Expression in Trichoderma reesei and characterisation of a thermostable family 3 beta-glucosidase from the moderately thermophilic fungus Talaromyces emersonii.
The 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 Cel3b (GenBank ) from T. reesei, and a beta-glucosidase from Aspergillus Niger (GenBank ), respectively. The heterologously expressed enzyme, Cel3a, 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. Cel3a was thermostable with an optimum temperature of 71.5 degrees C and half life of 62 min at 65 degrees C and was a specific beta-glucosidase with no beta-galactosidase side activity. Cel3a had a high specific activity against p-nitrophenyl-beta-D-glucopyranoside (Vmax, 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.
Murray
Patrick
P
Molecular Glycobiotechnology Group, Department of Biochemistry, National University of Ireland, Galway, Ireland.
Aro
Nina
N
Collins
Catherine
C
Grassick
Alice
A
Penttilä
Merja
M
Saloheimo
Markku
M
Tuohy
Maria
M
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D032901:Talaromyces / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D013696:Temperature; D014242:Trichoderma / Q000235:genetics*; D001617:beta-Glucosidase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; D001617:beta-Glucosidase