Carbonyl reductase mutant with improved thermal stability

A mutant and reductase technology, applied in the fields of genetic engineering and enzyme engineering, can solve problems such as pollution and low efficiency, and achieve the effect of improving catalytic efficiency and reducing production costs

Pending Publication Date: 2022-07-22
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the methods of synthesizing chiral alcohols include chemical catalysis and biocatalysis, but the traditional chemical synthesis process has bottlenecks such as low efficiency and serious pollution;

Method used

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  • Carbonyl reductase mutant with improved thermal stability
  • Carbonyl reductase mutant with improved thermal stability
  • Carbonyl reductase mutant with improved thermal stability

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Example 1. Prediction and screening of mutation sites

[0015] A) The recombinant plasmid pET-28a(+)-ChKRED20 constructed by carbonyl reductase ChKRED20 was previously constructed, the nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2 (Chinese Patent, CN103497911A ).

[0016] B) By analyzing the amino acid sequence and three-dimensional structure of carbonyl reductase ChKRED20, the potential stable mutation sites were predicted and screened. The selected sites are shown in Table 1.

[0017] First, we used the amino acid sequence SEQ ID NO. 2 of the wild-type carbonyl reductase ChKRED20 as a probe to search for sequence similarity in the NCBI database, and selected homology with sequence similarity between 30-60% and 60-90%, respectively. Sequence, and then through ClusterW multiple sequence alignment to obtain the amino acids with a frequency of more than 50% at different positions, which are used as the prediction of the ...

Embodiment 2

[0024] Example 2. Stability analysis of carbonyl reductase single point mutants

[0025] Thermodynamic stability test: using differential scanning fluorescence method to measure the melting temperature of enzyme protein (T m ) to measure: 10× SYPRO Orange staining solution and 20 mM mutant pure enzyme were mixed, and then 100 mM potassium phosphate buffer pH 8.0 was added to make the volume of the whole system 20 μL. In the CFX96 real-time quantitative PCR instrument, the temperature was increased from 5 °C to 95 °C at a speed of 1 °C / min, and the melting temperature of the enzyme protein was determined by observing the change of fluorescence intensity during the whole heating process (T m ).

[0026] Kinetic stability test: The catalytic system was set up with 3,5-bis-trifluoromethylacetophenone as the substrate, and the catalytic activity of the enzyme to the substrate was determined. The reaction system was 1 mL, which contained 1.6 mg / mL of crude enzyme solution, 30 mM s...

Embodiment 3

[0028] Example 3. Combination of single point mutation sites and stability analysis of combined mutants

[0029] According to the differences in the thermodynamic or kinetic stability of the single-point mutation sites, they are divided into three groups: thermodynamically stable mutants, kinetically stable mutants, and mutants with both kinetics and thermodynamics stability. Then, the mutation sites within or between groups were combined to obtain 5 combined mutants with significantly improved stability, namely M3TK, M4T, M6K, M8K, and M9TK, which were characterized as follows:

[0030] M3TK: Glycine at position 104 was mutated to serine, leucine at position 112 was mutated to isoleucine, and glutamic acid at position 131 was mutated to leucine.

[0031] M4T: Glycine at position 104 was mutated to serine, leucine at position 112 was mutated to isoleucine, glutamic acid at position 131 was mutated to leucine, and serine at position 232 was mutated to alanine.

[0032] M6K: Al...

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Abstract

The invention belongs to the technical field of gene engineering and enzyme engineering, and particularly discloses a thermal-stability carbonyl reductase mutant with high catalytic activity. Compared with a wild type enzyme, the mutant enzyme can tolerate higher temperature, and has higher catalytic efficiency at high temperature. The thermal inactivation half-life period of the combined mutant M8K containing 8 mutation sites at the high temperature of 90 DEG C can reach 110 min, the catalytic activity of the combined mutant M8K at the temperature of 50-60 DEG C is higher than that of a wild type enzyme, the optimal catalytic efficiency is achieved at the temperature of 55 DEG C, and the combined mutant M8K has wide industrial application prospects.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and enzyme engineering, and particularly relates to a carbonyl reductase mutant with high heat resistance and its application in synthesizing chiral alcohol at high temperature. Background technique [0002] Chiral alcohols are important intermediates in the synthesis of chiral drugs. The current methods for synthesizing chiral alcohols include chemical catalysis and biocatalysis, but the traditional chemical synthesis process has bottleneck problems such as low efficiency and serious pollution; the biosynthesis process based on enzyme catalysis has the advantages of green environmental protection and good stereoselectivity, especially It has been widely used in the industrial production of innovative drugs. As biological macromolecules, enzymes are naturally in a mild environment and have limited resistance to external factors such as temperature and organic solvents. Therefore, impr...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12P7/22C12P41/00
CPCC12N9/0006C12Y101/01184C12P7/22C12P41/002
Inventor 吴中柳杨玉洁刘艳裴小琼
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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