Cellobiases

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. 

Cellobiase can be measured by following the glucose production from cellobiose and cellodextrins, the saligenin from salicin, and the p-nitrophenol formation from its / -glucoside (2). 

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. 

Figure 8. Sephadex G-75 column chromatography of crude celluloses. (-O-) Protein (A 0im), (-X-) activity with respect to CMC (endoglu-canase), (- -) activity with respect to Avicel (cellobiohydrolase). Inset, top right (-O-) cellobiase activity.

Figure 9. SDS-gel electrophoresis of purified celluloses from T. viride grown in Avicel and lactose. (a) Crude celluloses (Avicel as growth medium), (b) crude celluloses (lactose as growth medium), (c) purified LMW endoglucanase (Avicel), (d) purified HMW endoglucanase (Avicel), (e) purified HMW endoglucanase (lactose), (f) mixture of (d) and (e), (g) purified cellobiohydrolase (Avicel), (h) purified cellobiohydrolase (lactose), (i) mixture of (g) and (h), (j) purified cellobiase.

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... 

Carbon Source Growth0, /Dry weight, gm 200 mL culture J % Cellobiase" Activity % Cellulase Activity... 

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. 

Preliminary experiments done in our laboratory showed that antibodies specific for cellobiohydrolase failed to cross react with either purified cellobiase, purified endoglucanase, or crude endoglucanase. These results, together with data reported in the literature, which show that endoglucanase and cellobiohydrolase have different physical structures, indicate that the three cellulases could be transcribed and translated by different genomes. In this context, then, the question arises as to whether cellulase production is regulated by a common regulatory circuit or by different circuits. 

When endoglucanase (C in Table VII), cellobiohydrolase (Ci), and cellobiase are combined and incubated with Avicel, a synergistic effect is observed see Table VII). Cellobiase and endoglucanase have... 

---