Purification and properties of an alkaline proteinase of Fusarium culmorum.
The disease Fusarium head blight (scab) causes severe problems for farmers and for the industries that use cereals. It is likely that the fungi that cause scab (Fusarium spp.) use various enzymes when they invade grains. We are studying enzymes that the fungi may use to hydrolyze grain proteins. To do this, Fusarium culmorum was grown in a gluten-containing medium from which an alkaline serine proteinase with a molecular mass of 28.7 kDa was purified by size-exclusion and cation exchange chromatographies. The enzyme was maximally active at pH 8.3-9.6 and 50 degrees C, but was unstable under these conditions. It hydrolyzed the synthetic substrates N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide and, to a lesser extent, N-succinyl-Ala-Ala-Pro-Leu p-nitroanilide. It was inhibited by phenylmethanesulfonyl fluoride and chymostatin, but not by soybean trypsin or Bowman-Birk inhibitors. Parts of the amino-acid sequence were up to 82% homologous with those of several fungal subtilisins. One of the active site amino acids was detected and it occupied the same relative position as in the other subtilisins. Therefore, on the basis of these characteristics, the proteinase is subtilisin-like. Purification of the enzyme was complicated by the fact that, when purified, it apparently underwent autolysis. The presence of extraneous protein stabilized the activity.
Pekkarinen
Anja I
AI
Department of Agronomy, University of Wisconsin-Madison, USA. apekkarinen@facstaff.wisc.edu
Jones
Berne L
BL
Niku-Paavola
Marja-Leena
ML
D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004795:Enzyme Stability; D005670:Fusarium / Q000201:enzymology*; D005780:Gelatin / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
D005780:Gelatin; D012697:Serine Endopeptidases; C026109:oryzin