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 Abd. Murad

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish
Pages (from-to)19-26
Number of pages8
JournalMalaysian Applied Biology
Volume44
Issue number4
Publication statusPublished - 1 Dec 2015

Fingerprint

Humicola insolens
Cellulose 1,4-beta-Cellobiosidase
cellulose 1,4-beta-cellobiosidase
Pichia pastoris
Pichia
Organism Cloning
molecular cloning
Enzymes
enzymes
Ions
thermophilic fungi
enzyme activity
Recombinant Proteins
ions
Cellulose
cellobiose
metal ions
recombinant proteins
Cellobiose
manganese

Keywords

  • Cellobiohydrolase
  • Humicola insolens
  • Pichia pastoris
  • Recombinant enzyme

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)

Cite this

@article{b0b7513713f24599833548600cf8c823,
title = "Cloning, heterologous expression and characterisation of a recombinant cellobiohydrolase from Humicola Insolens ATCC16454 IN Pichia Pastoris",
abstract = "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.",
keywords = "Cellobiohydrolase, Humicola insolens, Pichia pastoris, Recombinant enzyme",
author = "Ali, {Amatul Samahah Md} and {Abu Bakar}, {Farah Diba} and Illias, {Rosli Md} and Osman Hassan and {Abd. Murad}, {Abdul Munir}",
year = "2015",
month = "12",
day = "1",
language = "English",
volume = "44",
pages = "19--26",
journal = "Malaysian Applied Biology",
issn = "0126-8643",
publisher = "Malaysian Society of Applied Biology",
number = "4",

}

TY - JOUR

T1 - Cloning, heterologous expression and characterisation of a recombinant cellobiohydrolase from Humicola Insolens ATCC16454 IN Pichia Pastoris

AU - Ali, Amatul Samahah Md

AU - Abu Bakar, Farah Diba

AU - Illias, Rosli Md

AU - Hassan, Osman

AU - Abd. Murad, Abdul Munir

PY - 2015/12/1

Y1 - 2015/12/1

N2 - 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.

AB - 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.

KW - Cellobiohydrolase

KW - Humicola insolens

KW - Pichia pastoris

KW - Recombinant enzyme

UR - http://www.scopus.com/inward/record.url?scp=84955300378&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84955300378&partnerID=8YFLogxK

M3 - Article

VL - 44

SP - 19

EP - 26

JO - Malaysian Applied Biology

JF - Malaysian Applied Biology

SN - 0126-8643

IS - 4

ER -