Glucose oxidase mutant with improved acid resistance and heat resistance as well as construction method and application of glucose oxidase mutant

Abstract

The invention discloses a glucose oxidase mutant with improved acid resistance and heat resistance as well as a construction method and application of the glucose oxidase mutant, and belongs to the technical field of bioengineering. According to the present invention, glucose oxidase AsGODP derived from Aspergillus scleroticus is adopted as a female parent, and Glu148 and Val179 are subjected to synchronous mutation to form Lys148 and Ala179 so as to obtain the mutant E148K / V179A; experiments show that the pH stability, the thermal stability, the half-life period at 75 DEG C and the catalytic performance of the mutant E148K / V179A are obviously improved compared with those of wild AsGODP. According to the catalytic performance, when the mutant E148K / V179A is applied to bread baking, the gluten degree of dough can be effectively improved under the fermentation condition, the bread texture is improved, and therefore the mutant E148K / V179A shows huge 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 together 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 be hydrolyzed non-enzymatically to produce gluconic acid and water.

[0003] Glucose oxidase has been widely used in the fields of medicine, food, feed, etc. because of its special reaction mechanism and safe reaction products. Glucose oxidase is mainly derived from Aspergillus and Penicillium, but the currently known GOD has poor stability and low catalytic activity. When the naturally expressed glucose oxidase exceeds 55°C, the enzyme activity is seriously lost. These defects greatly limit the scope of application of GOD and cannot meet the needs of industry. At the same time, although domestic and foreign scientists have focused on the directed evolution and rational design of enzyme molecules, the molecular directed evolution has a large workload, strong randomness and high investment cost. Rational design requires a certain understanding of the functional structure of GOD. The current GOD has a single source and few resolved structures, making it difficult to carry out precise design, and the results achieved are still not significant enough. Therefore, by using more reliable bioinformatics techniques to simulate and analyze the structure and function of glucose oxidase, trying to fully analyze and improve the key structures and sites of the theory, and improving its catalytic activity and stability, it can greatly promote its application in industrial production.

[0004] With the increase in access to large experimental protein databases, as well as the advancement of high-throughput screening (HTS) methods and artificial intelligence algorithms, protein engineering has been greatly promoted. At present, a series of reasonable computer-assisted learning strategies have been developed, such as predicting functional changes caused by changes in enzyme protein sequences through machine learning or deep learning methods and identifying sequence or structural features from a wide range of protein data sets. In addition, deep neural networks and molecular evolution can also 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, and a construction method and application thereof. Specifically, the present invention obtains a glucose oxidase mutant with significantly improved acid and heat resistance by screening after mutating the key amino acid sites E148 and V179 of AsGODP from Aspergillus sclerotiicarbonarius, which can significantly improve the 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, which is obtained by taking the wild-type glucose oxidase AsGODP as the parent, and mutating the 148th Glu of the wild-type glucose oxidase AsGODP to Lys, and the 179th Val 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 invention also provides a recombinant strain containing the recombinant vector.

[0010] The present invention also provides application 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 or the gene or 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 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 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 the precipitate after fermentation of the recombinant strain and inducing culture with 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, the induction culture conditions are 25-35°C, 100-300rpm, and methanol is added 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 derived 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 optimum pH of the wild enzyme and the mutant was 6.0. In acidic environments (pH 2.0, 3.0, 4.0 and 5.0), the mutants could maintain 19%, 62%, 77% and 86% relative enzyme activities, which were 2.8 times, 2.9 times, 1.3 times and 21% higher than those of the wild type (5%, 16%, 34% and 71%) under the same conditions, respectively. In terms of pH stability, in acidic environments (pH 2.0, 3.0, 4.0 and 5.0), the mutants could maintain 21%, 60%, 79% and 88% residual enzyme activities, which were 2.0 times, 1.6 times, 68% and 21% higher than those of the wild type (7%, 23%, 47% and 73%) under the same conditions, respectively. That is, the activity and tolerance of the mutant enzyme E148K / V179A in acidic environments were significantly better than those of the wild 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 ) were increased by 6 times and 63% respectively. The present invention obtains a more acid-resistant and heat-resistant high catalytic activity mutant E148K / V179A through site-directed mutagenesis. Therefore, the application of the glucose oxidase mutant with improved acid and heat resistance in the bread baking industry has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 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 determination of the optimum pH for wild-type glucose oxidase AsGODP and its mutant E148K / V179A;

[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 optimum 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 Company, and peroxidase was purchased from Yuanye Company; other reagents were domestic analytical grade reagents (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] In order to improve the acid resistance and heat resistance of glucose oxidase AsGODP from Aspergillus sclerotiicarbonarius, the 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 the above two mutation sites was named E148K / V179A; the amino acid sequence of the above E148K / V179A is shown in SEQ ID NO.2. The primer sequences used are shown in Table 1, and the site-directed mutagenesis method and cloning method refer to the literature (Exploiting the activity-stability trade-off of glucose oxidase from Aspergillus niger using a simple approach to calculate thermosability of mutants. Jiang, et al., 2021), and 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 the DMT competent cell, and the nucleotide sequence encoding the glucose oxidase mutant E148K / V179A was obtained by colony PCR verification. 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 a 10 mL test tube containing 2 mL of BMGY medium, and cultured in a shaking incubator at 30°C and 220 rpm for 48 h. The culture solution was centrifuged at 3000 g for 5 min, the supernatant was discarded, and the precipitate was resuspended in 2 mL of BMMY medium containing 0.5% methanol, and induced again at 30°C and 220 rpm for 48 h. The supernatant was used for enzyme activity detection, and the mutant E148K / V179A (amino acid sequence as shown in SEQ ID NO.2, nucleotide sequence as shown in SEQ ID NO.3) with improved acid resistance and thermal stability compared with the wild enzyme was screened.

[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% inoculum and cultured at 30°C and 220rpm for 48h. 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 induced again 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 anion exchange method was used to purify the protein. 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), 3 mL of the reaction system includes 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 allowed to proceed for 3 min, and 2 mL of 2MH2SO4 is added to terminate the reaction. The OD value is measured at 540 nm. One enzyme activity unit (U) is defined as the amount of enzyme required to decompose 1 μmoL of substrate to generate hydrogen peroxide per minute under standard conditions.

[0047] 1. Method for determining the kinetic parameters of recombinant glucose oxidase mutants and wild type

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

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

[0050] Under standard conditions (30°C, pH 6) with glucose as substrate, the specific activity of glucose oxidase mutant E148K / V179A was 237.5 U / mg, which was 6 times higher than that of wild-type AsGODP (33.67 U / mg); the catalytic efficiency of mutant enzyme E148K / V179A was 26.3 mM -1 ·s -1 , compared with wild-type AsGODP (16.15 mM -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. Method for determining the optimal pH of recombinant glucose oxidase mutants and wild-type

[0054] The glucose oxidase mutant and wild-type glucose oxidase of Example 2 were subjected to enzymatic reaction at different pH values ​​(1.0-12.0) to determine their optimum pH values. The glucose oxidase activity of the substrate β-D-glucose was determined at 30° C. using 0.1 mol / L citric acid-sodium hydrogen phosphate buffer at different pH values ​​(1.0-12.0).

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

[0056] 3. pH stability determination method of recombinant glucose oxidase mutant and wild type

[0057] The glucose oxidase mutant 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 enzyme activity without treatment 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 optimum temperature of the recombinant glucose oxidase mutant and the wild-type glucose oxidase was determined by carrying out 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, 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 mutant and wild type

[0063] 40-80℃T 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°C for different time periods, up to 60 min, and their respective residual enzyme activities were detected.

[0065] 40-80℃T 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 the wild type (60℃); the half-life measurement results at 75℃ are as follows Figure 6 As shown, the half-life (t 1 / 2 ) is 30min, which is 24min longer than that of the wild enzyme AsGODP (6min), 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 dough with the mutant enzyme and the wild enzyme respectively, the dough was prepared by mixing the dough materials at room temperature for 10 minutes with a dough mixer. The dough was placed in a constant temperature incubator at 28°C for proofing for 60 minutes. The dough was kneaded again and placed in an incubator for further proofing for 60 minutes. After the dough was proofed, the dough was put into a mold and placed in an oven. The oven was set to 180°C for the upper fire and 200°C for the lower fire. It was baked for 30 minutes and then taken out. After baking, it was evenly cut into slices of 1 cm thickness under a slicer for subsequent determination of bread quality indicators. The bread slices were placed directly under the probe for texture profile analysis (TPA, repeated three times for each sample). A cylindrical probe was selected, 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 to determine the texture parameters of the bread slices.

[0068] The results are as follows Figure 7 As shown, when the mutant E148K / V179A is added at a level of 0.1%, the volume of bread can be significantly increased, and the chewiness, softness and elasticity of the bread can be improved, thereby improving the quality of the 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, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

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.

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 a leavening agent for improving dough texture; (2) Application in the preparation of bread baking improvers.

7. A leavening agent for improving dough texture, characterized in that: The effective ingredients are obtained from the recombinant strain described in claim 4.

8. A bread baking improver, characterized in that: The effective ingredients are obtained from the recombinant strain described in claim 4.

9. A method for producing glucose mutants 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, characterized in that 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 100-300rpm. Methanol is added every 12 hours to keep the methanol concentration in the bacterial liquid at 0.5%.

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