A glucose oxidase mutant with improved acid and heat resistance, and its construction method and application

Abstract

The present invention discloses a glucose oxidase mutant with improved acid and heat resistance, as well as a construction method and application thereof, and belongs to the field of bioengineering technology. The present invention uses glucose oxidase AsGODP derived from the fungus Aspergillus sclerotiicarbonarius as the parent, and synchronously mutates Glu148 and Val179 to Lys148 and Ala179 to obtain the mutant E148K / V179A. Experiments have found that the mutant E148K / V179A has significant improvements in pH stability, thermal stability, half-life at 75°C and catalytic performance compared with the wild-type AsGODP. Based on the catalytic performance, the mutant E148K / V179A is used in bread baking, which can effectively increase the dough gluten under fermentation conditions and improve the texture of bread, thereby showing great development potential in the bread baking industry.

Description

Technical Field

[0001] The present invention relates to the fields of genetic engineering and protein engineering, and in particular to a glucose oxidase mutant with improved acid and heat resistance, and a construction method and application thereof. Background Art

[0002] Glucose oxidase (GOD) is a well-characterized glycoprotein consisting of two identical 80 kDa subunits linked by disulfide bonds, each of which contains 1 mol of tightly bound but not covalently linked FAD fragments as cofactors. GOD can use molecular oxygen as an electron acceptor to catalyze the oxidation of d-glucose (C6H 12 O6) to d-gluconolactone (C6H 10 O6) and hydrogen peroxide. d-Gluconolactone can undergo non-enzymatic hydrolysis to produce gluconic acid and water.

[0003] Glucose oxidase (GOD) has been widely used in medicine, food, and feed due to its unique reaction mechanism and safe reaction products. Glucose oxidase is primarily sourced from Aspergillus and Penicillium. However, currently known GODs exhibit poor stability and low catalytic activity. Naturally expressed GODs experience significant loss of enzyme activity at temperatures exceeding 55°C. These limitations significantly limit the application of GODs, hindering their ability to meet industrial needs. Furthermore, while scientists both domestically and internationally have focused on directed evolution and rational design of enzyme molecules, molecular directed evolution is labor-intensive, highly random, and expensive. Rational design requires a solid understanding of the functional structure of GODs. Currently, the limited availability of GODs and the limited number of resolved structures make precise design difficult, and the results achieved remain limited. Therefore, using more reliable bioinformatics techniques to model and elucidate the structure and function of glucose oxidase, and attempting to fully analyze and refine key theoretical structures and sites, to improve its catalytic activity and stability, could significantly advance its industrial application.

[0004] Increased access to large experimental protein databases, coupled with advances in high-throughput screening (HTS) methods and artificial intelligence algorithms, has significantly advanced protein engineering. A range of rational computer-assisted learning strategies have been developed, such as predicting functional changes resulting from changes in enzyme protein sequences through machine learning or deep learning methods, and identifying sequence or structural features from extensive protein datasets. Furthermore, deep neural networks and molecular evolution can be used to extract and identify characteristic information such as protein sequence, structure, and function. Summary of the Invention

[0005] Technical problem to be solved: The present invention provides a glucose oxidase mutant with improved acid and heat resistance, as well as a construction method and application thereof. Specifically, the present invention obtains a glucose oxidase mutant with significantly improved acid and heat resistance by mutating the key amino acid sites E148 and V179 of AsGODP from Aspergillus sclerotiicarbonarius and screening. The mutant can significantly improve dough texture and has broad prospects for application in bread baking improvement.

[0006] Technical solution: A glucose oxidase mutant with improved acid and heat resistance is obtained by taking the wild-type glucose oxidase AsGODP as the parent, and simultaneously mutating the 148th Glu of the wild-type glucose oxidase AsGODP to Lys, and the 179th Val of the wild-type glucose oxidase AsGODP to Ala; the amino acid sequence of the wild-type glucose oxidase AsGODP is shown in SEQ ID NO.1, and the amino acid sequence of the glucose oxidase mutant is shown in SEQ ID NO.2.

[0007] The present invention also provides a gene encoding the glucose oxidase mutant, and the nucleotide sequence of the gene is shown in SEQ ID NO.3.

[0008] The invention also provides a recombinant vector containing the gene.

[0009] The present invention also provides a recombinant strain containing the recombinant vector.

[0010] The present invention also provides use of the glucose oxidase mutant, the gene, the recombinant vector or the recombinant strain in bread baking.

[0011] The present invention also provides the use of the glucose oxidase mutant, the gene, the recombinant vector, or the recombinant strain in any of the following:

[0012] (1) Application in the preparation of a leavening agent for improving dough texture;

[0013] (2) Application in the preparation of bread baking improvers.

[0014] The present invention also provides a leavening agent for improving dough texture, wherein the effective component of the leavening agent is prepared from the recombinant strain.

[0015] The present invention also provides a bread baking improver, the effective ingredients of which are prepared from the recombinant strain.

[0016] The present invention also provides a method for producing glucose mutants by fermentation using the recombinant strain, comprising the steps of collecting precipitates after fermentation of the recombinant strain and inducing culture using an induction medium containing methanol.

[0017] Preferably, the methanol-containing induction medium is prepared by adding 0.5% methanol by mass volume to the fermentation medium of the recombinant strain, and the induction culture conditions are 25-35°C and 100-300rpm. Methanol is added once every 12 hours to keep the methanol concentration in the bacterial liquid at 0.5%.

[0018] Beneficial effects: The present invention obtains a mutant by performing site-directed mutagenesis on the amino acids at positions 148 and 179 using glucose oxidase AsGODP from Aspergillus sclerotiicarbonarius as the parent. Specifically, a recombinant strain containing the mutant is constructed, and after induction culture, a glucose oxidase mutant E148K / V179A with significantly improved acid resistance and thermal stability is screened out. In terms of pH performance, the optimal pH for both the wild-type enzyme and the mutant was 6.0. In acidic environments (pH 2.0, 3.0, 4.0, and 5.0), the mutant could maintain 19%, 62%, 77%, and 86% relative enzyme activities, respectively, which were 2.8-fold, 2.9-fold, 1.3-fold, and 21% higher than those of the wild-type (5%, 16%, 34%, and 71%) under the same conditions. In terms of pH stability, in acidic environments (pH 2.0, 3.0, 4.0, and 5.0), the mutant could maintain 21%, 60%, 79%, and 88% residual enzyme activities, respectively, which were 2.0-fold, 1.6-fold, 68%, and 21% higher than those of the wild-type (7%, 23%, 47%, and 73%) under the same conditions. This indicates that the mutant enzyme E148K / V179A has significantly better activity and tolerance in acidic environments than the wild-type enzyme. In terms of thermal stability, the T 50 The half-life of mutant E148K / V179A at 75℃ (t 1 / 2 ) was 30 min, which was 5 times higher than that of the wild enzyme AsGODP (6 min); in terms of catalytic performance, the specific activity and catalytic efficiency of the mutant E148K / V179A were 237.5 U / mg and 26.3 mM, respectively. -1 ·s -1 , compared with wild enzyme AsGODP (specific activity 33.67U / mg, catalytic efficiency 16.15mM -1 s -1 ) increased by 6-fold and 63%, respectively. The present invention, through site-directed mutagenesis, obtained a more acid-resistant and heat-resistant mutant with high catalytic activity, E148K / V179A. Therefore, the application of the glucose oxidase mutant with improved acid and heat resistance of the present invention in the bread baking industry has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the polyacrylamide gel electrophoresis of wild-type glucose oxidase AsGODP and its mutant E148K / V179A; M is a marker, A and B are wild-type enzyme AsGODP and mutant enzyme E148K / V179A, respectively;

[0020] Figure 2 The results of the optimal pH determination of wild-type glucose oxidase AsGODP and its mutant E148K / V179A are shown;

[0021] Figure 3 The results of pH stability test of wild-type glucose oxidase AsGODP and its mutant E148K / V179A are shown;

[0022] Figure 4 The results of the optimal temperature determination of wild-type glucose oxidase AsGODP and its mutant E148K / V179A;

[0023] Figure 5 T is the wild-type glucose oxidase AsGODP and its mutant E148K / V179A 50 Measurement results;

[0024] Figure 6 is the half-life of wild-type glucose oxidase AsGODP and its mutant E148K / V179A at 75°C 1 / 2 Measurement results;

[0025] Figure 7 These are the results of the effects of wild-type glucose oxidase AsGODP and its mutant E148K / V179A on bread quality. DETAILED DESCRIPTION

[0026] The test materials used in the following examples are:

[0027] 1. Strains and vectors: The expression host is Pichiapastoris GS115, and the expression plasmid vector pPIC9r is prepared in the laboratory;

[0028] 2. Enzymes and other biochemical reagents: Taq enzyme was purchased from Quanshijin Company, endonuclease was purchased from Quanshijin Company, o-dianisidine was purchased from Sigma, and peroxidase was purchased from Yuanye Company; other reagents were domestic analytical grade (all purchased from Sinopharm Group);

[0029] 3. Culture medium:

[0030] (1) LB medium: 0.5% yeast extract, 1% peptone, 1% NaCl, pH 7.0;

[0031] (2) YPD medium: 1% yeast extract, 2% peptone, 2% glucose;

[0032] (3) MD solid medium: 2% glucose, 1.5% agarose, 1.34% YNB, 0.00004% Biotin;

[0033] (4) MM solid medium: 1.5% agarose, 1.34% YNB, 0.00004% Biotin, 0.5% methanol;

[0034] (5) BMGY medium: 1% yeast extract, 2% peptone, 1% glycerol (V / V), 1.34% YNB, 0.00004% Biotin;

[0035] (6) BMMY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 0.5% methanol (V / V).

[0036] Example 1 Synthesis of Glucose Oxidase Mutant Gene and Acquisition of Recombinant Plasmid

[0037] To improve the acid and heat resistance of glucose oxidase (AsGODP) from Aspergillus sclerotii carbonarius, amino acid residues at positions 148 and 179 were mutated: Glu at position 148 was mutated to Lys, and Val at position 179 was mutated to Ala. The glucose oxidase mutant containing these two mutation sites was named E148K / V179A; the amino acid sequence of E148K / V179A is shown in SEQ ID NO. 2. The primer sequences used are shown in Table 1. The site-directed mutagenesis and cloning methods were based on the literature (Exploiting the activity-stability trade-off of glucose oxidase from Aspergillus niger using a simple approach to calculate thermostability of mutants. Jiang, et al., 2021). The recombinant expression plasmid pPIC9r / E148K / V179A of the mutant E148K / V179A was obtained.

[0038] Table 1 Error-prone PCR primers for glucose oxidase AsGODP

[0039]

[0040] Example 2 Preparation of Glucose Oxidase Mutants

[0041] The linear recombinant expression vector obtained by PCR in Example 1 was directly transformed into a DMT competent cell and verified by colony PCR to obtain the nucleotide sequence encoding the glucose oxidase mutant E148K / V179A. The recombinant plasmid was linearized and transformed into Pichia pastoris GS115 to obtain the recombinant yeast strain GS115 / E148K / V179A.

[0042] The GS115 strain containing the recombinant plasmid was inoculated into 2 mL of BMGY medium in a 10 mL test tube and incubated at 30°C, 220 rpm, and shaken for 48 hours. The culture was then centrifuged at 3000 g for 5 minutes, the supernatant discarded, and the pellet resuspended in 2 mL of BMMY medium containing 0.5% methanol. The culture was again incubated at 30°C, 220 rpm, for 48 hours. The supernatant was used for enzyme activity testing, and the mutant E148K / V179A (amino acid sequence shown in SEQ ID NO. 2, nucleotide sequence shown in SEQ ID NO. 3) was identified, which exhibited improved acid resistance and thermostability compared to the wild-type enzyme.

[0043] The wild-type and mutant yeast strains were scaled up to form a fermentation system. The seed culture was first inoculated into YPD medium to obtain the seed culture solution, which was then inoculated into a 1L Erlenmeyer flask containing 300mL of BMGY medium at a 1% inoculation rate and cultured at 30°C and 220rpm. The culture solution was then centrifuged at 3000g for 5min, the supernatant was discarded, and the precipitate was resuspended in 100mL of BMMY medium containing 0.5% methanol and again induced at 30°C and 220rpm. 0.5mL of methanol was added every 12h to maintain the methanol concentration in the bacterial solution at 0.5%, and the supernatant was taken for enzyme activity detection. Finally, the supernatant was concentrated to 20mL for enzyme property determination and comparison. The protein was purified using the anion exchange method. After the expressed glucose oxidase was purified, polyacrylamide gel electrophoresis ( Figure 1 ) showed that the molecular weight of wild-type and mutant glucose oxidase mutants in the fermentation supernatant was approximately 80-90 kDa.

[0044] Example 3 Comparative analysis of enzymatic properties of recombinant glucose oxidase mutants with improved acid and heat resistance and wild type

[0045] 1. Determination by o-dianisidine method

[0046] The specific method is as follows: Under standard conditions (pH 6.0, 30°C), a 3 mL reaction system consists of 2.5 mL of o-dianisidine buffer, 300 μL of substrate, 100 μL of peroxidase (90 U / mL), and 100 μL of diluted enzyme solution. The reaction is incubated for 3 minutes and terminated by adding 2 mL of 2M H₂SO₄. The OD value is measured at 540 nm. One unit (U) of enzyme activity is defined as the amount of enzyme required to decompose 1 μmol of substrate per minute to produce hydrogen peroxide under standard conditions.

[0047] 1. Kinetic parameter determination method for recombinant glucose oxidase mutants and wild type

[0048] The detection method was referred to the literature (Improving the thermostability and catalytic efficiency of glucose oxidase from Aspergillus niger by molecular evolution. Tu, et al., 2019).

[0049] Glucose solutions of varying concentrations (3.125 mM-1000 mM) were prepared as substrates in 0.1 mol / L citric acid-sodium hydrogen phosphate buffer at pH 6.0. Enzyme activity was determined at 30°C and pH 6.0. The enzyme activity data were analyzed using GraphPad Prism 5.01 software to obtain the K values ​​of the wild-type recombinant high-catalytic glucose oxidase and its mutants. m Value and V max .

[0050] Under standard conditions (30°C, pH 6) with glucose as substrate, the specific activity of the glucose oxidase mutant E148K / V179A was 237.5 U / mg, which was 6 times higher than that of the wild-type AsGODP (33.67 U / mg); the catalytic efficiency of the mutant enzyme E148K / V179A was 26.3 mM -1 ·s -1 , compared with wild type AsGODP (16.15mM -1 ·s -1 ) was increased by 62%. The specific activities and kinetic parameters of the mutant and wild type are shown in Table 2.

[0051] Table 2 Comparison of specific activities and kinetic parameters of recombinant glucose oxidase mutants and wild type

[0052]

[0053] 2. Optimal pH determination method for recombinant glucose oxidase mutants and wild type

[0054] The glucose oxidase mutant and wild-type glucose oxidase from Example 2 were subjected to enzymatic reactions at various pH levels (1.0-12.0) to determine their optimal pH. Glucose oxidase activity was determined at 30°C using 0.1 mol / L citric acid-disodium hydrogen phosphate buffer at various pH levels (1.0-12.0) using the substrate β-D-glucose.

[0055] The results are as follows Figure 2 As shown, the optimal reaction pH of wild-type glucose oxidase and glucose oxidase mutants is 6.0.

[0056] 3. pH Stability Determination of Recombinant Glucose Oxidase Mutants and Wild Type

[0057] The glucose oxidase mutants and wild-type glucose oxidase were diluted with 0.1 mol / L citric acid-disodium hydrogen phosphate buffer at different pH values ​​(1.0-12.0) and placed in a 37°C constant temperature water bath for 1 h. The relative residual enzyme activity was then measured at pH 6.0 and 30°C. The untreated enzyme activity was set as 100% of the control.

[0058] The results are as follows Figure 3 As shown, the enzyme activity and stability of the glucose oxidase mutant E148K / V179A were better than those of the wild-type glucose oxidase in an acidic environment of pH 1.0-5.0.

[0059] 4. Method for determining the optimal temperature of recombinant glucose oxidase mutants and wild type

[0060] The optimal temperatures of the recombinant glucose oxidase mutant and the wild-type glucose oxidase were determined by performing the enzymatic reaction in a 0.1 mol / L citric acid-disodium hydrogen phosphate buffer (pH 6.0) buffer system at different temperatures (0-75°C).

[0061] The results are as follows Figure 4 As shown in the table, the optimum temperature of the recombinant wild-type glucose oxidase is 40°C, the optimum temperature of the high catalytic activity glucose oxidase mutant E148K / V179A is 45°C, and the relative enzyme activity under high temperature (50-75°C) conditions is significantly improved compared with the wild enzyme.

[0062] 5. Thermal stability determination method of recombinant glucose oxidase mutants and wild type

[0063] T at 40-80℃ 50 Value: After the mutant and wild-type enzymes were treated at different temperatures of 40-80℃ for 30min, the residual enzyme activities of each were detected.

[0064] Half-life at 75℃ (t 1 / 2):The mutant and wild-type enzymes were treated at 75℃ for different time periods, up to 60 min, and their residual enzyme activities were detected.

[0065] T at 40-80℃ 50 The results of the value determination are as follows Figure 5 As shown, the T of mutant E148K / V179A 50 The half-life value is 75℃, which is 15℃ higher than that of the wild type (60℃); the half-life determination results at 75℃ are as follows Figure 6 As shown, the half-life of mutant E148K / V179A at 75°C (t 1 / 2 ) is 30 min, which is 24 min longer than that of the wild enzyme AsGODP (6 min), that is, the thermal stability of the glucose oxidase mutant E148K / V179A is significantly improved.

[0066] 6. Analysis of the effects of recombinant glucose oxidase mutants and wild type on dough texture properties

[0067] After mixing the mutant enzyme and wild-type enzyme, the dough was mixed with a dough mixer at room temperature for 10 minutes. The dough was then proofed in a 28°C incubator for 60 minutes. The dough was kneaded again and then proofed in an incubator for another 60 minutes. After proofing, the dough was placed in a mold and baked in an oven set to 180°C (top heat) and 200°C (bottom heat) for 30 minutes. After baking, the dough was sliced ​​into 1cm thick slices using a slicer for subsequent bread quality assessment. The bread slices were placed directly under the probe for texture profile analysis (TPA, three replicates per sample). A cylindrical probe was used, the probe pause time was set to 1s, and after zeroing, the sample height was set to 2cm, the deformation was set to 30%, the detection speed was set to 30mm / min, and the starting force was set to 0.15N. Texture parameters of the bread slices were measured.

[0068] The results are as follows Figure 7 As shown, when the mutant E148K / V179A is added at a level of 0.1%, it can significantly increase the volume of bread, improve the chewiness, softness and elasticity of bread, thereby improving the quality of bread.

[0069] SEQ ID NO.1:

[0070] LPHYIRSSGIEASLLTDPEDVAGRTVDYIIAGGGLTGLTTAARLTENPDITVLVIESGFYESDQGPMVEDLNAYGEIFGSNVDHAYETVELATNNLTELIRSGNGLGGSTLVNGGTWTRPHKVQVDSWETVFGNEGWNWDNVAAYSLEAELARAPNAKQIAAGHYFDESCHGMNGTVHVGPRDTGEDYTPIIEALMTTVDKRGVPTKKDLGCGDPHGVSMFPNTLHEDQVRSDAAREWLLPNYQRPNLKVLTGQLVGKVLLNQNATVPKAVGVEFGTHRDNTFNVYAKHEVLLAAGSAISPTILEHSGIGMKSVLDSVGIDTVVDLPVGLNLQDQTTVAVSSRITSAGAGQGQAAYFATFNETFGDYAAEAHEMLNSKLEQWAEETVARGGFHNTTALLIQYENYRDWLVNHNVAYSELFLDTAGAVSFTIWDLIPFTRGYVHTSHKDPYLRLSAYDPQYFLNELDLYGQAAASQLARNLSNSDAMSTYFAGETTPGDNLAYDAGLSDWAEYIRYNFRPNYHGVGTCSMMPKEMGGVVDSSARVYGVDSLRVIDGSIPPTQVSSHVMTVFYAMALKISDAILADYAKSQ。

[0071] SEQ ID NO.2:

[0072] LPHYIRSSGIEASLLTDPEDVAGRTVDYIIAGGGLTGLTTAARLTENPDITVLVIESGFYESDQGPMVEDLNAYGEIFGSNVDHAYETVELATNNLTELIRSGNGLGGSTLVNGGTWTRPHKVQVDSWETVFGNEGWNWDNVAAYSLKAELARAPNAKQIAAGHYFDESCHGMNGTVHAGPRDTGEDYTPIIEALMTTVDKRGVPTK KDLGCGDPHGVSMFPNTLHEDQVRSDAAREWLLPNYQRPNLKVLTGQLVGKVLLNQNATVPKAVGVEFGTHRDNTFNVYAKHEVLLAAGSAISPTILEHSGIGMKSVLDSVGIDTVVDLPVGLNLQDQTTVAVSSRITSAGAGQGQAAYFATFNETFGDYAAEAHEMLNSKLEQWAEETVARGGFHNTTALLIQYENYRDWLVNHNVAYSELFLDTAGAVSFTIWDLIPFTRGYVHTSHKDPYLRLSAYDPQYFLNELDLYGQAAASQLARNLSNSDAMSTYFAGETTPGDNLAYDAGLSDWAEYIRYNFRPNYHGVGTCSMMPKEMGGVVDSSARVYGVDSLRVIDGSIPPTQVSSHVMTVFYAMALKISDAILADYAKSQ。

[0073] SEQ ID NO.3:

[0074]

[0075] TCTGCTGGTGCTGGTCAAGGTCAAGCTGCTTATTTTGCTACTTTTAATGAAA

[0076] CTTTCGGTGATTATGCTGCTGAAGCTCATGAAATGTTGAACTCTAAGTTGGA

[0077] ACAATGGGCTGAAGAAACTGTTGCTAGAGGTGGTTTCCATAACACTACTGC

[0078] TTTGTTGATTCAATACGAAAACTACAGAGATTGGTTGGTTAACCATAATGTT

[0079] GCTTACTCTGAATTGTTCTTGGATACTGCTGGTGCTGTTTCTTTCACTATTTG

[0080] GGATTTGATTCCTTTTACTAGAGGTTATGTTCATACTTCTCATAAGGACCCTT

[0081] ATTTGAGATTGTCTGCTTATGATCCACAATACTTTTTGAATGAACTGGATTTG

[0082] TACGGTCAAGCTGCCGCTTCTCAATTGGCTAGAAACTTGTCTAATTCTGATG

[0083] CTATGTCTACTTACTTTGCTGGTGAAACTACTCCTGGTGATAACTTGGCTTA

[0084] CGATGCTGGTTTGTCTGATTGGGCTGAATACATTAGATACAACTTTAGACCA

[0085] AACTACCATGGTGTTGGTACTTGTTCTATGATGCCTAAGGAAATGGGTGGTG

[0086] TTGTTGATTCTTCTGCTAGAGTTTACGGTGTTGATTCTTTGAGAGTTATTGAT

[0087] GGTTCTATTCCACCTACTCAAGTTTCTTCTCATGTTATGACTGTTTTTTACGC

[0088] TATGGCTTTGAAGATTTCTGATGCTATTTTGGCTGATTACGCTAAGTCTCAA。

Claims

1. A glucose oxidase mutant with improved acid and heat resistance, characterized in that: The wild-type glucose oxidase AsGODP is used as the parent, and the 148th Glu of the wild-type glucose oxidase AsGODP is mutated to Lys, and the 179th Val is mutated to Ala. The amino acid sequence of the wild-type glucose oxidase AsGODP is shown in SEQ ID NO.1, and the amino acid sequence of the glucose oxidase mutant is shown in SEQ ID NO.

2.

2. A gene encoding the glucose oxidase mutant according to claim 1.

3. A recombinant vector, characterized in that It contains the gene according to claim 2.

4. A recombinant strain, characterized in that It contains the recombinant vector according to claim 3.

5. Use of the glucose oxidase mutant according to claim 1, the gene according to claim 2, the recombinant vector according to claim 3, or the recombinant strain according to claim 4 in bread baking, characterized in that: The host bacteria of the recombinant strain is Pichia pastoris.

6. Use of the glucose oxidase mutant according to claim 1, the gene according to claim 2, the recombinant vector according to claim 3, or the recombinant strain according to claim 4 in any of the following: (1) Application in the preparation of leavening agents for improving dough texture; (2) Application in the preparation of bread baking improvers; The host bacteria of the recombinant strain is Pichia pastoris.

7. A leavening agent for improving dough texture, characterized in that: The effective component is prepared from the recombinant strain according to claim 4; the host bacteria of the recombinant strain is Pichia pastoris.

8. A bread baking improver, characterized in that The effective component is prepared from the recombinant strain according to claim 4; the host bacteria of the recombinant strain is Pichia pastoris.

9. A method for producing a glucose oxidase mutant by fermentation using the recombinant strain according to claim 4, characterized in that: The method comprises the steps of collecting the precipitate after the fermentation of the recombinant strain and inducing the culture with an induction medium containing methanol.

10. The method according to claim 9, wherein The methanol-containing induction medium is prepared by adding 0.5% methanol by mass volume to the fermentation medium of the recombinant strain. The induction culture conditions are 25-35°C and 100-300 rpm. Methanol is added every 12 hours to maintain the methanol concentration in the bacterial liquid at 0.5%.

Images