Identifying and characterizing the most significant β-glucosidase of the novel species  Aspergillus saccharolyticus.
The newly discovered fungal species  Aspergillus saccharolyticus  was found to produce a culture broth rich in β-glucosidase activity. In this present work, the main β-glucosidase of A. saccharolyticus responsible for the efficient hydrolytic activity was identified, isolated, and characterized. Ion exchange chromatography was used to fractionate the culture broth, yielding fractions with high β-glucosidase activity and only 1 visible band on an SDS-PAGE gel. Mass spectrometry analysis of this band gave peptide matches to β-glucosidases from aspergilli. Through a polymerase chain reaction approach using degenerate primers and genome walking, a 2919 bp sequence encoding the 860 amino acid BGL1 polypeptide was determined. BGL1 of A. saccharolyticus has 91% and 82% identity with BGL1 from  Aspergillus aculeatus  and BGL1 from  Aspergillus niger , respectively, both belonging to Glycoside Hydrolase family 3. Homology modeling studies suggested β-glucosidase activity with preserved retaining mechanism and a wider catalytic pocket compared with other β-glucosidases. The bgl1 gene was heterologously expressed in  Trichoderma reesei  QM6a, purified, and characterized by enzyme kinetics studies. The enzyme can hydrolyze cellobiose, p-nitrophenyl-β-d-glucoside, and cellodextrins. The enzyme showed good thermostability, was stable at 50 °C, and at 60 °C it had a half-life of approximately 6 h.
Section for Sustainable Biotechnology, Aalborg University Copenhagen, Denmark.
Annette
A
Sørensen
Birgitte K
BK
Ahring
Mette
M
Lübeck
Wimal
W
Ubhayasekera
Kenneth S
KS
Bruno
David E
DE
Culley
Peter S
PS
Lübeck
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics; D002475:Cellobiose / Q000378:metabolism; D002482:Cellulose / Q000031:analogs & derivatives / Q000378:metabolism; D003912:Dextrins / Q000378:metabolism; D006207:Half-Life; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013696:Temperature; D014242:Trichoderma / Q000235:genetics; D001617:beta-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D003912:Dextrins; D002475:Cellobiose; D002482:Cellulose; C068994:cellodextrin; D001617:beta-Glucosidase