title: High expression level of levansucrase from Bacillus licheniformis RN-01 and synthesis of levan nanoparticles.
abstract: LsRN from Bacillus licheniformis was cloned and expressed in Escherichia coli. From a 1793 bp genomic sequence, the lsRN gene was found to be composed of a single 1446 bp ORF with a putative promoter consensus boxes and a ribosome-binding site. This ORF was predicted to encode for 482 amino acid residues. The LsRN was constitutively expressed at a relatively high level without sucrose induction. The enzyme was highly purified and an apparent size of 52 kDa with an optimum temperature and pH of 50 °C and 6.0 were determined. The wide range of M(w) of levan (1-600 kDa) was synthesized in a controlled reaction with two variable parameters: temperature and ionic strength. At high temperature (50 °C), LsRN synthesized high M(w) levan (612 kDa) as a major product while at low temperature (30 °C), low M(w) levan (11 kDa) was mainly synthesized. When 0.5M NaCl was added into the reaction, the major products at both temperatures were of the size 11 kDa. Moreover we report for the first time, an enzymatic synthesis of levan nanoparticles (NPs) by a single step reaction. The LsRN synthesized levan NPs as agglomerate with average particle size of 50 nm. The encapsulation of O-acetyl-α-tocopherol was carried out to demonstrate the applicable use of levan NPs.
authors.affiliation: Department of Biochemistry, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Bangkok 10330, Thailand.
authors.forename: Santhana
authors.initials: S
authors.lastname: Nakapong
authors.forename: Rath
authors.initials: R
authors.lastname: Pichyangkura
authors.forename: Kazuo
authors.initials: K
authors.lastname: Ito
authors.forename: Masaru
authors.initials: M
authors.lastname: Iizuka
authors.forename: Piamsook
authors.initials: P
authors.lastname: Pongsawasdi
mesh_terms: D001407:Bacillus / Q000187:drug effects / Q000201:enzymology*; D003001:Cloning, Molecular; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005630:Fructans / Q000096:biosynthesis*; D006602:Hexosyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D053758:Nanoparticles / Q000737:chemistry / Q000648:ultrastructure; D017550:Spectroscopy, Fourier Transform Infrared; D024502:alpha-Tocopherol / Q000378:metabolism
chemical_list: D005630:Fructans; D008670:Metals; C072599:levan; D004492:Edetic Acid; D006602:Hexosyltransferases; C021513:levansucrase; D024502:alpha-Tocopherol