Applied Microbiology and Biotechnology, Vol.104, No.16, 7051-7066, 2020
Identification of a unique 1,4-beta-D-glucan glucohydrolase of glycoside hydrolase family 9 fromCytophaga hutchinsonii
Cytophaga hutchinsoniiis an aerobic cellulolytic soil bacterium that rapidly digests crystalline cellulose. The predicted mechanism by whichC. hutchinsoniidigests cellulose differs from that of other known cellulolytic bacteria and fungi. The genome ofC. hutchinsoniicontains 22 glycoside hydrolase (GH) genes, which may be involved in cellulose degradation. One predicted GH with uncertain specificity, CHU_0961, is a modular enzyme with several modules. In this study, phylogenetic tree of the catalytic modules of the GH9 enzymes showed that CHU_0961 and its homologues formed a new group (group C) of GH9 enzymes. The catalytic module of CHU_0961 (CHU_0961B) was identified as a 1,4-beta-d-glucan glucohydrolase (EC 3.2.1.74) that has unique properties compared with known GH9 cellulases. CHU_0961B showed highest activity against barley glucan, but low activity against other polysaccharides. Interestingly, CHU_0961B showed similar activity against rho-nitrophenyl beta-d-cellobioside (rho-NPC) and rho-nitrophenyl beta-d-glucopyranoside. CHU_0961B released glucose from the nonreducing end of cello-oligosaccharides, rho-NPC, and barley glucan in a nonprocessive exo-type mode. CHU_0961B also showed same hydrolysis mode against deacetyl-chitooligosaccharides as against cello-oligosaccharides. Thek(cat)/K(m)values for CHU_0961B against cello-oligosaccharides increased as the degree of polymerization increased, and itsk(cat)/K(m)for cellohexose was 750 times higher than that for cellobiose. Site-directed mutagenesis showed that threonine 321 in CHU_0961 played a role in hydrolyzing cellobiose to glucose. CHU_0961 may act synergistically with other cellulases to convert cellulose to glucose on the bacterial cell surface. The end product, glucose, may initiate cellulose degradation to provide nutrients for bacterial proliferation in the early stage of C. hutchinsonii growth.
Keywords:Cytophaga hutchinsonii;Glycoside hydrolase family 9;1,4-beta-D-glucan glucohydrolase;Glucose;Site-directed mutagenesis