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Monooxygenase mutant and application thereof

A technology of monooxygenase and mutants, applied in the direction of application, oxidoreductase, and introduction of foreign genetic material using vectors, etc., can solve the problems of low enzyme activity and poor selectivity of monooxygenase

Active Publication Date: 2019-07-26
JINLIN ASYMCHEM PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The present invention aims to provide a monooxygenase mutant and its application to solve the technical problems of poor monooxygenase selectivity and low enzyme activity in the prior art

Method used

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  • Monooxygenase mutant and application thereof

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Embodiment approach

[0050] 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.

[0051] 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.

[0052] The ...

Embodiment 1

[0066] Comparison of Reaction Properties of (R,R)-1-Oxo-3-Hydroxytetrahydrothiophene Prepared by Site-directed Mutagenesis of Monooxygenase

[0067] In a 50mL glass conical flask, add 100mg (R)-3-hydroxytetrahydrothiophene to 850μL isopropanol, mix well, adjust the pH to 6.0-9.0, then add 800mg of monooxygenase, isopropanol Dehydrogenase 0.12g, 50μL (20mg / mL) NADP + , in the crude enzyme solution of 0.1M Tris-HCl buffer, the total reaction volume is 10mL, the system pH is 6.0-9.0, and the reaction is carried out in a constant temperature shaker flask at 15°C-30°C. After 16 hours, take 700 μL of the reaction system, add 1.4 mL of absolute ethanol, centrifuge at 12,000 rpm for 5 min, add 0.5 g of anhydrous MgSO to the supernatant 4 Remove water, centrifuge at 12000rpm for 5min, take supernatant and use N 2 After drying, re-dissolve with 700 μL absolute ethanol and send to GC for analysis. The response characteristics of some mutants are shown in Table 1:

[0068] Table 1

...

Embodiment 2

[0073] Comparison of Reaction Properties of (R,R)-1-Oxo-3-Hydroxytetrahydrothiophene Prepared by Combined Mutation of Monooxygenase

[0074] In a 50mL glass conical flask, add 100mg (R)-3-hydroxytetrahydrothiophene to 850μL isopropanol, mix well, adjust the pH to 6.0-9.0, then add 800mg of monooxygenase, isopropanol Dehydrogenase 0.12g, 50μL (20mg / mL) NADP + , in the crude enzyme solution of 0.1M Tris-HCl buffer, the total reaction volume is 10mL, the system pH is 6.0-9.0, and the reaction is carried out in a constant temperature shaker flask at 15°C-30°C. After 16 hours, take 700 μL of the reaction system, add 1.4 mL of absolute ethanol, centrifuge at 12,000 rpm for 5 min, add 0.5 g of anhydrous MgSO to the supernatant 4 Remove water, centrifuge at 12000rpm for 5min, take supernatant and use N 2 After drying, re-dissolve with 700 μL absolute ethanol and send to GC for analysis. The response characteristics of some mutants are shown in Table 2:

[0075] Table 2

[0076] ...

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Abstract

The invention discloses a monooxygenase mutant and application thereof. The amino acid sequence of the monooxygenase mutant is the amino acid sequence obtained by mutating the amino acid sequence shown in SEQ ID NO: 1, and the mutation includes at least one of the following mutation loci: 49th, 60th, 61st, 144th, 145th, 146th, 147th, 167th, 169th, 189th, 246th, 247th, 280th, 284th, 285th, 286th, 287th, 328th, 330th, 332nd, 382rd, 427th, 428th, 429th, 430th, 431st, 432nd and the like. The monooxygenase mutant has the advantage of greatly increasing stereoselectivity and improving the enzyme activity correspondingly.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a monooxygenase mutant and its application. Background technique [0002] Most of the traditional chemical oxidants are toxic and / or explosive, and their stereoselectivity is very low. For the sake of environmental friendliness and the need to synthesize drugs and agricultural chemicals with high optical purity, people are constantly developing new oxidants. Cyclohexanone monooxygenase (CHMO) is a reduced coenzyme I (NADPH)-dependent oxidase that catalyzes the oxidation of ketones, aldehydes, and some sulfides and selenides. Monooxygenases are widely used in organic synthesis and have good selectivity, controllability and economy. For the synthesis of chiral drugs, usually the products have different configurations, and their functions and toxicity vary greatly. Therefore, obtaining chiral compounds with high optical purity is very important in pharmaceutical research and developmen...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/70C12P17/00
CPCC12N9/0004C12N15/70C12P17/00
Inventor 洪浩詹姆斯·盖吉卢江平张娜马玉磊程逸冰牟慧艳
Owner JINLIN ASYMCHEM PHARM CO LTD
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