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Esterase mutant and application thereof

A technology of mutants and esterases, applied in the fields of esterase mutants and their applications, can solve problems such as unsatisfactory reactivity, stability and selectivity, and achieve the goals of improving enzyme specificity, reducing costs, and improving enzyme activity Effect

Active Publication Date: 2021-03-02
ASYMCHEM LIFE SCI TIANJIN +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although wild-type biocatalysts usually have good reactivity and selectivity for their natural substrates, their reactivity, stability and selectivity for unnatural substrates are often unsatisfactory

Method used

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  • Esterase mutant and application thereof
  • Esterase mutant and application thereof
  • Esterase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0032] According to a typical embodiment of the present invention, a recombinant plasmid is provided. The recombinant plasmid contains any one of the above DNA molecules. The DNA molecules in the above recombinant plasmids are placed in appropriate positions of the recombinant plasmids, so that the above DNA molecules can be replicated, transcribed or expressed correctly and smoothly.

[0033] Although the qualifier used in the present invention is "contains" when limiting the above-mentioned DNA molecule, it does not mean that other sequences irrelevant to its function can be arbitrarily added to both ends of the DNA sequence. Those skilled in the art know that in order to meet the requirements of recombination operations, it is necessary to add suitable restriction endonuclease cutting sites at both ends of the DNA sequence, or additionally add start codons, stop codons, etc., therefore, if using A closed-ended statement will not truly cover these situations.

[0034]The t...

Embodiment 1

[0044] Add 20 mg of substrate 1, 1 mL reaction system, 2 mg esterase, 0.3 M potassium phosphate buffer pH 7.5. 30 o After reacting in C for 16 h, add 50 μL of 6 M HCl to the 1 mL reaction system, the pH is between 2 and 3, mix well, add 2 mL of ethyl acetate, shake well, centrifuge at 12000 rpm for 3 min, and take the Add an appropriate amount of anhydrous magnesium sulfate to the supernatant, centrifuge at 12,000 rpm for 3 minutes, take the supernatant for gas phase detection, and detect the conversion rate and e.e. value. Substrate 2 and substrate 3 are as described in substrate 1, and the reaction and processing method. The results are shown in Table 1.

[0045] Table 1

[0046]

[0047]

[0048] Compared with the mother parent, the activity is reduced and increased by 10-50 times, --- by 5-10 times, - by 1-5 times, + by 1-5 times, ++ by 5-10 times, +++ increased by 10-50 times, ++++ increased by more than 50 times.

[0049] The ee value is less than 0%*, the ee v...

Embodiment 2

[0051] Add 20 mg of substrate 4, 1 mL reaction system, 2 mg esterase, 0.3 M potassium phosphate buffer pH 7.5. 30 o After reacting in C for 16 h, add 50 μL of 6 M HCl to the 1 mL reaction system, the pH is between 2 and 3, mix well, add 2 mL of ethyl acetate, shake well, centrifuge at 12000 rpm for 3 min, and take the Add an appropriate amount of anhydrous magnesium sulfate to the supernatant, centrifuge at 12,000 rpm for 3 minutes, take the supernatant for gas phase detection, and detect the conversion rate and e.e. value. Substrate 5 is as described for substrate 4, and the reaction and treatment of the same system are established. The results are shown in Table 2.

[0052] Table 2

[0053]

[0054]

[0055] Compared with the mother parent, the activity is reduced and increased by 10-50 times, --- by 5-10 times, - by 1-5 times, + by 1-5 times, ++ by 5-10 times, +++ increased by 10-50 times, ++++ increased by more than 50 times.

[0056] The ee value is less than 0%*...

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Abstract

The invention discloses an esterase mutant and application thereof. The esterase mutant has a sequence with amino acid mutation of a sequence shown in SEQ ID NO: 1, and sites with amino acid mutationcomprise an N51G site. According to the esterase mutant, on the basis of esterase shown in SEQ ID NO: 1, mutation is conducted through a site-specific mutagenesis method, so the amino acid sequence ofthe esterase mutant is changed, the structure and function of protein are changed, and esterase with the mutation site is obtained through a directional screening method; therefore, the esterase mutants have the advantage that the enzyme specificity is greatly improved, and the enzyme activity is correspondingly improved, so the use amount of enzyme is greatly reduced, and the cost in industrialproduction is reduced.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an esterase mutant and its application. Background technique [0002] With the development of medicine, pesticide and other fine chemical industries, organic synthesis is facing more and more challenges. First of all, due to the chiral recognition ability in organisms, only one stereoisomer in a drug molecule often has a therapeutic effect, while the other stereoisomer has no therapeutic effect or even has side effects. Secondly, small production batches and high value-added products such as pharmaceutical products have structural complexity and diversity. If the production process is chemically and regioselective, unnecessary protection and deprotection steps can be avoided (in the traditional In organic synthesis, protection and deprotection steps are usually introduced to make up for the lack of reaction chemistry and regioselectivity), greatly optimizing the production process, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/18C12N15/55C12N15/70C12N15/81C12N1/21C12N1/19C12P7/40C12R1/19
CPCC12N9/18C12N15/70C12N15/81C12P7/40C12Y301/01C12P41/005
Inventor 洪浩詹姆斯·盖吉肖毅张娜焦学成杨益明王翔赵军旗
Owner ASYMCHEM LIFE SCI TIANJIN
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