title: Expression, crystal structure and cellulase activity of the thermostable cellobiohydrolase Cel7A from the fungus Humicola grisea var. thermoidea.
abstract: Glycoside hydrolase family 7 (GH7) cellobiohydrolases (CBHs) play a key role in biomass recycling in nature. They are typically the most abundant enzymes expressed by potent cellulolytic fungi, and are also responsible for the majority of hydrolytic potential in enzyme cocktails for industrial processing of plant biomass. The thermostability of the enzyme is an important parameter for industrial utilization. In this study, Cel7 enzymes from different fungi were expressed in a fungal host and assayed for thermostability, including Hypocrea jecorina Cel7A as a reference. The most stable of the homologues, Humicola grisea var. thermoidea Cel7A, exhibits a 10°C higher melting temperature (T(m) of 72.5°C) and showed a 4-5 times higher initial hydrolysis rate than H. jecorina Cel7A on phosphoric acid-swollen cellulose and showed the best performance of the tested enzymes on pretreated corn stover at elevated temperature (65°C, 24 h). The enzyme shares 57% sequence identity with H. jecorina Cel7A and consists of a GH7 catalytic module connected by a linker to a C-terminal CBM1 carbohydrate-binding module. The crystal structure of the H. grisea var. thermoidea Cel7A catalytic module (1.8 Å resolution; R(work) and R(free) of 0.16 and 0.21, respectively) is similar to those of other GH7 CBHs. The deviations of several loops along the cellulose-binding path between the two molecules in the asymmetric unit indicate higher flexibility than in the less thermostable H. jecorina Cel7A.
authors.affiliation: Department of Chemistry and Biotechnology, Swedish University of Agricultural Sciences, PO Box 7015, SE-750 07 Uppsala, Sweden.
authors.forename: Majid Haddad
authors.initials: MH
authors.lastname: Momeni
authors.affiliation: DuPont, Industrial Biosciences, Archimedesweg 30, 2333 CN Leiden, The Netherlands.
authors.forename: Frits
authors.initials: F
authors.lastname: Goedegebuur
authors.affiliation: Department of Chemistry and Biotechnology, Swedish University of Agricultural Sciences, PO Box 7015, SE-750 07 Uppsala, Sweden.
authors.forename: Henrik
authors.initials: H
authors.lastname: Hansson
authors.affiliation: Department of Chemistry and Biotechnology, Swedish University of Agricultural Sciences, PO Box 7015, SE-750 07 Uppsala, Sweden.
authors.forename: Saeid
authors.initials: S
authors.lastname: Karkehabadi
authors.affiliation: Department of Chemistry and Biotechnology, Swedish University of Agricultural Sciences, PO Box 7015, SE-750 07 Uppsala, Sweden.
authors.forename: Glareh
authors.initials: G
authors.lastname: Askarieh
authors.affiliation: DuPont, Industrial Biosciences, Page Mill Road, Palo Alto, CA 94304, USA.
authors.forename: Colin
authors.initials: C
authors.lastname: Mitchinson
authors.affiliation: DuPont, Industrial Biosciences, Page Mill Road, Palo Alto, CA 94304, USA.
authors.forename: Edmundo A
authors.initials: EA
authors.lastname: Larenas
authors.affiliation: Department of Chemistry and Biotechnology, Swedish University of Agricultural Sciences, PO Box 7015, SE-750 07 Uppsala, Sweden.
authors.forename: Jerry
authors.initials: J
authors.lastname: Ståhlberg
authors.affiliation: Department of Chemistry and Biotechnology, Swedish University of Agricultural Sciences, PO Box 7015, SE-750 07 Uppsala, Sweden.
authors.forename: Mats
authors.initials: M
authors.lastname: Sandgren
mesh_terms: D000595:Amino Acid Sequence; D002480:Cellulase / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D020037:Sordariales / Q000201:enzymology*
chemical_list: D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase
keywords: Cel7A; cellobiohydrolase