endo-beta-1,4-Mannanases from blue mussel, Mytilus edulis: purification, characterization, and mode of action.
Two variants of an endo-beta-1,4-mannanase from the digestive tract of blue mussel, Mytilus edulis, were purified by a combination of immobilized metal ion affinity chromatography, size exclusion chromatography in the absence and presence of guanidine hydrochloride and ion exchange chromatography. The purified enzymes were characterized with regard to enzymatic properties, molecular weight, isoelectric point, amino acid composition and N-terminal sequence. They are monomeric proteins with molecular masses of 39216 and 39265 Da, respectively, as measured by MALDI-TOF mass spectrometry. The isoelectric points of both enzymes were estimated to be around 7.8, however slightly different, by isoelectric focusing in polyacrylamide gel. The enzymes are stable from pH 4.0 to 9.0 and have their maximum activities at a pH about 5.2. The optimum temperature of both enzymes is around 50-55 degrees C. Their stability decreases rapidly when going from 40 to 50 degrees C. The N-terminal sequences (12 residues) were identical for the two variants. They can be completely renatured after denaturation in 6 M guanidine hydrochloride. The enzymes readily degrade the galactomannans from locust bean gum and ivory nut mannan but show no cross-specificity for xylan and carboxymethyl cellulose. There is no binding ability observed towards cellulose and mannan.
Xu
Bingze
B
Center for Surface Biotechnology, Biomedical Center, Uppsala University, Box 577, SE-751 23 Uppsala, Sweden.
Hägglund
Per
P
Stålbrand
Henrik
H
Janson
Jan-Christer
JC
D000595:Amino Acid Sequence; D000818:Animals; D001709:Biotechnology; D049872:Bivalvia / Q000201:enzymology* / Q000235:genetics; D002482:Cellulose; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008351:Mannans; D008361:Mannosidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013696:Temperature
D007527:Isoenzymes; D008351:Mannans; D002482:Cellulose; D008361:Mannosidases; C041867:endo-1,4-beta-D-mannanase