Roles of the Catalytic Domain and Two Cellulose Binding Domains of Thermomonospora fusca E4 in Cellulose Hydrolysis

• DOI: 10.1128/jb.180.7.1709-1714.1998
• Publication Date: 2019-12-31T16:31:40Z
• Authors: Diana Irwin; Dong-Hoon Shin; Sheng Zhang; Brian K. Barr; Joshua Sakon; P. Andrew Karplus; David B. Wilson
• Keywords: Carboxymethyl cellulose

ABSTRACT Thermomonospora fusca E4 is an unusual 90.4-kDa endocellulase comprised of a catalytic domain (CD), internal family IIIc cellulose binding (CBD), fibronectinlike domain, and II CBD. Constructs containing the CD alone (E4-51), plus CBD (E4-68), (E4-74) were made by using recombinant DNA techniques. The activities each purified protein on bacterial microcrystalline (BMCC), filter paper, swollen cellulose, carboxymethyl measured. Only whole enzyme, E4-90, could reach target digestion 4.5% paper. Removal (E4-51 E4-74) decreased activity markedly every substrate. E4-74 did bind to BMCC but had almost no hydrolytic activity, while E4-68 retained 32% even though it not bind. A low-activity mutant one bases, (Asp55Cys), BMCC, although E4-51 (Asp55Cys) not. ratios soluble insoluble reducing sugar produced after paper hydrolysis E4-68, E4-74, 6.9, 3.5, 1.3, 0.6, respectively, indicating that important for processivity.