Functional and structural characterization of synthetic cardosin B-derived rennet

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.

Plant Aspartic Proteases for Industrial Applications: Thistle Get Better

“…The single-chain recombinant cardosin A was active, with slight differences in its specificity for amino acids in the P1 position when compared to normal recombinant cardosin A [11]. The same strategy was used for the production of recombinant cardosin B, which also exhibited altered specificity [45]. The single-chain form showed a more stringent specificity and lower catalytic efficiency in comparison with the native cardosin B.…”

“…The single-chain form showed a more stringent specificity and lower catalytic efficiency in comparison with the native cardosin B. The authors explained this difference as being due to possible diminished flexibility of the enzyme in the C-terminal region due to the direct fusion of the heavy and light chains [45]. This fact is very important and could have a great impact in the successful application of synthetic cardosin B in cheese manufacturing at industrial scale.…”

Cynara cardunculus: Use in Cheesemaking and Pharmaceutical Applications

“…A laboratory in Spain has been selling a coagulant extract of plant origin for a number of years, claimed to be obtained from C. cardunculus. Almeida et al [130,131] have developed and characterized a new cardosin B-derived coagulant produced in the generally regarded as safe (GRAS) yeast K. lactis, which they claim to be effective in the manufacture of cheese from milk of different species. In 2015, a cyprosin was claimed to be authorized for use in Portugal, Spain, France and the Netherlands, and joined the list of authorized food enzymes in Canada, after favorable decision from Health Canada's Food Directorate [92], and since 2015 a number of applications by different entities for the inclusion of cardoon enzymes or extracts in the European list of food enzymes in accordance with Regulation (EC) No 1331/2008 are waiting for official approval.…”

Artichoke cv. Francés flower extract as a rennet substitute: effect on textural, microstructural, microbiological, antioxidant properties, and sensory acceptance of miniature cheeses

“…The most traditional milk‐clotting enzyme is chymosin, with its synthetic versions being by far the most common milk clotting enzymes used in cheese industry 6 . Rennet substitutes currently used in cheese production are aspartic endopeptidases (AP) from microbial and vegetable origin, the proteolytic properties of which are not exactly the same as those of chymosin 7 . An adequate RS is characterized by high specific hydrolytic activity and low general proteolytic activity 8 .…”

“…6 Rennet substitutes currently used in cheese production are aspartic endopeptidases (AP) from microbial and vegetable origin, the proteolytic properties of which are not exactly the same as those of chymosin. 7 An adequate RS is characterized by high specific hydrolytic activity and low general proteolytic activity. 8 In general, APs with milk clotting activity (MCA) meet both conditions, and are consequently favorite candidates in the search for RS.…”