In silico site-directed mutagenesis of Acinetobacter Haemolyticus Lipase KV1 for improved alkaline stability

The interest on alkaline-stable lipases by the scientific community is increasing due to its great potential use. As most industrial processes are performed under highly basic conditions, alkaline-stable lipases become hugely valued biocatalysts. In this study, three aspartic acid residues at positi...

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Main Author:	Khairul Anuar, Nurul Fatin Syamimi
Format:	Thesis
Language:	English
Published:	2020
Subjects:	Q Science (General)
Online Access:	http://eprints.utm.my/id/eprint/102067/1/NurulFatinSyamimiMFS2020.pdf.pdf
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spelling	my-utm-ep.1020672023-07-31T07:42:43Z In silico site-directed mutagenesis of Acinetobacter Haemolyticus Lipase KV1 for improved alkaline stability 2020 Khairul Anuar, Nurul Fatin Syamimi Q Science (General) The interest on alkaline-stable lipases by the scientific community is increasing due to its great potential use. As most industrial processes are performed under highly basic conditions, alkaline-stable lipases become hugely valued biocatalysts. In this study, three aspartic acid residues at positions 51, 122 and 247 in the outer loop of LipKV1 from Acinetobacter haemolyticus was computationally mutated into lysine using the SWISS-MODEL program, followed by energy minimization of the protein models. PROCHECK, ERRAT and Verify3D refined models of LipKV1 and Mut-LipKV1 indicated that the Mut-LipKV1 protein conformation is in a good condition. The study found that the overall electrostatic surface potentials and charge distributions of the Mut-LipKV1 model was more stable and better adapted to conditions of elevated pHs (pH 8.0 −10.0). Molecular dynamics (MD) simulation of Lip-KV1 and Mut-LipKV1 protein models under different alkaline pHs using GROMACS version 2018.6 revealed that Mut-LipKV1 was more stable at the high pH 9.0 (RMSD ~0.3 nm, RMSF ~0.05 – 0.2 nm), compared the optimal pH 8.0 of LipKV1 (RMSD 0.3 nm, RMSF 0.05 – 0.20 nm). Molecular docking using AutoDock Vina with tributyrin as the substrate identified detailed changes that occurred post mutation. The highest binding affinity (−4.1 kcal/mol) with Mut-LipKV1 which occurred at pH 9.0 was from a single hydrogen bond with His289. MD simulations showed that configurations which formed between Mut-LipKV1-tributyrin (RMSD 0.3 nm; RMSF 0.05 − 0.3 nm) and the LipKV1-tributyrin complexes (RMSD 0.35 nm; RMSF 0.05 − 0.4 nm) were comparably stable at pH 8.0. Furthermore, MM-PBSA calculation validated that the Mut-LipKV1-tributyrin complex at pH 8.0 (-44.01 kcal/mol) showed comparable binding free energy to LipKV1-tributyrin complex (−43.83 kcal/mol). Whereas the lowest binding free energy for Mut-LipKV1-tributyrin complex was simulated at pH 12.0 (−44.04 kcal/mol). Thus, adaptive strategy of replacing the outer loop surface aspartic acid to lysine in LipKV1 successfully broadened pH stability of Mut-LipKV1 towards higher pH, raising it from pH 8.0 − 11.0 to pH 8.0 − 12.0 in the mutant lipase. In a nutshell, this research offered a considerable insight for further improving the alkaline tolerance of lipases. 2020 Thesis http://eprints.utm.my/id/eprint/102067/ http://eprints.utm.my/id/eprint/102067/1/NurulFatinSyamimiMFS2020.pdf.pdf application/pdf en public http://dms.library.utm.my:8080/vital/access/manager/Repository/vital:146291 masters Universiti Teknologi Malaysia Faculty of Science
institution	Universiti Teknologi Malaysia
collection	UTM Institutional Repository
language	English
topic	Q Science (General)
spellingShingle	Q Science (General) Khairul Anuar, Nurul Fatin Syamimi In silico site-directed mutagenesis of Acinetobacter Haemolyticus Lipase KV1 for improved alkaline stability
description	The interest on alkaline-stable lipases by the scientific community is increasing due to its great potential use. As most industrial processes are performed under highly basic conditions, alkaline-stable lipases become hugely valued biocatalysts. In this study, three aspartic acid residues at positions 51, 122 and 247 in the outer loop of LipKV1 from Acinetobacter haemolyticus was computationally mutated into lysine using the SWISS-MODEL program, followed by energy minimization of the protein models. PROCHECK, ERRAT and Verify3D refined models of LipKV1 and Mut-LipKV1 indicated that the Mut-LipKV1 protein conformation is in a good condition. The study found that the overall electrostatic surface potentials and charge distributions of the Mut-LipKV1 model was more stable and better adapted to conditions of elevated pHs (pH 8.0 −10.0). Molecular dynamics (MD) simulation of Lip-KV1 and Mut-LipKV1 protein models under different alkaline pHs using GROMACS version 2018.6 revealed that Mut-LipKV1 was more stable at the high pH 9.0 (RMSD ~0.3 nm, RMSF ~0.05 – 0.2 nm), compared the optimal pH 8.0 of LipKV1 (RMSD 0.3 nm, RMSF 0.05 – 0.20 nm). Molecular docking using AutoDock Vina with tributyrin as the substrate identified detailed changes that occurred post mutation. The highest binding affinity (−4.1 kcal/mol) with Mut-LipKV1 which occurred at pH 9.0 was from a single hydrogen bond with His289. MD simulations showed that configurations which formed between Mut-LipKV1-tributyrin (RMSD 0.3 nm; RMSF 0.05 − 0.3 nm) and the LipKV1-tributyrin complexes (RMSD 0.35 nm; RMSF 0.05 − 0.4 nm) were comparably stable at pH 8.0. Furthermore, MM-PBSA calculation validated that the Mut-LipKV1-tributyrin complex at pH 8.0 (-44.01 kcal/mol) showed comparable binding free energy to LipKV1-tributyrin complex (−43.83 kcal/mol). Whereas the lowest binding free energy for Mut-LipKV1-tributyrin complex was simulated at pH 12.0 (−44.04 kcal/mol). Thus, adaptive strategy of replacing the outer loop surface aspartic acid to lysine in LipKV1 successfully broadened pH stability of Mut-LipKV1 towards higher pH, raising it from pH 8.0 − 11.0 to pH 8.0 − 12.0 in the mutant lipase. In a nutshell, this research offered a considerable insight for further improving the alkaline tolerance of lipases.
format	Thesis
qualification_level	Master's degree
author	Khairul Anuar, Nurul Fatin Syamimi
author_facet	Khairul Anuar, Nurul Fatin Syamimi
author_sort	Khairul Anuar, Nurul Fatin Syamimi
title	In silico site-directed mutagenesis of Acinetobacter Haemolyticus Lipase KV1 for improved alkaline stability
title_short	In silico site-directed mutagenesis of Acinetobacter Haemolyticus Lipase KV1 for improved alkaline stability
title_full	In silico site-directed mutagenesis of Acinetobacter Haemolyticus Lipase KV1 for improved alkaline stability
title_fullStr	In silico site-directed mutagenesis of Acinetobacter Haemolyticus Lipase KV1 for improved alkaline stability
title_full_unstemmed	In silico site-directed mutagenesis of Acinetobacter Haemolyticus Lipase KV1 for improved alkaline stability
title_sort	in silico site-directed mutagenesis of acinetobacter haemolyticus lipase kv1 for improved alkaline stability
granting_institution	Universiti Teknologi Malaysia
granting_department	Faculty of Science
publishDate	2020
url	http://eprints.utm.my/id/eprint/102067/1/NurulFatinSyamimiMFS2020.pdf.pdf
_version_	1776100837885476864

In silico site-directed mutagenesis of Acinetobacter Haemolyticus Lipase KV1 for improved alkaline stability

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