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

Lessons from the Discovery of Mitochondrial Fragmentation (Fission): A Review and Update

Thirty-five years ago, we described fragmentation of the mitochondrial population in a living cell into small vesicles (mitochondrial fission). Subsequently, this phenomenon has become an object of general interest due to its involvement in the process of oxidative stress-related cell death and having high relevance to the incidence of a pathological phenotype. Tentatively, the key component of mitochondrial fission process is segregation and further asymmetric separation of a mitochondrial body yielding healthy (normally functioning) and impaired (incapable to function in a normal way) organelles with subsequent decomposition and removal of impaired elements through autophagy (mitophagy). We speculate that mitochondria contain cytoskeletal elements, which maintain the mitochondrial shape, and also are involved in the process of intramitochondrial segregation of waste products. We suggest that perturbation of the mitochondrial fission/fusion machinery and slowdown of the removal process of nonfunctional mitochondrial structures led to the increase of the proportion of impaired mitochondrial elements. When the concentration of malfunctioning mitochondria reaches a certain threshold, this can lead to various pathologies, including aging. Overall, we suggest a process of mitochondrial fission to be an essential component of a complex system controlling a healthy cell phenotype. The role of reactive oxygen species in mitochondrial fission is discussed

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 β-glucosidase expressed in B1 host strain). Enzyme loading was 10 mg protein/g dry substrate and 40 U/g of β-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 27–30 g/L, 25–27 g/100 g; reducing sugars 37–42 g/L, 34–38 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 (92–96 g/L, 84–87 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 42–47% for SBH and 16–18% and 21–26% for EFB, respectively

Enzymatic Hydrolysis of Kraft and Sulfite Pulps: What Is the Best Cellulosic Substrate for Industrial Saccharification?

Sulfite and kraft pulping are two principal methods of industrial delignification of wood. In recent decades, those have been considered as possibilities to pretreat recalcitrant wood lignocellulosics for the enzymatic hydrolysis of polysaccharides and the subsequent fermentation of obtained sugars to valuable bioproducts. Current work compares chemistry and technological features of two different cooking processes in the preparation of polysaccharide substrates for deep saccharification with P. verruculosum glycosyl hydrolases. Bleached kraft and sulfite pulps were subjected to hydrolysis with enzyme mixture of high xylanase, cellobiohydrolase, and β-glucosidase activities at a dosage of 10 FPU/g of dry pulp and fiber concentration of 2.5, 5, and 10%. HPLC was used to analyze soluble sugars after hydrolysis and additional acid inversion of oligomers to monosaccharides. Kraft pulp demonstrated higher pulp conversion after 48 h (74–99%), which mostly resulted from deep xylan hydrolysis. Sulfite-pulp hydrolysates, obtained in similar conditions due to higher hexose concentration (more than 50 g/L), had higher fermentability for industrial strains producing alcohols, microbial protein, or organic acids. Along with saccharification, enzymatic modification of non-hydrolyzed residues occurred, which led to decreased degree of polymerization and composition changes in two industrial pulps. As a result, crystallinity of kraft pulp increased by 1.3%, which opens possibilities for obtaining new types of cellulosic products in the pulp and paper industry. The high adaptability and controllability of enzymatic and fermentation processes creates prospects for the modernization of existing factories