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KMID : 1094720090140030266
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Biotechnology and Bioprocess Engineering
2009 Volume.14 No. 3 p.266 ~ p.273
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Construction and Characterization of Novel Chimeric β-glucosidases with Cellvibrio gilvus (CG) and Thermotoga maritima (TM) by Over Overlapping PCR
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Kim Jong-Deog
Seo Hyo-Jin
Hayashi Kiyoshi
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Abstract
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In order to create novel β-glucosidase constructs, 8 kinds of chimeric β-glucosidases were constructed using overlapping polymerase chain reaction (PCR) based on Cellvibrio gilvus (CG) and Thermotoga maritima (TM) genes. Two kinds of novel chimeric β-glucosidases (No. 6 and No. 8 type) were selected and their properties characterized. SDS-PAGE analysis showed that both constructs had a molecular mass of 80 kDa. The optimum pH of No. 6 chimeric β-glucosidase was found to be 3.0 and 5.0, showing varying maximum activity according to the buffer used. No. 8 chimeric enzyme was found to be optimally active at a pH of 4.5 and the optimum temperature of No.6 and No.8 chimeric β-glucosidases was reported to be 60°C, respectively. The Km values of both novel chimeric enzymes were calculated to be 0.012 mM and 0.0082 mM, respectively and the characteristics of the novel chimeric enzymes were to lie between those of the parental enzymes.
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KEYWORD
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Chimeric β-glucosidase, overlapping PCR, Cellvibrio gilvus (CG), Thermotoga maritima (TM), kinetic parameter
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