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cDNA Cloning of the Cellobiohydrolase Gene cbh1 from hermoascus aurantiacus var. levisporus and Its Expression in Pichia pastoris
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| CHEN Jing;LI Duo-chuan;ZHANG Yu-qin;DU Xin-ke |
| Department of Environmental Biology, College of Plant Protection,Shandong Agricultural University, Tai’an 271018, China |
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Abstract The cellobiohydrolase gene cbh1 fragment (GenBank Accession No. AY840982) was amplified by RT-PCR from thermophilic fungus Thermoascus aurantiacus var. levisporus. RACE was used to obtain its full-length cDNA (1710 bp), encoding 457 amino acids. The first 19 amino acids of the deduced amino acid sequence were presumed to be a signal peptide. The fragment encoding mature cellobiohydrolase was inserted into Pichia pastoris vector pPIC9K to construct recombinant plasmid pPIC9K/cbh1. The pPIC9K/cbh1 was then introduced into Pichia pastoris GS115 and 61 transformants were obtained, After comfirmed by G418 risistance and PCR, and induced expression, one clone GSp-15 was selected from the 61 transformants. The expression level of GSp-15 was 1.17 mg/mL after induction for 144 h in methanol, and its activity was 20.3 U/mL with p-NPC as substrate.
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Received: 30 May 2005
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Corresponding Authors:
LI Duo-chuan
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