Cellulase after

Enzymatic liquefaction is a relatively new process for the production of juices from fruits and vegetables [1]. Essentially the process is as follows the material is crushed to obtun a pulp which is treated with a combination of pectinases and cellulases. After a certain incubation time, the material becomes a liquid and the Juice can be recovered by decantation. 

Azevedo, H., Bishop, D., and Cavaco-Paulo, A. 2002a. Possibilities for Recycling Cellulases After Use in Cotton Processing. Part I Effects of End-Product Inhibition, Thermal and Mechanical Deactivation, and Cellulase Depletion by Adsorption. Appl. Biochem. Biotechnol., 101, 61-75. 

Deactivation of cellulases after the desired effects have been achieved is very important. If the enzyme is not completely removed from the fabric, or is not effectively deactivated, the hydrolysis reaction will continue, although at a slower rate. As very large molecules, cellulases cannot diffuse into the crystalline parts of the cellulose fibres. They react on the fibre surface, so fibre damage takes time. But eventually enough hydrolysis will have taken place to weaken the affected fabrics or garments, leading to customer complaints and returns. 

Lemos, MA Teixeira, JA Mota, M Gama, FM. A simple method to separate cellulosebinding domains of fungal cellulases after digestion by a protease. Biotechnology Letters, 2000, 22, 703-7. 

Figure 4. Content of galacturonic acid in the soiuble fraction after hydrolysis of AIS with polygalacturonases [a], with polygalacturonases and pectin methylesterase [b], with pectinases and cellulases [c].

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). 

C. fluminea 34 Residues were 433 mg/kg DW soft parts in 30 days after feeding on periphyton containing 956-4369 mg Zn/kg DW growth reduced cellulase enzyme activity reduced 11... 

Pretreatment of Substrate. Several different lignocelluloses were pretreated with NaOH. This pretreatment partially solubilizes the hemicelluloses and lignin and swells the cellulose so that the organism can utilize it for its growth and for production of a cellulase system in SSF. The treated lignocelluloses were not washed. The NaOH treatment is done with a minimum amount of water so that, after the addition of nutrient solution and inoculum, the moisture content is less than 80% wt/wt and there is no free water in the medium. More water was added to make suspensions of different lignocellulosic substrates of the desired concentration (1% or 5%) for liquid-state (submerged) fermentation (LSF). 

A maximum FP cellulase of 6.3 lU/ml (191 lU/g cellulose or 126 lU/g substrate) was obtained on NaOH-treated CTMP after 20 days in SSF. On untreated CTMP, the FP cellulase remained about 5 lU/ml from 20 to 26 days of fermentation and then increased to 7.2 at 30 days of fermentation (Table III). This indicated that CTMP was a good substrate for cellulase production in SSF even without the mild NaOH treatment. 

The third group is that of compounds which may potentially be transported by the PTS and inhibit cAMP production. Cellulase synthesis is initiated after these compounds are consumed for cell growth. This group includes D-glucose, D-fructose, maltose, mannitol, glycerol, sorbitol, and -methyl glucoside. The presence of these compounds in Solka Floe fermentations, enhanced enzyme yields (132 to 254%) but the time required to complete cellulase synthesis took longer (106 to 148%) than the control. 

Endo-xylanases can be used industrially in two ways 1) to remove xylan from paper pulp to give purer cellulose, and 2) to convert xylan into D-xylose or xylo-oligo-sac jharides that can be further converted into useful materials. In the first case, at least, it is clear that the enzyme preparation should be free of cellulases, either by (hoosing, or producing by mutation, a strain that makes xylanases but not cellulases, or by separating the lattCT from the former after they are produced. In nearly all cases t tie use of a cellulase-firee strain is preferable. 

Figure 1. Analytical isoelectric focusing of cellulases from Trichodtrma ree-sei. Detection of CBH I and EG I activities using MeUmbLac, in the absence (A) and presence (B) of 10 mM cellobiose. Lane 1, EG I lane 2, EG I (iso-components) lane 3, CBH I (pi 3.9 component) lane 4, EG I-CBH I mixture). Gels were flooded with the fluorogenic substrate (pH 5.0) and after 5-10 min (room temperature) photographed (Polaroid 57, green filter) on a long wavelength UV-transilluminator (8).

Figure 2. Zymogram of gCenA (A-I) and ngCenA (J-Q) after incubation with C. fimi protease. Cellulases, bound to Avicel, were incubated with protease or control buffer for 72 hr at 30° C, then centrifuged to give cellulose-bound (A-E, J-N) and supernatant (F-I, O-Q) fractions. Products were separated on a SDS gel, replicated onto CMC-agarose and developed with Congo red. A,J. buffer control (4°C incubation) B,F,K,0, protease C,G,L,P, protease + PMSF control D,H,M,Q, buffer control E,I,N, buffer + PMSF control.

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). 

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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