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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 36 h of enzymatic treatment.C. homosphaera biomass (50 mg/mL) was incubated in 50 mM citrate buffer pH 4.8 at 50°C with 1.5 IU/g of endoglucanase load and β-glucosidase loads of 7.5, 15.0, and 22.5 IU/g biomass loads by adding partially purified β-glucosidase.
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Fig4: The hydrolysis yield ofC. homosphaerabiomass after 36 h of enzymatic treatment.C. homosphaera biomass (50 mg/mL) was incubated in 50 mM citrate buffer pH 4.8 at 50°C with 1.5 IU/g of endoglucanase load and β-glucosidase loads of 7.5, 15.0, and 22.5 IU/g biomass loads by adding partially purified β-glucosidase.

Mentions: The hydrolysis results from the control experiments were surprisingly high, accounting for over 40% and 50% of the total hexose and glucose released, respectively (Figure 3). The data were even higher than that presented in Figures 1 and 2, which could be related to differences in the uneven manual grinding of the algae biomass, affecting the particle sizes as we observed in preliminary assays in our laboratory (data not shown). The C. homosphaera biomass also showed a small amount of biomass hydrolysis not caused by enzymatic action as previously found (Figures 1 and 2). The different enzyme preparations contained different endoglucanase loads (Figure 3), which might have played an important role in the hydrolysis results, although no correlation between the endoglucanase load and initial rates and final yields was found. For this reason, all enzyme preparations in the second set of assays were set to a fixed endoglucanase load (1.5 IU/g dry biomass) and increasing β-glucosidase loads as follows: 7.5, 15.0, and 22.5 IU/g dry biomass, which were achieved through purified β-glucosidase supplementation. Figure 4 shows that after 36 h of incubation, all enzymes showed an increase in the hydrolysis yield, especially when the β-glucosidase load increased from 7.5 to the 15 IU/g that was significant for all enzyme preparations. The T. reesei preparation showed no significant increase for the hydrolysis yield of reducing sugar when the β-glucosidase load increased from 15.0 to 22.5 IU/g; however, a significant increase in the hydrolysis yield of glucose was found. The same pattern was observed for the A. cellulolyticus preparation. However, no significant increase in the glucose hydrolysis yield was found for the A. awamori enzyme and the A. awamori-T. reesei blend; nevertheless, the reducing sugar yield increase was significant. An analysis among the groups showed that the hydrolysis yields for A. awamori enzyme preparations were the highest even when compared with experiments presenting a higher β-glucosidase load. Moreover, the A. awamori preparation was more responsive to the increase in the β-glucosidase load (Figure 4), indicating a unique catalytic feature for the A. awamori enzymes.Figure 4


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 36 h of enzymatic treatment.C. homosphaera biomass (50 mg/mL) was incubated in 50 mM citrate buffer pH 4.8 at 50°C with 1.5 IU/g of endoglucanase load and β-glucosidase loads of 7.5, 15.0, and 22.5 IU/g biomass loads by adding partially purified β-glucosidase.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: The hydrolysis yield ofC. homosphaerabiomass after 36 h of enzymatic treatment.C. homosphaera biomass (50 mg/mL) was incubated in 50 mM citrate buffer pH 4.8 at 50°C with 1.5 IU/g of endoglucanase load and β-glucosidase loads of 7.5, 15.0, and 22.5 IU/g biomass loads by adding partially purified β-glucosidase.
Mentions: The hydrolysis results from the control experiments were surprisingly high, accounting for over 40% and 50% of the total hexose and glucose released, respectively (Figure 3). The data were even higher than that presented in Figures 1 and 2, which could be related to differences in the uneven manual grinding of the algae biomass, affecting the particle sizes as we observed in preliminary assays in our laboratory (data not shown). The C. homosphaera biomass also showed a small amount of biomass hydrolysis not caused by enzymatic action as previously found (Figures 1 and 2). The different enzyme preparations contained different endoglucanase loads (Figure 3), which might have played an important role in the hydrolysis results, although no correlation between the endoglucanase load and initial rates and final yields was found. For this reason, all enzyme preparations in the second set of assays were set to a fixed endoglucanase load (1.5 IU/g dry biomass) and increasing β-glucosidase loads as follows: 7.5, 15.0, and 22.5 IU/g dry biomass, which were achieved through purified β-glucosidase supplementation. Figure 4 shows that after 36 h of incubation, all enzymes showed an increase in the hydrolysis yield, especially when the β-glucosidase load increased from 7.5 to the 15 IU/g that was significant for all enzyme preparations. The T. reesei preparation showed no significant increase for the hydrolysis yield of reducing sugar when the β-glucosidase load increased from 15.0 to 22.5 IU/g; however, a significant increase in the hydrolysis yield of glucose was found. The same pattern was observed for the A. cellulolyticus preparation. However, no significant increase in the glucose hydrolysis yield was found for the A. awamori enzyme and the A. awamori-T. reesei blend; nevertheless, the reducing sugar yield increase was significant. An analysis among the groups showed that the hydrolysis yields for A. awamori enzyme preparations were the highest even when compared with experiments presenting a higher β-glucosidase load. Moreover, the A. awamori preparation was more responsive to the increase in the β-glucosidase load (Figure 4), indicating a unique catalytic feature for the A. awamori enzymes.Figure 4

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.