Glucose oxidase mutant and its application

Abstract

The present invention discloses a glucose oxidase mutant and its application. The glucose oxidase mutant has an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: 1, and contains one or more substitution mutations of V20Y, T30V, S53F, T276F, H277F, D315K, T389A, and V541T. The mutant exhibits significantly improved enzyme activity or thermostability. The application of the glucose oxidase mutant in the production of gluconic acid and psicose, or their co-production, has practical application value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a mutant of glucose oxidase and application thereof in the co-production of D-psicose and gluconic acid. Background Art

[0002] D-allulose is a rare monosaccharide found in trace amounts in foods such as figs, kiwis, raisins, and wheat. It is the 3-dimer of D-fructose. As a bulk sweetener, it closely resembles sucrose in processing properties, boasting 70% of its sweetness but only 0.4 kcal / g (one-tenth of sucrose). It has a similar taste and solubility to sucrose and possesses unique physiological functions, including regulating blood sugar and insulin levels, achieving a very low glycemic index (GI), and inhibiting fat accumulation. D-allulose, with its natural origin, low calories, and sweetness similar to sucrose, has become a rising star in the new generation of sugar substitutes, particularly in the health food sector. It has great potential and is already used in food production in countries and regions including the United States, Australia, and New Zealand. In industry, 90% high fructose syrup is obtained through saccharification, liquefaction, isomerization and separation of starch. The fructose in high fructose syrup is catalyzed by D-psicose 3-epimerase (DAE) to obtain D-psicose. The unconverted glucose cannot be used, which not only affects the separation and refining of sugar, but also increases the production cost of D-psicose.

[0003] Gluconic acid is a stable organic acid with a mild acidity. It is widely used in various fields and holds significant industrial and biological value. In the pharmaceutical field, its salt forms, such as calcium gluconate and zinc gluconate, are commonly used to supplement calcium, zinc, and other trace elements required by the human body, with good bioavailability and safety. In the food industry, gluconic acid is used as an acidity regulator, nutritional supplement, and preservative, effectively improving the taste and stability of food. In the building materials industry, sodium gluconate is widely used as a concrete retarder and cement dispersant, helping to extend setting time and enhance construction performance. Furthermore, due to its excellent chelating ability and biodegradability, gluconic acid also shows promising applications in metal cleaning agents, environmentally friendly water treatment agents, and agricultural chelated fertilizers. Enzymatic production of gluconic acid is an efficient and environmentally friendly method, primarily through the oxidation of glucose catalyzed by glucose oxidase (GOD). This process operates under mild reaction conditions and does not require high temperatures or strong oxidants, making it widely used in the food and pharmaceutical industries. However, natural GOD has defects such as poor thermal stability and low catalytic efficiency, which requires frequent addition of enzyme preparations during enzymatic production, pushing up production costs.

[0004] Therefore, it is of great significance to improve the stability and catalytic performance of GOD through molecular design and modification, optimize its expression and secretion process, and use it to convert 90% of the glucose in high fructose syrup into gluconic acid. The remaining pure fructose is used for the production of D-psicose, realizing the co-production of D-psicose and gluconic acid, improving substrate utilization and product yield, and reducing production costs. Summary of the Invention

[0005] The present invention adopts a semi-rational design strategy combining the FireProt 2.0 online tool with sequence consistency analysis to design mutation sites, obtaining a total of 83 designed sites, verifying single-site mutations, and then performing combined mutations to obtain the glucose oxidase mutant of the present invention.

[0006] The present invention provides a glucose oxidase mutant, wherein the amino acid sequence thereof corresponds to the amino acid sequence shown in SEQ ID NO: 1, and one or more of V20Y, T30V, S53F, T276F, H277F, D315K, T389A and V541T are present in the mutant.

[0007] Furthermore, the following mutations exist corresponding to the amino acid sequence shown in SEQ ID NO: 1: T276F / T34V, T276F / T34V / Q90R, T276F / T34V / Q90R, T276F / T34V / Q90R / S53F or T276F / T34V / Q90R / S53F / S74T.

[0008] The present invention provides a nucleic acid encoding the glucose oxidase mutant.

[0009] The present invention also provides a recombinant expression vector encoding the nucleic acid.

[0010] The present invention also provides a recombinant bacterium containing the encoding nucleic acid.

[0011] The present invention also provides use of the glucose oxidase mutant in the preparation of gluconic acid and / or psicose.

[0012] The present invention further provides a method for preparing gluconic acid, which comprises the steps of:

[0013] The step of coupling the glucose oxidase mutant and catalase to catalyze D-glucose substrate to generate gluconic acid.

[0014] Specifically, the catalytic reaction system uses acetic acid-sodium acetate buffer as the medium, the reaction temperature is 40° C., the pH is 5.6, the DO is 50%, and the concentrations of the glucose oxidase mutant and catalase are 8 U:80 U per 100 mg of glucose.

[0015] The present invention also provides a method for co-producing D-psicose and gluconic acid, comprising the following steps:

[0016] S1 is a step of coupling the glucose oxidase mutant and catalase to catalyze fructose and glucose substrates to produce gluconic acid;

[0017] S2 separates gluconic acid and fructose after completing the reaction in step S1;

[0018] S3 uses D-psicose-3-epimerase to catalyze the conversion of fructose in S2 into D-psicose.

[0019] Preferably, in step S3, a D-psicose-3-epimerase immobilized enzyme column reactor is used to react to obtain D-psicose; fructose and psicose are separated by a simulated moving bed, and the separated fructose solution is further passed through the D-psicose-3-epimerase immobilized enzyme column reactor for reaction, and this process is repeated multiple times to collect the psicose solution.

[0020] The glucose oxidase mutant obtained by the present invention has significantly improved enzyme activity or thermal stability. The application of the glucose oxidase mutant of the present invention to the production of gluconic acid and psicose or their co-production has practical application value. DETAILED DESCRIPTION

[0021] The present invention is further described in detail below in conjunction with examples. The examples are implemented under the premise of the technical solution of the present invention, and detailed implementation methods and specific operating procedures are given. The examples will help to understand the present invention, but the protection scope of the present invention is not limited to the following examples. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0022] Example 1 An Construction and screening of GOD recombinant strains

[0023] (1) Aspergillus niger Glucose oxidase ( An GOD) amino acid sequence is shown in SEQ ID NO.1, nucleotide sequence is shown in SEQ ID NO.2, primers were designed to amplify An The GOD nucleotide sequence was linearized with the pPICZαA vector and the purified An The GOD gene fragment was connected with the linearized vector pPICZαA by homologous recombination and transformed into E. coli In DH5α competent cells, the recombinant plasmid pPICZαA- An GOD.

[0024] (2) The recombinant plasmid pPICZαA- An After GOD electroporation into Pichia pastoris X33, positive transformants growing on resistance plates were randomly picked, transferred to YPD plates containing 2, 4, 8, and 16 mg / mL Zeocin resistance, and numbered. A mixture of horseradish peroxidase and o-dianisidine was poured onto the growth plates for color development. Strains that grew on high-concentration resistance plates, had larger color zones, and exhibited darker colors were selected.

[0025] Example 2: Protein expression optimization

[0026] (1) The recombinant strain was inoculated into BMMY medium containing different methanol concentrations (0-1.25%). The same concentration of methanol was added every 24 hours, and expression was induced at 30°C for 96 hours. The results showed that the enzyme activity of the fermentation broth reached 35 U / mL when 1.0% methanol was added, which was significantly higher than that of the uninduced group (enzyme activity <5 U / mL).

[0027] (2) The fermentation liquid is centrifuged to obtain the fermentation supernatant and the bacterial cells.

[0028] (3) Use a high-pressure homogenizer to crush the bacteria to obtain a broken bacteria liquid.

[0029] (4) The fermentation supernatant and the bacterial cell disrupted liquid are concentrated, bound to a nickel column, eluted, and purified to obtain pure protein.

[0030] (5) Through SDS-PAGE and enzyme activity determination, it was found that the enzyme activity in the fermentation broth supernatant accounted for >90%, indicating that An GOD was mainly secreted extracellularly. After purification, the specific activity of the extracellular enzyme was 188.29 U / mg, which was significantly higher than that of the intracellular enzyme (115.76 U / mg).

[0031] Example 3. Construction and screening of mutants

[0032] (1) Glucose oxidase mutation site design

[0033] The mutation sites were designed using a semi-rational design strategy combining the FireProt 2.0 online tool with sequence consistency analysis. An The GOD crystal structure (PDB ID: 3QVP) was submitted to the FireProt 2.0 online server for calculations. Potential mutation sites with FoldX and Rosetta folding free energies less than 0 and evolutionary conservation scores less than 7 were selected, and a total of 35 candidate sites were obtained. 250 GOD homologous sequences were obtained by alignment with the Uniprot database. Multiple alignments were performed using Clustal Omega, and sites with a single amino acid occurrence frequency greater than 40% were selected for further selection. An A total of 58 sites were obtained in the GOD sequence that were inconsistent with the most frequently occurring amino acid, 10 of which overlapped with the FireProt 2.0 predicted sites. A total of 83 designed sites were obtained by combining the two strategies.

[0034] (2) Construction and screening of single-point mutants

[0035] The designed sites were mutated using site-directed mutagenesis technology, and corresponding PCR primers were designed for each mutation site to obtain An The GOD single point mutation linearized vector fragment was electroporated into E. coli DH5α strains were screened for Zeocin resistance and verified by sequencing, yielding 83 recombinant strains with single-point mutations. Plasmids were extracted, linearized, and electroporated into Pichia pastoris X33. Expression was induced with methanol (1.0% v / v). The fermentation supernatant was collected and the mutant proteins purified by Ni-NTA affinity chromatography.

[0036] To test the specific enzyme activity and thermal stability of the single-point mutant, first, a hydrogen peroxide standard curve was drawn. Different concentrations of hydrogen peroxide standard solutions were reacted with o-dianisidine and the absorbance at 460 nm was measured. The standard curve was drawn with hydrogen peroxide concentration as the horizontal axis and absorbance as the vertical axis, and the slope k1 was calculated. An A GOD sample was mixed with the substrate glucose, and the absorbance was measured every 30 seconds for 4 minutes. A curve was plotted with time as the abscissa and absorbance as the ordinate, and the slope, k2, was calculated. Finally, the GOD enzyme activity was calculated using the formula (U / mL) = (k2 / k1) × (V1 / V2) × N × 1000, where V1 is the sample volume, V2 is the reaction volume, and N is the sample dilution factor. Specific enzyme activity (1 U / mg) is defined as the amount of enzyme (U / mg) required to generate 1 μmol H2O2 per minute. The enzyme solution was incubated at 55°C for 40 minutes, and samples were collected to determine the residual enzyme activity. The enzyme activity and stability of the dominant mutants obtained are shown in Table 1.

[0037] Table 1. Comparison of relative enzyme activity and residual enzyme activity between wild type and dominant mutant

[0038]

[0039] The enzyme activity of mutant T276F was the most significantly improved, reaching 2.1-fold that of the wild type. The enzyme activities of S74T, T34V, and D315K were 1.8-2.0 times that of the wild type, and those of T30V, S53F, Q90R, and T389A were 1.3-fold that of the wild type. No significant improvement was observed in the enzyme activities of V20Y, V106I, H277F, and V541T. In terms of stability, mutant S53F showed the most significant improvement, retaining over 80% of its initial activity at the half-life of the wild type. T30V, T34V, S74T, V106I, T276F, H277F, and V541T maintained over 60-70% of their initial activity. V20Y, Q90R, D315K, and T389A showed no significant improvement in thermal stability.

[0040] (3) Combinatorial mutant screening

[0041] In the first stage, the screening criteria were enzyme activity enhancement. Mutant T276F was the single-point dominant mutant with the highest specific enzyme activity, and its residual enzyme activity after treatment at 55°C for 40 minutes was 67.3%. This mutant was designated as mutant M1. Eleven other single-point mutants were added to M1 to generate two-point mutants. Mutant M2 (T276F / T34V) exhibited a further improvement in specific enzyme activity compared to M1, reaching 2.6 times that of the wild type, and its residual enzyme activity after treatment at 55°C for 40 minutes was further increased to 75%. The remaining 10 single-point mutants were added to M2 to generate three-point mutants. Mutant M3 (T276F / T34V / Q90R) exhibited a specific enzyme activity 1.7 times that of M2 and 4.3 times that of the wild type. Its residual enzyme activity after treatment at 55°C for 40 minutes dropped to 62.3%, but remained above the 51.1% residual enzyme activity of the wild type. Further additions of the remaining mutants did not result in further increases in specific enzyme activity. The results are shown in Table 2.

[0042] Table 2. Combination mutations based on the improvement of enzyme activity

[0043]

[0044] In the second stage, the screening criteria for improved thermostability while maintaining essentially unchanged enzyme activity were used. Additional single-point mutants were superimposed on M3. The results are shown in Table 3. Mutant M4 (T276F / T34V / Q90R / S53F) showed an increase in residual enzyme activity to 89.2% after treatment at 55°C for 40 min, with a slight decrease in specific activity. This may be because the S53F site is the single-point mutant with the most significant increase in thermostability, contributing significantly to the thermostability of M4. Further superimposition of additional single-point mutants on M4 resulted in mutant M5 (T276F / T34V / Q90R / S53F / S74T), which showed an increase in residual enzyme activity to 91.0% after treatment at 55°C for 40 min, while its specific activity was slightly lower than that of M4, reaching 4.1 times that of the WT.

[0045] Table 3. Combination mutations based on improved thermal stability as the screening criterion

[0046]

[0047] Example 4: Determination of enzymatic properties of mutant M5

[0048] Wild type and An The GOD-M5 mutant T276F / T34V / Q90R / S53F / S74T was expressed and prepared as described in Example 2 to obtain pure protein. The optimal reaction pH of M5 is 5.5 (the pH range for activity > 80% is 4.0-6.5), and the optimal temperature is 40°C, which is basically the same as that of the wild type. At 65°C, the half-life of M5 is 30 minutes, which is 30 times that of the wild type (1 minute). After treatment at 75°C for 10 minutes, the residual enzyme activity of M5 reaches 20% (the wild type is completely inactivated). The enzyme kinetic parameters show that the activity of M5 is k cat The value is 755.0 s -1 , catalytic efficiency ( k cat / K m ) is 34.52 mmol / L -1 s -1 , which were 3.67 times and 2.48 times that of the wild type, respectively.

[0049] Example 5: Preparation of gluconic acid by coupling glucose oxidase with catalase

[0050] The mutant M5 was coupled with catalase (CAT) and a 1 mL micro-reaction system was established using the BioLector XT microfluidic system. Reaction parameters such as reaction temperature, pH, DO, and enzyme concentration were optimized. 0.5 mol / L acetic acid-sodium acetate buffer (pH 5.5) was added to a 100 g / L D-glucose substrate system. An GOD M5 and commercial CAT were thoroughly mixed and reacted under different conditions for 10 hours. Samples were collected every hour and the reaction was terminated by boiling for 10 minutes. The residual glucose in the reaction solution was determined by the DNS method to calculate the conversion rate. The results showed that the optimal temperature for the dual-enzyme coupling to produce gluconic acid was 40°C, the optimal pH was 5.6, the optimal DO was 50%, and the optimal concentrations of GOD and CAT were 8 units:80 units per 100 mg of glucose.

[0051] Prepare 100 g / L glucose solution and place it in a reactor equipped with a stirring device. Control the temperature at 40°C, adjust the pH to 6.0, and add 8 U / mL of An GOD wild-type enzyme was added with 80 U / mL of catalase. Air was introduced to provide oxygen during the reaction. The reaction was continued for 6 h, and the conversion rate was determined to be approximately 63%.

[0052] Prepare 100 g / L glucose solution and place it in a reactor equipped with a stirring device. Control the temperature at 40°C, adjust the pH to 6.0, and add 8 U / mL of An GOD M5 mutant enzyme was added with 80 U / mL of catalase. Air was introduced to provide oxygen during the reaction. The reaction was continued for 6 h. The conversion rate was greater than 99%. An The conversion rate of GOD wild-type enzyme increased by more than 36%.

[0053] Example 6: Multi-enzyme cascade to achieve co-production of D-psicose and gluconic acid

[0054] Place 10 L of high fructose syrup solution (sugar concentration 500 g / L) containing 90% fructose and 10% glucose in a reactor equipped with a stirring device, control the temperature to 40°C, and adjust the pH to 5.6. Add AnThe GOD M5 mutant enzyme and catalase were added at 4 U / mL and 30 U / mL, respectively. Air was introduced to provide oxygen during the reaction. The reaction lasted approximately 6 hours and was terminated when the glucose conversion rate reached over 99%. Anionic resin was used to adsorb gluconic acid from the reaction solution, and the reaction solution was separated from the resin by filtration to obtain a pure fructose solution. The anionic resin was rinsed with sodium hydroxide solution, and then evaporated, concentrated, and crystallized to obtain pure gluconic acid with an initial yield of 95%. The resulting pure fructose solution was then reacted in a column reactor containing immobilized D-psicose-3-epimerase at 60°C and pH 7.0, yielding a D-psicose conversion solution of 30%. Fructose and psicose were separated by a simulated moving bed, and the separated fructose solution was further reacted in a D-psicose-3-epimerase immobilized enzyme column reactor. This process was repeated multiple times, and the psicose solution was collected for evaporation, concentration, and crystallization to obtain pure D-psicose with a yield of 85%. The total yield of D-psicose and gluconic acid reached 86.5%, which improved substrate utilization and product yield and reduced production costs.

Claims

1. A glucose oxidase mutant, characterized in that The amino acid sequence thereof is based on the amino acid sequence shown in SEQ ID NO: 1 and only has the mutations T276F, T276F / T34V, T276F / T34V / Q90R, T276F / T34V / Q90R / S53F or T276F / T34V / Q90R / S53F / S74T.

2. The nucleic acid encoding the glucose oxidase mutant according to claim 1.

3. The recombinant expression vector encoding the nucleic acid according to claim 2.

4. A recombinant bacterium containing the encoding nucleic acid according to claim 2.

5. Use of the glucose oxidase mutant according to claim 1 in the preparation of gluconic acid and / or psicose.

6. A method for preparing gluconic acid, characterized in that, The steps include: The step of coupling the glucose oxidase mutant according to claim 1 and catalase to catalyze D-glucose substrate to produce gluconic acid.

7. The method according to claim 6, wherein The catalytic reaction system uses acetic acid-sodium acetate buffer as the medium, the reaction temperature is 40°C, the pH is 5.6, the DO is 50%, and the concentrations of the glucose oxidase mutant and catalase are 8U:80U per 100mg of glucose.

8. A method for co-producing D-psicose and gluconic acid, characterized in that: The steps include: S1: a step of coupling the glucose oxidase mutant according to claim 1 and catalase to catalyze fructose and glucose substrates to produce gluconic acid; S2 separates gluconic acid and fructose after completing the reaction in step S1; S3 uses D-psicose-3-epimerase to catalyze the conversion of fructose in S2 into D-psicose.

9. The method according to claim 8, wherein In step S3, a D-psicose-3-epimerase immobilized enzyme column reactor is used to react to obtain D-psicose; fructose and psicose are separated by a simulated moving bed, and the separated fructose solution is further passed through the D-psicose-3-epimerase immobilized enzyme column reactor for reaction. This process is repeated multiple times to collect the psicose solution.

Images