Cloning and characterization of a levanbiohydrolase from Microbacterium laevaniformans ATCC 15953.
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.
Song
Eun-Kyung
EK
Division of Biological Sciences, College of Natural Sciences, Pusan National University, Busan 609-735, South Korea.
Kim
Hyunjin
H
Sung
Hee-Kyung
HK
Cha
Jaeho
J
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
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C403663:2,6-beta-fructan 6-levanbiohydrolase; C021512:levanase