Breeding Of Producing Transglutaminase Strains And Expression Of Transglutaminase Gene

Transglutaminase (protein-glutamine y-glutamyltransferase, EC 2.3.2.13) is an enzyme capable of catalyzing acyl-transfer reactions introducing covalent cross-links between proteins as well as peptides and various primary amines. They are widely distributed in living beings, including humans, plants, animals and micro organisms. These reactions catalysed by transglutaminase can be used to modify the functional properties of food proteins. So it has potential application in food industry.In order to obtain a high-production strain, we used ultraviolet to mutate the Streptomyces mobaraensis(CICC 11019). And optimize fermentation conditions of producing transglutaminase. At last study the expression of transglutaminase gene in Escherichia coli.One strain, whose transglutaminase activity is 0.66U/ml, was selected from 150 strains randomly. Then this strain was mutated with ultraviolet, and a stable and high-production strain was obtained, and its transglutaminase activity is 0.88U/ml, an increase of 33%.Single factor experiment and orthogonal design experiment was used to optimize fermentation conditions of producing MTG. The best components of medium is soluble starch 25g/L, glucose 20g/L, peptone 15g/L, soybean cake meal 30g/L. And the suitable culture conditions in shaking fask are seed age 48h, inoculum amount 8%, initial pH 7.0, culture time 48h, culture temperature 28℃, and shaker speed 200rpm.A 1224 bp of transglutaminase gene fragment from Streptomyces mobaraensis was obtained by PCR, and then was inserted into multi-cloning site of pGEX-6p-1 vector. The recombinant plasmid was then transformed into E.coli BL221(DE3). GST-TGase fusion protein was induced by IPTG, and the induction conditions were optimized. The fusion protein was absorbed on the Glutathione Sepharose. After cutting the fusion protein by thrombin, TGase can be purificated. The result of SDS-PAGE reveals the recombinant protein has a high level expression.