Cellobiose hydrolysis rate

Hydrolysis of delignified wheat straw up to 90% was obtained in 96 hours with the cellulase system produced in SSF with wheat straw. It took only 72 hours to obtain over 90% hydrolysis of wheat straw with cellulase system produced with SSF on Pro-cell (Table VII). It is interesting to note that the quantity of cellobiose in the hydrolysate obtained with the cellulase system produced on Pro-cell was higher than that of the cellulase system produced on wheat straw. This is consistent with the observation that cellulase system produced on Pro-cell had a lower ratio of FP cellulase )3-glucosidase (1 0.77) as compared to that produced on wheat straw (1 1.2). However, this cellulase system had a faster hydrolysis rate it took only 72 hours to obtain over 90% hydrolysis. This might be related to cotton hydrolyzing activity of this cellulase system (Table V). 

There are two reasons for the measurable cellobiose concentration in the T. viride cellulase hydrolyzed syrups. The most likely is that T. viride has rather poor / -glucosidase activity so that cellobiose accumulates. Evidence of this is that additions of / -glucosidase to the T. viride cellulase improves its activity. A second reason is that the / -glucosidase enzyme is strongly glucose inhibited. Hence the rate of cellobiose hydrolysis slows down as the glucose concentration rises, allowing cellobiose to accumulate. 

To measure kinetic parameters, hydrolysis rates were determined by varying the concentrations of pNPG (0.05-10 mM) and cellobiose (0.04-16 mM). The inhibition by glucose was evaluated with only pNPG (10 mM) as substrate, whereas the inhibitory effect of glucono-1,5-lactone was verified with both pNPG (10 mM) and cellobiose (1.0 mM) as substrates. Km, Vmax, and fC, values were calculated from Lineweaver-Burk plots. 

Karl-Erik Eriksson (42) reported that he had prepared an enzyme from Chrysosporium lignorum which, when combined with each of three CM-cellulase enzymes, increased the hydrolysis rate of cotton tenfold. When he incubated this enzyme, which he believed to be of the Ci type, with cellohexaose and observed cellobiose as the only product, he concluded that it was an exo-glucanase which split off cellobiose units. Later (27) Eriksson and Rzedowski reported that the three CM-cellulase enzymes from C. lignorum contained 13, 10, and 7% carbohydrate and catalyzed the hydrolysis of cotton and cellodextrins to produce cellobiose and glucose in the approximate ratio of 3 1. 

Enzymatic hydrolysis Cellulase and P-glucosidase were used Cellulose Hydrolysis rate equivalent to 100% obtained in the absence of an inhibitor (cellobiose) Ethanol Philippidis et al. (1993)... 

Freudenberg, Kuhn and their co-workers showed that both the velocity constants and the courses followed by the hydrolyses of these various polymers can be accounted for by postulating that one or the other or both of the terminal linkages, a and h of Table VIII, in these various species hydrolyze more rapidly than the internal c linkages. All of the latter can be assumed to hydrolyze at the same rate. If, for example, one of the two terminal linkages, a or 6, in an x-mer reacts at a rate equal to cellobiose, 1.07 X10, and the rate for each of the other X —2 linkages corresponds to the initial average rate, 0.305 X10, of hydrolysis of the bonds in cellulose then the calculated... 

Proceeding on the same line, Hagerdal et al. reported that perfluorinated resin supported sulfonic sites (NATION 501) can hydrolyze disaccharides [25]. In particular, these authors studied the effect of the addition of sodium chloride in the hydrolysis of cellobiose, a subunit of cellulose much more resistant to hydrolysis than sucrose. They observed that the presence of sodium chloride in water dramatically increased the conversion of cellobiose. Indeed, in the presence of 10 wt% of sodium chloride, 80% of cellobiose was converted at 95°C after 6 h. For comparison, when 1% of sodium chloride was added, only 50% of cellobiose was hydrolyzed. It should be noted that without addition of sodium chloride only 15% conversion was achieved, thus pushing forward the key role of sodium chloride on the reaction rate. Effect of salt on the reaction rate was attributed to a change of the pH caused by the release of proton in the reaction medium (due to an exchange of the supported proton by sodium). 

As expected, mesoporous silica-supported sulfonic sites were able to catalyze the hydrolysis of cellobiose. Indeed, at 448 K, 90% of cellobiose was hydrolyzed within 30 min of reaction with an apparent activation energy ( = 130 kJ moF ) similar to that of reactions promoted by homogeneous organic acid catalysts [33]. The hydrolysis reaction rate is proportional to the concentration of hydrated... 

The initial, rather rapid increase in CER found in the second stage of two-phase batch cultivation was somewhat unexpected. We decided to compare this value to the initial rates of glucose and cellobiose formation in in vitro enzymatic hydrolysis experiments (Figs. 4 and 5). The enzyme loadings were chosen to represent the actual enzyme-to-substrate ratio relevant to the point of cellulose addition and the point of maximum CER. 

The amount of mono- and disaccharides released (glucose, xylose, cellobiose, and arabinose) by pretreatment utilizing acid and enzymatic hydrolysis was analyzed by high-performance liquid chromatography (HPLC) (Shimadzu, Kyoto, Japan), using an Aminex HPX-87H column with a matching precolumn (Bio-Rad, Hercules, CA) at 65°C. The eluent was 5 mM H2S04 at a flow rate of 0.5 mL/min with detection by refractive index. 

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). 

A detailed study has been made of the action of pure triduoroacetic acid on cellulose and cellobiose (and their acetates). Dissolution of cellulose occurs, and swelling takes place with rupture of hydrogen bonds and with micellar dispersion esteridcation takes place without occurrence of degradation, the cellulose being fully recovered on hydrolysis of the triduoro-acetylated product. It appears that there is a more rapid rate of triduoro-acetylation of primary than of secondary alcohol groups. 

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