Exo‐mode of action of cellobiohydrolase Cel48C from Paenibacillus sp. BP‐23

Abstract

Sequence analysis of a Paenibacillus sp. BP‐23 recombinant clone coding for a previously described endoglucanase revealed the presence of an additional truncated ORF with homology to family 48 glycosyl hydrolases. The corresponding 3509‐bp DNA fragment was isolated after gene walking and cloned in Escherichia coli Xl1‐Blue for expression and purification. The encoded enzyme, a cellulase of 1091 amino acids with a deduced molecular mass of 118 kDa and a pI of 4.85, displayed a multidomain organization bearing a canonical family 48 catalytic domain, a bacterial type 3a cellulose‐binding module, and a putative fibronectin‐III domain. The cloned cellulase, unique among Bacillales and designated Cel48C, was purified through affinity chromatography using its ability to bind Avicel. Maximum activity was achieved at 45 °C and pH 6.0 on acid‐swollen cellulose, bacterial microcrystalline cellulose, Avicel and cellodextrins, whereas no activity was found on carboxy methyl cellulose, cellobiose, cellotriose, p NP‐glycosides or 4‐methylumbeliferyl α‐ d ‐glucoside. Cellobiose was the major product of cellulose hydrolysis, identifying Cel48C as a processive cellobiohydrolase. Although no chromogenic activity was detected from p NP‐glycosides, TLC analysis revealed the release of p ‐nitrophenyl‐glycosides and cellodextrins from these substrates, suggesting that Cel48C acts from the reducing ends of the sugar chain. Presence of such a cellobiohydrolase in Paenibacillus sp. BP‐23 would contribute to widen up its range of action on natural cellulosic substrates.