Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0272170 (SDS)
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Microbacterium sp. AL-210 producing a novel levan fructotransferase (LFTase) was screened from soil samples. The LFTase was purified to homogeneity by (NH4)2SO4 fractionation, column chromatography on Resource Q, and Superdex 200HR. The molecular weight of the purified enzyme was estimated to be approximately 46 kDa by both SDS-PAGE and gel filtration, and the enzyme's isoelectric point was pH 4.8. The major product produced from the levan hydrolysis by the enzyme reaction was identified by atmospheric pressure ionization mass spectrometry and NMR analysis as di-D-fructose-2,6':6,2'-dianhydride (DFA IV). The optimum pH and temperature for DFA IV production were 7.0 and 40 degrees C, respectively. The enzyme was stable at a pH range 7.0-8.0 and up to 40 degrees C. The enzyme activity was inhibited by FeCl2 and AgNO3. The enzyme converted the levan to DFA IV, with a conversion yield of approximately 44%. A gene encoding the LFTase (lftM) from Microbacterium sp. AL-210 was cloned and sequenced. The nucleotide sequence included an ORF of 1593 nucleotides, which is translated into a protein of 530 amino acid residues. The predicted amino acid sequence of the enzyme shared 79% of the identity and 86% of the homology with that of Arthrobacter nicotinovorans GS-9.
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PMID:Molecular and enzymatic characterization of a levan fructotransferase from Microbacterium sp. AL-210. 1152 62

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.
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PMID:Expression, purification and characterization of a recombinant levan fructotransferase. 1207 98

A bacterial strain, GS-9, isolated from soil as a levan-degrading microorganism produced an extracellular enzyme that converted levan into DFA IV. This strain was identified as Arthrobacter nicotinovorvans. The DFA IV-producing enzyme was specifically induced by levan. The enzyme was purified 60-fold from culture supernatant to give a single band on SDS-PAGE. The molecular weight of this enzyme was 52,000 by SDS-PAGE and a monomer by gel filtration. The enzyme gave DFA IV as a main product (> 75%), and fructose, levanbiose, and two unidentified oligosaccharides as minor products, and was identified as a novel levan fructotransferase.
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PMID:Purification of Levan Fructotransferase from Arthvobacter nicotinovoram GS-9 and Production of DFA IV from Levan by the Enzyme. 2739 68