tailieunhanh - Báo cáo Y học: Puri®cation and properties of an alkaline proteinase of Fusarium culmorum

The diseaseFusarium head blight (scab) causes severe problems for farmers and for the industries that use cereals. It is likely that the fungi that cause scab (Fusariumspp.) use various enzymes when they invade grains. We are studying enzymes that the fungi may use to hydrolyze grain proteins. To do this,Fusarium culmorumwas grown in a gluten-con-taining medium from which an alkaline serine proteinase with a molecular mass of kDa was puri®ed by size-exclusion and cation exchange chromatographies | Eur. J. Biochem. 269 798-807 2002 FEBS 2002 Purification and properties of an alkaline proteinase of Fusarium culmorum Anja I. Pekkarinen1 2 Berne L. Jones3 and Marja-Leena Niku-Paavola2 1 Department of Agronomy University of Wisconsin-Madison USA 2VTT Biotechnology Finland 3USDA-ARS Cereal Crops Research Unit Madison WI USA 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-con-taining medium from which an alkaline serine proteinase with a molecular mass of kDa was purihed by sizeexclusion and cation exchange chromatographies. The enzyme was maximally active at pH and 50 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. Purihcation of the enzyme was complicated by the fact that when purihed it apparently underwent autolysis. The presence of extraneous protein stabilized the activity. Keywords cereal fungus chymotrypsin subtilisin proteinase. Fusarium head blight FHB scab has for many years been a serious problem for cereal producers and for the various cereal industries. The majority of past FHB epidemics have been caused by the fungus Fusarium graminearum Gibberella zeae but some infestations have also been .