Comparison of Two Methods for Assaying Reducing Sugars in the Determination of Carbohydrase Activities

The Nelson-Somogyi (NS) and 3,5-dinitrosalicylic acid (DNS) assays for reducing sugars are widely used in measurements of carbohydrase activities against different polysaccharides. Using twelve commercial enzyme preparations, the comparison of the NS and DNS assays in determination of cellulase, β-glucanase, xylanase, and β-mannanase activities was carried out. When cellulase activities against CMC were measured, the DNS assay gave activity values, which were typically 40–50% higher than those obtained with the NS assay. In the analysis of the xylanase, β-mannanase, and β-glucanase activities, the overestimations by the DNS assay were much more pronounced (the observed differences in the activities were 3- to 13-fold). Reasons for preferential use of the NS assay for measuring activities of carbohydrases other than cellulases are discussed

Cellulases from Mycelial fungi <em>Penicillium verruculosum</em> as a Real Alternative to Trichoderma Enzymes in Industrial Hydrolysis of Cellulosic Biomass

Abstract The possibility of using the recipient strain Penicillium verruculosum B1-537 (ΔniaD) as a producer of laboratory and industrial enzymes was considered. The advantage of this strain is its ability to secrete a basic cellulase complex consisting of cellobiohydrolases, endoglucanases, and β-glucosidase, which exceeds in its hydrolytic ability the enzyme complex of Hypocrea (Trichoderma) strains. Using the expression system, the basic complex of cellulases of the recipient strain Piptochaetium verruculosum B1-537 (ΔniaD) was supplemented with new (booster) enzymes that are necessary to increase its hydrolytic activity. Enzyme preparations adapted to the processing of various types of renewable plant biomass were obtained

Engineering robust cellulases for tailored lignocellulosic degradation cocktails

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. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

Engineering Robust Cellulases for Tailored Lignocellulosic Degradation Cocktails

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. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

Effect of Novel Penicillium verruculosum Enzyme Preparations on the Saccharification of Acid- and Alkali-Pretreated Agro-Industrial Residues

This study aimed at evaluating different enzyme combinations in the saccharification of sugarcane bagasse (SCB), soybean husks (SBH) and oil palm empty fruit bunches (EFB) submitted to mild acid and alkaline pretreatments. Enzyme pools were represented by B1 host (crude cellulase/xylanase complexes of Penicillium verruculosum); B1-XylA (Penicillium canescens xylanase A expressed in P. verruculosum B1 host strain); and F10 (Aspergillus niger &beta;-glucosidase expressed in B1 host strain). Enzyme loading was 10 mg protein/g dry substrate and 40 U/g of &beta;-glucosidase (F10) activity. SCB was efficiently hydrolyzed by B1 host after alkaline pretreatment, yielding glucose and reducing sugars at 71 g/L or 65 g/100 g of dry pretreated substrate and 91 g/L or 83 g/100 g, respectively. B1 host performed better also for EFB, regardless of the pretreatment method, but yields were lower (glucose 27&ndash;30 g/L, 25&ndash;27 g/100 g; reducing sugars 37&ndash;42 g/L, 34&ndash;38 g/100 g). SBH was efficiently saccharified by the combination of B1 host and B1-XylA, yielding similar concentrations of reducing sugars for both pretreatments (92&ndash;96 g/L, 84&ndash;87 g/100 g); glucose recovery, however, was higher with alkaline pretreatment (81 g/L, 74 g/100 g). Glucose and reducing sugar yields from initial substrate mass were 42% and 54% for SCB, 36% and 42&ndash;47% for SBH and 16&ndash;18% and 21&ndash;26% for EFB, respectively

Using an Inducible Promoter of a Gene Encoding <i>Penicillium verruculosum</i> Glucoamylase for Production of Enzyme Preparations with Enhanced Cellulase Performance

<div><p>Background</p><p><i>Penicillium verruculosum</i> is an efficient producer of highly active cellulase multienzyme system. One of the approaches for enhancing cellulase performance in hydrolysis of cellulosic substrates is to enrich the reaction system with β -glucosidase and/or accessory enzymes, such as lytic polysaccharide monooxygenases (LPMO) displaying a synergism with cellulases.</p><p>Results</p><p>Genes <i>bglI</i>, encoding β-glucosidase from <i>Aspergillus niger</i> (AnBGL), and <i>eglIV</i>, encoding LPMO (formerly endoglucanase IV) from <i>Trichoderma reesei</i> (TrLPMO), were cloned and expressed by <i>P</i>. <i>verruculosum</i> B1-537 strain under the control of the inducible <i>gla1</i> gene promoter. Content of the heterologous AnBGL in the secreted multienzyme cocktails (hBGL1, hBGL2 and hBGL3) varied from 4 to 10% of the total protein, while the content of TrLPMO in the hLPMO sample was ~3%. The glucose yields in 48-h hydrolysis of Avicel and milled aspen wood by the hBGL1, hBGL2 and hBGL3 preparations increased by up to 99 and 80%, respectively, relative to control enzyme preparations without the heterologous AnBGL (at protein loading 5 mg/g substrate for all enzyme samples). The heterologous TrLPMO in the hLPMO preparation boosted the conversion of the lignocellulosic substrate by 10–43%; however, in hydrolysis of Avicel the hLPMO sample was less effective than the control preparations. The highest product yield in hydrolysis of aspen wood was obtained when the hBGL2 and hLPMO preparations were used at the ratio 1:1.</p><p>Conclusions</p><p>The enzyme preparations produced by recombinant <i>P</i>. <i>verruculosum</i> strains, expressing the heterologous AnBGL or TrLPMO under the control of the <i>gla1</i> gene promoter in a starch-containing medium, proved to be more effective in hydrolysis of a lignocellulosic substrate than control enzyme preparations without the heterologous enzymes. The enzyme composition containing both AnBGL and TrLPMO demonstrated the highest performance in lignocellulose hydrolysis, providing a background for developing a fungal strain capable to express both heterologous enzymes simultaneously.</p></div

The scheme of cloning pGA-BGL and pGA-EGIV plasmids.

<p>The scheme of cloning pGA-BGL and pGA-EGIV plasmids.</p