A mutant glucose oxidase Gox-M12-T108S, its encoding gene and applications

Abstract

The present invention relates to the field of agricultural biotechnology, and particularly relates to a mutant glucose oxidase Gox-M12-T108S, its encoding gene and applications. Glucose oxidase Gox-M12 with an amino acid sequence as shown in SEQ ID NO: 3 is subjected to T108S mutation to obtain glucose oxidase Gox-M12-T108S with high enzyme activity and excellent acid stability, which is suitable for applications in fields such as food, medicine, feed, and textile industry.

Description

Technical Field

[0001] The present invention relates to the field of agricultural biotechnology, and specifically relates to a mutant glucose oxidase Gox-M12-T108S, its coding gene and applications. Background Art

[0002] Glucose oxidase (Gox) is an aerobic dehydrogenase with an EC number of 1.1.3.4. It can highly specifically oxidize β-D-glucose into gluconolactone and hydrogen peroxide under aerobic conditions, so it is also called β-D-glucose oxidoreductase. Currently, most Gox on the market are heterologously expressed in the widely concerned Pichia pastoris eukaryotic expression system. The strains for industrial production of Gox mainly come from Aspergillus niger and Penicillium. Relatively speaking, the Gox produced by Aspergillus niger has better thermal stability, while the Gox produced by Penicillium has higher enzyme activity. As an important industrial enzyme in the food industry, Gox is widely used in food deoxidation such as wine, beer, fruit juice, milk powder, flour improvement, food browning prevention, etc., and also has a large number of applications in the quantitative determination of the human biochemical index glucose in the pharmaceutical industry and providing an anaerobic environment for probiotics in the feed industry.

[0003] Chinese Patent CN112301009A provides a Gox mutant of glucose oxidase derived from Aspergillus niger The optimal temperature and optimal pH of the mutant Gox-M10 are 45 °C and pH 5.5 respectively.

[0004] During the research process of the present invention, the inventors of the present invention first performed four-point mutations on the glucose oxidase mutant GoxM10, changing the amino acid at position 241 from Gln to Glu, the amino acid at position 499 from Arg to Glu, the amino acid at position 361 from Glu to Pro, and the amino acid at position 419 from Ala to Ile, to obtain the variant Gox-M12. Compared with the mutant parent GoxM10, its acid stability was effectively improved without affecting the enzyme activity and thermal stability of the parent.

[0005] To meet the application requirements of specific industries for Gox, that is, to obtain Gox with good acid stability, high activity, etc., the current common methods are to explore novel gene resources, protein engineering, and optimize the application environment, etc. The research on molecular improvement of Gox by means of protein engineering to improve its acid stability has achieved rapid development. At present, the principle of improving the acid stability of Gox is not clear and is mostly accompanied by improving stability. Counting the reported papers on the research of enzyme stability improvement at present, rational design strategies such as protein surface electrostatic interaction, B-factor value, hydrophobic interaction, hydrogen bond, salt bond, cation-π interaction, disulfide bond, etc. have been widely used in the improvement of protein stability. However, among the above-mentioned strategies, the vast majority improve the acid stability of proteins at the cost of losing enzyme catalytic activity. Therefore, it is very meaningful to explore a protein modification strategy that can improve the acid stability of proteins without losing enzyme activity. The present invention attempts to improve the thermal stability of Gox without losing enzyme activity, which has important theoretical research significance and application significance. Summary of the Invention

[0006] The object of the present invention is to provide a mutant glucose oxidase Gox-M12-T108S, whose enzyme activity and acid stability are further improved compared with the wild enzyme.

[0007] Another object of the present invention is to provide a gene encoding the above-mentioned mutant glucose oxidase Gox-M12-T108S.

[0008] Another object of the present invention is to provide a recombinant vector containing the above-mentioned mutant gene.

[0009] Another object of the present invention is to provide a recombinant strain containing the above-mentioned gene.

[0010] Another object of the present invention is to provide a method for preparing glucose oxidase.

[0011] According to the mutant glucose oxidase Gox-M12-T108S of the present invention, its amino acid sequence is as shown in SEQ ID NO:1 (site-directed mutation of the 108th amino acid of glucose oxidase Gox-M12, that is, Thr108Ser).

[0012] SEQ ID NO:1:

[0013] GIEASLLTDPKEVAGRTVDYIIAGGGLTGLVVAARLTENPDITVLVIESGSYESDRGPIIEDLNAYGKIFGSSVDHAYETVCLATNNRTALIRAGNGLGGSTLVNGG SWTRPHKAQVDSWETVFGNEGWNWDSVAAYSLQAERARAPNAKQIAAGHYFNASCHGINGTVHAGPRDTGDDYSPIVKALMSAVEDRGVPTKKDLGCGDPHGVSMFPNTLHEDQVRSDAAREWLLPNYQRPNLEVLTGQYVGKVLLSQNATTPRAVGVEFGTHKGNFHNVTAKHEVLLAAGSAVSPTILEYSGIGMKSILEPLGIKTVVDLPVGLNLQDQTTSTVRSRITSAGAGQGQAAWFATFNETFGDYTPKAHELLNTKLEQWAEEAVARGGFHNTTALLIQYENYRDWIVKDNVAYSELFLDTAGEISFDVWDLLPFTRGYVHILDKDPYLRHFAYDPQYFLNELDLLGQAAATQLARNISNSGAMQTYFAGETIPGDNLAYDADLEAWVEYIPYHFRPNYHGVGTCSMMPKEMGGVVDNAARVYGVQGLRVIDGSIPPTQMSSHVMTVFYAMALKIADAVLADYASMQ。

[0014] The nucleotide sequence of the encoding gene of the glucose oxidase mutant Gox-M12-T108S with improved enzyme activity and thermal stability is shown in SEQ ID NO:2.

[0015] SEQ ID NO:2:

[0016]

[0017]

[0018] The amino acid sequence of glucose oxidase Gox-M12 is shown in SEQ ID NO:3.

[0019] SEQ ID NO:3:

[0020] GIEASLLTDPKEVAGRTVDYIIAGGGLTGLVVAARLTENPDITVLVIESGSYESDRGPIIEDLNAYGKIFGSSVDHAYETVCLATNNRTALIRAGNGLGGSTLVNGGSWTRPHKAQVDSWETVFGNEGWNWDSVAAYSLQAERARAPNAKQIAAGHYFNASCHGINGTVHAGPRDTGDDYSPIVKALMSAVEDRGVPTKKDLGCGDPHGVSMFPNTLHEDQVRSDAAREWLLPNYQRPNLEVLTGQYVGKVLLSQNATTPRAVGVEFGTHKGNFHNVTAKHEVLLAAGSAVSPTILEYSGIGMKSILEPLGIKTVVDLPVGLNLQDQTTSTVRSRITSAGAGQGQAAWFATFNETFGDYTPKAHELLNTKLEQWAEEAVARGGFHNTTALLIQYENYRDWIVKDNVAYSELFLDTAGEISFDVWDLLPFTRGYVHILDKDPYLRHFAYDPQYFLNELDLLGQAAATQLARNISNSGAMQTYFAGETIPGDNLAYDADLEAWVEYIPYHFRPNYHGVGTCSMMPKEMGGVVDNAARVYGVQGLRVIDGSIPPTQMSSHVMTVFYAMALKIADAVLADYASMQ.

[0021] The method for preparing glucose oxidase with improved enzyme activity and thermal stability according to the present invention comprises the following steps:

[0022] 1) Transforming a host cell with the above recombinant vector to obtain a recombinant strain;

[0023] 2) Culturing the recombinant strain to induce the expression of recombinant glucose oxidase;

[0024] 3) Recovering and purifying the expressed mutant enzyme.

[0025] The present invention also provides the application of the above mutant.

[0026] 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 acid stability, which is suitable for applications in fields such as food, medicine, feed, and the textile industry. The enzyme activity of the mutant enzyme of the present invention has been increased from 485.68 U / mg to 521.75 U / mg, with an increase of 7.4%; after being treated at 37°C for 60 min in a citric acid-phosphate buffer (0.1 mM) with a pH of 3.0, the remaining enzyme activity of Gox-M12 is 286.70 U / mg, and the remaining enzyme activity of Gox-M12-T108S is 354.98 U / mg, with an increase in acid stability of 17.9%. After being treated at 37°C for 30 min in a citric acid-phosphate buffer (0.1 mM) with a pH of 2.5, the remaining enzyme activity of Gox-M12 is 37.46 U / mg, and the remaining enzyme activity of Gox-M12-T108S is 130.46 U / mg, with the stability increased by 2.24 times. It can well meet the application requirements in fields such as food, medicine, feed, and the textile industry, and has a very broad application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a comparison chart of the enzyme activities of the wild type and the mutant;

[0028] Figure 2 It shows the acid stability of the wild type and the mutant at pH 3.0;

[0029] Figure 3 It shows the acid stability of the wild type and the mutant at pH 2.5. DETAILED DESCRIPTION OF THE INVENTION

[0030] Experimental Materials and Reagents

[0031] 1. Strains and Vectors: Expression Host Pichia pastoris GS115, expression plasmid vector pPIC9;

[0032] 2. Enzymes and Other Biochemical Reagents: Restriction Enzymes, Ligases;

[0033] 3. Working Solutions: Ortho-dianisidine Working Solution: 0.1 g of ortho-dianisidine in 10 mL of methanol, shaken well before use to form a suspension;

[0034] 4. Media:

[0035] LB Medium: 0.5% yeast extract, 1% peptone, 1% NaCl, pH 7.0;

[0036] YPD Medium: 1% yeast extract, 2% peptone, 2% glucose;

[0037] MD solid medium: 2% glucose, 1.5% agarose, 1.34% YNB, 0.00004% Biotin;

[0038] MM solid medium: 1.5% agarose, 1.34% YNB, 0.00004% Biotin, 1% methanol

[0039] BMGY medium: 1% yeast extract, 2% peptone, 1% glycerol (V / V), 1.34% YNB, 0.00004% Biotin;

[0040] BMMY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 1% methanol (V / V);

[0041] For the molecular biology experimental methods not described in detail in this example, they are all carried out with reference to the specific methods listed in "Molecular Cloning: A Laboratory Manual" (Third Edition) by J. Sambrook, or according to the kits and product instructions.

[0042] Example 1 Site-directed Mutagenesis

[0043] The mutation site was designed to mutate threonine at position 108 to serine. The mutation site was introduced by the method of Over-lap PCR and verified by sequencing to obtain the mutant gene GOX-M12-T108S . The primers used in the design were:

[0044] N159D / A160P-F: 5'CTTTGGTTAACGGAGG TTC TTGGACCAGACCACACAAGG3';

[0045] N159D / A160P-R: 5'GTGGTCTGGTCCAA GAA CCTCCGTTAACCAAAGTAG3'.

[0046] The recombinant plasmid containing the mutant gene GOX-M12-T108S was subjected to PCR single-point mutation using the above primers, and pPIC9-GOX-M12-T108S was incubated at 37°C for 2 h to digest the template circular plasmid. Then Not II was used to linearize the plasmid and transform Pichia pastoris GS115 to obtain the recombinant yeast strain Bgl / GS115 / GOX-M12-T108S .

[0047] Take the GS115 strain containing the recombinant plasmid and inoculate it into a 1 L Erlenmeyer flask with 400 mL of BMGY medium. Incubate it at 30°C on a shaker at 220 rpm for 48 h. Then centrifuge the culture solution at 4500 g for 5 min, discard the supernatant, and resuspend the precipitate in 200 mL of BMMY medium containing 1% methanol. Incubate it again at 30°C and 220 rpm for induction culture. Add 2 mL of methanol every 24 h to keep the methanol concentration in the bacterial solution at 1%. At the same time, take the supernatant for enzyme activity detection.

[0048] Example 2 Activity Analysis of Mutants and Wild Types

[0049] I. The Gox enzyme activity was determined by spectrophotometry. The specific method is as follows: Under the given conditions, a 1.5 mL reaction system includes 50 μL of an appropriately diluted enzyme solution, 10 μL of o-dianisidine solution, 50 μL of 90 U / mL HRP solution, 150 μL of 1 M glucose solution, and 1.24 mL of disodium hydrogen phosphate-citric acid Buffer (0.1 M, pH 6.0). React the mixture in a 30°C water bath for 3 min, add 1 mL of 2 M sulfuric acid to terminate the reaction, take 250 uL of the reaction solution to measure the optical density at a wavelength of 540 nm, use the reaction solution without adding 50 μL of the enzyme solution as a control, and calculate the enzyme activity according to the Gox enzyme activity determination standard curve equation. The unit of Gox enzyme activity (U) is defined as: The amount of enzyme required to catalyze 1 μmol of β-d-glucose to produce gluconic acid and hydrogen peroxide per minute under the conditions of 30 °C and pH 6.0.

[0050] II. Property Determination of Mutants and Wild Types

[0051] 1. The method for determining the enzyme activities of mutants and wild types is as follows:

[0052] Perform an enzymatic reaction on the purified mutant enzyme and wild type from Example 1 at pH 6.5 and 30°C to determine their enzyme activities. As Figure 1 shown, the enzyme activity of the wild type is 485.68 U / mg, and the enzyme activity of the mutant enzyme is 521.75 U / mg, which is 7.4% higher than that of the wild enzyme.

[0053] 2. The method for determining the stability of mutants and wild types at pH 3.0 is as follows:

[0054] The pH stability determination of the mutants and wild types is carried out in 0.1 mol / L citric acid-disodium hydrogen phosphate buffer at different pH 3.0. After incubating at 37 °C for 1 h, the remaining enzyme activity is measured at 30°C. As Figure 2As shown, after the wild type was treated at pH 3.0 for 1 h, the enzyme activity decreased to 286.70 U / mg, and the remaining enzyme activity was equivalent to 60.75% of that before treatment; after the mutant was treated at pH 3.0 for 1 h, the remaining enzyme activity was 354.98 U / mg, and the remaining enzyme activity was equivalent to 71.61% of that before treatment, which was 17.9% higher than that of the wild type.

[0055] 3. The method for measuring the stability of the mutant and the wild type at pH 2.5 is as follows:

[0056] The pH stability measurement of the mutant and the wild type was carried out in 0.1 mol / L citric acid-disodium hydrogen phosphate buffer at different pH 2.5. After incubation at 37 °C for different times (10, 20, 30, 40, 50, 60 min), the remaining enzyme activity was measured at 30 °C. As Figure 3 shown, after the wild type was treated at pH 2.5 for 30 min, the enzyme activity decreased to 37.46 U / mg, and the remaining enzyme activity was equivalent to 7.71% of that before treatment. The remaining enzyme activity of Gox-M12-T108S was 130.46 U / mg, and the remaining enzyme activity was equivalent to 25.00% of that before treatment, and the stability was increased by 2.24 times.

[0057] 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. A glucose oxidase Gox-M12 mutant with improved enzyme activity and acid stability, characterized in that: The amino acid sequence of the mutant is shown in SEQ ID NO:

1.

2. A glucose oxidase gene, characterized in that: Encoding the glucose oxidase Gox-M12 mutant with improved enzyme activity and acid stability as claimed in claim 1.

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

2.

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

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

6. Use of the glucose oxidase Gox-M12 mutant with improved enzyme activity and acid stability according to claim 1 for hydrolyzing glucose for non-therapeutic and diagnostic purposes.

7. A method for preparing glucose oxidase, characterized in that: The method comprises the following steps: Transforming a host strain with the recombinant expression vector according to claim 4 to obtain a recombinant strain; Inducing the recombinant strain to express recombinant glucose oxidase; The glucose oxidase is isolated and purified.

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