Cloning, sequencing and extracellular expression of the alkaline protease gene from Bacillus stearothermophilus F1

The gene of a highly thermostable alkaline protease was amplified from Bacillus stearothermophilus F1 by polymerase chain reaction using consensus primers based on the sequences of serine protease genes from related species. Nucleotide sequence analysis of the gene revealed an open reading frame...

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Main Author:	Fu, Zhibiao
Format:	Thesis
Language:	English English
Published:	2001
Subjects:	Molecular cloning Proteolytic enzymes
Online Access:	http://psasir.upm.edu.my/id/eprint/9210/1/FSAS_2001_6_IR.pdf
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id	my-upm-ir.9210
record_format	uketd_dc
spelling	my-upm-ir.92102024-02-06T09:00:15Z Cloning, sequencing and extracellular expression of the alkaline protease gene from Bacillus stearothermophilus F1 2001-07 Fu, Zhibiao The gene of a highly thermostable alkaline protease was amplified from Bacillus stearothermophilus F1 by polymerase chain reaction using consensus primers based on the sequences of serine protease genes from related species. Nucleotide sequence analysis of the gene revealed an open reading frame containing 1,206 bp which encodes for a polypeptide of 401 amino acid residues. The polypeptide composed of a signal peptide (25 amino acid residues), a prosequence (97 amino acid residues), and a mature protein of 279 amino acid residues. Amino acid sequence comparison revealed that it shared high homology with those of other serine proteases from a number of Bacillus spp. The recombinant F1 protease was efficiently excreted into culture medium using E.coli XL1-Blue harbouring two vectors: pTrcHis bearing the protease gene and pJL3 containing the bacteriocin-release-protein (BRP). Both vectors contain the E.coli lac promoter-operator system. In the presence of 40 uM isopropyl-β-D thiogalactopyranoside (IPTG), the recombinant F1 protease and the BRP were expressed and the mature F1 protease was released into the culture medium. The enzyme was purified through a one-step heat treatment at 70°C for 3 h, and this method purified the protease to near homogeneity. The purified enzyme showed a pH optimum of 9.0, temperature optimum of 80°C, and was stable at 70°C for 24 h in pH ranges from 8.0 to 10.0. The enzyme exhibited a high degree of thermostability with half-lives of 3.5 h at 85°C, 25 min at 90°C, and was inhibited by the serine protease inhibitor, phenylmethanesulfonyl fluoride (PMSF). Molecular cloning Proteolytic enzymes 2001-07 Thesis http://psasir.upm.edu.my/id/eprint/9210/ http://psasir.upm.edu.my/id/eprint/9210/1/FSAS_2001_6_IR.pdf text en public masters Universiti Putra Malaysia Molecular cloning Proteolytic enzymes Faculty of Science and Environmental Studies Abdul Razak, Che Nyonya English
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English English
advisor	Abdul Razak, Che Nyonya
topic	Molecular cloning Proteolytic enzymes
spellingShingle	Molecular cloning Proteolytic enzymes Fu, Zhibiao Cloning, sequencing and extracellular expression of the alkaline protease gene from Bacillus stearothermophilus F1
description	The gene of a highly thermostable alkaline protease was amplified from Bacillus stearothermophilus F1 by polymerase chain reaction using consensus primers based on the sequences of serine protease genes from related species. Nucleotide sequence analysis of the gene revealed an open reading frame containing 1,206 bp which encodes for a polypeptide of 401 amino acid residues. The polypeptide composed of a signal peptide (25 amino acid residues), a prosequence (97 amino acid residues), and a mature protein of 279 amino acid residues. Amino acid sequence comparison revealed that it shared high homology with those of other serine proteases from a number of Bacillus spp. The recombinant F1 protease was efficiently excreted into culture medium using E.coli XL1-Blue harbouring two vectors: pTrcHis bearing the protease gene and pJL3 containing the bacteriocin-release-protein (BRP). Both vectors contain the E.coli lac promoter-operator system. In the presence of 40 uM isopropyl-β-D thiogalactopyranoside (IPTG), the recombinant F1 protease and the BRP were expressed and the mature F1 protease was released into the culture medium. The enzyme was purified through a one-step heat treatment at 70°C for 3 h, and this method purified the protease to near homogeneity. The purified enzyme showed a pH optimum of 9.0, temperature optimum of 80°C, and was stable at 70°C for 24 h in pH ranges from 8.0 to 10.0. The enzyme exhibited a high degree of thermostability with half-lives of 3.5 h at 85°C, 25 min at 90°C, and was inhibited by the serine protease inhibitor, phenylmethanesulfonyl fluoride (PMSF).
format	Thesis
qualification_level	Master's degree
author	Fu, Zhibiao
author_facet	Fu, Zhibiao
author_sort	Fu, Zhibiao
title	Cloning, sequencing and extracellular expression of the alkaline protease gene from Bacillus stearothermophilus F1
title_short	Cloning, sequencing and extracellular expression of the alkaline protease gene from Bacillus stearothermophilus F1
title_full	Cloning, sequencing and extracellular expression of the alkaline protease gene from Bacillus stearothermophilus F1
title_fullStr	Cloning, sequencing and extracellular expression of the alkaline protease gene from Bacillus stearothermophilus F1
title_full_unstemmed	Cloning, sequencing and extracellular expression of the alkaline protease gene from Bacillus stearothermophilus F1
title_sort	cloning, sequencing and extracellular expression of the alkaline protease gene from bacillus stearothermophilus f1
granting_institution	Universiti Putra Malaysia
granting_department	Faculty of Science and Environmental Studies
publishDate	2001
url	http://psasir.upm.edu.my/id/eprint/9210/1/FSAS_2001_6_IR.pdf
_version_	1794018802364579840

Cloning, sequencing and extracellular expression of the alkaline protease gene from Bacillus stearothermophilus F1

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