Glucose oxidase GoxM12 mutant G272T with improved thermal stability and its derivative mutants and applications

Abstract

The present invention belongs to the field of feed biotechnology, and specifically relates to a thermostable glucose oxidase GoxM12 mutant G272T and its derivative mutants and applications. The present invention uses high thermostable glucose oxidase GoxM12 as research material, conducts stability improvement research on it, obtains a mutant with improved stability, and thus promotes the application of Gox in the feed industry.

Description

Technical Field

[0001] The invention belongs to the field of feed biotechnology, and particularly relates to a glucose oxidase GoxM12 mutant G272T with improved thermal stability and derivative mutants thereof and applications thereof. Background Art

[0002] Glucose oxidase (Gox; EC1.1.3.4) is a well-characterized aerobic dehydrogenase that uses oxygen molecules as electron acceptors to catalyze β-d-glucose to produce d-gluconolactone and hydrogen peroxide under aerobic conditions, and then d-gluconolactone is hydrolyzed into gluconic acid and water. In recent years, Gox has received widespread attention in the feed industry as a new antibiotic alternative additive. As an alternative to antibiotics and growth promoters, Gox is essential to ensure animal health and production performance without compromising human health. A large number of application studies have shown that Gox, as a green feed additive, can be used to prevent gastrointestinal infections and diarrhea in livestock, and has the effect of promoting animal growth.

[0003] In patent CN 108893453 A, the Aspergillus niger The glucose oxidase GOD was used as the parent, and after multiple mutations, a mutant GOD-M5 with improved thermal stability was obtained, but it still could not meet industrial requirements.

[0004] Chinese patent application CN201910835766.4 provides a Aspergillus niger A series of mutants of the glucose oxidase mutant GOD-M5 were synthesized. The thermal stability of the glucose oxidase mutant was significantly improved. After heat treatment at 80℃ for 2min, the relative residual enzyme activity of GOD-M10 was increased by about 2.2 times compared with GODM5. However, the pH condition for the glucose oxidase mutant GoxM10 to exert the maximum enzyme activity was 6.0, and the enzyme activity was extremely low under the pH stability condition of pH 2.5-3.0.

[0005] Chinese patent application CN202210648703.X provides Aspergillus niger The optimal temperature and optimal pH of mutant E361P / A419I are 40°C and pH 6.0, respectively, and the half-life at 65°C is 578min, which is 1.5 times that of GoxM10. Aspergillus nigerThe optimal temperature and pH of mutant Q241E / R499E were 40 ℃ and 5.0, respectively. After treatment at pH 2.5 and 37℃ for 60 min, the enzyme activity of mutant Q241E / R499E was 15.89%, which was 144.46% higher than that of GoxM10.

[0006] During the research and development process of the present application, the inventors of the present application first subjected GoxM10 to a triple-point mutation Q241E / E361P / A419I, and then subjected it to a quadruple-point mutation Q241E / R499E / E361P / A419I, to obtain the mutant GoxM12. The acid stability and thermal stability of the mutant GoxM12 are significantly better than those of GoxM10. The triple-point mutation Q241E / E361P / A419I of GoxM10, and then subjected it to a quadruple-point mutation Q241E / R499E / E361P / A419I, to obtain the mutant GoxM12. The acid stability and thermal stability of the mutant GoxM12 are significantly better than those of GoxM10.

[0007] Currently, most animal feeds need to be pelletized at high temperatures, so improving the thermal stability of Gox is crucial for its widespread use in feed. In addition, it is also important to be able to maintain high activity in an acidic environment so that it can play the greatest role in the animal gastrointestinal tract. Therefore, in order to meet the application of Gox in the feed industry, Gox must have good thermal stability and acid stability, and it is necessary to further modify the glucose oxidase GoxM12. Summary of the invention

[0008] The object of the present invention is to provide a mutant of glucose oxidase GoxM12 with improved thermal stability.

[0009] Another object of the present invention is to provide a gene encoding the above mutant.

[0010] Another object of the present invention is to provide a recombinant vector comprising the mutant gene.

[0011] Another object of the present invention is to provide a recombinant strain comprising the mutant gene.

[0012] Another object of the present invention is to provide the application of the mutant.

[0013] Another object of the present invention is to provide a method for preparing glucose oxidase with improved thermal stability.

[0014] Another object of the present invention is to provide a method for improving the thermal stability of glucose oxidase GoxM12.

[0015] According to a specific embodiment of the present invention, site-directed mutagenesis is performed on the glucose oxidase GoxM12 with an amino acid sequence as shown in SEQ ID NO: 1.

[0016] SEQ ID NO:1

[0017] According to a specific embodiment of the present invention, the glucose oxidase with an amino acid sequence as shown in SEQ ID NO: 1 is subjected to single point mutations of T8K, S72T, V104I, A171V, V184M, S224A, G272S, T278Y, and E418V; and then subjected to single point mutations of G272S / T8K, G272S / S72T, G272S / V104I, G272S / A171V, G272S / V184M, G272S / S224A, G272S / T278 Y, G272S / E418V double-point combination mutation; then G272S / A171V / T8K, G272S / A171V / S72T, G272S / A171V / V104I, G272S / A171V / V184M, G272S / A171V / S224A, G272S / A171V / T278Y, G272S / A171V / E418V triple-point combination mutation.

[0018] The glucose oxidase with an amino acid sequence as shown in SEQ ID NO: 1 was subjected to single point mutations of E12V, R16T, T168S, S254D, G272T, L310V, L322V, T466S, D490F, P523S, and G541R; and then subjected to double point combination mutations of G272T / E12V, G272T / R16T, G272T / T168S, G272T / S254D, G272T / L310V, G272T / L322V, G272T / T466S, G272T / D490F, G272T / P523S, and G272T / G541R.

[0019] Then the best mutants G272S / A171V / T8K and G272T / D490F were selected for iterative mutation. However, because the G272 site overlapped, the mutant amino acids at the G272 site were swapped and compared to construct the G272T / A171V / T8K (GoxM13) and G272S / D490F mutants. After comparison, it was found that G272T / A171V / T8K (GoxM13) had the best stability. Therefore, using G272T / A171V / T8K (GoxM13) as a template, we performed G272T / A171V / T8K / E12V, G272T / A171V / T8K / R16T, G272T / A171V / T8K / S72T, G272T / A171V / T8K / V104I, G272T / A171V / T8K / T168S, G272T / A171V / T8K / V184M, G272T / A171V / T8K / S224A, G272T / A171V / T8K / S72T Four-point combination mutations, 171V / T8K / S254D, G272T / A171V / T8K / L310V, G272T / A171V / T8K / L322V (GoxM14), G272T / A171V / T8K / E418V, G272T / A171V / T8K / D490F, G272T / A171V / T8K / P523S (GoxM15), and G272T / A171V / T8K / G541R, were used to obtain glucose oxidase mutants with improved acid and thermal stability.

[0020] According to a specific embodiment of the present invention, the glucose oxidase with an amino acid sequence as shown in SEQ ID NO: 1 is subjected to a G272T mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO: 2.

[0021] According to a specific embodiment of the present invention, the glucose oxidase with an amino acid sequence as shown in SEQ ID NO: 1 is subjected to G272T / E12V mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO: 3 having improved thermal stability.

[0022] According to a specific embodiment of the present invention, the glucose oxidase with an amino acid sequence as shown in SEQ ID NO: 1 is subjected to G272T / R16T mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO: 4 having improved thermal stability.

[0023] According to a specific embodiment of the present invention, the glucose oxidase with an amino acid sequence as shown in SEQ ID NO:1 is subjected to G272T / T168S mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO:5.

[0024] According to a specific embodiment of the present invention, the glucose oxidase with an amino acid sequence as shown in SEQ ID NO: 1 is subjected to G272T / S254D mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO: 6.

[0025] According to a specific embodiment of the present invention, the glucose oxidase with an amino acid sequence as shown in SEQ ID NO:1 is subjected to G272T / L310V mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO:7.

[0026] According to a specific embodiment of the present invention, the glucose oxidase with an amino acid sequence as shown in SEQ ID NO: 1 is subjected to G272T / L322V mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO: 8.

[0027] According to a specific embodiment of the present invention, the glucose oxidase with an amino acid sequence as shown in SEQ ID NO:1 is subjected to G272T / T466S mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO:9.

[0028] According to a specific embodiment of the present invention, the glucose oxidase with an amino acid sequence as shown in SEQ ID NO: 1 is subjected to G272T / D490F mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO: 10.

[0029] According to a specific embodiment of the present invention, the glucose oxidase with an amino acid sequence as shown in SEQ ID NO: 1 is subjected to G272T / P523S mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO: 11.

[0030] According to a specific embodiment of the present invention, the glucose oxidase with an amino acid sequence as shown in SEQ ID NO:1 is subjected to G272T / G541R mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO:12.

[0031] According to a specific embodiment of the present invention, the glucose oxidase with an amino acid sequence as shown in SEQ ID NO:1 is subjected to G272T / A171V mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO:13.

[0032] According to a specific embodiment of the present invention, the glucose oxidase with an amino acid sequence as shown in SEQ ID NO:1 is mutated to G272T / A171V / T8K (GoxM13) to obtain a mutant with an amino acid sequence as shown in SEQ ID NO:14.

[0033] According to a specific embodiment of the present invention, the glucose oxidase with an amino acid sequence as shown in SEQ ID NO: 1 is subjected to G272T / A171V / T8K / E12V mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO: 15.

[0034] According to a specific embodiment of the present invention, the glucose oxidase with an amino acid sequence as shown in SEQ ID NO: 1 is subjected to G272T / A171V / T8K / R16T mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO: 16.

[0035] According to a specific embodiment of the present invention, the glucose oxidase with an amino acid sequence as shown in SEQ ID NO: 1 is subjected to G272T / A171V / T8K / S72T mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO: 17.

[0036] According to a specific embodiment of the present invention, the glucose oxidase with an amino acid sequence as shown in SEQ ID NO: 1 is subjected to G272T / A171V / T8K / V104I mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO: 18.

[0037] According to a specific embodiment of the present invention, the glucose oxidase with an amino acid sequence as shown in SEQ ID NO: 1 is subjected to G272T / A171V / T8K / T168S mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO: 19.

[0038] According to a specific embodiment of the present invention, the glucose oxidase with an amino acid sequence as shown in SEQ ID NO: 1 is subjected to G272T / A171V / T8K / V184M mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO: 20.

[0039] According to a specific embodiment of the present invention, the glucose oxidase with an amino acid sequence as shown in SEQ ID NO: 1 is subjected to G272T / A171V / T8K / S224A mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO: 21.

[0040] According to a specific embodiment of the present invention, the glucose oxidase with an amino acid sequence as shown in SEQ ID NO: 1 is subjected to G272T / A171V / T8K / S254D mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO: 22.

[0041] According to a specific embodiment of the present invention, the glucose oxidase with an amino acid sequence as shown in SEQ ID NO: 1 is subjected to G272T / A171V / T8K / L310V mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO: 23.

[0042] According to a specific embodiment of the present invention, the glucose oxidase with an amino acid sequence as shown in SEQ ID NO: 1 is mutated to G272T / A171V / T8K / L322V (GoxM14) to obtain a mutant with an amino acid sequence as shown in SEQ ID NO: 24.

[0043] According to a specific embodiment of the present invention, the glucose oxidase with an amino acid sequence as shown in SEQ ID NO: 1 is subjected to G272T / A171V / T8K / E418V mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO: 25.

[0044] According to a specific embodiment of the present invention, the glucose oxidase with an amino acid sequence as shown in SEQ ID NO: 1 is subjected to G272T / A171V / T8K / D490F mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO: 26.

[0045] According to a specific embodiment of the present invention, the glucose oxidase with an amino acid sequence as shown in SEQ ID NO: 1 is mutated to G272T / A171V / T8K / P523S (GoxM15) to obtain a mutant with an amino acid sequence as shown in SEQ ID NO: 27.

[0046] According to a specific embodiment of the present invention, the glucose oxidase with an amino acid sequence as shown in SEQ ID NO: 1 is subjected to G272T / A171V / T8K / G541R mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO: 28.

[0047] According to a specific embodiment of the present invention, a recombinant vector comprising the above-mentioned glucose oxidase mutant gene is also provided.

[0048] According to a specific embodiment of the present invention, a recombinant strain comprising the above-mentioned glucose oxidase mutant gene is also provided.

[0049] The method for preparing glucose oxidase with improved thermal stability according to the present invention comprises the following steps:

[0050] 1) preparing a recombinant vector containing the mutant gene;

[0051] 2) The recombinant vector is transformed into the Pichia pastoris GS115 expression host;

[0052] 3) fermenting and culturing the host and isolating glucose oxidase.

[0053] The present invention provides the application of the glucose oxidase mutant with acid stability or thermal stability, which can be specifically applied in the field of food and feed.

[0054] Compared with glucose oxidase GoxM12, the mutants of the present invention have significantly improved specific activity, thermal stability and acid stability. After being incubated at 75°C for 20 min, the residual enzyme activity of GoxM12 was 19%, and the residual enzyme activities of mutants T8K, S72T, V104I, A171V, V184M, S224A, G272S, T278Y and E418V were 36%, 25%, 26%, 29%, 24%, 48%, 58%, 22% and 30%, respectively. After being incubated at 85°C for 2 min later, the residual enzyme activity of GoxM12 was 17%, and the mutants G272S / T8K, G272S / S72T, G272S / V104I, G272S / A171V, G272S / V184M, G272S / S224A, G272S / T278Y, G272S / E418V, G272S / A171V / T8K, G272S / A171V / S72T, and G272 The residual enzyme activities of S / A171V / V104I, G272S / A171V / V184M, G272S / A171V / S224A, G272S / A171V / T278Y and G272S / A171V / E418V were 39%, 36%, 40%, 60%, 33%, 48%, 32%, 40%, 66%, 62%, 51%, 52%, 64%, 61% and 57%, respectively. min later, the residual enzyme activity of GoxM12 was 0%, and the mutants G272S / T8K, G272S / S72T, G272S / V104I, G272S / A171V, G272S / V184M, G272S / S224A, G272S / T278Y, G272S / E418V, G272S / A171V / T8K, G272S / A171V / S72T, G272 The residual enzyme activities of S / A171V / V104I, G272S / A171V / V184M, G272S / A171V / S224A, G272S / A171V / T278Y and G272S / A171V / E418V were 50%, 50%, 44%, 67%, 41%, 58%, 44%, 50%, 82%, 83%, 59%, 68%, 81%, 67% and 74%, respectively.

[0055] After incubation at 80 ℃ for 3 min, the residual enzyme activity of GoxM12 was 55%, and the residual enzyme activities of mutants E12V, R16T, T168S, S254D, G272T, L310V, L322V, T466S, D490F, P523S and G541R were 58%, 50%, 60%, 63%, 84%, 56%, 70%, 52%, 63%, 0% and 64%, respectively.

[0056] After incubation at 80 ℃ for 2 min, the residual enzyme activity of GoxM12 was 80%, and the residual enzyme activities of mutants G272T / E12V, G272T / R16T, G272T / T168S, G272T / S254D, G272T / L310V, G272T / L322V, G272T / T466S, G272T / D490F, G272T / P523S and G272T / G541R were 92%, 97%, 93%, 99%, 100%, 100%, 99%, 100%, 90% and 98%, respectively. After incubation at 80 ℃ for 5 min, the residual enzyme activity of GoxM12 was 0%, and the residual enzyme activities of the mutants G272T / E12V, G272T / R16T, G272T / T168S, G272T / S254D, G272T / L310V, G272T / L322V, G272T / T466S, G272T / D490F, and G272T / P523S were 10%, 15%, 12%, 27%, 23%, 31%, 10%, 41%, and 5%, respectively.

[0057] After incubation at 85 °C for 5 min, the residual enzyme activity of GoxM12 was 0%, and the mutants G272T / A171V, G272T / A171V / T8K (GoxM13), G272T / A171V / T8K / E12V, G272T / A171V / T8K / R16T, G272T / A171V / T8K / S72T, G272T / A171V / T8K / V104I, G272T / A171V / T8K / T168S, G272T / A171V / T8K / V184M, G272T / A171V / T8K / S224A, and G272T / A171V / T8K / S72T. The residual enzyme activities of K / S254D, G272T / A171V / T8K / L310V, G272T / A171V / T8K / L322V (GoxM14), G272T / A171V / T8K / E418V, G272T / A171V / T8K / D490F, G272T / A171V / T8K / P523S (GoxM15), and G272T / A171V / T8K / G541R were 35%, 58%, 21%, 15%, 26%, 30%, 31%, 28%, 35%, 0%, 0%, 45%, 28%, 34%, 46%, and 1%, respectively. The cells were kept at 90 °C for 2 h. min later, the residual enzyme activity of GoxM12 was 0%, and the mutants G272T / A171V, G272T / A171V / T8K (GoxM13), G272T / A171V / T8K / E12V, G272T / A171V / T8K / R16T, G272T / A171V / T8K / S72T, G272T / A171V / T8K / V104I, G272T / A171V / T8K / T168S, G272T / A171V / T8K / V184M, G272T / A171V / T8K / S224A, and G272T / A171V / T8K / The residual enzyme activities of S254D, G272T / A171V / T8K / L310V, G272T / A171V / T8K / L322V (GoxM14), G272T / A171V / T8K / E418V, G272T / A171V / T8K / D490F, G272T / A171V / T8K / P523S (GoxM15) and G272T / A171V / T8K / G541R were 40%, 61%, 43%, 38%, 43%, 33%, 56%, 48%, 53%, 31%, 28%, 61%, 49%, 50%, 62% and 17%, respectively.

[0058] When treated at pH 2.5 for 60 min, the relative residual enzyme activities of mutants G272T / A171V / T8K (GoxM13), G272T / A171V / T8K / L322V (GoxM14) and G272T / A171V / T8K / P523S (GoxM15) were 28%, 28% and 22%, respectively. When treated at pH 2.75 and 37 ℃ for 60 min, the relative residual enzyme activities of mutants G272T / A171V / T8K (GoxM13), G272T / A171V / T8K / L322V (GoxM14) and G272T / A171V / T8K / P523S (GoxM15) were 75%, 75% and 68%, respectively.

[0059] The glucose enzyme mutant with improved acid stability or thermal stability provided by the present invention is suitable for application in the field of food and feed and has very broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 The specific activities of GoxM12 and G272S series mutants are shown;

[0061] Figure 2 The specific activities of GoxM12 and G272T series mutants are shown;

[0062] Figure 3 The specific activities of GoxM12 and iterated mutants are shown;

[0063] Figure 4 Shows the comparison of thermal stability of GoxM12 and G272S series mutants treated at 80 °C for 5 min;

[0064] Figure 5 Shows the comparison of thermal stability of GoxM12 and G272S series mutants treated at 85 °C for 2 min;

[0065] Figure 6 Shows the comparison of thermal stability of GoxM12 and G272T series mutants treated at 80 °C for 3 min;

[0066] Figure 7 Shows the comparison of thermal stability of GoxM12 and G272T series mutants treated at 80 °C for 2 min;

[0067] Figure 8 Shows the comparison of thermal stability of GoxM12 and G272T series mutants treated at 80 °C for 5 min;

[0068] Fig. 9Shows the comparison of thermal stability of GoxM12 and iterated mutants treated at 85 °C for 5 min;

[0069] Fig.10 Shows the comparison of thermal stability of GoxM12 and iterated mutants treated at 90 °C for 2 min;

[0070] Fig.11 Shown is the comparison of acid stability (residual activity) of GoxM12 and iterated mutants treated at pH 2.5-8.0 for 60 min;

[0071] Fig.12 Shown is a comparison of the acid-stable residual specific activities of GoxM12 and iterated mutants treated at pH 2.5-8.0 for 60 min. DETAILED DESCRIPTION

[0072] Experimental materials and reagents

[0073] 1. Strains and vectors: The expression host is Pichia pastoris GS115, the expression plasmid vector is pPIC9.

[0074] 2. Enzymes and other biochemical reagents: endonucleases / high-fidelity DNA polymerases and recombinases.

[0075] 3. LB medium for Escherichia coli (1% peptone, 0.5% yeast extract, 1% NaCl, natural pH); YPD medium for Pichia pastoris (1% yeast extract, 2% peptone, 2% glucose, natural pH); BMGY (1% yeast extract, 2% peptone, 10% amino-free yeast nitrogen source, 0.1% biotin, natural pH); BMMY (1% yeast extract, 2% peptone, 10% amino-free yeast nitrogen source, 0.1% biotin, 1% methanol, natural pH).

[0076] Note: The molecular biology experimental methods not specifically described in the following examples are all carried out with reference 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.

[0077] According to a specific embodiment of the present invention, the glucose oxidase with the amino acid sequence shown in GoxM12 ID NO: 1 is subjected to single point mutations of T8K, S72T, V104I, A171V, V184M, S224A, G272S, T278Y, and E418V; and then subjected to single point mutations of G272S / T8K, G272S / S72T, G272S / V104I, G272S / A171V, G272S / V184M, G272S / S224A, G272S / T278Y, and E418V. 8Y, G272S / E418V double-point combination mutation; then G272S / A171V / T8K, G272S / A171V / S72T, G272S / A171V / V104I, G272S / A171V / V184M, G272S / A171V / S224A, G272S / A171V / T278Y, G272S / A171V / E418V triple-point combination mutation.

[0078] The glucose oxidase with the amino acid sequence shown in GoxM12 ID NO: 1 was subjected to single point mutations of E12V, R16T, T168S, S254D, G272T, L310V, L322V, T466S, D490F, P523S, and G541R; and then subjected to double point combination mutations of G272T / E12V, G272T / R16T, G272T / T168S, G272T / S254D, G272T / L310V, G272T / L322V, G272T / T466S, G272T / D490F, G272T / P523S, and G272T / G541R.

[0079] Then, the mutants G272S / A171V / T8K and G272T / D490F were selected for iterative mutation. However, because the G272 site overlapped, the mutant amino acids at the G272 site were swapped and compared, and the G272T / A171V / T8K (GoxM13) and G272S / D490F mutants were constructed. Finally, it was found that G272T / A171V / T8K (GoxM13) had the best stability. Therefore, G272T / A171V / T8K (GoxM13) was used as a template, the above single point was introduced, and G272T / A171V / T8K / E12V, G272T / A171V / T8K / R16T, G272T / A171V / T8K / S72T, G272T / A171V / T8K / V104I, G272T / A171V / T8K / T168S, G272T / A171V / T8K / V184M, G272T / A171V / T8K / S224A, G272 T / A171V / T8K / S254D, G272T / A171V / T8K / L310V, G272T / A171V / T8K / L322V (GoxM14), G272T / A171V / T8K / E418V, G272T / A171V / T8K / D490F, G272T / A171V / T8K / P523S (GoxM15), and G272T / A171V / T8K / G541R were used to obtain glucose oxidase mutants with improved acid and thermal stability.

[0080] Example 1. Preparation of recombinant strain GS115 (pPIC9-goxM12)

[0081] Amplification by PCR goxM12 Gene fragments, vector pPIC9 were obtained by inoculating the preserved strains with liquid LB medium and culturing overnight, and then extracting the plasmid. After amplification, the PCR product and the extracted plasmid were subjected to nucleic acid electrophoresis detection. goxM12 The band sizes of pPIC9 and pPIC9 were 1746 bp and 8088 bp respectively. Eco RI and Not After enzyme digestion, the PCR product and the enzyme digestion product were recovered and purified respectively, and the two were connected by recombinase to obtain the recombinant plasmid pPIC9- goxM12 , and transformed into Pichia pastoris GS115 to obtain the recombinant Pichia pastoris strain GS115 (pPIC9 -goxM12 ).

[0082] Example 2. Preparation of recombinant mutant strain GS115

[0083] 1. Construction of recombinant plasmid with improved stability

[0084] Specifically, the glucose oxidase with the amino acid sequence shown in GoxM12 ID NO: 1 was mutated as follows:

[0085] T8K, S72T, V104I, A171V, V184M, S224A, G272S, T278Y, E418V;

[0086] G272S / T8K, G272S / S72T, G272S / V104I, G272S / A171V, G272S / V184M, G272S / S224A, G272S / T278Y, G272S / E418V;

[0087] G272S / A171V / T8K, G272S / A171V / S72T, G272S / A171V / V104I, G272S / A171 V / V184M, G272S / A171V / S224A, G272S / A171V / T278Y, G272S / A171V / E418V;

[0088] E12V, R16T, T168S, S254D, G272T, L310V, L322V, T466S, D490F, P523S, G541R;

[0089] G272T / E12V, G272S / R16T, G272T / T168S, G272T / S254D, G272T / L310V, G272T / L322V, G272T / T466S, G272T / D490F, G272T / P523S, G272T / G541R;

[0090] G272T / A171V, G272T / A171V / T8K (GoxM13);

[0091] G272T / A171V / T8K / E12V, G272T / A171V / T8K / R16T, G272T / A171V / T8K / S72T, G272T / A171V / T8K / V 104I, G272T / A171V / T8K / T168S, G272T / A171V / T8K / V184M, G272T / A171V / T8K / S224A, G272T / A17 1V / T8K / S254D, G272T / A171V / T8K / L310V, G272T / A171V / T8K / L322V (GoxM14), G272T / A171V / T8K / E418V, G272T / A171V / T8K / D490F, G272T / A171V / T8K / P523S (GoxM15), G272T / A171V / T8K / G541R.

[0092] Design mutation primers according to the mutants and use plasmid pPIC9- goxM12 As a template, the mutant amino acid was introduced by a point mutation kit, and the PCR product containing the mutant amino acid was used Dpn The PCR products were transformed into competent E. coli Trans1_T1 cells and sequenced to obtain the E. coli pPIC9- goxm12 - Mutants .

[0093] 2. Construction of acid-stable and thermostable recombinant strain GS115

[0094] use Bgl Ⅱ The mutant plasmids with correct sequencing were digested by enzymes, and the digested products were recovered and transformed into Pichia pastoris GS115 competent cells by electroporation to obtain the recombinant expression strains GS115 (pPIC9- goxM12 - Mutants ).

[0095] Example 3. Obtaining glucose oxidase GoxM12 and highly acid-stable and thermostable mutant enzyme proteins

[0096] 1. Inducible expression of GoxM12 and its mutants

[0097] Each recombinant yeast expression strain obtained above was inoculated into 50 mL YPD medium at an inoculation rate of 1% for seed liquid culture. After culturing at 200 rpm and 30°C for 48 h, it was transferred to 400 mL BMGY medium at an inoculation rate of 1%, and cultured at 200 rpm and 30°C for 48 h. The cells were collected by centrifugation and transferred to 200 mL BMMY medium. 1% methanol was added every 24 h to induce expression, and the culture was carried out for a total of 48 h.

[0098] 2. Purification of GoxM12 and its mutants

[0099] The induced bacterial solution was centrifuged at 12000 rpm for 10 min, the supernatant was collected and concentrated using a 10kDa membrane package, and then dialyzed with 10 mM disodium hydrogen phosphate-citric acid buffer (pH 6.5). The dialyzed crude enzyme solution was then purified using an anion column. The purification solution A was 10 mM disodium hydrogen phosphate-citric acid buffer (pH 6.5), and the solution B was solution A plus 1 MNaCl. The protein was purified and the eluate was collected.

[0100] 3. Determination of enzymatic properties of GoxM12 and mutants

[0101] Enzyme activity assay of GoxM12 and mutants

[0102] The purified enzyme solution was diluted with 0.1 M sodium hydrogen phosphate-citrate buffer (pH 6.0) and incubated at 30 °C for 3 min to measure the Gox enzyme activity. The enzyme activities of GoxM12 and each mutant are shown in Table 1 and Figure 1~Figure 3 shown.

[0103] Table 1

[0104] Mutants Enzyme activity Compared with GoxM12, GoxM12 480 U / mg 0% G272S 496 U / mg 3% G272S / T8K 224 U / mg -53% G272S / S72T 507 U / mg 6% G272S / V104I 541 U / mg 13% G272S / A171V 552 U / mg 15% G272S / V184M 509 U / mg 6% G272S / S224A 482 U / mg 0.40% G272S / T278Y 457 U / mg -4.8% G272S / E418V 521 U / mg 9% G272S / A171V / T8K 501 U / mg 4% G272S / A171V / S72T 501 U / mg 4% G272S / A171V / V104I 411 U / mg -14% G272S / A171V / V184M 471 U / mg -2% G272S / A171V / S224A 489 U / mg 2% G272S / A171V / T278Y 431 U / mg -10% G272S / A171V / E418V 475 U / mg -1% G272T 483 U / mg 0.6% G272T / E12V 606 U / mg 26% G272T / R16T 610 U / mg 27% G272T / T168S 416 U / mg -13% G272T / S254D 482 U / mg 0.40% G272T / L310V 637 U / mg 33% G272T / L322V 524 U / mg 9% G272T / T466S 443 U / mg -8% G272T / D490F 551 U / mg 15% G272T / P523S 599 U / mg 25% G272T / G541R 609 U / mg 27% G272T / A171V / T8K (GoxM13) 534 U / mg 11% G272T / A171V / T8K / E12V 585 U / mg 22% G272T / A171V / T8K / R16T 498 U / mg 4% G272T / A171V / T8K / S72T 418 U / mg -13% G272T / A171V / T8K / V104I 295 U / mg -39% G272T / A171V / T8K / T168S 529 U / mg 10% G272T / A171V / T8K / V184M 478 U / mg -0.4% G272T / A171V / T8K / S224A 407 U / mg -15% G272T / A171V / T8K / S254D 434 U / mg -10% G272T / A171V / T8K / L310V 621 U / mg 29% G272T / A171V / T8K / L322V (GoxM14) 584 U / mg 22% G272T / A171V / T8K / E418V 493 U / mg 3% G272T / A171V / T8K / D490F 458 U / mg -5% G272T / A171V / T8K / P523S (GoxM15) 572 U / mg 18% G272T / A171V / T8K / G541R 516 U / mg 8%

[0105] There is a negative correlation between enzyme stability and activity, and the stability of the enzyme often leads to a decrease in its activity, which is the so-called activity-stability trade-off. The reason for this trade-off relationship mainly involves the interaction between the structural characteristics and functions of the enzyme. In the process of thermostability modification, the introduction of new mutations may weaken other functions, resulting in mutual constraints between stability and catalytic activity during evolution. Therefore, the thermostability of the GoxM12 mutant was higher than that of GoxM12 when kept in a water bath at high temperatures of 75℃, 80℃, 85℃ and 90℃ for different times, while at 30℃, after incubation for 3 min, the enzyme activity of some mutants was lower than that of GoxM12. The enzyme activity of some mutants in Table 1 was slightly lost, but their thermostability was improved, which broadened the scope of industrial application.

[0106] G272T / A171V / T8K (GoxM13), G272T / A171V / T8K / E12V, G272T / A171V / T8K / R16T, G272T / A171V / T8K / S72T, G 272T / A171V / T8K / V104I, G272T / A171V / T8K / T168S, G272T / A171V / T8K / V184M, G272T / A171V / T8K / S224A, G2 The specific activities of 72T / A171V / T8K / S254D, G272T / A171V / T8K / L310V, G272T / A171V / T8K / L322V (GoxM14), G272T / A171V / T8K / E418V, G272T / A171V / T8K / D490F, G272T / A171V / T8K / P523S (GoxM15), and G272T / A171V / T8K / G541R are 534 and 541, respectively. U / mg, 585 U / mg, 498 U / mg, 418 U / mg, 295 U / mg, 529 U / mg, 478 U / mg, 407 U / mg, 434 U / mg, 621 U / mg, 584 U / mg, 493 U / mg, 458 U / mg, 572 U / mg and 516 U / mg, compared with GoxM12, they increased by 11%, 22%, 4%, -13%, -39%, 10%, -0.4%, -15%, -10%, 29%, 22%, 3%, -5%, 18% and 8% respectively.

[0107] (2) Thermal stability determination of GoxM12 and its mutants

[0108] The purified enzyme solution was diluted to the same protein concentration with 0.1 M sodium hydrogen phosphate-citrate buffer (pH 6.0), and then incubated in a water bath at 75℃, 80℃, 85℃ and 90℃ for different time periods. The relative residual enzyme activity was determined according to the standard method. After incubation at 75℃ for 20 min, the residual enzyme activity of GoxM12 was 19%, and the residual enzyme activities of mutants T8K, S72T, V104I, A171V, V184M, S224A, G272S, T278Y and E418V were 36%, 25%, 26%, 29%, 24%, 48%, 58%, 22% and 30%, respectively, which were increased by about 89%, 32%, 37%, 53%, 26%, 153%, 205%, 16% and 58% compared with GoxM12 (19%).

[0109] like Figure 4As shown, the residual enzyme activity of GoxM12 was 0% after incubation at 80 °C for 5 min, and the mutants G272S / T8K, G272S / S72T, G272S / V104I, G272S / A171V, G272S / V184M, G272S / S224A, G272S / T278Y, G272S / E418V, G272S / A171V / T8K, G272S / A171V / S72T, G272S / A171V / V184M, G272S / A171V The residual enzyme activities of 71V / S224A, G272S / A171V / T278Y and G272S / A171V / E418V were 50%, 50%, 44%, 67%, 41%, 58%, 44%, 50%, 82%, 83%, 59%, 68%, 81%, 67% and 74%, respectively, which were increased by approximately 50%, 50%, 44%, 67%, 41%, 58%, 44%, 50%, 82%, 83%, 59%, 68%, 81%, 67% and 74% compared with GoxM12 (0%).

[0110] like Figure 5 As shown, the residual enzyme activity of GoxM12 was 17% after incubation at 85 °C for 2 min, and the mutants G272S / T8K, G272S / S72T, G272S / V104I, G272S / A171V, G272S / V184M, G272S / S224A, G272S / T278Y, G272S / E418V, G272S / A171V / T8K, G272S / A171V / S72T, G272S / A171V / V104I, G272S / A171V, G272S / V184M, G272S / S224A The residual enzyme activities of 24A, G272S / A171V / T278Y and G272S / A171V / E418V were 39%, 36%, 40%, 60%, 33%, 48%, 32%, 40%, 66%, 62%, 51%, 52%, 64%, 61% and 57%, respectively, which were approximately 129%, 112%, 135%, 253%, 94%, 182%, 88%, 135%, 288%, 265%, 200%, 206%, 276%, 259% and 235% higher than those of GoxM12 (17%).

[0111] like Figure 6As shown in the figure, after 3 min of incubation at 80 ℃, the residual enzyme activity of GoxM12 was 55%, and the residual enzyme activities of mutants E12V, R16T, T168S, S254D, G272T, L310V, L322V, T466S, D490F, P523S and G541R were 58%, 50%, 60%, 63%, 84%, 56%, 70%, 52%, 63%, 0% and 64%, respectively, which were increased by about 11%, 0%, 9%, 15%, 53%, 2%, 27%, 0%, 15%, 0% and 16% respectively compared with GoxM12 (55%). Although the thermal stability of single-point R16T, L310V and P523S was not improved, the thermal stability of double-point and quadruple-point mutants was improved after being superimposed with G272T, and the effect was very good.

[0112] like Figure 7 As shown, the residual enzyme activity of GoxM12 was 80% after incubation at 80 ℃ for 2 min, and the residual enzyme activities of mutants G272T / E12V, G272T / R16T, G272T / T168S, G272T / S254D, G272T / L310V, G272T / L322V, G272T / T466S, G272T / D490F, G272T / P523S and G272T / G541R were 92%, 97%, 93%, 99%, 100%, 100%, 99%, 100%, 90% and 98%, respectively, which were increased by about 12%, 21%, 16%, 24%, 25%, 25%, 24%, 25%, 12.5% ​​and 22.5% compared with GoxM12 (80%).

[0113] like Figure 8 As shown, after incubation at 80 ℃ for 5 min, the residual enzyme activity of GoxM12 was 0%, and the residual enzyme activities of mutants G272T / E12V, G272T / R16T, G272T / T168S, G272T / S254D, G272T / L310V, G272T / L322V, G272T / T466S, G272T / D490F, and G272T / P523S were 10%, 15%, 12%, 27%, 23%, 31%, 10%, 41%, 5%, and 0%, respectively, which were increased by about 10%, 15%, 12%, 27%, 23%, 31%, 10%, 41%, and 5%, respectively, compared with GoxM12 (0%).

[0114] like Fig. 9As shown, after incubation at 85 °C for 5 min, the residual enzyme activity of GoxM12 was 0%, while the mutants G272T / A171V, G272T / A171V / T8K (GoxM13), G272T / A171V / T8K / E12V, G272T / A171V / T8K / R16T, G272T / A171V / T8K / S72T, and G272T / A171 V / T8K / V104I, G272T / A171V / T8K / T168S, G272T / A171V / T8K / V184M, G272T / A171 V / T8K / S224A, G272T / A171V / T8K / S254D, G272T / A171V / T8K / L310V, G272T / A171 The residual enzyme activities of G272T / A171V / T8K / L322V (GoxM14), G272T / A171V / T8K / E418V, G272T / A171V / T8K / D490F, G272T / A171V / T8K / P523S (GoxM15) and G272T / A171V / T8K / G541R were 35%, 58%, 21%, 15%, 26%, 30%, 31%, 28%, 35%, 0%, 0%, 45%, 28%, 34%, 46% and 1%, respectively, which were increased by about 35%, 58%, 21%, 15%, 26%, 30%, 31%, 28%, 35%, 0%, 0%, 45%, 28%, 34%, 46% and 1% compared with GoxM12 (0%).

[0115] like Fig.10As shown, after incubation at 90 °C for 2 min, the residual enzyme activity of GoxM12 was 0%, and the mutants G272T / A171V, G272T / A171V / T8K (GoxM13), G272T / A171V / T8K / E12V, G272T / A171V / T8K / R16T, G272T / A171V / T8K / S72T, G272T / A171V / T8K / V104I, G272T / A171V / T8K / T168S, G272T / A171V / T8K / V184M, G272T / A171V / T8K / S224A, G272T / A171V / T8K / S254D, G272T / A171V / T8K / L310V, G272T / A171V / T The residual enzyme activities of G272T / A171V / T8K / E418V, G272T / A171V / T8K / D490F, G272T / A171V / T8K / P523S (GoxM15) and G272T / A171V / T8K / G541R were 40%, 61%, 43%, 38%, 43%, 33%, 56%, 48%, 53%, 31%, 28%, 61%, 49%, 50%, 62% and 17%, respectively, which were increased by about 40%, 61%, 43%, 38%, 43%, 33%, 56%, 48%, 53%, 31%, 28%, 61%, 49%, 50%, 62% and 17% compared with GoxM12 (0%). The residual enzyme activities of G272T / A171V / T8K / S254D and G272T / A171V / T8K / L310V were 0 after incubation at 85 °C for 5 min, which was the same as GoxM12, but they were improved compared with GoxM12 after treatment at 90 °C for 2 min.

[0116] (3) Acid stability assay of GoxM12 and its mutants

[0117] The same concentration of GoxM12 and mutant enzyme solution was placed in accurately prepared 0.1 M sodium dihydrogen phosphate-citrate buffer at pH 2.5, 2.75, 3.0, 4.0, 5.0, 6.0, 7.0 and 8.0, and then incubated at 37 °C for 1 h. After the incubation, the residual enzyme activity was measured under standard conditions, and the enzyme activity of the untreated sample was recorded as 100%. Fig.11As shown, when treated at pH 2.5 for 60 min, the relative residual enzyme activities of the mutants G272T / A171V / T8K (GoxM13), G272T / A171V / T8K / L322V (GoxM14) and G272T / A171V / T8K / P523S (GoxM15) were 28%, 28% and 22%, respectively, which were increased by about 87%, 87% and 47% compared with GoxM12 (15%). After treatment at pH 2.75 and 37 ℃ for 60 min, the relative residual enzyme activities of mutants G272T / A171V / T8K (GoxM13), G272T / A171V / T8K / L322V (GoxM14) and G272T / A171V / T8K / P523S (GoxM15) were about 75%, 75% and 68%, respectively, which were increased by about 74%, 74% and 58% compared with GoxM12 (43%).

[0118] like Fig.12 As shown, after treatment at pH 2.5 for 60 min, the residual specific activities of the mutants G272T / A171V / T8K (GoxM13), G272T / A171V / T8K / L322V (GoxM14) and G272T / A171V / T8K / P523S (GoxM15) were 157 U / mg, 164 U / mg and 128 U / mg, respectively, which were increased by approximately 112%, 122% and 73% compared with GoxM12 (74 U / mg). After treatment at pH 2.75 and 37 ℃ for 60 min, the relative residual specific activities of the mutants G272T / A171V / T8K (GoxM13), G272T / A171V / T8K / L322V (GoxM14) and G272T / A171V / T8K / P523S (GoxM15) were 415 U / mg, 436 U / mg and 391 U / mg, respectively, which were increased by about 99%, 110% and 88% compared with GoxM12 (208 U / mg).

[0119] The above embodiments are only used to explain the technical solution of the present application and do not limit the protection scope of the present application.

Claims

1. A glucose oxidase GoxM12 mutant with improved thermal stability, characterized in that: The amino acid sequence of the mutant is shown in SEQ ID NO:

2.

2. A glucose oxidase GoxM12 mutant with improved thermal stability, characterized in that: The amino acid sequence of the mutant is shown in any one of SEQ ID NO: 3 to SEQ ID NO:

26.

3. A glucose oxidase gene encoding the glucose oxidase GoxM12 mutant with improved thermal stability according to claim 1 or 2.

4. A method for improving the thermal stability of glucose oxidase, characterized in that: The method comprises the steps of performing G272T mutation on the glucose oxidase having an amino acid sequence as shown in SEQ ID NO:

1.

5. A method for improving the stability of glucose oxidase, characterized in that: The method comprises the following steps of subjecting the glucose oxidase having an amino acid sequence as shown in SEQ ID NO: 1 to the following mutations: G272T / E12V, G272T / R16T, G272T / T168S, G272T / S254D, G272T / L310V, G272T / L322V, G272T / T466S, G272T / D490F, G272T / P523S, or G272T / G541R.

6. A method for improving the thermal stability of glucose oxidase, characterized in that: The method comprises the following steps of subjecting the glucose oxidase having an amino acid sequence as shown in SEQ ID NO: 1 to the following mutations: G272T / A171V, G272T / A171V / T8K, G272T / A171V / T8K / E12V, G272T / A171V / T8K / R16T, G272T / A171V / T8K / S72T, G272T / A171V / T8K / V104I 41R, G272T / A171V / T8K / T168S, G272T / A171V / T8K / V184M, G272T / A171V / T8K / S224A, G272T / A171V / T8K / S254D, G272T / A171V / T8K / L310V, G272T / A171V / T8K / L322V, G272T / A171V / T8K / E418V, G272T / A171V / T8K / D490F, G272T / A171V / T8K / P523S, or G272T / A171V / T8K / G541R.

7. A method for improving the thermal stability and acid stability of glucose oxidase, characterized in that: The method comprises the following steps of subjecting the glucose oxidase having an amino acid sequence as shown in SEQ ID NO: 1 to the following mutations: G272T / A171V / T8K, G272T / A171V / T8K / L322V, or G272T / A171V / T8K / P523S.

8. A recombinant expression strain comprising the glucose oxidase gene of claim 3.

9. Use of the glucose oxidase GoxM12 mutant with improved thermal stability according to claim 1 or 2 in the food field or feed field.

10. A method for preparing glucose oxidase, characterized in that: The method comprises the following steps: Inducing the recombinant expression strain of claim 8 to express glucose oxidase; The glucose oxidase is isolated and purified.

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