Hydrolysis cellulose substrates

Cellulase enzyme complexes consist of three major types of proteins that synergistically catalyze the breakdown of a cellulosic substrate. Because the enzymes are strictly substrate-specific in their action, any change in the structure or accessibility of the substrate can have a considerable influence on the course of the hydrolysis reaction. A pretreatment method based on exposing cellulosic substrate to phosphoric acid solution [9] and addition of the nonionic... 

Ionisation of the hydroxy groups in cellulose is essential for the nucleophilic substitution reaction to take place. At neutral pH virtually no nucleophilic ionised groups are present and dye-fibre reaction does not occur. When satisfactory exhaustion of the reactive dye has taken place, alkali is added to raise the pH to 10-11, causing adequate ionisation of the cellulose hydroxy groups. The attacking nucleophile ( X ) can be either a cellulosate anion or a hydroxide ion (Scheme 7.8), the former resulting in fixation to the fibre and the latter in hydrolysis of the reactive dye. The fact that the cellulosic substrate competes effectively with water for the reactive dye can be attributed to three features of the reactive dye/ cellulosic fibre system ... 

Typically, the raw material for cellulose processing is a lignin-hemicellulose-cellulose (LHC) complex that is not very amenable to hydrolysis. Various pretreatments involving size reduction, separation of constituents of the complex, and processes to increase the accessibility of cellulose to hydrolytic agents may be required. These activities convert a relatively intractable raw material into a cellulosic substrate. 

A considerable amount of experimentation has been done on the kinetics of acid hydrolysis of pure cellulose substrates. Little experimentation has been done on natural cellulosic materials. Typical examples of kinetic studies of acid hydrolysis of cellulose can be found in the papers of Saeman (33) and Grethlein (13). These researchers depict the acid hydrolysis process as a pseudo-first-order sequential process, with the rate constants as a function of the acid concentration raised to a power, i.e.,... 

Figure 12. Relation of glucose selling price to delivered cellulose substrate costs for enzymatic hydrolysis process. Data for 250,000 t/yr plant 90% overall process efficiency, 50% enzyme recovery (reuse).

The relative influence of vibratory milling on the course of enzymatic and dilute acid hydrolysis of four cellulosic substrates was investigated. The four substrates—cotton linters, newsprint, Douglas fir, and red oak— were vacuum-dried and then milled for various time periods ranging up to 240 min. Assays were then made of rate and extent of hydrolysis, maximum yield of reducing sugar, and cellulose crystallinity. 

Further progress in understanding this effect, as well as others, of the physical structure of cellulose on enzymatic degradation may be expected from combining physicochemical and morphological techniques and from kinetic measurements in heterogeneous enzymatic hydrolysis, applying substrates of well-defined physical structure and isolated components of the enzyme systems. 

Figure 14. Paper chromatogram of the hydrolysis products from higher cellulose substrates by Ex-1. Developed by the descending technique for 96 hr at room temperature on Whatman No. 1 paper, using 1-butanol pyridine water (6 4 3, v/v) as a solvent (S) standard, (Gt) glucose, (Gz) cellobiose, (Gs) cellotriose, (Gu) cellotetraose, (G5) cellopentaose, (G6) cellohexaose final enzyme concentration 2.82 X 10 2%.

This session deals with recent progress on pretreatment of lignocellu-losic biomass, the peripheral reactions associated with pretreatment, and assessment of the effectiveness of pretreatment by enzymatic hydrolysis. Pretreatment is an essential element of the integral bioconversion process, and its objective is to enhance the susceptibility of cellulosic substrates to the action of cellulase enzymes. 

In the present study, we quantified the degree of inhibition on both P-glucosidase and cellulase mixtures by glucose and cellobiose at different concentrations. We also determined the inhibitory effects of mannose, galactose, and xylose on both P-glucosidase and cellulase activities and assessed the potential to increase the final sugar concentration by supplementing cellulosic substrate hydrolysis with hemicellulose-rich stream-obtained from steam exploded softwood (prehydrolysate). 

Grafting and hydrolysis proceed as efficiently for cellulosic substrates as previously found for starch and water soluble polysaccharides. The grafted and hydrolyzed cellulose-based products prepared by the methods described, are of potential technical interest for water retention in various applications. 

Various authors have shown that non-ionic surfactants have a beneficial effect on the hydrolysis of cellulosic and lignocellulosic substrates, whereas anionic and cationic surfactants alone interfere negatively (Castanon and Wilke, 1981 Helle et al, 1993 Park et al, 1992 Ooshima et al., 1986 Traore and Buschle-Diller, 1999 Ueda el al., 1994 Eriksson el al., 2002). Increases in the amount of reducing soluble sugars and substrate conversion were reported. The effect depends on the substrate and is not observed for soluble substrates, such as carboxymethylcellulose or cellobiose. Nonionic surfactants increased the initial rate of hydrolysis of Sigmacell 100, and when they were added later in the process they were less effective (Helle et al, 1993). They same authors found also that the addition of cellulose increases the critical micelle concentration of the surfactant, which indicates that the surfactant adsorbs to the substrate. Surfactants are more effective at lower enzyme loads and reduce the amount of adsorbed protein (Castanon and Wilke, 1981 Ooshima et al, 1986 Helle et al, 1993 Eriksson et al., 2002) which can be used to increase desorption of cellulase from the cellulosic substrate (Otter et al., 1989). Anyhow, the use of surfactants to enhance desorption of cellulases from textile substrates in order to recover and recycle cellulases was not successful (Azevedo et al., 2002b). 

One possible explanation for the effect of surfactants on cellulose hydrolysis is that surfactants adsorb to the cellulosic substrate, lower the surface... 

Application and Principle This assay is based on the enzymatic hydrolysis of the interior (3-1,4-glucosidic bonds of a defined carboxymethyl cellulose substrate at pH 4.5 and at 40°. The corresponding reduction in substrate viscosity is determined with a calibrated viscometer. 

Figure 4. Effect of pH on enzymatic hydrolysis of charged and uncharged cellulosic substrates (12), CMC = Carboxymethylcellulose...

The susceptibility of cellulose to enzymatic hydrolysis is determined largely by its accessibility to extracellular enzymes secreted by or bound on the surface of cellulolytic microorganisms. Direct physical contact between these enzymes and the cellulosic substrate molecules is an essential prerequisite to hydrolysis. Since cellulose is an insoluble and structurally complex substrate, this contact can be achieved only by diffusion of the enzymes from the organism into the complex structural matrix of the cellulose. Any structural feature that limits the accessibility of the cellulose to enzymes by diffusion within the fiber will diminish the susceptibility of the cellulose of that fiber to enzymatic degradation. In this review, the influence of eight such structural features have been discussed in detail. 

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