Purification, characterization, and partial primary sequence of a major-maltotriose-producing alpha-amylase, ScAmy43, from Sclerotinia sclerotiorum.
A novel alpha-amylase (alpha-1,4-alpha-D-glucan glucanohydrolase, E.C. 3.2.1.1), ScAmy43, was found in the culture medium of the phytopathogenic fungus Sclerotinia sclerotiorum grown on oats flour. Purified to homogeneity, ScAmy43 appeared as a 43 kDa monomeric enzyme, as estimated by SDS-PAGE and Superdex 75 gel filtration. The MALDI peptide mass fingerprint of ScAmy43 tryptic digest as well as internal sequence analyses indicate that the enzyme has an original primary structure when compared with other fungal alpha- amylases. However, the sequence of the 12 N-terminal residues is homologous with those of Aspergillus awamori and Aspergillus kawachii amylases, suggesting that the new enzyme belongs to the same GH13 glycosyl hydrolase family. Assayed with soluble starch as substrate, this enzyme displayed optimal activity at pH 4 and 55oC with an apparent Km value of 1.66 mg/ml and Vmax of 0.1 micromol glucose x min-1 x ml-1. ScAmy43 activity was strongly inhibited by Cu2+, Mn2+, and Ba2+, moderately by Fe2+, and was only weakly affected by Ca2+ addition. However, since EDTA and EGTA did not inhibit ScAmy43 activity, this enzyme is probably not a metalloprotein. DTT and beta-mercaptoethanol strongly increased the enzyme activity. Starting with soluble starch as substrate, the end products were mainly maltotriose, suggesting for this enzyme an endo action.
Ben Abdelmalek-Khedher
Imen
I
Institut National des Sciences Appliquees et de Technologie (INSAT), Centre Urbain Nord 676-1080 Tunis Cedex, Tunisia.
Urdaci
Maria Camino
MC
Limam
Ferid
F
Schmitter
Jean Marie
JM
Marzouki
M Nejib
MN
Bressollier
Philippe
P
D000595:Amino Acid Sequence; D000681:Amylases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001203:Ascomycota / Q000201:enzymology* / Q000235:genetics; D018554:Avena / Q000378:metabolism; D055162:Biocatalysis; D002413:Cations, Divalent / Q000494:pharmacology; D002845:Chromatography; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D010449:Peptide Mapping; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013213:Starch / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D002413:Cations, Divalent; D008670:Metals; D013213:Starch; D000681:Amylases; C086530:maltotriose-forming amylase; D000516:alpha-Amylases