caa a22 4500 605497974 CHVBK 20210128100543.0 cr unu---uuuuu 210128e20150101xx s 000 0 eng 10.1007/s00253-014-5902-5 doi (NATIONALLICENCE)springer-10.1007/s00253-014-5902-5 Engineering a cardosin B-derived rennet for sheep and goat cheese manufacture [Elektronische Daten] [Carla Almeida, David Gomes, Carlos Faro, Isaura Simões] Different sheep and goat cheeses with world-renowned excellence are produced using aqueous extracts of Cynara cardunculus flowers as coagulants. However, the use of this vegetable rennet is mostly limited to artisanal scale production, and no effective solutions to large-scale industrial applications have been reported so far. In this sense, the development of a synthetic rennet based on the most abundant cardoon milk-clotting enzymes (cardosins) would emerge as a solution for scalability of production and for application of these proteases as alternative rennets in dairy industry. In this work, we report the development of a new cardosin B-derived rennet produced in the generally regarded as safe (GRAS) yeast Kluyveromyces lactis. Using a stepwise optimization strategy—consisting of culture media screening, complemented with a protein engineering approach with removal of the plant-specific domain, and a codon optimization step—we successfully improved cardosin B production yield (35×) in K. lactis. We demonstrated that the secreted enzyme displays similar proteolytic properties, such as casein digestion profiles as well as optimum pH (pH 4.5) and temperature (40°C), with those of native cardosin B. From this optimization process resulted the rennet preparation Vegetable Rennet (VRen), requiring no downstream protein purification steps. The effectiveness of VRen in cheese production was demonstrated by manufacturing sheep, goat, and cow cheeses. Interestingly, the use of VRen resulted in a higher cheese yield for all three types of cheese when compared with synthetic chymosin. Altogether, these results clearly position VRen as an alternative/innovative coagulant for the cheese-making industry. Springer-Verlag Berlin Heidelberg, 2014 Rennet nationallicence Aspartic protease nationallicence Cheese nationallicence Vegetable nationallicence Cardosins nationallicence Cynara cardunculus nationallicence Plant nationallicence Kluyveromyces lactis nationallicence Almeida Carla Biocant, Biotechnology Innovation Center, Parque Tecnológico de Cantanhede, Núcleo 4, Lote 3, 3060-197, Cantanhede, Portugal aut Gomes David Instituto Politécnico de Coimbra—Escola Agrária, Bencanta, 3045-601, Coimbra, Portugal aut Faro Carlos Biocant, Biotechnology Innovation Center, Parque Tecnológico de Cantanhede, Núcleo 4, Lote 3, 3060-197, Cantanhede, Portugal aut Simões Isaura Biocant, Biotechnology Innovation Center, Parque Tecnológico de Cantanhede, Núcleo 4, Lote 3, 3060-197, Cantanhede, Portugal aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/1(2015-01-01), 269-281 0175-7598 99:1<269 2015 99 253 https://doi.org/10.1007/s00253-014-5902-5 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-014-5902-5 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Almeida Carla Biocant, Biotechnology Innovation Center, Parque Tecnológico de Cantanhede, Núcleo 4, Lote 3, 3060-197, Cantanhede, Portugal aut NATIONALLICENCE 700 1- Gomes David Instituto Politécnico de Coimbra—Escola Agrária, Bencanta, 3045-601, Coimbra, Portugal aut NATIONALLICENCE 700 1- Faro Carlos Biocant, Biotechnology Innovation Center, Parque Tecnológico de Cantanhede, Núcleo 4, Lote 3, 3060-197, Cantanhede, Portugal aut NATIONALLICENCE 700 1- Simões Isaura Biocant, Biotechnology Innovation Center, Parque Tecnológico de Cantanhede, Núcleo 4, Lote 3, 3060-197, Cantanhede, Portugal aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/1(2015-01-01), 269-281 0175-7598 99:1<269 2015 99 253