Procedure:-

• Assay of the enzyme activity – Invertase activity in the free and immobilized biocatalyst is determined by taking a known quantity of cells in a test tube and adding 5 mL of 0.1 M Sucrose solution (in 0.05 M acetate buffer, pH 5.0) and incubating at 300C in shaking water bath, the reaction is stopped after 2 minutes by adding 0.2 mL of 0.5 M Na2CO3. The reducing sugars (Glucose and fructose) formed is estimated by DNS method against a blank (reaction is terminated at zero time) Express enzyme activity in terms of I.U. mg-1 cells.

• Entrapment of permeabilized yeast cells (PYC) in calcium alginate beads. Mix a known quantity of PYC into a 2 % (w/v) solution of sodium alginate to get a 10% w/v slurry of cells. The mixture is pumped into a stirring 0.5 M Cacl2 solution for half an hour before use.

• Experimental approach Design suitable experiment (s) to collect following data to enable characterization of the IMB.

         a)    Reactor space time

               VR = Volume of the reactor
               Q = flow rate through the reactor

         b)   Fractional Conversion

               So & S are the inlet and outlet substrate concentrations respectively.

         c)    Reactor productivity

               Pr =  amount of product produced per unit reactor volume.

         d)   Reactor capacity         

CR = Vm  Є , VR

               Vm = maximum reactions rate
                      = k2 E0, k2 = rate constant
                 E0 = enzyme concentration in the reactor Є is porosity of the reactor Є is defined as the ratio of fluid volume to the total volume of the reactor (VR) Є may
                          vary from 0.5 to 0.7 in the case of plug flow reactor & is near 1.0 for a CSTR.

         e)    Yield of immobilization.

         f)     Kinetic parameters for free and immobilized biocatalyst.

         g)   Reactor performance in terms of S, X, T and Pr.

         h)   Operational stability.