Dissertation > Industrial Technology > Chemical Industry > Other chemical industries > Fermentation industry > Enzyme preparation ( enzyme )

Breeding of Producing Transglutaminase Strains and Expression of Transglutaminase Gene

Author HuangKe
Tutor JinZhiHua
School Zhejiang University
Course Biochemical Engineering
Keywords Transglutaminase Streptomyces mobaraensis Ferment Optimization Fusion expression
Type Master's thesis
Year 2010
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Transglutaminase ( protein - glutamate -γ - glutamyl transferase , EC ) is a catalyzed acyl transferase enzyme , which are widely distributed in a variety of biological , including human beings , animals, plants and a variety of microorganisms . This acyl transfer reaction can make a variety of proteins, peptides and amino acids to produce covalent cross -linking between in vitro , thus changing the nature of the functions of the various food proteins , and thus has a broad application prospects in the food industry . To obtain in this article glutamyl aminotransferase overproducing strain as the goal , overproducing strain of the Streptomyces mobaraensis UV mutagenesis to breeding , and production transglutaminase enzyme fermentation medium and fermentation conditions were optimized preliminary study The use of genetic engineering to build genetically engineered bacteria production transglutaminase . Strain after activation will later be obtained by natural breeding an enzyme production capacity was 0.66 U / ml of the original strain , and the strain spores ultraviolet treatment to obtain a relatively high yield and stable strains by mutagenesis screening , enzyme production capacity of 0.88 U / ml, and 33% higher than the original strain . Screening high-yield strains of the original strain by single factor and orthogonal experiment to determine the best production transglutaminase medium composition of the carbon and nitrogen sources : starch 25g / L glucose 20g / L , peptone 15g / L soybean flour 30g / L, the optimum culture conditions : 48h age liquids seed inoculation amount of 8% , initial pH 7.0 , culture temperature is 28 ℃, shaking speed of 200rpm , the incubation time was 48h . Transglutaminase enzyme reach 1.4U/ml . To the Streptomyces mobaraensis genome DNA as a template , a length of 1224bp of transglutaminase gene fragments obtained by PCR amplification successfully constructed prokaryotic expression vector pGEX -TGase and transformed into E. coli BL21 (DE3 ) , recombinant bacteria were IPTG induced fusion protein GST-TGase , and the expression conditions were optimized application affinity chromatography purification of the fusion protein by SDS-PAGE electrophoresis analysis of purified product , higher levels of expression obtained results show that the target protein .

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