Glucose oxidase mutant and application thereof

Abstract

The invention discloses a glucose oxidase mutant and application thereof. The amino acid sequence of the glucose oxidase mutant is a mutant obtained by substitution mutation of one or more of V20Y, T30V, S53F, T276F, H277F, D315K, T389A and V541T corresponding to the amino acid sequence as shown in SEQ ID NO: 1, and the glucose oxidase mutant has significantly improved enzymatic activity or thermal stability. The glucose oxidase mutant is applied to gluconic acid and psicose or co-production of gluconic acid and psicose, and has practical application value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to mutants of glucose oxidase and their application in the co-production of D-allulose and gluconic acid. Background Art

[0002] D-allulose is a monosaccharide that exists very rarely in nature. It is present in trace amounts in natural foods such as figs, kiwifruits, raisins, and wheat, and is the 3-epimer of D-fructose. As a bulking sweetener, it is very similar to sucrose in processing performance, with a sweetness equivalent to 70% of sucrose, but its calorie content is only 0.4 kcal / g (1 / 10 of sucrose). It has a taste and solubility similar to sucrose, and has unique physiological functions such as regulating blood glucose levels and insulin levels, a very low glycemic index (GI), and inhibiting fat accumulation. D-allulose has become a new-generation sugar substitute star with its "natural source, low calories, and sweetness close to sucrose", especially with great potential in the field of health foods. It has been used in food production in many countries and regions such as the United States, Australia, and New Zealand. Industrially, starch is saccharified, liquefied, isomerized, and separated to obtain 90% high fructose syrup. The fructose in the high fructose syrup is catalyzed by D-allulose 3-epimerase (DAE) to obtain D-allulose, and the unutilized glucose cannot be used, which not only affects sugar separation and purification but also increases the production cost of D-allulose.

[0003] Gluconic acid is an organic acid with a stable structure and mild acidity, and is widely used in many fields, with important industrial and biological values. In the pharmaceutical field, its salt forms such as calcium gluconate and zinc gluconate are commonly used to supplement trace elements such as calcium and zinc required by the human body, with good bioavailability and safety. In the food industry, gluconic acid is used as an acidity regulator, nutritional fortifier, and preservative, which can effectively improve the taste and stability of foods. In the building materials field, sodium gluconate is widely used as a concrete retarder and cement dispersant, which helps to extend the setting time and improve the construction performance. In addition, due to its excellent complexing ability and biodegradability, gluconic acid also shows broad application prospects in metal cleaning agents, environmentally friendly water treatment agents, and agricultural chelating fertilizers. Enzymatic production of gluconic acid is an efficient and environmentally friendly method, mainly through the oxidation reaction of glucose catalyzed by glucose oxidase (GOD) to produce gluconic acid. The reaction conditions of this process are mild and do not require high temperature or strong oxidants, so it is widely used in industries such as food and medicine. However, natural GOD has defects such as poor thermal stability and low catalytic efficiency, resulting in frequent supplementation of enzyme preparations in enzymatic production, which increases the production cost.

[0004] Therefore, it is of great significance to improve the stability and catalytic performance of GOD through molecular design modification, optimize its expression and secretion process, use it to convert glucose in 90% high fructose syrup into gluconic acid, and use the remaining pure fructose for the production of D-allulose, so as to realize the co-production of D-allulose and gluconic acid, improve the substrate utilization rate and product yield, and reduce the production cost. Summary of the Invention

[0005] In the present invention, mutation sites are designed by a semi-rational design strategy combining the online tool FireProt 2.0 and sequence identity analysis. A total of 83 design sites are obtained, and single-site mutations are verified for them, and then combinatorial mutations are carried out to obtain the glucose oxidase mutants of the present invention.

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

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

[0008] The present invention provides the coding nucleic acid of the glucose oxidase mutant described above.

[0009] The present invention further provides a recombinant expression vector of the coding nucleic acid.

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

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

[0012] The present invention further provides a method for preparing gluconic acid, which comprises the following steps: The step of coupling and catalyzing D-glucose substrate with the glucose oxidase mutant and catalase to generate gluconic acid.

[0013] Specifically, the catalytic reaction system uses acetic acid-sodium acetate buffer 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 100 mg of glucose.

[0014] The present invention also provides a method for co-producing D-allulose and gluconic acid, which comprises the following steps: S1 A step of coupling and catalyzing fructose and glucose substrates by the glucose oxidase mutant and catalase to generate gluconic acid; S2 Separating gluconic acid and fructose after the reaction in step S1; S3 Using D-allulose-3-epimerase to catalyze the fructose in S2 to generate D-allulose.

[0015] Preferably, in step S3, the reaction is carried out through a D-allulose-3-epimerase immobilized enzyme column reactor to obtain D-allulose; fructose and allulose are separated by simulated moving bed, and the separated fructose solution continues to pass through the D-allulose-3-epimerase immobilized enzyme column reactor for reaction. This process is repeated multiple times, and the allulose solution is collected.

[0016] 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 gluconic acid and allulose or their co-production has practical application value. Specific Embodiments

[0017] The present invention will be further described in detail below with reference to embodiments. The embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation manners and specific operation processes are given. The embodiments will help to understand the present invention, but the protection scope of the present invention is not limited to the following embodiments. Those skilled in the art should understand 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.

[0018] Example 1 A Construction and Screening of GOD Recombinant Strains (1) Aspergillus niger The amino acid sequence of the glucose oxidase ( A GOD) derived from the source is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2. Primers are designed to amplify and obtain A the GOD nucleotide sequence and the pPICZαA linearized vector. The purified A GOD gene fragment and the linearized vector pPICZαA are ligated by homologous recombination and transformed into E. coli DH5α competent cells. After screening with Zeocin resistance (100 μg / mL) and sequencing verification, the recombinant plasmid pPICZαA- A GOD is obtained.

[0019] (2) After electrotransforming the recombinant plasmid pPICZαA- A GOD containing the above glucose oxidase gene into Pichia pastoris X33, positive transformants growing on the resistant plate were randomly picked, transferred to YPD plates containing 2, 4, 8, 16 mg / mL Zeocin resistance and numbered. The chromogenic solution mixed with horseradish peroxidase and o-dianisidine was poured onto the plate with growing bacteria for color development. Strains growing on the high-concentration resistant plate, having a larger chromogenic circle and showing a deeper color were selected.

[0020] Example 2. Optimization of protein expression (1) The recombinant strain was inoculated into BMMY medium containing different methanol concentrations (0 - 1.25%), and methanol of the same concentration was supplemented every 24 h. Induced expression was carried out at 30 °C for 96 h. The results showed that at the methanol addition amount of 1.0%, the enzyme activity in the fermentation broth reached 35 U / mL, which was significantly higher than that of the uninduced group (enzyme activity < 5 U / mL).

[0021] (2) The above fermentation broth was centrifuged to obtain the fermentation supernatant and the bacterial cells.

[0022] (3) The bacterial cells were disrupted by a high-pressure homogenizer to obtain the disrupted cell solution.

[0023] (4) The fermentation supernatant and the disrupted cell solution were concentrated, bound to a nickel column, eluted, and purified to obtain the pure protein.

[0024] (5) Through SDS-PAGE and enzyme activity determination, it was found that the proportion of enzyme activity in the fermentation broth supernatant > 90%, indicating that A GOD was mainly secreted extracellularly. The specific activity of the extracellular enzyme after purification was 188.29 U / mg, which was significantly higher than that of the intracellular enzyme (115.76 U / mg).

[0025] Example 3. Construction and screening of mutants (1) Design of glucose oxidase mutation sites The mutation sites were designed by a semi-rational design strategy combining the online tool FireProt 2.0 and sequence identity analysis. Submitting the A GOD crystal structure (PDB ID: 3QVP) to the FireProt 2.0 online server for calculation, 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. Through sequence alignment in the Uniprot database, 250 GOD homologous sequences were obtained, and multiple alignment was performed using Clustal Omega. Sites with the frequency of a single amino acid > 40% were selected, and further AThere are 58 sites in the GOD sequence that are inconsistent with the most frequently occurring amino acids. Among them, 10 sites overlap with the prediction results of FireProt 2.0. A total of 83 design sites are obtained by combining the two strategies.

[0026] (2)Construction and screening of single-point mutants The design sites were mutated using site-directed mutagenesis technology. Corresponding PCR primers were designed for each mutation site, and the A linearized vector fragment of GOD single-point mutation was amplified and electrotransformed into E. coli DH5α. After screening with Zeocin resistance and sequencing verification, 83 recombinant strains of single-point mutants were obtained. The plasmids were extracted, linearized, and then electrotransformed into Pichia pastoris X33. After expression induced by methanol (1.0% v / v), the supernatant of the fermentation broth was collected and the mutant proteins were purified by Ni-NTA affinity chromatography.

[0027] The specific enzyme activity and thermal stability of the single-point mutants were tested. First, a standard curve of hydrogen peroxide was drawn. The standard solutions of hydrogen peroxide with different concentrations were reacted with o-dianisidine, and the absorbance at 460 nm was measured. The standard curve was drawn with the hydrogen peroxide concentration as the abscissa and the absorbance as the ordinate, and the slope k1 was calculated. Then, A the GOD sample was mixed with the substrate glucose, and the absorbance was measured every 30 s for 4 min. A curve was drawn with time as the abscissa and absorbance as the ordinate, and the slope k2 was calculated. Finally, according to the formula U / mL = (k2 / k1) × (V1 / V2) × N × 1000, the enzyme activity of GOD was calculated, where V1 is the sample volume, V2 is the reaction system volume, and N is the sample dilution factor. The specific enzyme activity is defined as (1 U / mg): the amount of enzyme required to generate 1 μmol H2O2 per minute (U / mg). The enzyme solution was incubated at 55 °C for 40 min, and samples were taken to measure the residual enzyme activity. The enzyme activities and stabilities of the obtained dominant mutants are shown in Table 1.

[0028] Table 1. Comparison of relative enzyme activities and residual enzyme activities between wild type and dominant mutants

[0029] The enzyme activity of the mutant T276F increased the most significantly, reaching 2.1 times that of the wild type. The specific enzyme activities of S74T, T34V, and D315K were 1.8 - 2.0 times that of the wild type. The enzyme activities of T30V, S53F, Q90R, and T389A were 1.3 times that of the wild type. The enzyme activities of V20Y, V106I, H277F, and V541T did not show significant improvement. In terms of stability, the stability of the mutant S53F increased the most significantly. When the wild type reached its half-life, it could still maintain more than 80% of its initial activity. T30V, T34V, S74T, V106I, T276F, H277F, and V541T could maintain more than 60 - 70% of their initial activity. The improvement effect of the thermal stability of V20Y, Q90R, D315K, and T389A was not obvious.

[0030] (3)Screening of combinatorial mutants In the first stage, the screening criterion was the increase in enzyme activity. The mutant T276F was the single-site dominant mutant with the highest specific enzyme activity. At the same time, the residual enzyme activity after treatment at 55 °C for 40 min was 67.3%. It was used as mutant M1. Based on M1, the other 11 single-site mutants were superimposed to obtain two-site mutants. The specific enzyme activity of mutant M2 (T276F / T34V) was further increased compared to M1, being 2.6 times that of the wild type. The residual enzyme activity after treatment at 55 °C for 40 min was further increased to 75%. Based on M2, the remaining 10 single-site mutants were superimposed to obtain three-site mutants. The specific enzyme activity of mutant M3 (T276F / T34V / Q90R) was 1.7 times that of M2 and 4.3 times that of WT. Its residual enzyme activity after treatment at 55 °C for 40 min decreased to 62.3%, but was higher than the residual enzyme activity of the wild type at 51.1%. After continuously superimposing the remaining mutants, the specific enzyme activity no longer increased. The results are shown in Table 2.

[0031] Table 2. Combinatorial mutations with the increase in specific enzyme activity as the screening criterion

[0032] In the second stage, the screening criterion was the improvement of thermal stability while maintaining the enzyme activity basically unchanged. Based on M3, the remaining single-site mutants were superimposed. The results are shown in Table 3. The residual enzyme activity of mutant M4 (T276F / T34V / Q90R / S53F) after treatment at 55 °C for 40 min was increased to 89.2%. The specific enzyme activity decreased slightly. The reason might be that the S53F site was the single-site mutant with the most obvious improvement in thermal stability and contributed greatly to the thermal stability of M4. Based on M4, other single-site mutants were continuously superimposed. The residual enzyme activity of mutant M5 (T276F / T34V / Q90R / S53F / S74T) after treatment at 55 °C for 40 min was increased to 91.0%. At the same time, the specific enzyme activity decreased slightly compared to M4 and was 4.1 times the specific enzyme activity of WT.

[0033] Table 3. Combinatorial mutations with improved thermal stability as the screening criterion

[0034] Example 4. Determination of the enzymatic properties of mutant M5 Wild type and A The GOD-M5 mutant T276F / T34V / Q90R / S53F / S74T was obtained as pure protein by the expression preparation method as in Example 2. The optimal reaction pH of M5 was 5.5 (the pH range with activity > 80% was 4.0 - 6.5), and the optimal temperature was 40 °C, which was basically the same as that of the wild type. At 65 °C, the half-life of M5 was 30 min, which was 30 times that of the wild type (1 min). After treatment at 75 °C for 10 min, the residual enzyme activity of M5 reached 20% (the wild type was completely inactivated). The enzyme kinetic parameters showed that the k cat value of M5 was 755.0 s -1 , and the catalytic efficiency ( k cat / K m ) was 34.52 mmol / L -1 s -1 , which were 3.67 times and 2.48 times that of the wild type, respectively.

[0035] Example 5. Preparation of gluconic acid by coupling glucose oxidase with catalase Mutant M5 was coupled with catalase (CAT), and a 1 mL microreaction system was established using a BioLector XT microfluidic system. Reaction parameters such as reaction temperature, pH, DO, and enzyme concentration ratio were optimized. In a 100 g / L D-glucose substrate system, 0.5 mol / L acetic acid-sodium acetate buffer (pH 5.5) was added, and A GOD M5 and commercial CAT were added in proportion. After thorough mixing, the reaction was carried out under different conditions for 10 h. Samples were taken every 1 h and boiled for 10 min to terminate the reaction. The residual amount of glucose in the reaction solution was measured by the DNS method to calculate the conversion rate. The results showed that the optimal temperature for the preparation of gluconic acid by double-enzyme coupling was 40 °C, the optimal pH was 5.6, the optimal DO was 50%, and the optimal concentration of GOD to CAT was 8U:80U per 100 mg glucose.

[0036] A 100 g / L glucose solution was prepared and placed in a reaction kettle equipped with a stirring device. The temperature was controlled at 40 °C, the pH was adjusted to 6.0, 8U / mL of A wild type GOD enzyme was added, and 80U / mL of catalase was added. Air was introduced during the reaction to provide oxygen, and the reaction was carried out for 6 h. The conversion rate was measured to be approximately 63%.

[0037] Prepare a 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 A GOD M5 mutant enzyme at 8 U / mL and catalase at 80 U / mL. During the reaction, introduce air to supply oxygen, react for 6 h, and measure that the conversion rate is greater than 99%, which is A more than 36% higher than the conversion rate of GOD wild-type enzyme.

[0038] Example 6: Multi-enzyme cascade for co-production of D-allulose and gluconic acid Place 10 L of a high-fructose syrup solution containing 90% fructose and 10% glucose (sugar concentration is 500 g / L) in a reactor equipped with a stirring device. Control the temperature at 40 °C and adjust the pH to 5.6. Add A GOD M5 mutant enzyme and catalase to the reaction system, with the addition amounts of 4 U / mL and 30 U / mL respectively. During the reaction, introduce air to supply oxygen, react for about 6 h, and terminate the reaction after the glucose conversion rate reaches more than 99%. Use an anion resin to adsorb gluconic acid in the reaction solution, filter to separate the reaction solution from the resin, and obtain a pure fructose solution. Rinse the anion resin with a sodium hydroxide solution, evaporate, concentrate, and crystallize to obtain pure gluconic acid, with a yield reaching 95%. Pass the obtained pure fructose solution through a D-allulose-3-epimerase immobilized enzyme column reactor for reaction, with a reaction temperature of 60 °C and a pH of 7.0, to obtain a conversion solution of D-allulose with a 30% conversion rate. Separate fructose and allulose by simulated moving bed, and continue to pass the separated fructose solution through the D-allulose-3-epimerase immobilized enzyme column reactor for reaction. Repeat this process multiple times, collect the allulose solution, evaporate, concentrate, and crystallize to obtain pure D-allulose, with a yield reaching 85%. The total yield of D-allulose and gluconic acid reaches 86.5%, improving the substrate utilization rate and product yield and reducing the production cost.

Claims

1. Glucose oxidase mutant, characterized in that, A mutant obtained by one or more substitution mutations of V20Y, T30V, S53F, T276F, H277F, D315K, T389A and V541T, whose amino acid sequence corresponds to the amino acid sequence shown in SEQ ID NO:

1.

2. The glucose oxidase mutant according to claim 1, wherein It has the following mutations 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.

3. The coding nucleic acid of the glucose oxidase mutant according to claim 1 or 2.

4. The recombinant expression vector of the coding nucleic acid according to claim 3.

5. A recombinant bacterium containing the coding nucleic acid according to claim 3.

6. The application of the glucose oxidase mutant according to claim 1 or 2 in the preparation of gluconic acid and / or allulose.

7. A method for preparing gluconic acid, characterized in that, Comprising the following steps: The step of coupling and catalyzing the D-glucose substrate to produce gluconic acid by the glucose oxidase mutant according to claim 1 or 2 and catalase.

8. The method according to claim 7, wherein The catalytic reaction system uses an acetic acid-sodium acetate buffer 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 100 mg of glucose.

9. A method for co-producing D-psicose and gluconic acid, characterized in that, Comprising the following steps: S1 The step of coupling and catalyzing the fructose and glucose substrates to produce gluconic acid by the glucose oxidase mutant according to claim 1 or 2 and catalase; S2 Separating gluconic acid and fructose after the reaction in step S1; S3 Using D-allulose-3-epimerase to catalyze the fructose in S2 to produce D-allulose.

10. The method according to claim 9, characterized in that In step S3, the reaction is carried out through a D-allulose-3-epimerase immobilized enzyme column reactor to obtain D-allulose; fructose and allulose are separated by simulated moving bed, and the separated fructose solution is continuously passed through the D-allulose-3-epimerase immobilized enzyme column reactor for reaction, and this process is repeated multiple times to collect the allulose solution.

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