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Identification and characterization of core cellulolytic enzymes from Talaromyces cellulolyticus (formerly Acremonium cellulolyticus) critical for hydrolysis of lignocellulosic biomass.

Inoue H, Decker SR, Taylor LE, Yano S, Sawayama S - Biotechnol Biofuels (2014)

Bottom Line: Cel5A seemed to be more effective in the synergistic hydrolysis of the PCS than Cel7B.Core cellulolytic enzymes in the T. cellulolyticus cellulase system were identified to Cel5A, Cel6A, Cel7A, Xyl10A, and Bgl3A and characterized.The optimized mixture of these five enzymes was highly effective for the hydrolysis of PCS glucan, providing a foundation for future improvement of the T. cellulolyticus cellulase system.

View Article: PubMed Central - PubMed

Affiliation: Biomass Refinery Research Center, National Institute of Advanced Industrial Science and Technology, 3-11-32 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046 Japan.

ABSTRACT

Background: Enzymatic hydrolysis of pretreated lignocellulosic biomass is an essential process for the production of fermentable sugars for industrial use. A better understanding of fungal cellulase systems will provide clues for maximizing the hydrolysis of target biomass. Talaromyces cellulolyticus is a promising fungus for cellulase production and efficient biomass hydrolysis. Several cellulolytic enzymes purified from T. cellulolyticus were characterized in earlier studies, but the core enzymes critical for hydrolysis of lignocellulosic biomass remain unknown.

Results: Six cellulolytic enzymes critical for the hydrolysis of crystalline cellulose were purified from T. cellulolyticus culture supernatant using an enzyme assay based on synergistic hydrolysis of Avicel. The purified enzymes were identified by their substrate specificities and analyses of trypsin-digested peptide fragments and were classified into the following glycosyl hydrolase (GH) families: GH3 (β-glucosidase, Bgl3A), GH5 (endoglucanase, Cel5A), GH6 (cellobiohydrolase II, Cel6A), GH7 (cellobiohydrolase I and endoglucanase, Cel7A and Cel7B, respectively), and GH10 (xylanase, Xyl10A). Hydrolysis of dilute acid-pretreated corn stover (PCS) with mixtures of the purified enzymes showed that Cel5A, Cel7B, and Xyl10A each had synergistic effects with a mixture of Cel6A and Cel7A. Cel5A seemed to be more effective in the synergistic hydrolysis of the PCS than Cel7B. The ratio of Cel5A, Cel6A, Cel7A, and Xyl10A was statistically optimized for the hydrolysis of PCS glucan in the presence of Bgl3A. The resultant mixture achieved higher PCS glucan hydrolysis at lower enzyme loading than a culture filtrate from T. cellulolyticus or a commercial enzyme preparation, demonstrating that the five enzymes play a role as core enzymes in the hydrolysis of PCS glucan.

Conclusions: Core cellulolytic enzymes in the T. cellulolyticus cellulase system were identified to Cel5A, Cel6A, Cel7A, Xyl10A, and Bgl3A and characterized. The optimized mixture of these five enzymes was highly effective for the hydrolysis of PCS glucan, providing a foundation for future improvement of the T. cellulolyticus cellulase system.

No MeSH data available.


Related in: MedlinePlus

Effect of enzyme loading on the hydrolysis of PCS glucan. Hydrolysis was carried out at pH 5, 45°C, with 3% (w/v) PCS. The percent hydrolysis of PCS glucan was calculated from the amount of glucose in the hydrolysate after 48 hours. PCS, pretreated corn stover.
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Fig5: Effect of enzyme loading on the hydrolysis of PCS glucan. Hydrolysis was carried out at pH 5, 45°C, with 3% (w/v) PCS. The percent hydrolysis of PCS glucan was calculated from the amount of glucose in the hydrolysate after 48 hours. PCS, pretreated corn stover.

Mentions: To compare the hydrolytic activity of the core enzyme mixture and crude enzymes, three different enzyme mixtures were evaluated at different total protein loadings for PCS glucan hydrolysis (Figure 5). The following enzyme preparations were compared: the core enzyme mixture Cel7A:Cel6A:Cel5A:Xyl10A:Bgl3A (46:25:13.5:4.5:11) approximating to the optimized composition (Figure 4), the crude enzyme from T. cellulolyticus CF-2612, and a commercial enzyme preparation (Novozymes NA., Franklinton, North Carolina, United States) from a genetically modified strain of T. reesei, Cellic CTec2, which has an increased level of β-glucosidase. The core mixture hydrolyzed PCS glucan more efficiently than the crude and commercial enzymes (Figure 5). The enzyme loading to achieve 75% hydrolysis was 7.5, 10.5, and 13 mg/g glucan for the core mixture, the crude enzymes, and the commercial preparation, respectively. This suggests that the core mixture was more potent for the degradation of cellulose in PCS than the crude and commercial enzymes, which contain extra enzymatic components. The lower degree of hydrolysis with the commercial enzyme preparation is probably due to the relatively low reaction temperature (45°C) because the optimum temperature for Cellic CTec2 is 50°C. To achieve 80% hydrolysis, which is necessary for an economical industrial process, further loading of 3.7 mg/g glucan was required for the core mixture, while an additional 2 mg/g glucan was required for the crude enzyme (Figure 5). This difference indicates that trace amounts of other enzymes such as hemicellulases are required for efficient hydrolysis of PCS glucan by the core enzymes.Figure 5


Identification and characterization of core cellulolytic enzymes from Talaromyces cellulolyticus (formerly Acremonium cellulolyticus) critical for hydrolysis of lignocellulosic biomass.

Inoue H, Decker SR, Taylor LE, Yano S, Sawayama S - Biotechnol Biofuels (2014)

Effect of enzyme loading on the hydrolysis of PCS glucan. Hydrolysis was carried out at pH 5, 45°C, with 3% (w/v) PCS. The percent hydrolysis of PCS glucan was calculated from the amount of glucose in the hydrolysate after 48 hours. PCS, pretreated corn stover.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4196096&req=5

Fig5: Effect of enzyme loading on the hydrolysis of PCS glucan. Hydrolysis was carried out at pH 5, 45°C, with 3% (w/v) PCS. The percent hydrolysis of PCS glucan was calculated from the amount of glucose in the hydrolysate after 48 hours. PCS, pretreated corn stover.
Mentions: To compare the hydrolytic activity of the core enzyme mixture and crude enzymes, three different enzyme mixtures were evaluated at different total protein loadings for PCS glucan hydrolysis (Figure 5). The following enzyme preparations were compared: the core enzyme mixture Cel7A:Cel6A:Cel5A:Xyl10A:Bgl3A (46:25:13.5:4.5:11) approximating to the optimized composition (Figure 4), the crude enzyme from T. cellulolyticus CF-2612, and a commercial enzyme preparation (Novozymes NA., Franklinton, North Carolina, United States) from a genetically modified strain of T. reesei, Cellic CTec2, which has an increased level of β-glucosidase. The core mixture hydrolyzed PCS glucan more efficiently than the crude and commercial enzymes (Figure 5). The enzyme loading to achieve 75% hydrolysis was 7.5, 10.5, and 13 mg/g glucan for the core mixture, the crude enzymes, and the commercial preparation, respectively. This suggests that the core mixture was more potent for the degradation of cellulose in PCS than the crude and commercial enzymes, which contain extra enzymatic components. The lower degree of hydrolysis with the commercial enzyme preparation is probably due to the relatively low reaction temperature (45°C) because the optimum temperature for Cellic CTec2 is 50°C. To achieve 80% hydrolysis, which is necessary for an economical industrial process, further loading of 3.7 mg/g glucan was required for the core mixture, while an additional 2 mg/g glucan was required for the crude enzyme (Figure 5). This difference indicates that trace amounts of other enzymes such as hemicellulases are required for efficient hydrolysis of PCS glucan by the core enzymes.Figure 5

Bottom Line: Cel5A seemed to be more effective in the synergistic hydrolysis of the PCS than Cel7B.Core cellulolytic enzymes in the T. cellulolyticus cellulase system were identified to Cel5A, Cel6A, Cel7A, Xyl10A, and Bgl3A and characterized.The optimized mixture of these five enzymes was highly effective for the hydrolysis of PCS glucan, providing a foundation for future improvement of the T. cellulolyticus cellulase system.

View Article: PubMed Central - PubMed

Affiliation: Biomass Refinery Research Center, National Institute of Advanced Industrial Science and Technology, 3-11-32 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046 Japan.

ABSTRACT

Background: Enzymatic hydrolysis of pretreated lignocellulosic biomass is an essential process for the production of fermentable sugars for industrial use. A better understanding of fungal cellulase systems will provide clues for maximizing the hydrolysis of target biomass. Talaromyces cellulolyticus is a promising fungus for cellulase production and efficient biomass hydrolysis. Several cellulolytic enzymes purified from T. cellulolyticus were characterized in earlier studies, but the core enzymes critical for hydrolysis of lignocellulosic biomass remain unknown.

Results: Six cellulolytic enzymes critical for the hydrolysis of crystalline cellulose were purified from T. cellulolyticus culture supernatant using an enzyme assay based on synergistic hydrolysis of Avicel. The purified enzymes were identified by their substrate specificities and analyses of trypsin-digested peptide fragments and were classified into the following glycosyl hydrolase (GH) families: GH3 (β-glucosidase, Bgl3A), GH5 (endoglucanase, Cel5A), GH6 (cellobiohydrolase II, Cel6A), GH7 (cellobiohydrolase I and endoglucanase, Cel7A and Cel7B, respectively), and GH10 (xylanase, Xyl10A). Hydrolysis of dilute acid-pretreated corn stover (PCS) with mixtures of the purified enzymes showed that Cel5A, Cel7B, and Xyl10A each had synergistic effects with a mixture of Cel6A and Cel7A. Cel5A seemed to be more effective in the synergistic hydrolysis of the PCS than Cel7B. The ratio of Cel5A, Cel6A, Cel7A, and Xyl10A was statistically optimized for the hydrolysis of PCS glucan in the presence of Bgl3A. The resultant mixture achieved higher PCS glucan hydrolysis at lower enzyme loading than a culture filtrate from T. cellulolyticus or a commercial enzyme preparation, demonstrating that the five enzymes play a role as core enzymes in the hydrolysis of PCS glucan.

Conclusions: Core cellulolytic enzymes in the T. cellulolyticus cellulase system were identified to Cel5A, Cel6A, Cel7A, Xyl10A, and Bgl3A and characterized. The optimized mixture of these five enzymes was highly effective for the hydrolysis of PCS glucan, providing a foundation for future improvement of the T. cellulolyticus cellulase system.

No MeSH data available.


Related in: MedlinePlus