Cellulase component requirements

Research to improve ethanol fermentation has also focused on the development of a high solids, continuous feeding process as well as improved yeast strains. Other related developments include processes for the use of biomass feedstocks, such as cellulosic waste material.40"44 The Genencor-NREL collaboration funded by the U.S. DOE has (a) put in place the tools required for continual improvement of biomass-derived cellulases, (b) built a suite of enzymes with enhanced thermostability and improved specific performance at elevated temperatures, (c) developed an improved production strain for the enhanced cellulase components and demonstrated an enhanced production process. Therefore, plans are in place to provide a developmental product(s) in support of continued industry development. 

The anaerobic biological conversion of the major polymeric components of MSW identified require appropriate microorganisms and hydrolytic enzyme systems. Extracellular hydrolytic enzymes, such as cellulases and lipases, have been shown to be effective in the post hydrolysis of anaerobic digester efQuent solids 34) or pretreatment of complex organic polymers before the digestion process 48),... 

Since protein adsorption to an anion exchange resin is reversible and does not constitute a classical immobilization, the ability of the resins to retain activity under various conditions must be determined. Macrosorb KAX DEAE bound -D-glucosidase was tested with solutions of primary interest for their final application. Several batch washes of a 1% w/v slurry were required to ensure complete equilibrium elution for a given concentration, as determined from the absence of pNPG units in subsequent washes. Several salt solutions of typical fermentation media components were tested. These included 3 mM to 50 mM solutions of MgSO, KHgPO, NaQ, and sodium acetate. Also, incubations with cellulase solutions were tested to determine if the proteins present in a cellulose hydrolysis would displace the -D-glucosidase. Both of these displacement studies were carried out at 22°C and 40 C. 

The scheme proposed above requires microbial colonization of the material and excludes degradation by amylases and cellulases that are present in soils (28), but are not newly synthesized or associated with microbial cells. Active polysaccharide hydrolases are found in nearly all soils, but these enzymes are primarily bound to soil organic matter or mineral components attachment is firm enough to severely limit migration of the enzymes from surrounding soil to the film. 

Assay of Endocellulase Activity. Cellulase is an enzyme complex a synergistic action between the components is required for a complete hydrolysis of the insoluble cellulose. There is no consensus about the substrate to be used for the cellulase activity measurements. 

Not all cellulase enzymes give identical results, even with similar fabrics in similar equipment. Cellulases derived from Trichoderma typically are the most aggressive in their action, whereas mono-component endo-glucanases often require the most mechanical action to achieve the desired effects. Slow deactivation of the cellulases during transport and storage can adversely affect the reproducibility of the resulting effects. If cotton is not washed carefully before bio-finishing,... 

As currently understood, the cellulase complex contains the following components (listed in the order in which their action on cellulose occurs) 1. Ci is an enzyme whose action is unspecified. It is required for the hydrolysis of highly oriented solid cellulose (Cotton, Avicel, etc.) by 0-1 — 4 glucanases. 

An active cellulase will find many uses other than production of sugar (34, 36). These include removal of unwanted cellulose impurities (33) and breakdown of cell walls to improve extraction of a cell component, or to increase digestibility or nutrient availability of feeds (6, 35). Such uses require a preparation free of enzymes that might attack the residue being purified or the substance being extracted. 

Although efficient and novel genetic methods have been employed by various researchers to overexpress a few components of cellulase and xylanase enzymes in several hosts, information on modification of the properties of the recombinant enzymes is rather scanty (cf. section 5.3). Furthermore, the enzyme components which have been overexpressed are unlikely to hydrolyze cellulose efficiently unless mixed with other components to have a complete enzyme system, which is required for efficient hydrolysis. This is perhaps the reason as to why such recombinant organisms have not been widely studied for production of the enzymes in bench or pilot-scale bioreactors, which is required to assess the economics of their large-scale production. The production of hydrolytic enzymes economically is of prime concern for their subsequent commercial application in bioproduct manufacture. For successful industrial... 

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