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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

SDS-PAGE analysis of purified enzymes. Lanes: M, protein marker; 1, Xyl10A; 2, Cel5A; 3, Cel7B; 4, Cel6A; 5, Bgl3A; 6, Cel7A.
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Fig2: SDS-PAGE analysis of purified enzymes. Lanes: M, protein marker; 1, Xyl10A; 2, Cel5A; 3, Cel7B; 4, Cel6A; 5, Bgl3A; 6, Cel7A.

Mentions: Cellulolytic enzymes involved in the hydrolysis of crystalline cellulose play a role as core enzymes in cellulase systems to degrade pretreated lignocellulosic biomass [8,12,13,28]. For purification of the core enzymes in the T. cellulolyticus cellulase system, we developed an assay for the synergistic hydrolysis of Avicel. First, synergistic activity for Avicel hydrolysis was determined for combinations of four groups (F1, F2, F3, and F4) of fractions obtained by Resource Q column chromatography of T. cellulolyticus culture supernatant (Figure 1). F2, containing Cel6A, showed synergism with F1, F3, and F4. F3, containing Cel7A and Bgl3A, was synergistic with F1 and F4, as well as F2. There was no synergism between F1 and F4. Thus, F2 and F3 were used to detect synergism for Avicel hydrolysis in fractions obtained by further chromatography of each group. Finally, six cellulolytic enzymes were purified to electrophoretically homogeneous forms (Figure 2). Tryptic peptide fragments obtained from the purified proteins were assigned to putative cellulolytic enzymes from Talaromyces marneffei or Talaromyces stipitatus in the National Center for Biotechnology Information (NCBI) database (Table 1) because putative genes from these fungi are known to have high similarity with those from T. cellulolyticus [29]. The genes corresponding to the T. cellulolyticus cellulolytic enzymes were found in our in-house T. cellulolyticus draft genome sequence. The glycosyl hydrolase (GH) families [30] of the six enzymes were determined from the deduced amino acid sequences and are as follows: GH10 for Xyl10A (from F1), GH6 for Cel6A (from F2), GH3 for Bgl3A (from F3), GH7 for Cel7A (from F3) and Cel7B (from F4), and GH5 for Cel5A (from F4).Figure 1


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)

SDS-PAGE analysis of purified enzymes. Lanes: M, protein marker; 1, Xyl10A; 2, Cel5A; 3, Cel7B; 4, Cel6A; 5, Bgl3A; 6, Cel7A.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: SDS-PAGE analysis of purified enzymes. Lanes: M, protein marker; 1, Xyl10A; 2, Cel5A; 3, Cel7B; 4, Cel6A; 5, Bgl3A; 6, Cel7A.
Mentions: Cellulolytic enzymes involved in the hydrolysis of crystalline cellulose play a role as core enzymes in cellulase systems to degrade pretreated lignocellulosic biomass [8,12,13,28]. For purification of the core enzymes in the T. cellulolyticus cellulase system, we developed an assay for the synergistic hydrolysis of Avicel. First, synergistic activity for Avicel hydrolysis was determined for combinations of four groups (F1, F2, F3, and F4) of fractions obtained by Resource Q column chromatography of T. cellulolyticus culture supernatant (Figure 1). F2, containing Cel6A, showed synergism with F1, F3, and F4. F3, containing Cel7A and Bgl3A, was synergistic with F1 and F4, as well as F2. There was no synergism between F1 and F4. Thus, F2 and F3 were used to detect synergism for Avicel hydrolysis in fractions obtained by further chromatography of each group. Finally, six cellulolytic enzymes were purified to electrophoretically homogeneous forms (Figure 2). Tryptic peptide fragments obtained from the purified proteins were assigned to putative cellulolytic enzymes from Talaromyces marneffei or Talaromyces stipitatus in the National Center for Biotechnology Information (NCBI) database (Table 1) because putative genes from these fungi are known to have high similarity with those from T. cellulolyticus [29]. The genes corresponding to the T. cellulolyticus cellulolytic enzymes were found in our in-house T. cellulolyticus draft genome sequence. The glycosyl hydrolase (GH) families [30] of the six enzymes were determined from the deduced amino acid sequences and are as follows: GH10 for Xyl10A (from F1), GH6 for Cel6A (from F2), GH3 for Bgl3A (from F3), GH7 for Cel7A (from F3) and Cel7B (from F4), and GH5 for Cel5A (from F4).Figure 1

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