Cellulase inactivation

Selective inactivation of cellulase activities is another method which has been applied to fungal culture filtrates to produce cellulase-free xylanase preparations. Cellulase inactivation of a crude enzymatic complex was achieved by Barnoud et al. (15) using a 1 mM mercuric chloride solution. In the presence of this sulfhydryl binding metal, complete inactivation of endocellulases was observed whereas the xylanases retained 80% of their activity. 

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... 

Isolation of mutations increasing pectinase synthesis led to the characterisation of the pecS locus (67). Inactivation of pecS results in derepressed synthesis of pectinases, of the cellulase CelZ, of the secretion machinery and of an extracellular insoluble blue pigment (Figure 4). 

Cellulases can also be eliminated fiom a mixture with xylanases by selective thermal inactivation. Cellulases are more thermolabile than xylanases in tiie cdlulolytic systems of the fungus Y-94 (79), T. harzianum 20), and Tkermoascus aurantiacus (77), but not in the Trickoderma reesei system (Biely, P. and Vrsanska, M., Slovak Academy of Sciences, Bratislava, unpublished results). Since cellulase thoixud inactivation causes a significant loss of xylanase also, a more convenient way to eliminate cellulase activity is by selective chemical or biological inhibition or inactivation. There appear, however, to be no reports on the existence of natural inhibitors that would be specific for cellulases. Such inhibitors of amylases and pectinases are known to occur in plants (27). 

Further progress can be expected in the area of selective inhibition and inactivat-iem of cellulases undesirable in xylanase preparations. There is a possibility of finding natural selective cellulase inhibitors or developing highly reactive derivatives of cello-biose and cellodextrins that inactivate cellulolytic enzymes. 

Oxidation of two out of 13 tryptophan residues in a cellulase from Penicillium notatum resulted in a complete loss of enzymic activity (59). There was an interaction between cellobiose and tryptophan residues in the enzyme. Participation of histidine residues is also suspected in the catalytic mechanism since diazonium-l-H-tetrazole inactivated the enzyme. A xylanase from Trametes hirsuta was inactivated by N-bromosuc-cinimide and partially inactivated by N-acetylimidazole (60), indicating the possible involvement of tryptophan and tyrosine residues in the active site. As with many chemical modification experiments, it is not possible to state definitively that certain residues are involved in the active site since inactivation might be caused by conformational changes in the enzyme molecule produced by the change in properties of residues distant from the active site. However, from a summary of the available evidence it appears that, for many / -(l- 4) glycoside hydrolases, acidic and aromatic amino acid residues are involved in the catalytic site, probably at the active and binding sites, respectively. 

Index Entries Amylase cellulase immobilization inactivation waste-paper. 

Immobilized cellulase and amylase are able to hydrolyze cellulose and starch. However, the immobilized enzymes possess only about 1-6% of the activity of the soluble forms. In addition, immobilization clearly enhanced the thermal stability of amylase. Immobilized amylase retained more than half of its activity, even after incubation at 125°C. By comparison, soluble amylase was almost completely inactivated under these conditions. Furthermore, kinetics modeling indicates that the susceptibility to product inhibition is dependent on the amylase source. Finally, immobilization can reduce the susceptibility to product inhibition fQ was less for each of the immobilized forms, compared with their soluble counterparts. 

Reese, E. T. 1980. Inactivation of cellulase by shaking and its prevention by surfactants. /. Appl. Biochem., 2,36-39. 

Cellulase action can be inhibited or inactivated by several classes of compounds, including strong oxidants or reducing agents, metal ions, salts, solvents, and surfactants. The binding of the enzyme to cellulose can protect the enzyme from these compounds, so it is not easy to generalize the relationship between concentration and degree of inhibition (which is concentration dependent) or inactivation (which is time dependent). 

A number of commercial cellulases, samples from other workers, and acetone precipitated powders were compared (Table II). All of these demonstrated good activity on carboxymethylcellulose. In fact a number of these, notably the enzyme from Poria, were more active on CMC than the T. viride enzyme. About half of the enzymes showed good activity on filter paper. But T. viride enzymes show the highest activity on cotton, the most resistant substrate. In case inactivation at 50 °C. affected the activity on cotton and filter paper, these enzymes were also tested for hydrolysis at 25°C. (data not shown). The relative activities were the same as those found at 50°C. Selby (31) made a similar study of a number of cellulase preparations. He also found that the cellulase of Trichoderma viride had the highest activity on cotton. 

Enzyme Studies. (1) Effect of Temperature and pH on the Inactivation of Cellulase. Details of experiments and results on the effects of temperature on the inactivation and adsorption of Tv cellulase, effect of pH on adsorption, activity, and inactivation of To cellulase, effect of enzyme concentration on saccharification of cellulose, and glucose inhibition of saccharification of cellulose have been reported in a separate communication (3). These data show that small enzyme losses (up to 10%) are difficult to detect. Tv cellulase alone does not appear to lose... 

Loss of Cellulase (Cx) Activity in the Presence of Highly Susceptible Substrate and Absorbent. Temperature and pH affect the inactivation and adsorption losses of T. viride cellulase in contact with Solka Floe and milled Solka Floe (3). Since several saccharification studies were conducted with highly reactive cellulose (Sweco 70 heated), a knowledge of the difference, if any, between adsorption and other losses of T. viride cellulase in contact with such substrates (at 50°C., pH—4.8) was necessary. Accordingly, T. viride cellulase was incubated with Sweco... 

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