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.
- Gene cloning
- Glycosyl hydrolase
- Heterologous expression
- Secondary structure prediction