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Expression, activation and processing of a novel plant milk-clotting aspartic protease in Pichia pastoris.
J Biotechnol ; 268: 28-39, 2018 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-29339117
ABSTRACT
Galium verum, also known as Lady's Bedstraw or Cheese Rennet, is an herbaceous perennial plant traditionally used in cheese-making. We used RACE PCR to isolate novel enzymes from Galium verum with the ability to clot milk. This approach generated two cDNA sequences (named preprogaline A and B) encoding proteins displaying the typical plant aspartic protease primary structure. Preprogaline B was expressed in the yeast Pichia pastoris, after deleting and replacing its original signal peptide with the yeast α-factor signal peptide from Saccharomyces cerevisiae. The secreted recombinant protein was obtained by growing P. pastoris in YPD medium and had the ability to clot milk. The mature form of progaline B is a heterodimeric glycosylated enzyme, with a molecular weight of approximately 48 kDa, that contains a heavy (30.7 kDa) and a light (13.5 kDa) polypeptide chains linked by disulfide bonds. Western blot analysis revealed that progaline B is activated by the acidification of the yeast culture medium and that enzymatic activation requires two steps. First the precursor protein is cleaved into two polypeptide chains by partial removal of the plant-specific insert (PSI) present in plant aspartic proteases; this is later followed by propeptide removal. By altering the pH of the P. pastoris culture medium, we were able to obtain either active or inactive forms of the enzyme. Recombinant progaline B displayed a κ-casein hydrolysis pattern analogous to those produced by the animal and microbial coagulants currently used in the dairy industry, but it exhibited a different digestion profile on α- and ß-caseins. The plant protease progaline B displays milk-clotting activities suitable for the production of novel dairy products.
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Texto completo: Disponível Coleções: Bases de dados internacionais Base de dados: MEDLINE Assunto principal: Pichia / Ácido Aspártico Endopeptidases / Galium / Leite Limite: Animais Idioma: Inglês Revista: J Biotechnol Assunto da revista: Biotecnologia Ano de publicação: 2018 Tipo de documento: Artigo País de afiliação: Espanha