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ARS Home » Midwest Area » Madison, Wisconsin » Cereal Crops Research » Research » Publications at this Location » Publication #126005
Title: PURIFICATION AND PROPERTIES OF AN ALKALINE PROTEINASE OF FUSARIUM CULMORUM

Author
item PEKKARINEN, A - VTT BIOTECHNOLOGY FINLAND
item Jones, Berne
item NIKU-PAAVOLA, M - VTT BIOTECHNOLOGY FINLAND

Submitted to: European Journal of Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/23/2001
Publication Date: N/A
Citation: N/A

Interpretive Summary: During the last several years, the disease Fusarium head blight (scab) has caused severe losses for farmers and brewers. It is caused by a fungus called Fusarium. The Fusarium probably uses a group of enzymes called proteinases to attack grain. We are studying these enzymes to establish the roles they play in how the fungus causes the disease. To do this, we grew Fusarium in a broth that contained wheat protein and purified an enzyme that digests protein from it. When we studied the properties of this enzyme, we found that it was quite similar to enzymes that have been isolated from other molds. It is likely that the fungus uses this enzyme to get into the grain, but this need to be confirmed by further studies. When we know enough about the enzymes of Fusarium we will be able to use that knowledge to tell plant researchers how they can make plants that are resistant to the Fusarium. Development of Fusarium resistant plants will allow producers to grow better crops, will ensure a supply of quality grain for processors and consumers.

Technical Abstract: 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-enriched 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 50C, but was unable under those 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 phenylmethylsulfonyl fluoride and chymostatin, but not by soybean trypsin or Bowman-Birk inhibitors. Parts of its amino acid sequence were up to 82% homologous with those of several fungal subtilisins. One of its active site amino acids was detected and it was same as in the other subtilisins. 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. This proteinase likely plays a role in helping the fungus to colonize and utilize the grain.