Cellobiohydrolase B of Aspergillus niger over-expressed in Pichia pastoris stimulates hydrolysis of oil palm empty fruit bunches

James Sy Keen Woon, Mukram M. Mackeen, Rosli M. Illias, Nor M. Mahadi, William J. Broughton, Abdul Munir Abdul Murad, Farah Diba Abu Bakar

Research output: Research - peer-reviewArticle

Abstract

Background. Aspergillus niger, along with many other lignocellulolytic fungi, has been widely used as a commercial workhorse for cellulase production. A fungal cellulase system generally includes three major classes of enzymes i.e.,β-glucosidases, endoglucanases and cellobiohydrolases. Cellobiohydrolases (CBH) are vital to the degradation of crystalline cellulose present in lignocellulosic biomass. However, A. niger naturally secretes low levels of CBH. Hence, recombinant production of A. niger CBH is desirable to increase CBH production yield and also to allow biochemical characterisation of the recombinant CBH from A. niger. Methods. In this study, the gene encoding a cellobiohydrolase B (cbhB) from A. niger ATCC 10574 was cloned and expressed in the methylotrophic yeast Pichia pastoris X-33. The recombinant CBHB was purified and characterised to study its biochemical and kinetic characteristics. To evaluate the potential of CBHB in assisting biomass conversion, CBHB was supplemented into a commercial cellulase preparation (Cellic ® CTec2) and was used to hydrolyse oil palm empty fruit bunch (OPEFB), one of the most abundant lignocellulosic waste from the palm oil industry. To attain maximum saccharification, enzyme loadings were optimised by response surface methodology and the optimum point was validated experimentally. Hydrolysed OPEFB samples were analysed using attenuated total reflectance FTIR spectroscopy (ATR-FTIR) to screen for any compositional changes upon enzymatic treatment. Results. Recombinant CBHB was over-expressed as a hyperglycosylated protein attached to N-glycans. CBHB was enzymatically active towards soluble substrates such as 4-methylumbelliferylβ-D-cellobioside (MUC), p-nitrophenyl-cellobioside (pNPC) and p-nitrophenyl-cellobiotrioside (pNPG3) but was not active towards crystalline substrates like Avicel ® and Sigmacell cellulose. Characterisation of purified CBHB using MUC as the model substrate revealed that optimum catalysis occurred at 50 °C and pH 4 but the enzyme was stable between pH 3 to 10 and 30 to 80 °C. Although CBHB on its own was unable to digest crystalline substrates, supplementation of CBHB (0.37%) with Cellic ® CTec2 (30%) increased saccharification of OPEFB by 27%. Compositional analyses of the treated OPEFB samples revealed that CBHB supplementation reduced peak intensities of both crystalline cellulose Iα and Iβ in the treated OPEFB samples. Discussion. Since CBHB alone was inactive against crystalline cellulose these data suggested that it might work synergistically with other components of Cellic R CTec2. CBHB supplements were desirable as they further increased hydrolysis of OPEFB when the performance of Cellic® CTec2 was theoretically capped at an enzyme loading of 34% in this study. Hence, A. niger CBHB was identified as a potential supplementary enzyme for the enzymatic hydrolysis of OPEFB.

LanguageEnglish
Article numbere3909
JournalPeerJ
Volume2017
Issue number10
DOIs
StatePublished - 2017

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cellulose 1,4-beta-cellobiosidase
Pichia pastoris
Elaeis guineensis
Aspergillus niger
hydrolysis
fruits
Cellulose 1,4-beta-Cellobiosidase
Palm oil
Aspergillus
Fruits
Hydrolysis
Pichia
Fruit
Oils
enzymes
Cellulose
Crystalline materials
Enzymes
cellulose
Cellulase

Keywords

  • 4-methylumbelliferyl-βD-cellobioside
  • Cellic® CTec2
  • Cellulase
  • Glycosylation
  • Lignocellulose
  • Saccharification

ASJC Scopus subject areas

  • Neuroscience(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Woon, J. S. K., Mackeen, M. M., Illias, R. M., Mahadi, N. M., Broughton, W. J., Murad, A. M. A., & Bakar, F. D. A. (2017). Cellobiohydrolase B of Aspergillus niger over-expressed in Pichia pastoris stimulates hydrolysis of oil palm empty fruit bunches. PeerJ, 2017(10), [e3909]. DOI: 10.7717/peerj.3909

Cellobiohydrolase B of Aspergillus niger over-expressed in Pichia pastoris stimulates hydrolysis of oil palm empty fruit bunches. / Woon, James Sy Keen; Mackeen, Mukram M.; Illias, Rosli M.; Mahadi, Nor M.; Broughton, William J.; Murad, Abdul Munir Abdul; Bakar, Farah Diba Abu.

In: PeerJ, Vol. 2017, No. 10, e3909, 2017.

Research output: Research - peer-reviewArticle

Woon, JSK, Mackeen, MM, Illias, RM, Mahadi, NM, Broughton, WJ, Murad, AMA & Bakar, FDA 2017, 'Cellobiohydrolase B of Aspergillus niger over-expressed in Pichia pastoris stimulates hydrolysis of oil palm empty fruit bunches' PeerJ, vol 2017, no. 10, e3909. DOI: 10.7717/peerj.3909
Woon JSK, Mackeen MM, Illias RM, Mahadi NM, Broughton WJ, Murad AMA et al. Cellobiohydrolase B of Aspergillus niger over-expressed in Pichia pastoris stimulates hydrolysis of oil palm empty fruit bunches. PeerJ. 2017;2017(10). e3909. Available from, DOI: 10.7717/peerj.3909
Woon, James Sy Keen ; Mackeen, Mukram M. ; Illias, Rosli M. ; Mahadi, Nor M. ; Broughton, William J. ; Murad, Abdul Munir Abdul ; Bakar, Farah Diba Abu. / Cellobiohydrolase B of Aspergillus niger over-expressed in Pichia pastoris stimulates hydrolysis of oil palm empty fruit bunches. In: PeerJ. 2017 ; Vol. 2017, No. 10.
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TY - JOUR

T1 - Cellobiohydrolase B of Aspergillus niger over-expressed in Pichia pastoris stimulates hydrolysis of oil palm empty fruit bunches

AU - Woon,James Sy Keen

AU - Mackeen,Mukram M.

AU - Illias,Rosli M.

AU - Mahadi,Nor M.

AU - Broughton,William J.

AU - Murad,Abdul Munir Abdul

AU - Bakar,Farah Diba Abu

PY - 2017

Y1 - 2017

N2 - Background. Aspergillus niger, along with many other lignocellulolytic fungi, has been widely used as a commercial workhorse for cellulase production. A fungal cellulase system generally includes three major classes of enzymes i.e.,β-glucosidases, endoglucanases and cellobiohydrolases. Cellobiohydrolases (CBH) are vital to the degradation of crystalline cellulose present in lignocellulosic biomass. However, A. niger naturally secretes low levels of CBH. Hence, recombinant production of A. niger CBH is desirable to increase CBH production yield and also to allow biochemical characterisation of the recombinant CBH from A. niger. Methods. In this study, the gene encoding a cellobiohydrolase B (cbhB) from A. niger ATCC 10574 was cloned and expressed in the methylotrophic yeast Pichia pastoris X-33. The recombinant CBHB was purified and characterised to study its biochemical and kinetic characteristics. To evaluate the potential of CBHB in assisting biomass conversion, CBHB was supplemented into a commercial cellulase preparation (Cellic ® CTec2) and was used to hydrolyse oil palm empty fruit bunch (OPEFB), one of the most abundant lignocellulosic waste from the palm oil industry. To attain maximum saccharification, enzyme loadings were optimised by response surface methodology and the optimum point was validated experimentally. Hydrolysed OPEFB samples were analysed using attenuated total reflectance FTIR spectroscopy (ATR-FTIR) to screen for any compositional changes upon enzymatic treatment. Results. Recombinant CBHB was over-expressed as a hyperglycosylated protein attached to N-glycans. CBHB was enzymatically active towards soluble substrates such as 4-methylumbelliferylβ-D-cellobioside (MUC), p-nitrophenyl-cellobioside (pNPC) and p-nitrophenyl-cellobiotrioside (pNPG3) but was not active towards crystalline substrates like Avicel ® and Sigmacell cellulose. Characterisation of purified CBHB using MUC as the model substrate revealed that optimum catalysis occurred at 50 °C and pH 4 but the enzyme was stable between pH 3 to 10 and 30 to 80 °C. Although CBHB on its own was unable to digest crystalline substrates, supplementation of CBHB (0.37%) with Cellic ® CTec2 (30%) increased saccharification of OPEFB by 27%. Compositional analyses of the treated OPEFB samples revealed that CBHB supplementation reduced peak intensities of both crystalline cellulose Iα and Iβ in the treated OPEFB samples. Discussion. Since CBHB alone was inactive against crystalline cellulose these data suggested that it might work synergistically with other components of Cellic R CTec2. CBHB supplements were desirable as they further increased hydrolysis of OPEFB when the performance of Cellic® CTec2 was theoretically capped at an enzyme loading of 34% in this study. Hence, A. niger CBHB was identified as a potential supplementary enzyme for the enzymatic hydrolysis of OPEFB.

AB - Background. Aspergillus niger, along with many other lignocellulolytic fungi, has been widely used as a commercial workhorse for cellulase production. A fungal cellulase system generally includes three major classes of enzymes i.e.,β-glucosidases, endoglucanases and cellobiohydrolases. Cellobiohydrolases (CBH) are vital to the degradation of crystalline cellulose present in lignocellulosic biomass. However, A. niger naturally secretes low levels of CBH. Hence, recombinant production of A. niger CBH is desirable to increase CBH production yield and also to allow biochemical characterisation of the recombinant CBH from A. niger. Methods. In this study, the gene encoding a cellobiohydrolase B (cbhB) from A. niger ATCC 10574 was cloned and expressed in the methylotrophic yeast Pichia pastoris X-33. The recombinant CBHB was purified and characterised to study its biochemical and kinetic characteristics. To evaluate the potential of CBHB in assisting biomass conversion, CBHB was supplemented into a commercial cellulase preparation (Cellic ® CTec2) and was used to hydrolyse oil palm empty fruit bunch (OPEFB), one of the most abundant lignocellulosic waste from the palm oil industry. To attain maximum saccharification, enzyme loadings were optimised by response surface methodology and the optimum point was validated experimentally. Hydrolysed OPEFB samples were analysed using attenuated total reflectance FTIR spectroscopy (ATR-FTIR) to screen for any compositional changes upon enzymatic treatment. Results. Recombinant CBHB was over-expressed as a hyperglycosylated protein attached to N-glycans. CBHB was enzymatically active towards soluble substrates such as 4-methylumbelliferylβ-D-cellobioside (MUC), p-nitrophenyl-cellobioside (pNPC) and p-nitrophenyl-cellobiotrioside (pNPG3) but was not active towards crystalline substrates like Avicel ® and Sigmacell cellulose. Characterisation of purified CBHB using MUC as the model substrate revealed that optimum catalysis occurred at 50 °C and pH 4 but the enzyme was stable between pH 3 to 10 and 30 to 80 °C. Although CBHB on its own was unable to digest crystalline substrates, supplementation of CBHB (0.37%) with Cellic ® CTec2 (30%) increased saccharification of OPEFB by 27%. Compositional analyses of the treated OPEFB samples revealed that CBHB supplementation reduced peak intensities of both crystalline cellulose Iα and Iβ in the treated OPEFB samples. Discussion. Since CBHB alone was inactive against crystalline cellulose these data suggested that it might work synergistically with other components of Cellic R CTec2. CBHB supplements were desirable as they further increased hydrolysis of OPEFB when the performance of Cellic® CTec2 was theoretically capped at an enzyme loading of 34% in this study. Hence, A. niger CBHB was identified as a potential supplementary enzyme for the enzymatic hydrolysis of OPEFB.

KW - 4-methylumbelliferyl-βD-cellobioside

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KW - Cellulase

KW - Glycosylation

KW - Lignocellulose

KW - Saccharification

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