Recombinant collagenase from Grimontia hollisae as a tissue dissociation enzyme for isolating primary cells.

Collagenase products are crucial to isolate primary cells in basic research and clinical therapies, where their stability in collagenolytic activity is required. However, currently standard collagenase products from Clostridium histolyticum lack such stability. Previously, we produced a recombinant 74-kDa collagenase from Grimontia hollisae, which spontaneously became truncated to ~60 kDa and possessed no stability.

-(Carboxymethyl)arginine Is One of the Dominant Advanced Glycation End Products in Glycated Collagens and Mouse Tissues.

Advanced glycation end products (AGEs) accumulate in proteins during aging in humans. In particular, the AGE structure -(carboxymethyl)arginine (CMA) is produced by oxidation in glycated collagen, accounting for one of the major proteins detected in biological samples. In this study, we investigated the mechanism by which CMA is generated in collagen and detected CMA in collagen-rich tissues.

The C-terminal segment of collagenase in binds collagen to enhance collagenolysis.

The collagenase secreted by strain 1706B is a 74 kDa protein that consists of two parts: the catalytic module and a C-terminal segment that includes the bacterial pre-peptidase C-terminal domain. Here, we produced a recombinant C-terminal segment protein and examined its ability to bind collagen and other characteristics as compared with collagen-binding domains (CBDs) derived from () collagenases; these CBDs are the only ones thus far identified in bacterial collagenases. We found that the C-terminal segment binds to collagen only when the collagen is in its triple-helical conformation.

Cloning of a novel collagenase gene from the gram-negative bacterium Grimontia (Vibrio) hollisae 1706B and its efficient expression in Brevibacillus choshinensis.

The collagenase gene was cloned from Grimontia (Vibrio) hollisae 1706B, and its complete nucleotide sequence was determined. Nucleotide sequencing showed that the open reading frame was 2,301 bp in length and encoded an 84-kDa protein of 767 amino acid residues. The deduced amino acid sequence contains a putative signal sequence and a zinc metalloprotease consensus sequence, the HEXXH motif.

Amount of N(omega)-(Carboxymethyl)arginine generated in collagen and bovine serum albumin during glycation reactions is significantly different.

N(omega)-(Carboxymethyl)arginine (CMA), an advanced glycation end product (AGE), is found in glycated type I collagen. The levels of CMA generated in collagen and bovine serum albumin (BSA) were compared during in vitro glycation reactions. CMA production increased in collagen during incubation with glucose or ribose, attaining a molar quantity approximately the same as that of N(epsilon)-(carboxymethyl)lysine (CML), the dominant AGE.