Glucose oxidase mutant AhGODA-M1, coding gene and application thereof, and method for improving enzymatic characteristics of glucose oxidase

Abstract

The invention relates to the technical field of agricultural biology, in particular to a glucose oxidase mutant AhGODA-M1, a coding gene and application thereof, and a method for improving enzymatic characteristics of glucose oxidase. According to the invention, T72R and A153P site-directed mutagenesis is carried out on 72th and 153th amino acids of glucose oxidase AhGODA with an amino acid sequence as shown in SEQ ID NO: 1. The enzyme activity of the mutant is improved from 112 U / mg to 166 U / mg, and the improvement amplitude is 48%; the optimum temperature is increased from 40 DEG C to 50 DEG C; after treatment at 70 DEG C for 30 min, about 33% of residual activity is reserved, the application requirements in the fields of food, feed, textile industry and the like can be better met, and the method has very wide application prospects.

Description

Technical Field

[0001] The invention relates to the field of agricultural biotechnology, and in particular to a glucose oxidase mutant AhGODA-M1 and its encoding gene, application, and a method for improving the enzymatic properties of glucose oxidase. Background Art

[0002] Glucose oxidase (GOD) is an aerobic dehydrogenase belonging to the glucose-methanol-choline oxidoreductase family. It can specifically catalyze β-D-glucose to produce hydrogen peroxide and gluconic acid. Glucose oxidase is widely present in animals, plants and microorganisms, and Aspergillus and Penicillium are its main production strains. Glucose oxidase has the functions of promoting digestion and absorption, maintaining intestinal health, improving the body's immunity, and improving production performance. It is a good substitute for antibiotics.

[0003] Glucose oxidase has a wide range of applications, mainly including feed, food, medicine and energy industries. Most of the actual application environments are under high temperature conditions. The existing glucose oxidase has poor thermal stability and low activity, and its production and application are limited. Therefore, it is very important to obtain glucose oxidase with excellent thermal stability. Generally, Aspergillus GOD has better stability, and Penicillium GOD has higher catalytic activity. Therefore, Aspergillus-derived GOD is often used for thermal stability improvement research.

[0004] Protein engineering is an important means of enzyme molecule improvement. It changes the enzyme function by changing the protein structure through gene mutation or protein modification. It mainly improves the enzymatic properties such as thermal stability, catalytic efficiency, pH stability and substrate specificity. Summary of the invention

[0005] The purpose of the present invention is to provide a glucose oxidase AhGODA mutant AhGODA-M1.

[0006] Another object of the present invention is to provide a glucose oxidase gene.

[0007] Another object of the present invention is to provide a recombinant vector comprising the above-mentioned glucose oxidase gene.

[0008] Another object of the present invention is to provide a recombinant strain comprising the above-mentioned glucose oxidase gene.

[0009] Another object of the present invention is to provide a method for preparing glucose oxidase.

[0010] Another object of the present invention is to provide a method for improving the enzyme activity, optimal temperature and thermal stability of glucose oxidase.

[0011] According to the technical solution of the present invention, the amino acid sequence of the wild-type glucose oxidase AhGODA is shown as SEQ ID NO: 1.

[0012] SEQ ID NO: 1

[0013] PHYARRSLLEANLLTDPSKVAEHTVDYIIIAGGGLTGLTAAARLSENPNITVLVIETGFYESERGPIIEDLNTYGDIFGTTVDHAYETVELATNNRTEVIRSGNGLGGSTLVNGGTWTRPHKAQIDSWESVFGNDGWNWDTLVAYSHQ VETVRAPNEKQIAAGHYFDPSCHGQNGSVHAGPRDTGESYSPIMKALMSTVQDQGVPIQQDLSCGNPHGVSMFPNSLNEEQTRSDAAREWLLPNFQRPNLQVLTGQRVGKVLLDQTGETPQAVGVEFGTHNNTKYNVYANHEVLLAA GSTVSPLILEYSGIGMKSILDSVGIETIVDLPVGLNLQDQTTTPVTSRITSAGDGQGQAAYFATFNETFGDYAPQAHELLNTKLAQWASETVSRGGFHNVTALTIQYENYRDWIVTHDVAYSELFLDTAGSISFTVWDLLPFTRGYV HILDKDPYLHRYAYNPQYLLNELDLLGQAAACKLARELSNSGEMQTYFAGEDVPGDNLAYDAGLDDWAEYIRYNFRPNYHGVGTCSMMPKELGGVVDSTARVYGVQGLRVIDGSIPPTQVSAHVMTVFYGMALKVSEAVLEDYAASL

[0014] The AhgodA nucleotide sequence of the glucose oxidase gene encoding the wild-type glucose oxidase AhGODA is shown in SEQ ID NO:2.

[0015] SEQ ID NO: 2

[0016]

[0017] The amino acid sequence of the glucose oxidase mutant AhGODA-M1 according to the present invention is shown in SEQ ID NO: 3, which is obtained by performing site-directed mutation on the 72nd and 153rd amino acids of glucose oxidase AhGODA, T72R&A153P.

[0018] SEQ ID NO: 3

[0019]

[0020] The nucleotide sequence of the glucose oxidase mutant encoding gene AhgodA-M1 is shown in SEQ ID NO:4.

[0021] SEQ ID NO: 4

[0022]

[0023] CTGTTTTGGAAGATTACGCTGCTTCTTTGTAA

[0024] According to a specific embodiment of the present invention, the present invention also provides a recombinant vector comprising the above-mentioned wild-type and mutant genes, amplifying the wild-type and mutant sequence fragments by the over-lap PCR method, cloning the wild-type and mutant sequence fragments into the expression vector pPICZαA, and constructing a recombinant vector; inserting the glucose oxidase gene of the present invention between appropriate restriction enzyme cutting sites of the expression vector so that its nucleotide sequence can be operably connected to the expression regulatory sequence.

[0025] As a most preferred embodiment of the present invention, the glucose oxidase gene is preferably inserted between the EcoR I and Not I restriction enzyme sites on the plasmid pPICZαA to obtain the recombinant expression plasmids pPICZαA-AhgodA and pPICZαA-AhgodA-M1.

[0026] The present invention also provides a recombinant strain containing the glucose oxidase gene, wherein the recombinant expression plasmid is linearized with BglⅡ restriction endonuclease, electroporated into Pichia pastoris GS115 competent cells, and cultured at 30°C for 48 hours. The transformants grown on the YPD plate are selected and induced to express on the his-MM plate, and then the glucose oxidase recombinant strains GS115 / AhgodA and AhgodA-M1 are obtained by colorimetric screening.

[0027] The present invention also provides a method for preparing glucose oxidase, comprising the following steps:

[0028] Transforming a host cell with the above-mentioned recombinant vector to obtain a recombinant strain;

[0029] Cultivating the recombinant strain and inducing the expression of recombinant glucose oxidase;

[0030] The expressed mutant enzyme was recovered and purified.

[0031] The present invention also provides application of the glucose oxidase mutant.

[0032] The method for improving the enzyme activity, optimum temperature and thermal stability of glucose oxidase according to the present invention comprises performing T72R and A153P site-directed mutations on the 72nd and 153rd amino acids of the glucose oxidase AhGODA with an amino acid sequence as shown in SEQ ID NO: 1.

[0033] The first technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a glucose oxidase with high enzyme activity and excellent thermal stability suitable for application in the fields of food, feed and textile industry. The mutant enzyme activity of the present invention is increased from 112U / mg to 166U / mg, with an increase of 48%; the optimum temperature is increased from 40°C to 50°C; after treatment at 70°C for 30 minutes, about 33% of the residual activity is retained, which can better meet the application needs in the fields of food, feed and textile industry, and has a very broad application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The results of electrophoresis analysis of wild-type and mutant proteins are shown, where M: protein marker; lane 1: WT; lane 2: AhGODA-M1;

[0035] Figure 2 Enzyme activities of wild type and mutants are shown;

[0036] Figure 3 The optimum temperatures for the wild type and mutants are shown;

[0037] Figure 4 The stability of wild type and mutants at 70°C is shown;

[0038] Figure 5 The optimal pH of wild type and mutants is shown;

[0039] Figure 6 Wild-type and mutant pH stability is shown. DETAILED DESCRIPTION

[0040] The experimental materials and reagents used in the following examples include:

[0041] Strains and vectors: expression host Pichiapastoris GS115, expression plasmid vector pPICZαA;

[0042] Enzymes and other biochemical reagents: restriction endonucleases, ligases;

[0043] Culture medium:

[0044] LB medium: 0.5% yeast extract, 1% peptone, 1% NaCL;

[0045] YPD medium: 1% yeast extract, 2% peptone, 2% glucose;

[0046] his-MM solid medium: 2% agarose, 13.4% YNB, 0.00004% Biotin, 1% methanol, 0.004% histidine;

[0047] BMGY medium: 1% yeast extract, 2% peptone, 1% glycerol, 13.4% YNB, 0.00004% Biotin;

[0048] BMMY medium: 1% yeast extract, 2% peptone, 13.4% YNB, 0.00004% Biotin, 1% methanol (V / V).

[0049] The molecular biology experimental methods not described in detail in this example are all carried out according to the specific methods listed in the book "Molecular Cloning Experiment Guide" (3rd edition) by J. Sambrook, or according to the kits and product instructions.

[0050] Example 1 Cloning and expression of wild-type glucose oxidase gene AhGODA

[0051] The AhgodA sequence of glucose oxidase gene from Aspergillus heteromorphus (GeneBank accession number QLI42895.1, SEQ ID NO: 2) was obtained from the NCBI database and synthesized. The expression vector pPICZαA was double-digested (EcoR I+Not I), and the gene AhgodA was connected to the expression vector pPICZαA after the enzyme-digested fragments were recovered by electrophoresis to obtain the recombinant plasmid pPICZαA-AhgodA and electroporated into Pichia pastoris GS115 competent cells to obtain the recombinant yeast strain GS115 / AhgodA.

[0052] Take the GS115 strain containing the recombinant plasmid, inoculate it in a 1L triangular flask with 300mL BMGY medium, and place it at 30°C, 220rpm shaking incubator for 48h; then centrifuge the culture solution at 4500rpm for 5min, discard the supernatant, resuspend the precipitate in 200mL BMMY medium containing 1% methanol, and place it again at 30°C, 220rpm for induction incubation for 48h, add 2mL methanol every 24h to keep the methanol concentration at about 0.5%. Centrifuge at 8000rpm for 15min, collect the supernatant of the recombinant glucose oxidase cultured in the shaking flask, concentrate the fermentation solution using a 10kDa membrane package, and then purify the enzyme solution by nickel column affinity chromatography.

[0053] Example 2 Cloning and expression of mutants

[0054] The threonine at position 72 and the alanine at position 153 of AhGODA were mutated to arginine and proline, respectively. The recombinant plasmid pPICZαA-AhgodA was used as a template, and the mutation site was introduced by Over-lap PCR, and then sequenced and verified to obtain the recombinant plasmid pPICZαA-AhgodA-M1. The primers used in the design include:

[0055] T72R-F: 5'GAGGATTTGAACagaTACGGTGACA3' (SEQ ID NO: 5);

[0056] T72R-R: 5'tctGTTCAAATCCTCAATAATTGGACC3' (SEQ ID NO: 6);

[0057] A153P-F: 5'GAAACTGTTAGAccaCCAAACGAAA3' (SEQ ID NO: 7);

[0058] A153P-R: 5'tggTCTAACAGTTTCAACTTGATGAGAGT3' (SEQ ID NO: 8).

[0059] The recombinant plasmid pPICZαA-AhgodA-M1 was linearized by BglⅡ restriction endonuclease and then transformed into Pichia pastoris GS115 competent cells by electroporation to obtain the recombinant yeast strain GS115 / AhgodA-M1. The GS115 strain containing the recombinant plasmid was inoculated into a 1L triangular flask containing 300mL of BMGY medium and cultured in a shaking incubator at 30°C and 220rpm for 48h; the culture solution was then centrifuged at 4500rpm for 5min, the supernatant was discarded, the precipitate was resuspended in 200mL of BMMY medium containing 1% methanol, and then induced and cultured again at 30°C and 220rpm for 48h, and 2mL of methanol was added every 24h to keep the methanol concentration at about 0.5%. The supernatant of the recombinant glucose oxidase cultured in the shaking flask was collected by centrifugation at 8000rpm for 15min, the fermentation broth was concentrated using a 10kDa membrane package, and then the enzyme solution was purified by nickel column affinity chromatography.

[0060] Example 3 Determination of properties of wild type and mutants

[0061] The GOD enzyme activity was determined by ultraviolet spectrophotometry. The specific method is as follows: 1.45 mL reaction system: 2.5 mg / mL dianisidine dissolved in 100 mM sodium dihydrogen phosphate-citrate buffer (pH 6.0); 18 mg / mL glucose solution; 9 U / mL horseradish peroxidase; 50 μL enzyme solution diluted to an appropriate concentration. The mixed reaction system was reacted under standard conditions (30°C, pH 6.0) for 3 minutes, and 1 mL of 2M sulfuric acid was added to terminate the reaction. The absorbance of the terminated reaction solution was measured at a wavelength of 540 nm. The enzyme activity unit is defined as the amount of enzyme consumed to oxidize 1 μmol of glucose per minute under the conditions of 30°C and pH 6.0.

[0062] The purification results of wild type and mutants are as follows Figure 1 The wild type and mutant purified in Examples 1 and 2 were subjected to enzyme activity assay at 30°C and pH 6.0. Figure 2 As shown in the figure, the specific activity of the wild type is 112U / mg, and the enzyme activity of the mutant is 166U / mg, which is 48% higher than that of the wild type. The enzyme activity was measured at pH 6.0 and different temperatures (30, 40, 50, 60, 70°C) to determine the optimal temperature. Figure 3 As shown, the optimum temperature of the wild type is 40°C, and it maintains 33% activity at 70°C; the optimum temperature of the mutant is 50°C, and it can still maintain 63% activity at 70°C.

[0063] The mutant and wild type were treated in a 0.1 mol / L citric acid-sodium hydrogen phosphate buffer (pH 6.0) at 70°C for 2, 5, 10, 20 and 30 min, respectively, and then the residual enzyme activity was measured. Figure 4As shown, after the wild type was treated at 70°C for 20 minutes, the remaining enzyme activity was about 2%, while the mutant still maintained about 43% of the remaining enzyme activity after being treated at 70°C for 20 minutes; the wild type was inactivated after being treated at 70°C for 30 minutes, while the mutant maintained about 33% of the remaining enzyme activity after being treated at 70°C for 30 minutes.

[0064] The mutant and wild type were tested for enzyme activity at 30°C and different pH (3.0-9.0) conditions to determine the optimal pH. Figure 5 As shown, the optimum pH of the wild type and mutants is 6.0; the wild type and mutant enzyme solutions were treated in different pH buffers for 1 hour, and then the residual enzyme activity was measured under standard conditions (30° C., pH 6.0), as shown in FIG. Figure 6 As shown, the wild type and mutants were relatively stable under pH 3.0-12.0 conditions, and the stability of the mutants was slightly better than that of the wild type.

[0065] The above embodiments are only used to understand the technical solution of the present application and do not limit the protection scope of the present application.

Claims

1. Glucose oxidase mutant AhGODA-M1, It is characterized in that The amino acid sequence of the glucose oxidase mutant AhGODA-M1 is shown in SEQ ID NO:

3.

2. A glucose oxidase gene, It is characterized in that Encoding the glucose oxidase mutant AhGODA-M1 according to claim 1.

3. The glucose oxidase gene according to claim 2, It is characterized in that The nucleotide sequence of the glucose oxidase gene is shown in SEQ ID NO:

4.

4. A recombinant expression plasmid comprising the glucose oxidase gene according to claim 2.

5. A recombinant strain comprising the glucose oxidase gene according to claim 2.

6. A method for improving the enzyme activity, optimum temperature and thermal stability of glucose oxidase, It is characterized in that The method comprises the steps of performing T72R and A153P site-directed mutations on the 72nd and 153rd amino acids of the glucose oxidase AhGODA with an amino acid sequence as shown in SEQ ID NO:

1.

7. A method for preparing glucose oxidase, It is characterized in that The method comprises the following steps: Transforming a host strain with the recombinant expression plasmid according to claim 4 to obtain a recombinant strain by screening; Cultivating the recombinant strain to induce expression of recombinant glucose oxidase; The expressed glucose oxidase is recovered and purified.

8. Use of the glucose oxidase mutant AhGODA-M1 according to claim 1.

9. Use of the glucose oxidase mutant AhGODA-M1 according to claim 1 for hydrolyzing glucose.

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