Expression of recombinant T1 lipase in E. coli strain W using fifth grade molasses as carbon source

T1 lipase is suitable for various industrial applications due to its thermoalkaliphilic property. However, for industrial purposes normally large scale cultures are used and the current plasmid-based expression system raised concerns for the cost of antibiotics, dissemination of antibiotics and meta...

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Main Author:	Yusoff, Siti Hajar
Format:	Thesis
Language:	English
Published:	2019
Subjects:	Lipase Escherichia coli Molasses
Online Access:	http://psasir.upm.edu.my/id/eprint/92795/1/FBSB%202021%205%20IR.pdf
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id	my-upm-ir.92795
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spelling	my-upm-ir.927952022-05-10T04:29:58Z Expression of recombinant T1 lipase in E. coli strain W using fifth grade molasses as carbon source 2019-11 Yusoff, Siti Hajar T1 lipase is suitable for various industrial applications due to its thermoalkaliphilic property. However, for industrial purposes normally large scale cultures are used and the current plasmid-based expression system raised concerns for the cost of antibiotics, dissemination of antibiotics and metabolic burden that could lead to reduction of recombinant protein productivity. Furthermore, the overall production cost depends greatly on the source of carbon. Therefore, many attempts have been made to produce recombinant proteins in E. coli from molasses, a cheap carbon source. However, most E. coli cannot utilize sucrose which is the major sugar in molasses. The aim of this study is to solve these problems by expressing the recombinant lipase using genome-based expression in E. coli W. E. coli W was chosen because it is the only safe laboratory strain that can utilize sucrose and shows fast, highly oxidative sucrose metabolism with low acetate production. The objectives of this study are to clone and integrate lipase gene into E. coli W genome, to express the recombinant lipase and to optimize the production of the recombinant product. The T1 lipase gene cassettes with different promoters (tac and trc promoters) were successfully cloned and integrated into E. coli W for plasmid based and genome based expression respectively. The expression of T1 lipase in E. coli W were higher in plasmid-based expression system compared to genome-based expression system but both systems have higher expression under trc promoter (thus, it was chosen for further study). Optimization study recorded the highest expression of T1 lipase at induction temperature of 16°C, 0.8 mM of IPTG concentration and 3% of molasses in M9 medium. A combination of the approaches described above may permit the industrial scale utilization of E. coli for bioconversion of low-cost starting materials (sucrose-molasses) into industrially important enzymes. Furthermore, production of recombinant lipase from the stable expression in the genome can be a model for production of other industrially important recombinant proteins. Lipase Escherichia coli Molasses 2019-11 Thesis http://psasir.upm.edu.my/id/eprint/92795/ http://psasir.upm.edu.my/id/eprint/92795/1/FBSB%202021%205%20IR.pdf text en public masters Universiti Putra Malaysia Lipase Escherichia coli Molasses Sabri, Suriana
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English
advisor	Sabri, Suriana
topic	Lipase Escherichia coli Molasses
spellingShingle	Lipase Escherichia coli Molasses Yusoff, Siti Hajar Expression of recombinant T1 lipase in E. coli strain W using fifth grade molasses as carbon source
description	T1 lipase is suitable for various industrial applications due to its thermoalkaliphilic property. However, for industrial purposes normally large scale cultures are used and the current plasmid-based expression system raised concerns for the cost of antibiotics, dissemination of antibiotics and metabolic burden that could lead to reduction of recombinant protein productivity. Furthermore, the overall production cost depends greatly on the source of carbon. Therefore, many attempts have been made to produce recombinant proteins in E. coli from molasses, a cheap carbon source. However, most E. coli cannot utilize sucrose which is the major sugar in molasses. The aim of this study is to solve these problems by expressing the recombinant lipase using genome-based expression in E. coli W. E. coli W was chosen because it is the only safe laboratory strain that can utilize sucrose and shows fast, highly oxidative sucrose metabolism with low acetate production. The objectives of this study are to clone and integrate lipase gene into E. coli W genome, to express the recombinant lipase and to optimize the production of the recombinant product. The T1 lipase gene cassettes with different promoters (tac and trc promoters) were successfully cloned and integrated into E. coli W for plasmid based and genome based expression respectively. The expression of T1 lipase in E. coli W were higher in plasmid-based expression system compared to genome-based expression system but both systems have higher expression under trc promoter (thus, it was chosen for further study). Optimization study recorded the highest expression of T1 lipase at induction temperature of 16°C, 0.8 mM of IPTG concentration and 3% of molasses in M9 medium. A combination of the approaches described above may permit the industrial scale utilization of E. coli for bioconversion of low-cost starting materials (sucrose-molasses) into industrially important enzymes. Furthermore, production of recombinant lipase from the stable expression in the genome can be a model for production of other industrially important recombinant proteins.
format	Thesis
qualification_level	Master's degree
author	Yusoff, Siti Hajar
author_facet	Yusoff, Siti Hajar
author_sort	Yusoff, Siti Hajar
title	Expression of recombinant T1 lipase in E. coli strain W using fifth grade molasses as carbon source
title_short	Expression of recombinant T1 lipase in E. coli strain W using fifth grade molasses as carbon source
title_full	Expression of recombinant T1 lipase in E. coli strain W using fifth grade molasses as carbon source
title_fullStr	Expression of recombinant T1 lipase in E. coli strain W using fifth grade molasses as carbon source
title_full_unstemmed	Expression of recombinant T1 lipase in E. coli strain W using fifth grade molasses as carbon source
title_sort	expression of recombinant t1 lipase in e. coli strain w using fifth grade molasses as carbon source
granting_institution	Universiti Putra Malaysia
publishDate	2019
url	http://psasir.upm.edu.my/id/eprint/92795/1/FBSB%202021%205%20IR.pdf
_version_	1747813768765636608

Expression of recombinant T1 lipase in E. coli strain W using fifth grade molasses as carbon source

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