PNPG

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.

To measure kinetic parameters, hydrolysis rates were determined by varying the concentrations of pNPG (0.05-10 mM) and cellobiose (0.04-16 mM). The inhibition by glucose was evaluated with only pNPG (10 mM) as substrate, whereas the inhibitory effect of glucono-1,5-lactone was verified with both pNPG (10 mM) and cellobiose (1.0 mM) as substrates. Km, Vmax, and fC, values were calculated from Lineweaver-Burk plots. 

Activities were measured with pNPG as substrate. 

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,... 

Activity on gentibiose (P-l,6-glucoside), methyl- 3-gluco-side, p-nitrophenyl-P-xyloside, salicin, maltose, sucrose, lactose, xylan (1.0% [w/v]), Avicel (1.0% [w/v]), or carboxy-methylcellulose (1.0% [w/v]) was <1.0% of that on pNPG. 

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

Procedure Measure absorbances of each of the three PNP Standard Solutions to calculate the molar extinction coefficient. Equilibrate the PNPG Solution in a 50° water bath for at least 15 min. For active samples, transfer 2.0 mL of the Sample Solution to a test tube. Loosely stopper, and place the tube in the water bath to equilibrate for 5 min. At zero time, add 2.0 mL of PNPG Solution, and mix at moderate speed on a vortex mixer. Return the mixture to the water bath. Exactly 10.0 min later, add 3.0 mL of the Sodium Carbonate Solution, mix on the vortex, and remove from the water bath. 

For sample blanks, transfer 2.0 mL of the Sample Solution and 3.0 mL of the Sodium Carbonate Solution into a test tube, and mix. Add 2.0 mL of PNPG Solution, and mix. Measure the absorbance of each sample and the blank versus water in a 10-mm cell. 

Calculations One unit of glucoamylase activity is defined as the amount of glucoamylase that will liberate 0.1 xmol/ min of / -niirophcnol from the PNPG Solution under the conditions of the assay. 

Tabk L Kinetic constants of the wild type and substituted ft-galactosidases with ONPG and PNPG as substrates... 

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]. 

Micro-organisms pH optimum Temperature optimum (°C) Km pNPG (mM) Ki glucose (mM) Molecular mass (kg/mol) monomer (kg/mol) pHi Ref... 

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