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Bacterial strain, amidohydrolase and encoding gene with single conversion function to S-napropamide and application thereof

A technology of amidohydrolase and napropamide, applied in the field of bioengineering, has achieved important theoretical value and application prospects

Active Publication Date: 2019-03-08
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no reports on the strains, enzymes and genes that have chiral isomer stereoselectivity for napropamide

Method used

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  • Bacterial strain, amidohydrolase and encoding gene with single conversion function to S-napropamide and application thereof
  • Bacterial strain, amidohydrolase and encoding gene with single conversion function to S-napropamide and application thereof
  • Bacterial strain, amidohydrolase and encoding gene with single conversion function to S-napropamide and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0031] 1.1 Isolation and screening of napropamide-degrading strain B2

[0032] Activated sludge was collected from a pesticide factory that produced napropamide technical in Nantong, Jiangsu. Take 4.0g of activated sludge and add it to 100mL basal salt medium, add Rac-Napropamide with a final concentration of 0.2mM, culture at 30°C and 180rpm / min for 6 days, and transfer with an inoculum size of 5% (V / V) Into fresh sterile basal salt medium, transfer twice in a row, use UV spectrophotometer and high performance liquid chromatography to detect the content of napropamide in the third-generation enrichment solution and whether there are new metabolites generated . Gradiently dilute the enrichment solution with degradation effect, take 10 -2 to 10 -5 Each 100 μL of gradient dilution enrichment solution was spread on 1 / 10LB solid medium with 0.8mM Rac-Napropamide, cultured at 30°C for 10 days, picked a single colony on the plate and cultured in LB test tube until exponential pha...

Embodiment 2

[0044] Cloning and functional verification of S-napropamide degradation gene snaH

[0045] 2.1 Sequencing analysis of bacterial genome total DNA

[0046] 2.1.1 Extraction of bacterial genome total DNA and analysis of sequencing results

[0047] The total genome DNA of strain B2 was extracted by the CTAB method, and the total genome DNA was dissolved in sterile ultrapure water and sent to Shenzhen Huada Gene Co., Ltd. for complete map sequencing of the bacterial genome. The complete genome map information of strain B2 is: the base number of the whole genome is 4078932bp, which contains 1 chromosome and 6 plasmids, and the G+C content is 62.41%. In the genome prediction analysis results, we searched with amidohydrolase as the key word, found 11 orfs annotated as amidohydrolase, compared and analyzed each orf on NCBI, and further verified the function of 3 amidohydrolase.

[0048] 2.1.2 Heterologous expression and functional verification of putative amidohydrolase genes

[004...

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Abstract

The invention discloses a bacterial strain, an amidohydrolase and an encoding gene with single conversion function to S-napropamide and application thereof. The bacteria strain Sphingobium sp.B2 for degrading napropamide is stored with number of CCTCC NO: M2018684 in China Center for Type Culture Collection on October 16, 2018. The amidohydrolase gene snaH with single conversion function to S-napropamide has the nucleotide sequence of SEQ ID No.1; the encoded protein sequence is shown in SEQ ID No.1. The SnaH pure enzyme can completely degrade 0.4mM S-napropamide within 1h and cannot degrade R-napropamide, so that the SnaH pure enzyme can be used for splitting napropamide raceme in an enzyme method to produce optical pure R-napropamide and can also be used for removing S-napropamide pollution in the environment, so that the theoretical value is high, and the application prospect is wide.

Description

technical field [0001] The invention belongs to the fields of bioengineering technology, microbial remediation technology applied to polluted environment, and enzymatic resolution of chiral pesticides, and relates to a method for specifically removing S-napropamide, which has low activity and high toxicity in the racemate of napropamide The strain, amidohydrolase, coding gene and its application. Background technique [0002] Chiral isomers refer to the enantiomers in which the real object and the mirror image cannot overlap, and there is a pair of enantiomers that are mirror images of each other but cannot overlap. At present, pesticides with chiral isomers account for about 28% of the global pesticide market, while the proportion of chiral pesticides in my country is even higher, reaching 40%. Due to differences in the recognition of chiral isomers in organisms, differences in the matching between isomers and target sites, etc., chiral isomers also have differences in bio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N15/70C12P41/00C12N15/55C12N9/50C12N1/21C12P13/00C12R1/01
CPCC12N9/0071C12N15/70C12P13/001C12P41/00C12N1/205C12R2001/01
Inventor 蒋建东黄俊伟陈典
Owner NANJING AGRICULTURAL UNIVERSITY
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