Identification, Expression And Sequence Analysis Of The Gene Encoding Transglutaminase Zymogen From Streptomyces Hygroscopicus

Microbial transglutaminase (MTG; protein-glutamineγ-glutamyltransferase, EC 2.3.2.13) is a recently developed enzyme and is capable of catalyzing acyl transfer reactions, which introduces covalent cross-links between proteins, peptides and various primary amines. So it has potential application in food processing, cosmetic, pharmacy and other industries.In order to identifiy microbial transglutaminase (MTGase) gene from Streptomyces hygroscopicus; cloning and expression of this gene in Escherichia coli; active sites sequence analysis of S. hygroscopicus MTGase through homologous sequence comparison,the following researches had been done:1. Wild-type microbial transglutaminase zymogen (pro-MTGase) was purified from liquid culture of S. hygroscopicus(CCTCC M203062).N-terminal amino acid sequence of this pro-MTGase was determined. According to the N-terminal sequence and the corresponding nucleotide sequence of MTGase from other three Streptomyces species, PCR primers of S. hygroscopicus pro-MTGase were designed and the completed gene of pro-MTGase was amplified and sequenced. This is the first report of the gene encoding microbial pro-transglutaminase from S. hygroscopicus.2. Pro-MTGase from S. hygroscopicus is composed of 1170bp, encoding 389 amino acids. The nucleotide sequence showed 92 % homologue with that of S. caniferus, S. platensis and S. paucisporogenes.About the amino acid sequence,1-58 is pro-region,59-389 is mature MTGase region,Cys122 is the important amino acid of catalytic center. The catalytic triad-'Cys64-His274-Asp255'is also similar to that from other Streptomyces sps.3. The gene was sub-cloned into pET-20b(+) vector,downstream pelB signal peptide,to construct the expression vector pET/pro-MTG. BL21(DE3) carrying the expression vector was induced for research its expression.Many methods have been tried but the expression always formed inclusion body. Rosetta(DE3)pLysS carrying the expression vector was induced with IPTG at 24℃and expressed pro-MTGase as extracellular soluble protein. SDS-PAGE showed the expressed recombinant pro-MTGase was about 44 kDa, similar to the wild-type pro-MTGase purified from S. hgroscopicus. Recombinant pro-MTGase was activated with trypsin and the enzyme activity reached to 0.35 U/mL. This is the first research of expression extracellular soluble pro-MTGase in E.coli in our country.4. Sequence anlysis,secondary structure prediction and three-dimensional structure model of MTGase gene from S. hygroscopicus had been done.There are manyα-helix,corner andlessβ-pleated sheet in its secondary structure; catalytic center amino acid Cys122 is in a hydrophbicity region what can make it stable;it has not transmembrane structure.The three-dimensional structure model prediction may show the ture crystal structure that can give a theory base for research the relation between function and structure, directed evolution of enzyme, chemical modification, and for finding some material increasing MTGase stability and industrial production.