Purification and characterisation of an extracellular fructan beta-fructosidase from a Lactobacillus pentosus strain isolated from fermented fish.
Lactobacillus pentosus B235, which was isolated as part of the dominant microflora from a garlic containing fermented fish product, was grown in a chemically defined medium with inulin as the sole carbohydrate source. An extracellular fructan beta-fructosidase was purified to homogeneity from the bacterial supernatant by ultrafiltration, anion exchange chromatography and hydrophobic interaction chromatography. The molecular weight of the enzyme was estimated to be approximately 126 kDa by gel filtration and by SDS-PAGE. The purified enzyme had the highest activity for levan (a beta(2-->6)-linked fructan), but also hydrolysed garlic extract, (a beta(2-->1)-linked fructan with beta(2-->6)-linked fructosyl sidechains), 1,1,1-kestose, 1,1-kestose, 1-kestose, inulin (beta(2-->1)-linked fructans) and sucrose at 60, 45, 39, 12, 9 and 3%, respectively, of the activity observed for levan. Melezitose, raffinose and stachyose were not hydrolysed by the enzyme. The fructan beta-fructosidase was inhibited by p-chloromercuribenzoate, EDTA, Fe2+, Cu2+, Zn2+ and Co2+, whereas Mn2+ and Cu2+ had no effect. The sequence of the first 20 N-terminal amino acids was: Ala-Thr-Ser-Ala-Ser-Ser-Ser-Gln-Ile-Ser-Gln-Asn-Asn-Thr-Gln-Thr-Ser-Asp-Val-Val. The enzyme had temperature and pH optima at 25 degrees C and 5.5, respectively. At concentrations of up to 12% NaCl no adverse effect on the enzyme activity was observed.
Paludan-Müller
Christine
C
Danish Institute for Fisheries Research, Department of Seafood Research, Søltofts Plads, Technical University of Denmark, Lyngby. cpm@dfu.min.dk
Gram
Lone
L
Rattray
Fergal P
FP
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis; D002847:Chromatography, Agarose / Q000379:methods; D003470:Culture Media; D005285:Fermentation; D005396:Fish Products / Q000382:microbiology*; D005737:Garlic / Q000737:chemistry / Q000235:genetics; D006026:Glycoside Hydrolases / Q000032:analysis* / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007778:Lactobacillus / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D012680:Sensitivity and Specificity; D013696:Temperature
D001426:Bacterial Proteins; D003470:Culture Media; D006026:Glycoside Hydrolases; C044118:fructan beta-fructosidase