scholarly journals Cellobiohydrolase B ofAspergillus nigerover-expressed inPichia pastorisstimulates hydrolysis of oil palm empty fruit bunches

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3909 ◽  
Author(s):  
James Sy-Keen Woon ◽  
Mukram M. Mackeen ◽  
Rosli M. Illias ◽  
Nor M. Mahadi ◽  
William J. Broughton ◽  
...  
Keyword(s):  
Oil Palm ◽  
Biomass Conversion ◽  
Methylotrophic Yeast ◽  
Cellulase Production ◽  
Crystalline Cellulose ◽  
Gene Encoding ◽  
Empty Fruit Bunches ◽  
Biochemical Characterisation ◽  
Fungal Cellulase ◽  
Hydrolysis Of

BackgroundAspergillus 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. nigernaturally secretes low levels of CBH. Hence, recombinant production ofA. nigerCBH is desirable to increase CBH production yield and also to allow biochemical characterisation of the recombinant CBH fromA. niger.MethodsIn this study, the gene encoding a cellobiohydrolase B (cbhB) fromA. nigerATCC 10574 was cloned and expressed in the methylotrophic yeastPichia pastorisX-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.ResultsRecombinant CBHB was over-expressed as a hyperglycosylated protein attached toN-glycans. CBHB was enzymatically active towards soluble substrates such as 4-methylumbelliferyl-β-D-cellobioside (MUC),p-nitrophenyl-cellobioside (pNPC) andp-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 Iαand Iβ in the treated OPEFB samples.DiscussionSince CBHB alone was inactive against crystalline cellulose, these data suggested that it might work synergistically with other components of Cellic®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. nigerCBHB was identified as a potential supplementary enzyme for the enzymatic hydrolysis of OPEFB.

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

2017 ◽  
Author(s):  
James Sy-Keen Woon ◽  
Mukram M Mackeen ◽  
Rosli M Illias ◽  
Nor M Mahadi ◽  
William J Broughton ◽  
...  
Keyword(s):  
Aspergillus Niger ◽  
Pichia Pastoris ◽  
Oil Palm ◽  
Biomass Conversion ◽  
Methylotrophic Yeast ◽  
Cellulase Production ◽  
Crystalline Cellulose ◽  
Empty Fruit Bunches ◽  
Biochemical Characterisation ◽  
Hydrolysis Of

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-methylumbelliferyl-β-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 oC and pH 4 but the enzyme was stable between pH 3 to 10 and 30 to 80 oC. 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 complementation 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® 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.

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

2017 ◽  
Author(s):  
James Sy-Keen Woon ◽  
Mukram M Mackeen ◽  
Rosli M Illias ◽  
Nor M Mahadi ◽  
William J Broughton ◽  
...  
Keyword(s):  
Aspergillus Niger ◽  
Pichia Pastoris ◽  
Oil Palm ◽  
Biomass Conversion ◽  
Methylotrophic Yeast ◽  
Cellulase Production ◽  
Crystalline Cellulose ◽  
Empty Fruit Bunches ◽  
Biochemical Characterisation ◽  
Hydrolysis Of

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-methylumbelliferyl-β-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 oC and pH 4 but the enzyme was stable between pH 3 to 10 and 30 to 80 oC. 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 complementation 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® 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.

scholarly journals Enzymatic hydrolysis of oil palm empty fruit bunch to produce reducing sugar and its kinetic Hidrolisis enzimatik tandan kosong kelapa sawit untuk menghasilkan gula pereduksi dan kinetikanya

2016 ◽  
Vol 83 (1) ◽  
Author(s):  
Vera BARLIANTI ◽  
Deliana DAHNUM ◽  
. MURYANTO ◽  
Eka TRIWAHYUNI ◽  
Yosi ARISTIAWAN ◽  
...  
Keyword(s):  
Enzymatic Hydrolysis ◽  
Oil Palm ◽  
Volume Ratio ◽  
Enzyme Concentration ◽  
Reducing Sugar ◽  
Economic Feasibility ◽  
First Order ◽  
Empty Fruit Bunches ◽  
Hydrolysis Process ◽  
Hydrolysis Of

Abstrak Sebagai salah satu Negara penghasil minyak kelapa sawit mentah (CPO), Indonesia juga menghasilkan tandan kosong kelapa sawit (TKKS) dalam jumlah besar. TKKS terdiri dari-tiga-komponen utama, yaitu selulosa, hemiselulosa, dan lignin. Pengolahan awal TKKS secara alkalindi ikuti dengan hidrolisis TKKS secara enzimatik menggunakan kombinasi enzim selulase dan β-glukosidase akan menghasilkan gula-gula yang mudah difermentasi.  Penelitian ini bertujuan untuk mempelajari pengaruh konsentrasi substrat, kon-sentrasi enzim, dan suhu selama proses hidrolisis berlangsung.  Hasil yang diperoleh menunjukkan bahwa konsentrasi gula maksimum (194,78 g/L) dicapai pada konsentrasi TKKS 20% (b/v), konsentrasi campuran enzim yang terdiri dari selulase dan β-1,4 glukosidase sebesar 3,85% (v/v), dan suhu 50oC. Perbandingan antara selulase dan β-1,4 glukosidase adalah 5:1 dengan masing-masing aktivitas enzim sebesar 144.5 FPU/mL dan 63 FPU/mL. Hasil penelitian juga menunjukkan bahwa model kinetika yang sesuai untuk proses hidrolisis TKKS secara enzimatik adalah model kinetika Shen dan Agblevor dengan reakside aktivasi enzim orde satu.  Hasil ini mendukung studi kelayakan ekonomi dalam pemanfaatan TKKS untuk produksi bioetanol.AbstractAs one of the crude palm oil producers, Indonesia also produces empty fruit bunches (EFB)in large quantities. The oil palm EFB consist of cellulose, hemicellulose and lignin. Alkaline pretreatment of EFB, followed by enzymatic hydro-lysis of cellulose using combination of cellulase and β-glucosidase enzymes produce fermentable sugars. This paper reported the effects of substrate loading, enzyme concentration, and temperature of hydrolysis process on reducing sugar production. The  maximum  sugar  concentration (194.78 g/L) was produced at 50oC using 20% (w/v) EFB and 3.85% (v/v) mixed enzymes of cellulase and β-1,4 glucosidase in volume ratio of 5:1 (v/v), with enzyme activity of 144.5 FPU/mL and 63 FPU/mL, respectively. The results also showed that the suitable kinetic model for enzymatic hydrolysis process of oil palm EFB follow Shen and Agblevor model with first order of enzyme deactivation. These results support the economic feasibility study in utilization of EFB of oil palm for bioethanol production.    

scholarly journals Xylose recovery from dilute-acid hydrolysis of oil palm (Elaeis guineensis) empty fruit bunches for xylitol production

2017 ◽  
Vol 16 (41) ◽  
pp. 1997-2008
Author(s):  
Manjarres-Pinzon Katherine ◽  
Arias-Zabala Mario ◽  
Correa-Londono Guillermo ◽  
Rodriguez-Sandoval Eduardo
Keyword(s):  
Acid Hydrolysis ◽  
Oil Palm ◽  
Elaeis Guineensis ◽  
Xylitol Production ◽  
Dilute Acid ◽  
Dilute Acid Hydrolysis ◽  
Empty Fruit Bunches ◽  
Hydrolysis Of

scholarly journals Production of Xylitol from Oil Palm Empty Friuts Bunch: A Case Study on Bioefinery Concept

2015 ◽  
Vol 9 (7) ◽  
pp. 206 ◽  
Author(s):  
MTAP Kresnowati ◽  
Efri Mardawati ◽  
Tjandra Setiadi
Keyword(s):  
Palm Oil ◽  
Oil Palm ◽  
Agricultural Waste ◽  
Crude Palm Oil ◽  
Empty Fruit Bunches ◽  
Starting Point ◽  
Good Starting Point ◽  
Hydrolysis Of ◽  
Low Glycemic Index

The concept of biorefinery offers the utilization of biomass, in particular agricultural waste, to be converted intoenergy, chemicals, materials, and food. In 2013 Indonesia produced about 27.4 thousand tons of crude palm oil(CPO) which corresponds to approximately 30 thousand tons of oil palm empty fruit bunches (EFB), the biomasswaste from palm oil industries. The huge availability of EFB in Indonesia may serve as a good starting point toimplement the concept of biorefinery. EFB mainly comprises of cellulose, hemicellulose and lignin. Thecellulosic components of EFB have been thoroughly studied, i.e. for the production of bioethanol. Thehemicellulosic component of EFB, which is a polymeric substance that comprises mainly of xylose, has beenbarely explored. This paper reviewed the potential utilization of hemicellulosic component of EFB to beconverted to xylitol, the 5-carbon-sugar-alcohol which is low calorie, low Glycemic Index, and anti-cariogenic.The pretreatment and hydrolysis of EFB and the following fermentation of EFB hydrolysate to xylitol will bediscussed further.

Sign in / Sign up

Export Citation Format

Share Document