MEASURING THE EFFECT OF DIFFERENT PARAMETERS ON THE CATALYTIC ACTIVITY OF IMMOBILIZED β-GALATOSIDASE FROM YEASTAbstract
Background: Enzyme catalysis is intermediate between homogeneous and heterogeneous catalysis. In general, it is similar to inorganic heterogeneous catalysis and sometimes it is called micro heterogeneous catalysis. Efficient enzyme catalysis depends on high specific nature of enzyme, pH, temperature, optimum temperature, colloidal nature, activator or co-enzyme, inhibitors or poisons. Immobilized β-galatosidase show high activity towards both pure lactose and lactose in skim milk, and a better thermal stability also.
Methods: This study was done in CDRI, Lucknow. In this study β-galatosidase was extracted by Kluyveromyces lactis and enzyme assay done by GOD/POD method. Partial purification of enzyme was done by precipitation with ammonium sulphate, DEAE-cellulose column chromatography followed by SDS-PAGE. Immobilization of K. lactis cells was done by Entrapment method in which we agarose beads, plyacrylamide beads and alginate beads were used to analyze the catalytic activity of β-galatosidase on different parameters.
Results: In this study the effect of temperature, pH, and amino acids on normal β-galatosidase and on immobilized β-galatosidase were analyzed. Immobilized enzyme showed the decreasing conversion level of lactose. We used three types of beads to analyze the activity of enzyme in which polyacrylamide beads showed decreasing conversion level of lactose at different concentration of lactose and same concentration of beads. Effect of temperature on β-galactosidase was maximum at 40oC for growth. pH profile (6.2 to 7.4) of β-galactosidase was studied at 28oC & 37oC. Highest lactose conversion (4.44%) was noted at pH-6.8.
Conclusion: It was found that the β-galactosidase from K.lactis showed an optimum activity at temperature 40oC, pH 6.8. The extracted sample was loaded in to the SDS page along with the marker. The fine band was observed. This showed the purity of the β-galactosidase. Permeabilization of the cell wall with chloroform increased the enzyme activity. The immobilized non-viable permeabilized cells give better result than viable immobilized cells.
Sandhya Hora*, Manoj Gupta and Mamta Gupta
Central Research Laboratory, Department of Biochemistry, Shri Ram Murti Samarak-Institute of Medical Sciences, Bareilly-243202, Uttar Pradesh, India
25 August, 2012
26 September, 2012
28 November, 2012
01 December, 2012