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Effects of lid 1 mutagenesis on lid displacement, catalytic performances and thermostability of cold-active Pseudomonas AMS8 lipase in toluene


Citation

Yaacob, Norhayati and Ahmad Kamarudin, Nor Hafizah and Leow, Adam Thean Chor and Salleh, Abu Bakar and Raja Abdul Rahman, Raja Noor Zaliha and Mohamad Ali, Mohd Shukuri (2019) Effects of lid 1 mutagenesis on lid displacement, catalytic performances and thermostability of cold-active Pseudomonas AMS8 lipase in toluene. Computational and Structural Biotechnology Journal, 17. pp. 215-228. ISSN 2001-0370

Abstract

Pseudomonas fluorescens AMS8 lipase lid 1 structure is rigid and holds unclear roles due to the absence of solvent-interactions. Lid 1 region was stabilized by 17 hydrogen bond linkages and displayed lower mean hydrophobicity (0.596) compared to MIS38 lipase. Mutating lid 1 residues, Thr-52 and Gly-55 to aromatic hydrophobic-polar tyrosine would churned more side-chain interactions between lid 1 and water or toluene. This study revealed that T52Y leads G55Y and its recombinant towards achieving higher solvent-accessible surface area and longer half-life at 25 to 37 °C in 0.5% (v/v) toluene. T52Y also exhibited better substrate affinity with long-chain carbon substrate in aqueous media. The affinity for pNP palmitate, laurate and caprylate increased in 0.5% (v/v) toluene in recombinant AMS8, but the affinity in similar substrates was substantially declined in lid 1 mutated lipases. Regarding enzyme efficiency, the recombinant AMS8 lipase displayed highest value of kcat/Km in 0.5% (v/v) toluene, mainly with pNPC. In both hydrolysis reactions with 0% and 0.5% (v/v) toluene, the enzyme efficiency of G55Y was found higher than T52Y for pNPL and pNPP. At 0.5% (v/v) toluene, both mutants showed reductions in activation energy and enthalpy values as temperature increased from 25 to 35 °C, displaying better catalytic functions. Only T52Y exhibited increase in entropy values at 0.5% (v/v) toluene indicating structure stability. As a conclusion, Thr-52 and Gly-55 are important residues for lid 1 stability as their existence helps to retain the geometrical structure of alpha-helix and connecting hinge.


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Additional Metadata

Item Type: Article
Divisions: Faculty of Biotechnology and Biomolecular Sciences
Institute of Bioscience
DOI Number: https://doi.org/10.1016/j.csbj.2019.01.005
Publisher: Elsevier
Keywords: Lid engineering; Cold-active lipase; Molecular dynamics simulation; Hydrophobic contacts; Site-directed mutagenesis; Solvent accessibility; Substrate affinity; Thermostability
Depositing User: Nurul Ainie Mokhtar
Date Deposited: 29 Jul 2021 02:09
Last Modified: 29 Jul 2021 02:09
Altmetrics: http://www.altmetric.com/details.php?domain=psasir.upm.edu.my&doi=10.1016/j.csbj.2019.01.005
URI: http://psasir.upm.edu.my/id/eprint/79602
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