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

The gene encoding a thermostable β-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 β-glucosidase falls into glycosyl hydrolase family 3, showing approximately 56 and 67% identity with Cel3b (GenBank AAP57755) from T. reesei, and a β-glucosidase from Aspergillus Niger (GenBank CAB75696), respectively. The heterologously expressed enzyme, Cel3a, was a dimer equal to 130 kDa subunits with 17 potential N-glycosylation sites and a previously unreported β-glucosidase activity produced extracellularly by Tal. emersonii. Cel3a was thermostable with an optimum temperature of 71.5 °C and half life of 62 min at 65 °C and was a specific β-glucosidase with no β-galactosidase side activity. Cel3a had a high specific activity against p-nitrophenyl-β- d-glucopyranoside ( V max, 512 IU/mg) and was competitively inhibited by glucose ( k i, 0.254 mM). Cel3a was also active against natural cellooligosacharides with glucose being the product of hydrolysis. It displayed transferase activity producing mainly cellobiose from glucose and cellotetrose from cellobiose.