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Purification and biochemical characteristics of β‐D‐glucosidase from a thermophilic fungus, Thermomyces lanuginosus– SSBP
Author(s) -
Lin Johnson,
Pillay Balakrishna,
Singh Suren
Publication year - 1999
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1111/j.1470-8744.1999.tb01163.x
Subject(s) - cellobiose , salicin , chemistry , enzyme , thermophile , hydrolysis , molecular mass , beta glucosidase , glucoside , residue (chemistry) , biochemistry , chromatography , enzyme assay , glucosidases , cysteine , cellulase , medicine , alternative medicine , pathology
The β‐D‐glucosidase produced by Thermomyces lanuginosus‐ SSBP was purified to apparent homogeneity. The purified enzyme consisted of two identical subunits with a native molecular mass of 200 kDa. The purified β‐D‐glucosidase only hydrolysed the glucoside substrates containing a terminal, non‐reducing β‐D‐glucose residue and was active on both aryl‐β‐glucoside and cellobiose. This enzyme also exhibited less, but significant α‐D‐glucosidase activity and was capable of hydrolysing β‐1,6‐linked diglucosides and gentiobiose. The K appm, V max and k cat values for p ‐nitrophenyl‐β‐D‐glucopyranoside were calculated to be 0.075 mM, 12.12 units/mg of protein and 44.44 glucose molecules released/s respectively. The β‐D‐glucosidase retained its full activity after a 30 min incubation at 50 °C but was inactive after the same treatment at 70 °C. The enzyme appeared to be stable when the pH of the storage buffer was above 5.0. Maximal β‐D‐glucosidase activity occurred at 65 °C and pH 6.0. This enzyme was competitively inhibited by glucose, cellobiose and salicin with K i values of 0.55, 0.52 and 0.81 mM respectively. The presence of Hg 2+ and N‐bromosuccinimide inhibited the enzyme activity completely at 2 mM, while cysteine enhanced β‐D‐glucosidase activity.