A kind of pullulanase enzyme-producing strain and the method for improving its enzyme-producing ability
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A technology of pullulanase and bacterial strains, applied in the field of bioengineering, can solve the problems of unrealized industrial production, difficulty in meeting urgent needs, low enzyme activity, etc.
Active Publication Date: 2017-11-14
河南新仰韶生物酶制剂有限公司
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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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Embodiment 1
[0052] The pullulanase engineered bacterium constructed in the present invention is Klebsiella mutabilis HN7 ( Klebsiella variicola HN7) is the original starting strain, which was screened from the soil near the starch production plant. It has been preserved in the General Microbiology Center (Beijing) of the China Committee for the Collection of Microbial Cultures on January 14, 2015. The preservation number is: CGMCC NO .10357.
[0053] The following is a brief introduction to the screening process of the bacteria.
[0054] screening method
[0055] The method of screening microorganisms from soil samples was used for screening. The soil samples were taken from different starch factories and production starch pool annexes in many provinces and cities in China.
[0056] Media used during screening
[0057] Strain enrichment screening medium (mass%): glutinous rice flour 1.0, peptone 0.5, yeast extract 0.5, KH 2 PO 4 0.05, MgSO 4 .7H 2 O 0.01, KH 2 PO 4 0.05, ag...
Embodiment 2
[0100] The high-yield pullulanase engineering bacterium constructed in this embodiment is Klebsiella mutabilis HN7 ( Klebsiella variicola HN7) is the original starting strain, which was constructed using the following steps:
[0101] (1) Extract DNA, amplify and culture Bacillus subtilis in LB medium, culture at 30°C, 220 rpm for 15 h, centrifuge at 8000 r / min for 20 min to collect bacteria, and then follow Ezup column bacterial genomic DNA Extract the DNA of the cultured bacteria according to the instructions of the extraction kit.
[0102] (2) PCR amplification, PCR amplification of tetracycline resistance gene Tet upstream fragment 、 tetracycline resistance gene Tet Downstream fragment, Bacillus subtilis P43 promoter, specifically as follows:
[0103] PCR amplification of tetracycline resistance gene Tet For upstream fragments, the primer sequences are designed as follows:
[0104] Primer Tet-F1 of the upstream fragment: 5'-GGGGGATGATTGCGCCCCGGAAAGCAAAAATATCTAA...
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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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