Expression in Trichoderma reesei and characterisation of a thermostable family 3 beta-glucosidase from the moderately thermophilic fungus Talaromyces emersonii

摘要

T he gene encoding a thermostable beta-glucosidase (cel3a) was isolated from the thermophilic fungus Talalaromyces emersonii by degenerate PCR and expressed in the filamentous fungus Trichoderma reesei. The cel3a gene encodes an 857 amino acid long protein with a calculated molecular weight of 90.59 kDa. Tal. emersonii beta-glucosidase falls into glycosyl hydrolase family 3, showing approximately 56 and 67% identity with Ce13b (GenBank AAP57755) from T reesei, and a beta-glucosidase from Aspergillus Niger (GenBank CAB75696), respectively. The heterologously expressed enzyme, Ce13a, was a dimer equal to 130 kDa subunits with 17 potential N-glycosylation sites and a previously unreported beta-glucosidase activity produced extracellularly by Tal. emersonii. Ce13a was thermostable with an optimum temperature of 71.5 degreesC and half life of 62 min at 65 degreesC and was a specific beta-glucosidase with no beta-galactosidase side activity. Ce13a had a high specific activity against p-nitrophenyl-beta-D-glucopyranoside (V-max 512IU/mg) and was competitively inhibited by glucose (k(i), 0.254 mM). Ce13a was also active against natural cello oligo sacharides with glucose being the product of hydrolysis. It displayed transferase activity producing mainly cellobiose from glucose and cellotetrose from cellobiose. (C) 2004 Elsevier Inc. All rights reserved.