PRODUCTION AND CHARACTERIZATION OF ALKALINE PROTEASE FROM HALOPHILIC BACTERIA Chromohalobacter japonicus BK-AB18

• Main Author: Isyana Achmad, Dwi
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/33985
• Institution: Institut Teknologi Bandung

Proteases are one of the hydrolysis enzymes that cleavage protein into shorter peptide. Proteases represent the largest industrial enzyme that has been applied in various types of industries, such as surfactants/detergents, leather, food, and pharmaceutical industries. These industries require proteases that have a high activity and good performance at various conditions ranging from mild to extreme ones, such as in organic solvent, in detergent and also in the high salinity. Proteases that stable and active in such harsh environment are typically derived from extremophiles microorganisms, which is microorganisms that live in extreme habitat, such as high temperature (thermophiles), low pH (acidophiles) and high salt concentrations (halophiles). The later bacteria are interesting because they can live in hypersaline environment or in other word in a low water activity condition. Extracellular enzymes produces by these bacteria will also have relatively high activity in the solution containing organic solvent or detergent. In the present study, we are interested to explore the potential halophilic bacteria from the genus of Halomonas and Chromohalobacter in producing extracellular protease. These bacteria were screened for its proteolytic activity on the medium containing 2% casein. Among five bacterial isolates able to Chromohalobacter japonicus BK-AB18 exhibited the highest protease activity, thus we used this bacteria in the subsequent work. Extracellular protease from Chromohalobacter japonicus BK-AB18 was produced by growing the bacteria in LB medium containing 5% of casein and 5% of NaCl. The crude of protease were partially purified by five level of amonium sulfate fractionation, which were 0?40, 40?50, 50?60, 60?70, and 70?80 % amonium sulfate concentration range. The activity assay of these five fractions was carried out using 1% casein as substrate and found that the highest specific activity exhibited by the fraction of 70?80%. This fraction also has 63 folds higher of purity compare to the crude enzyme. The activity of protease in this fraction was enhanced by the addition of 5mM CaCl2. The addition of EDTA and PMSF, however reduced the activity of protease significantly, thereby indicating as metallo and serine protease. The protease activity reached the highest activity at pH 9 (alkaline protease), 45 °C and 2.7 M of NaCl. The stability of protease in high salinity reveals its potential to be applied in industries requires high concentration of salt in their production process, such as in the cheese production. The thermal stability of protease that was examined at its optimum condition showed that the half live of native enzyme with and without additional Ca2+ ion was about 73 and 49 minutes, respectly. In addition, protease in the fraction of 70?80% was stable and active in some organic solvents such as acetone, n-hexane and ethylene glycol. The protease activity was increased in the solution of 0.01% SDS and it also quite stable in SDS solution with the concentration up to 5%. Our study showed that Chromohalobacter japonicus BK-AB18 is a potential source to produce alkaline serine protease that can be applied in industries involving organic solvent or high salinity in their process.