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KMID : 1094720120170030506
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Biotechnology and Bioprocess Engineering
2012 Volume.17 No. 3 p.506 ~ p.511
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Screening of stable cutinase from Fusarium solani pisi using plasmid display system
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Park Sang-Tae
Yoo Young-Je
Min Kyoung-Seon
Choi Yoo-Seong
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Abstract
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Although enzymes are potential candidates for industrial catalysts, their industrial applications have been limited because they are easily deactivated under harsh operational conditions. In this study, a plasmid display system was used for the screening of stable cutinase in organic solvent (20% acetonitrile) and at high temperature. The fusion proteins were expressed and bound to specific DNA sequences on the encoding plasmids. Proteolysis resistance was used as a selection tool, where well-folded proteins are more resistant to the protease digestion than poorly-folded proteins. Stable mutants, identified to be I183T, I183F, and A56V, were screened in the organic solvent and at high temperature. The I183T and I183F mutants were more stable than the A56V mutant in 20% acetonitrile, while the A56V mutant was superior to the I183T and I183F mutants at high temperature. Molecular modeling was performed in order to investigate the residual characteristics of the stable mutants; secondary structure, residual solvation energy, residual α-carbon flexibility, number of hydrogen bonds, number of neighboring amino acids, ratio of exposed/buried residue, and surface area. This analysis provided some guidelines for increased stability.
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KEYWORD
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plasmid display system, cutinase, thermostability, stability in organic solvent
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