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Gene engineering bacterium of beta - glucosaccharase, and application

A technology of genetically engineered bacteria and glucosidase, which is applied in the application field of preparing effective mycotoxamine A compounds, can solve the problem of being difficult to prepare and separate effective mycotoxamine A and the like, and achieve the target product effective mycotoxane A. The effect of large amount of base amine A, stable and single product, mild and easily controllable reaction conditions

Active Publication Date: 2007-10-10
SHANGHAI LAIYI BIOMEDICAL RES & DEV CENT +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It can be seen that the biotransformation with these microbial cells or their broken supernatants (crude enzymes) is a series of reactions. Enzymatic action generates valid mycoylidene amine A (validoxylamine A), which will be degraded by subsequent enzymes (validoxylamine A 3-dehydrogenase, C-N lyase, etc.) soon, so it is difficult to use the existing The strains directly degrade the substrate to produce and isolate a large amount and high-purity effective mycoylidene A product

Method used

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  • Gene engineering bacterium of beta - glucosaccharase, and application
  • Gene engineering bacterium of beta - glucosaccharase, and application
  • Gene engineering bacterium of beta - glucosaccharase, and application

Examples

Experimental program
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Effect test

Embodiment 1

[0016] Construction of engineering bacteria CGMCC No.1626

[0017] Specific PCR amplification primers were designed and synthesized according to a β-glucosidase gene sequence in the reported complete gene sequence of Agrobacterium tumefaciens str.C58 (GenBank accession no.NC_003063). The 5' ends of the two primers contained a single HindIII. and SacI cleavage site.

[0018] P1: 5'-GC AAGCTT TCACCCCTTCACCACACC-3' (the underlined HindIII restriction site), P2: 5'GCGG GAGCTC ATGGATAGAAAGGCCTCT-3' (SacI restriction site is underlined). Using the genome of Agrobacterium sp.HCCB-20052 as a template, the β-glucosidase gene of primers P1 and P2 was used for PCR amplification. The PCR reaction system was 50 μl, containing 5 ng of Agrobacterium sp. HCCB-20052 total DNA, 25 μl of Premix Taq, and 50 pmol of each primer. Reaction conditions: denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, extension at 72 °C for...

Embodiment 2

[0021] Cultivation of engineered bacteria

[0022] The recombinant engineered bacteria were inoculated into LB medium containing kanamycin (50 μg / ml) and cultured with shaking at 37°C to logarithmic growth phase (OD). 600 to about 0.6), add 1 mM IPTG or lactose, and induce culture at 37°C for 4 hours.

Embodiment 3

[0024] Obtaining of Crude Enzyme and Conversion of Crude Enzyme to Effective Validamycin A

[0025] The induced cultured recombinant bacteria were centrifuged at 4000rpm for 15min at room temperature to collect the bacteria, washed three times with PBS buffer, and then used 1 / 10 volume of 0.05M, pH5.5 citric acid / sodium citrate buffer The bacteria were resuspended, sonicated, and incubated at 4°C with Centrifuge for 25 min and take the supernatant as crude enzyme.

[0026] The crude enzyme solution was diluted 20 times, 20 ml was taken out, 0.1 g of effective valomycin A substrate was added, and the conversion reaction was carried out at 32°C. 54% conversion after 1 hour of conversion

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Abstract

This invention discloses engineering bacterium containing beta-glucosidase, Escherichia coli (CGMCC No.1626), and its application in preparing validoxylamine A compounds. The engineering bacterium can convert validamycin A into validoxylamine A, and has such advantages as high conversion efficiency, low cost, mild reaction conditions, high product purity, easy separation and purification, and little chemical pollution.

Description

technical field [0001] The invention relates to the technical field of applied microorganisms, in particular to a recombinant β-glucosidase genetically engineered bacteria---Escherichia coli CGMCC No. 1626, and the use of the strain in the preparation of effective mold subunits Use of Amine A Compounds. Background technique [0002] Several components of validamycin complex and a series of degradation products or many semi-synthetic compounds based on them have different degrees of glycosidase inhibitory activity, indicating that these substances have potential development value in anti-diabetic applications . The typical examples that have been successfully developed are Voglibose and Acarbose. [0003] Validoxylamines are a group of the above-mentioned glycosidase-inhibiting active substances, which are present in small amounts in the validoxylamine complexes produced by microbial fermentation. Among them, validoxylamine A (validoxylamine A, the structural formula is sh...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P1/04C12P13/00C12R1/19
Inventor 殷瑜朱丽杨志钧陶正利陈代杰王天娇
Owner SHANGHAI LAIYI BIOMEDICAL RES & DEV CENT
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