Effect of Addition of Ion Ba2+ On Activity and Stability of Extracell Lipase Product From Moderate Halophilic Bacteria Halomonas elongata BK-SM1

• Main Author: SAFITRI (NIM: 10512066), MAHARANI
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/28709
• Institution: Institut Teknologi Bandung

<p align="justify">Lipase (EC 3.1.1.3) is an enzyme that has role as a biocatalyst in either hydrolysis reaction and ester carboxyl synthesis from triacylglycerols. Lipase has been used widely in industrial filed such as a component in food, textile, pharmacy, and biodiesel process. This research aims to isolate and characterizing an extra-cell of moderate halophilic Halomonas elongate BK-SM1 from Bleduk Kuwu, Purwodadi, Central Java. H. elongate BK-SM1 is considered as moderate halophilic due to its salt tolerance level lies between 0.5&#9472;22.5%. Isolation and initial purification of this lipase was done partially byammonium sulphate fractionation with concentration ranges of 0&#9472;20%, 20&#9472;40%, 40&#9472;60%, and 60&#9472;80%. By measuring the enzyme activity and total protein concentration in each fractions, it showed that two of fractions exhibited high specific activity, namely 0&#9472;20% and 20&#9472;40% fractions with respective specific activity were 0.465±0.008 U/mg and 0.473±0.005 U/mg. These two fractions were then mixed together and were used for in the further characterizations. NativePAGE, SDS-PAGE, and zimography results on the mixed fractions revealed one active lipase with the estimated molecular weight was about 50 kDa. Adding various metal ions to the enzyme solution found Ba2+ ion prominently enhanced the enzyme activity up to 35%. Further characterization on the effect of Ba2+ ion in various pH, temperature, organic solvents, and inhibitors showed an activity enhancement in various range of those parameters. Interestingly, the activity enhancement due to the addition of Ba2+ ion did not affect the optimum parameter of the enzyme, in which similar to the condition without the addition of this ion, pH and temperature optimum were occured at pH 8 and 40 oC, respectively. Ba2+ ion was likely bound non-covalently with the enzyme because the addition of the chelating agent, such EDTA, did not significantly reduced the activity of its activity. Although Ba2+ ion was not strongly bound to the enzyme but the addition of this ion improve the stability of the enzyme against surfactant as SDS. The addition of PMSF also only slighty reduced the activity of the enzyme indicating that lipase produced by H. elongata BK-SM1 was not belong to the group of serine hydrolases. Furthermore, the addition of Ba2+ ion was also enhancing thermal stability of the enzyme, in which its melting temperature shifted from 60 oC to 68.9 oC. Kinetic analysis also showed that the addition of Ba2+ ion reduced the rate of thermal inactivation of the enzyme at 40 oC as indicated by the increase of its half life from 64.7 minutes to 121.9 minutes. Thermodynamically, the prolong of the half life of the enzyme correspond to the elevation of the transition barried towards denatured state after the addition of Ba2+ ion as indicated by the increase of activation energy (Ea) for thermal denaturation process from 0.7 kJ/mol to 6.3 kJ/mol. Overall, the addition of ion Ba2+ ion have successfully transformed lipase produced by Halomonas elongata BKSM1 to become multistable enzyme.<p align="justify">