PRODUCTION AND CHARACTERIZATION OF EXTRACELLULER LEVANSUKRASE SECRETED BY HALOPHILIC BACTERIUM OF HALOMONAS AND CHROMOHALOBACTER GENUSES ORIGINATED FROM BLEDUG KUWU MUD CRATER

• Main Author: Qodarisman Nasir, Daris
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/33972
• Institution: Institut Teknologi Bandung

Fructooligosaccharides (FOSs) are of increasing interest as “functional sweeteners” because of their low caloric content, their non-cariogenicity and their ability to stimulate the growth or activity of beneficial colonic lactic acid bacteria and to enhance the intestinal immune response. The levan-type-FOSs, with ?-(2- 6)-linked fructose and some ?-(2-1)-linked branching, are synthesized by bacterial levansucrase (EC 2.4.1.10) from sucrose. Indonesia has a wealth of high biodiversity, because it has many unique natural resources as a habitat for the growth of bacteria owing high potential in producing high market value bioproducts, such as levan. One of the unique bacterial habitats is found in the region of Central Java Purwodadi, Grobogan, Bledug Kuwu, where at this location there is a mud volcanic crater that periodically spit brine to the surface. The uniqueness of this habitat has encouraged us to explore the potential of bacteria in producing levan. Seven isolates have been characterized and identified from this mud crater comprising of five moderately halophilic bacteria from the genus Halomonas and Chromohalobacter and two low halophilic bacteria from those of Pseudomonas. In this study, five isolates bacterial isolates from the genus of Halomonas and Chromohalobacter were assayed their levansucrose activity. Initial screening was conducted on of modified Belgith medium with the composition of 1% tryptone, 0.5% yeast extract, 1.5% bacto agar, 0.25% K2HPO4, 10% NaCl, and 20% sucrose. In the presence of sucrose, the colonies had a slimy mucoid appearance indicating the production of levan from sucrose. The screening result showed only one bacterium, which was Chromohalobacter japonicus BK AB18 that potentially produce levansucrose. The optimum levansucrose produced by Chromohalobacter japonicus BK AB18 has the highest specific activity when the bacteria was grown in the medium containing 7.5% NaCl and 7.5% sucrose. The resulted crude extract of the enzyme was partially purified with the fractionation method using ammonium sulfate. The highest specific activity contained in the fraction of 60-80%. SDS PAGE and zymography estimated the molecular weight of the enzyme approximately about 50 kDa. The activity of levansucroase increased by the addition metal ion Co2+ and 5% NaCl about 18% and 14% respectively. We noted that the addition of EDTA sharply decreased its activity indicating that the isolated levansucrase is metalloenzymes. Levansucrose contained in this fraction has optimum activity at pH 6 and 55oC. At the optimum temperature and pH, the isolated levansucrose has the half-life of about 114 minutes. Levan produced by the enzyme has been confirmed its structure and termal stability with FTIR spectroscopy and TGA respectively.