Mutation Breeding Of A Transglutaminase Producing Strain By ARTP

Transglutaminase(EC 2.3.2.13,TGase in short)is a multifunctional enzyme.The cross-linking reactions catalyzed by TGase can be employed to improve the structure and functional properties of proteins.Therefore,TGase has been widely concerned and applied in a variety of fields like food and biomedicine industries.As so far,the mature TGase production methods were fermentation of the wild-type strains as well as recombinant strains.TGase used in the food industry was mainly produced by microbial fermentation of Streptomyces mobaraense.In order to achieve TGase mass production by this method,obtaining high-yielding strains with good performance is of great importance.Thus,a novel and efficient mutation breeding method,Atmospheric and Room Temperature Plasma(ARTP)mutation technology,was used to treat TGase-producing strain Streptomyces mobaraense ECU7480 for iterative mutagenesis in this study.Some investigations from molecular level were conducted in order to explain the possible reasons for the enhanced SmTGase production of mutants obtained by ARTP mutagenesis.The major work of this paper contained the followings:1)Characterization of SmTGase production of the wild-type strain S.mobaraense ECU7480.SmTGase production during fermentation of S.mobaraense ECU7480 was the type of growth unassociated.SmTGase was gradually produced and accumulated after the cell growth equilibrium reached.However,the SmTGase fermentation activity declined rapidly after achieving the highest in the later stage of fermentation,which was probably related to the proteolysis catalyzed by proteases existing in the fermentation broth.2)Gene cloning and recombinant expression in E.coli of SmTGase zymogen gene from S.mobaraense ECU7480.Recombinant SmTGase was obtained by molecular biological methods and the specific activity of recombinant SmTGase was 4.06 U/mgprot.Biochemical characterization of the recombinant SmTGase revealed apparent KM,kcat and kcat/KM values of 12.1 mM,4.6 s-1 and 0.38s-1mM-1.3)Iterative ARTP mutagenesis and screening of mutants that produced SmTGase with higher yield.In order to control the mutation library size reasonably,the lethal rate was restricted to about 90%by changing the ARTP treatment time.The first eight positive mutants screened by tube fermentation for each round of ARTP mutagenesis were used as the starting strains in the next round of ARTP mutagenesis.Through eight rounds of iterative ARTP mutagenesis,four positive mutants,Sm5-V1,Sm6-V13,Sm2-V10 and Sm7-V12,with increased SmTGase fermentation activity of 27%,24%,24%and 19%respectively,were finally identified.Moreover,all these four mutants exhibited well genetic stability after eight generations.4)Analysis of SmTGase zymogen gene sequences and expression levels of mutants.Firstly,amino acids sequence alignment showed that the SmTGase zymogen amino acids sequences of mutants remained unchanged compared to that of the wild-type strain,indicating that ARTP had no direct effects on the gene sequences of the target protein.Furthermore,results of the Quantitative Real-Time PCR(qRT-PCR)reflected that SmTGase zymogen gene expressions of mutants were all up-regulated compared to that of the wild-type strain.It is reasonable to infer that the increased expression level of the target gene contributed to the improved SmTGase fermentation production.