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Untreated Chlorella homosphaera biomass allows for high rates of cell wall glucan enzymatic hydrolysis when using exoglucanase-free cellulases.

Rodrigues MA, Teixeira RS, Ferreira-Leitão VS, da Silva Bon EP - Biotechnol Biofuels (2015)

Bottom Line: The initial hydrolysis rates when using A. cellulolyticus or T. reesei enzymes were significantly lower, whereas the results for the T. reesei-A. awamori and A. awamori-A. cellulolyticus blends were similar to that for the A. awamori enzymes.High rates of enzymatic hydrolysis were achieved for untreated C. homosphaera biomass with enzymes containing endoglucanase and β-glucosidase activities and devoid of cellobiohydrolase activity.These findings simplify the complexity of the enzyme pools required for the enzymatic hydrolysis of microalgal biomass decreasing the enzyme cost for the production of microalgae-derived glucose syrups.

View Article: PubMed Central - PubMed

Affiliation: Federal University of Rio de Janeiro, Institute of Chemistry, Department of Biochemistry, Applied Photosynthesis Laboratory, Athos Avenida da Silveria Ramos, 149-Technology Centre, Block A, Room 532, University City, Rio de Janeiro, RJ 21941-909 Brazil.

ABSTRACT

Background: Chlorophyte microalgae have a cell wall containing a large quantity of cellulose Iα with a triclinic unit cell hydrogen-bonding pattern that is more susceptible to hydrolysis than that of the cellulose Iβ polymorphic form that is predominant in higher plants. This study addressed the enzymatic hydrolysis of untreated Chlorella homosphaera biomass using selected enzyme preparations, aiming to identify the relevant activity profile for the microalgae cellulose hydrolysis. Enzymes from Acremonium cellulolyticus, which secretes a complete pool of cellulases plus β-glucosidase; Trichoderma reesei, which secretes a complete pool of cellulases with low β-glucosidase; Aspergillus awamori, which secretes endoglucanases and β-glucosidase; blends of T. reesei-A. awamori or A. awamori-A. cellulolyticus enzymes; and a purified A. awamori β-glucosidase were evaluated.

Results: The highest initial glucan hydrolysis rate of 140.3 mg/g/h was observed for A. awamori enzymes with high β-glucosidase, low endoglucanase, and negligible cellobiohydrolase activities. The initial hydrolysis rates when using A. cellulolyticus or T. reesei enzymes were significantly lower, whereas the results for the T. reesei-A. awamori and A. awamori-A. cellulolyticus blends were similar to that for the A. awamori enzymes. Thus, the hydrolysis of C. homosphaera cellulose was performed exclusively by the endoglucanase and β-glucosidase activities. X-ray diffraction data showing negligible cellulose crystallinity for untreated C. homosphaera biomass corroborate these findings. The A. awamori-A. cellulolyticus blend showed the highest initial polysaccharide hydrolysis rate of 185.6 mg/g/h, as measured by glucose equivalent, in addition to the highest predicted maximum glucan hydrolysis yield of 47% of total glucose (w/w). T. reesei enzymes showed the lowest predicted maximum glucan hydrolysis yield of 25% (w/w), whereas the maximum yields of approximately 31% were observed for the other enzyme preparations. The hydrolysis yields were proportional to the enzyme β-glucosidase load, indicating that the endoglucanase load was not rate-limiting.

Conclusions: High rates of enzymatic hydrolysis were achieved for untreated C. homosphaera biomass with enzymes containing endoglucanase and β-glucosidase activities and devoid of cellobiohydrolase activity. These findings simplify the complexity of the enzyme pools required for the enzymatic hydrolysis of microalgal biomass decreasing the enzyme cost for the production of microalgae-derived glucose syrups.

No MeSH data available.


The hydrolysis yield ofC. homosphaerabiomass after 3 h of enzymatic treatment.C. homosphaera biomass (50 mg/mL) was incubated in 50 mM citrate buffer pH 4.8 at 50°C with the indicated enzyme preparation at a final β-glucosidase activity of 23.3 IU/g d.w. biomass load. Biomass without enzyme was incubated under the same conditions as the control. Letters a, b, c, and d indicate differences in the means at 0.05 level.
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Fig3: The hydrolysis yield ofC. homosphaerabiomass after 3 h of enzymatic treatment.C. homosphaera biomass (50 mg/mL) was incubated in 50 mM citrate buffer pH 4.8 at 50°C with the indicated enzyme preparation at a final β-glucosidase activity of 23.3 IU/g d.w. biomass load. Biomass without enzyme was incubated under the same conditions as the control. Letters a, b, c, and d indicate differences in the means at 0.05 level.

Mentions: The use of the A. awamori-A. cellulolyticus enzyme blend resulted in a hydrolysis reaction medium with the highest β-glucosidase load (90.3 IU/g d.w. biomass) followed by the A. cellulolyticus enzymes (49.0 IU/g) and by both the A. awamori enzymes and the T. reesei-A. awamori enzyme blend (22.3 IU/g). The T. reesei enzymes showed the lowest β-glucosidase load (8.7 IU/g). With the exception of A. cellulolyticus, the initial hydrolysis rates and final yields correlated positively with the β-glucosidase. To understand further the importance of β-glucosidase activity, we conducted two sets of hydrolysis assays with T. reesei, A. awamori, A. cellulolyticus, and a blend of A. awamori-T. reesei enzyme preparations. In the first set, the assays were performed with the same β-glucosidase load for 3 h because more striking differences regarding the initial hydrolysis yields were observed within this time interval. A control experiment, with the same load of a partially purified β-glucosidase and devoid of endo- and exoglucanase activities, was conducted. Figure 3 shows that after 3 h of hydrolysis, no significant differences among the means of hydrolyzed hexose equivalents or glucose were observed for any enzyme preparation. However, the A. cellulolyticus enzymes showed lower mean values, mostly for glucose, which were significant when compared with the A. awamori enzyme, confirming the previous results shown in Figures 1 and 2, where despite having a higher β-glucosidase load, it showed a lower initial rate but a similar final hydrolysis yield.Figure 3


Untreated Chlorella homosphaera biomass allows for high rates of cell wall glucan enzymatic hydrolysis when using exoglucanase-free cellulases.

Rodrigues MA, Teixeira RS, Ferreira-Leitão VS, da Silva Bon EP - Biotechnol Biofuels (2015)

The hydrolysis yield ofC. homosphaerabiomass after 3 h of enzymatic treatment.C. homosphaera biomass (50 mg/mL) was incubated in 50 mM citrate buffer pH 4.8 at 50°C with the indicated enzyme preparation at a final β-glucosidase activity of 23.3 IU/g d.w. biomass load. Biomass without enzyme was incubated under the same conditions as the control. Letters a, b, c, and d indicate differences in the means at 0.05 level.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4356055&req=5

Fig3: The hydrolysis yield ofC. homosphaerabiomass after 3 h of enzymatic treatment.C. homosphaera biomass (50 mg/mL) was incubated in 50 mM citrate buffer pH 4.8 at 50°C with the indicated enzyme preparation at a final β-glucosidase activity of 23.3 IU/g d.w. biomass load. Biomass without enzyme was incubated under the same conditions as the control. Letters a, b, c, and d indicate differences in the means at 0.05 level.
Mentions: The use of the A. awamori-A. cellulolyticus enzyme blend resulted in a hydrolysis reaction medium with the highest β-glucosidase load (90.3 IU/g d.w. biomass) followed by the A. cellulolyticus enzymes (49.0 IU/g) and by both the A. awamori enzymes and the T. reesei-A. awamori enzyme blend (22.3 IU/g). The T. reesei enzymes showed the lowest β-glucosidase load (8.7 IU/g). With the exception of A. cellulolyticus, the initial hydrolysis rates and final yields correlated positively with the β-glucosidase. To understand further the importance of β-glucosidase activity, we conducted two sets of hydrolysis assays with T. reesei, A. awamori, A. cellulolyticus, and a blend of A. awamori-T. reesei enzyme preparations. In the first set, the assays were performed with the same β-glucosidase load for 3 h because more striking differences regarding the initial hydrolysis yields were observed within this time interval. A control experiment, with the same load of a partially purified β-glucosidase and devoid of endo- and exoglucanase activities, was conducted. Figure 3 shows that after 3 h of hydrolysis, no significant differences among the means of hydrolyzed hexose equivalents or glucose were observed for any enzyme preparation. However, the A. cellulolyticus enzymes showed lower mean values, mostly for glucose, which were significant when compared with the A. awamori enzyme, confirming the previous results shown in Figures 1 and 2, where despite having a higher β-glucosidase load, it showed a lower initial rate but a similar final hydrolysis yield.Figure 3

Bottom Line: The initial hydrolysis rates when using A. cellulolyticus or T. reesei enzymes were significantly lower, whereas the results for the T. reesei-A. awamori and A. awamori-A. cellulolyticus blends were similar to that for the A. awamori enzymes.High rates of enzymatic hydrolysis were achieved for untreated C. homosphaera biomass with enzymes containing endoglucanase and β-glucosidase activities and devoid of cellobiohydrolase activity.These findings simplify the complexity of the enzyme pools required for the enzymatic hydrolysis of microalgal biomass decreasing the enzyme cost for the production of microalgae-derived glucose syrups.

View Article: PubMed Central - PubMed

Affiliation: Federal University of Rio de Janeiro, Institute of Chemistry, Department of Biochemistry, Applied Photosynthesis Laboratory, Athos Avenida da Silveria Ramos, 149-Technology Centre, Block A, Room 532, University City, Rio de Janeiro, RJ 21941-909 Brazil.

ABSTRACT

Background: Chlorophyte microalgae have a cell wall containing a large quantity of cellulose Iα with a triclinic unit cell hydrogen-bonding pattern that is more susceptible to hydrolysis than that of the cellulose Iβ polymorphic form that is predominant in higher plants. This study addressed the enzymatic hydrolysis of untreated Chlorella homosphaera biomass using selected enzyme preparations, aiming to identify the relevant activity profile for the microalgae cellulose hydrolysis. Enzymes from Acremonium cellulolyticus, which secretes a complete pool of cellulases plus β-glucosidase; Trichoderma reesei, which secretes a complete pool of cellulases with low β-glucosidase; Aspergillus awamori, which secretes endoglucanases and β-glucosidase; blends of T. reesei-A. awamori or A. awamori-A. cellulolyticus enzymes; and a purified A. awamori β-glucosidase were evaluated.

Results: The highest initial glucan hydrolysis rate of 140.3 mg/g/h was observed for A. awamori enzymes with high β-glucosidase, low endoglucanase, and negligible cellobiohydrolase activities. The initial hydrolysis rates when using A. cellulolyticus or T. reesei enzymes were significantly lower, whereas the results for the T. reesei-A. awamori and A. awamori-A. cellulolyticus blends were similar to that for the A. awamori enzymes. Thus, the hydrolysis of C. homosphaera cellulose was performed exclusively by the endoglucanase and β-glucosidase activities. X-ray diffraction data showing negligible cellulose crystallinity for untreated C. homosphaera biomass corroborate these findings. The A. awamori-A. cellulolyticus blend showed the highest initial polysaccharide hydrolysis rate of 185.6 mg/g/h, as measured by glucose equivalent, in addition to the highest predicted maximum glucan hydrolysis yield of 47% of total glucose (w/w). T. reesei enzymes showed the lowest predicted maximum glucan hydrolysis yield of 25% (w/w), whereas the maximum yields of approximately 31% were observed for the other enzyme preparations. The hydrolysis yields were proportional to the enzyme β-glucosidase load, indicating that the endoglucanase load was not rate-limiting.

Conclusions: High rates of enzymatic hydrolysis were achieved for untreated C. homosphaera biomass with enzymes containing endoglucanase and β-glucosidase activities and devoid of cellobiohydrolase activity. These findings simplify the complexity of the enzyme pools required for the enzymatic hydrolysis of microalgal biomass decreasing the enzyme cost for the production of microalgae-derived glucose syrups.

No MeSH data available.