PNPG glucosidase

Since protein adsorption to an anion exchange resin is reversible and does not constitute a classical immobilization, the ability of the resins to retain activity under various conditions must be determined. Macrosorb KAX DEAE bound -D-glucosidase was tested with solutions of primary interest for their final application. Several batch washes of a 1% w/v slurry were required to ensure complete equilibrium elution for a given concentration, as determined from the absence of pNPG units in subsequent washes. Several salt solutions of typical fermentation media components were tested. These included 3 mM to 50 mM solutions of MgSO, KHgPO, NaQ, and sodium acetate. Also, incubations with cellulase solutions were tested to determine if the proteins present in a cellulose hydrolysis would displace the -D-glucosidase. Both of these displacement studies were carried out at 22°C and 40 C. 

Figure 4. Conversion of 50 g/L a-cellulose to glucose ly Genencor GC 123 cellulase supplemented to various ratios (r = pNPG U at 45°C/IFPU at 45 C) of free and immobilized -D-glucosidase. Nonsupplemented cellulase is included as a control Reactions were carried out in mixed tube with 10 mM sodium acetate pH 4.8 at 45°C.

Km, Ku and Vmax values for the secreted BglA were obtained from Lineweaver-Burk plots (Table 2). The Km value with pNPG as substrate at 40°C and pH 6.0 was 0.762 mM, higher than that (0.35 mM [3]) reported for the native P-glucosidase of the same fungus. However, the Km value with cellobiose as substrate in the range of 0.04—1.0 mM was 0.31 mM,... 

Fig. 2. Degree of Inhibition on (3-glucosidase activity caused by addition of glucose during hydrolysis of cellobiose and pNPG.

P-Glucosidase activity against />-nitrophenyl-P-D-glucopyranoside (pNPG) was determined by adding 0.1 ml of enzyme solution to 4.9 ml of citrate-phosphate buffer (0.1 M) containing pNPG (5 mM final) [4]. 

The activities on CMC, KC-floc, and PNPG of purified enzymes were determined as shown in Table 2. 3-Glucosidase 3II had a little activity on CMC, but 31 had none. Cellulase CV had a quite high activity on KC-floc, which was one-quarter of that on CMC, CII had some activity on PNPG. 

When Arbutin, MeG, Cellobiose and Salicin served as inducers, the 3-D-glucosidase activity (PNPG used as substrate) was localized in the cytoplasm and in the periplasm. With CMC and MC as inducers the enzyme activity was found mostly in medium. The enzyme(s) formed after induction with different inducers possess the same electrophoretic mobility in the 7,5% polyacrylamide gel. The possibility that the enzymic activities are... 

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