Applied Microbiology and Biotechnology

, Volume 101, Issue 18, pp 6951–6968 | Cite as

Functional and structural characterization of synthetic cardosin B-derived rennet

  • Carla Malaquias Almeida
  • José A. Manso
  • Ana C. Figueiredo
  • Liliana Antunes
  • Rui Cruz
  • Bruno Manadas
  • Daniel Bur
  • Pedro José Barbosa Pereira
  • Carlos Faro
  • Isaura Simões
Biotechnologically Relevant Enzymes and Proteins


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.


PSI Aspartic protease Glycosylation Kluyveromyces lactis Cardosin B Rennet Milk clotting Specificity 3D structure 



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 “Structured 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.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Human and Animal Rights and Informed Consent

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2017_8445_MOESM1_ESM.pdf (588 kb)
ESM 1 (PDF 587 kb)


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Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • Carla Malaquias Almeida
    • 1
  • José A. Manso
    • 2
    • 3
  • Ana C. Figueiredo
    • 2
    • 3
  • Liliana Antunes
    • 1
    • 4
  • Rui Cruz
    • 1
    • 5
  • Bruno Manadas
    • 5
  • Daniel Bur
    • 6
  • Pedro José Barbosa Pereira
    • 2
    • 3
  • Carlos Faro
    • 1
    • 5
  • Isaura Simões
    • 1
    • 5
  1. 1.Biocant, Biotechnology Innovation CenterParque Tecnológico de CantanhedeCantanhedePortugal
  2. 2.IBMC, Instituto de Biologia Molecular e CelularUniversidade do PortoPortoPortugal
  3. 3.Instituto de Investigação e Inovação em SaúdeUniversidade do PortoPortoPortugal
  4. 4.Wellcome Trust Sanger Institute, Wellcome Trust Genome CampusHinxtonUK
  5. 5.CNC-Center for Neuroscience and Cell BiologyUniversity of CoimbraCoimbraPortugal
  6. 6.Actelion Pharmaceuticals Ltd.AllschwilSwitzerland

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