Molecular and biochemical characterizations of a new cold-active and mildly alkaline β-Mannanase from Verrucomicrobiae DG1235

Mannanases catalyze the cleavage of beta-1,4-mannosidic linkages in mannans and have various applications in different biotechnological industries. In this study, a new beta-mannanase from Verrucomicrobiae DG1235 (ManDG1235) was biochemically characterized and its enzymatic properties were revealed. Amino acid alignment indicated that ManDG1235 belonged to glycoside hydrolase family 26 and shared a low amino acid sequence identity to reported beta-mannanases (up to 50% for CjMan26C from Cellvibrio japonicus). ManDG1235 was expressed in Escherichia coli. Purified ManDG1235 (rManDG1235) exhibited the typical properties of cold-active enzymes, including high activity at low temperature (optimal at 20 degrees C) and thermal instability. The maximum activity of rManDG1235 was achieved at pH 8, suggesting that it is a mildly alkaline beta-mannanase. rManDG1235 was able to hydrolyze a variety of mannan substrates and was active toward certain types of glucans. A structural model that was built by homology modeling suggested that ManDG1235 had four mannose-binding subsites which were symmetrically arranged in the active-site cleft. A long loop linking beta 2 and alpha 2 as in CjMan26C creates a steric border in the glycone region of active-site cleft which probably leads to the exo-acting feature of ManDG1235, for specifically cleaving mannobiose from the non-reducing end of the substrate.

Automated summary

A new beta-mannanase, ManDG1235 from Verrucomicrobiae DG1235, was characterized biochemically and its enzymatic properties were revealed. The purified rManDG1235 exhibited cold-active enzyme properties, with optimal activity at 20 degrees Celsius and high activity at pH 8. A structural model suggested that ManDG1235 has four mannose-binding subsites and an exo-acting feature for specifically cleaving mannobiose from the non-reducing end of the substrate.