PURIFICATION BY ION EXCHANGE CHROMATOGRAPHY AND ENZYME CHARACTERIZATION OF POTENTIAL DE-HAIRING ALKALINE PROTEASE FROM BACILLUS CEREUS LS2B

Abstract

Objective: Effort to develop an environmental-friendly oriented method of animal leather tanning, especially de-hairing process, becomes a concern of this research. The aim was to purify the alkaline protease from Bacillus cereus LS2B using a set of methods including ion exchange chromatography with DEAE Sepharose matrix. The enzyme was considered for having a significant potential in the de-hairing process in tanning industries.
Method: The step of alkaline protease enzyme purifications including enzyme productions, ammonium precipitation, membrane dialysis and ion exchange chromatography was performed in this research. The total proteins and specific activity of every fractionation resulted from ion exchange chromatography were measured in this research. Identification of molecular weight by SDS-PAGE of each fraction and measurement of optimum pH and temperature of the purified enzyme was also described.
Result: The data showed that there was difference enzyme activity in every fraction obtained from the chromatography indicated the position of the enzyme. Alkaline protease from Bacillus cereus LS2B purification using ion exchange chromatography with DEAE Sepharose matrix has resulted in 35 fractions, with each fraction containing about 1.5 ml enzyme. The research was performed with a flow rate of 1.5 ml min-1. Each enzyme fraction has different specific activity. The highest activity is shown at the 15th fraction that confirmed 64.4 U/mg. The balance condition between protein concentration and specific activity was observed at the 21st fraction (45.5 U/mg). The 21st fraction was become interesting due to the total of enzyme protein was almost same as the total enzyme activity. The correlation was considered becomes one of the indications of the effectiveness and efficiency of an enzyme purification. The result of SDS-PAGE determination showed that the 15th fraction has 3 bands of protein enzyme with a molecular weight of 72 kDa, 20 kDa, and 13 kDa. The 21st fraction has smaller protein bands, which are observed 20 kDa and 13 kDa.
Conclusion: Each fraction has dominant pure protein molecule around 20 kDa. Moreover, it is assumed that the molecular weight of alkaline protease enzyme specific protein of Bacillus cereus LS2B is 20 kDa. Optimal temperature and pH of the purified enzyme were 40°C and pH 8, respectively.