Potencial biotecnológico de uma protease cisteínica do látex de Calotropis procera para produção de queijo

• Autor(a) principal: Silva, Maria Zelândia Rocha
• Data de Publicação: 2019
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: Repositório Institucional da Universidade Federal do Ceará (UFC)
• Texto Completo: http://www.repositorio.ufc.br/handle/riufc/43429
• topic: coagulação do leite; microscopia de força atômica; protease de planta; expressão heteróloga

Although various plant proteases with milk-clotting activity have been described, most of them produces cheeses with bitter tastes. In contrast, different proteolytic extracts from Calotropis procera plant have been used successfully for cheesemaking. Because there are several disadvantages for using proteolytic extracts, the aims of this work were to characterize and evaluate the biotechnological potential of a cysteine protease purified from C. procera latex, named CpCP3. Thus, with the study of the milk coagulation process by the commercial chymosin and CpCP3 by atomic force microscopy (AFM).This enzyme was highly stable to different metal ions and was able to hydrolyse κ-casein similarly to bovine chymosin. Atomic force microscopy showed that the whole process of casein micelles aggregation induced by CpCP3 was very similar to that caused by chymosin. The cheeses made using CpCP3 showed higher moisture content than those made with chymosin, but protein, fat, and ash were similar. In silico analysis predicted the presence of only four allergenic peptides in CpCP3, but only two of these were present on its three-dimensional surface. CpCP3 was highly susceptible to digestive enzymes, pepsin and trypsin, including its allergenic peptides. Even using high doses, CpCP3 did not show toxicity on zebrafish embryos, an animal model widely used for in vivo toxicity test. In addition, CpCP3 was highly expressed in Escherichia coli cells, but in the insoluble form and without proteolytic activity. All results support the biotechnological potential of CpCP3 as an alternative enzyme to replace chymosin. Further studies should be performed to optimize the heterologous expression of CpCP3 to reach its proteolytic activity.