Purification and characterization of a novel levanoctaose-producing levanase from Pseudomonas strain K-52.
Levan-assimilating micro-organisms from soil samples were screened for levanoligosaccharide-generating enzyme production. The isolated strain K-52 produced an extracellular levanoctaose-generating enzyme and was identified as belonging to genus Pseudomonas. The levanase was purified to homogeneity by (NH4)2SO4 fractionation and successive column chromatography on DEAE-cellulose, phenyl-Toyopearl 650 M, Sephadex G-100 and hydroxyapatite. The molecular mass of the enzyme was estimated as approx. 38 kDa by both SDS/PAGE and gel filtration, and its isoelectric point was approx. pH 4.8. The optimum temperature and pH for the enzyme reaction were 35 and 7.0 respectively. The enzyme was stable at a pH range of 6.0-9.0 at 4 degrees C and up to 40 degrees C at pH 6.8. The enzyme activity was inhibited by Fe2+, Cu2+, Hg2+ and Ag2+. The levanase was specific toward 2,6-beta-D-fructosidic linkages of levan and did not hydrolyse other polysaccharides such as inulin and dextran. Chemical modification on the levanase suggested that cysteine and histidine residues are essential for enzyme activity. The levanoctaose liberated by levanase reaction was used selectively only by the intestinal beneficial micro-organisms, such as Bifidobacterium and Lactobacillus spp.
Kang
S K
SK
Department of Microbiology, College of Natural Sciences, Pusan National University, Kumjung-gu, Korea.
Lee
S O
SO
Lim
Y S
YS
Jang
K L
KL
Lee
T H
TH
D001426:Bacterial Proteins; D004795:Enzyme Stability; D005630:Fructans / Q000378:metabolism* / Q000494:pharmacology; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007422:Intestines / Q000382:microbiology; D007526:Isoelectric Point; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D011549:Pseudomonas / Q000201:enzymology* / Q000502:physiology; D012705:Serratia / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D017355:Zymomonas / Q000378:metabolism
D001426:Bacterial Proteins; D005630:Fructans; D008670:Metals; C112152:levanoctaose; C072599:levan; D006026:Glycoside Hydrolases; C021512:levanase