Abstract
Cutinase is an important biocatalyst for various industrial applications. This enzyme which has dual functionality comparable to esterases and lipases, is efficient in the hydrolysis of soluble esters and emulsified triacylglycerols. Naturally-occurring enzymes usually have disadvantages when applied in non-natural catalysis due to Glomerella cingulata cutinase enzyme thermostability. It is postulated that by increasing the rigidity at certain amino acid positions showing high mobility based on the three-dimensional structure of G. cingulata cutinase, the improvement in thermostability will be achieved. The amino acid N82 of G. cingulata cutinase was selected based on its high B-factor value determined via the B-FITTER program. Megaprimer PCR was employed to introduce mutations at the chosen site by randomization using NNK degenerate primers. About 300 transformants were selected for screening of positive cutinase variants. The N82-V14 cutinase variant was observed to be more thermostable at an almost 2-fold increase when exposed at 50°C for 1 hr as compared to the wild-type enzyme. This study may provide valuable information regarding thermal stability of cutinases denaturation at high temperatures.
| Original language | English |
|---|---|
| Title of host publication | 2015 UKM FST Postgraduate Colloquium: Proceedings of the Universiti Kebangsaan Malaysia, Faculty of Science and Technology 2015 Postgraduate Colloquium |
| Publisher | American Institute of Physics Inc. |
| Volume | 1678 |
| ISBN (Electronic) | 9780735413252 |
| DOIs | |
| Publication status | Published - 25 Sep 2015 |
| Event | 2015 Postgraduate Colloquium of the Universiti Kebangsaan Malaysia, Faculty of Science and Technology, UKM FST 2015 - Selangor, Malaysia Duration: 15 Apr 2015 → 16 Apr 2015 |
Other
| Other | 2015 Postgraduate Colloquium of the Universiti Kebangsaan Malaysia, Faculty of Science and Technology, UKM FST 2015 |
|---|---|
| Country | Malaysia |
| City | Selangor |
| Period | 15/4/15 → 16/4/15 |
Fingerprint
ASJC Scopus subject areas
- Physics and Astronomy(all)
Cite this
Site-saturation mutagenesis of Glomerella cingulata cutinase gene for enhanced enzyme thermostability. / Hanapi, Wan Nurhidayah Wan; Iuan-Sheau, Chin; Mahadi, Nor Muhammad; Abd. Murad, Abdul Munir; Abu Bakar, Farah Diba.
2015 UKM FST Postgraduate Colloquium: Proceedings of the Universiti Kebangsaan Malaysia, Faculty of Science and Technology 2015 Postgraduate Colloquium. Vol. 1678 American Institute of Physics Inc., 2015. 030021.Research output: Chapter in Book/Report/Conference proceeding › Conference contribution
}
TY - GEN
T1 - Site-saturation mutagenesis of Glomerella cingulata cutinase gene for enhanced enzyme thermostability
AU - Hanapi, Wan Nurhidayah Wan
AU - Iuan-Sheau, Chin
AU - Mahadi, Nor Muhammad
AU - Abd. Murad, Abdul Munir
AU - Abu Bakar, Farah Diba
PY - 2015/9/25
Y1 - 2015/9/25
N2 - Cutinase is an important biocatalyst for various industrial applications. This enzyme which has dual functionality comparable to esterases and lipases, is efficient in the hydrolysis of soluble esters and emulsified triacylglycerols. Naturally-occurring enzymes usually have disadvantages when applied in non-natural catalysis due to Glomerella cingulata cutinase enzyme thermostability. It is postulated that by increasing the rigidity at certain amino acid positions showing high mobility based on the three-dimensional structure of G. cingulata cutinase, the improvement in thermostability will be achieved. The amino acid N82 of G. cingulata cutinase was selected based on its high B-factor value determined via the B-FITTER program. Megaprimer PCR was employed to introduce mutations at the chosen site by randomization using NNK degenerate primers. About 300 transformants were selected for screening of positive cutinase variants. The N82-V14 cutinase variant was observed to be more thermostable at an almost 2-fold increase when exposed at 50°C for 1 hr as compared to the wild-type enzyme. This study may provide valuable information regarding thermal stability of cutinases denaturation at high temperatures.
AB - Cutinase is an important biocatalyst for various industrial applications. This enzyme which has dual functionality comparable to esterases and lipases, is efficient in the hydrolysis of soluble esters and emulsified triacylglycerols. Naturally-occurring enzymes usually have disadvantages when applied in non-natural catalysis due to Glomerella cingulata cutinase enzyme thermostability. It is postulated that by increasing the rigidity at certain amino acid positions showing high mobility based on the three-dimensional structure of G. cingulata cutinase, the improvement in thermostability will be achieved. The amino acid N82 of G. cingulata cutinase was selected based on its high B-factor value determined via the B-FITTER program. Megaprimer PCR was employed to introduce mutations at the chosen site by randomization using NNK degenerate primers. About 300 transformants were selected for screening of positive cutinase variants. The N82-V14 cutinase variant was observed to be more thermostable at an almost 2-fold increase when exposed at 50°C for 1 hr as compared to the wild-type enzyme. This study may provide valuable information regarding thermal stability of cutinases denaturation at high temperatures.
UR - http://www.scopus.com/inward/record.url?scp=85006230954&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85006230954&partnerID=8YFLogxK
U2 - 10.1063/1.4931242
DO - 10.1063/1.4931242
M3 - Conference contribution
AN - SCOPUS:85006230954
VL - 1678
BT - 2015 UKM FST Postgraduate Colloquium: Proceedings of the Universiti Kebangsaan Malaysia, Faculty of Science and Technology 2015 Postgraduate Colloquium
PB - American Institute of Physics Inc.
ER -