Cellulase and

Figure 3.10 Typical screening plate for cellulase and amylase positive clones from enrichment cultures. Clear halos indicate the presence of positive clones and the halos diameters give a first idea about the overall activities of the respective enzymes.

Cellulase and all chemicals used in this work were obtained from Sigma. Hydrolysis experiments were conducted by adding a fixed amount of 2 x 2 mm oflSce paper to flasks containing cellulase in 0.05 M acetate buffer (pH = 4.8). The flasks were placed in an incubator-shaker maintained at 50 °C and 100 rpm. A Box-Behnken design was used to assess the influence of four factors on the extent of sugar production. The four factors examined were (i) reaction time (h), (ii) enzyme to paper mass ratio (%), (iii) amount of surfactant added (Tween 80, g/L), and (iv) paper pretreatment condition (phosphoric add concentration, g/L), as shown in Table 1. Each factor is coded according to the equation... 

Table 1 shows the dry weight of substrate, and amounts of HCl aqueous solution for pretreatment, cellulase and suspension broth for the lactic acid fermentation with ESS. The initially supplied amount of bean curd refuse in dry weight basis was changed from 10 to 150 g to examine the influence of substrate loading. The amount of cellulase was increased against initial substrate loading. And also, the amoimt of 0.1 mol/1 HCl was increased against... 

Total pectinase, cellulase and lipase activities secreted by colonies were detected on BSM plates containing respectively 1% of citrus pectin, 2% Walseth cellulose and 1% olive oil + rhodamine. After few days at 30°C, pectin plates were covered by 1% CTAB for Ihour, positive colonies became surrounded by a clear halo walseth plates are not stained the halo is visible directly on positive clones lipase activity is revealed under UV on oil-rhodamine plates. 

The cambium was isolated firom spruce (Picea abies) logs felled in October-November. The bark was stripped off, and the cambium was removed by gentle scraping. The isolated cambium was immediately fiozen in liquid nitrogen and fi-eeze-dried. The carbohydrate composition of the isolated cambium was analysed by HPLC after enzymatic hydrolysis using Pectinex Ultra and a mixture of cellulases and hemicellulases (Buchert et al 1993). 

The precise effects obtained are dependent on the fabric quality, the type of cellulase enzyme and the application conditions, but no mechanical forces are involved in removal of the fibrils. The process has attracted considerable attention and is now one of the main methods of defibrillating lyocell fabrics [94,101-114]. Simultaneous treatment with cellulase and protease enzymes has been applied to the biofinishing of wool/cotton blends [115]. 

The traditional method of carbonising with sulphuric acid is environmentally undesirable and can easily lead to fibre damage. Hence it is not surprising that research has been directed towards alternatives in which enzymes are used to remove the cellulosic impurities from wool. Cellulases and lignases are mainly used but others have been proposed [116] ... 

Water removal on the paper machine has been shown to improve as a result of limited hydrolysis of the fibres in recycled paper. Mixtures of cellulases and hemicellulases were found to decrease the SR-value, which describes the drainage behaviour of the fibres (45), The effect is apparently due to fibrillation or change in the composition of fine particles. 

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),... 

A literature survey indicated that very little work has been done to produce an optimal cellulase system as described above. Here, we used solid-state fermentation (SSF) to achieve this objective. SSF processes, such as the "koji" process, have been used extensively for amylase production on wheat bran in Japan its application was extended to cellulase production on wheat bran and Ugnocellulosic materials by Toyama (13), Since then, wheat bran has become an important substrate for producing various products by SSF (14-20), In this study, we tested various lignocellulosic substrates for the production of cellulase and )3-glucosidase from T, reesei QMY-1 by SSF. 

Langsford et al. reported that Cellulomonas fimi culture supernatants contained cellulase and proteinase activities, for which there appeared to be a relationship. Glucose repressed the synthesis of both activities and cellulose induced both 60), Adding cellulose to Cellulomonas sp. (NRCC 2406) cultures stimulated growth and improved production of cellulases 61). Optimum conditions for growth and cellulase production were pH 6.5 and 30 C. The addition of glucose in the presence of cellulose inhibited growth. Several species of Cellulomonas have cellobiose phosphorylase. 

Most microorganisms grown under natural conditions produce both cellulases and xylanases. Biochemistry, microbiology, and molecular biology offer several approaches to obtain xylanase prq)arations largely or completely free of cellulases (77). 

Elimination of Cellulases from Xylanases. Classical methods of protein fractionation can be used for to separate cellulases and xylanases on a large scale only when they differ considerably in molecular weight or isoelectric point. The Tricho-derma harzianum enzymes were separated by ultrafiltration because the xylanase was smaller and passed through the membrane into the ultrafiltrate 18). Fractional precipitation with organic solvents is another possibility (7). 

Selective inhibition of cellulases by Hg in a mixture of cellulases and xylanases of Sporotrichum dimorphosporum used in a laboratory experiments (22) cannot be considered for industrial application for obvious reasons. 

Selective Production of Xylanases by Cellulolytic Microorganisms. Until recently there was little information on common or separate genetic control of cellulase and xylanase synthesis in microorganisms (60). Studies on this subject were complicated by the fact that numerous microbial ceUulases and xylanases are non-specific with respect to cellulose and xylan as substrates. As could be expected from a comparison of both polysaccharide structures, non-specificity is more frequently observed with cel-lulases, because their substrate binding sites can easily accommodate substrate using an unsubstituted p-(l 4)-linked chain of D-xylopyranosyl units. 

Another method which may become a useful technique for selective inactivation of cellulases in enzyme mixtures is the use of selective heat inactivation. While establishing the thermostability properties of crude xylanases from a fungal strain Y-94, Mitsuishi et al. (80) observed differential heat labilities of the cellulase and xylanase activities in the culture filtrate. After an incubation period of 20 minutes at 65°C, the xylanase activity was reduced by 5-10% whereas the Avicelase and /3-glucosidase activities were reduced by 100% and 60%, respectively. We have observed a similar temperature dependency of xylanase and cellulase activities in T. auranti-acus. As indicated in Figure 2, treatment of the culture filtrate at 70°C for 20 minutes resulted in less than a 5% loss in xylanase activity whereas cellulase activities were reduced by 40-50%. A similar effect has also been observed for the xylanases and cellulase enzymes produced in culture filtrates from T. harzianum (93). Further work in the area of heat treatments may improve the effectiveness of cellulase inactivation. Since the cellulase activities of some enzyme preparations can be more rapidly inactivated on... 

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