Breeding of Producing Transglutaminase Strains and Expression of Transglutaminase Gene
|Keywords||Transglutaminase Streptomyces mobaraensis Ferment Optimization Fusion expression|
Transglutaminase ( protein - glutamate -γ - glutamyl transferase , EC 220.127.116.11 ) 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 .