A kind of pullulanase enzyme-producing strain and the method for improving its enzyme-producing ability

Abstract

The invention belongs to the technical field of bioengineering, and in particular relates to a method for constructing high-yield pullulanase engineering bacteria and the constructed high-yield pullulanase engineering strain. The method comprises the steps of DNA extraction, PCR amplification, PCR fusion, electric shock transformation, screening and identification, and the like. Klebsiella variicola HN7 (Klebsiella variicola HN7), whose preservation number is CGMCC NO.10357, is the original starting strain. The invention transforms the bacillus subtilis P43 promoter into the pullulanase-producing original bacterial strain through the genetic engineering technology, and the operation mode is easy to realize. Because the pullulanase can be expressed in the body, the synthesis and secretion pathways are not changed, and the plasmid loss phenomenon does not occur, so the genetic stability is high; the high-yield pullulanase engineering strain constructed by the present invention is compared with the original starting strain, The enzyme activity and enzyme expression of the pullulanase are better improved, so it has better popularization and application value.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a pullulanase-producing strain, a method for improving its enzyme-producing ability and a new high-yield pullulanase engineering strain constructed. Background technique [0002] Most of the sugary raw materials formed in nature and agricultural production exist in the form of polysaccharides, including starchy raw materials (corn, wheat, potatoes, etc.), lignocellulose (straw, forest trees, etc.) and chitosan (insects, shrimps, etc.) crab shell), etc. Due to various reasons, starchy raw materials can be effectively utilized at present. my country's food and fermentation industry consumes more than 50 million tons of starchy raw materials such as corn, rice, wheat and potatoes every year. [0003] When starchy raw materials are used in food and fermentation production, they often need to undergo enzymatic hydrolysis to generate glucose. Enzymes used in amyla...

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Problems solved by technology

[0004] In the existing technology, my country's pullulanase market is monopolized by a few large multinational companies, and the price of enzymes is expensive, which is difficult to meet the urgent needs of domestic food and fermentation industries

Although my country has made some progress in the screening of pullulanase-producing bacteria resources, molecular transformation and genetic engineering, both the strains screened from nature and engineering strains have low enzyme activity and poor enzymatic properties. However, industrial production has not been realized so far, so further exploration and research are needed

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