Cloning, heterologous expression and characterisation of a recombinant cellobiohydrolase from Humicola insolens ATCC16454 in Pichia pastoris

A cellobiohydrolase gene from the thermophilic fungus Humicola insolens ATCC 16454 was expressed in the methylotrophic yeast Pichia pastoris X-33, and the biochemical properties of the recombinant protein were characterised. The full-length cDNA of the cellobiohydrolase gene avi2 was cloned into t...

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Main Authors:	Amatul Samahah Md Ali, Farah Diba Abu Bakar, Rosli Md Illias, Osman Hassan, Abdul Munir Abdul Murad
Format:	Article
Language:	English
Published:	Malaysian Society of Applied Biology 2015
Online Access:	http://journalarticle.ukm.my/11786/ http://journalarticle.ukm.my/11786/ http://journalarticle.ukm.my/11786/1/44_4_03.pdf

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spelling	ukm-117862018-06-29T07:54:18Z http://journalarticle.ukm.my/11786/ Cloning, heterologous expression and characterisation of a recombinant cellobiohydrolase from Humicola insolens ATCC16454 in Pichia pastoris Amatul Samahah Md Ali, Farah Diba Abu Bakar, Rosli Md Illias, Osman Hassan, Abdul Munir Abdul Murad, A cellobiohydrolase gene from the thermophilic fungus Humicola insolens ATCC 16454 was expressed in the methylotrophic yeast Pichia pastoris X-33, and the biochemical properties of the recombinant protein were characterised. The full-length cDNA of the cellobiohydrolase gene avi2 was cloned into the P. pastoris expression vector pPICZαC and expressed extracellularly as a recombinant cellobiohydrolase protein with a molecular weight of approximately 52.3 kDa. The purified recombinant Avi2 enzyme displayed an optimal activity at 50°C and was found stable between temperatures of 30°C and 60°C. The optimal pH of the enzyme was pH 5.0. More than 80% of the enzyme activity was retained at pH values ranging from pH3.0 to pH9.0. Recombinant Avi2 enzyme showed its highest activity towards the substrates Avicel (0.075 U mg-1) and Sigmacell-cellulose (0.018 U mg-1). Very low or undetectable hydrolysis was observed with cellobiose and filter paper. Metal ions, such as Mn2+, Co2+, and Ba2+, increased the activity of the recombinant enzyme. Manganese ions caused the highest increase in activity of approximately 1.38-fold compared to the control assay. Other ions such as Pd2+, Cu2+, Zn2+, Fe2+, and SDS, however, inhibited Avi2 enzyme activity. Interestingly, this recombinant enzyme showed high pH stability when it was incubated in either acidic or basic solutions. Malaysian Society of Applied Biology 2015-12 Article PeerReviewed application/pdf en http://journalarticle.ukm.my/11786/1/44_4_03.pdf Amatul Samahah Md Ali, and Farah Diba Abu Bakar, and Rosli Md Illias, and Osman Hassan, and Abdul Munir Abdul Murad, (2015) Cloning, heterologous expression and characterisation of a recombinant cellobiohydrolase from Humicola insolens ATCC16454 in Pichia pastoris. Malaysian Applied Biology, 44 (4). pp. 19-26. ISSN 0126-8643 http://www.mabjournal.com/index.php?option=com_content&view=article&id=554&catid=59:current-view&Itemid=56
repository_type	Digital Repository
institution_category	Local University
institution	Universiti Kebangasaan Malaysia
building	UKM Institutional Repository
collection	Online Access
language	English
description	A cellobiohydrolase gene from the thermophilic fungus Humicola insolens ATCC 16454 was expressed in the methylotrophic yeast Pichia pastoris X-33, and the biochemical properties of the recombinant protein were characterised. The full-length cDNA of the cellobiohydrolase gene avi2 was cloned into the P. pastoris expression vector pPICZαC and expressed extracellularly as a recombinant cellobiohydrolase protein with a molecular weight of approximately 52.3 kDa. The purified recombinant Avi2 enzyme displayed an optimal activity at 50°C and was found stable between temperatures of 30°C and 60°C. The optimal pH of the enzyme was pH 5.0. More than 80% of the enzyme activity was retained at pH values ranging from pH3.0 to pH9.0. Recombinant Avi2 enzyme showed its highest activity towards the substrates Avicel (0.075 U mg-1) and Sigmacell-cellulose (0.018 U mg-1). Very low or undetectable hydrolysis was observed with cellobiose and filter paper. Metal ions, such as Mn2+, Co2+, and Ba2+, increased the activity of the recombinant enzyme. Manganese ions caused the highest increase in activity of approximately 1.38-fold compared to the control assay. Other ions such as Pd2+, Cu2+, Zn2+, Fe2+, and SDS, however, inhibited Avi2 enzyme activity. Interestingly, this recombinant enzyme showed high pH stability when it was incubated in either acidic or basic solutions.
format	Article
author	Amatul Samahah Md Ali, Farah Diba Abu Bakar, Rosli Md Illias, Osman Hassan, Abdul Munir Abdul Murad,
spellingShingle	Amatul Samahah Md Ali, Farah Diba Abu Bakar, Rosli Md Illias, Osman Hassan, Abdul Munir Abdul Murad, Cloning, heterologous expression and characterisation of a recombinant cellobiohydrolase from Humicola insolens ATCC16454 in Pichia pastoris
author_facet	Amatul Samahah Md Ali, Farah Diba Abu Bakar, Rosli Md Illias, Osman Hassan, Abdul Munir Abdul Murad,
author_sort	Amatul Samahah Md Ali,
title	Cloning, heterologous expression and characterisation of a recombinant cellobiohydrolase from Humicola insolens ATCC16454 in Pichia pastoris
title_short	Cloning, heterologous expression and characterisation of a recombinant cellobiohydrolase from Humicola insolens ATCC16454 in Pichia pastoris
title_full	Cloning, heterologous expression and characterisation of a recombinant cellobiohydrolase from Humicola insolens ATCC16454 in Pichia pastoris
title_fullStr	Cloning, heterologous expression and characterisation of a recombinant cellobiohydrolase from Humicola insolens ATCC16454 in Pichia pastoris
title_full_unstemmed	Cloning, heterologous expression and characterisation of a recombinant cellobiohydrolase from Humicola insolens ATCC16454 in Pichia pastoris
title_sort	cloning, heterologous expression and characterisation of a recombinant cellobiohydrolase from humicola insolens atcc16454 in pichia pastoris
publisher	Malaysian Society of Applied Biology
publishDate	2015
url	http://journalarticle.ukm.my/11786/ http://journalarticle.ukm.my/11786/ http://journalarticle.ukm.my/11786/1/44_4_03.pdf
first_indexed	2023-09-18T20:01:08Z
last_indexed	2023-09-18T20:01:08Z
_version_	1777406856354856960

Cloning, heterologous expression and characterisation of a recombinant cellobiohydrolase from Humicola insolens ATCC16454 in Pichia pastoris

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