Purification And Characterization Of Organic Solvent Tolerant Protease From Pseudomonas Aeruginosa Strain K

A bacterium known as Strain K was identified as Pseudomonas aeruginosa since it 16S rRNA sequence exhibited similarity of up to 99 % with P. aeruginosa from the NCBI database. Protease from the P. aeruginosa strain K was purified to homogeneity by 80.6 fold and 107 % recovery using a combination of...

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Main Author:	Yusoff, NorulAiman
Format:	Thesis
Language:	English English
Published:	2007
Subjects:	Proteolytic enzymes
Online Access:	http://psasir.upm.edu.my/id/eprint/4886/1/FBSB_2007_9.pdf
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spelling	my-upm-ir.48862013-05-27T07:18:55Z Purification And Characterization Of Organic Solvent Tolerant Protease From Pseudomonas Aeruginosa Strain K 2007 Yusoff, NorulAiman A bacterium known as Strain K was identified as Pseudomonas aeruginosa since it 16S rRNA sequence exhibited similarity of up to 99 % with P. aeruginosa from the NCBI database. Protease from the P. aeruginosa strain K was purified to homogeneity by 80.6 fold and 107 % recovery using a combination of ultrafiltration and ion exchange chromatography on Q-Sepharose. In second method, protease K-01 was purified to homogeneity by 116.2 fold purification and 199 % recovery, using a combination of ultrafiltration and hydrophobic interaction chromatography on Butyl-Sepharose. The purified protease was named as protease K-01. The apparent molecular mass of the purified protease K-01 was estimated to be 33 kDa on gel filtration Sephadex G-100, and SDS PAGE. The purified protease hydrolyzed azocasein at optimum temperature of 55 ºC. However, the enzyme lost its activity with a half life of more than 60 min at 55 and 60 ºC. The optimum activity of the protease was observed at pH 8.0 and it was stable in the pH range of pH 6 to 13. The protease activity was completely inhibited by EDTA and 1,10-phenantroline, while 70 and 30 % reduction of protease activity was observed in the presence of DTT and 2-mercaptoethanol respectively. Among the metal ions tested, Mg2+ and Ca2+ ions increased enzyme activity by 8 %. Protease activity was completely inhibited by Fe2+, Ni2+, Cu2+, Ag2+, Zn2+, and Hg2+ ions. Fe3+ and Co2+ ions were found to restore the activity of inactivated protease by 1mM EDTA. Protease K-01 was more stable in water miscible organic solvents (DMSO, methanol, ethanol, 2-propanol, n-butanol, and 1-decanol) than water immiscible organic solvent. For substrate specificity, protease K-01 was able to hydrolyze several native proteins such as casein, haemoglobin, albumin and gelatin. Proteolytic enzymes 2007 Thesis http://psasir.upm.edu.my/id/eprint/4886/ http://psasir.upm.edu.my/id/eprint/4886/1/FBSB_2007_9.pdf application/pdf en public masters Universiti Putra Malaysia Proteolytic enzymes Faculty of Biotechnology and Biomolecular sciences English
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English English
topic	Proteolytic enzymes
spellingShingle	Proteolytic enzymes Yusoff, NorulAiman Purification And Characterization Of Organic Solvent Tolerant Protease From Pseudomonas Aeruginosa Strain K
description	A bacterium known as Strain K was identified as Pseudomonas aeruginosa since it 16S rRNA sequence exhibited similarity of up to 99 % with P. aeruginosa from the NCBI database. Protease from the P. aeruginosa strain K was purified to homogeneity by 80.6 fold and 107 % recovery using a combination of ultrafiltration and ion exchange chromatography on Q-Sepharose. In second method, protease K-01 was purified to homogeneity by 116.2 fold purification and 199 % recovery, using a combination of ultrafiltration and hydrophobic interaction chromatography on Butyl-Sepharose. The purified protease was named as protease K-01. The apparent molecular mass of the purified protease K-01 was estimated to be 33 kDa on gel filtration Sephadex G-100, and SDS PAGE. The purified protease hydrolyzed azocasein at optimum temperature of 55 ºC. However, the enzyme lost its activity with a half life of more than 60 min at 55 and 60 ºC. The optimum activity of the protease was observed at pH 8.0 and it was stable in the pH range of pH 6 to 13. The protease activity was completely inhibited by EDTA and 1,10-phenantroline, while 70 and 30 % reduction of protease activity was observed in the presence of DTT and 2-mercaptoethanol respectively. Among the metal ions tested, Mg2+ and Ca2+ ions increased enzyme activity by 8 %. Protease activity was completely inhibited by Fe2+, Ni2+, Cu2+, Ag2+, Zn2+, and Hg2+ ions. Fe3+ and Co2+ ions were found to restore the activity of inactivated protease by 1mM EDTA. Protease K-01 was more stable in water miscible organic solvents (DMSO, methanol, ethanol, 2-propanol, n-butanol, and 1-decanol) than water immiscible organic solvent. For substrate specificity, protease K-01 was able to hydrolyze several native proteins such as casein, haemoglobin, albumin and gelatin.
format	Thesis
qualification_level	Master's degree
author	Yusoff, NorulAiman
author_facet	Yusoff, NorulAiman
author_sort	Yusoff, NorulAiman
title	Purification And Characterization Of Organic Solvent Tolerant Protease From Pseudomonas Aeruginosa Strain K
title_short	Purification And Characterization Of Organic Solvent Tolerant Protease From Pseudomonas Aeruginosa Strain K
title_full	Purification And Characterization Of Organic Solvent Tolerant Protease From Pseudomonas Aeruginosa Strain K
title_fullStr	Purification And Characterization Of Organic Solvent Tolerant Protease From Pseudomonas Aeruginosa Strain K
title_full_unstemmed	Purification And Characterization Of Organic Solvent Tolerant Protease From Pseudomonas Aeruginosa Strain K
title_sort	purification and characterization of organic solvent tolerant protease from pseudomonas aeruginosa strain k
granting_institution	Universiti Putra Malaysia
granting_department	Faculty of Biotechnology and Biomolecular sciences
publishDate	2007
url	http://psasir.upm.edu.my/id/eprint/4886/1/FBSB_2007_9.pdf
_version_	1747810304494927872

Purification And Characterization Of Organic Solvent Tolerant Protease From Pseudomonas Aeruginosa Strain K

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