[Cloning and expressing of cellulase gene (cbh2) from thermophilic fungi Chaetomium thermophilum CT2].
Chaetomium thermophilum CT2 can produce extracellular cellulase with industrial value. We designed two degenerate primers to amplify catalytic domain sequence of cellobiohydrolase II ( CBH II). Full length of cDNA was obtained by rapid amplification of cDNA ends technologies. DNA sequencing revealed that cbh2 has an open reading frame of 1428bp, which encodes a putative polypeptide of 476 amino acids. The deduced amino acid sequence shows that the predicted molecular mass is 53 kD and the cbh2 consists of a fungal-type carbohydrate binding domain (CBD) separated from a catalytic domain by a linker region rich in proline/serine/threonine. PCR product consisting of the entire CBH II coding region without its signal sequences was cloned into the yeast secretive plasmid pPIC9K, which was then transformed into Pichia pastoris GS115. Highly efficient production of the cellobiohydrolase II was achieved in P. pastoris under the control of the AOX1 promoter, and the expressing level was 1.2 mg/mL by small-scale culturing. The recombinant cellobiohydrolase II was purified by using ammonium sulfate fraction, DEAE-Sepharose Fast flow chromatography. A molecular mass of the purified enzyme is 67 kD determined by SDS-PAGE and this is similar to the native cellobiohydrolase II purified from C. thermophilum CT2. The recombinant enzyme exhibited optimum catalytic activity at pH 4.0 and 50 degrees C respectively. It was thermostable at 50 degrees C and retained 50% of its original activity after 30 min at 70 d degrees C . The high level of fully active recombinant cellobiohydrolase II got from P. pastoris makes this expression system attractive for fermentor and industrial applications.
Liu
Shou-An
SA
Department of Environmental Biology, Shandong Agriculture University, Tai' an 271018, China.
Li
Duo-Chuan
DC
E
Shi-Jin
SJ
Zhang
Yan
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000096:biosynthesis* / Q000235:genetics; D002597:Chaetomium / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D018076:DNA, Complementary; D005656:Fungal Proteins; D011994:Recombinant Proteins; D043366:Cellulose 1,4-beta-Cellobiosidase