PRODUCTION AND CHARACTERIZATION OF INULIN FROM HALOPHILIC BACTERIA SALT LAKE GILl MENO LOMBOK ISOLATE APPLIED FOR IMMOBILIZATION LIPASE AND LYSOZYME NANOPARTICLES

• Main Author: Miftahul Aini, Diah
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/38562
• Institution: Institut Teknologi Bandung

Exopolysaccharide (EPS) is a polymer consisting of straight and branched chains, consisting of sugar units and their derivatives. This sugar uni t consists of glucose, gal actose, mannose, N-acetylglucosamine, N-acetyl galactosamine, ramnosa, and fructose. EPS can be divided into homopolysaccharide (HoPS) and heteropol ysaccharide (HePS). Polymers which are homopolysaccharide are dextran , inulin, levan , and amylose, while heterosaccharide polymers are pentose, hexose, gellan, and xanthan. EPS is very interestin g to study because it has propet1ies that are not toxic, soluble in water, and very safe. EPS i s utilized in various industrial fields, for example in food used for making cheese, bread, and milk; for pharmaceuticals as a drug delivery; and for bioindustry it is used for enzyme immobilization. EPS producing organisms include plants, namely lnula helentum, chtCO!)' root, Jerusalem arttchoke, dal1lia; from fungi namely A5pergillus sydowi andAspergt/Lus mger; and from mesophyll bacteria including Streptococcu5 mwans and !.actobaci/lus ;ohnsonu, as well as from halophilic bacteria such as Bact/Ius ltchen!formts and Salmtcoc:c:us sp. In this study, sampling of new EPS­ producing halophilic bacteria sampl es from halofil habitat of salt water Lake Gili Meno Lombok, NTB, Indonesia was conducted. The aim of this st udy is to obtained potential halophilic bacteria producing EPS, identification of potential halophilic bacterial species, biochemistry characterization of inulosucrase, production of inulin, and its application for the manufacture of nanoparticles for protein immobilization. The results showed sal t water Lake Gili Meno are known to have pH 7.75, temperature 26.5 °C, salinity 4.4%, and density 1.127 g/m l. The mineral content contained in salt water lake Gili Meno consists of 44% Na+, 2.2% Mg2 0.7% K+, 2.83% Ca2+, 1 3% Mn2+, and 2.7% zr+. The bacteria contained in saltwater samples obtained from three sampling locations in salt water Gili Meno were grown in liquid LB media containing I 0% NaCI. The bacteria that grow then spread on solid LB media containing I Oo NaCI. Single colony from three sites were tested for exopolysaccharide production potential in solid media containing sucrose. Based on three samples obtained one isolate that has the most potential to produce exopolysaccharide. The exopolysaccharide verified its stmcture by using FTIR and NMR. Exopolysaccharide produced by these bacteria was inulin. Potential bacterial species were determined by the 16s rONA sequence analysis method and it was found that the species was Salml\'lbrio co.wcola GMOI. This bacteria is a new strain bacteria that produces EPS, namely inulin which has never been fonned before. This bacteria was also a moderate halophilic bacteria with optimum growth at 7% (b/v) NaCI concentration. lnulosucrase is produced by this bacteria by growing bacteria for 24 hours at 37 oc in liquid LB med ia containing 10° o (w/w) NaCI and 20% (w/w) sucrose. The molecular weight of inulosucrase is about 50 kDa. Then inulin from Salinivibrio costicola GMO I was growed at 37 oc for 24 hours in liquid media containing 20% (b/v) sucrose and I 0% (b/v) NaCI. Obtained from the cell by centrifugation , the supematant obtained was presi pi tated with 95°/o (v/v) cold ethanol. The precipitate washed with ddH20 and dried by the lyophilization method. Inulin isolated then used as an immobilization medium for nanoparticles. Inulin was dissolved in ddH:O, then protein was added and stirred with a stining speed of 500 rpm using a magnetic stirrer for 19 hours. The immobilized protein in this study were lipase and lysozyme. The nanoparticles obtained were detennined for their immobilisation efficiency and characterized using SEM and PSA. The efficiency of lipase and lysozyme immobilization on nanoparticles were 81% and 87%, respectively. The SEM results attract relatively rounded nanoparticles for both inulin-lipase and inulin-lysozyme complexes with particle size distributions based on PSA measurements between 218,3-886.5 nm and 538,6-797,5 nm respectively. The surface loads of inulin-lipase and inulin­ lysozyme nanoparticles are +0.03 m V and +0.13 m V, respectively. For comparison nanoparticles using commercial inulin (standard). The immobilization efficiency obtained were 73% and 87% for each inulin-lipase and inulin-lysozyme nanoparticles. The shape of the nanoparticles obtained were relatively round with a distribution size of 162,6-16-l2,8 nm with a potential zeta of +0.36 m V for standard lipase-inulin and 691 ,9-1219,8 nm with a potential zeta +0,13 m V for standard inulin lysozyme. Lipase activity before and after immobilization was 0.36 units/mg and 0.28 units/mg. These results showed that nanoparticles was good enough to be used as a carrier for lipase immobilization because they can still maintain 80% of their activity.