Spezyme Cellulase

Enzymes were immobilized onto silica gel by covalent crosslinking with glutaraldehyde in a procedure similar to that of Kondo et al. (3). Briefly, 1-4 g of silica gel was incubated in 1-13 wt% glutaraldehyde and in 100-1000 mL of enzyme solution for up to 48 h. The immobilized enzyme was recovered by filtration and washed to remove loosely bound enzyme. Samples of the soluble enzyme were collected before and after immobilization and assayed for activity, to provide an estimate of enzyme uptake onto the support. The a-amylases studied were Spezyme Fred (Genencor), Allyzme (Alltech), and Liquozyme (Novozymes). The cellulases studied were Spezyme CP and Spezyme CE (Genencor). 

Figure 5 illustrates the performance of immobilized cellulase in comparison with that of soluble cellulase. The small difference between the "crude" and "used" Spezyme CP profiles illustrates the very low uptake of soluble cellulase onto the support. Furthermore, soluble cellulase is much more active the dose of soluble enzyme is approx one-twentieth that of immobilized cellulase, once enzyme uptake is taken into account. Nonetheless, it is important to note that the immobilized enzyme is able to hydrolyze this insoluble substrate, albeit at a much lower rate. 

Fig. 5. Comparison of cellulose hydrolysis with soluble and immobilized cellulase. "Crude" and "Used" designations refer to the soluble enzyme collected before and after immobilization, respectively. CP, Spezyme CP.

Spezyme CP was loaded at 5 FPU/g cellulose for cellulase-only digestions and 2.5 FPU/g cellulose when assayed with P-glucosidase present, p-glucosidase was added at 15 CBU/g cellulose. Flasks were preincubated at 45 °C on the rotary shaker at 150 rpm for 10 min, and... 

To evaluate the practical application of immobilized (J-glucosidase for hydrolysis of pretreated com stover, we have carried out enzymatic hydrolysis in flasks with the use of cellulase and immobilized 3-glucosidase. Taking into account the fact that Spezyme CP contains a certain quantity of p-glucosidase, we used reduced quantities of Spezyme CP in our experiments—5 U/g of glucan instead of normally used 15 0 U/1 g of glucan. This allowed us to better evaluate the effect of immobilized 3-glucosidase in the process of hydrolysis of com stover. The obtained results are presented in Fig. 4. 

Considering that the microorganism source for the Spezyme CP enzyme preparation is also H. jecorina, our results using a different approach confirm previous work showing that low molecular weight phenolic compounds are inhibitory to the action of cellulases from H. jecorina. Vohra et al. [35] demonstrate that at least some of H. jecorina cellulases are inhibited in the presence of different concentrations of ferulic acid. H. jecorina itself does not produce feruloyl esterase activity [12], so it may not be surprising that the cellulases do not seem to act in synergism with feruloyl esterases and are inhibited by the esterase... 

A commercial preparation produced by Trichoderma reesei (Spezyme CP) and supplied by Genencor (Rochester, NY, USA) was used as the cellulase enzyme in this study. This preparation was supplemented with a (3-glucosidase preparation (Novozym 188) produced by Aspergillus niger and supplied by Sigma (C6105). Various commercial xylanase preparations were kindly provided by various North American suppliers, and their cellulolytic and hemicellulolytic activities were evaluated (Table 1). BioCat xylanase was in powder form and was suspended in 100 mM sodium acetate buffer (pH 5) at a concentration of 10 mg/ml before use. 

Cellulase (Spezyme CP, Lot No. 301-00348-257, Genencor, Palo Alto, CA) at 28 FPU/mL supplemented with P-glucosidase (Novozyme 188 at 250 P-glucosidase lU/mL, Sigma, St. Louis, MO) at a ratio of l 1.75FPUase CBUase was used for all hydrolysis experiments. Enzymatic Ueatments were performed at 60 FPU/g cellulose with the activity as calculated by adding the activities of both the Spezyme CP and Novozyme 188 (66),... 

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