A novel thermostable α-galactosidase from the thermophilic fungus Thermomyces lanuginosus CBS 395.62/b: Purification and characterization

• Published in: Biochimica et biophysica acta. General subjects Vol. 1770; no. 1; pp. 55 - 62
• Main Authors: Rezessy-Szabó, Judit M., Nguyen, Quang D., Hoschke, Ágoston, Braet, Christophe, Hajós, Gyöngyi, Claeyssens, Marc
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 2007
• Subjects: Purification; Thermomyces lanuginosus; Thermophilic fungus; α-Galactosidase; Purification; α-Galactosidase; Thermophilic fungus; Thermomyces lanuginosus
• ISSN: 0304-4165; 1872-8006
• DOI: 10.1016/j.bbagen.2006.06.022

High levels of an extracellular α-galactosidase are produced by the thermophilic fungus Thermomyces lanuginosus CBS 395.62/b when grown in submerse culture and induced by sucrose. The enzyme was purified 114-fold from the culture supernatant by (NH 4) 2SO 4 fractionation, and by chromatographical steps including Sepharose CL-6B gel filtration, DEAE-Sepharose FF anion-exchange, Q-Sepharose FF anion-exchange and Superose 12 gel filtration. The purified enzyme exhibits apparent homogeneity as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and iso-electric focusing (IEF). The native molecular weight of the monomeric α-galactosidase is 93 kDa with an isoelectric point of 3.9. The enzyme displays a pH and temperature optimum of 5–5.5 and 65 °C, respectively. The purified enzyme retains more than 90% of its activity at 45 °C in a pH range from 5.5 to 9.0. The enzyme proves to be a glycoprotein and its carbohydrate content is 5.3%. Kinetic parameters were determined for the substrates p-nitrophenyl-α-galactopyranoside, raffinose and stachyose and very similar K m values of 1.13 mM, 1.61 mM and 1.17 mM were found. Mn ++ ions activates enzyme activity, whereas inhibitory effects can be observed with Ca ++, Zn ++ and Hg ++. Five min incubation at 65° with 10 mM Ag + results in complete inactivation of the purified α-galactosidase. Amino acid sequence alignment of N-terminal sequence data allows the α-galactosidase from Thermomyces lanuginosus to be classified in glycosyl hydrolase family 36.