title: Enhancement of extracellular cellobiase activity by reducing agents in the filamentous fungus Termitomyces clypeatus.
abstract: Extracellular cellobiase activity of Termitomyces clypeatus increased from 2.9 U ml(-1) to 4.4 and 4.1 in presence of dithiothreitol (DTT) and β-mercaptoethanol (ME), respectively, with a decrease in Km from 0.4 to 0.3 mM (DTT) and 0.35 mM (ME). Catalysis was further enhanced if the reduced enzyme was alkylated and activity increased from 11.4 U ml(-1) (control) to 15.2 (DTT+N-ethylmaleimide) and 15.3 (DTT+iodoacetamide) using p-nitrophenyl-β-D-glucopyranoside and from 14.6 U ml(-1)(control) to 21.9 (DTT+N-ethylmaleimide) and 18.7 (DTT+iodoacetamide) using cellobiose. The reduced enzyme showed 17 % lesser glucose inhibition. CD and tryptophan fluorescence showed no change in secondary structure was caused by DTT up to 50 mM. Cysteine content of the enzyme was 24 %. It is postulated that reduction of disulphide bonds allows better substrate affinity for cellobiase. The studies describe a novel and simple method to increase cellobiase activity for industrial applications.
authors.affiliation: Department of Microbiology, Maulana Azad College, 8 Rafi Ahmed Kidwai Road, Kolkata, 700013, India.
authors.forename: Samudra Prosad
authors.initials: SP
authors.lastname: Banik
authors.forename: Soumya
authors.initials: S
authors.lastname: Mukherjee
authors.forename: Swagata
authors.initials: S
authors.lastname: Pal
authors.forename: Shakuntala
authors.initials: S
authors.lastname: Ghorai
authors.forename: Rajib
authors.initials: R
authors.lastname: Majumder
authors.forename: Suman
authors.initials: S
authors.lastname: Khowala
mesh_terms: D004229:Dithiothreitol / Q000737:chemistry / Q000494:pharmacology; D005110:Extracellular Space / Q000187:drug effects / Q000201:enzymology*; D005656:Fungal Proteins / Q000187:drug effects / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D007700:Kinetics; D008623:Mercaptoethanol / Q000737:chemistry / Q000494:pharmacology; D019163:Reducing Agents / Q000737:chemistry / Q000494:pharmacology*; D055434:Termitomyces / Q000187:drug effects / Q000201:enzymology*; D001617:beta-Glucosidase / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D019163:Reducing Agents; D008623:Mercaptoethanol; D001617:beta-Glucosidase; D005947:Glucose; D004229:Dithiothreitol