Functional characterisation of cellobiohydrolase I (Cbh1) from Trichoderma virens UKM1 expressed in Aspergillus niger

Anis Farhan Fatimi Ab Wahab, Noor Adila Abdul Karim, Jonathan Guyang Ling, Nurain Shahera Hasan, Hui Yee Yong, Izwan Bharudin, Shazilah Kamaruddin, Farah Diba Abu Bakar, Abdul Munir Abd. Murad

Research output: Contribution to journalArticle

Abstract

Cellobiohydrolases catalyze the processive hydrolysis of cellulose into cellobiose. Here, a Trichoderma virens cDNA predicted to encode for cellobiohydrolase (cbhI) was cloned and expressed heterologously in Aspergillus niger. The cbhI gene has an open reading frame of 1518 bp, encoding for a putative protein of 505 amino acid residues with a calculated molecular mass of approximately 54 kDa. The predicted CbhI amino acid sequence has a fungal type carbohydrate binding module separated from a catalytic domain by a threonine rich linker region and showed high sequence homology with glycoside hydrolase family 7 proteins. The partially purified enzyme has an optimum pH of 4.0 with stability ranging from pH 3.0 to 6.0 and an optimum temperature of 60 °C. The partially purified CbhI has a specific activity of 4.195 Umg−1 and a low Km value of 1.88 mM when p-nitrophenyl-β-D-cellobioside (pNPC) is used as the substrate. The catalytic efficiency (kcat/Km) was 5.68 × 10−4 mM−1s−1, which is comparable to the CbhI enzymes from Trichoderma viridae and Phanaerochaete chrysosporium. CbhI also showed activity towards complex substrates such as Avicel (0.011 Umg−1), which could be useful in complex biomass degradation. Interestingly, CbhI also exhibited a relatively high inhibition constant (Ki) for cellobiose with a value of 8.65 mM, making this enzyme more resistant to end-product inhibition compared to other fungal cellobiohydrolases.

LanguageEnglish
Pages52-61
Number of pages10
JournalProtein Expression and Purification
Volume154
DOIs
Publication statusPublished - 1 Feb 2019

Fingerprint

Cellulose 1,4-beta-Cellobiosidase
Trichoderma
Aspergillus niger
Glycoside Hydrolases
Cellobiose
Cellulose
Enzymes
Chrysosporium
Threonine
Sequence Homology
Biomass
Open Reading Frames
Amino Acid Sequence
Catalytic Domain
Proteins
Hydrolysis
Complementary DNA
Carbohydrates
Amino Acids
Temperature

Keywords

  • Aspergillus niger
  • Cellobiohydrolase I
  • Glycoside hydrolase
  • Trichoderma virens

ASJC Scopus subject areas

  • Biotechnology

Cite this

Functional characterisation of cellobiohydrolase I (Cbh1) from Trichoderma virens UKM1 expressed in Aspergillus niger. / Wahab, Anis Farhan Fatimi Ab; Abdul Karim, Noor Adila; Ling, Jonathan Guyang; Hasan, Nurain Shahera; Yong, Hui Yee; Bharudin, Izwan; Kamaruddin, Shazilah; Abu Bakar, Farah Diba; Abd. Murad, Abdul Munir.

In: Protein Expression and Purification, Vol. 154, 01.02.2019, p. 52-61.

Research output: Contribution to journalArticle

Wahab, Anis Farhan Fatimi Ab ; Abdul Karim, Noor Adila ; Ling, Jonathan Guyang ; Hasan, Nurain Shahera ; Yong, Hui Yee ; Bharudin, Izwan ; Kamaruddin, Shazilah ; Abu Bakar, Farah Diba ; Abd. Murad, Abdul Munir. / Functional characterisation of cellobiohydrolase I (Cbh1) from Trichoderma virens UKM1 expressed in Aspergillus niger. In: Protein Expression and Purification. 2019 ; Vol. 154. pp. 52-61.
@article{aca0b761030040c28e51807182c50a5e,
title = "Functional characterisation of cellobiohydrolase I (Cbh1) from Trichoderma virens UKM1 expressed in Aspergillus niger",
abstract = "Cellobiohydrolases catalyze the processive hydrolysis of cellulose into cellobiose. Here, a Trichoderma virens cDNA predicted to encode for cellobiohydrolase (cbhI) was cloned and expressed heterologously in Aspergillus niger. The cbhI gene has an open reading frame of 1518 bp, encoding for a putative protein of 505 amino acid residues with a calculated molecular mass of approximately 54 kDa. The predicted CbhI amino acid sequence has a fungal type carbohydrate binding module separated from a catalytic domain by a threonine rich linker region and showed high sequence homology with glycoside hydrolase family 7 proteins. The partially purified enzyme has an optimum pH of 4.0 with stability ranging from pH 3.0 to 6.0 and an optimum temperature of 60 °C. The partially purified CbhI has a specific activity of 4.195 Umg−1 and a low Km value of 1.88 mM when p-nitrophenyl-β-D-cellobioside (pNPC) is used as the substrate. The catalytic efficiency (kcat/Km) was 5.68 × 10−4 mM−1s−1, which is comparable to the CbhI enzymes from Trichoderma viridae and Phanaerochaete chrysosporium. CbhI also showed activity towards complex substrates such as Avicel (0.011 Umg−1), which could be useful in complex biomass degradation. Interestingly, CbhI also exhibited a relatively high inhibition constant (Ki) for cellobiose with a value of 8.65 mM, making this enzyme more resistant to end-product inhibition compared to other fungal cellobiohydrolases.",
keywords = "Aspergillus niger, Cellobiohydrolase I, Glycoside hydrolase, Trichoderma virens",
author = "Wahab, {Anis Farhan Fatimi Ab} and {Abdul Karim}, {Noor Adila} and Ling, {Jonathan Guyang} and Hasan, {Nurain Shahera} and Yong, {Hui Yee} and Izwan Bharudin and Shazilah Kamaruddin and {Abu Bakar}, {Farah Diba} and {Abd. Murad}, {Abdul Munir}",
year = "2019",
month = "2",
day = "1",
doi = "10.1016/j.pep.2018.09.014",
language = "English",
volume = "154",
pages = "52--61",
journal = "Protein Expression and Purification",
issn = "1046-5928",
publisher = "Academic Press Inc.",

}

TY - JOUR

T1 - Functional characterisation of cellobiohydrolase I (Cbh1) from Trichoderma virens UKM1 expressed in Aspergillus niger

AU - Wahab, Anis Farhan Fatimi Ab

AU - Abdul Karim, Noor Adila

AU - Ling, Jonathan Guyang

AU - Hasan, Nurain Shahera

AU - Yong, Hui Yee

AU - Bharudin, Izwan

AU - Kamaruddin, Shazilah

AU - Abu Bakar, Farah Diba

AU - Abd. Murad, Abdul Munir

PY - 2019/2/1

Y1 - 2019/2/1

N2 - Cellobiohydrolases catalyze the processive hydrolysis of cellulose into cellobiose. Here, a Trichoderma virens cDNA predicted to encode for cellobiohydrolase (cbhI) was cloned and expressed heterologously in Aspergillus niger. The cbhI gene has an open reading frame of 1518 bp, encoding for a putative protein of 505 amino acid residues with a calculated molecular mass of approximately 54 kDa. The predicted CbhI amino acid sequence has a fungal type carbohydrate binding module separated from a catalytic domain by a threonine rich linker region and showed high sequence homology with glycoside hydrolase family 7 proteins. The partially purified enzyme has an optimum pH of 4.0 with stability ranging from pH 3.0 to 6.0 and an optimum temperature of 60 °C. The partially purified CbhI has a specific activity of 4.195 Umg−1 and a low Km value of 1.88 mM when p-nitrophenyl-β-D-cellobioside (pNPC) is used as the substrate. The catalytic efficiency (kcat/Km) was 5.68 × 10−4 mM−1s−1, which is comparable to the CbhI enzymes from Trichoderma viridae and Phanaerochaete chrysosporium. CbhI also showed activity towards complex substrates such as Avicel (0.011 Umg−1), which could be useful in complex biomass degradation. Interestingly, CbhI also exhibited a relatively high inhibition constant (Ki) for cellobiose with a value of 8.65 mM, making this enzyme more resistant to end-product inhibition compared to other fungal cellobiohydrolases.

AB - Cellobiohydrolases catalyze the processive hydrolysis of cellulose into cellobiose. Here, a Trichoderma virens cDNA predicted to encode for cellobiohydrolase (cbhI) was cloned and expressed heterologously in Aspergillus niger. The cbhI gene has an open reading frame of 1518 bp, encoding for a putative protein of 505 amino acid residues with a calculated molecular mass of approximately 54 kDa. The predicted CbhI amino acid sequence has a fungal type carbohydrate binding module separated from a catalytic domain by a threonine rich linker region and showed high sequence homology with glycoside hydrolase family 7 proteins. The partially purified enzyme has an optimum pH of 4.0 with stability ranging from pH 3.0 to 6.0 and an optimum temperature of 60 °C. The partially purified CbhI has a specific activity of 4.195 Umg−1 and a low Km value of 1.88 mM when p-nitrophenyl-β-D-cellobioside (pNPC) is used as the substrate. The catalytic efficiency (kcat/Km) was 5.68 × 10−4 mM−1s−1, which is comparable to the CbhI enzymes from Trichoderma viridae and Phanaerochaete chrysosporium. CbhI also showed activity towards complex substrates such as Avicel (0.011 Umg−1), which could be useful in complex biomass degradation. Interestingly, CbhI also exhibited a relatively high inhibition constant (Ki) for cellobiose with a value of 8.65 mM, making this enzyme more resistant to end-product inhibition compared to other fungal cellobiohydrolases.

KW - Aspergillus niger

KW - Cellobiohydrolase I

KW - Glycoside hydrolase

KW - Trichoderma virens

UR - http://www.scopus.com/inward/record.url?scp=85054294661&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85054294661&partnerID=8YFLogxK

U2 - 10.1016/j.pep.2018.09.014

DO - 10.1016/j.pep.2018.09.014

M3 - Article

VL - 154

SP - 52

EP - 61

JO - Protein Expression and Purification

T2 - Protein Expression and Purification

JF - Protein Expression and Purification

SN - 1046-5928

ER -