Enhancement of extracellular cellobiase activity by reducing agents in the filamentous fungus Termitomyces clypeatus

• Published in: Biotechnology letters 2015-01, Vol.37 (1), p.175-181
• Main Authors: Banik, Samudra Prosad, Mukherjee, Soumya, Pal, Swagata, Ghorai, Shakuntala, Majumder, Rajib, Khowala, Suman
• Format: Article
• Language: English
• Subjects: Affinity; Applied Microbiology; beta-glucosidase; beta-Glucosidase - antagonists & inhibitors; beta-Glucosidase - drug effects; beta-Glucosidase - metabolism; Biocatalysts; Biochemistry; Biomedical and Life Sciences; Biotechnology; Catalysis; catalytic activity; Cellobiase; cellobiose; Cysteine; disulfide bonds; Disulfides; dithiothreitol; Dithiothreitol - chemistry; Dithiothreitol - pharmacology; Enzymes; Extracellular Space - drug effects; Extracellular Space - enzymology; Fluorescence; Fungal Proteins - drug effects; Fungal Proteins - metabolism; Fungi; Glucose; Glucose - metabolism; industrial applications; Kinetics; Life Sciences; Mercaptoethanol - chemistry; Mercaptoethanol - pharmacology; Microbiology; Original Research Paper; reducing agents; Reducing Agents - chemistry; Reducing Agents - pharmacology; Termitomyces; Termitomyces - drug effects; Termitomyces - enzymology; Termitomyces clypeatus; tryptophan
• ISSN: 0141-5492; 1573-6776
• DOI: 10.1007/s10529-014-1669-0

Extracellular cellobiase activity of Termitomyces clypeatus increased from 2.9 U ml⁻¹to 4.4 and 4.1 in presence of dithiothreitol (DTT) and β-mercaptoethanol (ME), respectively, with a decrease in Kₘfrom 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.