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 results show that after methionine at the 144th site is mutated into isoleucine (M144I), glutamine at the 253rd site is mutated into isoleucine (Q253I), and threonine at the 327th site is mutated into phenylalanine (T327F), the catalytic efficiency and the catalytic activity are obviously improved, which is of great significance to industrial application.

Description

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] How to efficiently capture and utilize carbon dioxide (CO2) has become a key scientific issue for achieving carbon neutrality and sustainable development. Compared with traditional methods such as physical adsorption or chemical fixation, biological carbon sequestration technology has attracted widespread attention due to its low energy consumption, environmental friendliness, and ability to produce high-value-added products.

[0003] Among various biological carbon fixation strategies, malic enzymes (MEs) possess unique advantages. This enzyme catalyzes the synthesis of L-malic acid from pyruvate and CO2, thereby achieving efficient carbon dioxide conversion and resource utilization. Compared to other carbon fixation products, L-malic acid is not only an important organic acid widely found in nature, but also has significant application value in the food, pharmaceutical, and chemical industries. For example, in the food industry, L-malic acid serves as an acidulant and preservative, improving flavor and extending shelf life; in the pharmaceutical field, it is an important intermediate in the synthesis of various drugs; and in the chemical industry, it can be used as a raw material to prepare a variety of functional chemicals. Therefore, L-malic acid biosynthesis based on malic enzymes is considered a green technology pathway with both environmental benefits and economic potential.

[0004] However, naturally derived malic enzymes still face challenges in industrial applications: on the one hand, their catalytic efficiency is limited, making it difficult to achieve high-level L-malic acid synthesis; on the other hand, the enzymes lack environmental stability and are sensitive to temperature and pH changes, limiting their application in complex production systems. To address these issues, recent research has gradually shifted towards the targeted modification of enzymes (MEs) through enzyme engineering. Traditional site-directed mutagenesis and directed evolution have made some progress in improving enzyme performance, but they suffer from bottlenecks such as long experimental cycles and low screening efficiency.

[0005] With the development of artificial intelligence and computational biology, molecular modification strategies based on protein language models (PLMs) have provided new opportunities for enzyme performance optimization. This method can predict potential functional mutation sites based on large-scale sequence and structural information, thereby enabling more efficient enzyme design and modification. Optimizing malate enzymes using this emerging technology is expected to significantly improve their catalytic activity and environmental adaptability, thereby promoting CO2 carbon fixation efficiency and laying the foundation for the green synthesis of L-malic acid. Summary of the Invention

[0006] This invention provides a malic acid oxidase mutant and its application, which solves the problem of malic acid oxidase derived from Escherichia coli (E. coli).Escherichia coli To address the issue of low catalytic efficiency of the original malic enzyme for pyruvate and carbon dioxide, this paper provides a malic enzyme mutant and its application in the catalytic synthesis of L-malic acid from CO2 fixation.

[0007] This invention relates to the source Escherichia coli The malicase (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) was used for fitness assessment using a zero-shot prediction method based on a protein big language model to identify mutation sites with potential functional improvements. Four widely used protein big language models, including ESM2, ProGen2, SaProt, and ProSST, were employed in this study, identifying a total of 118 potential mutation sites through comprehensive analysis. Based on the prediction results of these 118 mutation sites, the SaProt model was fine-tuned, and the top 10 sequences with fitness scores for multi-site combined mutations were output for subsequent characterization analysis. Screening using this method revealed that the triple mutant combination M144I+Q253I+T327F (methionine at position 144 is mutated to isoleucine, glutamine at position 253 is mutated to isoleucine, and threonine at position 327 is mutated to phenylalanine) performed exceptionally well in fitness scores. Prediction results indicate that this mutant combination has the potential to significantly enhance enzyme catalytic activity.

[0008] Further experimental verification showed that not all enzyme mutants with high model prediction scores exhibited improved catalytic performance in actual enzyme kinetic experiments. However, it was found that the catalytic activity of the M144I+Q253I+T327F mutant was 1.46 times that of the wild type, which is 46% higher than that of the wild type, significantly improving the efficiency of CO2 fixation to synthesize L-malic acid.

[0009] 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 amino acid sequences at positions 144, 253, and 327 of the amino acid sequence shown in SEQ ID NO: 1 as follows: M144I (mutation from methionine to isoleucine), Q253I (mutation from glutamine to isoleucine), and T327F (mutation from threonine to phenylalanine).

[0010] In one aspect, the present invention provides a polynucleotide encoding the malic acid enzyme mutant described herein.

[0011] 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.

[0012] In one aspect, the recombinant vector of the present invention is a pET28a-SUMO vector containing the polynucleotides described in the present invention.

[0013] In one aspect, the present invention provides a host cell comprising the polynucleotides described herein or the recombinant vectors described herein.

[0014] In one aspect, the host cell described in this invention is a fungal cell, bacterial cell, or plant cell. It should be noted that the plant cell described in this invention is not intended to protect any specific plant, and this invention does not disclose any method for developing plant cells into a complete plant. The plant cell described in this invention is only used as an engineered cell for expressing the enzyme mutant of this invention.

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

[0016] In one aspect, the bacterial cell described in this invention is an *Escherichia coli* cell. Preferably, the host cell of the genetically engineered bacteria is *Escherichia coli* (…). E. coli BL21(DE3) cells, this strain has good protein expression ability and genetic stability.

[0017] In one aspect, the Escherichia coli cells of the present invention are E. coli BL21(DE3) cells.

[0018] 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.

[0019] 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.

[0020] In another aspect, the present invention also provides a method for producing the malic acid enzyme mutant described herein, the method comprising: (a) The host cells of the present invention are cultured under conditions suitable for the expression of the malicase mutant; and (b) Recover the malic acidase mutant.

[0021] Compared with the prior art, the present invention has at least the following beneficial effects: This invention utilizes the zero-shot prediction method of protein language models to obtain the malic acid oxidase mutant M144I+Q253I+T327F. The catalytic efficiency of this mutant (…) k cat / K m From the wild type 1.32 s - ¹·mM - ¹Increased to 1.93 s - ¹·mM - ¹, the improvement was as high as 46.2%. This result indicates that the present invention can significantly improve the technical defect of low catalytic efficiency of wild-type malic enzyme, thereby effectively increasing the generation rate of the target product.

[0022] The M144I+Q253I+T327F mutant obtained in this invention also exhibits excellent performance in terms of substrate affinity. K m The value decreased from 4.51 mM to 2.55 mM (a reduction of 43.5%). This improvement demonstrates that the mutant of this invention can maintain high catalytic efficiency under low CO2 concentration conditions, exhibiting effects that cannot be expected by existing technologies, thereby greatly expanding its application prospects in industrial production.

[0023] This invention employs a biocatalytic carbon fixation method, which, compared to traditional chemical catalysis processes, offers advantages such as being green and environmentally friendly, producing fewer reaction byproducts, and having a lower environmental burden, thus meeting the requirements of sustainable development. Furthermore, this invention not only reduces carbon dioxide emissions but also converts them into high-value-added chemicals, achieving a balance between environmental and economic benefits and demonstrating significant comprehensive advantages. Attached Figure Description

[0024] Figure 1 Top 30 fitness scores of malic acidase mutants predicted by four protein big language models.

[0025] Figure 2 This represents the three-dimensional structural distribution of malic acid oxidase mutation sites predicted based on a protein big language model.

[0026] Figure 3 The relative enzyme activities of 118 malic acidase mutants are shown.

[0027] Figure 4 Michaelis equation curves for wild-type and mutant M144I+Q253I+T327F malic acid oxidase.

[0028] Figure 5 Comparison of L-malate conversion rates between wild-type and mutant M144I+Q253I+T327F malate enzymes. Detailed Implementation

[0029] 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.

[0030] 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.

[0031] Reagents used in upstream genetic engineering: those used in the embodiments of this invention Dpn I was purchased from TaKaRa, Takara Bio Engineering (Dalian) Co., Ltd.; plasmid extraction kit and DNA recovery and purification kit were purchased from Axygen Hangzhou Co., Ltd. E. coli BL21(DE3) and plasmid pET28a-SUMO were purchased from Novagen; DNA markers, low molecular weight standard proteins, and agarose gel electrophoresis reagents were purchased from Beijing TransGen Biotech Co., Ltd.; primer synthesis and sequencing were performed by Qingke Biotechnology Co., Ltd. Refer to the product instructions for the usage of the above reagents.

[0032] 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.

[0033] Example 1: Design of Mutants This invention first constructs a complete system for designing malicase mutants, which 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. Firstly, AlphaFold 2.3.2 was used to predict the structure of malicase. The average pLDDT score for the predicted structure reached 92.99, indicating that the predicted structure has an accuracy close to that of experimental resolution.

[0034] 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.

[0035] Specifically, the four models adopted by this system and their characteristics are as follows: ESM2-650M: A protein language model developed by Meta AI based on the Transformer architecture. It was trained on over 60 million protein sequences from the UniRef50 database and serves as the current standard base model in the field, demonstrating outstanding sequence feature representation capabilities.

[0036] 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).

[0037] 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.

[0038] 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.

[0039] This invention employs the zero-shot prediction method of four protein big language models to calculate the fitness of each site in the malic enzyme sequence after replacing it with 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, and high-potential mutation sites are screened as candidates for subsequent experimental characterization.

[0040] like Figure 1As shown, four different protein big language models were used to predict the fitness scores of single-point mutants of malic acid oxidase. The top 25 mutation sites with the highest scores were selected for each model, and duplicate sites were removed (resulting in a total of 90 sites). These results indicate that different protein big language models differ in their training data, modeling objectives, and feature representation methods. This invention does not rely on the consistent prediction results of a single model, but rather expands the screening range of potential beneficial mutation sites through parallel prediction using multiple models, thereby reducing the impact of a single model on the mutation design results.

[0041] Based on predictions from four different protein big language models, high-scoring mutation sites were displayed in the three-dimensional structure of malicase ME (see [link]). Figure 2 These mutation sites are partly located in the active site and substrate / coenzyme binding region, and partly located in distal regions (such as conformational stability-related domains and other sites with unknown functions).

[0042] Meanwhile, based on the prediction results of four different protein big language models, this invention screened, constructed, and tested 90 malic acid oxidase mutants (see [link to relevant documentation]). Figure 3 Experimental results showed that, despite differences in the predictions of various models, a number of mutation sites that positively contribute to enzyme activity could still be effectively screened out.

[0043] Based on the predicted and experimental data of 90 malicase mutants, the parameters of the SaProt protein big language model were adjusted. The adjusted model was then used to predict the fitness scores of multi-site combination mutants, and the top 10 mutant sequences with the highest fitness scores were output as candidates for subsequent construction and characterization analysis. Through the above screening method, it was found that triple mutation combinations containing the M144I, Q253I, and T327F mutation sites had high fitness scores in the predicted multi-site mutant results, and are worthy of further experimental research as a potential direction for modification.

[0044] Example 2 Construction of mutant expression system Multi-site combinatorial mutations were performed on wild-type malicase. The specific method is as follows: Whole plasmid PCR: The wild-type plasmid was transferred to Qingke Biotechnology Co., Ltd. ME Gene fragments were synthesized in the MCS region of the pET-28a-SUMO plasmid. Bam H Ⅰ and Hind III. pET28a-SUMO- is obtained between the two restriction sites. ME plasmid, then with pET28a-SUMO- ME Using plasmids as templates, upstream and downstream primers covering the mutation sites were designed (Table 1) for whole-plasmid PCR, sequentially introducing the target mutation sites: Table 1. Primer Design for Malicase ME Mutants

[0045] Table 2 PCR Reaction Sample Addition System

[0046] Table 3 PCR reaction conditions

[0047] After PCR is complete, use QuickCut™ Dpn I. The GmATC sequence containing adenine methylation in the template is cleaved to remove template DNA. During this process, the PCR products are unaffected. The specific reaction system is as follows: Table 4 Dpn Ⅰ. Components of the digestion reaction system

[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, used for protein expression, are a widely used T7-expressing Escherichia coli strain. The specific transformation steps are 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 cells in the remaining medium, and gently spread evenly on a kanamycin-resistant plate using a sterile spreader; incubate upside down at 37 ℃ for 12-16 h.

[0050] through Dpn Enzyme I digestion and transformation yielded a recombinant plasmid containing a single-point mutation. Using a second pair of mutation primers, full-plasmid PCR was repeated. Dpn The first enzyme digestion and transformation step yields a recombinant plasmid containing a double mutation. Subsequently, using the double-mutant plasmid as a template, a third mutation is introduced using the same method, ultimately yielding a recombinant plasmid containing mutations at three sites: M144I, Q253I, and T327F.

[0051] The obtained recombinant plasmids were verified by sequencing to confirm the correctness of the target mutation site. Transformants that were verified to be correct could be used for subsequent induction expression experiments and preservation of the bacterial strain in glycerol tubes.

[0052] Example 3 Expression and purification of mutants The purification process for malic acid oxidase and its mutants is as follows: (1) Induced expression Thaw the glycerol tubes on ice, take 10-20 μL, streak on LB agar plates containing kanamycin resistance, and incubate at 37 °C for 12 h to activate.

[0053] 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 induction temperature 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.

[0054] (2) Protein purification The harvested cell pellet was resuspended in lysis buffer and sonicated on ice under the following conditions: Two probes were used. The enzyme was broken down for 2 seconds, paused for 3 seconds, and the power was set to 50-60% for a total duration of 10 minutes. After breaking down the enzyme, the supernatant containing soluble protein was collected by centrifugation at 12,000 rpm, which is the crude enzyme solution.

[0055] 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.

[0056] 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.

[0057] (3) SDS-PAGE electrophoresis verification During purification, crude enzyme, purified enzyme, and labeled purified enzyme are sampled separately. The SDS-PAGE electrophoresis buffer uses a pre-made gel (tear off the seal before use) and its accompanying buffer. An additional 180 kDa protein marker needs to be loaded into one well. Electrophoresis parameters: 120V, 400 mA, time 1 h, or until the bromophenol blue indicator in the loading buffer reaches the bottom of the gel.

[0058] Example 4 Enzyme activity and enzyme kinetics determination (1) Enzyme activity assay First, add 10 μL of NADH solution (70 mM) and 100 μL of enzyme solution (enzyme 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 mixture to each well of the microplate (4 wells). After the NADH reading stabilizes, add 2.5 μL each of pyruvate (250 mM) and sodium bicarbonate (250 mM). Incubate the reaction system at 30°C for 5 min, and then measure the absorbance change at 340 nm using a microplate reader. Using a pre-plotted NADH standard curve (concentration range 0-1000 μM), convert the measured absorbance values ​​to NADH concentration, thereby quantitatively analyzing the NADH production / consumption 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. Specifically, the absorbance (A) of NADH solutions at 340 nm wavelength was measured using a microplate reader, with NADH concentration as the x-axis. 340 The 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.

[0059] (2) Determination of enzyme kinetic constants To determine the kinetic parameters of malic acid oxidase, this study employed a two-substrate enzymatic reaction system, using a method of fixing one substrate concentration while varying the concentration of the other substrate to determine the Michaelis constant. K 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 The changed 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

[0060] After obtaining the reaction rates at different substrate concentrations, the maximum reaction rate was obtained by nonlinear fitting using the Michaelis-Menten equation. V max and 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.

[0061] Table 6 Kinetic parameters of each mutant

[0062] The results showed that the three single-point mutations, M144I, Q253I, and T327F, exhibited different characteristics from the wild-type malic acid enzyme in terms of catalytic efficiency and substrate affinity. The catalytic constant of the single mutant M144I (…) k cat ) from wild type 5.96 s - ¹Increased to 7.36 s - ¹ indicates that its catalytic rate is enhanced; however, its K m The concentration increased to 8.28 μM, leading to a decrease in substrate affinity; however, the catalytic rate of the single mutant Q253I further increased to 9.11 s. - ¹, but its K m The value increased significantly to 40.66 μM, indicating a significant decrease in substrate affinity; the single mutant T327F... K m The concentration decreased to 3.27 μM, a reduction of approximately 27.4% compared to the wild type, indicating higher substrate affinity, and its catalytic efficiency improved to 1.40 s⁻¹. - ¹·mM - ¹, slightly higher than wild type, indicating that the T327F mutation helps improve enzyme-substrate binding performance and achieves an overall improvement in catalytic efficiency (see [link to relevant documentation]). Figure 4 ).

[0063] The results of the three-site combination mutation showed that the M144I+Q253I+T327F mutant integrated the synergistic effects of multiple sites and also exhibited excellent performance in substrate affinity. K m The value decreased from 4.51 mM to 2.55 mM (a reduction of 43.5%). This improvement indicates that the mutant can maintain high catalytic efficiency under low CO2 conditions, exhibiting effects that cannot be expected with existing technologies, thus greatly expanding its application prospects in industrial production. Furthermore, the catalytic efficiency of the M144I+Q253I+T327F mutant (… k cat / K m From the wild type 1.32 s - ¹·mM - ¹Increased to 1.93 s - ¹·mM - ¹, the improvement was as high as 46.2%. This result indicates that the present invention can significantly improve the technical defect of low catalytic efficiency of wild-type malic enzyme, thereby effectively increasing the generation rate of the target product.

[0064] Furthermore, as shown in Table 6, the mutant fitness score obtained based on the protein big language model does not necessarily correspond to an improvement in the catalytic performance of the mutant in actual enzyme kinetic experiments. A variety of multi-site combination mutants were constructed and characterized. While some mutants achieved high scores in the prediction stage, they did not show superior catalytic performance compared to the wild type in actual enzyme activity and kinetic assays. For example, the mutants Q173T+Q253I+R388V and E11K+Q173T+Q253I are both multi-site combination mutants obtained through model prediction screening, but their catalytic efficiency (…) k cat / K m The values ​​were 0.1099 and 0.3742, respectively, which were significantly lower than the average catalytic efficiency of wild-type malic acid oxidase (1.3199).

[0065] In contrast, the three-site combination mutant M144I+Q253I+T327F obtained by screening in this invention showed significant consistency between the predicted score and experimental verification, and its catalytic efficiency ( k cat / K m The score reached 1.9324, which was higher than that of wild-type enzymes and other mutant combinations with high prediction scores but poor experimental results.

[0066] Example 5: Determination of L-malate conversion rate by malicase synthesis This invention provides a method for the production of L-malic acid from pyruvate using malic acid catalysis. 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 malic acid catalysis and its mutants.

[0067] (1) Establishment of the reaction system The reaction system was prepared according to the proportions shown in Table 7, and the total reaction volume was 1 mL. All reagents were pre-cooled on ice before the reaction.

[0068] Table 7. Reaction system for the production of L-malic acid catalyzed by malic acid oxidase.

[0069] 2) Reaction conditions The reaction steps are as follows: Add reaction buffer, MnCl2, NADH, and pyruvate sequentially to a 1.5 mL centrifuge tube, mix gently, and place in a 30 °C 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 in a 30 °C constant temperature metal bath shaker and gently shake at 300 rpm for 30 min.

[0070] 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.

[0071] (3) Reaction termination and sample processing After the reaction is complete, immediately heat the reaction tube at 100 °C for 5 min to inactivate the enzyme. Then centrifuge at 12000 rpm for 5-10 min to remove the protein precipitate. Filter the supernatant through a 0.22 μm filter membrane and store it in a liquid chromatography sample vial for later use.

[0072] (4) Detection of L-malic acid 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 partially overlapped at approximately 9.26 min, affecting the interpretation 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.

[0073] (5) Establishment of standard curve and calculation of content 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. Peak areas were recorded, and a standard curve was plotted. A linear regression equation was obtained by plotting L-malic acid concentration as the x-axis and peak area as the y-axis. y = 20670x - 397.51, R 2 =0.9999 Therefore, 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.

[0074] 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.

[0075] (6) Calculation of L-malic acid yield The reaction system uses pyruvate as the substrate, and the reaction equation is as follows: Pyruvate + CO2 + NADH → L-malic acid + NAD + (Molar ratio 1:1) The conversion rate of L-malic acid was calculated based on pyruvate. The formula is as follows: 1. Initial moles 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 ,0) × 100% Based on the test results, the pyruvate conversion rate of the wild-type malic acid oxidase was 16%, while the conversion rate of the mutant M144I+Q253I+T327F was 54% (see [link to study]). Figure 5 This indicates that the catalytic efficiency of the mutant is significantly higher than that of the wild-type enzyme.

[0076] 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.

[0077] sequence list SEQ ID NO: 1 Wild-type malic acid sequence: MDIQKRVSDMEPKTKKQRSLYIPYAGPVLLEFPLLNKGSAFSMEERRNFNLLGLLPEVVETIEEQAERAWIQYQGFKTEIDKHIYLRNIQDTNETLFYRLVNNHLDEMMPVIYTPTVGAACERFSEIYRRSRGVFISYQNRHNMDDILQNVPNHNIKVIVVTDGERILGLGDQGIGGMGIPIGKLSLYTACGGISPAYTLPVVLDVGTNNQQLLNDPLYMGWRNPRITDDEYYEFVDEFIQAVKQRWPDVLLQFEDFAQKNAMPLLNRYRNEICSFNDDIQGTAAVTVGTLIAASRAAGGQLSEKKIVFLGAGSAGCGIAEMIISQTQREGLSEEAARQKVFMVDRFGLLTDKMPNLLPFQTKLVQKRENLSDWDTDSDVLSLLDVVRNVKPDILIGVSGQTGLFTEEIIREMHKHCPRPIVMPLSNPTSRVEATPQDIIAWTEGNALVATGSPFNPVVWKDKIYPIAQCNNAFIFPGIGLGVIASGASRITDEMLMSASETLAQYSPLVLNGEGMVLPELKDIQKVSRAIAFAVGKMAQQQGVAVKTSAEALQQAIDDNFWQAEYRDYRRTSI SEQ ID NO: 2 Nucleic acid coding sequence corresponding to wild-type malic enzyme:

Claims

1. A malic acid oxidase mutant, characterized in that, The mutant was obtained by mutating the amino acid sequences at positions 144, 253, and 327 of SEQ ID NO: 1 as follows: M144I, Q253I, T327F.

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; 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 includes: (a) The host cells of any one of claims 4-7 are cultured under conditions suitable for the expression of the malicase mutant; and (b) Recover the malic acidase mutant.

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