Tuesday, 6 January 2015

Immobilization of Yeast Cells


Objective

To Immobilize active Yeast Cells in the Calcium Alginate Gel and to check the viability of cell by 
invertase activity


 
Introduction

The term ‘immobilization’ was first proposed at the first enzyme engineering conference in 1971.
 Immobilization often causes a dramatic change in the apparent measuring parameter of the enzyme
 catalyzed by Michalis – Menton constant, temp optima, pH optima, and effect of inhibitors may be 
changed when an enzyme in immobilized. The degree and nature of these changes not only depends
 on the immobilization but also on the enzyme reaction.
There are various methods available for immobilization of enzyme
  • Absorption
  • Covalent binding
  • Cross matching
  • Micro encapsulation
  • Polymerization
  • Gel entrapment
Advantages of enzyme- immobilization
  • Stability of the enzyme immobilization increases even in adverse condition.
  • Resistance of enzyme molecules against metal ions and other inhibitors can also be increased.
  • Enzyme can be used repeatedly and continuously for the conversion of substrate into products.
  • In immobilized condition enzyme can be stored for longer time
Requirement
  • Yeast potato Dextrose (YPD)/ Potato Dextrose Agar (PDA) medium (for cultivation of Yeast cells)
  • Sodium alginate solution (2.5% w/v in 0.1% NaCl)
  • Calcium Chloride (CaCl2 ) solution (0.05 N)
  • Gluteraldehyde (2.5% v/v)
  • Sucrose solution (1% w/v)
Procedure for immobilization of Yeast Cells
  • Grow the yeast cells in YPD/PDA medium
  • Keep it in shaker for 24hours at 100 rpm
  • filter out the yeast cells with the help of Whattman filter paper
  • Take 20 ml sodium alginate solution and add 3 ml yeast cell in it. Mix properly and incubate at room temp for 30 min. then add 3 ml of gluteraldehyde solution incubate at room temperature for 90 min. With the help of 10 ml pipette, drop wise add this mixture into the beaker containing 100 ml CaCl2 solution
  • Filter out the beads with the help of normal filter paper
  • Wash the beads 2-3 times with sterile D/W
  • Load these beads into thoroughly washed glass column/ beaker
  • Add 50ml of sterile 1% sucrose solution
  • After every 30 min interval collect the 1 ml of sample and estimate the amount of glucose by using Dinitro salilcylic acid method (DNSA) method
Procedure for Estimation of Glucose by Dinitro salilcylic acid method (DNSA) method
  • Prepare a standard solution of carbohydrate (here glucose) having concentration of 1.0 mg/ml
  • Take different volumes of glucose solution like 0.5, 1.0, 1.5 & 2.0 ml etc. into various tubes previously labeled as S1, S2, S3, S4 etc. respectively
  • One tube should be labeled as blank
  • Now, take three tubes and labels as U1, U2 & U3 & pipette out 3 different volumes of sucrose solution from reaction mixture from above experiment in this tube
  • Add distilled water in all tubes in such a way that the total volume will be 2.0 ml
  • Add 2.0 ml of DNSA reagent in all tubes. Mix it properly by reversing the tubes or by using magnetic stirrer
  • Keep all the tubes in boiling water bath for 10 minutes. Then allow it to cool down
  • Take absorbance at 520 nm (using green filter) and plot a standard curve
  • Calculate out the concentration of glucose produced in the reaction mixture of above experiment

Immobilization of Yeast Cells


Result
Increase in the concentration of  glucose from the sucrose with respect totime by Active yeast cell (Invertase) indicates that Yeast Cells immobilized and they are viable

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