Partial purification of an endoglucanase from Biomphalaria glabrata.
Previous studies have shown that Biomphalaria glabrata contains a complete cellulolytic system which includes an endoglucanase, an exoglucanase and a beta-glucosidase. In the present report, a scheme for the purification of the endoglucanase from this invertebrate is proposed. Two major problems were encountered during the study: 1) the presence of a green-brownish pigment, which could not be eliminated by thermal shock or ammonium sulfate precipitation and 2) relative instability of enzymatic activity. Various alternatives were tested and the best sequence of steps was: 1) a sample of the crude extract, obtained by homogenization of the digestive glands in 50 mM Tris-HCl buffer, pH 8.4, and ultracentrifugation, was applied to a Q-Sepharose FPLC column (50 mM Tris-HCl buffer, pH 8.4; 10 mm x 22.2 cm column; flow rate 1.5 ml/min; 0.1 to 0.5 M NaCl gradient); 2) the eluate peak containing activity was dialyzed, lyophilized and eluted from a Superdex-75 gel filtration FPLC column (50 mM ammonium acetate buffer, pH 4.8; 16 mm x 60 cm column; flow rate 1.0 ml/min). A low degree of purification (about 36-fold) and recovery (about 12%) were observed, probably due to enzyme instability. SDS-electrophoresis of the active fraction showed a major peak of 30 kDa. In order to improve the purification scheme, further studies are required to stabilize this enzyme during purification and storage.
Andrade
A M
AM
Departamento de Bioquímica-Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, Brasil.
Alves
A C
AC
Nicoli
J R
JR
Santoro
M M
MM
D000818:Animals; D001700:Biomphalaria / Q000201:enzymology*; D002847:Chromatography, Agarose; D006026:Glycoside Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D014461:Ultracentrifugation
D006026:Glycoside Hydrolases