CN110628738A

CN110628738A - Method for improving activity of glucose oxidase, mutant and application thereof

Info

Publication number: CN110628738A
Application number: CN201910922705.1A
Authority: CN
Inventors: 钱江潮; 张文玉; 周虎志; 周航成
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2019-09-27
Filing date: 2019-09-27
Publication date: 2019-12-31
Anticipated expiration: 2039-09-27
Also published as: CN110628738B

Abstract

The invention provides a method for improving the activity of glucose oxidase, a mutant and application thereof. Compared with wild type, the mutant has higher enzyme activity, catalytic efficiency and affinity to a substrate. The glucose oxidase mutant provided by the invention has important value for widening the practical application of glucose oxidase.

Description

Method for improving activity of glucose oxidase, mutant and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for improving activity of glucose oxidase, a mutant and application thereof.

Background

Glucose oxidase (. beta. -D-glucose: oxygen 1-oxide glucose, GOD, EC 1.1.3.4) is a flavoprotein that oxidizes glucose to hydrogen peroxide and glucono-delta-lactone using molecular oxygen as an electron acceptor, followed by spontaneous hydrolysis to gluconic acid (Pluschkell S et al, Kinetics of glucose oxidase interaction by recombinant Aspergillus niger. Biotechnology & bioengineering 2015,51(2): 215-. GOD has wide application in the Food industry as a Food preservative and color stabilizer (Parpinello GP et al. preferably Study on Glucose Oxidase-calcium Enzyme System to control the Browning of application and Pear Press, LWT-Food Science and technology.2002,35(3):239-243), and Gluconic acid (Cui C et al. genipin Cross-Linked Glucose Oxidase and calcium Multi-Enzyme for Glucose acid synthesis. applied Biochem Biotechnology.2017, 181(2): 526; Rico-Rodrigue F. Imperial of Glucose Oxidase and Glucose Oxidase, Glucose, hu Q et al, simulation of Liquid-Crystal-Based Optical Sensing Platform for Detection of Hydrogen Peroxide and blood glucose analytical chemistry.2018,90(19) 11607-; ho J A et al, development of a long-life enzyme biosensor for the determination of the degree of bloodglucose. Talanta.2007,71(1): 391) 396.

GOD has wide application, and has important practical significance for qualitatively evolving GOD protein so as to improve the catalytic activity and stability of enzyme along with the success of exogenous expression of GOD in yeast. The amino acid sequence and protein structure of the enzyme are changed through gene mutation, and the molecular modification is carried out on the enzyme, so that the characteristic of the protein can be possibly improved.

In conclusion, since GOD has a good application prospect, there is a need in the art to improve the performances of enzyme activity, stability and the like, so as to generate higher application value.

Disclosure of Invention

The invention aims to provide a method for improving the activity of glucose oxidase, a mutant of the glucose oxidase and application thereof.

In the first aspect of the invention, a glucose oxidase mutant is provided, the amino acid sequence of which corresponds to the amino acid sequence shown in SEQ ID NO. 1, Val is mutated into Trp at the 20 th position, and Thr is mutated into Val at the 30 th position; mutation of Val to Trp at position 20 and mutation of Thr to Trp at position 30; mutation from Val to Met at position 20 and mutation from Thr to Ser at position 30; or the 20 th position is mutated from Val to Cys, and the 30 th position is mutated from Thr to Ser.

In a preferred embodiment, the amino acid sequence of the mutant corresponds to SEQ ID NO 1, with a mutation from Val to Trp at position 20 and Thr to Val at position 30.

In a further aspect of the invention there is provided an isolated polynucleotide encoding a mutant as described in any one of the preceding.

In another aspect of the invention, there is provided a vector comprising said polynucleotide.

In another aspect of the invention, there is provided a genetically engineered host cell comprising said vector, or having said polynucleotide integrated into its genome.

In another preferred embodiment, the host cell comprises: a prokaryotic cell or a eukaryotic cell; preferably, the eukaryotic host cell comprises: yeast cells, fungal cells, insect cells, mammalian cells, and the like; the prokaryotic host cells comprise escherichia coli, bacillus subtilis and the like; more preferably, the eukaryotic cells comprise yeast cells.

In another preferred embodiment, in the vector, the 3' end of the polynucleotide further comprises a signal peptide and/or a promoter.

In another preferred embodiment, the host cell is a yeast cell, and the signal peptide includes but is not limited to: saccharomyces cerevisiae alpha mating factor (alpha-MF), Pichia acid phosphatase (PHO1) signal peptide, etc.

In another preferred embodiment, the host cell is a yeast cell, and the promoter includes but is not limited to: AOX promoter, and the like.

In another aspect of the present invention, there is provided a method for producing the mutant of glucose oxidase, comprising the steps of: (1) culturing said host cell to obtain a culture; and (2) isolating the glucose oxidase mutant from the culture.

In another aspect of the invention there is provided the use of a glucose oxidase mutant as described in any preceding claim, to catalyse the oxidation of glucose to hydrogen peroxide and glucono-delta-lactone, or to gluconic acid.

In another aspect of the present invention, there is provided a method of increasing an enzymatic activity, a catalytic efficiency, or a substrate affinity of glucose oxidase, the method comprising: subjecting glucose oxidase to a mutation selected from the group consisting of: mutation from Val to Trp at position 20 and mutation from Thr to Val at position 30; mutation of Val to Trp at position 20 and mutation of Thr to Trp at position 30; mutation from Val to Met at position 20 and mutation from Thr to Ser at position 30; or the 20 th position is mutated from Val to Cys, and the 30 th position is mutated from Thr to Ser.

In a preferred embodiment, compared with wild-type glucose oxidase, the enzymatic activity of the glucose oxidase mutant is improved by more than 1.5 times; preferably improved by more than 2.5 times; more preferably 2.9 times or 3.9 times.

In another preferred example, the glucose oxidase mutant has significantly improved catalytic efficiency or substrate affinity compared to wild-type glucose oxidase.

In another aspect of the present invention, there is provided a composition for oxidizing glucose, comprising a glucose oxidase mutant as described in any of the above, and a dietetically, pharmaceutically or industrially acceptable carrier.

In another aspect of the present invention, there is provided a kit for oxidizing glucose, comprising the composition; or, a mutant comprising any of the foregoing glucose oxidases.

Other aspects of the invention will be apparent to those skilled in the art in view of this disclosure.

Drawings

FIG. 1, high throughput screening procedure.

FIG. 2, GOD mutation library construction process.

FIG. 3, colony PCR verification of mutant recombinant bacteria. Lane M: 2000; lane 1-12: pPIC9K-GODF/pPIC9K-GODR is used as a primer, and the mutant recombinant bacteria are verified by PCR.

FIG. 4 shows the relative extracellular enzyme activity of the recombinant bacteria.

FIG. 5 shows the growth curve of GOD high-enzyme-activity mutant

FIG. 6 shows the extracellular GOD yield of GOD-high-enzyme-activity mutant bacteria

FIG. 7 is a protein electrophoresis chart of a sample before and after column chromatography. Lane M: 200 kDa; lane 1, 2: G/GOD samples; lane 3, 4: G/G30B sample; lane 1, 3: before column chromatography; lane 2, 4: after column chromatography.

Detailed Description

The present inventors have conducted intensive studies and have revealed a method for improving the enzymatic activity of glucose oxidase by performing amino acid mutations at a part of the sites, i.e., 20 th and 30 th positions. On the basis, the glucose oxidase mutant is obtained, and compared with a wild type, the mutant has higher enzyme activity, catalytic efficiency and affinity to a substrate. The glucose oxidase mutant provided by the invention has important value for widening the practical application of glucose oxidase.

As used herein, unless otherwise indicated, the terms "glucose oxidase mutant", "MGOD", "mutant glucose oxidase", "mutant of the invention" are used interchangeably and refer to a mutation from Val to Trp at position 20 and Thr to Val at position 30, corresponding to wild-type glucose oxidase (e.g., SEQ ID NO: 1); mutation of Val to Trp at position 20 and mutation of Thr to Trp at position 30; mutation from Val to Met at position 20 and mutation from Thr to Ser at position 30; or a protein formed by mutating Val to Cys at the 20 th position and Thr to Ser at the 30 th position. Preferably, the protein is formed by mutating Val to Trp at position 20 and Thr to Val at position 30.

If desired, a wild-type glucose oxidase, which will be designated as "wild-type glucose oxidase", "GOD" or "wild-type protein", has the amino acid sequence of SEQ ID NO: 1.

As used herein, the "glucose oxidase (or mutant) activity" is defined as: the enzyme amount for catalyzing 1 mu mol of beta-D-glucose to generate gluconic acid per minute at the temperature of 37 ℃ is one enzyme activity unit U.

As used herein, "isolated" refers to a substance that is separated from its original environment (which, if it is a natural substance, is the natural environment). If the polynucleotide or protein in the natural state in the living cell is not isolated or purified, the same polynucleotide or protein is isolated or purified if it is separated from other substances coexisting in the natural state. By "isolated glucose oxidase mutant" is meant a glucose oxidase mutant that is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. One skilled in the art can purify glucose oxidase mutants using standard protein purification techniques. Substantially pure proteins produce a single major band on a non-reducing polyacrylamide gel.

As used herein, "recombinant" refers to a protein, a genetically engineered vector or cell, or the like, that is obtained (or prepared in large quantities) by means of genetic engineering.

The protein of the present invention may be a recombinant protein, a natural protein, a synthetic protein, preferably a recombinant protein. The proteins of the invention may be naturally purified products, or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plant, insect, and mammalian cells).

The invention also includes fragments, derivatives and analogues of the glucose oxidase mutants. As used herein, the terms "fragment," "derivative," and "analog" refer to a protein that retains substantially the same biological function or activity as the native glucose oxidase mutant of the present invention. A protein fragment, derivative or analog of the invention may be (i) a protein in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a protein having a substituent group in one or more amino acid residues, or (iii) a protein in which an additional amino acid sequence is fused to the protein sequence (e.g., a leader or secretory sequence or a sequence used to purify the protein or a pro-protein sequence, or a fusion protein). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the definitions herein. However, in the amino acid sequences of the glucose oxidase mutant and the fragments, derivatives and analogs thereof, the 20 th position is mutated from Val to Trp, Met or Cys corresponding to wild-type glucose oxidase; the 30 th position is mutated from Thr to Val, Trp or Ser.

In the present invention, the term "glucose oxidase mutant" also includes (but is not limited to): deletion, insertion and/or substitution of several (usually 1 to 20, more preferably 1 to 10, still more preferably 1 to 8, 1 to 5, 1 to 3, or 1 to 2) amino acids, and addition or deletion of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. The term also includes active fragments and active derivatives of glucose oxidase mutants. However, in these variants, Val is mutated to Trp, Met or Cys at position 20, corresponding to wild-type glucose oxidase; the 30 th position is mutated from Thr to Val, Trp or Ser.

The invention also provides a polynucleotide sequence for encoding the glucose oxidase mutant or conservative variant protein thereof.

The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.

The polynucleotides encoding the mature proteins of the mutants include: a coding sequence that encodes only the mature protein; the coding sequence for the mature protein and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature protein.

The term "polynucleotide encoding a protein" may include a polynucleotide encoding the protein, and may also include additional coding and/or non-coding sequences.

The invention also relates to variants of the above polynucleotides which encode proteins having the same amino acid sequence as the present invention or fragments, analogues and derivatives of the proteins. The variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the protein encoded thereby.

The full-length nucleotide sequence or the fragment of the glucose oxidase mutant can be obtained by a PCR amplification method, a recombination method or an artificial synthesis method. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly open reading frame sequences, and the sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

At present, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) have been obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

The invention also relates to a vector containing the polynucleotide of the invention, a host cell produced by genetic engineering by using the vector or the coding sequence of the glucose oxidase mutant of the invention, and a method for producing the protein of the invention by using a recombinant technology.

The polynucleotide sequences of the present invention may be used to express or produce recombinant glucose oxidase mutants by conventional recombinant DNA techniques (Science, 1984; 224: 1431). Generally, the following steps are performed:

(1) transforming or transducing a suitable host cell with a polynucleotide (or variant) of the invention encoding a glucose oxidase mutant, or with a recombinant expression vector comprising the polynucleotide;

(2) a host cell cultured in a suitable medium;

(3) isolating and purifying the protein from the culture medium or the cells.

In the present invention, the glucose oxidase mutant polynucleotide sequence may be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector well known in the art. In general, any plasmid or vector can be used as long as it can replicate and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translation control elements.

Methods well known to those skilled in the art can be used to construct expression vectors containing DNA sequences encoding glucose oxidase mutants and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or kanamycin or ampicillin resistance for E.coli.

Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. Representative examples are: escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; plant cells, and the like. In higher eukaryotic cells, transcription is enhanced if enhancer sequences are inserted into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs, that act on a promoter to increase transcription of a gene.

In a preferred form of the invention, the polynucleotide is expressed in a yeast cell, preferably a Pichia cell. Preferably, the 3' end of the polynucleotide further comprises a signal peptide and/or a promoter. Such signal peptides include, but are not limited to: saccharomyces cerevisiae alpha mating factor (alpha-MF), Pichia acid phosphatase (PHO1) signal peptide, etc. Such promoters include, but are not limited to: AOX promoter, and the like.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl₂Methods, the steps used are well known in the art. Another method is to use MgCl₂. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The obtained transformant can be cultured by a conventional method to express the protein encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The recombinant protein in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

In the specific embodiment of the invention, the GOD protein is modified, and a strategy of combining irrational design and semi-rational design is adopted to carry out directed evolution on the GOD gene so as to improve the activity of the GOD. GOD mutant bacteria with high enzyme activity and corresponding mutant GOD genes are obtained by a high-throughput screening method. Experimental results show that a high-throughput screening method for rapidly and efficiently screening GOD mutant bacteria is established by using a pore plate culture method, and the feasibility and the accuracy of the high-throughput screening method are verified by comparing with a shake flask culture result. The GOD gene is artificially evolved in error-prone PCR and site-directed saturation mutagenesis modes respectively, and an expression vector and a recombinant cell library containing the mutant GOD gene are constructed. A large number of mutant transformants are screened by a high-throughput method, and mutant bacteria with enzyme activity obviously higher than that of original bacteria (G/GOD) are obtained. Selecting high-enzyme-activity mutant strains, sequencing to verify mutant sequences and copy number determination, and obtaining GOD mutant bacteria G/G30A, G/G30B, G/G31A and G/G31B with improved enzyme activity. Among them, the recombinant strain G/G30B with the mutation (V20W + T30V) has the highest extracellular enzyme yield. The characteristics of the 4 high-enzyme-activity mutant bacteria are examined in a shake flask, the unit cell enzyme activities of G/G30A, G/G31A and G/G31B are 1686.7, 2338.0, 2419.7 and 3004.3U/g.DCW respectively, and are 2.2, 3.1, 3.2 and 4.0 times of G/GOD. The mutant MGOD (V20W + T30V) is separated from the fermentation liquor of G/G30B, the Km value is reduced by 7.8%, the Kcat and Kcat/Km values are respectively improved by 7.4% and 16.4%, and the affinity and catalytic efficiency of the enzyme are slightly improved compared with the non-mutated GOD.

Glucose oxidase can catalyze the oxidation of glucose to produce hydrogen peroxide and glucono-delta-lactone, or to produce gluconic acid. Thus, the glucose oxidase mutants of the present invention can be applied in a wide range of fields including, but not limited to: producing gluconic acid; as food preservatives and color stabilizers; for the production of hydrogen peroxide for textile bleaching; used for manufacturing a glucometer for detecting the blood sugar concentration of a diabetic patient and the like, and has more ideal enzyme activity, catalytic efficiency or substrate affinity compared with the wild type.

The invention also provides a composition for oxidizing glucose, which comprises the glucose oxidase mutant disclosed by the invention and a dietetic, pharmaceutical or industrial acceptable carrier.

The invention also provides a kit for oxidizing glucose, comprising the composition; or a mutant containing the glucose oxidase.

In conclusion, the mutant strain with significantly improved GOD enzyme activity is obtained by various directed evolution strategies, and a novel strain with high productivity is provided for industrial large-scale production through subsequent fermentation optimization and amplification, so that the mutant strain has industrial application value.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Materials and methods

1. Plasmids and strains

The plasmids and strains used and constructed in the present invention are shown in Table 1.

TABLE 1 strains and plasmids

The amino acid sequence encoded by the wild-type GOD gene (Uniprot: P13006, without the signal peptide sequence at position 22) is shown in SEQ ID NO:1 (from Aspergillus niger):

SNGIEASLLTDPKDVSGRTDYIIAGGGLGLTTAARLTENPNISVLVIESGSYESDRGPIIEDLNAYGDIFGSSVDHAYETVELATNNQTALIRSGNGLGGSTLVNGGTWTRPHKAQVDSWETVFGNEGWNWDNVAAYSLQAERARAPNAKQIAAGHYFNASCHGVNGTVHAGPRDTGDDYSPIVKALMSAVEDRGVPTKKDFGCGDPHGVSMFPNTLHEDQVRSDAAREWLLPNYQRPNLQVLTGQYVGKVLLSQNGTTPRAVGVEFGTHKGNTHNVYAKHEVLLAAGSAVSPTILEYSGIGMKSILEPLGIDTVVDLPVGLNLQDQTTATVRSRITSAGAGQGQAAWFATFNETFGDYSEKAHELLNTKLEQWAEEAVARGGFHNTTALLIQYENYRDWIVNHNVAYSELFLDTAGVASFDVWDLLPFTRGYVHILDKDPYLHHFAYDPQYFLNELDLLGQAAATQLARNISNSGAMQTYFAGETIPGDNLAYDADLSAWTEYIPYHFRPNYHGVGTCSMMPKEMGGVVDNAARVYGVQGLRVIDGSIPPTQMSSHVMTVFYAMALKISDAILEDYASMQ

2. culture medium

LB culture medium: 10g/L yeast extract, 20g/L peptone and 10g/L NaCl.

YPD medium: tryptone 20g/L, glucose 20g/L, yeast extract 10 g/L.

BMGY growth medium: tryptone 20g/L, YNB 13.4g/L, glycerol 10g/L, biotin 4X 10^-4K of g/L, 0.1M pH 6.0₂HPO₄/KH₂PO₄And (4) a buffer solution.

BMMY induction medium: tryptone 20g/L, YNB 13.4g/L, biotin 4X 10^-4g, K at pH 6.0 at 0.1M₂HPO₄/KH₂PO₄And (4) a buffer solution. When the expression is induced, 1% methanol is supplemented every 24 h.

3. Reagents and primers

The standard GOD was obtained from Sigma and the o-dianisidine was obtained from Merrel Chemicals, Inc., Shanghai. Other conventional reagents are imported or domestic analytical purifiers.

Super-Fidelity DNA Polymerase, Clonexpress II One Step Cloning Kit was purchased from Biotech Inc. of Nanjing Nodezam. The column type yeast total RNA extraction and purification kit is purchased from Shanghai Biotechnology Limited. The plasmid extraction kit, the PCR purification kit and the gel recovery kit are purchased from AXYGEN company.

The DNA primers are shown in Table 2.

TABLE 2 primers

Note: n in the table represents base A, T, C, G; k represents base T, G; m represents base A, C.

4. Culture method

Coli seed culture: selecting single colony to culture seed in test tube, liquid content of culture medium is 15%, culturing overnight at 37 deg.C and 220 r/min.

Culturing passtoris seeds: single colonies were picked from the plates and inoculated into 3mL YPD tubes and cultured at 30 ℃ at 220r/min for 16-18 h.

And P, culturing passtoris by fermentation: inoculating 25mL BMGY medium from the seed solution, and culturing at 30 deg.C and 220r/min to OD₆₀₀Centrifuging for 5min between 4 and 6 to collect all thalli, resuspending the thalli by BMMG culture medium, and adjusting OD₆₀₀About 1, then transferred to a 500mL shake flask containing 50mL BMMY medium for induction culture, sampled every 24h and supplemented with 1% methanol.

5. Cloning of the target Gene

The PCR reaction system is as follows: 2 × Max Buffer 25 μ L, 10mmol/L dNTP Mix 1 μ L, Phanta Max Super-Fidelity DNA Polymerase 1 μ L(or rTaq enzyme), 2. mu.L of each of 10. mu. mol/L of the forward and reverse primers, 1. mu.L of the template (50-100 ng/. mu.L), ddH₂O 18μL。

The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 15s, annealing at 58 ℃ for 30s, extension at 72 ℃ (1min/kb), 30 cycles. PCR product purification, gel cutting recovery procedures refer to kit instructions.

6. Construction of GOD mutant plasmids

And obtaining the GOD mutant gene by adopting site-directed saturation mutation. Primers containing NNK degenerate bases were synthesized for selected sites using plasmid pPIC9K-GOD as template, e.g., saturation mutagenesis for V20 and T30-I94, and the primers used are shown in Table 2. Using the designed degenerate primers, the target plasmid was PCR-amplified using Phanta Max Super-Fidelity DNA Polymerase, and the PCR amplification product was digested with Dpn I restriction enzyme to remove the methylated template plasmid. Recombination reaction can occur at the 5 'end and the 3' end of the amplification product under the catalysis of ExnaseMultiS, and the cyclization process of the amplification product is completed to obtain the mutant plasmid (the kit is Mut)Multis Fast Mutagenesis Kit V2)。

7. Construction and expression of recombinant GOD engineering bacteria

The linearized mixed plasmid after enzyme digestion is transferred into pichia pastoris GS115 competent cells by electric shock, and an electrotransfer instrument (Micro Pluser) is used^TMBio-Rad), electrotransfer conditions: 1500V, 5-6 ms. After electric shock transformation, the transformant was obtained by plating on YPD plates having hygromycin resistance and culturing in a 30 ℃ incubator for 48 hours. Positive transformants were picked and subjected to high throughput screening. Culturing the mutant strain obtained by screening at 30 ℃ for 24h at 220r/min, and transferring to a shake flask for P.

8. Measurement of Dry weight of cells

Taking fermentation liquor, diluting to a certain multiple, and measuring OD of thallus₆₀₀According to OD₆₀₀And dry weight relationship: DCW (g/L) ═ 0.24 XOD₆₀₀+1.23(R²0.994), DCW is calculated.

9. GOD enzyme activity assay

Collecting 1mL of liquid Yeast cells, centrifuging to remove supernatant, adding precooled sterile water into the precipitate to resuspend and wash the bacteria, centrifuging to remove supernatant, then adding 25 muL of Yeast Protein Extraction Reagent into the bacteria to resuspend the precipitate, putting the precipitate into a 30 ℃ water bath for warm bath for 1h, gently oscillating for 2-3 times during the warm bath, then fixing the volume to 1mL, centrifuging, taking the supernatant, and determining the intracellular enzyme activity.

GOD enzyme activity assay reference is made to Gao Z, et al Biotechnol Lett.2012,34(3):507- "514). GOD enzyme activity definition: the enzyme amount for catalyzing 1 mu mol of beta-D-glucose to generate gluconic acid per minute at the temperature of 37 ℃ is one enzyme activity unit U.

10. Yeast genome DNA extraction

And (3) centrifugally collecting thalli, extracting the yeast genome DNA by using a column type yeast genome DNA extraction and purification kit, and operating the operation method according to the kit specification.

Diluting the obtained genome DNA to a proper concentration, performing fluorescent quantitative PCR as a template, and determining the copy number of the GOD gene by using a primer RT-ARG4-F/RT-ARG4-R, RT-GOD-F/RT-GOD-R. The kit used in the reaction is TBGreen^TMPremix Ex Taq^TMII kit (TaKaRa), the primers used are shown in Table 2, the apparatus is CFX96 Real-Time PCR of Bio-Rad, the reaction condition is pre-denaturation at 95 ℃ for 30 s; 5s at 95 ℃, 30s at 57 ℃ and 40 cycles; 95 ℃ for 10s and finally 60 ℃ for 30 s. The transcription level is calculated byARG4 was used as an internal reference gene.

11. Separation and purification of recombinant glucose oxidase GOD

(1) Ethanol precipitation

The cultured fermentation supernatant was subjected to refrigerated centrifugation at 12,000rpm at 4 ℃ for 5min, and the supernatant was collected. Mixing the supernatant with 4 times of anhydrous ethanol, standing, centrifuging to remove supernatant, collecting precipitate, resuspending with deionized water, and filtering with 0.22 μm microporous membrane.

(2) DEAE Aogalose FF ion exchange chromatography

After column pretreatment of DEAE Aquarose FF resin, the ion exchange column was equilibrated by washing about 10 column volumes with 0.05mol/L Tris-HCl buffer pH 7.1. The enzyme solution obtained in the previous step was slowly loaded at a flow rate of 1mL/min, and the permeant protein on the unadsorbed column was collected. And then carrying out gradient elution by using 0-0.3 mol/L NaCl solution (prepared by using 0.05mol/L Tris-HCl buffer solution with the pH value of 7.1) at the flow rate of 1mL/min, collecting effluent liquid, collecting 10mL of effluent liquid in each tube, freezing and storing at 4 ℃ for later use, and measuring the GOD enzyme activity in each tube.

Example 1 Strain screening

1. Establishment and validation of high throughput screening methods

In order to rapidly and efficiently screen GOD mutant strains, the inventor utilizes a multi-plate culture to establish a high-throughput method suitable for screening GOD secretion expression recombinant bacteria. The screening process designed by the inventor is shown in figure 1, after obtaining mutant bacteria secreting and expressing GOD, single colonies are picked from a transformation plate of MD, and high-throughput screening is carried out according to two stages of pre-culture and main culture, wherein the culture media are BMGY and BMMY respectively. In the preculture stage, the mutant strain was inoculated into a 96-well plate containing 300. mu.L of BMGY culture medium, cultured at 30 ℃ for 24 hours at 250rpm, 150. mu.L of the strain was aspirated for main culture, and the preculture plate was temporarily stored in a refrigerator at 4 ℃. The main culture was performed in 48-well plates containing 900. mu.L of BMMY induction medium per well, inoculated and cultured at 30 ℃ for 24h at 220rpm, and the culture medium was collected to determine the concentration of the bacteria and the extracellular enzyme activity. After the high-enzyme-activity strains are obtained by screening, corresponding mutant strains are selected from a pre-culture plate preserved at 4 ℃ for culture and seed preservation.

When the GOD gene is subjected to directed evolution, the inventor carries out a large amount of sequence analysis, comparison and verification work, considers and analyzes key amino acids around a binding site of coenzyme FAD and a substrate in a GOD structure, and carries out mutation on the amino acids to introduce new salt bridges and other strategies.

Firstly, according to the literature report, the T554M mutation of GOD can introduce a sulfur-pi action in the protein structure, and the Y509E and H172K mutations can help to introduce a new salt bridge near the interface of the dimer protein structure, thereby improving the activity and the thermal stability of the enzyme. The inventor designs primers T554M-F/T554M-R, Y509E-F/Y509E-R and H172K-F/H172K-R respectively, carries out site-directed mutagenesis on the 3 sites of GOD, and constructs corresponding mutant strains. Through the designed high-throughput screening process and shake flask fermentation, the influence of the mutation site on the GOD activity is inspected, and the feasibility of the high-throughput screening method is verified.

After the mutant strain is constructed, selecting monoclonal mutant bacteria for culturing, verifying that the recombinant bacteria genome has integrated GOD genes through PCR, sequencing to confirm that designed mutation occurs, extracting yeast genome DNA, and determining the copy number of the GOD genes by taking ARG4 as an internal reference gene (the used primer is RT-ARG4-F/RT-ARG4-R, RT-GOD-F/RT-GOD-R).

Then, mutant bacteria which verify that the GOD gene is single copy and are subjected to sequencing verification are selected, and the mutant bacteria concentration and the GOD extracellular enzyme activity are determined through a high-throughput screening pore plate culture process, and the results are shown in table 3. And comparing the relative enzyme activity of the extracellular GOD of the mutant by taking the G/GOD as a control bacterium, wherein the volume enzyme activity of the extracellular GOD of the mutant is G/Y509E > G/H172K > G/T554M, but the extracellular enzyme activity of the three mutant strains is less than that of the G/GOD (0.450U/mL). Therefore, the results of the well plate screening show that the extracellular enzyme activity of the three mutant bacteria is not improved.

Meanwhile, the three mutant strains are subjected to induced fermentation in a shake flask by taking G/GOD as a control, and the results are shown in Table 3. According to the results of shake flask culture, the growth trends of the three mutant strains are basically consistent with those of a control, the extracellular enzyme activities of the strains are all lower than G/GOD, the volume enzyme activity yield is reduced according to the sequence of G/Y509E, G/H172K and G/T554M, and compared with the G/GOD, the relative level change trend is close to the pore plate screening trend.

TABLE 3 extracellular enzyme activity of site-directed mutants cultured in well plates and shake flasks

Bacterial concentration and enzyme activity were both results at the end of the culture, relative enzyme activity was calculated using G/GOD as a control.

The extracellular enzyme activity yield obtained by shake flask culture of the recombinant strain is consistent with the result of pore plate culture, which indicates that the constructed high-throughput method can be used for screening GOD mutant strains.

2. GOD gene mutation and construction of recombinant bacteria cell bank

The above results demonstrate the effectiveness of the established high throughput screening method, but also indicate that the reported site-directed mutagenesis site is not necessarily effective in increasing the activity of a particular GOD. Thus, the inventors selected other sites to perform single site or combination of different sites to modify the GOD gene (Table 4).

TABLE 4 selection of mutation sites

After obtaining the mutant GOD gene, the mutant gene is used for replacing the original GOD gene in a secretion expression vector pPIC9K-GOD, GS115 is transformed, and a recombinant bacterial cell bank containing the mutant GOD gene is constructed (figure 2).

After transforming P.pastoris GS115 competent cells by electric shock, plating on an MD plate, carrying out inverted culture for 3-4 days, selecting positive transformants for screening mutant bacteria, carrying out colony PCR verification by taking pPIC9K-GODF/pPIC9K-GODR as primers to obtain a strip with the size of 1956bp, and according with an expected result (figure 3), thus proving that the construction of a mutant GOD cell bank inserted in recombinant bacteria is successful.

3. Screening of high-enzyme-activity mutant bacteria

After obtaining the recombinant strain containing the mutant GOD, screening the mutant strain with high enzyme activity by using a high-throughput method, and screening 3009 mutant strains together. After the activity of the recombinant bacterium is determined, the single copy strain G/GOD containing the non-mutated GOD gene is used as a reference bacterium, the extracellular GOD enzyme activity of the single copy strain G/GOD is used as a reference standard, the screened mutant bacterium enzyme activity is normalized, and the relative enzyme activity is calculated. The extracellular GOD relative enzyme activity of all mutant bacteria is counted, and the relative enzyme activity of more than 90% of recombinant bacteria is below 1.0, which indicates that most of mutations do not obtain positive results. And 51 strains with relative enzyme activity of more than 1.5 account for 1.7 percent of the total screening quantity, wherein the relative enzyme activity of 31 strains with relative enzyme activity of more than 1.7 and 11 strains reaches more than 2 (figure 4).

In order to eliminate the possibility of enzyme activity increase caused by GOD gene copy number increase in mutant bacteria, GOD gene copy number determination is required. And (3) determining the copy number of the GOD gene of the mutant bacteria with the relative enzyme activity value of more than 1.5. 4 single copy GOD recombinant bacteria with the highest yield are selected from high enzyme activity mutant bacteria and named as G/G30A, G/G30B, G/G31A and G/G31B, wherein the relative enzyme activity of G/G31B is the highest and reaches 2.17 times of that of G/GOD, and G/G30A is slightly lower and is 1.83 times of that of G/GOD (Table 5). After genotype verification, the mutants are all obtained by simultaneous saturation mutation of V20 and T30, wherein the V20 and T30 sites are replaced by different amino acids. Therefore, the GOD enzyme activity of the mutant bacteria is improved due to the GOD gene sequence mutation, but not due to the increase of the copy number of the GOD gene.

TABLE 5 relative enzyme Activity and genotype of the mutant bacteria

Bacterial strains	Relative extracellular enzyme activity	Mutation site	GOD Gene copy number
				G/GOD(control)	1	-	1
G/G30A	1.83	V20C+T30S	0.74
				G/G30B	2.15	V20W+T30V	0.89
G/G31A	2.05	V20M+T30S	0.96
				G/G31B	2.17	V20W+T30W	0.81

4. Investigation of physiological characteristics of mutant bacteria

And (3) taking the recombinant strain G/GOD containing the non-mutated GOD gene as a control strain, and inspecting the growth and enzyme production characteristics of the recombination in a shake flask. The growth trends of the mutant G/G30B are basically consistent, while the growth of the mutant G/G30A, G/G31A and G/G31B is slightly lower than that of the control bacteria, but the difference is not large (figure 5), which shows that the GOD gene mutation has no obvious influence on the growth of thalli.

And (3) determining the extracellular GOD enzyme activity of the recombinant bacteria in the culture process, wherein the extracellular GOD enzyme activity of the mutant bacteria G/G30B is obviously higher than that of other mutant bacteria as shown in figure 6. When the mutant strain G/G30B is induced for 168 hours, the unit bacterial enzyme activity of the mutant strain G/G30B reaches 3004.3U/g.DCW, which is 4.0 times of the GOD gene non-mutant strain G/GOD. The unit bacterial enzyme activities of mutant bacteria G/G30A, G/G31A and G/G31B reach the maximum at 144h of induction, are respectively 1686.7, 2338.0 and 2419.7U/g.DCW, and are 2.2, 3.1 and 3.2 times of the development bacteria G/GOD (754.3U/g.DCW).

And when the mutant strain is induced for 168 hours, the enzyme activity of the extracellular unit thallus of the G/GOD reaches the maximum value, and the extracellular volume enzyme production level of each mutant strain is compared. As shown in Table 6, the mutant G/G30B showed the highest extracellular volume enzyme activity, 31.89U/mL, which was 3.9 times higher than that of the non-mutant G/GOD. The GOD mutant of V20W + T30V is obtained by adopting double-site saturation mutation, and is expressed in recombinant strain G/G30B, and the extracellular enzyme activity of unit thallus can reach 3004.3U/g.DCW, which is 4.0 times of that of G/GOD.

TABLE 6 extracellular GOD volume enzyme Activity of 168h mutant bacteria

Example 2 investigation of basic Properties of high enzyme-Activity GOD mutant enzymes

The extracellular enzyme activity of the mutant G/G30B reaches about 4 times of that of G/GOD, and the reason is that the specific activity of the enzyme is improved because the GOD has mutation of V20W + T30V in the amino acid sequence. To clarify this, the present inventors selected G/G30B and G/GOD, cultured them, and preliminarily purified them to obtain enzymes before and after mutation, analyzed the change in kinetic parameters of the mutant enzymes, and named the enzyme containing the mutation site (V20W + T30V) as MGOD.

And collecting fermentation liquor of the recombinant bacteria, purifying the recombinant bacteria, and performing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis on samples before and after purification, wherein the results are shown in figure 7, the extracellular proteins of the recombinant bacteria G/GOD and G/G30B have obvious bands at about 80kDa, the molecular weight of the extracellular proteins is the same as that of the target protein GOD, the extracellular proteins are judged to be target bands, and the impurity proteins in the purified samples are obviously reduced. The gray scale semi-quantitative analysis of the image is carried out by using software SmartView, the content of GOD protein obtained by G/GOD and G/G30B respectively accounts for 94.1 percent and 95.3 percent of the total protein, and the fact that the protein with higher purity can be obtained after purification is proved, and the protein can be used for subsequent enzyme kinetic parameter determination.

Protein concentrations of the purified enzyme solutions were determined using the Bradford kit, with GOD and MGOD protein concentrations of 0.0877 and 0.0583mg/mL, respectively. Subsequently, protease activity was measured using glucose at various concentrations as a restriction substrate, and Vmax and Km values were calculated using a double reciprocal plot of the mie equation (equation 1-1): v is V_max*[S]/(Km+[S]) (ii) a Wherein V represents the initial reaction rate, V_maxRepresents the maximum reaction rate, [ S ]]Representing the substrate concentration.

From the protein concentration and Km value, the Kcat and Kcat/Km values of the protein were calculated (Table 7).

TABLE 7 kinetic parameters of MGOD and GOD

Enzyme	Km(mM)	Kcat(s^-1)	Kcat/Km(mM^-1·s^-1)
				GOD	17.9	599.2	33.5
MGOD	16.5	643.8	39.0

The results showed that the Km value of MGOD was reduced by 7.8% compared to GOD, and that Kcat and Kcat/Km were increased by 7.4% and 16.4%, respectively. Compared with GOD, MGOD has higher Kcat and Kcat/Km, which shows that the MGOD has higher catalytic efficiency on substrate glucose, and has lower Km value, which shows that the affinity of MGOD on the substrate is obviously improved.

Meanwhile, the inventor observes that the stability of the mutant enzyme is also very ideal, and the mutant enzyme is suitable for the application of industrial systems.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

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Claims

1. A glucose oxidase mutant characterized in that the amino acid sequence thereof corresponds to the amino acid sequence shown in SEQ ID NO. 1,

mutation from Val to Trp at position 20 and mutation from Thr to Val at position 30;

mutation of Val to Trp at position 20 and mutation of Thr to Trp at position 30;

mutation from Val to Met at position 20 and mutation from Thr to Ser at position 30; or

Val is mutated to Cys at position 20, and Thr is mutated to Ser at position 30.

2. The mutant according to claim 1, wherein the amino acid sequence of the mutant corresponds to SEQ ID NO 1, with a mutation from Val to Trp at position 20 and Thr to Val at position 30.

3. An isolated polynucleotide encoding a mutant according to any one of claims 1 to 2.

4. A vector comprising the polynucleotide of claim 3.

5. A genetically engineered host cell comprising the vector of claim 4, or having the polynucleotide of claim 3 integrated into its genome.

6. A method of producing a mutant of glucose oxidase of claim 1, comprising the steps of:

(1) culturing the host cell of claim 5 to obtain a culture; and

(2) isolating the glucose oxidase mutant of claim 1 from the culture.

7. Use of a glucose oxidase mutant according to any of claims 1-2 for catalyzing the oxidation of glucose to produce hydrogen peroxide and glucono-delta-lactone, or to produce gluconic acid.

8. A method of increasing the enzymatic activity, catalytic efficiency, or substrate affinity of glucose oxidase, comprising: subjecting glucose oxidase to a mutation selected from the group consisting of:

Val is mutated to Cys at position 20, and Thr is mutated to Ser at position 30.

9. A composition for oxidizing glucose, comprising the glucose oxidase mutant according to any one of claims 1 to 2, and a pharmaceutically or industrially acceptable carrier.

10. A kit for oxidizing glucose comprising the composition of claim 9; or, comprising the glucose oxidase mutant of any of claims 1-2.