Glucose oxidase GoxM12 mutant G272S 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 glucose oxidase GoxM12 mutant G272S with improved thermal stability and its derivative mutants and applications. The present invention uses high thermal stability glucose oxidase GoxM12 as research material, conducts thermal improvement research on it, obtains a mutant with improved thermal stability, thereby promoting 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 G272S with improved thermal stability and a derivative mutant thereof and application 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] CN112301009A 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 2 min, 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] CN114736879A provides a 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 made a triple mutation Q241E / E361P / A419I to GoxM10, and then made a quadruple 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] 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.

[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 a G272S mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO: 2.

[0018] 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 G272S / S72T mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO:3.

[0019] 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 G272S / V104I mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO:4.

[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 G272S / A171V mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO:5.

[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 G272S / V184M mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO: 6.

[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 G272S / S224A mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO:7.

[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 G272S / T278Y mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO: 8.

[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 G272S / E418V mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO:9.

[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 G272S / A171V / T8K mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO: 10.

[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 G272S / A171V / S72T mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO:11.

[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 G272S / A171V / V104I mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO:12.

[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 G272S / A171V / V184M mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO:13.

[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 G272S / A171V / S224A mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO:14.

[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 G272S / A171V / T278Y mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO:15.

[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 G272S / A171V / E418V mutation to obtain a mutant with an amino acid sequence as shown in SEQ ID NO: 16.

[0032] According to a specific embodiment of the present invention, a recombinant vector comprising the above-mentioned glucose oxidase mutant gene is also provided, and the starting vector of the recombinant expression vector is pPIC9.

[0033] According to a specific embodiment of the present invention, a recombinant strain comprising the above-mentioned glucose oxidase mutant gene is also provided.

[0034] The method for preparing glucose oxidase with improved thermal stability according to the present invention comprises the following steps:

[0035] 1) preparing a recombinant vector containing the mutant gene;

[0036] 2) The recombinant vector is transformed into the Pichia pastoris GS115 expression host;

[0037] 3) fermenting and culturing the host and isolating glucose oxidase.

[0038] The present invention provides the application of the above-mentioned glucose oxidase mutant with acid stability or thermal stability, which can be specifically applied in the field of food and feed.

[0039] 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.

[0040] The invention provides a glucose enzyme mutant with improved thermal stability, which is suitable for application in the field of food and feed and has very broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The specific activities of GoxM12 and G272S series mutants are shown;

[0042] Figure 2The residual enzyme activities of GoxM12 and G272S series mutants after incubation at 75 °C for 20 min are shown;

[0043] Figure 3 The residual enzyme activities of GoxM12 and G272S series mutants after incubation at 80 °C for 5 min are shown;

[0044] Figure 4 Shown is the comparison of thermal stability of GoxM12 and G272S series mutants treated at 85 °C for 2 min. DETAILED DESCRIPTION

[0045] Experimental materials and reagents

[0046] 1. Strains and vectors: The expression host is Pichia pastoris GS115, the expression plasmid vector is pPIC9.

[0047] 2. Enzymes and other biochemical reagents: endonucleases / high-fidelity DNA polymerases and recombinases.

[0048] 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).

[0049] 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.

[0050] 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.

[0051] Example 1. Preparation of recombinant strain GS115 (pPIC9-goxM12)

[0052] 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 ).

[0053] Example 2. Preparation of recombinant mutant strain GS115

[0054] 1. Construction of recombinant plasmid with improved stability

[0055] Specifically, the glucose oxidase with the amino acid sequence shown in GoxM12 ID NO: 1 was mutated as follows:

[0056] T8K, S72T, V104I, A171V, V184M, S224A, G272S, T278Y, E418V;

[0057] G272S / T8K, G272S / S72T, G272S / V104I, G272S / A171V, G272S / V184M, G272S / S224A, G272S / T278Y, G272S / E418V;

[0058] G272S / A171V / T8K, G272S / A171V / S72T, G272S / A171V / V104I, G272S / A171 V / V184M, G272S / A171V / S224A, G272S / A171V / T278Y, G272S / A171V / E418V.

[0059] 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 .

[0060] 2. Construction of acid-stable and heat-stable recombinant strain GS115

[0061] 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 ).

[0062] Example 3. Obtaining glucose oxidase GoxM12 and highly thermostable mutant enzyme proteins

[0063] 1. Inducible expression of GoxM12 and its mutants

[0064] 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.

[0065] 2. Purification of GoxM12 and its mutants

[0066] 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.

[0067] 3. Determination of Enzymatic Properties of GoxM12 and Mutants

[0068] (1) Enzyme activity assay of GoxM12 and mutants

[0069] 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 shown.

[0070] Table 1

[0071] 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%

[0072] 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.

[0073] (2) Thermal stability determination of GoxM12 and its mutants

[0074] 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°C, 80°C, 85°C and 90°C for different time periods. The relative residual enzyme activity was determined according to the standard method. After incubation 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, which were increased by about 89%, 32%, 37%, 53%, 26%, 153%, 205%, 16% and 58% compared with GoxM12 (19%). Figure 2 ).

[0075] like Figure 3 As 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%).

[0076] like Figure 4As 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%).

[0077] 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:

16.

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 step of subjecting the glucose oxidase having an amino acid sequence as shown in SEQ ID NO: 1 to a G272S mutation.

5. A method for improving the thermal stability of glucose oxidase, characterized in that: The method comprises the steps of mutating the glucose oxidase having an amino acid sequence as shown in SEQ ID NO: 1 as follows: G272S / T8K, G272S / S72T, G272S / V104I, G272S / A171V, G272S / V184M, G272S / S224A, G272S / T278Y, or G272S / E418V.

6. A method for improving the thermal stability of glucose oxidase, characterized in that: The method comprises the steps of mutating the glucose oxidase having an amino acid sequence as shown in SEQ ID NO: 1 as follows: G272S / A171V / T8K, G272S / A171V / S72T, G272S / A171V / V104I, G272S / A171V / V184M, G272S / A171V / S224A, G272S / A171V / T278Y, or G272S / A171V / E418V.

7. A recombinant expression vector comprising the glucose oxidase gene of claim 3.

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 for non-therapeutic purposes.

10. 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 7 to obtain a recombinant expression strain; Inducing the recombinant expression strain to express glucose oxidase; isolating and purifying the glucose oxidase.

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