Limits...
Suitability of magnetic nanoparticle immobilised cellulases in enhancing enzymatic saccharification of pretreated hemp biomass.

Abraham RE, Verma ML, Barrow CJ, Puri M - Biotechnol Biofuels (2014)

Bottom Line: Cellulase from Trichoderma reesei was immobilised on an activated magnetic support by covalent binding and its activity was compared with that of the free enzyme to hydrolyse microcrystalline cellulose and hemp hurds on the basis of thermostability and reusability.The immobilised enzyme retained 50% enzyme activity up to five cycles, with thermostability at 80°C superior to that of the free enzyme.With pretreated hemp hurd biomass (HHB), the free and immobilised enzymes resulted in maximum hydrolysis in 48 h of 89% and 93%, respectively.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Chemistry and Biotechnology (CCB), Geelong Technology Precinct, Waurn Ponds, Deakin University, Geelong, Victoria 3217, Australia.

ABSTRACT

Background: Previous research focused on pretreatment of biomass, production of fermentable sugars and their consumption to produce ethanol. The main goal of the work was to economise the production process cost of fermentable sugars. Therefore, the objective of the present work was to investigate enzyme hydrolysis of microcrystalline cellulose and hemp hurds (natural cellulosic substrate) using free and immobilised enzymes. Cellulase from Trichoderma reesei was immobilised on an activated magnetic support by covalent binding and its activity was compared with that of the free enzyme to hydrolyse microcrystalline cellulose and hemp hurds on the basis of thermostability and reusability.

Results: Up to 94% protein binding was achieved during immobilisation of cellulase on nanoparticles. Successful binding was confirmed using Fourier transform infrared spectroscopy (FTIR). The free and immobilised enzymes exhibited identical pH optima (pH 4.0) and differing temperature optima at 50°C and 60°C, respectively. The K M values obtained for the free and immobilised enzymes were 0.87 mg/mL and 2.6 mg/mL respectively. The immobilised enzyme retained 50% enzyme activity up to five cycles, with thermostability at 80°C superior to that of the free enzyme. Optimum hydrolysis of carboxymethyl cellulose (CMC) with free and immobilised enzymes was 88% and 81%, respectively. With pretreated hemp hurd biomass (HHB), the free and immobilised enzymes resulted in maximum hydrolysis in 48 h of 89% and 93%, respectively.

Conclusion: The current work demonstrated the advantages delivered by immobilised enzymes by minimising the consumption of cellulase during substrate hydrolysis and making the production process of fermentable sugars economical and feasible. The activity of cellulase improved as a result of the immobilisation, which provided a better stability at higher temperatures. The immobilised enzyme provided an advantage over the free enzyme through the reusability and longer storage stability properties that were gained as a result of the immobilisation.

No MeSH data available.


Binding efficiency of cellulase onto nanoparticle with varying concentration of protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4061456&req=5

Figure 2: Binding efficiency of cellulase onto nanoparticle with varying concentration of protein.

Mentions: The binding efficiency and protein loading of cellulase onto the magnetic nanoparticles were confirmed using the Bio-Rad protein assay kit. The immobilisation was done for different protein:nanoparticle ratios, as shown in Figure 2. The activation of nanoparticle supports was tested for 1 h as optimised earlier [18]. The quantity of protein loading and the binding time were studied over 3.5 h (data not shown). The binding rate of protein onto the nanoparticle supports increased for 1.5 to 2 h, and thereafter protein elution slowed, indicating the onset of equilibrium. Therefore, 2 h was the optimum time for protein:nanoparticle immobilisation at 25°C. The experiment was performed in a broad range of protein:nanoparticle concentrations; however, only the best result of the study is presented. Moreover, the minimal binding of 86% was observed with a protein:nanoparticle ratio of 0.2. The binding of protein onto nanoparticles showed a broad range of binding efficiencies, varying in the protein:nanoparticle ratio between 1 to 1.8, and indicating that the activated nanoparticle had attained protein loading saturation. The protein elution concentration was observed to be comparatively high when the protein:nanoparticle ratio increased to 2.2, indicating that the protein concentration was high to bind on the surface of the nanoparticle.


Suitability of magnetic nanoparticle immobilised cellulases in enhancing enzymatic saccharification of pretreated hemp biomass.

Abraham RE, Verma ML, Barrow CJ, Puri M - Biotechnol Biofuels (2014)

Binding efficiency of cellulase onto nanoparticle with varying concentration of protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4061456&req=5

Figure 2: Binding efficiency of cellulase onto nanoparticle with varying concentration of protein.
Mentions: The binding efficiency and protein loading of cellulase onto the magnetic nanoparticles were confirmed using the Bio-Rad protein assay kit. The immobilisation was done for different protein:nanoparticle ratios, as shown in Figure 2. The activation of nanoparticle supports was tested for 1 h as optimised earlier [18]. The quantity of protein loading and the binding time were studied over 3.5 h (data not shown). The binding rate of protein onto the nanoparticle supports increased for 1.5 to 2 h, and thereafter protein elution slowed, indicating the onset of equilibrium. Therefore, 2 h was the optimum time for protein:nanoparticle immobilisation at 25°C. The experiment was performed in a broad range of protein:nanoparticle concentrations; however, only the best result of the study is presented. Moreover, the minimal binding of 86% was observed with a protein:nanoparticle ratio of 0.2. The binding of protein onto nanoparticles showed a broad range of binding efficiencies, varying in the protein:nanoparticle ratio between 1 to 1.8, and indicating that the activated nanoparticle had attained protein loading saturation. The protein elution concentration was observed to be comparatively high when the protein:nanoparticle ratio increased to 2.2, indicating that the protein concentration was high to bind on the surface of the nanoparticle.

Bottom Line: Cellulase from Trichoderma reesei was immobilised on an activated magnetic support by covalent binding and its activity was compared with that of the free enzyme to hydrolyse microcrystalline cellulose and hemp hurds on the basis of thermostability and reusability.The immobilised enzyme retained 50% enzyme activity up to five cycles, with thermostability at 80°C superior to that of the free enzyme.With pretreated hemp hurd biomass (HHB), the free and immobilised enzymes resulted in maximum hydrolysis in 48 h of 89% and 93%, respectively.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Chemistry and Biotechnology (CCB), Geelong Technology Precinct, Waurn Ponds, Deakin University, Geelong, Victoria 3217, Australia.

ABSTRACT

Background: Previous research focused on pretreatment of biomass, production of fermentable sugars and their consumption to produce ethanol. The main goal of the work was to economise the production process cost of fermentable sugars. Therefore, the objective of the present work was to investigate enzyme hydrolysis of microcrystalline cellulose and hemp hurds (natural cellulosic substrate) using free and immobilised enzymes. Cellulase from Trichoderma reesei was immobilised on an activated magnetic support by covalent binding and its activity was compared with that of the free enzyme to hydrolyse microcrystalline cellulose and hemp hurds on the basis of thermostability and reusability.

Results: Up to 94% protein binding was achieved during immobilisation of cellulase on nanoparticles. Successful binding was confirmed using Fourier transform infrared spectroscopy (FTIR). The free and immobilised enzymes exhibited identical pH optima (pH 4.0) and differing temperature optima at 50°C and 60°C, respectively. The K M values obtained for the free and immobilised enzymes were 0.87 mg/mL and 2.6 mg/mL respectively. The immobilised enzyme retained 50% enzyme activity up to five cycles, with thermostability at 80°C superior to that of the free enzyme. Optimum hydrolysis of carboxymethyl cellulose (CMC) with free and immobilised enzymes was 88% and 81%, respectively. With pretreated hemp hurd biomass (HHB), the free and immobilised enzymes resulted in maximum hydrolysis in 48 h of 89% and 93%, respectively.

Conclusion: The current work demonstrated the advantages delivered by immobilised enzymes by minimising the consumption of cellulase during substrate hydrolysis and making the production process of fermentable sugars economical and feasible. The activity of cellulase improved as a result of the immobilisation, which provided a better stability at higher temperatures. The immobilised enzyme provided an advantage over the free enzyme through the reusability and longer storage stability properties that were gained as a result of the immobilisation.

No MeSH data available.