title: Influence of a mannan binding family 32 carbohydrate binding module on the activity of the appended mannanase.
abstract: In general, cellulases and hemicellulases are modular enzymes in which the catalytic domain is appended to one or more noncatalytic carbohydrate binding modules (CBMs). CBMs, by concentrating the parental enzyme at their target polysaccharide, increase the capacity of the catalytic module to bind the substrate, leading to a potentiation in catalysis. Clostridium thermocellum hypothetical protein Cthe_0821, defined here as C. thermocellum Man5A, is a modular protein comprising an N-terminal signal peptide, a family 5 glycoside hydrolase (GH5) catalytic module, a family 32 CBM (CBM32), and a C-terminal type I dockerin module. Recent proteomic studies revealed that Cthe_0821 is one of the major cellulosomal enzymes when C. thermocellum is cultured on cellulose. Here we show that the GH5 catalytic module of Cthe_0821 displays endomannanase activity. C. thermocellum Man5A hydrolyzes soluble konjac glucomannan, soluble carob galactomannan, and insoluble ivory nut mannan but does not attack the highly galactosylated mannan from guar gum, suggesting that the enzyme prefers unsubstituted β-1,4-mannoside linkages. The CBM32 of C. thermocellum Man5A displays a preference for the nonreducing ends of mannooligosaccharides, although the protein module exhibits measurable affinity for the termini of β-1,4-linked glucooligosaccharides such as cellobiose. CBM32 potentiates the activity of C. thermocellum Man5A against insoluble mannans but has no significant effect on the capacity of the enzyme to hydrolyze soluble galactomannans and glucomannans. The product profile of C. thermocellum Man5A is affected by the presence of CBM32.
authors.affiliation: Applied Microbiology Laboratory, Graduate School of Bioresources, Mie University, Tsu, Japan.
authors.forename: Kimiya
authors.initials: K
authors.lastname: Mizutani
authors.forename: Vânia O
authors.initials: VO
authors.lastname: Fernandes
authors.forename: Shuichi
authors.initials: S
authors.lastname: Karita
authors.forename: Ana S
authors.initials: AS
authors.lastname: Luís
authors.forename: Makiko
authors.initials: M
authors.lastname: Sakka
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Kimura
authors.forename: Adam
authors.initials: A
authors.lastname: Jackson
authors.forename: Xiaoyang
authors.initials: X
authors.lastname: Zhang
authors.forename: Carlos M G A
authors.initials: CM
authors.lastname: Fontes
authors.forename: Harry J
authors.initials: HJ
authors.lastname: Gilbert
authors.forename: Kazuo
authors.initials: K
authors.lastname: Sakka
mesh_terms: D000595:Amino Acid Sequence; D002241:Carbohydrates / Q000737:chemistry; D048013:Acetivibrio thermocellus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D005685:Galactans / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D008351:Mannans / Q000378:metabolism*; D008361:Mannosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D053149:Plant Gums / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D002241:Carbohydrates; D005685:Galactans; D008351:Mannans; D053149:Plant Gums; D011994:Recombinant Proteins; C022901:(1-6)-alpha-glucomannan; C007894:guar gum; D006026:Glycoside Hydrolases; D008361:Mannosidases; C412205:beta-1,4-mannosidase