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Esterase mutant and its application

A kind of mutant, esterase technology, applied to the esterase mutant and its application field, can solve the problems of unsatisfactory reactivity, stability and selectivity, etc.

Active Publication Date: 2021-04-27
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 its application
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  • Esterase mutant and its application

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 at 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

[0043] 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. Set up the same system reaction and processing methods. The results are shown in Table 1.

[0044] Table 1

[0045]

[0046]

[0047] 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.

[0048] The ee value is less th...

Embodiment 2

[0050] 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 min, 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 methods of the same system are established. The results are shown in Table 2.

[0051] Table 2

[0052]

[0053]

[0054] 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.

[0055] The ee value is less than...

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Abstract

The invention discloses an esterase mutant and application thereof. Wherein, the esterase mutant has an amino acid mutation sequence shown in SEQ ID NO: 1, and the amino acid mutation site includes N51G site. The esterase mutant of the present invention is based on the esterase shown in SEQ ID NO: 1, and is mutated by site-directed mutation, so as to change its amino acid sequence, realize the change of protein structure and function, and then pass directional screening Method, obtain the esterase with above-mentioned mutation site, therefore these esterase mutants have the advantage that enzyme specificity improves greatly, and enzyme activity also has corresponding improvement, thereby greatly reduces the usage amount of enzyme, reduces the industrial production the cost of.

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 Patents(China)
IPC IPC(8): C12N9/18C12N15/55C12N15/70C12N15/81C12N1/21C12N1/19C12P7/40C12R1/19
CPCC12N9/18C12N15/70C12N15/81C12P7/40C12N15/52C12Y301/01
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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