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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.


Hydrolysis of CMC and pretreated hemp hurd using free and immobilised enzyme for 48 h.
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Figure 7: Hydrolysis of CMC and pretreated hemp hurd using free and immobilised enzyme for 48 h.

Mentions: Free and immobilised cellulase were used up to 48 h to hydrolyse varying concentrations of untreated synthetic substrate (CMC), ranging in enzyme:substrate ratio from 1:1 to 1:4, as shown in Figure 7. Hydrolysis increased with enzyme:substrate ratios from 1:1 to 1:3 for both free and immobilised enzymes, with no significant increase observed at a 1:4 ratio of enzyme:substrate. The immobilised and free enzymes showed optima at a 1:3 enzyme:substrate ratio, with the immobilised enzyme hydrolysing 83% of substrate and the free enzyme hydrolysing 88% of substrate over 48 h. The level of hydrolysis at a 1:1 ratio was 26% for the immobilised enzyme, increasing to 57% with higher levels of CMC. With the free enzyme at the same 1:1 enzyme to substrate ratio, the level of hydrolysis was 17%. When the level of CMC was increased to a 2:1 ratio of substrate to enzyme, hydrolysis increased to 81%. During a previous study, researchers obtained 85% glucose yields using free enzymes, and a maximum of 83% glucose yield with immobilised cellulase when hydrolysing CMC pretreated with ionic liquid [35]. An earlier study hydrolysing CMC resulted in a lower yield with immobilised compared with free enzyme and concluded that dilution of enzyme on the support accounted for the lower yield after immobilisation [36]. Another study conducted using immobilised cellulase to hydrolyse ionic liquid treated cellulose found that the addition of 1-ethyl-3-methylimidazolium diethyl phosphate (EMIM-DEP) increased the hydrolysis rate by a factor of 2.7, resulting in 0.95 g glucose/g cellulose in 8 h of hydrolysis with the addition of 4% of EMIM-DEP [37].


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)

Hydrolysis of CMC and pretreated hemp hurd using free and immobilised enzyme for 48 h.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Hydrolysis of CMC and pretreated hemp hurd using free and immobilised enzyme for 48 h.
Mentions: Free and immobilised cellulase were used up to 48 h to hydrolyse varying concentrations of untreated synthetic substrate (CMC), ranging in enzyme:substrate ratio from 1:1 to 1:4, as shown in Figure 7. Hydrolysis increased with enzyme:substrate ratios from 1:1 to 1:3 for both free and immobilised enzymes, with no significant increase observed at a 1:4 ratio of enzyme:substrate. The immobilised and free enzymes showed optima at a 1:3 enzyme:substrate ratio, with the immobilised enzyme hydrolysing 83% of substrate and the free enzyme hydrolysing 88% of substrate over 48 h. The level of hydrolysis at a 1:1 ratio was 26% for the immobilised enzyme, increasing to 57% with higher levels of CMC. With the free enzyme at the same 1:1 enzyme to substrate ratio, the level of hydrolysis was 17%. When the level of CMC was increased to a 2:1 ratio of substrate to enzyme, hydrolysis increased to 81%. During a previous study, researchers obtained 85% glucose yields using free enzymes, and a maximum of 83% glucose yield with immobilised cellulase when hydrolysing CMC pretreated with ionic liquid [35]. An earlier study hydrolysing CMC resulted in a lower yield with immobilised compared with free enzyme and concluded that dilution of enzyme on the support accounted for the lower yield after immobilisation [36]. Another study conducted using immobilised cellulase to hydrolyse ionic liquid treated cellulose found that the addition of 1-ethyl-3-methylimidazolium diethyl phosphate (EMIM-DEP) increased the hydrolysis rate by a factor of 2.7, resulting in 0.95 g glucose/g cellulose in 8 h of hydrolysis with the addition of 4% of EMIM-DEP [37].

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.