Exoproteome analysis of Bacillus lehensis G1 in ph regulation and starch utilization for screening of signal peptides for cyclodextrin glucanotransferase secretion in Escherichia coli

Bacillus lehensis G1 is an alkaliphilic bacterium that is capable of surviving in environment as extreme as pH 11. Gram-positive Bacillus species are among the bacterial winners in the production of secreted enzymes. However, until recently, there has been no study related to exoproteome profiling o...

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Main Author:	Ling, How Lie
Format:	Thesis
Language:	English
Published:	2018
Subjects:	TP Chemical technology
Online Access:	http://eprints.utm.my/id/eprint/79352/1/LingHowLiePFChE2018.pdf
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spelling	my-utm-ep.793522018-10-14T08:44:48Z Exoproteome analysis of Bacillus lehensis G1 in ph regulation and starch utilization for screening of signal peptides for cyclodextrin glucanotransferase secretion in Escherichia coli 2018 Ling, How Lie TP Chemical technology Bacillus lehensis G1 is an alkaliphilic bacterium that is capable of surviving in environment as extreme as pH 11. Gram-positive Bacillus species are among the bacterial winners in the production of secreted enzymes. However, until recently, there has been no study related to exoproteome profiling of B. lehensis under different cultural conditions. The aim of this study was to analyze exoproteome profile of B. lehensis G1 in alkaline and starch-supplemented medium to elucidate adaptive mechanism and to identify potential secretion aid molecules (i.e. signal peptides). The exoproteome was extracted from the cell culture supernatant, separated by two-dimensional electrophoresis, excised, digested with trypsin and analyzed using MALDI-TOF/TOF approach. A total of 85 proteins were identified in the extracellular space of B. lehensis G1 cultured under different conditions (i.e. pH8, pH10, pH11 and starch-supplemented medium). Alkaline adaptation that occurred in B. lehensis G1 includes 33 differentially expressed proteins such as GlcNAc-binding protein A, chitinase, endopeptidase lytE, cell surface protein, flagellar hook-associated proteins and enolase. At pH 8, 5 proteins were upregulated and 13 proteins were down-regulated, whereas at pH 11, 14 proteins were up-regulated and 8 were down-regulated. In response to medium pH changes, majority of the differentially expressed proteins were involved in cell wall, main glycolytic pathways, metabolism of amino acids and related molecules, and some proteins of unknown function. The bacteria exhibited thinner flagella at pH 11 compared to pH 10. Meanwhile, when the cells were grown in starch-supplemented medium, 23 proteins were specifically expressed including carbohydrate-degrading enzymes (e.g. cyclodextrin glucanotransferase (CGTase), glucanases and prokaryote trehalose catabolism protein ThuA). The identified extracellular proteins and their associated signal peptide could serve as an alternative to solve the problems encountered during the extracellular secretion in E. coli such as low secretion and cell lysis. Therefore, 14 signal peptides obtained through exoproteome analysis of B. lehensis G1 were selected and cloned together with CGTase in E. coli to assist its secretion across the cytoplasmic membrane. All clones were found to allow CGTase to be excreted into the medium as observed and measured by iodine plate assay and enzyme activity assay. Compared to native signal peptide of CGTase (G1), signal peptide of GlcNAc-binding protein A (GAP) significantly improved CGTase activities by 735% and 205% in extracellular and periplasmic compartment, respectively, with an increase of only ~1.7 fold the amount of β-galactosidase (cell lysis indicator) in the medium. Moreover, GAP has the highest secretion rate of 45.6 U/ml/hr among all clones, where physicochemical characteristics of signal peptide play significant role. GAP from this study secreted the highest amount of CGTase extracellularly (637.5 U/ml) in E. coli compared to E. coli signal peptides such as pelB and OmpA. 2018 Thesis http://eprints.utm.my/id/eprint/79352/ http://eprints.utm.my/id/eprint/79352/1/LingHowLiePFChE2018.pdf application/pdf en public phd doctoral Universiti Teknologi Malaysia, Faculty of Chemical & Energy Engineering Faculty of Chemical & Energy Engineering
institution	Universiti Teknologi Malaysia
collection	UTM Institutional Repository
language	English
topic	TP Chemical technology
spellingShingle	TP Chemical technology Ling, How Lie Exoproteome analysis of Bacillus lehensis G1 in ph regulation and starch utilization for screening of signal peptides for cyclodextrin glucanotransferase secretion in Escherichia coli
description	Bacillus lehensis G1 is an alkaliphilic bacterium that is capable of surviving in environment as extreme as pH 11. Gram-positive Bacillus species are among the bacterial winners in the production of secreted enzymes. However, until recently, there has been no study related to exoproteome profiling of B. lehensis under different cultural conditions. The aim of this study was to analyze exoproteome profile of B. lehensis G1 in alkaline and starch-supplemented medium to elucidate adaptive mechanism and to identify potential secretion aid molecules (i.e. signal peptides). The exoproteome was extracted from the cell culture supernatant, separated by two-dimensional electrophoresis, excised, digested with trypsin and analyzed using MALDI-TOF/TOF approach. A total of 85 proteins were identified in the extracellular space of B. lehensis G1 cultured under different conditions (i.e. pH8, pH10, pH11 and starch-supplemented medium). Alkaline adaptation that occurred in B. lehensis G1 includes 33 differentially expressed proteins such as GlcNAc-binding protein A, chitinase, endopeptidase lytE, cell surface protein, flagellar hook-associated proteins and enolase. At pH 8, 5 proteins were upregulated and 13 proteins were down-regulated, whereas at pH 11, 14 proteins were up-regulated and 8 were down-regulated. In response to medium pH changes, majority of the differentially expressed proteins were involved in cell wall, main glycolytic pathways, metabolism of amino acids and related molecules, and some proteins of unknown function. The bacteria exhibited thinner flagella at pH 11 compared to pH 10. Meanwhile, when the cells were grown in starch-supplemented medium, 23 proteins were specifically expressed including carbohydrate-degrading enzymes (e.g. cyclodextrin glucanotransferase (CGTase), glucanases and prokaryote trehalose catabolism protein ThuA). The identified extracellular proteins and their associated signal peptide could serve as an alternative to solve the problems encountered during the extracellular secretion in E. coli such as low secretion and cell lysis. Therefore, 14 signal peptides obtained through exoproteome analysis of B. lehensis G1 were selected and cloned together with CGTase in E. coli to assist its secretion across the cytoplasmic membrane. All clones were found to allow CGTase to be excreted into the medium as observed and measured by iodine plate assay and enzyme activity assay. Compared to native signal peptide of CGTase (G1), signal peptide of GlcNAc-binding protein A (GAP) significantly improved CGTase activities by 735% and 205% in extracellular and periplasmic compartment, respectively, with an increase of only ~1.7 fold the amount of β-galactosidase (cell lysis indicator) in the medium. Moreover, GAP has the highest secretion rate of 45.6 U/ml/hr among all clones, where physicochemical characteristics of signal peptide play significant role. GAP from this study secreted the highest amount of CGTase extracellularly (637.5 U/ml) in E. coli compared to E. coli signal peptides such as pelB and OmpA.
format	Thesis
qualification_name	Doctor of Philosophy (PhD.)
qualification_level	Doctorate
author	Ling, How Lie
author_facet	Ling, How Lie
author_sort	Ling, How Lie
title	Exoproteome analysis of Bacillus lehensis G1 in ph regulation and starch utilization for screening of signal peptides for cyclodextrin glucanotransferase secretion in Escherichia coli
title_short	Exoproteome analysis of Bacillus lehensis G1 in ph regulation and starch utilization for screening of signal peptides for cyclodextrin glucanotransferase secretion in Escherichia coli
title_full	Exoproteome analysis of Bacillus lehensis G1 in ph regulation and starch utilization for screening of signal peptides for cyclodextrin glucanotransferase secretion in Escherichia coli
title_fullStr	Exoproteome analysis of Bacillus lehensis G1 in ph regulation and starch utilization for screening of signal peptides for cyclodextrin glucanotransferase secretion in Escherichia coli
title_full_unstemmed	Exoproteome analysis of Bacillus lehensis G1 in ph regulation and starch utilization for screening of signal peptides for cyclodextrin glucanotransferase secretion in Escherichia coli
title_sort	exoproteome analysis of bacillus lehensis g1 in ph regulation and starch utilization for screening of signal peptides for cyclodextrin glucanotransferase secretion in escherichia coli
granting_institution	Universiti Teknologi Malaysia, Faculty of Chemical & Energy Engineering
granting_department	Faculty of Chemical & Energy Engineering
publishDate	2018
url	http://eprints.utm.my/id/eprint/79352/1/LingHowLiePFChE2018.pdf
_version_	1747818207555616768

Exoproteome analysis of Bacillus lehensis G1 in ph regulation and starch utilization for screening of signal peptides for cyclodextrin glucanotransferase secretion in Escherichia coli

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