Abstract

PURIFICATION AND CHARACTERIZATION OF PECTINASE (PECTIN METHYL ESTERASE) FROM APPLE POMACE

Niharika Sood*, Abhishek Mathur

ABSTRACT

Pectinases have extensive applications in extraction, clarification, and cloud stabilization of fruit juices, in degumming and retting of natural fibers. In the present study, Pectinases/ Pectin Methyl Esterase (PME) was purified from apple pomace by consecutive steps of enzyme extraction and further precipitated by saturated concentration of ammonium sulphate. The results confirm that the enzyme can activated at 45 to 55 °C and work well at a pH of 4.5 to 5.5. The enzyme was further purified and characterized by HPLC. The purified pectinase showed the retention time, 1.812 minutes with reference to standard pectinases which showed the retention time, 1.790 minutes. Purified pectinases were further utilized to observe the degradation of pectin in pectin-agar plate. The zone of degradation of pectin by pectinases was observed by overlaying 1% Iodine solution prepared in 95% ethyl alcohol. Purified pectinases degrade the pectin in pectin-agar plates in comparison to control. The enzyme purified was loaded on agarose gel and further stained with ruthenium red and coomassie brilliant blue separately. The enzyme purified showed molecular weight 43 kDa as compared to standard marker. The enzyme purified was qualitatively assessed for pectin degradation in fruits. It was observed that in case of test, orange slice incubated with pectinases/PME, the pectin degrades and converts the orange slice into juicy lump in comparison to control, in which orange slice was treated with distilled water but not with enzyme. The juice/extract was purified and viscosity was determined. It was found that enzyme treated sample (test) was more viscous in comparison to control. The protein content in pectinases was determined by Bradford method. It was found that total protein content per gram of apple was 1.7 g/apple sample. The antimicrobial activity of purified pectinases was determined by well diffusion method. The results confirmed the strong antimicrobial potential against all the pathogens studied. The results were found to be more surprising when the purified enzyme showed the maximum activity against MRSA amongst all the pathogens studied.

Keywords: Pectin, apple pomace, Pectin Methyl Esterase (PME)/Pectinase, protein content, electrophoresis, HPLC, antimicrobial activity.