title: Identification of a β-glucosidase from the Mucor circinelloides genome by peptide pattern recognition.
abstract: Mucor circinelloides produces plant cell wall degrading enzymes that allow it to grow on complex polysaccharides. Although the genome of M. circinelloides has been sequenced, only few plant cell wall degrading enzymes are annotated in this species. We applied peptide pattern recognition, which is a non-alignment based method for sequence analysis to map conserved sequences in glycoside hydrolase families. The conserved sequences were used to identify similar genes in the M. circinelloides genome. We found 12 different novel genes encoding members of the GH3, GH5, GH9, GH16, GH38, GH47 and GH125 families in M. circinelloides. One of the two GH3-encoding genes was predicted to encode a β-glucosidase (EC 3.2.1.21). We expressed this gene in Pichia pastoris KM71H and found that the purified recombinant protein had relative high β-glucosidase activity (1.73U/mg) at pH5 and 50°C. The Km and Vmax with p-nitrophenyl-β-d-glucopyranoside as substrate was 0.20mM and 2.41U/mg, respectively. The enzyme was not inhibited by glucose and retained 84% activity at glucose concentrations up to 140mM. Although zygomycetes are not considered to be important degraders of lignocellulosic biomass in nature, the present finding of an active β-glucosidase in M. circinelloides demonstrates that enzymes from this group of fungi have a potential for cellulose degradation.
authors.affiliation: Section for Sustainable Biotechnology, Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University Copenhagen, DK-2450 Copenhagen SV, Denmark; Environmental Microbiology Key Laboratory of Sichuan Province, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, Sichuan 610041, China; University of the Chinese Academy of Sciences, Beijing 100049, China. Electronic address: huyu@bio.aau.dk.
authors.forename: Yuhong
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Section for Sustainable Biotechnology, Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University Copenhagen, DK-2450 Copenhagen SV, Denmark. Electronic address: pkb@bio.aau.dk.
authors.forename: Peter Kamp
authors.initials: PK
authors.lastname: Busk
authors.affiliation: Section for Sustainable Biotechnology, Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University Copenhagen, DK-2450 Copenhagen SV, Denmark. Electronic address: mgrell90@gmail.com.
authors.forename: Morten Nedergaard
authors.initials: MN
authors.lastname: Grell
authors.affiliation: Environmental Microbiology Key Laboratory of Sichuan Province, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, Sichuan 610041, China. Electronic address: zhaohai@cib.ac.cn.
authors.forename: Hai
authors.initials: H
authors.lastname: Zhao
authors.affiliation: Section for Sustainable Biotechnology, Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University Copenhagen, DK-2450 Copenhagen SV, Denmark. Electronic address: lla@adm.aau.dk.
authors.forename: Lene
authors.initials: L
authors.lastname: Lange
mesh_terms: D001673:Biodegradation, Environmental; D017124:Conserved Sequence; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016681:Genome, Fungal; D007700:Kinetics; D008031:Lignin / Q000378:metabolism; D008958:Models, Molecular; D009089:Mucor / Q000201:enzymology* / Q000235:genetics*; D010363:Pattern Recognition, Automated / Q000379:methods; D010802:Phylogeny; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020539:Sequence Analysis, Protein / Q000379:methods; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; C036909:lignocellulose; D008031:Lignin; D001617:beta-Glucosidase
keywords: Glycoside hydrolase 3; Mucor circinelloides; Peptide pattern recognition; β-Glucosidase