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Alkaline pectinase genetic engineering bacteria and construction method thereof

A technology of genetically engineered bacteria and construction method, which is applied to the field of alkaline pectinase genetically engineered bacteria and its construction, can solve problems such as increasing production costs, and achieve the effects of broadening research ideas and fields, and facilitating separation, purification, and stable preservation.

Inactive Publication Date: 2009-09-16
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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Problems solved by technology

More and more research units have carried out effective attempts on the molecular biology operation of alkaline pectinase under the general trend of genetically engineered bacteria being applied to industrial production. Most of the molecular biology operations mainly focus on the expression system of Escherichia coli, or The Bacillus subtilis expression system is used as the research object, but most of the selection of vectors and hosts show some shortcomings when the purpose of production is for example. increase production cost

Method used

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  • Alkaline pectinase genetic engineering bacteria and construction method thereof
  • Alkaline pectinase genetic engineering bacteria and construction method thereof
  • Alkaline pectinase genetic engineering bacteria and construction method thereof

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Embodiment Construction

[0025] Below in conjunction with the examples, the present invention is further described, the following examples are illustrative, not limiting, and the protection scope of the present invention cannot be limited by the following examples.

[0026] 1. Construction of Alkaline Pectinase Genetic Engineering Bacteria

[0027] Alkaline pectinase gene was isolated from Bacillus subtilis (B.subtilis) (deposit unit: China Industrial Microorganism Culture Collection Management Center, deposit number: CICC 10027), and primers were designed (primers were commissioned to synthesize by Dalian Bao Biological Engineering Co., Ltd. ) uses pWB980 as the vector and WB600 as the expression host.

[0028] 1. PCR amplification of the target gene

[0029] GE UP 5′-CCG GCATGC CATGAAAAAAGTGATGTTAGCTACG-3′

[0030] GE DOWN 5′-CACGC GTC GAC TTAATTTAATTTACCCGCACC-3

[0031] According to NCBI report, primers were designed for the 1263 bp of the alkaline pectinase gene of Bacillus subtilis B. sub...

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Abstract

The invention relates to an alkaline pectinase genetic engineering bacteria and a construction method thereof. The construction method comprises the steps: separating chromosomal DNA from a strain for producing alkaline pectinase, designing a primer, obtaining a target gene by PCR, constructing a recombinant plasmid, expressing in Bacillus subtilis, measuring gene sequence and carrying out comparative analysis. The gene obtained and expressed in the method is different from the enzyme gene sequences which have been reported and has the differences on the composition of individual amino acids;the culture medium adopted for fermentation of the engineering bacteria is applicable to the industrial production, an expression host is protease deficient which is conducive to the separation, the purification and the stable preservation of enzyme in the late stage, and the enzyme activity of fermentation liquid of the engineering bacteria is improved by 22 times compared with the starting strain, which can achieve 330U / ml.

Description

technical field [0001] The invention belongs to the field of bioengineering, in particular to an alkaline pectinase genetically engineered bacterium and a construction method thereof. Background technique [0002] Pectinase is a general term for a class of enzymes that decompose pectin, and is a compound enzyme containing multiple components. Pectinase is widely distributed in higher plants and microorganisms, but the extraction from plants has been limited by resources and technical level and has not been truly industrialized. Therefore, pectinase from microorganisms has become a research hotspot. The research on acid pectinase at home and abroad has a certain basis, but there are few research reports on alkaline pectinase, especially pectate lyase. [0003] Alkaline pectinase refers to the pectinase with the most suitable pH in the alkaline range. It is an emerging content in the field of pectinase research. It is mainly used in the textile industry as an environmentally ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/75C12R1/125
Inventor 路福平肖静王春霞郝育杰
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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