Conformational design of minilipase

Protein of small size, mini protein, containing 30 to 80 amino acid residues are particularly attractive scaffold for protein design. They possess a great potential in overcoming difficulties associated with synthesis, or unfavorable physical properties. Therefore, a small zinc binding domain of T1...

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Main Author:	Baharum, Hafidza
Format:	Thesis
Language:	English
Published:	2013
Subjects:	Protein engineering Proteins - Chemistry Protein Conformation
Online Access:	http://psasir.upm.edu.my/id/eprint/67608/1/IB%202013%2026%20IR.pdf
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spelling	my-upm-ir.676082019-03-14T08:32:39Z Conformational design of minilipase 2013-07 Baharum, Hafidza Protein of small size, mini protein, containing 30 to 80 amino acid residues are particularly attractive scaffold for protein design. They possess a great potential in overcoming difficulties associated with synthesis, or unfavorable physical properties. Therefore, a small zinc binding domain of T1 lipase was used as scaffold protein for grafting functional motifs coupled with disulphide bonds to form a stable miniprotein. Several constructs of increasing solubility were made by substituting charged and hydrophilic amino acids to the engineered scaffold. From the predicted structure, this newly designed miniprotein contains two short α-helix connected by loops. Several substrates with different carbon length were used to recognize (docking) the interaction between ligand and receptor where acetate, butyrate and caprylate fitted perfectly within the binding pocket of the ligand. Applying molecular approach the optimized sequenced was synthesized and cloned in the pET32b vector. The minilipase was purified 4.97-fold with 78.4% yield using affinity chromatography and the molecular weight of minilipase was determined to be 17 kDa by SDS-PAGE. The minilipase exhibited maximum activity at 35⁰C and was stable at temperature below 50⁰C. This mini protein favored substrates containing short (pNP acetate, C2) and medium (pNP butyrate C4, pNP caprylate C8, pNP decanoate C10) carbon chain of acyl group, which correlated with docking result. The kinetic assay of minilipase for pNP-butyrate produced Km of 0.3 mM and Vmax 0.9 μmole/min/mL indicated the high binding affinity than T1 lipase on the same substrate. The lipolytic activity of purified lipase was slightly increased with 105.72% by the addition of 0.1% SDS, while Tween 60 and Tween 80 were the most effective inhibitors against the minilipase. Interestingly, the minilipase activities were increased by 130% and 115% in the presence of 1 mM Mg2+ and Mn2+, respectively. However when Ca2+ concentration was increased from 1 mM to 10 mM, the activity of purified minilipase was completely inhibited. Based on the finding of the present study, the designed minilipase has potential as an alkaline lipase and a candidate for industrial and pharmaceutical applications. Protein engineering Proteins - Chemistry Protein Conformation 2013-07 Thesis http://psasir.upm.edu.my/id/eprint/67608/ http://psasir.upm.edu.my/id/eprint/67608/1/IB%202013%2026%20IR.pdf text en public masters Universiti Putra Malaysia Protein engineering Proteins - Chemistry Protein Conformation
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English
topic	Protein engineering Proteins - Chemistry Protein Conformation
spellingShingle	Protein engineering Proteins - Chemistry Protein Conformation Baharum, Hafidza Conformational design of minilipase
description	Protein of small size, mini protein, containing 30 to 80 amino acid residues are particularly attractive scaffold for protein design. They possess a great potential in overcoming difficulties associated with synthesis, or unfavorable physical properties. Therefore, a small zinc binding domain of T1 lipase was used as scaffold protein for grafting functional motifs coupled with disulphide bonds to form a stable miniprotein. Several constructs of increasing solubility were made by substituting charged and hydrophilic amino acids to the engineered scaffold. From the predicted structure, this newly designed miniprotein contains two short α-helix connected by loops. Several substrates with different carbon length were used to recognize (docking) the interaction between ligand and receptor where acetate, butyrate and caprylate fitted perfectly within the binding pocket of the ligand. Applying molecular approach the optimized sequenced was synthesized and cloned in the pET32b vector. The minilipase was purified 4.97-fold with 78.4% yield using affinity chromatography and the molecular weight of minilipase was determined to be 17 kDa by SDS-PAGE. The minilipase exhibited maximum activity at 35⁰C and was stable at temperature below 50⁰C. This mini protein favored substrates containing short (pNP acetate, C2) and medium (pNP butyrate C4, pNP caprylate C8, pNP decanoate C10) carbon chain of acyl group, which correlated with docking result. The kinetic assay of minilipase for pNP-butyrate produced Km of 0.3 mM and Vmax 0.9 μmole/min/mL indicated the high binding affinity than T1 lipase on the same substrate. The lipolytic activity of purified lipase was slightly increased with 105.72% by the addition of 0.1% SDS, while Tween 60 and Tween 80 were the most effective inhibitors against the minilipase. Interestingly, the minilipase activities were increased by 130% and 115% in the presence of 1 mM Mg2+ and Mn2+, respectively. However when Ca2+ concentration was increased from 1 mM to 10 mM, the activity of purified minilipase was completely inhibited. Based on the finding of the present study, the designed minilipase has potential as an alkaline lipase and a candidate for industrial and pharmaceutical applications.
format	Thesis
qualification_level	Master's degree
author	Baharum, Hafidza
author_facet	Baharum, Hafidza
author_sort	Baharum, Hafidza
title	Conformational design of minilipase
title_short	Conformational design of minilipase
title_full	Conformational design of minilipase
title_fullStr	Conformational design of minilipase
title_full_unstemmed	Conformational design of minilipase
title_sort	conformational design of minilipase
granting_institution	Universiti Putra Malaysia
publishDate	2013
url	http://psasir.upm.edu.my/id/eprint/67608/1/IB%202013%2026%20IR.pdf
_version_	1747812488206876672

Conformational design of minilipase

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