Currently, the relatively high cost of enzymes such as glycoside hydrolases that catalyze cellulose hydrolysis represents a barrier to commercialization of a biorefinery capable of producing renewable transportable fuels such as ethanol from abundant lignocellulosic biomass. Among the many families of glycoside hydrolases that catalyze cellulose and hemicellulose hydrolysis, few are more enigmatic than family 61 (GH61), originally classified based on measurement of very weak endo-1,4-beta-d-glucanase activity in one family member. Here we show that certain GH61 proteins lack measurable hydrolytic activity by themselves but in the presence of various divalent metal ions can significantly reduce the total protein loading required to hydrolyze lignocellulosic biomass. We also solved the structure of one highly active GH61 protein and find that it is devoid of conserved, closely juxtaposed acidic side chains that could serve as general proton donor and nucleophile/base in a canonical hydrolytic reaction, and we conclude that the GH61 proteins are unlikely to be glycoside hydrolases. Structure-based mutagenesis shows the importance of several conserved residues for GH61 function. By incorporating the gene for one GH61 protein into a commercial Trichoderma reesei strain producing high levels of cellulolytic enzymes, we are able to reduce by 2-fold the total protein loading (and hence the cost) required to hydrolyze lignocellulosic biomass.

Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61: structure and function of a large, enigmatic family

The demand for fossil derivate fuels and chemicals has increased, augmenting concerns on climate change, global economic stability, and sustainability on fossil resources. Therefore, the production of fuels and chemicals from alternative and renewable resources has attracted considerable and growing attention. Ethanol is a promising biofuel that can reduce the consumption of gasoline in the transportation sector and related greenhouse gas (GHG) emissions. Lignocellulosic biomass is a promising feedstock to produce bioethanol (cellulosic ethanol) because of its abundance and low cost. Since the conversion of lignocellulose to ethanol is complex and expensive, the cellulosic ethanol price cannot compete with those of the fossil derivate fuels. A promising strategy to lower the production cost of cellulosic ethanol is developing a biorefinery which produces ethanol and other high-value chemicals from lignocellulose. The selection of such chemicals is difficult because there are hundreds of products that can be produced from lignocellulose. Multiple reviews and reports have described a small group of lignocellulose derivate compounds that have the potential to be commercialized. Some of these products are in the bench scale and require extensive research and time before they can be industrially produced. This review examines chemicals and materials with a Technology Readiness Level (TRL) of at least 8, which have reached a commercial scale and could be shortly or immediately integrated into a cellulosic ethanol process.

/pdf/a-review-on-commercial-scale-high-value-products-that-can-be-22ttee469w.pdf

A review on commercial-scale high-value products that can be produced alongside cellulosic ethanol.

https://www.cell.com/trends/biotechnology/pdf/S0167-7799(11)00070-9.pdf

Alternatives to Trichoderma reesei in biofuel production.

An extremely highly active cellobiohydrolase (CBH IIb or Cel6B) was isolated from Chrysosporium lucknowense UV18-25 culture filtrate. The CBH IIb demonstrated the highest ability for a deep degradation of crystalline cellulose amongst a few cellobiohydrolases tested, including C. lucknowense CBH Ia, Ib, IIa, and Trichoderma reesei CBH I and II. Using purified C. lucknowense enzymes (CBH Ia, Ib, and IIb; endoglucanases II and V; beta-glucosidase, xylanase II), artificial multienzyme mixtures were reconstituted, displaying an extremely high performance in a conversion of different cellulosic substrates (Avicel, cotton, pretreated Douglas fir wood) to glucose. These mixtures were much or notably more effective in hydrolysis of the cellulosic substrates than the crude multienzyme C. lucknowense preparation and other crude cellulase samples produced by T. reesei and Penicillium verruculosum. Highly active cellulases are a key factor in bioconversion of plant lignocellulosic biomass to ethanol as an alternative to fossil fuels.

Design of highly efficient cellulase mixtures for enzymatic hydrolysis of cellulose.

Cellulolytic bacteria and fungi have been shown to use two different approaches to degrade cellulose. Most aerobic microbes secrete sets of individual cellulases, many of which contain a carbohydrate binding molecule (CBM), which act synergistically on native cellulose. Most anaerobic microorganisms produce large multienzyme complexes called cellulosomes, which are usually attached to the outer surface of the microorganism. Most of the cellulosomal enzymes lack a CBM, but the cohesin subunit, to which they are bound, does contain a CBM. The cellulases present in each class show considerable overlap in their catalytic domains, and processive cellulases (exocellulases and processive endocellulases) are the most abundant components of both the sets of free enzymes and of the cellulosomal cellulases. Analysis of the genomic sequences of two cellulolytic bacteria, Cytophaga hutchinsonii, an aerobe, and Fibrobacter succinogenes, an anaerobe, suggest that these organisms must use a third mechanism. This is because neither of these organisms, encodes processive cellulases and most of their many endocellulase genes do not encode CBMs. Furthermore, neither organism appears to encode the dockerin and cohesin domains that are key components of cellulosomes.

Three microbial strategies for plant cell wall degradation.

A method for analysis of the component composition of multienzyme complexes secreted by the filamentous fungus Trichoderma reesei was developed. The method is based on chromatofocusing followed by further identification of protein fractions according to their substrate specificity and molecular characteristics of the proteins. The method allows identifying practically all known cellulases and hemicellulases of T. reesei: endoglucanase I (EG I), EG II, EG III, cellobiohydrolase I (CBH I), CBH II, xylanase I (XYL I), XYL II, β-xylosidase, α-L-arabinofuranosidase, acetyl xylan esterase, mannanase, α-galactosidase, xyloglucanase, polygalacturonase, and exo-β-1,3-glucosidase. The component composition of several laboratory and commercial T. reesei preparations was studied and the content of the individual enzymes in these preparations was quantified. The influence of fermentation conditions on the component composition of secreted enzyme complexes was revealed. The characteristic features of enzyme preparations obtained in “cellulase” and “xylanase” fermentation conditions are shown.

New effective method for analysis of the component composition of enzyme complexes from Trichoderma reesei.

Commercial and pilot enzyme preparations from fungi of the genera Penicillium and Trichoderma have been compared with regard to their action on conifer wood pretreated with acidified aqueous ethanol (organosolve). In most experiments, enzymes from the genus Penicillium allowed higher yields of reducing sugars and glucose than those from Trichoderma. High beta-glucosidase activity is essential for deep pulp hydrolysis.

New cellulases efficiently hydrolyzing lignocellulose pulp

Six xylan-hydrolyzing enzymes have been isolated from the preparations Celloviridin G20x and Xybeten-Xyl, obtained earlier based on the strain 1 Trichoderma longibrachiatum (Trichoderma reesei) TW-1. The enzymes isolated were represented by three xylanases (XYLs), XYL I (20 kDa, pI 5.5), XYL II (21 kDa, pI 9.5), XYL III (30 kDa, pI 9.1); endoglucanase I (EG I), an enzyme exhibiting xylanase activity (57 kDa, pI 4.6); and two exodepolymerases, β-xylosidase (β-XYL; 80 kDa, pI 4.5) and α-L-arabinofuranosidase I (α-L-AF I; 55 kDa, pI 7.4). The substrate specificity of the enzymes isolated was determined. XYL II exhibited maximum specific xylanase activity (190 U/mg). The content of the enzymes in the preparation was assessed. Maximum contributions to the total xylanase activities of preparations Celloviridin G20x and Xybeten-Xyl were made by EG I and XYL II, respectively. Effects of temperature and pH on the enzyme activities, their stabilities under various conditions, and the kinetics of exhaustive hydrolysis of glucuronoxylan and arabinoxylan were studied. Combinations of endodepolymerases (XYL I, XYL II, XYL III, or EG I) and exodepolymerases (α-L-AF I or β-XYL) produced synergistic effects on arabinoxylan cleavage. The reverse was the case when endodepolymerases, such as XYL I or EG I, were combined with α-L-AF I.

Properties of hemicellulases of the enzyme complex from Trichoderma longibrachiatum

An enzyme preparation has been produced on the basis of Penicillium canescens strains with the activity of cellibiohydrolase I, II; endo-1,4-beta-gluconase of Penicillium verruculosum; and beta-glucosidase of Aspergillus niger. It was shown that for the most effective hydrolysis of aspen wood pulp the optimal ratio of cellobiohydrolase and endo- 1,4-3-gluconase in enzyme preparations was 8 : 2 (by protein). It was also established that the homologous xylanase secreted by the Penicillium canescens fungus is a required component for the enzyme complex for hydrolysis of the hemicellulose matrix of aspen wood.

Production of enzyme preparations on the basis of Penicillum canescens recombinant strains with a high ability for the hydrolysis of plant materials

Cellobiase (β-D-glucosidase) with a molecular weight of 100 kDa and pI 5.2 was isolated from the cellulolytic system of Penicillium verruculosum. Kinetic parameters of enzymatic hydrolysis of cellobiose, gentiobiose, sophorose, and synthetic substrates, i. e. methylumbelliferyl and p-nitrophenyl sugar derivatives were determined. Glucose and D-glucose-δ-lactone competitively inhibited cellobiase (K
i0.19 mM and 17 μM, respectively). Glucosyl transfer reactions were studied with cellobiose as a single substrate and in the mixture of cellobiose and methylumbelliferyl cellobioside. The product composition was determined in these systems. The ratio of hydrolysis and transfer reaction rates for cellobiose conversion was calculated.

A. O. Bekkarevich

Papers

New effective method for analysis of the component composition of enzyme complexes from Trichoderma reesei.

New cellulases efficiently hydrolyzing lignocellulose pulp

Properties of hemicellulases of the enzyme complex from Trichoderma longibrachiatum

Production of enzyme preparations on the basis of Penicillum canescens recombinant strains with a high ability for the hydrolysis of plant materials

Isolation and properties of cellobiase from Penicillium verruculosum