A dual-enzyme fusion protein against hydrogen peroxide inhibition, polynucleotide, expression vector and application

Abstract

This invention belongs to the fields of biotechnology and genetic engineering, and relates to a dual-enzyme fusion protein resistant to hydrogen peroxide inhibition, a polynucleotide, an expression vector, and its applications. The dual-enzyme fusion protein is composed of a glutamate oxidase mutant, a catalase mutant, and a linker peptide; the amino acid sequences of the glutamate oxidase mutant and the catalase mutant are shown in SEQ ID NO. 2 and 4, respectively, in the sequence listing. This invention improves the performance of single enzymes through enzyme molecule modification, achieves spatial synergy through fusion protein design, and enables industrial application through a continuous reaction process; it fundamentally solves the problem of hydrogen peroxide's inhibition of glutamate oxidase products; and the fusion expression simultaneously improves the stability and reusability of the dual enzymes.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and genetic engineering, and relates to a dual-enzyme fusion protein, polynucleotide, expression vector, and application that resists hydrogen peroxide inhibition. Background Technology

[0002] Glutamate oxidase (LGOX) is a flavoprotein protease that specifically catalyzes the oxidative deamination of L-glutamate to produce α-ketoglutarate and hydrogen peroxide (H₂O₂). α-Ketoglutarate (α-KG) is an important dicarboxylic acid in the tricarboxylic acid cycle and amino acid metabolism, with a promising market outlook. In the pharmaceutical field, it is not only used as a diagnostic reagent for liver function, but recent research has also revealed its breakthrough potential in cardiac regeneration and repair, and delaying aging. In sports nutrition, it effectively removes ammonia, relieves fatigue, and promotes muscle synthesis. Furthermore, it is widely used as a functional food additive, a novel feed improver, and a fine chemical intermediate. Its applications span the pharmaceutical and healthcare, green agriculture, and chemical industries.

[0003] The international market demand for food-grade α-ketoglutarate is currently increasing. However, existing production technologies are insufficient to fill this significant gap, resulting in a severe supply-demand imbalance that falls far short of meeting the growing market demand. Existing production technologies suffer from inherent technical bottlenecks. On the one hand, traditional chemical synthesis and microbial fermentation methods present safety hazards, byproduct accumulation, and long fermentation cycles, hindering large-scale industrial production. On the other hand, conventional enzymatic catalysis exhibits a significant product inhibition effect—accumulated hydrogen peroxide rapidly oxidizes and inactivates glutamate oxidase, leading to a sharp decline in enzyme catalytic efficiency and preventing the reaction from proceeding sustainably. Furthermore, the difficulty in recovering and reusing free enzymes further increases production costs, restricting its industrial application. Summary of the Invention

[0004] The purpose of this invention is to overcome the defects in the existing enzymatic catalytic production of α-ketoglutarate, such as product inhibition (i.e., hydrogen peroxide inactivates glutamate oxidase), inability to sustain the reaction, difficulty in enzyme recovery, and poor stability. This invention provides a dual-enzyme fusion protein that is resistant to hydrogen peroxide inhibition, has high catalytic efficiency, and good operational stability, so as to achieve efficient and continuous conversion of L-glutamate to α-ketoglutarate and meet the needs of large-scale industrial production of food-grade α-ketoglutarate.

[0005] The technical solution provided by the present invention is as follows: a dual-enzyme fusion protein resistant to hydrogen peroxide inhibition, which is composed of a glutamate oxidase mutant, a catalase mutant and a linker peptide; the amino acid sequences of the glutamate oxidase mutant and the catalase mutant are shown in SEQ ID NO.2 and SEQ ID NO.4 in the sequence listing, respectively.

[0006] Furthermore, the linker peptide is a flexible peptide that enables the glutamate oxidase unit and the catalase unit to form a spatial proximity effect; the amino acid sequence of the linker peptide is shown in SEQ ID NO.5 of the sequence listing.

[0007] Furthermore, the present invention also provides a DNA molecule encoding the said dual-enzyme fusion protein.

[0008] Furthermore, the present invention also provides a recombinant expression vector comprising the aforementioned DNA molecule.

[0009] Furthermore, the present invention also provides an engineered strain comprising the recombinant expression vector and the host bacterium.

[0010] Furthermore, the host bacterium is Escherichia coli.

[0011] Furthermore, the present invention also provides the application of the aforementioned dual-enzyme fusion protein, the aforementioned DNA molecule, the aforementioned recombinant expression vector, and the aforementioned engineered strain in the preparation of α-ketoglutarate; wherein the aforementioned DNA molecule, the aforementioned recombinant expression vector, and the aforementioned engineered strain are used to prepare the aforementioned dual-enzyme fusion protein.

[0012] Furthermore, the present invention also provides a method for preparing α-ketoglutarate, wherein the method utilizes the aforementioned dual-enzyme fusion protein, with L-glutamic acid / monosodium glutamate as a substrate, and reacts under the following conditions: 25–40°C, pH 6.0–8.5, aeration rate of 0.5–2.0 vvm, and stirring speed of 200–500 rpm for 45–90 min to obtain α-ketoglutarate; in the reaction system, the substrate concentration is 100 g / L–200 g / L; and the enzyme addition amount is 1–10 U / mL.

[0013] Furthermore, the present invention also provides a glutamate oxidase mutant, the amino acid sequence of which is shown in SEQ ID NO.2 in the sequence listing.

[0014] Furthermore, the present invention also provides a catalase mutant, the amino acid sequence of which is shown in SEQ ID NO.4 of the sequence listing.

[0015] This invention focuses on solving the product inhibition problem. It enhances single-enzyme performance through enzyme molecule modification, achieves spatial synergy through fusion protein design, and realizes industrial application through a continuous reaction process. The innovation of this approach lies in: firstly, introducing the spatial proximity effect into the synergistic catalytic system of glutamate oxidase and catalase, fundamentally solving the product inhibition problem of hydrogen peroxide on glutamate oxidase; secondly, simultaneously improving the stability and reusability of both enzymes through fusion expression; and thirdly, providing a complete technical solution for the large-scale industrial production of food-grade α-ketoglutarate, potentially filling the current significant supply-demand gap in the market.

[0016] The implementation of this invention will completely change the predicament of the current enzymatic catalysis method, which cannot achieve continuous production due to product inhibition, and promote the development of α-ketoglutaric acid production technology towards green, efficient, and low-cost directions, which has important industrial value and application prospects. Attached Figure Description

[0017] Figure 1 This is a comparison chart of the product accumulation process in Example 3; Figure 2 The results show the storage stability evaluation of the dual-enzyme fusion protein in Example 4; Figure 3 The results of pH stability evaluation of the dual-enzyme fusion protein in Example 4 are shown. Figure 4 The results show the reusability evaluation of the dual-enzyme fusion protein in Example 5. Detailed Implementation

[0018] To facilitate understanding of the present invention, the specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and specific examples. The following examples or drawings are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0019] Example 1: Screening and Modification of Enzyme Molecules 1. Screening of primitive enzymes This invention screened the original sequence of glutamate oxidase (named LGOX-WT, SEQ ID NO.1) from *Streptomyces* sp. and the original sequence of catalase (named CAT-WT, SEQ ID NO.3) from *Micrococcus lysodeikticus*. Activity assays showed that LGOX-WT had a specific activity of 12.5 U / mL and CAT-WT had a specific activity of 850 U / mL.

[0020] 2. Molecular modification of glutamate oxidase An LGOX mutant library was constructed using error-prone PCR, and approximately 5000 single clones were screened. After three rounds of screening, the optimal mutant LGOX-M (SEQ ID NO.2) was obtained, with mutation sites including: D10R, R13A, D27K, K41D, R76N, R101G, K137P, R176P, R179S, R185E, R197D, R209E, R212P, R253A, R307A, K308D, P324K, P327R, K337D, K418S, and R422A.

[0021] The specific activity of the mutant LGOX-M was increased to 65 U / mL, which is 5.2 times that of the original enzyme.

[0022] 3. Molecular modification of catalase A site-directed saturation mutagenesis strategy was employed to modify the amino acid near the active site of CAT. The mutation site was Y485H, yielding the optimal mutant CAT-M (SEQ ID NO.4). The specific activity of mutant CAT-M was increased to 2150 U / mL, which is 2.5 times that of the original enzyme.

[0023] Example 2: Design and Construction of Fusion Proteins 1. Optimized filtering of Linker length To determine the optimal linker peptide length, a series of flexible linker peptides were designed and fusion proteins were constructed from them. Screening was conducted using the retention rate of dual enzyme activity and the expression level of the fusion protein as indicators. Ultimately, (GGGGS)3 (SEQ ID NO. 5) was selected as the final linker peptide, as it maintains the optimal spatial distance between the two enzyme units, achieving both substrate channel effects and avoiding mutual interference.

[0024] 2. Expression and purification of fusion proteins The LGOX-M-(GGGGS)3-CAT-M fusion gene was cloned into the pET-28a vector and transformed into Escherichia coli BL21. After induction with 0.5 mM IPTG at 18°C ​​for 16 hours, the cell wet weight reached 8.5 g / L. Purification by Ni-NTA affinity chromatography yielded a fusion protein with a purity >95%, with a final yield of 65 mg / L fermentation broth.

[0025] Example 3: Verification of Anti-product Inhibition Effect The following four experimental groups were set up for comparison, with each group having the same total LGOX enzyme activity: Group A: The fusion protein LGOX-M-CAT-M of this invention; Group B: Modified free mixed enzymes LGOX-M + CAT-M (equimolar); Group C: Original free mixed enzymes LGOX-M + CAT-M (equimolar); Group D: Alone modified enzyme LGOX-M.

[0026] 1. Analysis of reaction process and product accumulation Product accumulation analysis was performed on the four experimental groups under the same experimental conditions, and the results are as follows: Figure 1 As shown in the figure. The results showed that the reaction in group D stopped after 15 minutes, with a final yield of only 4.3 g / L, confirming the strong inhibitory effect of hydrogen peroxide. The yield of group A (the fusion protein of this invention) reached 48.2 g / L after 90 minutes of reaction, with a conversion rate of 96.4%, which was significantly higher than that of group B (64.2%) and group C (24.8%).

[0027] 2. Hydrogen peroxide concentration monitoring Real-time monitoring of the H2O2 concentration during the reaction process showed that: Group A: The H2O2 concentration was consistently maintained at <0.3 mM, proving that the fusion protein achieved in-situ immediate removal of H2O2; Group B: H2O2 peak value 1.8 mM (occurred at 15 min); Group C: H2O2 peak value 4.2 mM (occurred at 30 min); Group D: H2O2 accumulated rapidly, reaching a peak of 8.6 mM in 15 min.

[0028] Example 4: Stability Evaluation 1. Thermal stability The fusion protein of this invention and the free mixed enzyme (LGOX-M+CAT-M) were incubated at 45℃, 50℃, and 55℃, respectively, and residual activity was measured at regular intervals. The results are shown in Table 1. The half-life (t1 / 2) of the fusion protein at 50℃ was 3.2 h, while that of the free mixed enzyme was only 0.8 h.

[0029] Table 1 Results of thermal stability test .

[0030] 2. Storage stability The fusion protein was stored in 50 mM Tris-HCl buffer at 4°C, and its specific activity was measured periodically. The experimental results are as follows: Figure 2 As shown.

[0031] 3. pH stability The residual activity of the fusion protein was measured after treatment at 4°C for 12 hours in different pH buffers. The results are as follows: Figure 3 As shown.

[0032] Example 5: Performance Evaluation of Reusability The residual activity of the recovered enzyme was determined after each batch of reaction (with the initial activity as 100%), and the results are as follows: Figure 4 As shown, the enzyme activity of the fusion protein decayed at a significantly slower rate than that of the free mixed enzyme, retaining more than 80% of its activity after the fourth batch of reactions, while the free mixed enzyme had lost nearly 80% of its activity.

[0033] Example 6: Immobilization and Performance Evaluation of Fusion Proteins To improve the reusability and operational stability of the enzyme, the fusion protein LGOX-M-CAT-M prepared in Example 2 was immobilized on an epoxy-modified immobilization resin (LX-1000EP).

[0034] 1. Immobilization methods Weigh 5 g of epoxy resin and wash it with 100 mM phosphate buffer (pH 8.0) to equilibrate. Add the purified fusion protein solution (protein concentration 10 mg / mL) at a carrier-to-protein ratio of 1:20, and couple with shaking at 25°C for 16 h. After immobilization, recover the resin and wash with the above buffer until no protein is detected in the supernatant. Measure the residual enzyme activity in the supernatant and the enzyme activity in the washing buffer, and calculate the immobilization rate and activity recovery.

[0035] 2. Immobilized enzyme activity recovery assay The immobilization rate of the fusion protein on the carrier was determined to be 92.5%. Based on the initial total activity of the free fusion protein as 100%, the activity recovery rate of the immobilized enzyme was 85.3%. The results indicate that the immobilization process had minimal impact on enzyme activity, and the carrier provided a suitable microenvironment for the enzyme.

[0036] Example 7: Application in the preparation of food-grade α-ketoglutaric acid 1. Fermentation tank scale-up test Reaction system: 3 L, initial concentration of substrate L-glutamate 110 g / L, added with the fusion protein of this invention (LGOX total activity 5000 U). Reaction conditions: 35℃, pH 7.0 (maintained by automatic addition of 2 M NaOH), aeration rate 1 vvm, stirring speed 300 rpm. After 90 min of reaction, the L-glutamate conversion rate reached 98.2%, and the α-ketoglutarate accumulation concentration was 108.02 g / L.

[0037] 2. Product purification The reaction solution was treated as follows: decolorization with activated carbon (1% w / v, 80℃ for 30 minutes): decolorization rate 92%; concentrated to 1 / 3 of the original volume; crystallized overnight at 4℃, and the crystals were collected by filtration; vacuum dried (40℃, -0.08 MPa, 12 hours); 294.9 g of white α-ketoglutaric acid crystals were obtained (based on 3 L of reaction solution), with an overall yield of 91%.

Claims

1. A dual-enzyme fusion protein resistant to hydrogen peroxide inhibition, characterized in that: It is composed of a glutamate oxidase mutant, a catalase mutant and a linker peptide; the amino acid sequences of the glutamate oxidase mutant and the catalase mutant are shown in SEQ ID NO.2 and SEQ ID NO.4 in the sequence listing, respectively.

2. The hydrogen peroxide-resistant dual-enzyme fusion protein according to claim 1, characterized in that: The linker peptide is a flexible peptide that enables the glutamate oxidase unit and the catalase unit to form a spatial proximity effect; the amino acid sequence of the linker peptide is shown in SEQ ID NO.5 in the sequence listing.

3. A polynucleotide encoding the dual-enzyme fusion protein of claim 1 or 2.

4. A recombinant expression vector, characterized in that: It comprises the polynucleotide of claim 3.

5. An engineered bacterial strain, characterized in that, It includes the recombinant expression vector and host bacteria as described in claim 4.

6. The preparation method according to claim 5, characterized in that: The host bacterium is Escherichia coli.

7. The use of the dual-enzyme fusion protein of claim 1 or 2, the polynucleotide of claim 3, the recombinant expression vector of claim 4, and the engineered strain of claim 5 or 6 in the preparation of α-ketoglutaric acid; wherein, The polynucleotide, the recombinant expression vector, and the engineered strain are used to prepare the dual-enzyme fusion protein.

8. A method for preparing α-ketoglutaric acid, characterized in that: This method utilizes the dual-enzyme fusion protein described in claim 1 or 2, with L-glutamic acid / monosodium glutamate as a substrate, and reacts under the following conditions: 25–40°C, pH 6.0–8.5, aeration rate of 0.5–2.0 vvm, and stirring speed of 200–500 rpm for 45–90 min to obtain α-ketoglutarate; the substrate concentration in the reaction system is 100 g / L–200 g / L; and the enzyme addition amount is 1–10 U / mL.

9. A glutamate oxidase mutant, characterized in that: Its amino acid sequence is shown in SEQ ID NO.2 in the sequence listing.

10. A catalase mutant, characterized in that: Its amino acid sequence is shown in SEQ ID NO.4 in the sequence listing.

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