Thermal stability engineering of Glomerella cingulata cutinase

• 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
• ISSN: 1741-0126; 1741-0134
• DOI: 10.1093/protein/gzt007

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.