Improvement of cyclodextrin glucanotranferase excretion and cell viability of recombinant Escherichia coli immobilized on hollow fiber membrane

The excretion of a recombinant enzyme into culture medium presents significant advantages over cytoplasmic expression. However, cell lysis is one of the major drawbacks during the excretion of recombinant enzyme when using Escherichia coli (E. coli) as a host. Cell immobilization is a promising solu...

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Main Author:	Rohaida, Che Man
Format:	Thesis
Language:	English
Published:	2016
Subjects:	QR Microbiology
Online Access:	http://umpir.ump.edu.my/id/eprint/25221/1/Improvement%20of%20cyclodextrin%20glucanotranferase%20excretion%20and%20cell%20viability.pdf
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spelling	my-ump-ir.252212021-09-01T04:13:53Z Improvement of cyclodextrin glucanotranferase excretion and cell viability of recombinant Escherichia coli immobilized on hollow fiber membrane 2016-03 Rohaida, Che Man QR Microbiology The excretion of a recombinant enzyme into culture medium presents significant advantages over cytoplasmic expression. However, cell lysis is one of the major drawbacks during the excretion of recombinant enzyme when using Escherichia coli (E. coli) as a host. Cell immobilization is a promising solution for the enhancement of enzyme excretion with reduction of cell lysis. In the present study, a recombinant E. coli was immobilized using hollow fiber membrane to improve the enzyme excretion, cell viability and plasmid stability. The effects of different polymers of hollow fiber membrane and culture conditions on the cyclodextrin glucanotransferase (CGTase) excretion, cell lysis and plasmid stability of immobilized E. coli were investigated. The immobilized cells on a polyvinylidene fluoride polymer exhibited a 2.0-4.5-fold increase in the CGTase excretion with 18-95% reduction of cell lysis and over 100% increment of plasmid stability compared to the free cells. The CGTase excretion was successfully optimized by response surface methodology. Under the optimized conditions [25 °C of post- induction temperature, 0.011 mM isopropyl β-D-1-thiogalactopyronoside and pH 8.8], the CGTase excretion was 3.8-fold higher with 80% reduction of cell lysis compared to the value before optimization process. The use of low tryptone concentration (5 g/1) reduced the occurrence of cell lysis (90% reduction) and increased the plasmid stability (86% increment) without significant change in CGTase excretion in comparison with initial tryptone concentration (20 g/1). This approach (5 g/1) produced an approximately two times higher CGTase excretion (compared with 20 g/1 during) recycle process. The membrane bioreactor also showed 2.5-fold increase in the CGTase excretion (473 x 103 U/ml) with 75% reduction of cell lysis compared to shake flask culture (190 x 103 U/ml of CGTase activity). Hence, the immobilization of E. coli on hollow fiber membrane proved to be valuable for the excretion of recombinant proteins in E. coli with high cell stability. 2016-03 Thesis http://umpir.ump.edu.my/id/eprint/25221/ http://umpir.ump.edu.my/id/eprint/25221/1/Improvement%20of%20cyclodextrin%20glucanotranferase%20excretion%20and%20cell%20viability.pdf pdf en public phd doctoral Universiti Teknologi Malaysia Faculty of Chemical and Energy Engineering
institution	Universiti Malaysia Pahang Al-Sultan Abdullah
collection	UMPSA Institutional Repository
language	English
topic	QR Microbiology
spellingShingle	QR Microbiology Rohaida, Che Man Improvement of cyclodextrin glucanotranferase excretion and cell viability of recombinant Escherichia coli immobilized on hollow fiber membrane
description	The excretion of a recombinant enzyme into culture medium presents significant advantages over cytoplasmic expression. However, cell lysis is one of the major drawbacks during the excretion of recombinant enzyme when using Escherichia coli (E. coli) as a host. Cell immobilization is a promising solution for the enhancement of enzyme excretion with reduction of cell lysis. In the present study, a recombinant E. coli was immobilized using hollow fiber membrane to improve the enzyme excretion, cell viability and plasmid stability. The effects of different polymers of hollow fiber membrane and culture conditions on the cyclodextrin glucanotransferase (CGTase) excretion, cell lysis and plasmid stability of immobilized E. coli were investigated. The immobilized cells on a polyvinylidene fluoride polymer exhibited a 2.0-4.5-fold increase in the CGTase excretion with 18-95% reduction of cell lysis and over 100% increment of plasmid stability compared to the free cells. The CGTase excretion was successfully optimized by response surface methodology. Under the optimized conditions [25 °C of post- induction temperature, 0.011 mM isopropyl β-D-1-thiogalactopyronoside and pH 8.8], the CGTase excretion was 3.8-fold higher with 80% reduction of cell lysis compared to the value before optimization process. The use of low tryptone concentration (5 g/1) reduced the occurrence of cell lysis (90% reduction) and increased the plasmid stability (86% increment) without significant change in CGTase excretion in comparison with initial tryptone concentration (20 g/1). This approach (5 g/1) produced an approximately two times higher CGTase excretion (compared with 20 g/1 during) recycle process. The membrane bioreactor also showed 2.5-fold increase in the CGTase excretion (473 x 103 U/ml) with 75% reduction of cell lysis compared to shake flask culture (190 x 103 U/ml of CGTase activity). Hence, the immobilization of E. coli on hollow fiber membrane proved to be valuable for the excretion of recombinant proteins in E. coli with high cell stability.
format	Thesis
qualification_name	Doctor of Philosophy (PhD.)
qualification_level	Doctorate
author	Rohaida, Che Man
author_facet	Rohaida, Che Man
author_sort	Rohaida, Che Man
title	Improvement of cyclodextrin glucanotranferase excretion and cell viability of recombinant Escherichia coli immobilized on hollow fiber membrane
title_short	Improvement of cyclodextrin glucanotranferase excretion and cell viability of recombinant Escherichia coli immobilized on hollow fiber membrane
title_full	Improvement of cyclodextrin glucanotranferase excretion and cell viability of recombinant Escherichia coli immobilized on hollow fiber membrane
title_fullStr	Improvement of cyclodextrin glucanotranferase excretion and cell viability of recombinant Escherichia coli immobilized on hollow fiber membrane
title_full_unstemmed	Improvement of cyclodextrin glucanotranferase excretion and cell viability of recombinant Escherichia coli immobilized on hollow fiber membrane
title_sort	improvement of cyclodextrin glucanotranferase excretion and cell viability of recombinant escherichia coli immobilized on hollow fiber membrane
granting_institution	Universiti Teknologi Malaysia
granting_department	Faculty of Chemical and Energy Engineering
publishDate	2016
url	http://umpir.ump.edu.my/id/eprint/25221/1/Improvement%20of%20cyclodextrin%20glucanotranferase%20excretion%20and%20cell%20viability.pdf
_version_	1783732090700824576

Improvement of cyclodextrin glucanotranferase excretion and cell viability of recombinant Escherichia coli immobilized on hollow fiber membrane

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