Cellulase activities

In the treatment of cellulose pulps one essential criterion for a suitable enzyme preparation is that its cellulase activity should be as low as possible, or preferably absent completely. As even extremely low cellulase activities may ruin pulp quality, Trichoderma enzyme preparations are unlikely to be suitable for these applications. Many bacterial and fungal enzymes with low cellulase activity have been shown to be suitable for treatment of pulps 14, 15, 16,17), Regulation of the often synchronous production of cellulolytic and hemicellulolytic enzymes in micro-organisms is not well understood, and is further complicated by substrate cross-specificity of these enzymes. Enzymes with both endoglucanase and xylanase activity have been reported for bacteria 18, 19) and fungi 20, 21, 22), In addition to selection of strain and... 

The degree of polymerization (DP) of pulp treated with a cellulase-free xylanase was found to increase, apparently due to the selective removal of xylan, which has a lower DP (47). TTius, treatment of pulps with xylanases has been shown to increase their viscosities (27, 48, 49). However, even low cellulase activities in the enzyme preparations result in decreased viscosity. 

The titers of cellulase activities found in anaerobic digesters, when compared to the few other "hydrolytic environments" for which analytical data are available, are strikingly low. Table III shows such values for filter paper and carboxymethyl cellulose degrading activities. This evidence seems to indicate that the cellulose degrading enzymes in ... 

Table III. Comparison of Cellulase Activities found in Selected Natural and Conunercial Systems ...

Assays for endo-l,4- -glucanase [EC 3.2.1.4] (i.e., CMCase) and saccharifying cellulase (i.e., international filter paper U, IFPU) activities partially followed the methods recommended in the 1987 lUPAC report (65). When even undiluted enzyme samples fail to give the required glucose yield under prescribed assay conditions, the lUPAC committee recommends a less precise method. In the current study, cellulase activities in digester extracts were so low that the CMCU could only be defined as follows one CMC unit of activity was that amount of enzyme required to liberate one Hg glucose from CMC in 60 min. 

Both digester systems exhibit extremely low levels of detectable cellulase activities (exoglucanase, endoglucanase, and -glucosidase) when compared to industrial saccharifying processes (See Table III) in which the hydrolysis of cellulose in the feedstock is optimized with respect to enzyme loading. Therefore, the data indicate the level of improvement that may be made to attain maximum rates for cellulose hydrolysis in the anaerobic reactor system. 

Wheat straw. Wheat straw ground to 20 mesh was treated with 2% NaOH solution (wt/vol) in 1 2 (solidiliquid) ratio at 121 C for 0.5 h (i.e., 4 g NaOH/100 g wheat straw). Trichoderma reesei QMY-1 was grown on pretreated wheat straw in SSF as well as in LSF under otherwise identical culture conditions. The SSF was carried out with full nutrient concentrations in one set and with one-half nutrient concentrations in the other set to evaluate the possible deleterious effects of elevated osmotic pressure. T reesei QMY-1 produced FP cellulase of 8.6 lU/ml (430 lU/g cellulose or 172 lU/g substrate) in 22 days. This showed that the organism was able to tolerate the high salt concentrations required in the SSF. In contrast, when the nutrients were supplied in one-half concentration, FP cellulase activity dropped to 6.7 lU/ml (335 lU/g cellulose or 134 lU/g substrate). However, the maximum enzyme activity was obtained one week earlier (14 days) than that obtained with full salt concentrations (Table I). 

International filter paper cellulase activity unit. hiang, M. Linden, J.C. Mohagheghi, A. Grohmann, K. Himmel, M.E. AppL Microbiol TechnoL, submitted. 

Figure 2. Time course of cellulase activity accumulation in A. cellulofyticus cultures containing 15 g/L Solka Floe and various concentrations of glucose-l-phosphate.

Xylanolytic enzymes free of cellulases can be applied in the pulp and paper, textile, and food industries and in basic research. However, most microorganisms grown under natural conditions produce both xylanases and cellulases. Strategies to produce xylanolytic systems free of cellulases are elimination of cellulase activity by separation or inhibition, selection and construction of cellulase-negative strains, and finding conditions for separate production of xylanolytic systems by cellulolytic strains. 

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). 

Acidothermus cellulolyticus cellulase activity, 334,335/,341,342/ cellulase production parameters, 341,343r cellulase synthesis regulation, model development, 341-346 concentration vs. rate of cellulase synthesis, 344,345/... 

The second area of application involves the use of cellulase-free xylanases for removal of hemicellulose from pulps (10-20) and plant fibres (21). It is essential that these xylanase preparations are free of contaminating cellulase activity or damage to the cellulose fibres and consequently the product quality will result. 

I. Unlike most other cloned systems, the xylanase preparation from B. succinogenes was contaminated with cellulase activity. 

It has been proposed that the production of xylanases and cellulases is under separate regulatory control in some filamentous fungi (1). Hrmova et al. (42) reached a similar conclusion after monitoring the daily production of these enzymes in Trichoderma reesei QM 9414. Xylanase and cellulase activities followed independent production profiles during fungal growth. The same effect has been observed in batch cultures of T. harzianum. We have observed peak xylanase activity on the third day of growth whereas the cellulase activity peaked after day five or six (unpublished). 

Cellulase activity determined as activity on filter paper. c MCC = Microcrystalline cellulose. d Not determined. 

Several methods to prepare high-purity xylanases for potential industrial applications have focused on eliminating the cellulase contamination instead of purifying the xylanase components. This appears to be a very effective approach as it precludes the need for very expensive biochemical procedures and focuses, rather, on a limited number of simple steps to eliminate cellulase activity. Since the remaining materials in the enzyme preparation are essentially inert with respect to the cellulose, their presence may often be ignored. 

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... 

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