An alkaline thermostable recombinant Humicola grisea var. thermoidea cellobiohydrolase presents bifunctional (endo/exoglucanase) activity on cellulosic substrates.
Humicola grisea var. thermoidea is a deuteromycete which secretes a large spectrum of hydrolytic enzymes when grown on lignocellulosic residues. This study focused on the heterologous expression and recombinant enzyme analysis of the major secreted cellulase when the fungus is grown on sugarcane bagasse as the sole carbon source. Cellobiohydrolase 1.2 (CBH 1.2) cDNA was cloned in Pichia pastoris under control of the AOX1 promoter. Recombinant protein (rCBH1.2) was efficiently produced and secreted as a functional enzyme, presenting a molecular mass of 47 kDa. Maximum enzyme production was achieved at 96 h, in culture medium supplemented with 1.34 % urea and 1 % yeast extract and upon induction with 1 % methanol. Recombinant enzyme exhibited optimum activity at 60 °C and pH 8, and presented a remarkable thermostability, particularly at alkaline pH. Activity was evaluated on different cellulosic substrates (carboxymethyl cellulose, filter paper, microcrystalline cellulose and 4-para-nitrophenyl β-D-glucopyranoside). Interestingly, rCBH1.2 presented both exoglucanase and endoglucanase activities and mechanical agitation increased substrate hydrolysis. Results indicate that rCBH1.2 is a potential biocatalyst for applications in the textile industry or detergent formulation.
Laboratório de Biotecnologia de Fungos, Departamento de Bioquímica e Biologia Molecular, Instituto de Ciências Biológicas, Campus II, Universidade Federal de Goiás, Goiânia, GO, CEP: 74001-970, Brazil.
G S
GS
Oliveira
C J
CJ
Ulhoa
M H L
MH
Silveira
J
J
Andreaus
I
I
Silva-Pereira
M J
MJ
Poças-Fonseca
F P
FP
Faria
D002482:Cellulose / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods; D003470:Culture Media / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D003904:Mitosporic Fungi / Q000201:enzymology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D013696:Temperature
D003470:Culture Media; D005656:Fungal Proteins; D011994:Recombinant Proteins; D002482:Cellulose; D043366:Cellulose 1,4-beta-Cellobiosidase