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CN-121991907-A - Malatase mutant and application thereof

CN121991907ACN 121991907 ACN121991907 ACN 121991907ACN-121991907-A

Abstract

The invention relates to the technical field of enzyme engineering, in particular to a malic enzyme mutant and application thereof. Specifically, the invention performs site mutation screening on malic enzyme derived from ESCHERICHIA COLI, and as a result, the invention discovers that after methionine at 144 is mutated into isoleucine (M144I), glutamine at 253 is mutated into isoleucine (Q253I) and threonine at 327 is mutated into phenylalanine (T327F), the catalytic efficiency and catalytic activity are obviously improved, which has important significance for industrial application.

Inventors

  • LI KECHENG
  • Pu Zhongji
  • GUAN JIAMING
  • Lai Guanxue

Assignees

  • 上海微观纪元数字科技有限公司

Dates

Publication Date
20260508
Application Date
20260122

Claims (10)

  1. 1. A malic enzyme mutant, characterized in that the mutant is obtained by mutating the amino acid sequences at position 144, 253 and 327 shown in SEQ ID NO.1, wherein M144I, Q253 and I, T F are obtained.
  2. 2. A polynucleotide encoding the malic enzyme mutant according to claim 1.
  3. 3. A recombinant vector comprising the polynucleotide of claim 2.
  4. 4. A host cell comprising the polynucleotide of claim 2 or the recombinant vector of claim 3.
  5. 5. The host cell of claim 4, wherein the host cell is a fungal cell, a bacterial cell, or a plant cell.
  6. 6. The host cell of claim 5, wherein the host cell is a bacterial cell.
  7. 7. The host cell of claim 6, wherein the bacterial cell is an E.coli cell.
  8. 8. Use of a malic enzyme mutant according to claim 1 or a host cell according to any of claims 4-7 in the preparation of L-malic acid.
  9. 9. The use according to claim 8, wherein the pathway comprises contacting pyruvic acid with the malic enzyme mutant in a reaction system comprising CO 2 for carboxylation; Wherein the CO 2 is derived from sodium bicarbonate.
  10. 10. A method for producing a malic enzyme mutant according to claim 1, wherein the method comprises: (a) Culturing the host cell of any one of claims 4-7 under conditions suitable for expression of the malic enzyme mutant, and (B) Recovering the malic enzyme mutant.

Description

Malatase mutant and application thereof Technical Field The invention relates to the technical field of enzyme engineering, in particular to a malic enzyme mutant and application thereof in catalyzing CO 2 to fix and synthesize L-malic acid. Background How to efficiently capture and utilize carbon dioxide (CO 2) has become a key scientific issue in achieving carbon neutralization and sustainable development. Compared with the traditional modes such as physical adsorption or chemical fixation, the biological carbon fixation technology is widely paid attention to because of low energy consumption, environmental friendliness and capability of producing high-added-value products. Among the various biocarbon strategies, malic Enzyme (ME) has unique advantages. The enzyme can catalyze pyruvic acid and CO 2 to synthesize L-malic acid, thereby realizing efficient conversion and resource utilization of carbon dioxide. Compared with other carbon-fixing products, the L-malic acid is not only an important organic acid widely existing in the nature, but also has significant application value in the fields of food, medicine, chemical industry and the like. For example, L-malic acid is used as a sour agent and a preservative in the food industry, can improve flavor and prolong shelf life, is an important intermediate for synthesizing various medicines in the medicine field, and can be used as a raw material for preparing various functional chemicals in the chemical industry. Therefore, malic enzyme-based L-malate biosynthesis is considered as a green technical route with both environmental benefits and economic potential. However, naturally derived malic enzymes are still challenging in industrial applications, on the one hand, they have limited catalytic efficiency and are difficult to achieve high levels of L-malic acid synthesis, and on the other hand, they have insufficient environmental stability, are sensitive to temperature and pH variations, and limit their use in complex production systems. In order to address these problems, recent studies have gradually turned to the targeted engineering of ME by enzyme engineering. Traditional site-directed mutagenesis and directed evolution have advanced to some extent in improving enzyme performance, but have bottlenecks of long experimental period, low screening efficiency and the like. With the development of artificial intelligence and computational biology, molecular engineering strategies based on large protein language models (Protein Language Models, pLMs) provide new opportunities for enzyme performance optimization. The method can predict potential functional mutation sites based on large-scale sequence and structure information, thereby realizing more efficient enzyme design and transformation. The application of the emerging technology to optimize the malic enzyme is expected to obviously improve the catalytic activity and environmental adaptability, thereby promoting the improvement of the CO 2 carbon fixation efficiency and laying a foundation for the green synthesis of the L-malic acid. Disclosure of Invention The invention provides a malic enzyme mutant and application thereof, solves the problem of low catalytic efficiency of original malic enzyme derived from escherichia coli (ESCHERICHIA COLI) on pyruvic acid and carbon dioxide, and provides the malic enzyme mutant and application of the same in catalyzing CO 2 to be immobilized and synthesized to prepare L-malic acid. The invention adopts a zero sample prediction method of a protein large language model to evaluate adaptability (fitness) to malic enzyme (ME, NCBI accession number: NP-415996.1, the amino acid sequence of which is shown as SEQ ID NO:1, the nucleotide sequence of which is shown as SEQ ID NO: 2) derived from ESCHERICHIA COLI so as to identify mutation sites with potential function improvement. Four widely used large language models of proteins were used in the study, including ESM2, proGen, saProt, and ProSST, and 118 potential mutation sites were identified by comprehensive analysis. Based on the predicted results of these 118 mutation sites, the SaProt model was re-trimmed and the top 10 fitness scoring sequence of the multi-site combinatorial mutation was output for subsequent characterization analysis. Screening by the method shows that triple mutation combination M144I+Q253I+T327F (the 144 th methionine is mutated to isoleucine, the 253 th glutamine is mutated to isoleucine and the 327 th threonine is mutated to phenylalanine) shows prominence in the adaptability score, and the prediction result shows that the mutation combination has the potential of remarkably improving the catalytic activity of enzyme. Further, experiments prove that not all the enzyme mutants with high model prediction scores have improved catalytic performance in practical enzyme dynamics experiments, but the catalytic activity of the M144I+Q253I+T327F mutant is found to be 1.46 times that of a wild type, namely, the