High-efficient expression and application of amidohydrolase

A technology of amidohydrolase and hydrolase, which is applied in the fields of molecular biology and enzyme engineering, can solve problems such as the research of gene expression systems, achieve the effects of reducing energy consumption, overcoming harsh conditions, and realizing green environmental protection

Inactive Publication Date: 2011-11-23
ZHEJIANG HISUN PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in this study, the researchers did not further study the expression system of the gene

Method used

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  • High-efficient expression and application of amidohydrolase
  • High-efficient expression and application of amidohydrolase
  • High-efficient expression and application of amidohydrolase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1: clone stereoselective amidohydrolase gene (DAM gene) from Delphi bacterium

[0050] 1. Genomic DNA acquisition

[0051] Genomic DNA of Delftia tsuruhatensis (CCTCCNo.M205114) was extracted using a genome extraction kit.

[0052] 2. Primer design and amplification of target gene DAM

[0053] According to the amidohydrolase gene from Delftia acidovorans reported in GenBank (gi|160361034:5879135-5880535, researchers speculate that the gene has the function of amidohydrolase), two PCR primers were designed:

[0054] Primer A (5'to 3'): TCT CAT ATG ATG AAC GAC AGC GAA CTGCAC CAT CTC GAA CT

[0055] Primer B (5'to 3'): CTA GAA TTC TCA GGC AGC AGG GTG CTGTCT GTG CCA GT

[0056] A primer (primer A) at the 5' end of the designed gene introduced an Nde I restriction site; a primer at the 3' end of the gene (primer B) introduced an EcoR I restriction site.

[0057] Using genomic DNA as a template, add a certain amount of primers A and B, Taq DNA polymerase ...

Embodiment 2

[0069] Embodiment 2: Construction of expression vector PET24a (+)-DAM and genetically engineered bacteria Escherichia coli (Escherichia coli) BL21 (DE3) / PET24a (+) DAM

[0070]After the pUC118-DAM plasmid and PET24a(+) were digested by EcoR I and Nde I respectively, the digested products were separated by 0.8% agarose gel electrophoresis, and the DNA fragments of about 1400bp and 5260bp were recovered from the gel, and T4DNA Ligase ligation, the resulting plasmid is called PET24a(+)-DAM plasmid, the ligation product is transformed into competent Escherichia coli (Escherichiacoli) BL21(DE3), and positive clones are obtained by screening on a kanamycin-resistant plate and subjected to EcoR I And Nde I double enzyme digestion identification, proved to contain the correct insert (such as Figure 4 shown), thereby obtaining the genetic engineering strain Escherichia coli (Escherichiacoli) BL21(DE3) / PET24a(+) DAM containing amidohydrolase of the present invention. The construction ...

Embodiment 3

[0071] Example 3: Fermentative expression of exogenous stereoselective amidohydrolase engineered bacteria BL21(DE3) / PET24a(+) DAM

[0072] The seed medium is LB medium, and the inoculation amount is 0.5-6%. The fermentation medium is TB medium: 24g / L yeast extract, 12g / L peptone, 4g / L industrial glycerol, 0.1M phosphate buffer, pH=7.5. When the seed OD600=1 or so, add 150ml seed fermentation liquid to 3000ml fermentation medium, start culturing at 37°C, when OD600=2, add 1% α-lactose for induction, induction temperature is 28°C, after 5 hours , add 1% α-lactose for the second time, when the dissolved oxygen rises to a higher value and no longer drops, put the tank.

[0073] The growth curve of the fermentation process is shown in Image 6 , Compared with the fermentation process of the host strain Delftia tsuruhatensis, the fermentation time is shortened and the biomass is increased. Under the above culture conditions, the enzyme activity can reach about 3000U / L.min when pu...

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Abstract

The invention relates to the clone of amidohydrolase, and the construction, zymoprotein expression and industrial application of engineering bacteria, belongs to the field of bioengineering technology and mainly aims to solve the problems that amidohydrolase produced by natural bacteria is low in enzyme activity, low in bacterial concentration and difficult for production application. On the basis that the hydrolase is produced by fermenting Delftiatsuruhatensis in Delftia, an amidohydrolase gene is cloned to establish a full set of process of the fermentation, and inducible expression, and industrial production of the Escherichia coli gene engineering bacteria. Through the gene engineering modification, the amidase activity is over 3000 u/l, much higher than 300 u/l of the original strain; the actual application shows that the enzyme has a wide application scope, can be applied the production of side chains of statins, the production cost is greatly reduced and the realization of green chemistry is promoted.

Description

technical field [0001] The present invention relates to molecular biology and enzyme engineering, specifically, the present invention relates to utilizing DNA recombination technology to construct the new bacterial strain of E. Amide compounds. Background technique [0002] Amide compounds are a class of substances widely distributed in nature. Among them, chiral amide compounds and their derivatives play an important role in the synthesis of statins. For example, S-(+)-2,2-dimethylcyclopropylcarboxamide is an important intermediate of cilastatin; (R)-4-carboxylic acid-3-hydroxy-butyric acid ethyl ester is (R)- 4-cyano-3-hydroxy-butyric acid ethyl ester is obtained through the two-step action of nitrile hydratase and amidohydrolase, and the former is an important intermediate in the synthesis of super statins. Cilastatin is the first clinical renal dehydrogenase inhibitor, which can effectively prevent imipenem from being degraded by renal dehydrogenase in vivo and enhanc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/55C12N15/66C12N1/21C12N9/78C12P41/00C12P7/42C12R1/19
Inventor 吕中原杨仲毅徐期李丛撑王献周甘春晖彭春龙张小英白骅
Owner ZHEJIANG HISUN PHARMA CO LTD
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