TY - JOUR
T1 - Purification and characterisation of an extracellular fructan beta-fructosidase from a Lactobacillus pentosus strain isolated from fermented fish
AU - Paludan-Müller, Christine
AU - Gram, Lone
AU - Rattray, Fergal P
PY - 2002/4
Y1 - 2002/4
N2 - 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.
AB - 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.
KW - Amino Acid Sequence
KW - Animals
KW - Bacterial Proteins
KW - Chromatography, Agarose
KW - Culture Media
KW - Fermentation
KW - Fish Products
KW - Garlic
KW - Glycoside Hydrolases
KW - Hydrogen-Ion Concentration
KW - Lactobacillus
KW - Sensitivity and Specificity
KW - Temperature
M3 - Journal article
C2 - 12086179
SN - 0723-2020
VL - 25
SP - 13
EP - 20
JO - Systematic and Applied Microbiology
JF - Systematic and Applied Microbiology
IS - 1
ER -