A Novel Cellobiohydrolase I (CBHI) from Penicillium digitatum: Production, Purification, and Characterization

Santos, Fabiane Cristina dos; Oliveira, Marco Aurélio Schüler de; Seixas, Flávio Augusto Vicente; Barbosa-Tessmann, Ione Parra

doi:10.1007/s12010-020-03307-9

Cited by 9 publications

(6 citation statements)

References 48 publications

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“…This possibly suggests that CBH1 of P. funiculosum might also utilize the amorphous CMC as substrate in addition to the crystalline Avicel. The activity against CMC was also reported by Santos et al while characterizing CBH1 of Penicillium digitatum [30].…”

Section: Discussionsupporting

confidence: 74%

Blocking drug efflux mechanisms facilitate genome engineering process in hypercellulolytic fungus, Penicillium funiculosum NCIM1228

Randhawa

Pasari

Sinha

et al. 2021

Biotechnol Biofuels

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Background Penicillium funiculosum NCIM1228 is a non-model filamentous fungus that produces high-quality secretome for lignocellulosic biomass saccharification. Despite having desirable traits to be an industrial workhorse, P. funiculosum has been underestimated due to a lack of reliable genetic engineering tools. Tolerance towards common fungal antibiotics had been one of the major hindrances towards development of reliable transformation tools against the non-model fungi. In this study, we sought to understand the mechanism of drug tolerance of P. funiculosum and the provision to counter it. We then attempted to identify a robust method of transformation for genome engineering of this fungus. Results Penicillium funiculosum showed a high degree of drug tolerance towards hygromycin, zeocin and nourseothricin, thereby hindering their use as selectable markers to obtain recombinant transformants. Transcriptome analysis suggested a high level expression of efflux pumps belonging to ABC and MFS family, especially when complex carbon was used in growth media. Antibiotic selection medium was optimized using a combination of efflux pump inhibitors and suitable carbon source to prevent drug tolerability. Protoplast-mediated and Agrobacterium-mediated transformation were attempted for identifying efficiencies of linear and circular DNA in performing genetic manipulation. After finding Ti-plasmid-based Agrobacterium-mediated transformation more suitable for P. funiculosum, we improvised the system to achieve random and homologous recombination-based gene integration and deletion, respectively. We found single-copy random integration of the T-DNA cassette and could achieve 60% efficiency in homologous recombination-based gene deletions. A faster, plasmid-free, and protoplast-based CRISPR/Cas9 gene-editing system was also developed for P. funiculosum. To show its utility in P. funiculosum, we deleted the gene coding for the most abundant cellulase Cellobiohydrolase I (CBH1) using a pair of sgRNA directed towards both ends of cbh1 open reading frame. Functional analysis of ∆cbh1 strain revealed its essentiality for the cellulolytic trait of P. funiculosum secretome. Conclusions In this study, we addressed drug tolerability of P. funiculosum and developed an optimized toolkit for its genome modification. Hence, we set the foundation for gene function analysis and further genetic improvements of P. funiculosum using both traditional and advanced methods.

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Section: Discussionsupporting

confidence: 74%

Blocking drug efflux mechanisms facilitate genome engineering process in hypercellulolytic fungus, Penicillium funiculosum NCIM1228

Randhawa

Pasari

Sinha

et al. 2021

Biotechnol Biofuels

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“…Thus, observations further suggest that this is a processive enzyme containing highest exo-catalytic activity on crystalline cellulose. The specific activity of this enzyme on avicel is higher than those reported for other CBHs (Dos Santos et al 2020 ; Gu et al 2019 ) making it an efficient Cbh. The rMtCel6A specifically attacks β-1,4-glycosidic bonds from the non-reducing ends of the crystalline cellulose and is remarkable in hydrolyzing a diverse range of polysaccharides containing β-1,4-linkages; this conclusion is based on the fact that Cel6 CBHs act from non-reducing ends of cellulose (Rouvinen et al 1990 ).…”

Section: Discussionmentioning

confidence: 60%

“…The K m value of this enzyme is lower than those of other reported fungal CBHs (Han et al 2020 ; Dos Santos et al 2020 ), suggesting that it has a higher substrate affinity and possesses a superior catalytic activity. It is known that Cel6A (CBHII) of Hypocrea jecorina is more effective when substrate is abundant, while Cel7A (CBHI) works efficiently when substrate is limiting (Badino et al 2017 ).…”

Section: Discussionmentioning

confidence: 63%

Recombinant cellobiohydrolase of Myceliophthora thermophila: characterization and applicability in cellulose saccharification

2021

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A codon optimized cellobiohydrolase (CBH) encoding synthetic gene of 1188 bp from a thermophilic mold Myceliophthora thermophila (MtCel6A) was cloned and heterologously expressed in Escherichia coli for the first time. In silico analysis suggested that MtCel6A is a GH6 CBH and belongs to CBHII family, which is structurally similar to Cel6A of Humicola insolens. The recombinant MtCel6A is expressed as active inclusion bodies, and the molecular mass of the purified enzyme is ~ 45 kDa. The rMtCel6A is active in a wide range of pH (4–12) and temperatures (40–100 °C) with optima at pH 10.0 and 60 °C. It exhibits T1/2 of 6.0 and 1.0 h at 60 and 90 °C, respectively. The rMtCel6A is an extremozyme with organic solvent, salt and alkali tolerance. The Km, Vmax, kcat and kcat/Km values of the enzyme are 3.2 mg mL−1, 222.2 μmol mg−1 min−1, 2492 s−1 and 778.7 s−1 mg−1 mL−1, respectively. The product analysis of rMtCel6A confirmed that it is an exoenzyme that acts from the non-reducing end of cellulose. The addition of rMtCel6A to the commercial cellulase mix (Cellic CTec2) led to 1.9-fold increase in saccharification of the pre-treated sugarcane bagasse. The rMtCel6A is a potential CBH that finds utility in industrial processes such as in bioethanol, paper pulp and textile industries. Graphical Abstract

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“…Unlike what was observed for another species of the same genus, such as P. verruculosum BS3 isolated from the wood-yards on Kallai river belts, which in the zymogram showed a low molecular weight cellulase estimated at 17 kDa [35]. Other species of Penicillium sp have been described presenting proteins with cellulase activities close to those observed for P. citrinum LMI03, such as P. ochrochloron with a cellulase of 55kDa [36], P. echinulatum in which two bands of apparent molecular weight of approximately 80 kDa and 250 kDa were observed [37] and P. digitatum, which presented CMCase activity with an estimated molecular weight of 74 kDa [38].…”

Section: Production Of the Enzymatic Extracts Protein Profile In Sds-page And Zimogrammentioning

confidence: 93%

Enzymatic Hydrolysis of Lignocellulosic Biomass Using an Optimized Enzymatic Cocktail Prepared From Secretomes of Filamentous Fungi Isolated From Amazonian Biodiversity

Rosa

Oliveira

Filho

et al. 2021

Preprint

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The use of lignocellulosic biomass (LCB) has emerged as one of the main strategies for generating renewable biofuels. For the efficient use of such feedstock, pretreatments are essential. The hydrolysis of cellulose – major component of LCB - demands enzymatic cocktails with improved efficiency to generate fermentable sugars. In this scenario, lignocellulolytic fungi have enormous potential for the development of efficient enzyme platforms. In this study, two enzymatic cocktails were developed for hydrolysis of two lignocellulosic biomasses: industrial cellulose pulp and cassava peel. The solid biomass ratio in relation to the protein content of the enzyme cocktail were performed by experimental design. The optimized cocktail for the hydrolysis of cellulose pulp (AMZ 1) was composed, in protein base, by 43% of Aspergillus sp LMI03 enzyme extract and 57% of T. reesei QM9414, while the optimal enzyme cocktail for cassava peel hydrolysis (AMZ 2) was composed by 50% of Aspergillus sp LMI03 enzyme extract, 25% of the extract of P. citrinum LMI01 and 25% of T. reesei. The ratio between solids and protein loading for AMZ 1 cocktail performance was 52 g/L solids and 30mg protein/g solids, resulting in a hydrolytic efficiency of 93%. For the AMZ 2 cocktail, the hydrolytic efficiency was 78% for an optimized ratio of 78g/L solids and 19mg protein/g solids. These results indicate that cocktails formulated with enzymatic extracts of P. citrinum LMI01, Aspergillus sp LMI03 and T. reesei QM9414 are excellent alternatives for efficient hydrolysis of plant biomass and for other processes that depend on biocatalysis.

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A Novel Cellobiohydrolase I (CBHI) from Penicillium digitatum: Production, Purification, and Characterization

Cited by 9 publications

References 48 publications

Blocking drug efflux mechanisms facilitate genome engineering process in hypercellulolytic fungus, Penicillium funiculosum NCIM1228

Blocking drug efflux mechanisms facilitate genome engineering process in hypercellulolytic fungus, Penicillium funiculosum NCIM1228

Recombinant cellobiohydrolase of Myceliophthora thermophila: characterization and applicability in cellulose saccharification

Enzymatic Hydrolysis of Lignocellulosic Biomass Using an Optimized Enzymatic Cocktail Prepared From Secretomes of Filamentous Fungi Isolated From Amazonian Biodiversity

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