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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.


(a) Thermostability study of free and immobilised enzymes and (b) free enzyme at 80°C.
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Figure 5: (a) Thermostability study of free and immobilised enzymes and (b) free enzyme at 80°C.

Mentions: An improvement in the stability of the immobilised cellulase at higher temperatures is shown in Figure 5a. The results obtained from the thermal stability studies show that the immobilised enzyme was stable for approximately 4 h at 80°C. It retained about 66% of its initial activity in the first 2 h of incubation, but this gradually decreased in the next 4 h of incubation, providing a maximum of 26% activity after 4 h. After an incubation of 4 h, the enzyme had lost about 73% of its activity. The immobilised preparation completely lost activity by 6 h of incubation at 80°C. The loss of activity for the immobilised cellulase preparation was slower than that for the free enzyme. The activity of free cellulase was found to decrease in the first half hour of incubation, which suggests that the free enzyme denatured at 80°C, as shown in Figure 5b. The loss in activity of the free cellulase over 2 h of incubation at 80°C is shown in Figure 5b. The immobilised enzyme retained 72% of its initial activity for up to 6 h of incubation at 60°C. The activity was 90% after the first 2 h of incubation and gradually reduced to 75% after 4 h of incubation, thereafter reducing to a negligible rate up to 6 h. The enzyme was more stable at 60°C after immobilisation than it was at 80°C. The free enzyme activity reduced to 61% after 2 h of incubation, and thereafter no significant decrease in the activity from 4 to 6 h was observed. This indicated that the free enzyme retained about 60% of its initial activity for up to 6 h of incubation at 60°C. These results indicate that the properties of the enzyme were not impacted by immobilisation, with the exception of higher stability at elevated temperatures. A similar study demonstrated the stability of the immobilised preparation at 80°C for an hour; however, the natural cellulase lost its activity during the same time period, similar to our observation [27]. In previous reports it has been seen that the stability of the enzyme preparation on magnetic nanosized supports has reduced to about 50% after an incubation of 2 h [23].


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)

(a) Thermostability study of free and immobilised enzymes and (b) free enzyme at 80°C.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: (a) Thermostability study of free and immobilised enzymes and (b) free enzyme at 80°C.
Mentions: An improvement in the stability of the immobilised cellulase at higher temperatures is shown in Figure 5a. The results obtained from the thermal stability studies show that the immobilised enzyme was stable for approximately 4 h at 80°C. It retained about 66% of its initial activity in the first 2 h of incubation, but this gradually decreased in the next 4 h of incubation, providing a maximum of 26% activity after 4 h. After an incubation of 4 h, the enzyme had lost about 73% of its activity. The immobilised preparation completely lost activity by 6 h of incubation at 80°C. The loss of activity for the immobilised cellulase preparation was slower than that for the free enzyme. The activity of free cellulase was found to decrease in the first half hour of incubation, which suggests that the free enzyme denatured at 80°C, as shown in Figure 5b. The loss in activity of the free cellulase over 2 h of incubation at 80°C is shown in Figure 5b. The immobilised enzyme retained 72% of its initial activity for up to 6 h of incubation at 60°C. The activity was 90% after the first 2 h of incubation and gradually reduced to 75% after 4 h of incubation, thereafter reducing to a negligible rate up to 6 h. The enzyme was more stable at 60°C after immobilisation than it was at 80°C. The free enzyme activity reduced to 61% after 2 h of incubation, and thereafter no significant decrease in the activity from 4 to 6 h was observed. This indicated that the free enzyme retained about 60% of its initial activity for up to 6 h of incubation at 60°C. These results indicate that the properties of the enzyme were not impacted by immobilisation, with the exception of higher stability at elevated temperatures. A similar study demonstrated the stability of the immobilised preparation at 80°C for an hour; however, the natural cellulase lost its activity during the same time period, similar to our observation [27]. In previous reports it has been seen that the stability of the enzyme preparation on magnetic nanosized supports has reduced to about 50% after an incubation of 2 h [23].

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.