Cloning and Expression of a Thermostable-α Glucosidase

Yeast is considered as a good host for large scale production of enzymes. This is the first report of α-glucosidase obtained from bacterial source to be expressed in yeast.Seven bacterial isolates were successfully obtained from water sample of Telaga Air Hangat, Langkawi. The optimum growth tempera...

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Main Author:	A. Mohamed, Rauda
Format:	Thesis
Language:	English English
Published:	2010
Subjects:	Cloning. Glycosides.
Online Access:	http://psasir.upm.edu.my/id/eprint/19437/1/FBSB_2010_24.pdf
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id	my-upm-ir.19437
record_format	uketd_dc
spelling	my-upm-ir.194372024-07-17T08:32:08Z Cloning and Expression of a Thermostable-α Glucosidase 2010-12 A. Mohamed, Rauda Yeast is considered as a good host for large scale production of enzymes. This is the first report of α-glucosidase obtained from bacterial source to be expressed in yeast.Seven bacterial isolates were successfully obtained from water sample of Telaga Air Hangat, Langkawi. The optimum growth temperature for these bacterial isolates (L2,L3, L4, GBB1, SR 38, SR 40 and SR 96) was at 55oC. Screening using an α-MUG plate overlay method indicated that 4 out of 7 isolates gave positive α-glucosidase activity (L2, L3, L4 and GBB1). The highest activity was 1.47 U/mL at 55°C from sample L3. This isolate was identified using 16S rRNA as a universal primer and from the BLAST result, the isolate showed 99% similarity to Geobacillus stearothermophilus. The gene encoding α-glucosidase was isolated from this identified bacterium using degenerate primers. A complete gene sequence encoding α-glucosidase (~1.7 kb) was obtained by a DNA walking approach. This gene fragment was successfully cloned and expressed into Escherichia coli Top10 cells using pBAD and pTrcHis2@TOPO TA expression vectors. The intracellular α-glucosidase production by recombinant E. coli was increased 3.4-fold and 2-fold in pBAD and pTrcHis2 compared to the wild type isolate, respectively. The restriction enzymes (RE) based primers were designed to clone the gene into a yeast expression vector pPICZαA and to allow transformation into P. pastoris. Transformation was successfully achieved with the α-glucosidase expression level at 3.3 U/mL before optimization. After optimization, the highest activity obtained was ~10 U/mL. This is about 2-fold higher than the expression by E. coli and 6-fold higher than the wild type isolate. P. pastoris expression system was shown to be effective in increasing the expression yield of the heterologous protein Cloning. Glycosides. 2010-12 Thesis http://psasir.upm.edu.my/id/eprint/19437/ http://psasir.upm.edu.my/id/eprint/19437/1/FBSB_2010_24.pdf text en public masters Universiti Putra Malaysia Cloning. Glycosides. Faculty of Biotechnology and Biomolecular Sciences Salleh, Abu Bakar English
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English English
advisor	Salleh, Abu Bakar
topic	Cloning. Glycosides.
spellingShingle	Cloning. Glycosides. A. Mohamed, Rauda Cloning and Expression of a Thermostable-α Glucosidase
description	Yeast is considered as a good host for large scale production of enzymes. This is the first report of α-glucosidase obtained from bacterial source to be expressed in yeast.Seven bacterial isolates were successfully obtained from water sample of Telaga Air Hangat, Langkawi. The optimum growth temperature for these bacterial isolates (L2,L3, L4, GBB1, SR 38, SR 40 and SR 96) was at 55oC. Screening using an α-MUG plate overlay method indicated that 4 out of 7 isolates gave positive α-glucosidase activity (L2, L3, L4 and GBB1). The highest activity was 1.47 U/mL at 55°C from sample L3. This isolate was identified using 16S rRNA as a universal primer and from the BLAST result, the isolate showed 99% similarity to Geobacillus stearothermophilus. The gene encoding α-glucosidase was isolated from this identified bacterium using degenerate primers. A complete gene sequence encoding α-glucosidase (~1.7 kb) was obtained by a DNA walking approach. This gene fragment was successfully cloned and expressed into Escherichia coli Top10 cells using pBAD and pTrcHis2@TOPO TA expression vectors. The intracellular α-glucosidase production by recombinant E. coli was increased 3.4-fold and 2-fold in pBAD and pTrcHis2 compared to the wild type isolate, respectively. The restriction enzymes (RE) based primers were designed to clone the gene into a yeast expression vector pPICZαA and to allow transformation into P. pastoris. Transformation was successfully achieved with the α-glucosidase expression level at 3.3 U/mL before optimization. After optimization, the highest activity obtained was ~10 U/mL. This is about 2-fold higher than the expression by E. coli and 6-fold higher than the wild type isolate. P. pastoris expression system was shown to be effective in increasing the expression yield of the heterologous protein
format	Thesis
qualification_level	Master's degree
author	A. Mohamed, Rauda
author_facet	A. Mohamed, Rauda
author_sort	A. Mohamed, Rauda
title	Cloning and Expression of a Thermostable-α Glucosidase
title_short	Cloning and Expression of a Thermostable-α Glucosidase
title_full	Cloning and Expression of a Thermostable-α Glucosidase
title_fullStr	Cloning and Expression of a Thermostable-α Glucosidase
title_full_unstemmed	Cloning and Expression of a Thermostable-α Glucosidase
title_sort	cloning and expression of a thermostable-α glucosidase
granting_institution	Universiti Putra Malaysia
granting_department	Faculty of Biotechnology and Biomolecular Sciences
publishDate	2010
url	http://psasir.upm.edu.my/id/eprint/19437/1/FBSB_2010_24.pdf
_version_	1804888711927693312

Cloning and Expression of a Thermostable-α Glucosidase

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