Isolation and Characterization of Hydroxyectoine from Halomonas elongata TG-1 and its Application as Protein Stabilizer

• Main Author: DWILISTYANI (NIM : 10513059), ANNISA
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/25658
• Institution: Institut Teknologi Bandung

Halophilic bacteria are bacteria that can survive in environments with high salinity level. To adapt to these environments, halophilic bacteria build strategies by accumulating an organic compound that has high solubility and low molecular weight called a compatible solutes. A total of about twenty different types of compatible solutes have been successfully characterized. Compatible solutes are grouped according to their structure, such as sugars, polyols, diamino acids N-acetylated, betaine, amino acids, and their derivatives. The last group is the group ectoine (ectoine and hydroxyectoine). Hydroxyectoine is a compatible solute that has many applications, such as stabilizing enzymes, DNA, and entire cells against different forms of stress, including heating, drying, and freezing. This study aimed to isolate and characterize hydroxyectoine from halophilic bacteria isolated from Gresik salt work. Screening results obtained halophilic bacteria that potentially produce hydroxyectoine is Halomonas elongata TG-1. Optimum hydroxyectoine production in MM63 medium containing 9% NaCl (w/v) concentration was reached after 45 hours of incubation at 37 °C in 150 rpm of a rotary shaking incubator. The verification of hydroxyectoine sample was carried out by HPLC method and the result showed similar retention time between isolated hydroxyectoine and its standard, which were about 1.291 min and 1.248 min, respectively. Spectroscopic structural verfification was also carried out by FTIR. The FTIR spectrum showed the presence of functional groups for N-H, O-H, C=N, and C-N groups at wave numbers 3446, 3055, 1653, and 1618 cm-1, which were macthed to the functional grups presence in hydroexctoine structure. The hydroxyectoine function as a protein stabilizer was studied by fluorescence spectroscopy using lysozyme as the target protein. Measurements were made at a excitation wavelength of 280 nm and its emissions was observed at 340 nm. The effect of hydroxyectoine on the protein stability is studied by observing the intensity of emissions at different temperatures. The results showed that the addition of 0.01 M hydroxyectoine to lysozyme solution could withstand the decrease in fluorescence intensity due to the thermal perturbation when compared to the lysozyme solution without the addition of hydroxyectoine. However, when NaCl was added to final concentration of 20%, the effect of hydroxyectoine protection was reduced. These results suggest that hydroxyectoine can only protect proteins from thermal perturbation under conditions of low salinity.