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Method for recombinant expression of beta-amylase in bacillus subtillis in integrated mode

A technology of Bacillus subtilis and amylase, which is applied in the biological field, can solve the problems of breaking cell extraction, containing endotoxin, difficult to secrete and express, etc.

Inactive Publication Date: 2011-07-20
南宁邦尔克生物技术有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the E. coli expression system has some defects, such as containing endotoxin, enterotoxin, difficult to secrete and express, and the product needs to be extracted by cell destruction, etc., and in order to maintain the stability of the plasmid, it is generally necessary to use antibiotics in the production process

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] This example will include the overlapping promoter P43 promoter derived from Bacillus subtilis (Bacillus subtilis), the signal peptide DNA fragment of the α-acetolactate decarboxylase gene derived from Bacillus brevis (Bacillus brevis) and the H2S-producing fusiform derived from high temperature Clostridium thermosulfurogenes β-amylase gene monocistronic β-amylase expression element was cloned into the integrated plasmid pMLK83, then transformed into host strain WB600 to construct integrated recombinant Bacillus subtilis, and finally fermented in LB liquid medium Produces beta-amylase.

[0022] 1. Construction of recombinant plasmid pMLK83-P43

[0023] According to the promoter P43 sequence annotated in Genbank, the upstream primer was designed as 5'attgctggacgcttatggac 3' and the downstream primer was 5'cgggatccattcctctcttacctataat 3'. PCR reaction system 100ul: DNA template (Bacillus subtilis 1A751 total DNA) 1ul (about 20ng), 5×PrimeSTAR Buffer 20ul, 10pmol / ul dNTP ...

Embodiment 2

[0076] This example will include the promoter derived from the amyM maltose amylase gene of Bacillus lincheniformis (Baclicus lincheniformis), the signal peptide DNA fragment of the α-acetolactate decarboxylase gene derived from Bacillus brevis (Bacillus brevis) and the DNA fragment derived from barley (Hordeum vulgare ) The monocistronic β-amylase expression element of the β-amylase gene was cloned into the integrated plasmid pMLK83, and then transformed into the host strain WB600 to construct the integrated recombinant Bacillus subtilis, and finally fermented in LB liquid medium to produce β-amylase enzyme.

[0077] 1. Construction of recombinant plasmid pMLK83-amyM

[0078] According to the promoter amyM sequence annotated in Genbank, the upstream primer was designed as 5'cccaagcttctgtacacttgcgtcctcca 3' and the downstream primer was 5'cgggatcctctcctcccctttcaatgtg 3'. PCR reaction system 100ul: DNA template (Baclicus licheniformis ATCC 14580 total DNA) 1ul (about 20ng), 5×...

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Abstract

The invention discloses a method for the recombinant expression of beta-amylase in bacillus subtillis in an integrated mode. Specifically, a beta-amylase expression element is integrated into the chromosome of the bacillus subtillis to construct integrated recombinant bacillus subtillis, and the beta-amylase is produced by fermentation in a nutrient medium by taking the recombinant bacillus subtillis as a strain. The method has the advantages that: the beta-amylase is produced by expression through a food safety expression system, a foreign gene contained in the integrally expressed strain can be stably generated and expressed, and a product meets food requirements and does not have antimicrobial activity.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for recombinantly expressing beta-amylase in Bacillus subtilis in an integrated manner. Background technique [0002] β-amylase (EC3.2.1.2), also known as starch 1,4-maltosidase, can cut off the α-1,4 glycosidic bond at the non-reducing terminal when acting on starch and glycogen, and decompose to obtain maltose. β-amylase is mainly used to replace part of malt to produce beer, or to produce starch syrup for beer fermentation, and to produce high maltose syrup, ultra-high maltose syrup, crystalline maltose, medical injection maltose, maltitol, etc. [0003] β-amylase can be obtained from plants and microorganisms. Currently commercialized β-amylases are mainly produced from plants. Plant-derived β-amylases are mostly extracted from some higher plants such as barley, wheat, rye, soybean seeds and sweet potato tubers. The main disadvantage of extracting β-amylase from barle...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N1/21C12N9/42C12N15/56C12R1/125
Inventor 李晓明蒙健宗周礼芹张云光梁莲华廖东庆
Owner 南宁邦尔克生物技术有限责任公司
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