Expression and characterization of a cellobiohydrolase (CBH7B) from the thermophilic fungus Thielavia terrestris in Pichia pastoris

James Sy Keen Woon, Muhammad Mukram Mohamed Mackeen, Nor Muhammad Mahadi, Rosli Md Illias, Abdul Munir Abd. Murad, Farah Diba Abu Bakar

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4 Citations (Scopus)

Abstract

The gene encoding a cellobiohydrolase 7B (CBH7B) of the thermophilic fungus Thielavia terrestris was identified, subcloned, and expressed in Pichia pastoris. CBH7B encoded 455 amino acid residues with a molecular mass of 51.8 kDa. Domain analysis indicated that CBH7B contains a family 7 glycosyl hydrolase catalytic core but lacks a carbohydrate-binding module. Purified CBH7B exhibited optimum catalytic activity at pH 5.0 and 55 °C with 4-methylumbelliferryl-cellobioside as the substrate and retained 85% of its activity following 24 H incubation at 50 °C. Despite the lack of activity toward microcrystalline substrates, this enzyme worked synergistically with the commercial enzyme cocktail Cellic® CTec2 to enhance saccharification by 39% when added to a reaction mixture containing 0.25% alkaline pretreated oil palm empty fruit bunch (OPEFB). Attenuated total reflectance Fourier transform infrared spectroscopy suggested a reduction of lignin and crystalline cellulose in OPEFB samples supplemented with CBH7B. Scanning electron microscopy revealed greater destruction extent of OPEFB strands in samples supplemented with CBH7B as compared with the nonsupplemented control. Therefore, CBH7B has the potential to complement commercial enzymes in hydrolyzing lignocellulosic biomass.

Original languageEnglish
JournalBiotechnology and Applied Biochemistry
DOIs
Publication statusAccepted/In press - 2015

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Cellulose 1,4-beta-Cellobiosidase
Palm oil
Pichia
Fruits
Fungi
Enzymes
Hydrolases
Saccharification
Gene encoding
Fruit
Molecular mass
Substrates
Oils
Carbohydrates
Lignin
Fourier transform infrared spectroscopy
Amino acids
Cellulose
Catalyst activity
Biomass

Keywords

  • 4-methyumbelliferryl-cellobioside
  • Cellic CTec2
  • Glycosylation
  • Lignin
  • Oil palm empty fruit bunch

ASJC Scopus subject areas

  • Biotechnology
  • Molecular Medicine
  • Bioengineering
  • Process Chemistry and Technology
  • Biomedical Engineering
  • Applied Microbiology and Biotechnology
  • Drug Discovery

Cite this

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title = "Expression and characterization of a cellobiohydrolase (CBH7B) from the thermophilic fungus Thielavia terrestris in Pichia pastoris",
abstract = "The gene encoding a cellobiohydrolase 7B (CBH7B) of the thermophilic fungus Thielavia terrestris was identified, subcloned, and expressed in Pichia pastoris. CBH7B encoded 455 amino acid residues with a molecular mass of 51.8 kDa. Domain analysis indicated that CBH7B contains a family 7 glycosyl hydrolase catalytic core but lacks a carbohydrate-binding module. Purified CBH7B exhibited optimum catalytic activity at pH 5.0 and 55 °C with 4-methylumbelliferryl-cellobioside as the substrate and retained 85{\%} of its activity following 24 H incubation at 50 °C. Despite the lack of activity toward microcrystalline substrates, this enzyme worked synergistically with the commercial enzyme cocktail Cellic{\circledR} CTec2 to enhance saccharification by 39{\%} when added to a reaction mixture containing 0.25{\%} alkaline pretreated oil palm empty fruit bunch (OPEFB). Attenuated total reflectance Fourier transform infrared spectroscopy suggested a reduction of lignin and crystalline cellulose in OPEFB samples supplemented with CBH7B. Scanning electron microscopy revealed greater destruction extent of OPEFB strands in samples supplemented with CBH7B as compared with the nonsupplemented control. Therefore, CBH7B has the potential to complement commercial enzymes in hydrolyzing lignocellulosic biomass.",
keywords = "4-methyumbelliferryl-cellobioside, Cellic CTec2, Glycosylation, Lignin, Oil palm empty fruit bunch",
author = "Woon, {James Sy Keen} and {Mohamed Mackeen}, {Muhammad Mukram} and Mahadi, {Nor Muhammad} and Illias, {Rosli Md} and {Abd. Murad}, {Abdul Munir} and {Abu Bakar}, {Farah Diba}",
year = "2015",
doi = "10.1002/bab.1431",
language = "English",
journal = "Biotechnology and Applied Biochemistry",
issn = "0885-4513",
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TY - JOUR

T1 - Expression and characterization of a cellobiohydrolase (CBH7B) from the thermophilic fungus Thielavia terrestris in Pichia pastoris

AU - Woon, James Sy Keen

AU - Mohamed Mackeen, Muhammad Mukram

AU - Mahadi, Nor Muhammad

AU - Illias, Rosli Md

AU - Abd. Murad, Abdul Munir

AU - Abu Bakar, Farah Diba

PY - 2015

Y1 - 2015

N2 - The gene encoding a cellobiohydrolase 7B (CBH7B) of the thermophilic fungus Thielavia terrestris was identified, subcloned, and expressed in Pichia pastoris. CBH7B encoded 455 amino acid residues with a molecular mass of 51.8 kDa. Domain analysis indicated that CBH7B contains a family 7 glycosyl hydrolase catalytic core but lacks a carbohydrate-binding module. Purified CBH7B exhibited optimum catalytic activity at pH 5.0 and 55 °C with 4-methylumbelliferryl-cellobioside as the substrate and retained 85% of its activity following 24 H incubation at 50 °C. Despite the lack of activity toward microcrystalline substrates, this enzyme worked synergistically with the commercial enzyme cocktail Cellic® CTec2 to enhance saccharification by 39% when added to a reaction mixture containing 0.25% alkaline pretreated oil palm empty fruit bunch (OPEFB). Attenuated total reflectance Fourier transform infrared spectroscopy suggested a reduction of lignin and crystalline cellulose in OPEFB samples supplemented with CBH7B. Scanning electron microscopy revealed greater destruction extent of OPEFB strands in samples supplemented with CBH7B as compared with the nonsupplemented control. Therefore, CBH7B has the potential to complement commercial enzymes in hydrolyzing lignocellulosic biomass.

AB - The gene encoding a cellobiohydrolase 7B (CBH7B) of the thermophilic fungus Thielavia terrestris was identified, subcloned, and expressed in Pichia pastoris. CBH7B encoded 455 amino acid residues with a molecular mass of 51.8 kDa. Domain analysis indicated that CBH7B contains a family 7 glycosyl hydrolase catalytic core but lacks a carbohydrate-binding module. Purified CBH7B exhibited optimum catalytic activity at pH 5.0 and 55 °C with 4-methylumbelliferryl-cellobioside as the substrate and retained 85% of its activity following 24 H incubation at 50 °C. Despite the lack of activity toward microcrystalline substrates, this enzyme worked synergistically with the commercial enzyme cocktail Cellic® CTec2 to enhance saccharification by 39% when added to a reaction mixture containing 0.25% alkaline pretreated oil palm empty fruit bunch (OPEFB). Attenuated total reflectance Fourier transform infrared spectroscopy suggested a reduction of lignin and crystalline cellulose in OPEFB samples supplemented with CBH7B. Scanning electron microscopy revealed greater destruction extent of OPEFB strands in samples supplemented with CBH7B as compared with the nonsupplemented control. Therefore, CBH7B has the potential to complement commercial enzymes in hydrolyzing lignocellulosic biomass.

KW - 4-methyumbelliferryl-cellobioside

KW - Cellic CTec2

KW - Glycosylation

KW - Lignin

KW - Oil palm empty fruit bunch

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