CHARACTERIZATION ION OF LEVANSUCRASE AND ITS BIOSYNTHESIS PRODUCT AND CLONING OF LEVANSUCRASE GENE FROM HALOMONASE SALINA BK3

• Main Author: CELLYANA , JESSICA
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/28269
• Institution: Institut Teknologi Bandung

Levan is a fructose polymer with fructofuranosyl monomer interconnected with ?-(2,6)- <br /> <br /> glycoside and ?-(2,1)-glycoside bonds on its branches. Levan is extensively studied <br /> <br /> because of its benefits, as anti-tumor, anti-inflammatory, alternative artificial sweetener, <br /> <br /> and prebiotics. Levan production by several types of plants, fungi, and bacteria is <br /> <br /> catalyzed by levansucrase. The levan-producing bacteria can be identified through the <br /> <br /> production of extracellular mucosa observed on sucrose containing growth medium. <br /> <br /> Halomonas salina BK3, a Gram-negative halophilic bacteria isolated from the saline mud <br /> <br /> crater in Bledug Kuwu, Central Java, Indonesia, is one of the bacteria identified as a <br /> <br /> levan producer. In this study, isolation and characterization of levan and levansucrase <br /> <br /> from H. salina BK3 were performed. The FTIR and C-NMR spectrum of the levan <br /> <br /> produced by H. salina BK3 are similar to those of a standard one from E. herbicola and <br /> <br /> its H-NMR spectrum is similar to that of the standard one from B. licheniformis. <br /> <br /> Biochemical characterization of levansucrase using RSM (Response Surface Method) <br /> <br /> suggested the optimum pH and temperature were estimated around pH 10 and 37.6ºC, <br /> <br /> respectively. The optimum pH of levansucrase of H. salina BK3 was higher than that of <br /> <br /> levansucrase produced by other bacteria, such as B. licheniformis (pH 6), Z. mobilis (pH <br /> <br /> 6), and L. sanfranciscencis (pH 5.4), whereas its optimum temperature is similar to that of <br /> <br /> levansucrase from R. aquatilis (35ºC) and L. sanfranciscencis (35-45ºC). The kinetic of <br /> <br /> levansucrase from H. salina BK3 did not obey the Michaelis-Menten kinetic and showed <br /> <br /> a sigmoid profile, therefore its kinetic parameter were determined by allosteric kinetics <br /> <br /> equation, which was Hill equation. The result revealed that the maximum reaction rate <br /> <br /> (Vm) of the enzyme was 6.33 x 103 U/min, its dissociation constant was (Kd) 26.8, and its <br /> <br /> Hill constant was (h) 1.85. One unit of levansucrase activity is defined as 1?mol of <br /> <br /> sucrose that is converted to product every one minute. These results suggest that the <br /> <br /> kinetic of levansucrase from H. salina BK3 is equivalent to that of allosteric enzymes <br /> <br /> with two cooperative units. Levansucrase from L. reuteri was identified to have similar <br /> <br /> allosteric profile to that of levansucrase from H. salina BK3. In order to enhance <br /> <br /> levansucrase production by overexpression method, the genetic engineering procedures <br /> <br /> have been conducted to isolate and to clone levansucrase gene from H. salina BK3. <br /> <br /> However, the obtained clone was verified to not encode the levansucrase protein.