Enhanced activity and stability of cellobiase (beta-glucosidase: EC 3.2.1.21) produced in the presence of 2-deoxy-D-glucose from the fungus Termitomyces clypeatus.
Generally less glycosylation or deglycosylation has a detrimental effect on enzyme activity and stability. Increased production and secretion of cellobiase was earlier obtained in the presence of the glycosylation inhibitor 2-deoxy-d-glucose in filamentous fungus Termitomyces clypeatus [Mukherjee, S.; Chowdhury, S.; Ghorai, S.; Pal, S.; Khowala, S. Biotechnol. Lett.2006, 28, 1773-1778]. In this study the enzyme was purified from the culture medium by ultrafiltration and gel-permeation, ion-exchange and high-performance liquid chromatography, and its catalytic activity was six times higher compared to the control enzyme. K(m) and V(max) of the purified enzyme were measured as 0.187 mM and 0.018 U mg(-1), respectively, using pNPG as the substrate. The enzyme had temperature and pH optima at 45 degrees C and pH 5.4, respectively, and retained full activity in a pH range of 5-8 and temperatures of 30-60 degrees C. Interestingly less glycosylated cellobiase was resistant towards proteolytic as well as endoglycosidase-H digestion and showed higher stability than native enzyme due to increased aggregation of the protein. The enzyme also showed higher specific activity in the presence of cellobiose and pNPG and less susceptibility towards salts and different chemical agents. The beta-glucosidase can be considered as a potentially useful enzyme in various food-processing, pharmaceutical and fermentation industries.
Ghorai
Shakuntala
S
Indian Institute of Chemical Biology (Unit of CSIR, Govt. of India), Drug Development and Biotechnology Division, 4, Raja S. C. Mullick Road, Kolkata 700 032, India.
Chowdhury
Sudeshna
S
Pal
Swagata
S
Banik
Samudra Prosad
SP
Mukherjee
Sumana
S
Khowala
Suman
S
D015153:Blotting, Western; D003847:Deoxyglucose / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013696:Temperature; D055434:Termitomyces / Q000187:drug effects* / Q000201:enzymology*; D001617:beta-Glucosidase / Q000378:metabolism*
D003847:Deoxyglucose; D001617:beta-Glucosidase