RESUMO
Ficus carica produces, in addition to the cysteine protease ficin, a serine protease. Earlier study on a serine protease from F. carica cultivar Brown Turkey showed that it specifically degraded collagen. In this study, we characterized the collagenolytic activity of a serine protease in the latex of F. carica cultivar Masui Dauphine. The serine protease degraded denatured, but not undenatured, acid-solubilized type I collagen. It also degraded bovine serum albumin, while the collagenase from Clostridium histolyticum did not. These results indicated that the serine protease in Masui Dauphine is not collagen-specific. The protease was purified to homogeneity by two-dimensional gel electrophoresis, and its partial amino acid sequence was determined by liquid chromatography-tandem mass spectrometry. BLAST searches against the Viridiplantae (green plants) genome database revealed that the serine protease was a subtilisin-like protease. Our results contrast with the results of the earlier study stating that the serine protease from F. carica is collagen-specific.
Assuntos
Colágeno/química , Ficus/química , Látex/química , Proteínas de Plantas/metabolismo , Serina Proteases/metabolismo , Subtilisinas/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Eletroforese em Gel Bidimensional , Ficus/enzimologia , Expressão Gênica , Temperatura Alta , Látex/metabolismo , Extratos Vegetais/química , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Desnaturação Proteica , Proteólise , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Serina Proteases/química , Serina Proteases/genética , Serina Proteases/isolamento & purificação , Especificidade por Substrato , Subtilisinas/química , Subtilisinas/genética , Subtilisinas/isolamento & purificaçãoRESUMO
In this study, we characterized protease activities of 23 Ficus carica cultivars. Extracts of fruit, branch, and leaf of Masui Dauphine, one of the most representative F. carica cultivars in Japan, exhibited gelatin-hydrolyzing activity, both in the absence and presence of a cysteine protease-specific inhibitor, E-64, suggesting that not only ficin (classified as cysteine protease) but also collagenase (classified as serine protease) were involved in the digestion of gelatin. In the hydrolysis of (7-methoxycoumarin-4-yl)acetyl-l-Lys-l-Pro-l-Leu-Gly-l-Leu-[N3 -(2,4-dinitrophenyl)-l-2,3-diaminopropionyl]-l-Ala-l-Arg-NH2 , all branch extracts of 23 F. carica cultivars exhibited the activity both in the absence and presence of cysteine protease-specific inhibitor E-64, indicating that they contain ficin and collagenase. During digestion of acid-solubilized type I collagen by the branch extract of Masui Dauphine at 40-55 °C, collagen was completely digested in the absence of E-64, while it was partially digested in the presence of the inhibitor, indicating that the manner of digestion differed between ficin and collagenase contained in the extract. These results suggest that F. carica is attractive for industrial use to digest collagen. PRACTICAL APPLICATION: The industrial use of F. carica might be enhanced by efficiently utilizing these proteases and/or selecting the appropriate F. carica cultivar. Collagen is one of the targets to which our results might be applied. It is widely accepted today that collagen and its digestion products could be useful as functional food. F. carica is a potential candidate for use in not only complete but also partial digestion of collagen.