Purification and Properties of a New Type of Protease Produced byMicrobacterium liquefaciens

Abstract

A bacterium, identified as Microbacterium liquefaciens MIM-CG-9535-I, was isolated from a soil sample taken from the industrial site of a gelatin manufacturer. A new type of protease, which restrictively decomposes gelatin at one or two positions, was purified from the bacterial culture. The molecular mass of the purified enzyme was 21 kDa by SDS-polyacrylamide gel electrophoresis. The purified enzyme specifically degraded the -chain of gelatin with a molecular weight of 100 kDa into two peptides of 60 kDa and 40 kDa. Native collagen was not a substrate for the enzyme.

Activity, molecular mass and hydrolysis on baker's yeast protein of extracellular proteases from the putative probiotic bacteria Microbacterium sp. strain 8L and Exiguobacterium mexicanum strain 8N

“…1). Extracellular non‐serine proteases from the genera Microbacterium have been reported by Kanayama and Sakai (2005), who characterized from Microbacterium liquefasciens a metalloendopeptidase (EC 3.4.24) of 21 kDa, likely a Zn‐dependent protein not inhibited by 20 mM PMSF. Although it degrades gelatin it is not a gelatinase (EC 3.4.24.24, EC 3.4.24.35).…”

Allosteric Regulation of the Carbohydrate-binding Ability of a Novel Conger Eel Galectin by d-Mannoside

“…Briefly, the pTVCon II-tagged plasmid vector, which contained the recognition sequence (FAGP) of the Microbacterium liquefaciens protease (MLP) isolated from the MIM-CG-9535-I strain (29) and an StuI site in the linker region, was prepared by PCR using pTVCon II-tagged BPII as a template and the following primers: P1, 5Ј-CGACTAGGCCTCGGTACTAGGATTGGCCCCGCGA-ACCCCGCTGG-3Ј, and P2, 5Ј-TGCAAGAAGGCCTATAC-ATGCTAA-3Ј. PCR was performed using KOD Plus DNA polymerase (Toyobo Co., Ltd., Osaka, Japan) and the following conditions: an initial denaturing step at 94°C for 2 min, followed by 30 cycles of a 15-s denaturing step at 94°C, a 30-s annealing step at 55°C, and a final extension step at 68°C for 4 min.…”