In vitro assessment of two novel Cellulases from Trabulsiella odontotermitis for agricultural waste utilization.

BACKGROUND: The production of agricultural wastes still growing as a consequence of the population growing. However, the majority of these residues are under-utilized due their chemical composition, which is mainly composed by cellulose. Actually, the search of cellulases with high efficiency to degrade this carbohydrate remains as the challenge. In the present experiment, two genes encoding an endoglucanase (EC 3.2.1.4) and ß-glucosidase (EC 3.2.1.21) were overexpressed in Escherichia coli and their recombinant enzymes (egl-FZYE and cel-FZYE, respectively) characterized. Those genes were found in Trabulsiella odontermitis which was isolated from the gut of termite Heterotermes sp. Additionally, the capability to release sugars from agricultural wastes was evaluated in both enzymes, alone and in combination. RESULTS: The results have shown that optimal pH was 6.0 and 6.5, reaching an activity of 1051.65 ± 47.78 and 607.80 ± 10.19 U/mg at 39 °C, for egl-FZYE and cel-FZYE, respectively. The Km and Vmax for egl-FZYE using CMC as substrate were 11.25 mg/mL and 3921.57 U/mg, respectively, whereas using Avicel were 15.39 mg/mL and 2314.81 U/mg, respectively. The Km and Vmax for cel-FZYE using Avicel as substrate were 11.49 mg/mL and 2105.26 U/mg, respectively, whereas using CMC the enzyme did not had activity. Both enzymes had effect on agricultural wastes, and their effect was improved when they were combined reaching an activity of 955.1 ± 116.1, 4016.8 ± 332 and 1124.2 ± 241 U/mg on corn stover, sorghum stover and pine sawdust, respectively. CONCLUSIONS: Both enzymes were capable of degrading agricultural wastes, and their effectiveness was improved up to 60% of glucose released when combined. In summary, the results of the study demonstrate that the recombinant enzymes exhibit characteristics that indicate their value as potential feed additives and that the enzymes could be used to enhance the degradation of cellulose in the poor-quality forage generally used in ruminant feedstuffs.

Chloroplasts as Cellular Factories for the Cost-effective Production of Cellulases.

Chloroplasts are vital photosynthetic organelles in plant cells that carry out several important cellular functions including synthesis of amino acids, fatty acids, and lipids and metabolism of nitrogen, starch, and Sulphur to sustain the homeostasis in plants. These organelles have got their own genome, and related genetic machinery to synthesize required proteins for various plant functions. Genetic manipulations of the chloroplast genome for different biotech applications has been of great interest due to desired features including the availability of operonal mode of gene expression, high copy number, and maternal mode of inheritance (in the most field crops). Their capacity to often express transgenes at high levels make it a cost-effective platform for the production of foreign proteins, particularly high-value targets of industrial importance, at large scale. This article reviews briefly the research work carried out to produce cellulolytic enzymes in higher plant chloroplasts. The challenges and future opportunities for the same are also discussed.

Advances in improving the performance of cellulase in ionic liquids for lignocellulose biorefinery.

Ionic liquids (ILs) have been considered as a class of promising solvents that can dissolve lignocellulosic biomass and then provide enzymatic hydrolyzable holocellulose. However, most of available cellulases are completely or partially inactivated in the presence of even low concentrations of ILs. To more fully exploit the benefits of ILs to lignocellulose biorefinery, it is critical to improve the compatibility between cellulase and ILs. Various attempts have been made to screen natural IL-tolerant cellulases from different microhabitats. Several physical and chemical methods for stabilizing cellulases in ILs were also developed. Moreover, recent advances in protein engineering have greatly facilitated the rational engineering of cellulases by site-directed mutagenesis for the IL stability. This review is aimed to provide the first detailed overview of the current advances in improving the performance of cellulase in non-natural IL environments. New ideas from the most representative progresses and technical challenges will be summarized and discussed.

Exploration of extremophiles for high temperature biotechnological processes.

Industrial processes often take place under harsh conditions that are hostile to microorganisms and their biocatalysts. Microorganisms surviving at temperatures above 60°C represent a chest of biotechnological treasures for high-temperature bioprocesses by producing a large portfolio of biocatalysts (thermozymes). Due to the unique requirements to cultivate thermophilic (60-80°C) and hyperthermophilic (80-110°C) Bacteria and Archaea, less than 5% are cultivable in the laboratory. Therefore, other approaches including sequence-based screenings and metagenomics have been successful in providing novel thermozymes. In particular, polysaccharide-degrading enzymes (amylolytic enzymes, hemicellulases, cellulases, pectinases and chitinases), lipolytic enzymes and proteases from thermophiles have attracted interest due to their potential for versatile applications in pharmaceutical, chemical, food, textile, paper, leather and feed industries as well as in biorefineries.

A novel and fully scalable Agrobacterium spray-based process for manufacturing cellulases and other cost-sensitive proteins in plants.

Transient transfection of plants by vacuum infiltration of Agrobacterium vectors represents the state of the art in plant-based protein manufacturing; however, the complexity and cost of this approach restrict it to pharmaceutical proteins. We demonstrated that simple spraying of Nicotiana plants with Agrobacterium vectors in the presence of a surfactant can substitute for vacuum inoculation. When the T-DNA of Agrobacterium encodes viral replicons capable of cell-to-cell movement, up to 90% of the leaf cells can be transfected and express a recombinant protein at levels up to 50% of total soluble protein. This simple, fast and indefinitely scalable process was successfully applied to produce cellulases, one of the most volume- and cost-sensitive biotechnology products. We demonstrate here for the first time that representatives of all hydrolase classes necessary for cellulosic biomass decomposition can be expressed at high levels, stored as silage without significant loss of activity and then used directly as enzyme additives. This process enables production of cellulases, and other potential high-volume products such as noncaloric sweetener thaumatin and antiviral protein griffithsin, at commodity agricultural prices and could find broad applicability in the large-scale production of many other cost-sensitive proteins.

Assessment of the enzymatic hydrolysis profile of cellulosic substrates based on reducing sugar release.

The activity profile of a 1:0.30 mixture of Celluclast 1.5L FG and Novozym 188 (Novozymes) was investigated using Whatman #1 filter paper (W1FP) as a single substrate for hydrolysis. The procedure was based on the ability of the enzymes to release total (RS(Tot)), insoluble (RS(Insol)) and soluble (RS(Sol)) reducing sugars from W1FP. RS(Insol) was used to estimate endoglucanase (EnG) activity whereas exoglucanases (ExG) were assessed by measuring RSSol in the presence of δ-gluconolactone. Finally, the ß-glucosidase (ßG) activity was derived from the difference between RS(Sol) measurements in the presence and absence of δ-gluconolactone. When this analytical procedure was applied to W1FP using 9.64 mg mL(-1) of the enzyme mixture, the relative contributions of EnG, ExG and ßG to the total cellulase activity were 63.28%, 12.02% and 24.70%, respectively. Also, this ratio changed with changes in the enzyme loading, giving a new insight into the synergy that exists among the enzymes.

Characterization of commercial cellulases and their use in the saccharification of a sugarcane bagasse sample pretreated with dilute sulfuric acid.

This study aimed to correlate the efficiency of enzymatic hydrolysis of the cellulose contained in a sugarcane bagasse sample pretreated with dilute H(2)SO(4) with the levels of independent variables such as initial content of solids and loadings of enzymes and surfactant (Tween 20), for two cellulolytic commercial preparations. The preparations, designated cellulase I and cellulase II, were characterized regarding the activities of total cellulases, endoglucanase, cellobiohydrolase, cellobiase, ß-glucosidase, xylanase, and phenoloxidases (laccase, manganese and lignin peroxidases), as well as protein contents. Both extracts showed complete cellulolytic complexes and considerable activities of xylanases, without activities of phenoloxidases. For the enzymatic hydrolyses, two 2(3) central composite full factorial designs were employed to evaluate the effects caused by the initial content of solids (1.19-4.81%, w/w) and loadings of enzymes (1.9-38.1 FPU/g bagasse) and Tween 20 (0.0-0.1 g/g bagasse) on the cellulose digestibility. Within 24 h of enzymatic hydrolysis, all three independent variables influenced the conversion of cellulose by cellulase I. Using cellulase II, only enzyme and surfactant loadings showed significant effects on cellulose conversion. An additional experiment demonstrated the possibility of increasing the initial content of solids to values much higher than 4.81% (w/w) without compromising the efficiency of cellulose conversion, consequently improving the glucose concentration in the hydrolysate.

Assessment of saccharification efficacy in the cellulase system of the brown rot fungus Gloeophyllum trabeum.

Brown rot fungi uniquely degrade wood by creating modifications thought to aid in the selective removal of polysaccharides by an incomplete cellulase suite. This naturally successful mechanism offers potential for current bioprocessing applications. To test the efficacy of brown rot cellulases, southern yellow pine wood blocks were first degraded by the brown rot fungus Gloeophyllum trabeum for 0, 2, 4, and 6 weeks. Characterization of the pine constituents revealed brown rot decay patterns, with selective polysaccharide removal as lignin compositions increased. G. trabeum liquid and solid state cellulase extracts, as well as a commercial Trichoderma reesei extract (Celluclast 1.5 L), were used to saccharify this pretreated material, using beta-glucosidase amendment to remove limitation of cellobiose-to-glucose conversion. Conditions varied according to source and concentration of cellulase extract and to pH (3.0 vs. 4.8). Hydrolysis yields were maximized using solid state G. trabeum extracts at a pH of 4.8. However, the extent of glucose release was low and was not significantly altered when cellulase loading levels were increased threefold. Furthermore, Celluclast 1.5 L continually outperformed G. trabeum cellulase extracts, although extent of glucose release never exceeded 22.0%. Results suggest methodological advances for utilizing crude G. trabeum cellulases and imply that the suboptimal hydrolysis levels obtained with G. trabeum and Celluclast 1.5 L cellulases, even at high loading levels, may be due to brown rot modifications insufficiently distributed throughout the pretreated material.

[Industrial exploitation of renewable resources: from ethanol production to bioproducts development]. / Valorisation des ressources renouvelables : de la production d'éthanol au développement de nouveaux bioproduits.

Plants, which are one of major groups of life forms, are constituted of an amazing number of molecules such as sugars, proteins, phenolic compounds etc. These molecules display multiple and complementary properties involved in various compartments of plants (structure, storage, biological activity etc.). The first uses of plants in industry were for food and feed, paper manufacturing or combustion. In the coming decades, these renewable biological materials will be the basis of a new concept: the "biorefiner" i.e. the chemical conversion of the whole plant to various products and uses. This concept, born in the 90ies, is analogous to today's petroleum refinery, which produces multiple fuels and derivative products from petroleum. Agriculture generates lots of co-products which were most often wasted. The rational use of these wasted products, which can be considered as valuable renewable materials, is now economically interesting and will contribute to the reduction of greenhouse has emissions by partially substituting for fossil fuels. Such substructures from biological waste products and transforming them into biofuels and new industrial products named "bioproducts". These compounds, such as bioplastics or biosurfactants, can replace equivalent petroleum derivatives. Towards that goal, lots of filamentous fungi, growing on a broad range of vegetable species, are able to produce enzymes adapted to the modification of these type of substrates. The best example, at least the more industrially developed to date, is the second generation biofuel technology using cellulose as a raw material. The process includes an enzymatic hydrolysis step which requires cellulases secreted from Trichoderma fungal species. This industrial development of a renewable energy will contribute to the diversification of energy sources used to transport and to the development of green chemistry which will partially substitute petrochemicals.

Consolidated bioprocessing of cellulosic biomass: an update.

Biologically mediated processes seem promising for energy conversion, in particular for the conversion of lignocellulosic biomass into fuels. Although processes featuring a step dedicated to the production of cellulase enzymes have been the focus of most research efforts to date, consolidated bioprocessing (CBP)--featuring cellulase production, cellulose hydrolysis and fermentation in one step--is an alternative approach with outstanding potential. Progress in developing CBP-enabling microorganisms is being made through two strategies: engineering naturally occurring cellulolytic microorganisms to improve product-related properties, such as yield and titer, and engineering non-cellulolytic organisms that exhibit high product yields and titers to express a heterologous cellulase system enabling cellulose utilization. Recent studies of the fundamental principles of microbial cellulose utilization support the feasibility of CBP.

Industrial scale-up of pH-controlled liquid hot water pretreatment of corn fiber for fuel ethanol production.

The pretreatment of cellulose in corn fiber by liquid hot water at 160 degrees C and a pH above 4.0 dissolved 50% of the fiber in 20 min. The pretreatment also enabled the subsequent complete enzymatic hydrolysis of the remaining polysaccharides to monosaccharides. The carbohydrates dissolved by the pretreatment were 80% soluble oligosaccharides and 20% monosaccharides with <1% of the carbohydrates lost to degradation products. Only a minimal amount of protein was dissolved, thus enriching the protein content of the undissolved material. Replication of laboratory results in an industrial trial at 43 gallons per minute (163 L/min) of fiber slurry with a residence time of 20 min illustrates the utility and practicality of this approach for pretreating corn fiber. The added costs owing to pretreatment, fiber, and hydrolysis are equivalent to less than 0.84 dollars/gal of ethanol produced from the fiber. Minimizing monosaccharide formation during pretreatment minimized the formation of degradation products; hence, the resulting sugars were readily fermentable to ethanol by the recombinant hexose and by pentose-fermenting Saccharomyces cerevisiae 424A(LNH-ST) and ethanologenic Escherichia coli at yields >90% of theoretical based on the starting fiber. This cooperative effort and first successful trial opens the door for examining the robustness of the pretreatment system under extended run conditions as well as pretreatment of other cellulose-containing materials using water at controlled pH.