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Mutant aspartate aminotransferase and its preparing process and application

A technology of aspartate transaminase and aspartate transaminase, which is applied in the field of aspartate transaminase, can solve the problems of reduced activity of dicarboxylate substrates and reduced transamination activity, and achieve low production cost, good effect, and high thermal efficiency. The effect of stability

Inactive Publication Date: 2007-06-27
NANJING UNIV OF TECH
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[19] 3 W140H, I17H and V37H The transamination activity of aromatic amino acids was reduced approximately 10-100 times, however, for two The activity of carboxyamino acid substrates was reduced by 20% to 60%, respectively
[20] 4 W140 / F / G Greatly reduced activity towards dicarboxylate substrates

Method used

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  • Mutant aspartate aminotransferase and its preparing process and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Improvement of aspartate aminotransferase activity and substrate specificity

[0050] 1. Determine the mutation site: use a rational design method to connect the three-dimensional structure and the one-dimensional structure of Escherichia coli aspartate aminotransferase (eAspAT) through sequence analysis software (Clustal W) and structure analysis software (Rasmol), according to The comparison of the amino acid sequence of the enzyme and the aspartate aminotransferase of the thermophilic bacteria and the comparison of the substrate specificity of the two enzymes (the substrate specificity of the enzyme in Escherichia coli and thermophilic bacteria are also different) using the software DeepView to select mutations The position and the amino acid residue of replacement determine that the amino acid residue phe at the 338th position in the Escherichia coli aspartate aminotransferase sequence (SEQ IN No.2) is closely related to the activity and substrate specific...

Embodiment 2

[0078] Embodiment 2: the improvement of aspartate aminotransferase thermostability

[0079] 1. Using a rational design method, the three-dimensional structure and one-dimensional structure of Escherichia coli aspartate aminotransferase were linked by sequence analysis software (Clustal W) and structure analysis software (Rasmol), and the software DeepView was used to determine the aspartate aminotransferase of Escherichia coli. The 221st amino acid residue Leu in the acid transaminase sequence is closely related to the stability of the enzyme, and replacing the amino acid residue at this position with an Asn residue can improve the thermostability of the enzyme.

[0080] 2. The establishment of the mutant gene library includes the following steps:

[0081] (1) Insert the Escherichia coli aspartate aminotransferase gene into pUC18 to construct a recombinant plasmid, and the corresponding genetically engineered bacterium is named A2, and the construction route is shown in FIG. 1...

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Abstract

The present invention discloses mutant aspartate aminotransferase and its preparation process and application. The mutant aspartate aminotransferase has the amino acid sequence as shown in SEQ IN No.2 with the 221st position of substituted Leu by Asn or the 338th position mutated from Phe to Lys. The mutant aspartate aminotransferase has raised biological function and is used in producing L-phenylalanine.

Description

technical field [0001] The invention relates to aspartate aminotransferase, in particular to a mutant enzyme of aspartate aminotransferase, and also relates to a preparation method and application of the mutant enzyme. Background technique [0002] Rational design technology is the process of designing a new amino acid sequence based on the relationship between protein structure and function, and then using recombinant DNA technology to change the primary sequence of the protein, modifying the natural protein or designing a new protein according to actual needs. Based on the knowledge of catalytic chemistry and structural chemistry, these studies have an in-depth understanding of the fine structure and functional relationship of proteins, and then use the principles and techniques of genetic engineering to carry out protein transformation. [0003] In the rational design of enzymes, it is necessary to design the enzyme molecules to be modified based on the information of the...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12P13/22
Inventor 许琳严明林涛张宏志
Owner NANJING UNIV OF TECH
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