Purification and characterization of a thermostable intra-cellular beta-glucosidase with transglycosylation properties from filamentous fungus Termitomyces clypeatus.
An intra-cellular beta-glucosidase was purified to homogeneity by gel filtration, ion exchange chromatography and HPGPLC from mycelial extract of Termitomyces clypeatus in the presence of the glycosylation inhibitor 2-deoxy-d-glucose. CD spectroscopy demonstrated that the purified enzyme exhibited alpha-helical conformation. MALDI-TOF identified the enzyme's molecular weight as 6688Daltons, but SDS-PAGE and immunoblotting indicated that the enzyme formed aggregates. The enzyme also showed unique properties of co-aggregation with sucrase in the fungus. The enzyme showed around 80% stability up to 60 degrees C and residual activity was 80-100% between pH ranges 5-8. The enzyme had higher specific activity against p-nitrophenyl-d-glucopyranoside than cellobiose and HPLC showed that the enzyme possesses transglycosylation activity and synthesizes cello-oligosaccharides by addition of glucose. The enzyme will be useful in synthetic biology to produce complex bioactive glycosides and to avoid chemical hazards. This is the first report of a beta-glucosidase enzyme with such a low monomeric unit size.
Pal
Swagata
S
Indian Institute of Chemical Biology, Drug Development and Biotechnology, Kolkata 700 032, India.
Banik
Samudra Prosad
SP
Ghorai
Shakuntala
S
Chowdhury
Sudeshna
S
Khowala
Suman
S
D000596:Amino Acids / Q000032:analysis; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000187:drug effects; D006031:Glycosylation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D042541:Intracellular Space / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature; D055434:Termitomyces / Q000187:drug effects / Q000201:enzymology*; D001617:beta-Glucosidase / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D000596:Amino Acids; D008670:Metals; D001617:beta-Glucosidase