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Heat-resistant and acid-resistant alpha-amylase gene engineering bacterium recombinase and application thereof

A technology of genetically engineered bacteria and amylase, applied in the exploratory application research field of biotechnology in food enzymology, can solve the problems of easy contamination of enzyme production, difficult control of fermentation conditions, reverse mutation, etc.

Inactive Publication Date: 2015-03-18
JILIN BUSINESS & TECH COLLEGE
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Problems solved by technology

Bacillus licheniformis ( Bacillus licheniformis) is recognized as an excellent strain for producing high-temperature-resistant α-amylase, but the strain itself is a high-yield strain obtained through multiple generations of mutagenesis, and there are conditions for enzyme production Unstable, prone to reverse mutations, difficult to control fermentation conditions, easy to pollute enzyme production, etc.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0013] 1 Through RT-PCR reaction, the structural gene of amylase was successfully cloned, and then the yeast secretion expression vector was successfully constructed, and 5 recombinant yeast strains with relatively high amylase expression and enzyme activity were screened.

[0014] 2 Take the recombinant Pichia pastoris ( P. pastoris GS115) genetically engineered bacteria were inoculated into fresh YPD medium for about 14 h at 30 °C and 200 r / min in shake flasks, and then transferred to BMGY medium with 3% inoculation amount for 30 Cultivate at 150 r / min for 24 h, centrifuge to discard the supernatant, transfer the cell pellet to BMMY, ferment at 30 °C at 200 r / min, add methanol every 24 h to make the final concentration 0.4% (per bottle 100 mL of medium was packed in a 500 mL Erlenmeyer flask). After culturing for 72 h, the fermentation broth was centrifuged (4 °C, 10000 r / min, 20 min), and the supernatant was the crude enzyme solution, and then the enzyme activity was ...

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Abstract

The invention discloses a heat-resistant and acid-resistant alpha-amylase gene engineering bacterium recombinase and an application method thereof, belonging to the technical field of food biology. The method comprises the following steps: gene engineering recombination, transformation, fermentation process optimization and enzyme activity determination. The heat-resistant and acid-resistant alpha-amylase gene engineering bacterium recombinase disclosed by the invention has the advantages of high enzyme activity, high tolerable temperature, wide tolerable pH value and convenience in use.

Description

technical field [0001] The invention belongs to the field of food biotechnology, and is an exploratory application research of biotechnology in food enzymology. Background technique [0002] High temperature resistant α-amylase belongs to hydrolytic enzymes, which can randomly hydrolyze the α-D-1,4 glycosidic bonds inside starch, glycogen and degradation products. It can reduce the viscosity of starch solution rapidly, produce soluble dextrin, oligosaccharide and a small amount of maltose and glucose. At present, industrial enzyme preparations represented by α-amylase have occupied 25% of the market share, and are widely used in the liquefaction of starch hydrolysis to generate glucose syrup, brewing, modified starch and improving the baking process of flour. There are hundreds of thermophilic fungi and thousands of bacteria in microorganisms that can produce amylase, only Bacillus ( Bacillus ) has 48 species that can produce amylase, but there are fewer microorganisms th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/28C12N15/56C12N15/81C12N1/19C12R1/84
CPCC12N9/2417C12Y302/01001
Inventor 孟宪梅潘风光柳增善
Owner JILIN BUSINESS & TECH COLLEGE
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