Surface display of endoglucanase and β-glucosidase using ice nucleation protein a from Erwinia ananas on Escherichia coli

Surface display of endoglucanase and β-glucosidase using ice nucleation protein a from Erwinia ananas on Escherichia coli

Cells disruption for obtainment of targeted protein has increased the cost for expression of recombinant protein in Escherichia coli. Cell surface display system is one of the approaches to resolve this issue. The objective of this research is to study expression and characterization of surface disp...

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Main Author: Yeng, Min Yi
Format: Thesis
Language:English
Published: 2019
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TP Chemical technology
Online Access:http://eprints.utm.my/id/eprint/96624/1/YengMinYiMSKT2019.pdf
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spelling my-utm-ep.966242022-08-08T05:00:30Z Surface display of endoglucanase and β-glucosidase using ice nucleation protein a from Erwinia ananas on Escherichia coli 2019 Yeng, Min Yi TP Chemical technology Cells disruption for obtainment of targeted protein has increased the cost for expression of recombinant protein in Escherichia coli. Cell surface display system is one of the approaches to resolve this issue. The objective of this research is to study expression and characterization of surface display cellulases on E. coli using ice nucleation protein A (InA) anchor protein from Erwinia ananas. Cellulases such as endoglucanase Cel5A (EC 3.2.1.4), exoglucanase Cel9E (EC 3.2.1.91) and Pglucosidase BglC (EC 3.2.1.21) fused to the C-terminal of InA were expressed in E. coli. InA-Cel5A and InA-BglC were shown to be displayed on the cell surface by analyzing outer membrane protein on SDS-PAGE and Western blot. Enzyme assay and immunofluorescence microscopy analysis showed that InA-Cel5A and InA-BglC were functionally expressed on the cell membrane. InA-Cel9E was not successfully displayed on cell surface. The optimum temperature and pH of surface display InACel5A and InA-BglC were 60 °C and pH 7, respectively. Optimization of cultivation conditions of InA-Cel5A and InA-BglC were carried out at different post induction time, medium, post induction temperature and isopropyl P-D-1- thiogalactopyranoside (IPTG) concentration. The optimized conditions obtained for expression of InA-Cel5A were M9 medium, 15 °C induction temperature, 0.1 mM IPTG and 14 hours, which gave endoglucanase activity of 0.6537 U/mL. The optimized conditions obtained for expression of InA-BglC were M9 medium, 30 °C induction temperature, 0.1 mM IPTG and 6 hours, which gave P-glucosidase activity of 198.439 U/mL. InA-Cel5A and InA-BglC were used for cellulose hydrolysis. Surface display InA-Cel5A and InA-BglC were used to degrade 4 % (w/v) Avicel at 50 °C and 200 rpm, producing 0.204 mg/mL reducing sugars but with low glucose concentration. The optimum ratio of InA-Cel5A and InA-BglC used in the hydrolysis was found to be 4: 1. Results indicated that InA-Cel5A and InA-BglC were successfully displayed on E. coli using InA. Nevertheless, such recombinant E. coli showed a low hydrolytic activity and only low glucose concentration can be detected. The success of displaying enzyme on E. coli using ice nucleation protein showed great potential to be used in whole-cell biocatalysis. 2019 Thesis http://eprints.utm.my/id/eprint/96624/ http://eprints.utm.my/id/eprint/96624/1/YengMinYiMSKT2019.pdf application/pdf en public http://dms.library.utm.my:8080/vital/access/manager/Repository/vital:131867 masters Universiti Teknologi Malaysia Faculty of Engineering - School of Chemical & Energy Engineering
institution Universiti Teknologi Malaysia
collection UTM Institutional Repository
language English
topic TP Chemical technology
spellingShingle TP Chemical technology
Yeng, Min Yi
Surface display of endoglucanase and β-glucosidase using ice nucleation protein a from Erwinia ananas on Escherichia coli
description Cells disruption for obtainment of targeted protein has increased the cost for expression of recombinant protein in Escherichia coli. Cell surface display system is one of the approaches to resolve this issue. The objective of this research is to study expression and characterization of surface display cellulases on E. coli using ice nucleation protein A (InA) anchor protein from Erwinia ananas. Cellulases such as endoglucanase Cel5A (EC 3.2.1.4), exoglucanase Cel9E (EC 3.2.1.91) and Pglucosidase BglC (EC 3.2.1.21) fused to the C-terminal of InA were expressed in E. coli. InA-Cel5A and InA-BglC were shown to be displayed on the cell surface by analyzing outer membrane protein on SDS-PAGE and Western blot. Enzyme assay and immunofluorescence microscopy analysis showed that InA-Cel5A and InA-BglC were functionally expressed on the cell membrane. InA-Cel9E was not successfully displayed on cell surface. The optimum temperature and pH of surface display InACel5A and InA-BglC were 60 °C and pH 7, respectively. Optimization of cultivation conditions of InA-Cel5A and InA-BglC were carried out at different post induction time, medium, post induction temperature and isopropyl P-D-1- thiogalactopyranoside (IPTG) concentration. The optimized conditions obtained for expression of InA-Cel5A were M9 medium, 15 °C induction temperature, 0.1 mM IPTG and 14 hours, which gave endoglucanase activity of 0.6537 U/mL. The optimized conditions obtained for expression of InA-BglC were M9 medium, 30 °C induction temperature, 0.1 mM IPTG and 6 hours, which gave P-glucosidase activity of 198.439 U/mL. InA-Cel5A and InA-BglC were used for cellulose hydrolysis. Surface display InA-Cel5A and InA-BglC were used to degrade 4 % (w/v) Avicel at 50 °C and 200 rpm, producing 0.204 mg/mL reducing sugars but with low glucose concentration. The optimum ratio of InA-Cel5A and InA-BglC used in the hydrolysis was found to be 4: 1. Results indicated that InA-Cel5A and InA-BglC were successfully displayed on E. coli using InA. Nevertheless, such recombinant E. coli showed a low hydrolytic activity and only low glucose concentration can be detected. The success of displaying enzyme on E. coli using ice nucleation protein showed great potential to be used in whole-cell biocatalysis.
format Thesis
qualification_level Master's degree
author Yeng, Min Yi
author_facet Yeng, Min Yi
author_sort Yeng, Min Yi
title Surface display of endoglucanase and β-glucosidase using ice nucleation protein a from Erwinia ananas on Escherichia coli
title_short Surface display of endoglucanase and β-glucosidase using ice nucleation protein a from Erwinia ananas on Escherichia coli
title_full Surface display of endoglucanase and β-glucosidase using ice nucleation protein a from Erwinia ananas on Escherichia coli
title_fullStr Surface display of endoglucanase and β-glucosidase using ice nucleation protein a from Erwinia ananas on Escherichia coli
title_full_unstemmed Surface display of endoglucanase and β-glucosidase using ice nucleation protein a from Erwinia ananas on Escherichia coli
title_sort surface display of endoglucanase and î²-glucosidase using ice nucleation protein a from erwinia ananas on escherichia coli
granting_institution Universiti Teknologi Malaysia
granting_department Faculty of Engineering - School of Chemical & Energy Engineering
publishDate 2019
url http://eprints.utm.my/id/eprint/96624/1/YengMinYiMSKT2019.pdf
_version_ 1747818670750433280

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