A method for large scale purification of turnip peroxidase and its characterization.
Purification of peroxidase has been carried out since 1960 from different sources and with different methods. Ion exchange, affinity, hydrophobic, and metal affinity chromatography are known, to our knowledge. The present method, developed in this study, is three-phase partitioning, a novel technique to separate protein directly from a large volume of crude suspension. It has been observed that interfacing phase with a metal makes this technique highly selective. Turnip peroxidase purified with this method has 512 units/mg with 20.3% recovery. The natural proteins containing histidine or cystine are often purified by immobilized metal affinity chromatography. The purification of turnip peroxidase with the three-phase partitioning technique is based on immobilized metal affinity chromatography and is used for large-scale purification. The present method, described here, would prove its value in purifying an industrially important enzyme on a large scale from a crude suspension. The enzyme purified with this technique showed two bands on SDS- PAGE, which showed a molecular weight of approx. 39KD. Enzyme showed maximum purification with Cu++ metal and had a maximum activity at pH 6.0. The enzyme has an affinity towards hydrogen peroxide as its substrate in the presence of orthodianisidine as a chromogenic substrate. Enzyme activity was enhanced with calcium and magnesium, whereas sodium, potassium, and manganese inhibit the enzyme activity.
Singh
Naresh
N
Center for Biochemical Technology (CSIR), Delhi University Campus, Delhi, India. snaresh@hotmail.com
Singh
Jai
J
D000643:Ammonium Chloride / Q000494:pharmacology; D029688:Brassica napus / Q000201:enzymology*; D004492:Edetic Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009195:Peroxidase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D018517:Plant Roots / Q000201:enzymology; D019810:Sodium Azide / Q000494:pharmacology; D012969:Sodium Fluoride / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013696:Temperature; D014508:Urea / Q000494:pharmacology
D000643:Ammonium Chloride; D014508:Urea; D012969:Sodium Fluoride; D019810:Sodium Azide; D004492:Edetic Acid; D009195:Peroxidase