A novel tannase from Aspergillus niger with beta-glucosidase activity.
An extracellular tannase was produced from solid-state cultures of Aspergillus niger. The enzyme was purified to homogeneity from the cell-free culture broth by preparative isoelectric focusing and by FPLC using anion-exchange and gel-filtration chromatography. SDS-PAGE analysis as well as gel localization studies of purified tannase indicated the presence of two enzyme forms, with molecular masses of 90 kDa and 180 kDa. The tannase had an isoelectric point of 3.8, a temperature optimum of 60-70 degrees C and a pH optimum of 6.0. The substrate specificity of the tannase was determined by HPLC analysis of tannin substrates and products. The enzyme was able to remove gallic acid from both condensed and hydrolysable tannins. Internal sequences were obtained from each of the gel-purified and trypsin-digested tannase forms. The peptide sequences obtained from both forms were identical to sequences within a beta-glucosidase from Aspergillus kawachii. The purified tannase was tested for beta-glucosidase activity and was shown to hydrolyse cellobiose efficiently. However, no beta-glucosidase activity was detected when the enzyme was assayed in the presence of tannic acid.
Ramírez-Coronel
M Ascención
MA
IRD-México, Cicerón 609, Los Morales, CP 11530, México DF, Mexico.|Departamento de Biotecnología, Universidad Autónoma Metropolitana, Av. Michoacán y la Purísima s/n, Colonia Vicentina, Iztapalapa, México DF, CP 09340, Mexico.
Viniegra-González
Gustavo
G
Departamento de Biotecnología, Universidad Autónoma Metropolitana, Av. Michoacán y la Purísima s/n, Colonia Vicentina, Iztapalapa, México DF, CP 09340, Mexico.
Darvill
Alan
A
Complex Carbohydrate Research Center, The University of Georgia, 220 Riverbend Road, Athens, GA 30602-4712, USA.
Augur
Christopher
C
IRD-México, Cicerón 609, Los Morales, CP 11530, México DF, Mexico.
D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology*; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D001617:beta-Glucosidase / Q000302:isolation & purification* / Q000378:metabolism
D002265:Carboxylic Ester Hydrolases; C022711:tannase; D001617:beta-Glucosidase