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Aspartate-β-semialdehyde dehydrogenase mutant and its application

A semialdehyde dehydrogenase and aspartic acid technology, applied in the field of aspartate-β-semialdehyde dehydrogenase mutants, can solve the problems of affecting the rapid growth of bacterial cells, energy consumption and cell metabolic pressure, etc.

Active Publication Date: 2019-12-27
风火轮(上海)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the overexpression of transhydrogenase in engineering bacteria also has certain defects. The overexpression of transhydrogenase will bring energy consumption and cell metabolic pressure, which will affect the rapid growth of bacteria.

Method used

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  • Aspartate-β-semialdehyde dehydrogenase mutant and its application
  • Aspartate-β-semialdehyde dehydrogenase mutant and its application
  • Aspartate-β-semialdehyde dehydrogenase mutant and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0125] Example 1, construction of a single-point saturation mutation library

[0126] Using ASADH derived from E.coli, the asd gene was amplified from the genome of MG1655, and 6 His were added to its N-terminus, and then linked into pET28a vector for expression. The wild-type ASADH derived from Escherichia coli was obtained by purifying with a nickel column.

[0127] The three-dimensional structure of ASADH protein derived from Escherichia coli was searched through the RCSB PDB database, and two different forms of ASADH were retrieved from the data, including the open form and the closed form. The structure whose PDB ID is 1GL3 is a three-dimensional complex of ASADH, NADP and its substrate analogs, and this structure is used as the basis for subsequent analysis.

[0128] Use Molegro Molecular Viewer (Thomsen, R. et al., Journal of medicinalchemistry 49, 3315-3321) to analyze the crystal structure of ASADH (ecASADH) derived from Escherichia coli, select NADP + binding activ...

Embodiment 2

[0131] Example 2. Identification of cofactor-dependent enzymatic properties of ecASADH-Q350N and ecASADH-Q350N / H171A

[0132] NAD + The two mutant proteins with the highest -dependent enzyme activity were tested for their enzymatic properties, and the results are shown in Table 2. The results of the assay showed that the two mutant proteins had an effect on NAD + The affinity of K m values ​​are reduced by a factor of 4.6 and 35, respectively. At the same time, the catalytic efficiency was improved, compared with the wild-type protein, the effect on NAD + conversion number k cat The values ​​are increased by 9 and 12 times, respectively.

[0133] NADP + The affinity of the binding mutant protein to NADP was also greatly improved + The decline of catalytic performance, the inventor speculates that after protein mutation, it strengthens the ability to NADP + binding force, leading to NADP + The dissociation becomes rate-limiting, resulting in decreased responsiveness. ...

Embodiment 3

[0136] Embodiment 3, wild-type ecASADH and mutant protein pair cofactor NADP + Determination of binding capacity

[0137] NADP was determined by isothermal titration calorimetry (ITC) + Dissociation constant k with ecASADH, ecASADH-Q350N, ecASADH-Q350N / H171A d . The result is as Figure 4 , it can be seen from the change of the dissociation constant, which is consistent with the enzymatic parameters, and the protein is mutated with NADP + The binding force has been greatly improved. Excessively high binding force may lead to the slow dissociation of the product NADPH and affect the ability of NADP + catalytic ability.

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Abstract

The invention relates to an aspartic acid-beta-semialdehyde dehydrogenase (ASADH) mutant and applications thereof. The invention provides an ASADH mutant that can effectively utilize NAD(H) and a synthesis route that can be used to establish NAD(H) dependent amino acids of aspartic acid family. The problem that cofactor is imbalance in engineering bacteria is solved therefore.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically, the invention relates to aspartic acid-β-semialdehyde dehydrogenase mutants and applications thereof. Background technique [0002] According to dietary requirements, 20 protein amino acids can be divided into non-essential amino acids and essential amino acids. Essential amino acids cannot be synthesized by humans and animals, and can only be obtained through diet. The four amino acids in the aspartic acid family are all essential amino acids and play an important role in the amino acid industry. [0003] Aspartic acid family amino acids include L-lysine, L-methionine, L-threonine and L-isoleucine, which are important components of the nine essential amino acids for humans and animals, and are used as additive ingredients Widely used in the fields of feed, food, medicine and cosmetics, the huge market size makes its production technology progress attracts attention. The aspa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N15/53C12P13/04
CPCC12N9/0008C12P13/04C12Y102/01011
Inventor 杨晟徐晓庶
Owner 风火轮(上海)生物科技有限公司
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