Tissue-Type Transglutaminase from Red Sea Bream (Pagrus major). Sequence Analysis of the cDNA and Functional Expression in Escherichia coli

Abstract

A cDNA clone encoding a tissue-type transglutaminase (TGase) was isolated from a cDNA library prepared from the liver of red sea bream (Pagrus major). The cDNA sequence had an open reading frame coding for a protein of 695 amino acids and showed 43% identity to the sequence of guinea pig liver TGase, reveahg a relatively low overall similarity. However, the 25-amino-acid sequence containing the putative active site (Cys272) of the enzyme was completely conserved between the two species, and was also identical to the corresponding regions of human and bovine endothelial cell TGases. In addition, the critical residues (His332 and Asp355) thought to form the catalytic-center triad together with Cys272, were found in the highly conserved region. The red sea bream TGase had an extension of 11 amino acids in the C-terminal region and some differences in the N-terminal region when compared with guinea pig TGase. From the cloned cDNA, a semi-synthetic TGase gene suitable for overexpression in Escherichia coli was constructed (pTTG2-22). At a reduced temperature (28"C), E. coli cells transformed with pTTG2-22 could produce soluble TGase which exhibited catalytic activity in the presence of calcium. E. coli extracts containing the recombinant red sea bream TGase induced gelation of actomyosin solutions, accompanied by a significant increase of E-(y-glutamy1)lysine bonds, which are predominantly derived from the cross-linking of myosin heavy chains. These results indicate that this fish TGase should be useful for further analysis of TGase structure/function relationships and that it could also be employed to enhance the viscoelastic properties of proteinaceous materials.

A Novel Operon Encoding Formaldehyde Fixation: the Ribulose Monophosphate Pathway in the Gram-Positive Facultative Methylotrophic Bacterium Mycobacterium gastri MB19

“…Each PCR product contained the open reading frame (ORF) of rmpA or rmpB, and the upstream and downstream ends of the PCR products of both rmpA and rmpB, and the upstream and downstream ends of the PCR products of both rmpA and rmpB contained the ClaI and XbaI sites (italicized sequence in the primer), respectively. The PCR product of rmpA and that of rmpB were each purified and cloned into the ClaI/XbaI site of pT13sNco (22), and the resultant plasmid was designated pT-rmpA or pT-rmpB, respectively. A pBR322-derived vector, pTTNco (22), was used as the control plasmid.…”

“…The PCR product of rmpA and that of rmpB were each purified and cloned into the ClaI/XbaI site of pT13sNco (22), and the resultant plasmid was designated pT-rmpA or pT-rmpB, respectively. A pBR322-derived vector, pTTNco (22), was used as the control plasmid. E. coli JM109 was transformed with one of the three plasmids.…”

Transglutaminases - a review with special reference to microbial transglutaminase and its application in food processing

“…και ζΰμες, οι οποίοι χρησιμοποιούνται ως κΰττα-ρα-ξενιστές (Ikura et al 1988, Bishop et al 1990, Takehana et al 1994, Yasueda et al 1995. Εντούτοις, καμία από αυτές τις TG δεν έχει διατεθεί στο εμπόριο, κυρίως λόγω της χαμηλής απο-δεκτικότητάς τους από το καταναλωτικό κοινό.…”

Evaluation of sorghum grain hydrolysates and dried distillers grains with solubles for the production of microbial transglutaminase

“…TGases have been found in animal tissues and body fluids, fish, plants, and microorganisms (Ando et al, 1989;Falcone, Serafini-Fracassini, & del Duca, 1993;Folk, 1980;Lilley, Skill, Griffin, & Bonner, 1998;Yasueda et al, 1995). In food processing, this enzyme has been used to promote polymerization of proteins, texture improvement, meat restructuring, gel formation, and to increase nutritional value of proteins as well (Alves-Macedo, Duraes, & Hurami, 2007;Griffin, Casadio, & Bergamini, 2002;Motoki & Seguro, 1998).…”