Cloning, Sequencing and Expression of an Organic Solvent Tolerant Protease from Bacillus Pumilus 115B

Five out of the nine isolated bacteria species screened earlier, demonstrated high protease production on Skim Milk Agar and were also to Benzene Toulene Xylene Ethylbenzene (BTEX). Among these, isolate 115b was found to produce the highest protease production. Besides being stable in 25% (v/v) benz...

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Main Author:	Mahamad, Shalihah
Format:	Thesis
Language:	English English
Published:	2006
Subjects:	Bacillus (Bacteria) - Cloning
Online Access:	http://psasir.upm.edu.my/id/eprint/503/1/600362_fbsb_2006_17_abstrak_je__dh_pdf_.pdf
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spelling	my-upm-ir.5032013-05-27T06:48:48Z Cloning, Sequencing and Expression of an Organic Solvent Tolerant Protease from Bacillus Pumilus 115B 2006-02 Mahamad, Shalihah Five out of the nine isolated bacteria species screened earlier, demonstrated high protease production on Skim Milk Agar and were also to Benzene Toulene Xylene Ethylbenzene (BTEX). Among these, isolate 115b was found to produce the highest protease production. Besides being stable in 25% (v/v) benzene and toluene, protease from isolate 115b was found to be activated by n-dodecane and n-tetradecane by 1.7 and 2.5 folds respectively. Isolate 115b was identified as Bacillus pumilus 115b via biochemical tests and 16S rDNA sequencing analysis. The gene encoding protease of Bacillus pumilus 115b was amplified via polymerase chain reaction (PCR) using consensus primers based on the sequences of alkaline serine protease genes from related species. The complete nucleotide sequence of the protease from Bacillus pumilus 115b was determined. Sequence analysis showed an open reading frame (ORF) of 1149 that encoded a polypeptide of 383 amino acid residues and the calculated protein molecular mass of 39,448 Da. The ORF also encoded a single peptide consisting of 29 residues and a propeptide of 79 residues. The mature protein comprised 275 amino acids with a calculated molecular mass of 27,846 Da. Homology searches revealed that the amino acid residues from B. pumilus 115b protease shared a high homology (90%) with the alkaline serine protease from B. pumilus TYO-67 and B. pumilus UN-31-C-42. The gene coding for an organic solvent tolerant 115b protease gene was cloned into pQE-30 UA expression vector. The recombinant plasmid was then transformed into E. coli M15[pREP4]. The organic solvent tolerant 115b protease gene was successfully expressed by induction with IPTG and was detected by SDS-PAGE analysis with a molecular weight of around 35 kDa. The expression of recombinant E. coli M15[pREP4] was optimized by inducing it with 1.0 mM of IPTG at 4 hour of induction time. Bacillus (Bacteria) - Cloning 2006-02 Thesis http://psasir.upm.edu.my/id/eprint/503/ http://psasir.upm.edu.my/id/eprint/503/1/600362_fbsb_2006_17_abstrak_je__dh_pdf_.pdf application/pdf en public masters Universiti Putra Malaysia Bacillus (Bacteria) - Cloning Faculty of Biotechnology and Biomolecular Sciences English
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English English
topic	Bacillus (Bacteria) - Cloning
spellingShingle	Bacillus (Bacteria) - Cloning Mahamad, Shalihah Cloning, Sequencing and Expression of an Organic Solvent Tolerant Protease from Bacillus Pumilus 115B
description	Five out of the nine isolated bacteria species screened earlier, demonstrated high protease production on Skim Milk Agar and were also to Benzene Toulene Xylene Ethylbenzene (BTEX). Among these, isolate 115b was found to produce the highest protease production. Besides being stable in 25% (v/v) benzene and toluene, protease from isolate 115b was found to be activated by n-dodecane and n-tetradecane by 1.7 and 2.5 folds respectively. Isolate 115b was identified as Bacillus pumilus 115b via biochemical tests and 16S rDNA sequencing analysis. The gene encoding protease of Bacillus pumilus 115b was amplified via polymerase chain reaction (PCR) using consensus primers based on the sequences of alkaline serine protease genes from related species. The complete nucleotide sequence of the protease from Bacillus pumilus 115b was determined. Sequence analysis showed an open reading frame (ORF) of 1149 that encoded a polypeptide of 383 amino acid residues and the calculated protein molecular mass of 39,448 Da. The ORF also encoded a single peptide consisting of 29 residues and a propeptide of 79 residues. The mature protein comprised 275 amino acids with a calculated molecular mass of 27,846 Da. Homology searches revealed that the amino acid residues from B. pumilus 115b protease shared a high homology (90%) with the alkaline serine protease from B. pumilus TYO-67 and B. pumilus UN-31-C-42. The gene coding for an organic solvent tolerant 115b protease gene was cloned into pQE-30 UA expression vector. The recombinant plasmid was then transformed into E. coli M15[pREP4]. The organic solvent tolerant 115b protease gene was successfully expressed by induction with IPTG and was detected by SDS-PAGE analysis with a molecular weight of around 35 kDa. The expression of recombinant E. coli M15[pREP4] was optimized by inducing it with 1.0 mM of IPTG at 4 hour of induction time.
format	Thesis
qualification_level	Master's degree
author	Mahamad, Shalihah
author_facet	Mahamad, Shalihah
author_sort	Mahamad, Shalihah
title	Cloning, Sequencing and Expression of an Organic Solvent Tolerant Protease from Bacillus Pumilus 115B
title_short	Cloning, Sequencing and Expression of an Organic Solvent Tolerant Protease from Bacillus Pumilus 115B
title_full	Cloning, Sequencing and Expression of an Organic Solvent Tolerant Protease from Bacillus Pumilus 115B
title_fullStr	Cloning, Sequencing and Expression of an Organic Solvent Tolerant Protease from Bacillus Pumilus 115B
title_full_unstemmed	Cloning, Sequencing and Expression of an Organic Solvent Tolerant Protease from Bacillus Pumilus 115B
title_sort	cloning, sequencing and expression of an organic solvent tolerant protease from bacillus pumilus 115b
granting_institution	Universiti Putra Malaysia
granting_department	Faculty of Biotechnology and Biomolecular Sciences
publishDate	2006
url	http://psasir.upm.edu.my/id/eprint/503/1/600362_fbsb_2006_17_abstrak_je__dh_pdf_.pdf
_version_	1747810236202221568

Cloning, Sequencing and Expression of an Organic Solvent Tolerant Protease from Bacillus Pumilus 115B

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