Malic enzyme mutant and application thereof

Abstract

The invention relates to the technical field of enzyme engineering, in particular to a malic enzyme mutant and application thereof. Specifically, the malic enzyme derived from Escherichia coli is subjected to site mutation screening, and the result shows that after valine at the 7th site is mutated into lysine (V7K), the catalytic efficiency and the catalytic activity are obviously improved, which is of great significance to industrial application.

Description

A malic acid enzyme mutant and its application Technical Field

[0001] This invention relates to the field of enzyme engineering technology, and in particular to a malic acid enzyme mutant and its application in the catalytic CO2 fixation synthesis of L-malic acid. Background Technology

[0002] Carbon sequestration technology aims to convert atmospheric carbon dioxide (CO2) into useful chemicals and fuels, thereby reducing greenhouse gas emissions and achieving carbon recycling. Malic enzyme (ME) plays a key role in the carbon sequestration process, catalyzing the synthesis of L-malic acid from pyruvate and carbon dioxide. This reaction not only helps reduce atmospheric CO2 but also produces high-value-added chemicals.

[0003] L-malic acid is an important organic acid widely used in the food, pharmaceutical, and chemical industries. In the food industry, L-malic acid is used as an acidulant, preservative, and food additive to improve the taste and shelf life of food. In the pharmaceutical field, L-malic acid is an important intermediate in the synthesis of certain drugs and possesses important physiological functions. Furthermore, L-malic acid also serves as a raw material for the synthesis of other chemicals in the chemical industry, showing broad application prospects. Therefore, improving the production efficiency and yield of L-malic acid has significant economic and social implications.

[0004] Although malic acid enzymes play an important role in carbon fixation and L-malic acid production, wild-type malic acid enzymes have certain limitations in terms of catalytic efficiency and substrate specificity, specifically: (1) Low catalytic efficiency: Wild-type malic acid enzymes have low catalytic activity, resulting in low L-malic acid production efficiency, which is difficult to meet the needs of industrial production.

[0005] (2) Weak environmental adaptability: Wild-type malic acid enzymes have poor stability under different environmental conditions (such as temperature, pH, etc.), which affects their application effect in actual production.

[0006] To address these issues and improve the activity and substrate adaptability of malicase, modifying it using enzyme engineering techniques has become an effective strategy. Molecular modification methods based on the protein big language model can significantly enhance the catalytic activity of malicase, thereby increasing the production efficiency and yield of L-malic acid and providing strong support for carbon fixation technology and the industrial production of L-malic acid. Summary of the Invention

[0007] This invention provides a malic acid oxidase mutant and its application. This malic acid oxidase mutant solves the problem of low catalytic efficiency of the original malic acid oxidase (amino acid sequence shown in SEQ ID NO: 1) derived from Escherichia coli for pyruvate and carbon dioxide. It provides a malic acid oxidase mutant and its application in the catalytic synthesis of L-malic acid by CO2 fixation.

[0008] This invention relates to a malicase derived from *Escherichia coli* (ME, NCBI accession number: NP_415996.1, amino acid sequence as shown in SEQ ID NO.1, nucleotide sequence as shown in SEQ ID NO.2). Using a protein big language model with zero-shot prediction, fitness was calculated to predict potential mutation sites. Based on a comprehensive analysis of prediction results from multiple models, it was found that the mutation of valine at position 7 to lysine (V7K) showed high fitness scores in multiple models, suggesting that this mutation may significantly enhance the enzyme's catalytic activity.

[0009] Experiments verified that the V7K mutant exhibits 1.35 times the catalytic activity of the wild type, representing a 35% increase in catalytic activity and significantly improving the efficiency of CO2 fixation for L-malate synthesis. This mutation site is located near the enzyme's active pocket, and by introducing a positively charged lysine residue, it may improve the enzyme's ability to capture HCO3- in solution. - This ability improves catalytic efficiency.

[0010] In this regard, the present invention includes, but is not limited to, the following: In one aspect, the present invention provides a malicase mutant obtained by mutating the 7th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 1 as follows: V7K (mutating valine to lysine).

[0011] In one aspect, the present invention provides a polynucleotide encoding the malic acid enzyme mutant described herein.

[0012] In one aspect, the present invention provides a recombinant vector comprising the polynucleotides described herein. Preferably, the original expression vector of the recombinant vector is pET28a-SUMO, which enables efficient expression of the target protein and enhances soluble protein expression through the SUMO tag.

[0013] In one aspect, the recombinant vector of the present invention is a pET28a-SUMO vector containing the polynucleotides described in the present invention.

[0014] In one aspect, the present invention provides a host cell comprising the polynucleotides described herein or the recombinant vectors described herein.

[0015] In one aspect, the host cell described in this invention is a fungal cell, a bacterial cell, or a plant cell.

[0016] In one aspect, the host cell described in this invention is a bacterial cell.

[0017] In one aspect, the bacterial cell described in this invention is an *Escherichia coli* cell. Preferably, the host cell for the genetically engineered bacteria is *Escherichia coli* BL21(DE3) cells, which exhibit good protein expression capabilities and genetic stability.

[0018] In one aspect, the Escherichia coli cells described in this invention are E. coli BL21(DE3) cells.

[0019] In another aspect, the present invention also provides the use of the malicase mutant or the host cell described herein in the preparation of L-malic acid.

[0020] In one aspect, the application of the present invention includes: in a CO2-containing reaction system, contacting pyruvate with the malic acid enzyme mutant to carry out a carboxylation reaction; wherein the CO2 is derived from sodium bicarbonate.

[0021] In another aspect, the present invention also provides a method for producing the malicase mutant of the present invention, the method comprising: (a) culturing the host cell of the present invention under conditions suitable for expression of the malicase mutant; and (b) recovering the malicase mutant.

[0022] Compared with the prior art, the present invention has at least the following beneficial effects: the malic acid enzyme mutant V7K obtained by the protein language model zero-shot prediction method of the present invention significantly improves the catalytic efficiency for pyruvate and carbon dioxide, and increases the catalytic efficiency of malic acid enzyme (kJ / kJ / kE) cat / K m From 1.32 s -1 ·mM -1 Increased to 1.78 s -1 ·mM -1 This improvement increased efficiency by 34.8%. This improvement solved the problem of low catalytic efficiency of wild-type malic acid oxidase, greatly improving production efficiency.

[0023] The V7K mutant significantly reduces the K-type response of the substrate. m The value decreased from 4.51 mM in the wild type to 3.24 mM, a reduction of 28.2%, indicating that the mutant showed improved resistance to the substrate HCO3-. - The affinity is significantly improved, and high catalytic efficiency can be achieved at lower CO2 concentrations, which is of great significance for industrial applications.

[0024] This invention employs a biocatalytic method, which is green and environmentally friendly. Compared to traditional chemical catalysis methods, biocatalysis produces fewer byproducts during the reaction process, resulting in a smaller environmental impact and meeting the requirements of sustainable development. Furthermore, by converting carbon dioxide into high-value-added chemicals through carbon sequestration technology, not only are greenhouse gas emissions reduced, but additional economic benefits are also created, achieving a win-win situation for both the environment and the economy.

[0025] This invention identifies V7, a key amino acid residue affecting malic acid oxidase activity, providing important guidance for further improvement of malic acid oxidase. The mutation of valine at position 7 to lysine introduces a positive charge into the randomly coiled region at the N-terminus, altering the electrostatic environment of that region and significantly enhancing the enzyme's interaction with HCO3-. - The binding ability. The positively charged N-terminal region may act as a negatively charged substrate, HCO3. - The initial recognition site promotes substrate recruitment and orientation through electrostatic interactions, while simultaneously increasing the local effective concentration of the substrate, thereby reducing K0. m This discovery reveals the important regulatory role of the N-terminal flexible region, far from the active site, in substrate binding, overcoming the limitation of traditional enzyme engineering that focuses solely on modifying the active site. Attached Figure Description

[0026] Figure 1 illustrates the catalytic mechanism of malic acid oxidase.

[0027] Figure 2 shows the electrophoretic analysis of purified malic acid enzyme on SDS-PAGE; where 1 represents crude malic acid enzyme solution; 2 represents pure malic acid enzyme solution; and 3 represents pure malic acid enzyme solution with the SUMO tag removed.

[0028] Figure 3 shows a comparison of the specific activities of wild-type and mutant malic acidase.

[0029] Figure 4 shows the Michaelis-Menten equation curves for wild-type and mutant V7K malic acid oxidase, where A represents the comparison of specific activities at different substrate concentrations; and B represents the Michaelis-Menten equation fitting.

[0030] Figure 5 shows a comparison of L-malate conversion rates between wild-type and mutant V7K malate enzymes. Detailed Implementation

[0031] The present invention will be further described below with reference to specific embodiments. The following are only specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto.

[0032] Unless otherwise specified, the experimental methods in this invention are conventional methods. For specific gene cloning operations, please refer to "Molecular Cloning: A Laboratory Manual" edited by J. Sambrook et al.

[0033] Reagents used in upstream genetic engineering: PrimeSTAR used in the embodiments of this invention. ® Max DNA Polymerase and QuickCut™ DpnI were purchased from TaKaRa, Takara Bio Engineering (Dalian) Co., Ltd.; plasmid extraction kits and DNA recovery and purification kits were purchased from Axygen Hangzhou Co., Ltd.; E. coli BL21(DE3), plasmid pET28a-SUMO, etc., were purchased from Novagen; DNA markers, low molecular weight standard proteins, and agarose gel electrophoresis reagents were purchased from TransGen Biotech Ltd.; primer synthesis and sequencing were performed by Qingke Biotechnology Co., Ltd. Refer to the product instructions for the usage of the above reagents.

[0034] Reagents used in the catalytic reaction: pyruvate, sodium bicarbonate, NAD. + NADH was purchased from Maclean's. The standard method for detecting crude enzyme activity in the malic acid carboxylation reaction is as follows: First, add 10 μL of NADH solution (70 mM) and 100 μL of enzyme solution to 865 μL of acetate-sodium acetate buffer (100 mM, pH=5.9), mix well, and add 195 μL of the mixture to each well of the microplate (4 wells). After the NADH reading stabilizes, add 2.5 μL each of pyruvate (500 mM) and sodium bicarbonate (500 mM). Incubate at 30 ℃ for 5 min. Quantitatively analyze the NADH generated / consumed in the sample using a microplate reader, taking a data point every 30 s. Definition of enzyme activity unit (U): Under standard reaction conditions, enzyme activity unit (U) is defined as the amount of enzyme required to consume or produce 1 μmol of NADH per minute.

[0035] Example 1: Mutant Design This invention first constructs a complete system for designing malicase mutants. This system combines advanced protein structure prediction methods with Protein Language Models (PLMs). PLMs effectively capture the fitness information of mutations during protein evolution by learning the probability distribution of evolutionarily preserved protein sequences in nature. First, AlphaFold 2.3.2 was used to predict the structure of malicase. The average pLDDT score of the predicted structure reached 92.99, indicating that the predicted structure has an accuracy close to experimental resolution. Through structural analysis, we identified the following key functional regions: substrate, Glu255, Asp256, Asp279, and a water molecule and Mn. 2+ / Mg 2+ Coordination forms a six-coordination structure.

[0036] The catalytic mechanism of ME is shown in Figure 1. (1) Lys184 is deprotonated by the nearby Asp278, and the two residues exist in a neutral form in a closed conformation. (2) Basic Lys184 deprotonates the hydroxyl group of L-malic acid. (3) The hydrogen atom on the C2 atom of L-malic acid is transferred to NAD(P). + On the C4N atom, L-malic acid is oxidized to oxaloacetic acid; (4) The generated oxaloacetic acid is unstable and rapidly undergoes β-decarboxylation with the assistance of acidic metal ions to generate enol pyruvate and release carbon dioxide; (5) Tyr113, as a generalized acid protonated C3, causes enol pyruvate to tautomerize into a ketone; (6) The protonated Lys184 transfers a proton to Tyr113, and the two are restored to the initial protonated state.

[0037] This invention constructs a prediction system based on a multimodal protein large language model for the efficient identification and screening of beneficial mutation sites in malic enzymes. The system employs four advanced PLMs, and through independent predictions by each model, comprehensively assesses the impact of potential mutations on enzyme function.

[0038] Specifically, the four models used in this system and their characteristics are as follows: ESM2-650M: A protein language model based on the Transformer architecture developed by Meta AI, trained on more than 60 million protein sequences in the UniRef50 database. As the current standard base model in the field, it demonstrates excellent sequence feature representation capabilities.

[0039] SaProt: An advanced model integrating sequence and structural information, achieving multimodal protein language modeling through an innovative structure-aware vocabulary design. After pre-training on approximately 40 million protein structure datasets, it achieved leading performance on the ProteinGym benchmark (October 2024).

[0040] ProSST is a structure quantization model based on geometric vector perceptrons. It effectively captures the intrinsic correlation between complex protein sequence-structure features using a sequence-structure untangling attention mechanism and demonstrates excellent performance on the ProteinGym dataset through a joint modeling strategy.

[0041] ProGen2: An autoregressive protein language model developed by Salesforce Research with a parameter size of 6.4B. It is trained on a dataset of more than 1 billion protein sequences from genomic, metagenomic, and immune repertoire datasets and has outstanding sequence distribution modeling capabilities and zero-sample fitness prediction accuracy.

[0042] This invention employs the zero-shot prediction method of four protein big language models to calculate the fitness of the first nine amino acids of a malic acid oxidase sequence after each site is replaced with one of 20 possible amino acids. Each model makes predictions independently, and by comparing the prediction results of different models, the functional impact of each mutation site is systematically evaluated. High-potential mutation sites (R6D, R6P, V7K, V7P, V7E, D9P) are then selected as candidates for subsequent experimental characterization.

[0043] Example 2: Construction of Single-Point Mutation A single-point mutation was performed on the wild-type malic enzyme. The specific method is as follows: The ME gene fragment was synthesized by Qingke Biotechnology Co., Ltd., between the BamH I and HindIII restriction sites in the MCS region of the pET-28a-SUMO plasmid.

[0044] Whole plasmid PCR: Using pET28a-SUMO-ME plasmid as a template, upstream and downstream primers covering the mutation site were designed (Table 1) for whole plasmid PCR: Table 1 Design of malicase ME mutation primers

[0045] Table 2 PCR Reaction Sample Addition System

[0046] Table 3 PCR reaction conditions

[0047] After PCR, the adenine-methylated GmATC sequence in the template was cleaved using QuickCut™ DpnI to remove the template DNA. The PCR products were unaffected during this process. The specific reaction system is shown in Table 4: DpnI Digestion Reaction System Components.

[0048] After adding the sample, mix gently and centrifuge briefly, then react at 37 °C for 30 min.

[0049] After removing the template DNA, the reverse PCR product was transformed into E. coli BL21(DE3) competent cells for protein expression. This is a widely used T7-expressing E. coli strain. The specific transformation steps were as follows: Thaw the cloned competent cells on ice; add 10 μL of recombinant product to 100 μL of competent cells, gently tap the tube wall to mix (do not shake), and incubate on ice for 30 min; heat shock at 42 ℃ for 45 s, then immediately cool on ice for 2-3 min; add 900 μL of SOC or LB medium (without antibiotics), and incubate at 37 ℃ for 1 h (200-250 rpm); centrifuge at 5000 rpm for 5 min, discard 900 μL of supernatant, resuspend the bacterial cells in the remaining medium, and gently spread on a kanamycin-resistant plate using a sterile spreader; incubate upside down at 37 ℃ for 12-16 h.

[0050] After overnight culture, hundreds of single colonies should form on the transformation plate. Pick a few, extract plasmids, and sequence them to confirm whether they are the correct mutations. Transformants with correct mutations can be used for subsequent induction expression experiments and for preservation of the bacterial strain in glycerol tubes.

[0051] Example 3 Based on the activity determination of malic acid enzyme and its mutant, the purification process of malic acid enzyme and its mutant is as follows: (1) Induction expression: thaw the glycerol tube on ice, take 10~20 μL, streak on LB plate containing kanamycin resistance, and incubate at 37 ℃ for 12h to activate.

[0052] Single bacteria were picked from activation plates and inoculated into LB liquid medium containing kanamycin. The culture was incubated at 37 °C and 200 rpm for 12–16 h to prepare a seed culture. The seed culture was then inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin at an inoculum size of 1–2%, and cultured at 37 °C with shaking at 200 rpm until OD (open-circuit retrieval). 600 The concentration was 0.6–0.8 (approximately 2 hours). IPTG was added to a final concentration of 0.5 mM, and expression was induced at 37 °C and 200 rpm for 6 hours. After induction, the cells were harvested by centrifugation at 7500 rpm for 10 minutes at 4 °C, and the supernatant was discarded. The cells were resuspended in PBS buffer (pH 7.0–7.5), centrifuged at 7500 rpm for 5 minutes at 4 °C, and the supernatant was discarded. This washing process was repeated three times.

[0053] (2) Protein purification: The harvested cell pellet was resuspended in lysis buffer and sonicated on ice under the following conditions: Six probes, 2 seconds of disruption, 3 seconds of pause, power 50-60%, total duration 10 minutes. After disruption, centrifuge at 12000 rpm to collect the supernatant containing soluble protein, i.e., crude enzyme solution.

[0054] The supernatant containing soluble protein was loaded onto a Ni-NTA 6FF pre-filled gravity column (purchased from Shanghai Sangon Biotech). Impurities were eluted with a low concentration of imidazole (20 mM) buffer for 10-20 column volumes, and the target protein was eluted with a high concentration of imidazole (500 mM) buffer to obtain the His-tagged protein.

[0055] Following a SUMOase:purified enzyme elution buffer ratio of 1:10, add SUMOase to the elution buffer, transfer to a 10 kDa MW ultrafiltration tube, centrifuge at 4 °C and below 5000 g until 200-500 μL of liquid remains, add PBS buffer to 15 mL, repeat 3 times to remove imidazole from the purified enzyme, and simultaneously remove the SUMO and His tags. After ultrafiltration, load the sample onto a Ni-NTA 6FF pre-packed gravity column, collect the elution buffer, which is the purified enzyme with the SUMO tag removed.

[0056] (3) SDS-PAGE electrophoresis verification: During the purification process, crude enzyme, pure enzyme, and labeled pure enzyme were sampled separately. The SDS-PAGE electrophoresis buffer used a pre-made gel (the seal strip was torn off before use) and the matching buffer. An additional 180 kda protein marker was applied to one well. Electrophoresis parameters: 120V, 400 mA, time 1 h. The results are shown in Figure 2.

[0057] (4) Enzyme activity assay: First, add 10 μL of NADH solution (70 mM) and 100 μL of enzyme solution (concentration 200 μg / mL) to 865 μL of acetate-sodium acetate buffer (100 mM, pH=5.9), mix well, and add 195 μL of the mixed solution to each well of the microplate, for a total of 4 wells. After the NADH reading stabilizes, add 2.5 μL each of pyruvate (250 mM) and sodium bicarbonate (250 mM). After incubating the reaction system at a constant temperature of 30°C for 5 min, measure the absorbance change at a wavelength of 340 nm using a microplate reader. Using a pre-plotted NADH standard curve (concentration range 0-1000 μM), convert the measured absorbance value into NADH concentration to quantitatively analyze the amount of NADH generated / consumed in the sample (Δ). NADH The standard curve was analyzed using linear regression to ensure a correlation coefficient R² ≥ 0.99, and all measurements were performed within the linear range of the standard curve. The absorbance (A) of NADH solutions at 340 nm was measured using a microplate reader with NADH concentration as the x-axis. 340The standard curve plotted on the y-axis is y = 2.8259x + 0.2108, R² = 0.9997. The definition of enzyme activity unit (U): Under standard reaction conditions, enzyme activity unit (U) is defined as the amount of enzyme required to consume or produce 1 μmol of NADH per minute. Specific activity is enzyme activity (U) divided by the enzyme concentration in the reaction system.

[0058] The specific activities of the purified enzymes were measured, showing that the specific activity of the wild-type enzyme (WT) was 1.495±0.034 U / mg, the specific activity of mutant R6D was 1.006±0.035 U / mg, R6P was 1.573±0.051 U / mg, V7K was 1.890±0.101 U / mg, V7P was 1.298±0.031 U / mg, D9P was 1.651±0.072 U / mg, and V7E was 1.720±0.063 U / mg (see Figure 3). One-way ANOVA was used to statistically test the differences among the mutants, and the results showed a significant difference in specific activity among the different mutants (F = 58.061, p<0.001). Further post-hoc multiple comparison tests using Tukey HSD revealed the following significant differences among the mutants: compared to the wild-type enzyme (ME WT), the specific activity of mutant R6D was significantly decreased (p<0.001), the specific activity of mutant V7K was significantly increased (p<0.001), and the specific activity of mutant V7P was slightly lower than that of wild-type (p<0.05); while the specific activities of mutants R6P, V7E, and D9P were not significantly different from those of wild-type (p>0.05).

[0059] The statistical results above indicate that the enzyme activity of mutant V7K is significantly higher than that of wild-type, classifying it as an activity-enhancing mutation; while the specific activity of mutant R6D is significantly lower than that of wild-type, classifying it as an activity-degrading mutation. The remaining mutants showed no significant difference from wild-type. More specifically, the V7K mutant exhibited the highest specific activity (1.890 U / mg), an increase of approximately 26.4% compared to wild-type, indicating that the mutation at this site is beneficial for enhancing enzyme activity. The V7E (1.720 U / mg) and D9P (1.651 U / mg) mutants also showed higher activities than wild-type, increasing by 15.0% and 10.4%, respectively. Conversely, the specific activity of the R6D mutant decreased significantly to 1.006 U / mg, only 67.3% of that of wild-type, indicating that this mutation had a negative impact on enzyme activity. The activities of the R6P and V7P mutants were similar to those of wild-type, at 105.2% and 86.8% of wild-type, respectively. These results suggest that different amino acid sites have varying degrees of influence on the catalytic activity of malic acid oxidase. Some mutations at the 7th and 9th amino acids can enhance enzyme activity, while mutations at the 6th arginine to aspartic acid can significantly reduce enzyme activity.

[0060] (4) Determination of enzyme kinetic constant To determine the kinetic parameters of malic acid oxidase, this study employed a two-substrate enzyme-catalyzed reaction system. The Michaelis constant (Km) was determined by fixing the concentration of one substrate while changing the concentration of the other. m ) and maximum reaction rate (V max The reaction system was prepared according to the enzyme activity assay method shown in Table 5. 190 μL of reaction solution was added to each well of the ELISA plate, and then 5 μL of pyruvate or sodium bicarbonate solution with different concentration gradients was added to each well of the experimental group. The amount of enzyme used was appropriately reduced to avoid excessive substrate consumption leading to nonlinearity. For both pyruvate and sodium bicarbonate, one substrate concentration was fixed at 12.5 mM, while the concentration of the other substrate [S] was varied (0.25 mM, 0.5 mM, 1.25 mM, 2.5 mM, 5 mM, 12.5 mM). The ΔΔC at different substrate concentrations was measured over a 5-minute reaction time. NADH Calculate the reaction rate V using the formula V = Δ NADH / t, where t is the reaction time (5 min). When the substrate concentration is too high, A will occur. 340 An increase in absorbance readings (at 340 nm) can be used to determine the effect of different substrate concentrations on A without the addition of enzyme. 340 Changes in numerical values ​​should be subtracted when calculating enzyme activity and reaction rate. The specific reaction system is as follows: Table 5 Reaction system for malic acid oxidase activity assay.

[0061] After obtaining the reaction rates at different substrate concentrations, the maximum reaction rate V was obtained by nonlinear fitting using the Michaelis-Menten equation. max and the Mi constant K m According to V max Calculate the transformation number k cat =V max / [E], where [E] is the molar concentration of the enzyme. Catalytic efficiency is determined by k cat / K m Calculations show that this parameter reflects the enzyme's catalytic ability on its substrate.

[0062] The results showed that the V7K mutant significantly reduced the K-response to the substrate. m The value decreased from 4.51 mM in the wild type to 3.24 mM, a reduction of 28.2%, indicating that the mutant showed improved resistance to the substrate HCO3-. - The affinity for catalytic activity is significantly enhanced, achieving high catalytic efficiency even at lower CO2 concentrations, which is of great significance for industrial applications (see Figure 4A). Furthermore, the V7K mutation significantly improves the catalytic efficiency of malic acid oxidase for pyruvate and carbon dioxide, increasing the catalytic efficiency (kJ / mL) of malic acid oxidase.cat / K m From the wild type 1.32s -1 ·mM -1 Increased to 1.78 s in the mutant. -1 ·mM -1 The catalytic efficiency was increased by 34.8%. This improvement solved the problem of low catalytic efficiency of wild-type malic enzyme and greatly improved production efficiency (see Figure 4B).

[0063] Example 4 Determination of L-malic acid conversion by malicase This invention provides a method for the conversion of pyruvate to L-malic acid using malicase. This method has the advantages of simple reaction system, controllable operation, and accurate product detection. It can be used for the determination and application research of the catalytic performance of malicase and its mutants.

[0064] (1) Establishment of the reaction system The reaction system was prepared according to the proportions shown in Table 6, and the total volume of the reaction system was 1 mL. All reagents were pre-cooled on ice before the reaction.

[0065] Table 6. Reaction system for the production of L-malic acid catalyzed by malic acid oxidase

[0066] 2) Reaction conditions and steps: Add reaction buffer, MnCl2, NADH, and pyruvate sequentially to a 1.5 mL centrifuge tube, mix gently, and place in a 30 ℃ metal bath to equilibrate the temperature. Then quickly add malic acid enzyme solution and sodium bicarbonate solution, and tighten the cap. Place the reaction tube on a 30 ℃ constant temperature metal bath shaker and gently shake at 300 rpm for 30 min.

[0067] Furthermore, to avoid gas-liquid interface disturbances leading to bubble formation in the system, the reaction is kept in a gentle shaking state rather than a violent oscillation.

[0068] (3) Reaction termination and sample processing After the reaction is completed, immediately place the reaction tube at 100 °C and heat for 5 min to inactivate the enzyme. Then centrifuge at 12000 rpm for 5~10 min to remove the protein precipitate. Take the supernatant, filter it through a 0.22 μm filter membrane, and put it into a liquid phase sample bottle for later use.

[0069] (4) The amount of L-malic acid generated in the reaction system was determined by high performance liquid chromatography (HPLC). The detection conditions were as follows: Column: Aminex HPX-87H; Column temperature: 55 ℃; Mobile phase: 5 mM H2SO4; Flow rate: 0.6 mL / min; Detector: Ultraviolet absorption (UV) detector; Detection wavelength: 210 nm; Injection volume: 15 μL; Elution method: isoclinic elution; Total running time: 20 min (including elution and equilibration phases). During the detection process, it was found that the substrate pyruvate and the product L-malic acid had partially overlapping peaks at approximately 9.26 min, which affected the determination of the results. To ensure the accuracy of the quantitative results, this invention selected the characteristic small peak of L-malic acid at approximately 13.35 min for quantitative analysis.

[0070] (5) Establishment of Standard Curve and Content Calculation: A series of L-malic acid standard solutions of different concentrations (0, 0.5, 1, 2, 5, 10, 25 mM) were prepared and injected under the same chromatographic conditions. The peak areas were recorded, and a standard curve was plotted. A graph was plotted with L-malic acid concentration as the abscissa (x) and peak area as the ordinate (y), and linear fitting was performed to obtain the regression equation: y = 20670x - 397.51, R0 2 =0.9999 This shows that the detection system established by this method has good linearity, with a correlation coefficient close to 1, and is suitable for the accurate quantification of L-malic acid.

[0071] The concentration of L-malic acid in the sample was calculated by substituting the measured peak area into the regression equation of the standard curve.

[0072] (6) Calculation of L-malic acid yield: Pyruvic acid was used as the substrate in the reaction system, and the reaction equation is as follows: Pyruvic acid + CO2 + NADH → L-malic acid + NAD + (Molar ratio 1:1) Calculate the conversion rate of L-malic acid based on pyruvate. The calculation formula is as follows: 1. Initial molar number of pyruvate: n p ,0= C p ,0× V xn 2. Moles of L-malic acid after the reaction: n mal = C mal × V xn 3. Conversion rate (%) = (n mal / n p According to the measurement results, the pyruvate conversion rate of wild-type malic acid enzyme was 16%, while that of mutant V7K was 32% (see Figure 5), indicating that the catalytic efficiency of mutant was significantly higher than that of wild-type enzyme.

[0073] Furthermore, this method can be used to compare the catalytic activity of different malic acid enzyme mutants and evaluate their catalytic performance by measuring the amount of L-malic acid produced.

[0074]

Claims

1. A malic acid oxidase mutant, characterized in that, The mutant was obtained by mutating the amino acid sequence shown in SEQ ID NO: 1 as follows: V7K.

2. A polynucleotide, characterized in that, The code is based on the malic acid enzyme mutant according to claim 1.

3. A recombinant vector, characterized in that, It contains the polynucleotide as described in claim 2.

4. A host cell, characterized in that, It contains the polynucleotide according to claim 2 or the recombinant vector according to claim 3.

5. The host cell according to claim 4, characterized in that, The host cell is a fungal cell, a bacterial cell, or a plant cell.

6. The host cell according to claim 5, characterized in that, The host cell is a bacterial cell.

7. The host cell according to claim 6, characterized in that, The bacterial cells are Escherichia coli cells.

8. The use of the malic acid enzyme mutant according to claim 1 or the host cell according to any one of claims 4-7 in the preparation of L-malic acid.

9. The application according to claim 8, characterized in that, The application method includes: in a CO2-containing reaction system, contacting pyruvate with the malic acid enzyme mutant to carry out a carboxylation reaction; wherein the CO2 is derived from sodium bicarbonate.

10. A method for producing the malic acid enzyme mutant according to claim 1, characterized in that, The method comprises: (a) culturing a host cell according to any one of claims 4-7 under conditions suitable for expression of the malicase mutant; and (b) recovering the malicase mutant.

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