THREE PHASE PARTITIONING FOR THE RECOVERY AND PURIFICATION OF PROTEASE FROM BACILLUS SUBTILIS STRAIN-24 ISOLATED FROM KINEMA AND ITS APPLICATIONS

Abstract

The primary objective of the present study was to isolate, purify and characterize the protease from Bacillus strain-24 and find its application on protein hydrolysis and destaining of cloth. The dialyzed enzyme was subjected to three phase partitioning (TPP) and different parameters such as ammonium sulphate (30-90%, w/v), crude extract to tert-butanol ratio (1.05-1.5, v/v), pH (6-9) and temperature (10-37⁰C) were varied. Protease activity and protein content were used to determine the purification fold and % yield for both lower aqueous phase and interfacial phase. The TPP purified protease was studied for general characteristics. Furthermore, the protease was subjected to milk protein hydrolysis at different E/S ratio, the results were compared with commercial enzyme trypsin including degree of hydrolysis, and antioxidant activity. The blood destaining assay on white cloth pieces were also conducted. The protease was found to be concentrated in the lower aqueous phase in step 1 TPP with a 30% ammonium sulphate concentration, a crude extract to tert-butanol ratio of 1.0:075 (v/v), pH 8 and temperature (20⁰C). In step 2 TPP, the enzyme was found in interfacial phase with a 50% ammonium sulphate precipitation and lower aqueous to tert-butanol of 1.0:075 (v/v) giving the 3.05-fold purification and 53.7% yield. The partitioned enzyme showed maximum activity at 50⁰C and pH 8.0, was active over different substrate, with the highest activity on casein. The protease activity was significantly enhanced by Zn++, Ba++, Ca++, Na+ and largely inhibited by Hg++ and retained activity in the presence of different solvents. The protease was stable with different surfactants however, the presence of SDS, PSMF and EDTA reduced the activity. The enzyme exhibited the Km and Vmax values of 1.153mg/ml and 1.143 μmole/ml/min respectively. The protease showed 29.5% and 26.802% ABTS radical scavenging activity at E/S ratio 10% and 5%. Similarly, 7.57% and 20.3% DPPH radical scavenging activity at E/S ratio 10% and 0.25% and 0.07 and 0.0361 reducing power at E/S ratio 10% and 5% respectively in whey and casein hydrolysate. The protease also demonstrated strong blood destaining capability showing its potential for use in commercial detergent. Thus, these finding indicate the protease has enormous application.