Method for producing alpha-acetolacetate decearboxylase by using integrated recombinant bacillus subtilis as strain

A technology of acetolactate decarboxylase and Bacillus subtilis, which is applied in the field of producing α-acetolactate decarboxylase, can solve the problems of using antibiotics, restricting the high-efficiency expression of foreign genes, and unstable replication of plasmids, etc., and achieve the effect of no antibacterial activity

Active Publication Date: 2011-01-19
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

2 There is no obvious codon preference, and the expression product is not easy to form inclusion bodies
However, replicating plasmids are usually not very stable in Baci

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] This example will include the overlapping promoter P43 promoter derived from Bacillus subtilis (Bacillus subtilis), the α-acetolactate decarboxylase gene signal peptide DNA fragment derived from Bacillus brevis and the DNA fragment derived from Agrobacterium Klebsiella (Klebsiella terrigena) α-acetolactate decarboxylase gene monocistronic α-acetolactate decarboxylase expression elements were cloned into the integrated plasmid pMLK83, and then transformed into host bacteria 1A751 to construct the integrated recombinant Bacillus subtilis, and finally in LB liquid medium Fermentative production of α-acetolactate decarboxylase.

[0024] 1. Construction of recombinant plasmid pMLK83-P43

[0025] 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...

Embodiment 2

[0037] This example will contain the promoter derived from the Bacillus lincheniformis maltose amylase gene amyM and the α-acetolactate decarboxylase gene (including signal peptide) monocistronic α derived from Bacillus brevis. - The acetolactate decarboxylase expression element was cloned into the integrated plasmid pMLK83, and then the host strain 1A751 was transformed to construct an integrated recombinant Bacillus subtilis, and finally α-acetolactate decarboxylase was produced by fermentation in LB liquid medium.

[0038] 1. Construction of recombinant plasmid pMLK83-amyM

[0039] 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 (Bacillus licheniformis ATCC 14580 total DNA) 1ul (about 20ng), 5×PrimeSTARBuffer 20ul, 10pmol / ul dNTP 2ul, 10pmol / ul forward and reverse primers 2ul each, 2.5U / ...

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Abstract

The invention discloses a method for producing alpha-acetolacetate decearboxylase by using integrated recombinant bacillus subtilis as a strain. The method comprises the following steps of: integrating an alpha-acetolacetate decearboxylase expression element to a bacillus subtilis chromosome to construct the integrated recombinant bacillus subtilis which serves as the strain, and producing the alpha-acetolacetate decearboxylase by fermentation in a nutrient medium. The method has the advantages that: the alpha-acetolacetate decearboxylase is produced by using a food safety expression system, the exogenous gene contained in the strain of integrated expression can stabilize passage and expression, and the product meets the food requirement and has no antibacterial activity.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for producing α-acetolactate decarboxylase by using an integrated recombinant bacillus subtilis as a strain. Background technique [0002] Diacetyl is a flavor substance in beer fermentation, but if the content of diacetyl in beer exceeds 0.15mg / L, it will produce an unpleasant smell of rancid rice, which seriously affects the flavor of beer. The national standard (GB4927-2008) stipulates that in premium beer Diacetyl content shall not be higher than 0.10mg / L. α-Acetolactate decarboxylase is a class of enzymes that can effectively reduce the content of diacetyl in beer and improve the flavor of beer. It can directly convert the precursor of diacetyl-acetolactate produced during beer fermentation into acetoin. It does not go through the stage of forming bad flavor substance diacetyl, accelerates the maturation of beer, shortens the production cycle, improves the utilization ...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/63C12N1/21C12R1/125
Inventor 李晓明廖东庆岳田芳王青艳黄日波蒙健宗林海鹏李丛王子龙马少敏陈发忠张云光梁莲华罗兆飞
Owner 南宁邦尔克生物技术有限责任公司
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