Purification and properties of an alkaline proteinase of Fusarium culmorum

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-containing medium from which an alkaline serine proteinase with a molecular mass of 28.7 kDa was puri®ed by sizeexclusion and cation exchange chromatographies. The enzyme was maximally active at pH 8.3±9.6 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¯u oride 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. Puri®cation of the enzyme was complicated by the fact that, when puri®ed, it apparently underwent autolysis. The presence of extraneous protein stabilized the activity.

Enhanced production of industrial enzymes in Mucoromycotina fungi during solid-state fermentation of agricultural wastes/by-products

“…The secreted proteolytic enzyme exhibited the highest activity towards the chymotrypsin substrate Suc-AAPF-pNA, indicating that it has endoproteolytic activity, and a preference for phenylalanine. Similar substrate specificities have been described for alkaline proteases from A. fumigatus [18] and Fusarium culmorum [23]. The enzyme from F. culmorum was considered to be a subtilisin-like protease, which implies that the extracellular proteolytic activity of Rh.…”

Purification and properties of an alkaline protease of Aspergillus clavatus

“…The protease was unstable above this temperature. Several alkaline proteases from Aspergillus species have an optimum temperature at 40°C (Pekkarinen et al 2002). Calcium at concentration of 5 mM increased the thermal stability of the enzyme.…”

A New Subtilase-Like Protease Deriving from Fusarium equiseti with High Potential for Industrial Applications

“…The enzyme is unstable after purification, especially in alkaline solutions and it appears to be prone to autoproteolysis. The enzyme loses its activity completely after incubation under slightly alkaline pH conditions at 40°C for 90 min [25].…”