Cellulase enhancement

Montenecourt and Eveleigh (37), using a special agar screening technique, have also isolated a cellulase enhanced mutant (NG-14). They suggest, however, that the cellobiohydrolase and the endoglucanase biosynthesis are regulated by different controls. They base this assumption on data for the relative ratios of cellulases obtained from this mutant needed to hydrolyze different substrates. 

In one study [90], enzyme pretreatment increased colour yield without affecting fastness properties. However, pretreatment of cellulosic fibres with cellulase lowered the subsequent fixation of homobifunctional triazine reactive dyes but did not impair the fixation of other types of reactive dyes [91]. Another study suggested that the enhanced brightness of reactive dyeings was greater with triazine dyes than with vinylsulphone types when cotton was pretreated or aftertreated with cellulase [92]. 

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. 

Novo Nordisk has also introduced another application of the laccase formulation, namely for stonewashing in combination with cellulase. It enhances the abrasion effect. In this case, a lower dose of the enzyme/mediator slurry is sufficient. Low doses have a mild bleaching effect though the end-result doesn t look like bleaching. Instead the denim looks as if it has been subject to more abrasion. Cellulases can give a stonewashed look to jeans but they have certain limitations when used on their own. If denim finishers want a highly abraded effect, they usually prefer a combination of cellulases and stones to obtain the desired level of abrasion. With the laccase slurry stones are no longer needed. 

Chakrabarti, A.C. and Storey, K., Enhanced glucose production from cellulose using coimmobilized cellulase and beta-glucosidase, Appl. Biochem. Biotechnol., (1989) 22, 3, 263-78. 

Cell-surface BI cellulase is envisaged as the form which is active against cellulose in peas in vivo, with a function that may be constructive in that it can act synergistically with plasma membrane-bound / -glucan synthetase complexes to enhance the rate of cellulose deposition (7,8,9). BS cellulase never appears to reach the wall in vivo in a form recognized by a BS antiserum (II). BS cellulase does not even bind readily to wall material in homogenates (Table III) despite its ability to bind to cellulose (3) and hydrolyze it (Table I). It is possible that BS cellulase functions intracellularly to hydrolyze a noncellulosic organelle-bound polysac-... 

There is still much to be learned about the structure and mechanism of action of this class of enzymes. Their mode of attack in terms of gross effects on substrates is now fairly well understood, especially in the cellulases, and this has resulted in a clearer classification of the purified components of the cellulase system. In order to explain the catalytic effects at a molecular level, it will be necessary first to obtain more information on the primary and, eventually, tertiary structures of the enzymes. The molecular mechanism, defined as a description of the number and structures of intermediates lying on the reaction path (6), then can be fully identified and from this the origin of the observed catalytic rate enhancements can be sought. 

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. 

This session deals with recent progress on pretreatment of lignocellu-losic biomass, the peripheral reactions associated with pretreatment, and assessment of the effectiveness of pretreatment by enzymatic hydrolysis. Pretreatment is an essential element of the integral bioconversion process, and its objective is to enhance the susceptibility of cellulosic substrates to the action of cellulase enzymes. 

Furthermore, enzymatic hydrolysis of the corn stover treated under optimal AFEX conditions showed almost 98% glucan conversion and 80% xylan conversion vs 29 and 16% for untreated com stover, respectively (at an enzyme loading of 60 FPU/g of glucan). Unlike acidic pretreatments, AFEX does not generate sugar monomers. The cellulase mixture in our study was developed for hydrolysis of acid-pretreated materials and has about 1% by weight xylanase activity (J. Cherry, personal communication, Nov. 2002). Enzyme cocktails with enhanced xylanase activity would presumably completely hydrolyze AFEX-treated xylans. 

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