Homologous cloning, purification and characterization of highly active cellobiohydrolase I (Cel7A) from Penicillium canescens

Volkov, Pavel V.; Рожкова, А. М.; Gusakov, Alexander V.; Sinitsyn, A. P.

doi:10.1016/j.pep.2014.08.011

Cited by 24 publications

(14 citation statements)

References 30 publications

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“…Another reason for the high efficiency of cellulase complexes based on Penicillium is the extremely high specific activity of their key enzymes such as CBH I and CBH II when compared with the corresponding enzymes from T. reesei (the difference in specific activity can reach 2-2.5 times). In particular, these properties were demonstrated for CBHs from P. funiculosum, P. pulvirrolum, P. verruculosum, and P. canescens [23,24]. It should be noted that one of the reasons for such a high…”

Section: Advantages Of the Enzymatic Complex Of Filamentous Fungi Penmentioning

confidence: 90%

Engineering Robust Cellulases for Tailored Lignocellulosic Degradation Cocktails

Contreras

Pramanik

Рожкова

et al. 2020

IJMS

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Lignocellulosic biomass is a most promising feedstock in the production of second-generation biofuels. Efficient degradation of lignocellulosic biomass requires a synergistic action of several cellulases and hemicellulases. Cellulases depolymerize cellulose, the main polymer of the lignocellulosic biomass, to its building blocks. The production of cellulase cocktails has been widely explored, however, there are still some main challenges that enzymes need to overcome in order to develop a sustainable production of bioethanol. The main challenges include low activity, product inhibition, and the need to perform fine-tuning of a cellulase cocktail for each type of biomass. Protein engineering and directed evolution are powerful technologies to improve enzyme properties such as increased activity, decreased product inhibition, increased thermal stability, improved performance in non-conventional media, and pH stability, which will lead to a production of more efficient cocktails. In this review, we focus on recent advances in cellulase cocktail production, its current challenges, protein engineering as an efficient strategy to engineer cellulases, and our view on future prospects in the generation of tailored cellulases for biofuel production.

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Section: Advantages Of the Enzymatic Complex Of Filamentous Fungi Penmentioning

confidence: 90%

Engineering Robust Cellulases for Tailored Lignocellulosic Degradation Cocktails

Contreras

Pramanik

Рожкова

et al. 2020

IJMS

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“…Proteins from the crude enzyme samples, containing rPvCel7A forms, were preliminary precipitated with ammonium sulfate (80% saturation at 0°C) followed by a desalting procedure on a Bio‐Gel P‐2 column (Bio‐Rad Laboratories, Hercules, CA). Then, the rPvCel7A forms were purified by anion‐exchange chromatography on a Source 15Q column followed by hydrophobic interaction chromatography on a Source 15 Isopropyl column (Pharmacia, Uppsala, Sweden) as described elsewhere (Volkov et al, ). The enzyme purity was characterized by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS‐PAGE) in a 12% gel using a Mini Protean II equipment (Bio‐Rad Laboratories, Hercules, CA).…”

Section: Methodsmentioning

confidence: 99%

“…chromatography on a Source 15 Isopropyl column (Pharmacia, Uppsala, Sweden) as described elsewhere (Volkov et al, 2014). The enzyme purity was characterized by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) in a 12% gel using a Mini Protean II equipment (Bio-Rad Laboratories, Hercules, CA).…”

Section: Enzyme Purificationmentioning

confidence: 99%

N‐linked glycosylation of recombinant cellobiohydrolase I (Cel7A) from Penicillium verruculosum and its effect on the enzyme activity

Dotsenko

Gusakov

Volkov

et al. 2015

Biotech & Bioengineering

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Cellobiohydrolase I from Penicillium verruculosum (PvCel7A) has four potential N-glycosylation sites at its catalytic module: Asn45, Asn194, Asn388, and Asn430. In order to investigate how the N-glycosylation influences the activity and other properties of the enzyme, the wild type (wt) PvCel7A and its mutant forms, carrying Asn to Ala substitutions, were cloned into Penicillium canescens PCA10 (niaD-) strain, a fungal host for production of heterologous proteins. The rPvCel7A-wt and N45A, N194A, N388A mutants were successfully expressed and purified for characterization, whereas the expression of N430A mutant was not achieved. The MALDI-TOF mass spectrometry fingerprinting of peptides, obtained as a result of digestion of rPvCel7A forms with specific proteases, showed that the N-linked glycans represent variable high-mannose oligosaccharides and the products of their sequential enzymatic trimming, according to the formula (Man)0-13 (GlcNAc)2 , or a single GlcNAc residue. Mutations had no notable effect on pH-optimum of PvCel7A activity and enzyme thermostability. However, the mutations influenced both the enzyme adsorption ability on Avicel and its activity against natural and synthetic substrates. In particular, the N45A mutation led to a significant increase in the rate of Avicel and milled aspen wood hydrolysis, while the substrate digestion rates in the case of N194A and N388A mutants were notably lower relative to rPvCel7A-wt. These data, together with data of 3D structural modeling of the PvCel7A catalytic module, indicate that the N-linked glycans are an important part of the processive catalytic machinery of PvCel7A.

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“…elsewhere [8,9]. The mutant P. canescens RN3-11-7 strain (VKPM F-436), deficient in the nitrate reductase gene (niaD-), was used as a host strain for transformation and chromosomal DNA preparation.…”

Section: Fungal Strains and Fermentation Mediamentioning

confidence: 99%

Glucoamylases from Penicillium verruculosum and Myceliophthora thermophila: Analysis of differences in activity against polymeric substrates based on 3D model structures of the intact enzymes

et al. 2015

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Homologous cloning, purification and characterization of highly active cellobiohydrolase I (Cel7A) from Penicillium canescens

Cited by 24 publications

References 30 publications

Engineering Robust Cellulases for Tailored Lignocellulosic Degradation Cocktails

Engineering Robust Cellulases for Tailored Lignocellulosic Degradation Cocktails

N‐linked glycosylation of recombinant cellobiohydrolase I (Cel7A) from Penicillium verruculosum and its effect on the enzyme activity

Glucoamylases from Penicillium verruculosum and Myceliophthora thermophila: Analysis of differences in activity against polymeric substrates based on 3D model structures of the intact enzymes

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