Purification, characterization and kinetic properties of alpha-naphthyl acetate esterase from wheat flour (Triticum aestivum)

Plant-esterase from wheat flour was purified via aqueous two-phase system (ATPS). The enzyme crude extract was first filtered through a polyethersulfone OmegaTM ultrafiltration membrane, followed by two-step ATPS with PEG1000, NaH2PO4 and (NH4)2SO4. Based on previous studies, the first ATPS cons...

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Main Authors:	Jameel, Ahmad Tariq, Soropogui, Keamou M.
Format:	Article
Language:	English
Published:	Elsevier 2018
Subjects:	TP Chemical technology
Online Access:	http://irep.iium.edu.my/32983/ http://irep.iium.edu.my/32983/ http://irep.iium.edu.my/32983/1/32983_Purification%2C%20characterization%20and%20kinetic.pdf

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spelling	iium-329832018-10-23T02:39:24Z http://irep.iium.edu.my/32983/ Purification, characterization and kinetic properties of alpha-naphthyl acetate esterase from wheat flour (Triticum aestivum) Jameel, Ahmad Tariq Soropogui, Keamou M. TP Chemical technology Plant-esterase from wheat flour was purified via aqueous two-phase system (ATPS). The enzyme crude extract was first filtered through a polyethersulfone OmegaTM ultrafiltration membrane, followed by two-step ATPS with PEG1000, NaH2PO4 and (NH4)2SO4. Based on previous studies, the first ATPS consisting of 27.0% PEG1000 and 13.0% NaH2PO4 (w/w) was performed at room temperature and pH 5.0. The top phase was carefully separated, and 6.0% (NH4)2SO4 (w/w) was added to form the second phase system under similar conditions, to obtain the pure enzyme that partitioned in the bottom salt phase. The purification thus obtained was 10.35 fold with an enzyme yield of 72.90%. Sodium dodecylsulphate polyacrymamide gel electrophoresis (SDS-PAGE) was used to estimate the molecular weight of the target esterase to be around 68 kilo Daltons (kDa). Using a 16 mM solution of alpha naphthyl acetate as substrate, the maximum activity of the purified enzyme, alpha naphthyl acetate esterase (ANAE) was found to be 0.78 U at the optimum conditions of 40 ◦C and pH 8.0 for 15 min of incubation. The Michaelis–Menten parameters Km and Vmax of the purified enzyme were estimated using Langmuir linear plot as 22.5 mM and 4.71 U mg−1, respectively. Further purification was obtained by dialyzing the bottom ANAE rich phase in a solution of 0.04 M phosphate buffer with pH 6.5 to remove the salt, which was then concentrated using centrifugal tubes with OmegaTM membrane. The highly pure ANAE so obtained was immobilized on the multiwalled carbon nanotube for the development of inhibition based enzyme biosensors for pesticides detection. Elsevier 2018 Article PeerReviewed application/pdf en http://irep.iium.edu.my/32983/1/32983_Purification%2C%20characterization%20and%20kinetic.pdf Jameel, Ahmad Tariq and Soropogui, Keamou M. (2018) Purification, characterization and kinetic properties of alpha-naphthyl acetate esterase from wheat flour (Triticum aestivum). New Biotechnology, 44S. S75-S75. ISSN 1871-6784 https://www.sciencedirect.com/science/article/pii/S1871678418312111
repository_type	Digital Repository
institution_category	Local University
institution	International Islamic University Malaysia
building	IIUM Repository
collection	Online Access
language	English
topic	TP Chemical technology
spellingShingle	TP Chemical technology Jameel, Ahmad Tariq Soropogui, Keamou M. Purification, characterization and kinetic properties of alpha-naphthyl acetate esterase from wheat flour (Triticum aestivum)
description	Plant-esterase from wheat flour was purified via aqueous two-phase system (ATPS). The enzyme crude extract was first filtered through a polyethersulfone OmegaTM ultrafiltration membrane, followed by two-step ATPS with PEG1000, NaH2PO4 and (NH4)2SO4. Based on previous studies, the first ATPS consisting of 27.0% PEG1000 and 13.0% NaH2PO4 (w/w) was performed at room temperature and pH 5.0. The top phase was carefully separated, and 6.0% (NH4)2SO4 (w/w) was added to form the second phase system under similar conditions, to obtain the pure enzyme that partitioned in the bottom salt phase. The purification thus obtained was 10.35 fold with an enzyme yield of 72.90%. Sodium dodecylsulphate polyacrymamide gel electrophoresis (SDS-PAGE) was used to estimate the molecular weight of the target esterase to be around 68 kilo Daltons (kDa). Using a 16 mM solution of alpha naphthyl acetate as substrate, the maximum activity of the purified enzyme, alpha naphthyl acetate esterase (ANAE) was found to be 0.78 U at the optimum conditions of 40 ◦C and pH 8.0 for 15 min of incubation. The Michaelis–Menten parameters Km and Vmax of the purified enzyme were estimated using Langmuir linear plot as 22.5 mM and 4.71 U mg−1, respectively. Further purification was obtained by dialyzing the bottom ANAE rich phase in a solution of 0.04 M phosphate buffer with pH 6.5 to remove the salt, which was then concentrated using centrifugal tubes with OmegaTM membrane. The highly pure ANAE so obtained was immobilized on the multiwalled carbon nanotube for the development of inhibition based enzyme biosensors for pesticides detection.
format	Article
author	Jameel, Ahmad Tariq Soropogui, Keamou M.
author_facet	Jameel, Ahmad Tariq Soropogui, Keamou M.
author_sort	Jameel, Ahmad Tariq
title	Purification, characterization and kinetic properties of alpha-naphthyl acetate esterase from wheat flour (Triticum aestivum)
title_short	Purification, characterization and kinetic properties of alpha-naphthyl acetate esterase from wheat flour (Triticum aestivum)
title_full	Purification, characterization and kinetic properties of alpha-naphthyl acetate esterase from wheat flour (Triticum aestivum)
title_fullStr	Purification, characterization and kinetic properties of alpha-naphthyl acetate esterase from wheat flour (Triticum aestivum)
title_full_unstemmed	Purification, characterization and kinetic properties of alpha-naphthyl acetate esterase from wheat flour (Triticum aestivum)
title_sort	purification, characterization and kinetic properties of alpha-naphthyl acetate esterase from wheat flour (triticum aestivum)
publisher	Elsevier
publishDate	2018
url	http://irep.iium.edu.my/32983/ http://irep.iium.edu.my/32983/ http://irep.iium.edu.my/32983/1/32983_Purification%2C%20characterization%20and%20kinetic.pdf
first_indexed	2023-09-18T20:47:39Z
last_indexed	2023-09-18T20:47:39Z
_version_	1777409782271967232

Purification, characterization and kinetic properties of alpha-naphthyl acetate esterase from wheat flour (Triticum aestivum)

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