Enzymes cellobiase

Enzyme concentration may be expressed in mass units instead of molar units. However, the amount of enzyme is not well quantified in mass units because the actual contents of an enzyme can differ widely depending on its purity. Therefore, enzyme concentration is expressed as an arbitrarily defined unit based on its catalytic ability. For example, one unit of an enzyme, cellobiase, is defined as the amount of enzyme required to hydrolyze cellobiose to produce 1 amol of glucose per minute. Whatever unit is adopted for Ceo, the unit for k CEo should be the same as that of r, kmole/mVs. Care should be taken for the consistency of units when enzyme concentration is not expressed in molar units. 

Cellulose is a very robust substance. However, it must be capable of being degraded rapidly, otherwise a cellulose film would cover the surface of the earth in a short time. This degradation is carried out by different microorganisms. With the help of cellulases, they break down the cellulose chains to cellobiose. By means of the appropriate enzymes, cellobiases, cellobiose can then be broken down to the fundamental building block jS-glucose. 

Enzymes Amylase, invertase, cellobiase, desoxyribonuclease, ribonu-clease, acid phosphatase, phyta.se, pyrophosphatase apy-rase, peroxidase, protease... 

FIG. 1 Effect of different enzymes or combination of enzymes (cellulases, xylanases, cellobiases, 3-glucanases, and proteases) on starch yield in the conventional corn wet milling process. 

Much effort has been expended over the years on increasing enzyme production of T. reesei by isolation of high yielding mutants and optimizing media and fermentation conditions. Strains have been isolated that produce 2-6 times the cellulase productivity of the parent wild strain (QM 6a) in batch culture. The mutants produce higher levels of cellulase protein but the specie activity of the enzymes and the proportions of the individual components (ca. 30% endo- -glucanase, 70% o- -glucanase, and less 1% cellobiase) are similar to those of the parent. 

As currently understood, cell-free enzyme preparations that can solubilize native cellulose contain at least two types of enzyme, so-called Ci and Cx (I). A third enzyme, a / -glucosidase or cellobiase, is normally,... 

Based on the work done in our laboratory, we believe that multiple enzymes of the same type are derived from the same enzyme and potentially arise from partial proteolysis of such an enzyme (10). In previous studies, we have purified three distinct cellobiases from T. reesei which are chromatographically distinct yet kinetically similar. 

Previously, both in our laboratory and elsewhere, cellulases subjected to purification procedures were obtained from commercial sources (5,6, 8,9,10,13,39,46). Three cellobiases and several endoglucanases and cellobiohydrolases from commercial preparations were purified in our laboratory. While use of protease inhibitors in the fractionation procedures minimized proteolysis during enzyme purification, the existence of enzymes proteolytically modified, presumedly during prolonged fermentation (required for obtaining high titres for commercial production), was a source of confusion, as previously explained. Therefore, we prepared T. reesei cellulase harvested from young culture broth. This was used to carry out the enzyme purification procedures described below. 

During purification procedures cellobiase activity was monitored by measuring nitrophenol (at A42onm) release for p-nitrophenyl-/ -D-glucoside (JO). Kinetic studies and enzyme characterization were carried out using / -D-cellobiose as substrate with the product, glucose, measured with a Beckman Glucose Analyzer (JO). Assay conditions were pH 4.8 and 50°C. 

These methods for enzyme activity determination are easy and convenient for screening enzymes during purification procedures, especially for cellobiase and endoglucanase. Up to 72 of these types of samples can be run at the same time. For cellobiohydrolase it is more difficult, but it is still possible to run up to 24 samples at the same time. In this procedure, the samples in Corex tubes are continuously stirred while kept in a constant-temperature water bath. After incubation the tubes are removed, put on ice, and 5% TCA is added to stop the reaction. The solid substrate is subsequently removed by centrifugation. 

Gel filtration of crude cellulase on Sephadex G-75 gave one cellobiase, a major HMW endoglucanase, a LMW endoglucanase, and one cellobiohydrolase (Figure 8). The purified enzyme and the crude enzyme protein was analyzed by SDS-gel electrophoresis and resulted in the gels shown in Figure 9. 

The cellobiase activity in culture filtrates of T. reesei was small relative to that of cellobiohydrolase and endoglucanase. The possibility that cellobiase of T. reesei is either an intracellular or membrane-bound enzyme was indicated by experiments in which cellobiose or other carbon sources were used as the substrate for culture growth. While cellobiose can be taken up rapidly by the fungus, very little cellulase activity could be detected in the filtrate see Table IV—cellobiose as carbon source). Furthermore, the appearance of cellobiase did not parallel the appearance of cellulase activity. Increasing culture incubation time did, however, result in increasing cellobiase activity in the filtrate. This data suggested that at least some of the cellobiase present in the filtrate might have been an intracellular cellobiase which was, perhaps, released when some of... 

These results, taken together with the data reported previously in this chapter, indicate that T. reesei produces one major endoglucanase and one cellobiohydrolase. Cellobiase-hydrolyzing enzymes are probably located intracellularly as well as extracellularly. More work is needed to determine whether or not the intracellular cellobiase is the same as the extracellular cellobiase. 

T. reesei yields one major endoglucanase and one major cellobiohydrolase. The multiplicity of these components is minimal when culture conditions are carefully controlled. The widely reported multiplicity of these components might possibly be due to post-translational modification of one endoglucanase and one cellobiohydrolase. The third major component produced by T. reesei, cellobiase, is present intracellularly as well as extracellularly. Whether or not these two enzyme fractions are the same enzyme, remains to be determined. 

In addition to catabolite repression, the cellulase enzymes themselves are subject to end-product inhibition. For example, as glucose accumulates during saccharification, it interacts noncompetively with cellobiase to inhibit further activity of this enzyme (6). Similar inhibition of endoglucanases occurs when cellobiose accumulates in a saccharification reactor (18,19,20). 

This chapter deals with three aspects of the cellulolytic enzyme system of Thermoactinomyces sp. the location of the CM-cellulase, Avicelase, and / -glucosidase (cellobiase) activities in the culture, the multiplicity of the extracellular enzyme system, and the stability of the different activities as a function of pH, temperature, and time. The results are discussed with reference to saccharification of cellulosic materials. 

Cellobiase enzyme (Novozym 188, Novo Nordisk Bioindustrials Inc., Danbury, CT) or other cellulase enzyme... 

Dilute the cellobiase-enzyme solution so that it contains approximately 20 units of enzyme per mL of solution. One unit of cellobiase is defined as the amount of enzyme needed to produce 1 ji-mol of glucose per min. 

Pour 100 mL of cellobiose solution with a certain concentration (20, 10, 5, 2, or ImM) into the reactor, turn on the stirrer, and wait until the solution reaches 50°C. Initiate the enzyme reaction by adding 1 mL of cellobiase solution to the reaction mixture and start to time. 

Concerning the enzyme acted on the substrate, some researchers considered all three components of the cellulase enzyme system (endoglucanases, cellobiohydrolases, and cellobiase). Since the kinetic behavior of the three enzyme system are different and not fully understood, simplified models were also suggested by assuming the cellulase system can be represented quantitatively by a single enzyme. 

Fungal cellulase enzyme systems capable of efficiently catalyzing the hydrolytic degradation of crystalline cellulose are typically composed of endo-acting cellulases (EGs), exo-acting cellulases (CBHs), and at least one cellobiase (1-6). The CBHs are typically the predominant enzymes, on a mole fraction basis, in such systems (7). Consequently, the CBHs have been the focus of many studies (8). The three-dimensional structure of prototypical CBHs is known (9-12) and their specificities are, in general, well characterized (13,14). However, mechanism-based kinetic analyses of CBH-catalyzed cellulose saccharification are rather limited (15,16). Studies of this latter type are particularly difficult owing to the inherent complexity of native cellulose substrates. 

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