title: Expression of novel β-glucanase Cel12A from Stachybotrys atra in bacterial and fungal hosts.
abstract: β-glucanase Cel12A from Stachybotrys atra has been cloned and expressed in Aspergillus niger. The purified enzyme showed high activity of β-1,3-1,4-mixed glucans, was also active on carboxymethylcellulose (CMC), while it did not hydrolyze crystalline cellulose or β-1,3 glucans as laminarin. Cel12A showed a marked substrate preference for β-1,3-1,4 glucans, showing maximum activity on barley β-glucans (27.69 U mg(-1)) while the activity on CMC was much lower (0.51 U mg(-1)). Analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focussing (IEF), and zymography showed the recombinant enzyme has apparent molecular weight of 24 kDa and a pI of 8.2. Optimal temperature and pH for enzyme activity were 50°C and pH 6.5. Thin layer chromatography analysis showed that major hydrolysis products from barley β-glucan and lichean were 3-O-β-cellotriosyl-D-glucose and 3-O-β-cellobiosyl-D-glucose, while glucose and cellobiose were released in smaller amounts. The amino acid sequence deduced from cel12A revealed that it is a single domain enzyme belonging to the GH12 family, a family that contains several endoglucanases with substrate preference for β-1,3-1,4 glucans. We believe that S. atra Cel12A should be considered as a lichenase-like or nontypical endoglucanase.
authors.affiliation: Department of Microbiology, University of Barcelona, Avinguda Diagonal 645, 08028 Barcelona, Spain. perepicart@hotmail.com
authors.forename: Pere
authors.initials: P
authors.lastname: Picart
authors.forename: Frits
authors.initials: F
authors.lastname: Goedegebuur
authors.forename: Pilar
authors.initials: P
authors.lastname: Díaz
authors.forename: F I
authors.initials: FI
authors.lastname: Javier Pastor
mesh_terms: D001234:Aspergillus niger / Q000235:genetics; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D015870:Gene Expression; D005936:Glucans / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013191:Stachybotrys / Q000201:enzymology* / Q000235:genetics*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D004271:DNA, Fungal; D005936:Glucans; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C028020:licheninase