Cloning, heterologous expression and characterisation of a recombinant cellobiohydrolase from Humicola insolens ATCC16454 in Pichia pastoris
A cellobiohydrolase gene from the thermophilic fungus Humicola insolens ATCC 16454 was expressed in the methylotrophic yeast Pichia pastoris X-33, and the biochemical properties of the recombinant protein were characterised. The full-length cDNA of the cellobiohydrolase gene avi2 was cloned into t...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Malaysian Society of Applied Biology
2015
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| Online Access: | http://journalarticle.ukm.my/11786/ http://journalarticle.ukm.my/11786/ http://journalarticle.ukm.my/11786/1/44_4_03.pdf |
| Summary: | A cellobiohydrolase gene from the thermophilic fungus Humicola insolens ATCC 16454 was expressed in the methylotrophic
yeast Pichia pastoris X-33, and the biochemical properties of the recombinant protein were characterised. The full-length
cDNA of the cellobiohydrolase gene avi2 was cloned into the P. pastoris expression vector pPICZαC and expressed
extracellularly as a recombinant cellobiohydrolase protein with a molecular weight of approximately 52.3 kDa. The purified
recombinant Avi2 enzyme displayed an optimal activity at 50°C and was found stable between temperatures of 30°C and
60°C. The optimal pH of the enzyme was pH 5.0. More than 80% of the enzyme activity was retained at pH values ranging
from pH3.0 to pH9.0. Recombinant Avi2 enzyme showed its highest activity towards the substrates Avicel (0.075 U mg-1)
and Sigmacell-cellulose (0.018 U mg-1). Very low or undetectable hydrolysis was observed with cellobiose and filter paper.
Metal ions, such as Mn2+, Co2+, and Ba2+, increased the activity of the recombinant enzyme. Manganese ions caused the
highest increase in activity of approximately 1.38-fold compared to the control assay. Other ions such as Pd2+, Cu2+, Zn2+,
Fe2+, and SDS, however, inhibited Avi2 enzyme activity. Interestingly, this recombinant enzyme showed high pH stability
when it was incubated in either acidic or basic solutions. |
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