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Cytochrome P450 enzyme mutant and application thereof

A cytochrome, mutant technology, applied in applications, enzymes, bacteria, etc., to achieve the effect of improving activity and selectivity

Active Publication Date: 2021-02-12
ASYMCHEM LIFE SCI TIANJIN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The main purpose of the present invention is to provide a cytochrome P450 enzyme mutant and its application to solve the problem that there is no P450 enzyme in the prior art that can efficiently catalyze olefinic compounds to carry out anti-Markov oxidation reaction.

Method used

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  • Cytochrome P450 enzyme mutant and application thereof
  • Cytochrome P450 enzyme mutant and application thereof
  • Cytochrome P450 enzyme mutant and application thereof

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preparation example Construction

[0051] In a typical embodiment of the present invention, there is also provided a method for preparing a carbonyl compound or an alcohol compound, the preparation method comprising: using any of the above-mentioned cytochrome P450 enzyme mutants to catalyze the alkene compound Carries out direct reverse Markov oxidation to carbonyl compounds or alcohol compounds , wherein R represents an optionally substituted or unsubstituted alkyl group, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group.

[0052] Preferably, R represents an optionally substituted or unsubstituted alkyl group, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group with 1-20 carbon atoms, more preferably Yes, R represents an optionally substituted or unsubstituted alkyl group, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl...

Embodiment 1

[0066] Add 1.5 mM raw material 1, raw material 2, raw material 3 and raw material 4 to a 10 mL glass bottle, add 1 eq NADP + , 20eq glucose, 3 wt glucose dehydrogenase, 0.1 g P450 enzyme, make up to 4 mL with Tris-HCl buffer (pH 8.0, 100 mM), mix well, and react at 30°C, 200 rpm shaker for 3h. After the reaction, add 2 mL of ethyl acetate, mix well and then centrifuge at 12,000 rpm for 5 min, take the upper layer, and detect it by HPLC with a wavelength of 210 nm. The response characteristics of some mutants are shown in Table 1.

[0067] Table 1:

[0068]

[0069] The above female parent is SEQ ID NO: 1; the multiples of activity and selectivity improvement are represented by +, + means an increase of 0-1 times, ++ means an increase of 1-2 times, +++ means an increase of 2-3 times, ++ ++ indicates an increase of 3-5 times, and +++++ indicates an increase of 5-10 times. Selectivity in the present invention / selectivity of anti-Markov oxidation is defined as: aldehyde prod...

Embodiment 2

[0071] Add 1.5 mM raw material 1, raw material 2, raw material 3 and raw material 4 to a 10 mL glass bottle, add 1 eq NADP + , 20eq glucose, 3 wt glucose dehydrogenase, 0.1 g P450 enzyme, make up to 4 mL with Tris-HCl buffer (pH 8.0, 100 mM), mix well, and react at 30°C, 200 rpm shaker for 3h. After the reaction, add 2 mL of ethyl acetate, mix well and then centrifuge at 12,000 rpm for 5 min, take the upper layer, and detect it by HPLC with a wavelength of 210 nm. The response characteristics of some mutants are shown in Table 2.

[0072] Table 2:

[0073]

[0074] The above female parent is SEQ ID NO: 1; the multiples of activity and selectivity improvement are represented by +, + means an increase of 0-1 times, ++ means an increase of 1-2 times, +++ means an increase of 2-3 times, ++ ++ indicates an increase of 3-5 times, and +++++ indicates an increase of 5-10 times.

[0075] It can be seen from the results in Table 2 that the directed evolution method using random mu...

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Abstract

The invention provides a cytochrome P450 enzyme mutant and application thereof. Protein modification is carried out on P450 enzyme activity and reverse Markov oxidation selectivity from a Bacillus megaterium wild type strain by means of directed evolution, so that the enzyme activity and selectivity are improved, and a series of P450 enzyme mutants capable of being used for industrial production are developed.

Description

technical field [0001] The invention relates to the technical field of enzymes, in particular to a cytochrome P450 enzyme mutant and its application. Background technique [0002] Catalytic anti-Markov oxidation reaction of olefin raw materials can simplify the synthetic route of many important chemical raw materials. The direct anti-Markov oxidation of alkenes to the corresponding carbonyl compounds is a very important challenge in organic synthesis, and this process often requires the participation of efficient catalysts. In the current synthesis methods, the use of noble metals as catalysts has low conversion efficiency and enantioselectivity, requires multi-step catalysis, and has many wastes [G. Dong, P.Teo, Z. K. Wickens, R. H. Grubbs, Primary alcohols from terminal olefins: Formal anti-Markovnikov hydration via triple relay catalysis. Science 333, 1609–1612(2011)]. [0003] Cytochrome P450 monooxygenases (P450s) are a family of heme-dependent enzymes that use oxygen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N1/21C12P7/24C12P7/02C12R1/19
CPCC12N9/0081C12Y114/15006C12N15/70C12P7/24C12P7/02C07K14/415C12N9/0071C12Y111/02004
Inventor 洪浩詹姆斯·盖吉张娜焦学成马玉磊曹珊程逸冰孙凯华
Owner ASYMCHEM LIFE SCI TIANJIN
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