title: Analysis of functional domains of endoglucanases from Clostridium cellulovorans by gene cloning, nucleotide sequencing and chimeric protein construction.
abstract: The nucleotide sequence of engD, an endo-beta-1,4-glucanase gene from Clostridium cellulovorans was determined (Genbank Accession No. M37434). The COOH-terminal part of the gene product, EngD, contained a Thr-Thr-Pro repeated sequence followed by a region that has homology to the exoglucanase of Cellulomonas fimi. EngD and EngB, another C. cellulovorans endoglucanase, show 75% amino acid sequence homology at their NH2-termini, in contrast to their carboxyterminal domains which show no homology. EngD had endoglucanase activity on carboxymethylcellulose (CMC), cellobiosidase activity on p-nitrophenyl-cellobioside (p-NPC), and partial hydrolytic activity on crystalline cellulose (Avicel), while EngB showed hydrolytic activity against only CMC. Chimeric proteins between EngB and EngD were constructed by exchanging the non-homologous COOH-terminal regions. Chimeric proteins that contained the NH2-terminus of EngD retained cellobiosidase activity but chimeras with the EngB NH2-terminus showed no cellobiosidase activity. Hydrolysis of crystalline cellulose (Avicelase activity) was observed only with the enzyme containing the EngD NH2-terminus and EngD COOH-terminus.
authors.affiliation: Department of Biochemistry and Biophysics, University of California, Davis 95616.
authors.forename: T
authors.initials: T
authors.lastname: Hamamoto
authors.forename: F
authors.initials: F
authors.lastname: Foong
authors.forename: O
authors.initials: O
authors.lastname: Shoseyov
authors.forename: R H
authors.initials: RH
authors.lastname: Doi
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002480:Cellulase / Q000235:genetics*; D002678:Chimera; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics*; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity
chemical_list: D002480:Cellulase