Expression, purification and characterization of a recombinant levan fructotransferase.
A 1.6 kb DNA fragment including the lftM gene, encoding a levan fructotransferase (LFTase) of Microbacterium sp. AL-210, was subcloned into a high-expression vector, pET-29b, and the recombinant enzyme was overexpressed in Escherichia coli. Most of the LFTase activity was detected in the cytoplasmic fraction after induction with isopropyl beta-d-thiogalactoside. The recombinant enzyme with a tag of six histidine residues at the C-terminus was purified 132-fold by affinity and gel-filtration chromatography. Analysis of the N-terminal amino acid sequence revealed that the first 42 amino acids were post-translationally cleaved off. The molecular mass of the purified LftM was approx. 54 kDa as determined by SDS/PAGE, which corresponded well with a predicted size from the nucleotide sequence of the lftM gene lacking 42 amino acids. The enzyme converted levan into difructose anhydride IV (DFA IV) with a K(m) of 2 mg/ml and a V(max) of 40.6 micromol/min at pH 7.0 and 40 degrees C. The pH-dependence study of the enzyme for DFA IV production showed that LftM had a broad pH optimum (5.0-8.0) and the pK(a) values obtained were 4.5 and 8.9 at 40 degrees C. These results suggest that the acidic residues at the active site may play important roles for the catalytic mechanism of the LFTase.
Yang
Sung Jae
SJ
Department of Microbiology, College of Natural Sciences, Pusan National University, Jangjeon-dong, Kumjung-ku, Busan, Korea.
Park
Na Hee
NH
Lee
Tae Ho
TH
Cha
Jaeho
J
D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics*; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D024861:Genetic Enhancement / Q000379:methods; D006602:Hexosyltransferases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
D011994:Recombinant Proteins; D006602:Hexosyltransferases; C040074:levan fructotransferase