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Styrene monooxygenase mutant with improved enzyme activity and application thereof

A technology of monooxygenase and styrene, which is applied in the fields of genetic engineering and microbial engineering, can solve the problems of low yield of styrene monooxygenase and limit the industrialization process of synthetic chiral epoxy compounds, and achieve temperature stability and pH The effect of stability improvement

Active Publication Date: 2019-02-22
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, since the production of styrene monooxygenase depends on NADH, it is difficult for styrene monooxygenase producing strains such as Pseudomonas putida, Pseudomonasputida, Pseudomonas sp, Pseudomonas fluorescens to produce styrene monooxygenase in large quantities, so that styrene Monooxygenase has the defect of low yield, which greatly limits the industrialization of biocatalytic synthesis of chiral epoxy compounds

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1: Construction of the recombinant vector containing styrene monooxygenase mutant

[0052] Specific steps are as follows:

[0053] (1) Obtaining the D305G mutant: using the nucleotide sequence shown in SEQ ID NO.4 as a template, Fprimer (sequence shown in SEQ ID NO.5), Rprimer (sequence shown in SEQ ID NO.6) As a primer, perform PCR to obtain the recombinant gene A shown in SEQ ID NO.3;

[0054] (2) Obtaining of the D305L mutant: using the nucleotide sequence shown in SEQ ID NO.4 as a template, Fprimer (sequence shown in SEQ ID NO.7), Rprimer (sequence shown in SEQ ID NO.8) As a primer, carry out PCR to obtain the recombinant gene B;

[0055] (3) Obtaining of the D305T mutant: using the nucleotide sequence shown in SEQ ID NO.4 as a template, Fprimer (sequence shown in SEQ ID NO.9), Rprimer (sequence shown in SEQ ID NO.10) As a primer, carry out PCR to obtain the recombinant gene C;

[0056] (4) Recombinant genes A-C and pETDute-1 were digested with BamH I...

Embodiment 2

[0057] Embodiment 2: the construction of the Escherichia coli engineering bacterium that produces styrene monooxygenase mutant

[0058] The recombinant plasmids pETDute-1-D305G, pETDute-1-D305L and pETDute-1-D305T obtained in Example 1 were respectively transformed into E.coli BL21 competent cells. The specific steps are as follows:

[0059] (1) Pick a single colony of Escherichia coli after activation on the LB plate (made from LB solid medium) and inoculate it in 10 mL of fresh LB liquid culture medium, and culture it at 37°C for 12 hours to obtain the seed solution;

[0060] (2) Inoculate the seed liquid into 50mL of fresh medium according to the inoculum amount of 1%, shake and culture at 37°C and 180rpm for 1.5h-2h, until OD 600 When it reaches about 0.5, the bacterial liquid is obtained;

[0061] (3) CaCl 2 The solution (sterilized) was pre-cooled on ice for 15 minutes;

[0062] (4) Divide 100mL of the bacterial solution into four 50mL centrifuge tubes and cool on ice...

Embodiment 3

[0067] Embodiment 3: the mensuration of styrene monooxygenase mutant enzyme activity

[0068] Specific steps are as follows:

[0069] The recombinant bacteria E.coli / pETDute-1-D305G, E.coli / pETDute-1-D305L and E.coli / pETDute-1-D305T constructed in Example 2 were compared with the control strain E.coli / pETDute-1-D305T expressing unmutated enzymes pETDute-1-SMO was respectively inoculated in 10 mL of LB medium containing ampicillin, and cultured with shaking at 37°C for 12 hours to obtain seed liquid; the seed liquid was transferred to E. Cultivate for 4 hours to obtain a fermentation broth; take the fermentation broth and centrifuge at 4°C and 10,000 r / min for 20 minutes, and the supernatant of the broken cells is the intracellular crude enzyme solution, which is then purified by a column to obtain a pure enzyme solution for the determination of enzyme activity.

[0070] The test results are as follows: the enzyme activity of mutant D305G is 99.12±1.5U / mL, which is 1.7 times h...

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Abstract

The invention discloses a styrene monooxygenase mutant with improved enzyme activity and application thereof, and belongs to the technical fields of gene engineering and microorganism engineering. Themutant is obtained by mutating amino acid, at a 305 position, of styrene monooxygenase with an initial amino acid sequence of SEQ ID NO.1 from aspartic acid to glycine; the enzyme activity of the mutant can be up to 99.12+ / -1.5 U / mL, and Kcat / Km can be up to 268.2+ / -19.5 mM<-1>*min<-1>, wherein the enzyme activity and the Kcat / Km are improved by 1.7 and 3.4 times respectively compared with thoseof a wild type; the temperature stability and the pH stability of the mutant are obviously improved compared with those of the wild type, by maintaining the mutant at 60 DEG C for 24 h, 59% of the enzyme activity can still remain, and in a state of a pH of 5.0, 71% of the enzyme activity can still remain when the mutant is maintained for 12 h.

Description

technical field [0001] The invention relates to a mutant of styrene monooxygenase with improved enzyme activity and application thereof, belonging to the technical fields of genetic engineering and microbial engineering. Background technique [0002] Epoxy compounds are a class of compounds with a three-membered cyclic ether structure. Because they can undergo ring-opening reactions with various reagents, they can be used as important precursors for the synthesis of many optically active drugs, pesticides and some fine chemicals. Organic synthesis, pharmaceutical industry, perfume industry and other fields have important applications. [0003] The traditional production of chiral epoxy compounds mostly adopts the chemical catalysis method. However, due to the chemical catalysis method, the crystalline titanosilicate catalyst with the MFI structure needs to be prepared before it can be in the liquid phase. At the same time, the noble metal catalyst and the MFI structure The ...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N1/21C12P17/02
CPCC12N9/0071C12N15/70C12P17/02C12Y114/14011
Inventor 饶志明谭春林张显杨套伟徐美娟
Owner JIANGNAN UNIV
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