BIOSYNTHESIS LEVAN BY INTRACELLULAR LEVANSUCRASE FROM MODERATE HALOPHILIC BACTERIA Bacillus licheniformis BK AG21

• Main Author: Mamay
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/34074
• Institution: Institut Teknologi Bandung

Levan is an extracellular polysaccharide as result from convertion sucrose by levansucrase (E.C. 2.4.1.10). Biosynthetis levan started with hydrolysis of sucrose into glucose and fructose, then transfered fructose to another sucrose accompanied by the release of glucose. In polymerization of levan, each monomer fructose are linked with glicosidic ?-(2,6) bond in formation linear chain and ?-(2,1) linked branches chain. Production of levan increasing intensively for research because of the very wide application in food industries cosmetic, pharmaceutical and environmental sector. The key production efficiency depends on the activity of levansucrase wich produced by bacteria producing levan. Most of the study about isolation and characterization of levansucrase has been published limited to mesophilic bacteria and plants. In this study, halophilic bacteria BK AG21 which isolated from salt water mud crater Bledug Kuwu, Purwodadi in Central Java used as a research object. Phylogenetic analysis based on 16S rRNA gene sequences identified that halophilic bacteria BK AG21 has a closeness with Bacillus licheniformis. The bacteria is "halophilic moderate” that can survive in the medium with 0-17% NaCl. The use of this bacteria as it gives a positive test result as levan-producing potential in screening media containing 5% sucrose. These bacteria produced levansucrase optimally at medium with composition 2% sucrose, 0,5% yeast extract, 2% NaCl, 0,05% K2HPO4.3H2O and MgSO4.7H2O with incubation for 18 hours, 37°C and rate of aeration 150 rpm. Crude extract intracellular levansucrase fractionated using ammonium sulfate with the fraction 0-40%, 40-50%, 50-60%, 60-70% and 70-80%, which the fraction 70-80% is the highest specific activity. The molecular weight determined by SDS PAGE and zimography where levan sucrase obtained has a molecular weight of about 50 kDa. Levansucrase activity and levan biosynthesis increased by Ca2+, Fe3+, Ba2+, Mg2+, Zn 2+ and Ti2+ but descreased by Si2+ and Fe2+. The addition of EDTA less descreased significantly levansucrase activity indicated that that the enzyme is not included in metalloenzyme group. Levansucrase showed optimum activity at 55 oC when presence or absence of Ca2+. This enzyme has good activity in the pH range 4-8 when absence of Ca2+ and has optimal activity at pH 6.5 when the presence of Ca2+. Optimum activity of biosynthesis levan at 45 oC and pH 6. Optimum activity of levansucrase and biosynthesis levan at a concentration of 1% NaCl. Levansucrase with addition Ca2+ has a half-life of about 157,5 minutes while absence of Ca2+ has a half-life of about 24,4 minutes in incubation at 55 oC. FT-IR spectrum analysis showed that levan identified as having an O-H group at 3600-3200 cm-1, C-H at 3000-2800 cm-1, C-O group at 1,600 cm-1 and a finger print for polysaccharide in some sharp peaks between 1200-900 cm-1. TGA analysis showed that the levan has degradation at 224oC. Based on the above results, it can be concluded that potential of halophilic bacteria Bacillus licheniformis BK AG21 as producing levan determined by the optimum conditions intracellular levansucrase wich depend on pH, temperature, Ca2+, and concentration of NaCl.