Thermal stability engineering of Glomerella cingulata cutinase

Cutinase has been ascertained as a biocatalyst for biotechnological and industrial bioprocesses. The Glomerella cingulata cutinase was genetically modified to enhance its enzymatic performance to fulfill industrial requirements. Two sites were selected for mutagenesis with the aim of altering the su...

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Bibliographic Details
Published in:Protein engineering, design and selection design and selection, 2013-05, Vol.26 (5), p.369-375
Main Authors: Chin, Iuan-Sheau, Murad, Abdul Munir Abdul, Mahadi, Nor Muhammad, Nathan, Sheila, Bakar, Farah Diba Abu
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Carboxylic Ester Hydrolases - chemistry
Carboxylic Ester Hydrolases - genetics
Carboxylic Ester Hydrolases - metabolism
Models, Molecular
Molecular Sequence Data
Mutagenesis, Site-Directed
Phyllachorales - chemistry
Phyllachorales - enzymology
Phyllachorales - genetics
Protein Engineering
Protein Unfolding
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Sequence Alignment
Temperature
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Summary:Cutinase has been ascertained as a biocatalyst for biotechnological and industrial bioprocesses. The Glomerella cingulata cutinase was genetically modified to enhance its enzymatic performance to fulfill industrial requirements. Two sites were selected for mutagenesis with the aim of altering the surface electrostatics as well as removing a potentially deamidation-prone asparagine residue. The N177D cutinase variant was affirmed to be more resilient to temperature increase with a 2.7-fold increase in half-life at 50°C as compared with wild-type enzyme, while, the activity at 25°C is not compromised. Furthermore, the increase in thermal tolerance of this variant is accompanied by an increase in optimal temperature. Another variant, the L172K, however, exhibited higher enzymatic performance towards phenyl ester substrates of longer carbon chain length, yet its thermal stability is inversely affected. In order to restore the thermal stability of L172K, we constructed a L172K/N177D double variant and showed that these two mutations yield an improved variant with enhanced activity towards phenyl ester substrates and enhanced thermal stability. Taken together, our study may provide valuable information for enhancing catalytic performance and thermal stability in future engineering endeavors.
ISSN:1741-0126
1741-0134
DOI:10.1093/protein/gzt007