title: Cloning and characterization of a levanbiohydrolase from Microbacterium laevaniformans ATCC 15953.
abstract: An extracellular levanbiohydrolase gene, levM, from Microbacterium laevaniformans ATCC 15953 was cloned and its nucleotide sequence was determined. Nucleotide sequence analysis of this gene revealed a 1863 bp open reading frame coding for a protein of 621 amino acids. The deduced amino acid sequence of the levM gene exhibited 28-47% sequence identities with levanases, levanfructotransferases, and inulinases. The LevM was overexpressed by using a T7 promoter in Escherichia coli BL21 (DE3) and purified 24-fold from culture supernatant. The molecular weight of this enzyme was 68,800 Da based on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pH and temperature of this enzyme for levan degradation was pH 6.0 and 30 degrees C, respectively. Thin-layer and high-performance liquid chromatography analyses proved that the enzyme produced mostly levanbiose from levan in an exo-acting manner. The recombinant enzyme also hydrolyzed inulin, 1-kestose, and nystose, indicating that the enzyme cleaves not only beta-2,6-linkage of levan but also beta-2,1-linkage of fructooligosaccharides. This is the first report on a gene encoding a levanbiohydrolase that produces levanbiose as a major degradation product.
authors.affiliation: Division of Biological Sciences, College of Natural Sciences, Pusan National University, Busan 609-735, South Korea.
authors.forename: Eun-Kyung
authors.initials: EK
authors.lastname: Song
authors.forename: Hyunjin
authors.initials: H
authors.lastname: Kim
authors.forename: Hee-Kyung
authors.initials: HK
authors.lastname: Sung
authors.forename: Jaeho
authors.initials: J
authors.lastname: Cha
mesh_terms: D000192:Actinomycetales / Q000201:enzymology / Q000235:genetics*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C403663:2,6-beta-fructan 6-levanbiohydrolase; C021512:levanase