@article{10902/38719,
year = {2017},
url = {https://hdl.handle.net/10902/38719},
abstract = {The potential of using a synthetic cardosin-based rennet in cheese manufacturing was recently demonstrated with the development and optimization of production of a recombinant form of cardosin B in Kluyveromyces lactis. With the goal of providing a more detailed characterization of this rennet, we herein evaluate the impact of the plant-specific insert (PSI) on cardosin B secretion in this yeast, and provide a thorough analysis of the specificity requirements as well as the biochemical and structural properties of the isolated recombinant protease. We demonstrate that the PSI domain can be substituted by different linker sequences without substantially affecting protein secretion and milk clotting activity. However, the presence of small portions of the PSI results in dramatic reductions of secretion yields in this heterologous system. Kinetic characterization and specificity profiling results clearly suggest that synthetic cardosin B displays lower catalytic efficiency and is more sequence selective than native cardosin B. Elucidation of the structure of synthetic cardosin B confirms the canonical fold of an aspartic protease with the presence of two high mannose-type, N-linked glycan structures; however, there are some differences in the conformation of the flap region when compared to cardosin A. These subtle variations in catalytic properties and the more stringent substrate specificity of synthetic cardosin B help to explain the observed suitability of this rennet for cheese production.},
organization = {This work was funded by Fundo Europeu de
Desenvolvimento Regional (FEDER) Funds through the Operational
Competitiveness Programme (COMPETE) and by National Funds
through Fundação para a Ciência e a Tecnologia (FCT) under projects
PTDC/AGR-ALI/102540/2008 and POCI-01-0145-FEDER-007274.
Support by the BStructured program on bioengineered therapies for infectious diseases and tissue regeneration^ (Norte-01-0145-FEDER-000012),
funded by Norte Portugal Regional Operational Programme (NORTE
2020), under the PORTUGAL 2020 Partnership Agreement, through
FEDER is also acknowledged. The authors would like to acknowledge
the strategic project UID/NEU/04539/2013 and the National Mass
Spectrometry Network (RNEM) under the contract REDE/1506/REM/
2005. Edman sequencing data were obtained by the Analytical
Laboratory, Analytical Services Unit, Instituto de Tecnologia Quıímica
e Biológica, Universidade Nova de Lisboa. We acknowledge the ESRF
for provision of synchrotron radiation facilities and thank the ESRF staff
for the help with data collection. The authors would like to thank Sandra
Isabel Anjo for her inputs on acquisition of MS data.},
publisher = {Springer Nature},
publisher = {Applied Microbiology and Biotechnology, 2017, 101, 6951-6968},
title = {Functional and structural characterization of synthetic cardosin B-derived rennet},
author = {Malaquias Almeida, Carla and Manso García, José Antonio and Figueiredo, Ana C. and Antunes, Liliana and Cruz, Rui and Manadas, Bruno and Bur, Daniel and Barbosa Pereira, Pedro José and Faro, Carlos and Simoes, Isaura},
}