and horseradish Peroxidase (Peroxidase) to produce developed quinone imine, and the content of triglyceride can be reflected according to the absorption change of the quinone imine under visible light, so as to achieve the aim of detection. The specific reaction principle is as follows:
the majority of glycerophosphate 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 the TG reagent detected by an enzyme method are limited, and meanwhile, the stability of the glycerophosphate oxidase can greatly influence the detection accuracy of the reagent.
At present, the wild type stability of the glycerophosphate oxidase (G3 PO) from the balloon fungus is deficient, and a glycerophosphate oxidase mutant with improved stability needs to be developed so as to improve the application stability.
[1] Bingde, jihua, zhang Yusheng, wang Yi, zhang Shuai, zhang Yong. Glycerol-3-phosphate oxidase with good thermostability and its use in a kit, CN 110938607A.
[2] Wang Teng methods for producing phosphoglycerate oxidase from Streptococcus in induction medium, CN 101070529A.
[3] Gao Guosheng, dong Feibo, sun Yi, hua Xin, zhang Wenyu, yang Jinxun. A recombinant glycerophosphate oxidase expression vector and a method for its construction, 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 how to develop a glycerophosphate oxidase (G3 PO) mutant with improved stability, thereby improving its stability in application.
The first aspect of the invention provides a glycerophosphate oxidase mutant (G3 PO mutant), wherein the glycerophosphate oxidase mutant is subjected to site-directed mutagenesis based on the amino acid sequence of wild type glycerophosphate oxidase, the amino acid sequence of the wild type glycerophosphate oxidase (wild type G3 PO) is shown as SEQ ID No.:4, and the mutation mode is selected from the group consisting of: D102N, T90V, A363S, F555E, F L, G5483 399K, G399E, A376K, G399P, H92Q, A341T, E D, V209N, S218T, L131V, V198V, V221V, V227V, V228V, V370V, V179V, V113V, V474V, V492V, V464N and combinations comprising the foregoing mutations.
In some preferred embodiments of the present invention, the combined mutation mode is selected from D102N, T90V, A363S, F555E, F L, G P, G399E, A79376K, G399P, H Q, A341T, E D, V218T, L V, V198V, V221V, V227V, V228V, V392V, V370V, V179V, V113V, V474V, V492V, V82 and V, V464N.
In some preferred embodiments of the invention, the mutation pattern is selected from the group consisting of: D102N, T90V, A363S, F555E and combinations comprising the foregoing.
In some preferred embodiments of the invention, the combinatorial mutation pattern is selected from any one of the following combinatorial mutation patterns:
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.
In a second aspect, the invention provides a fusion protein of a glycerophosphate oxidase mutant, wherein the fusion protein of the glycerophosphate oxidase mutant is connected with a fusion tag at the N-terminal and/or the C-terminal of the glycerophosphate 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 invention provides a nucleic acid construct having a nucleotide sequence selected from any one of the following:
(i) A nucleotide sequence encoding the glycerol phosphate oxidase mutant of the first aspect or the fusion protein of the second aspect; and
(ii) A nucleotide sequence complementary to the nucleotide sequence set forth in (i).
The nucleic acid construct may be DNA, mRNA, or a combination thereof.
In a fourth aspect, the present invention provides a recombinant vector comprising the nucleic acid construct of 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 recombinant vector is selected from the group consisting of: pET 28a,pET 30a,pANY1,pQE30,pG-KJE, pGRO7, pKJE7, pG-Tf2, pTf16, pUC18, pUC19 and pGEX.
In a fifth aspect, the invention provides an engineered bacterium, the genome of which is integrated with the nucleic acid construct of the third aspect, or which comprises the recombinant vector of the fourth aspect.
In some preferred embodiments of the invention, the engineered bacterium is derived from E.coli.
In a sixth aspect, the present invention provides a genetically engineered cell having the nucleic acid construct of the third aspect integrated into its genome or comprising the recombinant vector of the fourth aspect.
In some preferred embodiments of the invention, the genetically engineered cell is derived from E.coli.
In a seventh aspect, the invention provides a method of screening a mutant glycerophosphate oxidase from a library of mutations, comprising the steps of:
(1) A mutant library of wild type G3PO as shown in SEQ ID No. 4 was constructed, the mutation sites comprising: 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 obtain a library of mutations consisting of G3PO mutants; mutation modes comprise a single-point mutation mode and a combined mutation mode;
(2) Taking a wild G3PO gene as a template to obtain a coding sequence of each G3PO mutant in the mutation library;
(3) Respectively constructing recombinant vectors (such as the recombinant vectors in the fourth aspect) for the G3PO mutants in the mutation library, transforming the recombinant vectors into host bacteria to obtain recombinant expression strains, inducing the recombinant expression strains, and expressing the G3PO mutants or fusion proteins thereof to obtain the mutation library (corresponding to the G3PO mutants in the first aspect or the fusion proteins in 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 a fusion protein of the G3PO mutant of the mutation library;
(4) And (3) carrying out protein specific activity and stability analysis on the G3PO mutant or the fusion protein thereof, and screening to obtain the G3PO mutant or the fusion protein of the second aspect.
The eighth aspect of the present invention provides a method for producing a glycerophosphate oxidase mutant, comprising the steps of:
(1) Cloning the nucleic acid construct of the third aspect onto an expression vector to obtain a recombinant vector containing the coding sequence of the G3PO mutant or 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 egg thereof;
(3) Inducing the recombinant expression strain, and expressing under proper expression conditions to obtain the G3PO mutant or the fusion protein (corresponding to the G3PO mutant of the first aspect or the fusion protein of the second aspect).
In some preferred embodiments of the invention, the recombinant expression strain is E.coli, and in this case, the suitable expression conditions include: 0.1mM isopropyl-beta-D-thiogalactoside (IPTG), 0.1% by weight of an induction precursor substance, induction for 16 hours at 25 ℃. The inducing precursor substance may be Flavin Adenine Dinucleotide (FAD), a nuclease, a riboflavin analog, or a combination thereof.
The ninth aspect of the invention provides the glycerophosphate oxidase mutant, the fusion protein of the second aspect, the nucleic acid construct of the third aspect, the recombinant vector of the fourth aspect, the engineering bacterium of the fifth aspect and the application of the genetically engineered cell of the sixth aspect in serum triglyceride content detection.
The technical scheme disclosed by the invention has the beneficial effects that:
1. the invention constructs a stability mutation library of wild type glycerophosphate oxidase, clones G3PO mutant genes onto an expression vector, then converts the expression vector into host bacteria to construct corresponding unit point mutant strains, carries out stability analysis on expression products after induced expression, screens to obtain 20 more G3PO mutants with obviously improved thermal stability (including but not limited to four single-point mutants of D102N, T90V, A363S and F555E), screens to obtain 40 more preferable combination mutants containing the single-point mutation (such as six-point combination mutation of D102N, T90V, A363S, Q464N, F555E and E574K), and can be used for catalyzing glycerol-3-phosphate reaction to generate dihydroxyacetone phosphate.
2. The invention discloses a G3PO single-point mutant with more than 20 heat stability improved by at least 40%, and quantitative analysis of activity is carried out according to specific activity of protein or the residual activity percentage of incubation for 20min at 46 ℃. Wherein, the specific activities of the F6L, G P and G399K mutant proteins are slightly improved relative to the wild type G3PO, and the thermal stability is improved by 0.5-1.6 times; the specific protein activity of the D102N, T90V, A363S, F555E mutant can reach more than 90% of that of the wild G3PO, and the heat stability is improved by more than 2 times; the specific activity of the protein of the G399E, A376K, G399P, H92Q, A341T, E D and V209N mutants reaches more than 90% of that of the wild G3PO, and the heat stability is improved by 0.8-2.4 times; the heat stability of the S501A, K113G, Q474F, V492T, S A and Q464N mutants is improved by 1.5-3.1 times compared with that of the wild type G3PO, 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 the single point mutation mode, including the G3PO mutant with the single point mutation mode of D102N, T90V, A363S and F555E, with obviously improved comprehensive performance.
4. The invention also discloses a combined mutant which is formed by combining the following single-point mutation modes and has obviously improved stability on the basis of keeping certain activity: L4F, E574K, D102N, T90V, E574K, T370S, F555E, A363 82348 492T, G399P, E D, A376A, G P and the like.
5. In addition, the invention specifically discloses a G3PO mutant with more than 40 combined mutation modes, wherein the thermal stability of the G3PO mutant is improved by at least 60 percent, and the quantitative analysis of the activity is based on the specific activity of protein or the residual activity percentage of incubation at 48 ℃ for 20 min. Wherein, the specific activities of L4F+E574K, D102N+T90V+E574K and T370S+F555E+E574K proteins are improved by more than 10%, and the stability is improved by more than 80%; mutants such as D102N+T90V+A363S+Q464 N+F255E+E574K, T90V+A363S+F375E+I179 S+V198A, D102N+T90V+Q364N+V492+F550E+E574K, D102N+T90V+A367S+F550E+F6L, D102N+T90V+A370K+G399+S501 A+F555E, D102N+T90V+E574K, D102N+T90V+A341T+T370S+Q474F+E574K, D102N+T90V+A360S+F5E, F6L+G397T+A367S+F555 E+E574K are improved by 400% or more while maintaining protein specific activity; mutant stability such as D102N+T90V+G399K+S501A+Q464N, T90 V+L315V+A341 T+Q464N, T90V+F555E+A376K+G399E+Q474F, K21G+I179 S+S214T+Q474 F+V492T, V198 A+E2004 D+Q464N+S501A, T90V+A363S+Q464N, T90V+A363 S+Q200F+F555E, S82A+A360S+Q464N, S82A+Q464N, D102N+T90V+Q464N is improved by 400%, and protein specific activity is reduced to 30% -80% of wild type.
6. By comprehensively considering the activity and stability of the G3PO mutant, the invention obtains the combined mutant with very obvious improvement of comprehensive performance, including the six-point combined mutant of D102N+T90V+A363S+Q464N+F555E+E574K G3PO, the specific activity of the protein is improved to 109.3 percent of the wild type G3PO, and the half life at 48 ℃ is improved to 822.9 percent (namely, the half life is improved by more than 7 times) of the wild type G3 PO.
7. The invention also provides a batch preparation method and a purification method of the glycerophosphate oxidase and the mutant thereof which are recombined and heterologously expressed in the escherichia coli cells, and the G3PO mutant with better heat stability, especially the G3PO mutant with good heat stability and high enzyme activity is screened out by combining with a stability mutation library.
The advantages enable the glycerophosphate oxidase mutant to have great application potential in the aspect of detecting the content of serum triglyceride.
The conception, specific technical scheme and technical effects of the present invention will be further described with reference to the accompanying drawings so as to fully understand the objects, features and effects of the present invention.
Drawings
Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments, given by reference to the accompanying drawings in which:
FIG. 1 shows a comparison of properties of wild type G3PO and G3PO mutants, including protein specific activity (U/mg), half-life at 46℃for (min), and percent residual activity of incubation at 46℃for 20min, the comparison data being shown in normalized fashion based on wild type G3 PO; wherein WT represents wild type;
FIG. 2 shows a comparison of properties of wild type G3PO and G3PO combination mutants, including comparison of protein specific activity (U/mg), half-life at 48℃for (min), and percent residual activity at 48℃for 20min, the comparison data being shown in normalized manner based on wild type G3 PO; wherein WT represents wild type;
FIG. 3 shows the SDS-PAGE test result of each sample during purification of the recombinant expressed G3PO mutant exemplified by T90V; wherein M is Marker; lanes 1-9 correspond to the following samples: lane 1, broken supernatant; lane 2, sediment after disruption; lane 3, fluid flow; lane 4, edulcoration; lane 5, 200mM elution; lane 6, 400mM elution; lane 7, dialysate (mix 5 and 6); lane 8, 500mM wash column 1; lane 9, 500mM wash column 2.
Detailed Description
The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
The following description of the preferred embodiments and preferred examples of the present invention will make the technical contents thereof more clear and easier to understand. The present invention may be embodied in many different forms of embodiments and examples, and the scope of the present invention is not limited to only the embodiments and examples set forth herein.
G3PO: glycerophosphate oxidase, EC 1.1.3.21.
G3PO mutant: glycerol phosphate oxidase mutant.
Table 1 shorthand for some terms and Chinese and English full names
Table 2 middle-to-outer translation of partial terms
In the present invention, the G3PO mutant, that is, the glycerophosphate oxidase mutant, is a mutant obtained by genetic engineering techniques based on wild-type G3PO, and is not particularly limited, but refers to a mutant obtained by amino acid substitution.
In the present invention, the fusion protein of the G3PO mutant refers to a protein which is further connected with a fusion tag at the N-terminal and/or the C-terminal of the G3PO mutant and can still exert the G3PO activity. The fusion protein may be a peptide tag or a protein tag in terms of tag size. Functionally, the fusion tag may be at least one of an affinity tag (which may be used for protein purification), a fluorescent tag (which may be used for fluorescent labeling), and the like.
In the present invention, words such as "preferred," "particularly," "specifically," and the like, merely denote preferred or more specific embodiments or examples, and it is to be understood that the invention is not limited to the scope of the claims.
In the present invention, "and/or" means any one or any combination of the listed items.
In the present invention, "combination thereof", "any combination thereof" means any suitable combination of the listed items, so long as the present invention can be smoothly carried out. The number of related items constituting the combination is at least 2.
In the present invention, "combinatorial mutation" refers to a mutation pattern in which at least 2 sites are mutated. The mutants corresponding to the "combinatorial mutations" are designated as "combinatorial mutants". Examples of the combined mutation pattern include two-point, three-point, four-point, five-point, six-point and the like. The combination mutation pattern may be a combination of mutation patterns in which "+" indicates the mutation sites involved. For example, "L4F+E574K" means a two-point mutation obtained by combining the mutation at position 4 L.fwdarw.F and the mutation at position 574 E.fwdarw.K.
The percentage concentration referred to in the present invention, unless otherwise specifically limited or indicated, refers to the final concentration in the relevant system.
In the present invention, wt% refers to mass% concentration.
The technical features mentioned above and below of the present invention may be combined in any suitable manner, as long as there is no contradiction, and the combined technical solution can be implemented smoothly and the technical problem of the present invention can be solved. The technical scheme of the invention can be realized by any suitable mode, so that the technical problem of the invention is solved, and the corresponding technical effect can be realized.
In a first aspect of the present invention, there is provided a glycerophosphate oxidase mutant, which is subjected to site-directed mutagenesis based on the amino acid sequence of a wild type glycerophosphate oxidase, the amino acid sequence of which is shown in SEQ ID No. 4, and in some preferred embodiments of the present invention, the mutagenesis is selected from the group consisting of: D102N, T90V, A363S, F555E, F L, G5483 399K, G399E, A376K, G399P, H92Q, A341T, E D, V209N, S218T, L131V, V198V, V221V, V227V, V228V, V370V, V179V, V113V, V474V, V492V, V464N 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 combined mutation mode is selected from D102N, T90V, A363S, F555E, F L, G P, G399E, A79376K, G399P, H Q, A341T, E D, V218T, L V, V198V, V221V, V227V, V228V, V392V, V370V, V179V, V113V, V474V, V492V, V82 and V, V464N.
In some preferred embodiments of the invention, the mutation pattern is selected from the group consisting of: D102N, T90V, A363S, F555E and combinations comprising the foregoing.
In some preferred embodiments of the invention, the combinatorial mutation pattern is selected from any one of the following combinatorial mutation patterns:
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 (SEQ ID No.: 4) of the wild type G3PO 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 at 46℃ (min), and residual activity percentage at 46℃for 20min of the D102N mutant are 97.35%, 343.92%, 383.41% of the wild-type G3PO, respectively.
In some preferred embodiments of the invention, the protein specific activity (U/mg) of the T90V, A363S and F555E mutants is 92.29% -93.25% of that of the wild type G3PO, the half-life (min) at 46 ℃ is 271.96% -321.69% of that of the wild type G3PO, and the residual activity percentage is 343.78% -378.80% of that of the wild type G3PO after incubation at 46 ℃ for 20 min.
In some preferred embodiments of the invention, the F6L, G P and G399K mutants have protein specific activities (U/mg) of 101.45% -104.82% of wild type G3PO, a half-life (min) at 46℃of 116.40% -182.01% of wild type G3PO, and a residual activity percentage of 153.46% -256.22% of wild type G3PO after incubation at 46℃for 20 min.
In some preferred embodiments of the invention, the protein specific activities (U/mg) of the G399E, A376K, G399P, H Q, A341T, E D and V209N mutants are 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 percent residual activity at 46℃for 20min 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, L V, V198A, T221S, H227R, V228R, E392K, T370S and I179S mutants is 68.67% to 88.67% of wild type G3PO, the half-life (min) at 46℃is 124.34% to 291.53% of wild type G3PO, and the percent residual activity at 46℃for 20min is 183.87% to 337.33% of wild type G3 PO.
In some preferred embodiments of the invention, the protein specific activities (U/mg) of the S501A, K G, Q474F, V492T, S A and Q464N mutants are 21.20% -59.76% of the wild-type G3PO, the half-life at 46℃ (min) is 242.3% -451.85% of the wild-type G3PO, and the residual activity percentage at 46℃for 20min is 247.93% -413.82% of the 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+F555 E+E574K) is increased by more than 10% and the stability is increased by more than 80% compared to wild type G3 PO.
In preferred embodiments of the invention, the stability of some combination 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+A363 S+F363 E+E574K, etc.) is increased by more than 400% while the protein specific activity is maintained substantially unchanged (e.g.g., at least 90% compared to wild type G3 PO).
In some preferred embodiments of the invention, the stability of some combination mutants (e.g., D102N+T90V+G399K+S501A+Q464N, T90V+L315V+A341 T+Q464N, T90V+F555E+A376K+G399E+Q474F, K21G+I179 S+S217T+Q474 F+V492T, V198 A+Edriving +Q364N+S501A, T90V+A363S+Q464N, T90V+A363S+Q474F+F555E, S217A+A360S+Q464N, S267A+Q464N, D102N+T90V+Q464N) is improved by more than 400% compared to wild type G3PO, and the protein specific activity is 30% -80% of wild type.
In some preferred embodiments of the invention, the protein specific activity of some combination mutants (e.g., 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+F555 E+E574K) is increased by a factor of 106.0% to 127.6% compared to wild-type WT.
In some preferred embodiments of the invention, the half-life of some combination mutants (e.g., D102N+T90V+A360S+Q464 N+F555E+E574K, T90V+A363S+E574K, D102N+T90V+Q464N+V492T+F555E+E574K, D102N+T90V+E574K, D102N+T90V+A341T+T370S+Q474F+E574K, A36S+T370K, F6L+G29P+A341 T+A360S+F555 E+E574K) is increased to 386.7% -822.9% at 48℃compared to wild type and the protein specific activity is above 100% of wild type; some combination 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 ℃ increased to 422.7% -695.2% compared to the wild type and protein specific activities of 90% -100% of the wild type; some combined mutants (such as T90V+F255E+A376K+G399E+Q474F, T90V+T2000S+G399E179S, D102N+H25Q+H27R+K113G, E574K+Q464N, D102N+T90V+V492T, G29P+I179 S+V2000A+V228 R+A363S) have a half-life of 343.4-580.7% and a protein specific activity of 80-90% of that of the wild type at 48 ℃; some combined mutants (such as D102N+T90V+G399K+S501A+Q464N, T90V+A363S+Q474F+F555E, D102N+T90V+Q464N) have half lives at 48 ℃ increased to 434.9-627.7% compared with the wild type, and have protein specific activities of 70-80% of the wild type; some combined mutants (such as S82A+A363S+A341T+T370S+Q464N, T90V+Q474F, D102N+V492T) have half lives at 48 ℃ increased to 374.7-518.1% compared with wild type, and have specific protein activities of 60-70%; some combined mutants (such as T90V+L31V+A341 T+Q464N, T90V+A363S+Q464N, D102N+Q464N, S501A+F555E, A363 S+Q464N) have half lives at 48 ℃ increased to 386.7-562.7% compared with the wild type, and have specific activities of 50-60% of the wild type; some combination mutants (e.g., k21g+i179 s+s218t+q474f+v492T, s82a+q464N) have increased half-lives at 48 ℃ to 434.9.7% to 467.5% compared to the wild type, but protein specific activity is less than 50% of the wild type.
In some preferred embodiments of the invention, some single-point or combined mutants such as D102N, T90V and the like have a significant improvement in stability, while the adverse effect on viability is not great, and the specific protein viability can be kept substantially unchanged or changed to a small extent.
In some preferred embodiments of the invention, the protein specific activity, 48 ℃ half-life, and residual activity after 20min incubation of some combination mutants are all greatly improved relative to wild type G3 PO. For example, the six-point combined mutant of D102N+T90V+A363S+Q464N+F555E+E574K improves the specific activity of the protein to 109.3% compared with the wild type, improves the half life at 48 ℃ to 822.9% of the wild type, and greatly improves the residual activity after 20min incubation compared with the wild type.
It should be noted that the application effect of the decrease of the specific activity of the protein on the G3PO mutant can be overcome or alleviated by increasing the enzyme dosage, so that even if the specific activity of the protein is decreased to a certain extent under the condition of remarkably improving the stability, the application is within the protection scope of the invention. For example, the acceptable degree of change in the specific activity of the protein is, for example, 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" herein includes this number. In some preferred embodiments of the invention, the preferred G3PO mutant has a protein specific activity of 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 has a fusion tag attached to the N-terminus and/or C-terminus of the glycerophosphate oxidase mutant 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, reference may also be made to documents Protein Expression and Purification,41 (2005) 98-105 for selection of affinity tags, including but not limited to resin types and elution reagents.
In a third aspect, the invention provides a nucleic acid construct having a nucleotide sequence selected from any one of the following:
(i) A nucleotide sequence encoding the glycerol phosphate oxidase mutant (G3 PO mutant) of the first aspect or the fusion protein of the second aspect; and
(ii) A nucleotide sequence complementary to the nucleotide sequence set forth in (i).
The nucleic acid construct may be DNA, mRNA, or a combination thereof.
The gene of the wild G3PO is used as a template, and a nucleic acid construct encoding the G3PO mutant can be obtained.
The coding sequence of the G3PO mutant of the first aspect, and the coding sequence of the fusion protein of the second aspect are all within the scope of the nucleic acid construct of the third aspect of the invention.
In a fourth aspect, the present invention provides a recombinant vector comprising the nucleic acid construct of the third aspect.
In some preferred embodiments of the invention, the nucleic acid construct of the third aspect is cloned into an expression vector, resulting in a recombinant vector comprising the coding sequence of the G3PO mutant or fusion protein thereof.
In some preferred embodiments of the invention, a nucleic acid construct encoding a G3PO mutant is cloned into an expression vector, resulting in 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 '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 or a combination of His-tag, GST, flag-tag, MBP and other affinity tags.
In the present invention, various vectors known in the art, such as commercially available vectors including plasmids, cosmids, etc., may be used. In producing the G3PO mutants of the invention, the coding sequence of the G3PO mutant may be operably linked to an expression control sequence, thereby forming an expression vector for the G3PO mutant. Such 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 recombinant vector is selected from the group consisting of: pET 28a,pET 30a,pANY1,pQE30,pG-KJE, pGRO7, pKJE7, pG-Tf2, pTf16, pUC18, pUC19 and pGEX, reference is made to the relevant literature Biotechnol Lett (2008) 30:755-762; appl Environ Microb (2002) 68 (1): 263-270, available from the Biovector China center for type culture Collection of plasmid vector strains. These are all known plasmid vectors in the art, and can be known, understood and used by those of ordinary skill in the art.
In a fifth aspect, the invention provides an engineered bacterium, the genome of which is integrated with the nucleic acid construct of the third aspect, or which comprises the recombinant vector of the fourth aspect.
The recombinant vector of the fourth aspect may be used in a episomal form or may be integrated into the genome.
In a sixth aspect, the present invention provides a genetically engineered cell having the nucleic acid construct of the third aspect integrated into its genome or comprising the recombinant vector of the fourth aspect. In some preferred embodiments of the invention, the genetically engineered cell is derived from E.coli.
The recombinant vector of the fourth aspect may be used in a episomal form or may be integrated into the genome.
In some preferred embodiments of the invention, the engineered bacterium is derived from a prokaryote.
In some preferred embodiments of the invention, the engineered bacterium is derived from E.coli.
In some preferred embodiments of the invention, the engineering bacteria are derived from E.coli BL21 (DE 3).
In some preferred embodiments of the 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 vector containing the coding sequence of the G3PO mutant is transformed into a host bacterium, and a recombinant expression strain capable of gene expression of the G3PO mutant can be obtained. The obtained recombinant expression strain contains a nucleic acid construct encoding the G3PO mutant and contains the recombinant vector.
In a seventh aspect, the invention provides a method of screening a mutant glycerophosphate oxidase from a library of mutations, comprising the steps of:
(1) Constructing a mutant library of the wild type G3PO shown as SEQ ID No. 4, designing more than 50 mutants based on rationality and semi-rationality on the basis of the wild type, 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 obtain a library of mutations consisting of G3PO mutants; mutation modes comprise a single-point mutation mode and a combined mutation mode;
(2) Taking a wild G3PO gene as a template to obtain a coding sequence of each G3PO mutant in the mutation library;
(3) Respectively constructing recombinant vectors for each G3PO mutant in the mutation library, transforming into host bacteria to obtain recombinant expression strains, inducing the recombinant expression strains, and expressing to obtain the G3PO mutant of the mutation library or fusion protein (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 a fusion protein of the G3PO mutant of the mutation library;
(4) And (3) carrying out protein specific activity and stability analysis on the G3PO mutant or the fusion protein thereof, and screening to obtain the G3PO mutant or the fusion protein of the second aspect.
The eighth aspect of the present invention provides a method for producing a glycerophosphate oxidase mutant (G3 PO mutant), comprising the steps of:
(1) Cloning the nucleic acid construct of the third aspect onto an expression vector to obtain a recombinant vector containing the coding sequence of the G3PO mutant or 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, and expressing under proper expression conditions to obtain the G3PO mutant or the fusion protein (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 method for preparing a G3PO mutant further comprises performing an isolation and purification step after step (3) to prepare a purified G3PO mutant. The separation and purification may 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 E.coli, and the recombinant expression strain obtained in step (2) is E.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 the inducing precursor, induction at 25℃for 16 hours. Further, in some preferred embodiments of the invention, the inducing precursor substance may be FAD, a nuclease, a riboflavin analog, or a combination thereof. In some preferred embodiments of the present invention, examples of the riboflavin analog include, but are not limited to: riboflavin sodium phosphate, riboflavin potassium phosphate, riboflavin iodide, and the like.
In some preferred embodiments of the invention, an affinity tag may also be added to the 5 '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 or a combination of His-tag, GST, flag-tag, MBP and other affinity tags. And after the connection is correct, the recombinant expression strain (which is the engineering bacterium of the fifth aspect) is obtained by transforming the recombinant expression strain into host bacteria, and the G3PO mutant of the second aspect can be obtained by expression. The expression product can be purified using an affinity tag using an affinity chromatography column. In some preferred embodiments of the invention, G3PO mutants having a purity of greater than 90% can be purified using simple affinity chromatography methods.
The principle of activity detection of the glycerophosphate oxidase mutant product is as follows:
the amount of quinoneimine produced by the reaction can be detected at 555nm using a spectrophotometer.
The enzyme activity of glycerophosphate oxidase and mutants thereof is defined as: 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 invention provides the glycerophosphate oxidase mutant, the fusion protein of the second aspect, the nucleic acid construct of the third aspect, the recombinant vector of the fourth aspect, the engineering bacterium of the fifth aspect and the application of the genetically engineered cell of the sixth aspect in serum triglyceride content detection.
In the present invention, the serum triglyceride content can be detected by a detection method known to those skilled in the art, for example, a detection method described in patent document CN 107991477A can be referred to.
The embodiments of the present invention are described in detail below. The embodiment of the invention is implemented on the premise of the technical scheme of the invention, and a detailed implementation mode and a specific operation process are provided, but the protection scope of the invention is not limited to the following embodiment. Experimental methods (such as PCR amplification, chemical conversion methods, etc.) in which specific conditions are not noted in the following examples are preferably performed according to the methods and conditions of the detailed description section; molecular cloning is then generally carried out according to conventional methods and conditions, for example, as described in Sambrook et al: methods and conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer.
Example 1: construction of glycerophosphate oxidase stability mutation library
Molecular directed evolution is to create a great number of mutant homologous gene libraries by using a modern molecular biology method to artificially simulate a natural evolution mechanism, and to use a sensitive directed screening strategy to create mutant proteins or other biomolecules which do not exist in nature or have some characteristics which are significantly changed. Molecular directed evolution has been widely used for molecular engineering of proteins and is considered as the most efficient method for improving novel protein properties or regulatory sequences. Based on the protein structure and bioinformatics related information of Aerococcus viridans glycerophosphate oxidase, a site-directed saturation mutation library was constructed by applying Consensus Concept theory, and the mutation sites are shown in table 3 and correspond to a series of glycerophosphate oxidase mutants (G3 PO mutants), wherein WT represents a wild type. And (3) respectively designing primers according to the sites, determining the coding sequence of each G3PO mutant by taking wild G3PO as a template, performing PCR amplification, transferring the obtained mutated PCR product into an expression system, constructing a recombinant expression strain, and carrying out sequencing verification to obtain the mutant strain with correct sequencing verification. The mutant strain, i.e., the recombinant expression strain of the G3PO mutant.
TABLE 3 primer sequences for construction of stable mutant libraries
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TABLE 4 Activity and stability test data for single point mutants of G3PO in stability mutant library, normalized data based on data for wild type G3PO
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Example 2: construction of recombinant expression strains of glycerophosphate oxidase (including wild type and mutant)
Cloning glycerophosphate oxidase gene to the expression vector to construct a recombinant vector and a recombinant expression strain.
Wild type G3PO: based on literature investigation and sequence analysis, aerococcus viridans strain contains a wild type gene sequence of glycerol-3-phosphate oxidase (G3 PO), and PCR amplification is carried out by taking the strain genome as a template and designing a primer to obtain a target gene sequence. The expression plasmid is constructed by adopting an enzyme digestion connection method, a BamH I and Xho I double enzyme digestion PCR product is adopted, the purified fragment is connected with a plasmid pET28a which is subjected to BamH I and Xho I double enzyme digestion, and the plasmid pET28a is transformed into E.coli BL21 (DE 3) by adopting a chemical transformation method, wherein the N end of the constructed plasmid is provided with 6 His, and kanamycin resistance is selected for positive cloning. The plasmid of positive clone is extracted by using a commercial plasmid extraction kit, and is subjected to double enzyme digestion identification by BamH I and Xho I to obtain DNA fragments of about 1900bp, and the DNA fragments are subjected to sequencing identification to determine that the gene sequences are correct, so that the construction is successful. Induced expression is carried out under the condition of riboflavin phosphate with the mass concentration of 0.1 percent at 25 ℃ and 0.1mM IPTG, glycerophosphate oxidase is produced, and activity detection and SDS-Page expression verification are carried out.
G3PO mutant: the coding sequences of the G3PO mutants of the mutation library determined in example 1 are cloned on the expression vector independently by the same method, and after constructing the recombinant vector, recombinant expression strains are constructed, so that the recombinant expression strains of the respective G3PO mutants can be obtained.
Example 3: expression, purification and SDS-PAGE validation of glycerophosphate oxidase (including wild type and mutant)
Glycerol phosphate oxidase is a target protein, including wild type G3PO and the G3PO mutants in the mutant library of example 1.
3.1. Expression of glycerophosphate oxidase
Inoculating recombinant expression cell bacterial liquid of the recombinant expression strain constructed in the embodiment 2 into a 10mL LB culture medium test tube with an inoculum size of 0.1%, culturing overnight at 37 ℃, inoculating the seed liquid after overnight culturing into a 100mL LB cone flask culture medium with a ratio of 1:100, shake culturing for 3 hours at 37 ℃, cooling to 25 ℃ by a shaker until the OD600 grows to 0.8-1.0, adding IPTG with a final concentration of 0.1mM, and collecting thalli after induction overnight. The collected thalli are resuspended in cell thalli according to the mass-to-volume ratio of the strains to the resuspension buffer (50 mM Tris-HCl, pH 8.0,0.1M NaCl) of 1:10, and the breaking condition is that the ultrasonic treatment is carried out for 4s, the ultrasonic treatment is stopped for 2s, the power is 40%, and the ultrasonic treatment is carried out for 15min. Centrifuging the liquid after ultrasonic disruption, and obtaining supernatant liquid which is the enzyme liquid for expressing the obtained target protein.
3.2. Purification of glycerophosphate oxidase
Filtering the enzyme solution obtained after ultrasonic disruption in the step 3.1 with a 0.22 mu m filter membrane for later use, preparing a 2mL NTA affinity chromatography packing column, balancing with 10 times of column volume balancing buffer (50 mM Tris-HCl, pH 8.0 and 0.1M NaCl), loading the enzyme solution subjected to membrane filtration, flushing the column with 5 times of column volume of impurity removal buffer (10 mM imidazole, 50mM Tris-HCl and pH 8.7 and 400mM NaCl), eluting target protein with 5 times of column volume of 200mM imidazole eluent, then washing the column with 5 times of column volume of 500mM imidazole buffer, and collecting a purified process sample of each step, wherein the obtained eluent containing the target protein is the imidazole eluent, so that the subsequent analysis is convenient.
3.3. SDS-PAGE test of glycerophosphate oxidase
The wild type glycerophosphate oxidase consists of 611 amino acids, the amino acid sequence is shown as SEQ ID No. 4, and the Molecular Weight (Molecular Weight) is about 66kDa. The 200mM imidazole eluent of the target protein in the above step 3.2 was dialyzed twice against 50mM Tris-HCl at 4℃to remove NaCl and imidazole in buffer. Then, the purified sample and the dialyzed enzyme solution were subjected to 12% SDS-PAGE running test, and the samples were crushed and resuspended in 10mL of buffer corresponding to 1g of cells, and the supernatant and the pellet were diluted 10-fold respectively and then loaded. FIG. 3 shows the SDS-PAGE test of one of the G3PO mutants T90V, wherein lanes 1 to 9 correspond respectively to: lane 1, broken supernatant; lane 2, sediment after disruption; lane 3, fluid flow; lane 4, edulcoration; lane 5, 200mM elution; lane 6, 400mM elution; lane 7, dialysate (mix 5 and 6); lane 8, 500mM wash column 1; lane 9, 500mM wash column 2. As can be seen from FIG. 3, there is a distinct band of the target protein at 66kDa, indicating that the G3PO mutant was successfully expressed in E.coli by heterologous recombination.
Example 4: activity and half-life detection of glycerophosphate oxidase (wild type and mutant)
The reaction solution: 50mM Tris-HCl buffer, pH 8.0, 0.1M disodium glycerophosphate, 75U/mL POD,7.5mM TOOS,7.5mM 4-Aminoantantipyrine.
Enzyme dilution samples: the enzyme solution to be tested is released to 0.15-0.35U/mL by using 20mM Tris-HCl buffer solution, pH 7.5 and 0.2% BSA by mass concentration for detection.
Half-life detection: diluting the enzyme solution purified by the wild type and the mutant to the protein concentration of 1mg/ml by using an enzyme diluent, taking out the single-point mutation in a 46 ℃ water bath kettle for different times, and then placing the single-point mutation on 4 ℃ or ice, and placing the combined mutant in a 48 ℃ water bath for different times and then placing the combined mutant on 4 ℃ or ice; the specific activities of the residual proteins after different times of the water bath were detected according to the following activity detection methods, respectively, and the residual activities after 20 minutes of the water bath were calculated.
The enzyme activity determination method comprises the following steps: after 1mL of the reaction solution was incubated in a spectrophotometer at 37℃for 2min, 0.02mL of enzyme was added to dilute the sample, and a pipette was used to blow the sample uniformly, followed by measurement of the OD change value at 555nm for 1 min. The enzyme activity calculation formula is as follows:
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: mass specific activity (U/mg)
Δa: absorbance change
1.02: total volume of reaction solution (mL);
0.02: enzyme liquid volume (mL);
1: reaction time (min);
1/2:1mol hydrogen peroxide to produce 1/2mol quinone imine dye;
df: dilution factor;
c: enzyme concentration (mg/mL);
39.2: under standard reaction conditions, the chromophore has a millimolar absorbance (cm) at 555nm 2 /μmol)。
Example 5: stable site-directed saturation mutant library screening
The sequenced mutant strain was expressed, purified and validated as per the steps described in example 3. The whole eluted enzyme solution after dialysis was diluted with 50mM Tris-HCl buffer solution of 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 with an ultrafiltration tube. The following three parameters of each mutant were detected according to the viability assay: protein specific activity, half-life at 46 ℃, residual activity after incubation for 20min 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 are normalized based on the value of the wild type G3PO in table 4 for comparison, and the normalized data comparison result is also referred to fig. 1; wherein WT represents a wild type.
Protein specific activity (U/mg), half-life (min) at 46℃and residual activity percentage at 46℃for 20min of the D102N mutant were 97.35%, 343.92% and 383.41% of the wild-type G3PO, respectively. The thermal stability is improved by more than 2 times.
The specific protein activity (U/mg) of the T90V, A363S and F555E mutants is 92.29% -93.25% of that of the wild type G3PO, the half life (min) at 46 ℃ is 271.96% -321.69% of that of the wild type G3PO, and the residual activity percentage of incubation at 46 ℃ for 20min is 343.78% -378.80% of that of the wild type G3 PO. The thermal stability is improved by more than 2 times.
The specific protein activities (U/mg) of F6L, G P and G399K mutants are 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 of incubation at 46 ℃ for 20min 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 compared with 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 the G399E, A376K, G399P, H92Q, A341T, E D and V209N mutants is 90.60% -99.76% of that of the wild type G3PO, the half life (min) at 46 ℃ is 145.50% -210.58% of that of the wild type G3PO, and the residual activity percentage of the incubation at 46 ℃ for 20min is 144.24% -258.99% of that of the wild type G3 PO. Under the condition of obviously improving the thermal stability, the specific activity of the protein is only slightly reduced.
The protein specific activities (U/mg) of S218T, L131V, V198A, T221S, H227R, V228R, E392K, T S and I179S mutants are 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 of incubation at 46 ℃ for 20min is 183.87% -337.33% of wild type G3 PO. The heat stability is improved obviously, but the specific activity of the protein is reduced.
The specific protein activities (U/mg) of the S501A, K113G, Q474F, V492T, S A and Q464N mutants are 21.20% -59.76% of the wild type G3PO, the half life (min) at 46 ℃ is 242.3% -451.85% of the wild type G3PO, and the residual activity percentage at 46 ℃ is 247.93% -413.82% of the wild type G3PO after 20min incubation. Although the improvement in thermostability is very remarkable, the protein specific activity is reduced relatively more.
Wherein, the specific activity of D102N, T, 90V, A363S and F555E mutant reaches more than 90% of wild type G3PO, the half life is increased from 18.9min of wild type G3PO to 51.4-65 min at 46 ℃, and the residual activity of incubation for 20min is increased from 21.7% to 74.6% -83.2%. Among them, D102N is a relatively preferred mutant.
Example 6: construction and screening of a combinatorial mutation library
From the single point mutation results of example 5, it can be seen that mutant D102N, T, 90V, A363S, F, E, F6L, G, 399, E, A, 376, 399, P, H, Q, A, 341, T, E, D, V, 209, N, S, 218T, L131V, V198V, V221V, V227V, V228V, V392V, V370V, V179V, V52113V, V474V, V492V, V464N and the like are improved in activity or half-life compared with wild type G3PO, thus, a series of mutants as shown in Table 5 below were designed, constructed and directly genetically synthesized, and these mutants were characterized according to the expression, purification and detection methods of example 2, example 3 and example 4.
By taking wild type WT as a control, the half-life detection temperature of the detection mutant is increased to 48 ℃, and whether the mutants with different combination modes can further improve the thermal stability of G3PO is observed, meanwhile, the data of each combination mutant of G3PO is normalized for comparison, and the normalized data comparison results are shown in Table 5 and FIG. 2.
From the experimental results, the protein specific activities of the combination mutants of 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 were all improved by more than 1.8 times compared with those of the wild type WT.
From the experimental results, it was also found 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, D200N+T90V+V492T, T90V+F555E+A376K+G399E+Q474F, S501A+F555E, D200N+T90V+E574K, D200N+T90V+A363 S+F555E, S82A+Q464N, S82A+A363S+A341T+T370S+Q464N, K21G+I179 S+S364T+Q474 F+V492T, D200N+T90V+A376 K+G399K+S501A+F555E, t90v+l310v+a341 t+q464N, d102n+h92q+h227r+k113G, a363s+q464N, d102n+t90v+a341t+t370s+q474f+e574K, v198 a+e164d+q464 n+s501A, t90v+f555e+g399p+q464n+s501a+e574K, t90v+a363s+f555e+i179s+v198A, f6l+g29p+a341t+a363s+f555e+e574K, the half-life of the combined mutant of G29P+I217S+V360A+V228 R+A363S, D102N+T90V+A363S+Q464N+F555E+E574K, T90V+T221 S+G399E179S, D102N+T90V+Q464N+V492T+F555E+E574K is improved to 374.7-822.9% compared with the wild type, and the specific activity of the protein is 38.2-109.3% compared with the wild type. Some mutants have more stability improvement, but have relatively reduced activity, for example, the improvement of the stability of the site Q464N is very obvious, but the activity is relatively more reduced; E574K has obvious improvement on enzyme activity, but the stability is drastically reduced; some mutants such as D102N, T90V and the like have obvious stability improvement, have no obvious adverse effect on activity, and can basically keep unchanged or have smaller change amplitude of specific activity.
From the results shown in tables 4, 5, and fig. 1 and 2, it can be seen that the combination mutation containing the preferred single point mutation, in particular, the combination mutation obtained by combining the preferred single point mutations can exert a better effect on the improvement of stability.
The following combinatorial mutants made on the basis of the single point mutations described above do not list all possible permutations, but can be summarized to summarize some sites of paramount importance for stability and protein specific activity, in combination with the data presented in Table 5. In the combination mutation modes shown in Table 5 and FIG. 2, the optimal example is a six-point combination mutant of D102N+T90V+A363S+Q464N+F555E+E574K, the protein specific activity is improved to 109.3% of wild type G3PO, the half-life at 48 ℃ is improved to 822.9% of wild type G3PO, and the stability is greatly improved.
TABLE 5 Activity of partial combinatorial mutants in stability mutant library and half-life at 48℃thereof
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The foregoing describes in detail preferred embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention by one of ordinary skill in the art without undue burden. Therefore, all technical solutions which can be obtained by logic analysis, reasoning or limited experiments based on the prior art by the person skilled in the art according to the inventive concept shall be within the scope of protection defined by the claims.
Claims (10)
1. The glycerophosphate oxidase mutant is characterized in that the glycerophosphate oxidase mutant is subjected to site-directed mutagenesis based on the amino acid sequence of wild type glycerophosphate oxidase, the amino acid sequence of the wild type glycerophosphate oxidase is shown as SEQ ID No. 4, and the mutation mode is selected from the group consisting of: T90V, A363S, F555E, F6L, G P, G399K, G399E, A399 376K, G399P, H Q, A T, E79341T, E79204 209N, S T, L V, V A, T221S, H227R, V228R, E370R, E179R, E113R, E474R, E492R, E464N and combinations comprising the foregoing mutations.
2. The glycerophosphate oxidase mutant of claim 1, wherein the combined mutation pattern is selected from the group consisting of a combination of at least two mutation patterns of T90V, A363S, F555E, F6L, G P, G399E, A376 399 6323 399P, H92Q, A341T, E204D, V209 32218T, L V, V198 221S, H227R, V228R, E392R, E370R, E179R, E501 113R, E474R, E492R, E492N; or alternatively
The mutation mode is selected from the group consisting of: T90V, A363S, F555E and combinations comprising the foregoing.
3. A fusion protein of a glycerophosphate oxidase mutant, characterized in that the fusion protein of the glycerophosphate oxidase mutant is linked with a fusion tag at the N-terminal and/or C-terminal of the glycerophosphate oxidase mutant according to any one of claims 1-2.
4. A nucleic acid construct, wherein the nucleotide sequence of the nucleic acid construct is any one of the following group of nucleotide sequences:
(i) A nucleotide sequence encoding the glycerophosphate oxidase mutant of any of claims 1-2 or the fusion protein of claim 3; and
(ii) A nucleotide sequence complementary to the nucleotide sequence set forth in (i).
5. A recombinant vector comprising the nucleic acid construct of claim 4.
6. The recombinant vector of claim 5, wherein the recombinant vector is of a type selected from the group consisting of: pET 28a,pET 30a,pANY1,pQE30,pG-KJE, pGRO7, pKJE7, pG-Tf2, pTf16, pUC18, pUC19 and pGEX.
7. An engineering bacterium, wherein the genome of the engineering bacterium is integrated with the nucleic acid construct of claim 4, or the engineering bacterium contains the recombinant vector of claim 5 or 6.
8. A genetically engineered cell having the nucleic acid construct of claim 4 integrated into its genome or comprising the recombinant vector of claim 5 or 6.
9. A method for preparing a glycerophosphate oxidase mutant, comprising the steps of:
(1) Cloning the nucleic acid construct of claim 4 onto an expression vector to obtain a recombinant vector containing the coding sequence of the glycerophosphate oxidase mutant or the fusion protein thereof;
(2) Transforming the recombinant vector into host bacteria to obtain recombinant expression strains containing coding sequences of the glycerophosphate oxidase mutant or fusion proteins thereof;
(3) Inducing the recombinant expression strain, and expressing under proper expression conditions to obtain the glycerophosphate oxidase mutant or the fusion protein 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 or 6, the engineering bacterium according to claim 7, and the genetically engineered cell according to claim 8 for detecting serum triglyceride content.
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