ISOLATION AND CHARACTERIZATION OF LEVAN FROM HALOMONAS SMYRNENSIS BK4 AND ITS APPLICATION AS NANOPARTICLES

• Main Author: MAHBUBA SALEHA AMARI NIM: 10514057, MUZAYANA
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/29430
• Institution: Institut Teknologi Bandung

Levan is homopolymer of fructose synthesized through enzymatic reaction by <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> levansucrase (E.C. 2.4.1.10). This bioplymer is produced by several types of plants, fungi, <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> and bacteria. Currently, levan has been developed as nanoparticle systems, as a carrier <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> for metal ions, catalyst, and drugs. Halophilic bacteria has been known as one of the <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> potential levan producers. Halomonas smyrnensis BK4 was identified as one of halophilic <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> bacteria potentially produces levan. This study aims to evaluate the use of levan produced <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> by Halomonas smyrnensis BK4 as a material for immobilization of enzymes in the form of <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> nanoparticle. The bacterium was reconfirmed by ribotyping, followed by levan production <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> and isolation, enzyme immobilization with levan as nanoparticles, measurement of <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> enzyme activity in free and immobilized states, and cloning of levansucrase genes. <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> Ribotyping and phylogenetic analysis reconfirmed that the halophilic bacterium used is <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> Halomonas smyrnensis. Production of levan by this bacterium that cultivated in LB <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> medium containing 20% (w/v) sucrose by agitation rate of 150 rpm at 37 oC for 18 hours <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> produced levan of 3.02 g/L. The FTIR spectrum of levan sample from H. smyrnensis BK4 <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> appeared to be similar to levan from Erwinia herbicola, whereas its H-NMR spectrum, was <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> similar to levan from Bacillus licheniformis. This levan was then used to immobilize two <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> different enzymes, levansucrase and lipase, in the form of nanoparticle. The scanning <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> electron microscope (SEM) image showed irregular geometry of levan nanoparticle used <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> to immobilize levansucrase, while levan nanoparticles used to immobilize lipase exhibited <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> more spherical geometry. Particle size analyzer (PSA) indicated size distribusion range of <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> 347.3 &#822; 947.2 nm diameter of levan nanoparticles used to immobilize levansucrase with <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> zeta potential of about &#822; 43.59 mV, whereas for those used to immobilize lipase have <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> diameter distribution of 164.2 &#822; 635.7 nm with zeta potential of about &#822; 24.99 mV. The <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> specific activity of immobilized levansucrase was about 2.98 unit/mg, which was relatively <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> higher compare to levansucrase activity in the free state, which was about 1.51 unit/mg. <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> Contrary, the specific activity of immobilized lipase was almost similar to that of the free <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> state one, which was about 5.67 and 5.72 unit/mg, respectively. Efforts to clone the <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> levansucrase gene using pGEM-T plasmid and E. coli TOP10 as host cell has also been <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> carried out, but did not give to desired result.