ISOLATION AND CHARACTERIZATION OF ALKALINE PROTEASE SECRETED BY THE PSEUDOMONAS BACTERIUM ORIGINATED FROM MUD CRATER BLEDUG KUWU

• Main Author: Siti S, Dian
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/33976
• Institution: Institut Teknologi Bandung

Proteases which catalyze the hydrolysis of proteins are an industrial enzyme that has been applied in wide range of production process, such as bioactive component of detergent, biocatalyst in cheese production, bread production, medicines production, leather processing, etc. In order to fulfill wide variety of industrial needs, proteases have been isolated from various microorganisms, ranging from mesophiles to extremophiles. In this study, proteases have been isolated from halophilic bacteria originated from the mud crater located at Bledug Kuwu village, Purwodadi, Central Java. Two halophilic bacteria from the genus Pseudomonas, which is Pseudomonas stutzeri BK AB-12 and Pseudomonas alcaliphila BK AG-13 have been evaluated their potential in producing extracellular proteases by inoculating the free cell supernatant from the overnight culture of those bacteria on casein containing medium at 37 °C. Both bacteria produced proteases as exhibited by the appearance of clear zones resulted from casein hydrolysis, but protease of P. stutzeri BK AB-12 generated clear zone faster than that P. alcaliphila BK AG-13. It is suggesting that the amount and activity of protease produced by P. stutzeri BK AB-12 was higher than those of P. alcaliphila BK AG-13. It is therefore in the further study, protease was isolated only from P. stutzeri BK AB-12. The growth and extracellular protease activity profiles of P. stutzeri BK AB-12 showed that the maximum production of this enzyme was occured after 17 hours of incubation time. Protein collected at 17th hour of incubation were purified by ammonium sulfate fractionation method using three ammonium sulfhate concentration range i.e 0?60, 60?70, and 70?80%. Based on the protease specific activity, 70?80% fraction exhibited the highest activity. Protease in this fraction has highest activity at pH 8.0 and 55 °C and its activity of protease was enhanced by the addition of several metal ions. The highest enhancement in protease activity was observed upon the addition of Fe3+ ion. The addition of Fe3+ ion also resulted in a shifting of the optimum pH from 8 to 9 and the optimum temperature from 55 to 60 °C. Thus, the addition of Fe3+ ion not only improved the activity of protease but also its stability. Addition of inhibitors, such as ethylenediaminetetraaceticacid (EDTA) and ?-merkaptoetanol was able to reduce protease activity about 16 and 7%, respectively. Nevertheless the addition of phenylmethysulfonyl fluoride (PMSF) did not give significant effect on the activity of protease. It is, thus suggested that protease of P. stutzeri BK AB-12 in the fraction of 70?80% is not likely metallo-, cysteine-, or serine- protease. Proteases of this fraction was sensitive to ionic strength, where the highest activity was observed at concentrations at NaCl concentrations of 2.5 M. Beside influenced by the ionic strength, protease activity was also sensitive to solvent polarity. It was observed that protease activity was enhanced in various polarities of solvents, such as n-propanol, ethylacetate, n-butanol, n-hexane, and benzene. The activity of protease, however, decreased about 30% in medium polarity of solvents, such as isopropanol and chloroform. Potential protease as detergent bioactive components was assayed by measuring its activity in different concentrations of Sodium dodecyl sulphate (SDS) solutions and it showed that the activity was measurables up to 50% at 1% SDS. All of above suggest that P. stutzeri BK AB-12 is a potential source for extracellular proteases that have stability in various environmental conditions.