The function of the milk-clotting enzymes bovine and camel chymosin studied by a fluorescence resonance energy transfer assay.

Enzymatic coagulation of bovine milk can be divided in 2 steps: an enzymatic step, in which the Phe105-Met106 bond of the milk protein bovine κ-casein is cleaved, and an aggregation step. The aspartic peptidases bovine and camel chymosin (EC 3.4.23.4) are typically used to catalyze the enzymatic step. The most commonly used method to study chymosin activity is the relative milk-clotting activity test that measures the end point of the enzymatic and aggregation step. This method showed that camel chymosin has a 2-fold higher milk-clotting activity toward bovine milk than bovine chymosin. To enable a study of the enzymatic step independent of the aggregation step, a fluorescence resonance energy transfer assay has been developed using a peptide substrate derived from the 98-108 sequence of bovine κ-casein. This assay and Michaelis-Menten kinetics were employed to determine the enzymatic activity of camel and bovine chymosin under milk clotting-like conditions (pH 6.65, ionic strength 80 mM). The results obtained show that the catalytic efficiency of camel chymosin is 3-fold higher than bovine chymosin. The substrate affinity and catalytic activity of bovine and camel chymosin increase at lower pH (6.00 and 5.50). The glycosylation of bovine and camel chymosin did not affect binding of the fluorescence resonance energy transfer substrate, but doubly glycosylated camel chymosin seems to have slightly higher catalytic efficiency. In the characterization of the enzymes, the developed assay is easier and faster to use than the traditionally used relative milk-clotting activity test method.

Camel and bovine chymosin: the relationship between their structures and cheese-making properties.

Bovine and camel chymosin are aspartic peptidases that are used industrially in cheese production. They cleave the Phe105-Met106 bond of the milk protein κ-casein, releasing its predominantly negatively charged C-terminus, which leads to the separation of the milk into curds and whey. Despite having 85% sequence identity, camel chymosin shows a 70% higher milk-clotting activity than bovine chymosin towards bovine milk. The activities, structures, thermal stabilities and glycosylation patterns of bovine and camel chymosin obtained by fermentation in Aspergillus niger have been examined. Different variants of the enzymes were isolated by hydrophobic interaction chromatography and showed variations in their glycosylation, N-terminal sequences and activities. Glycosylation at Asn291 and the loss of the first three residues of camel chymosin significantly decreased its activity. Thermal differential scanning calorimetry revealed a slightly higher thermal stability of camel chymosin compared with bovine chymosin. The crystal structure of a doubly glycosylated variant of camel chymosin was determined at a resolution of 1.6 Šand the crystal structure of unglycosylated bovine chymosin was redetermined at a slightly higher resolution (1.8 Å) than previously determined structures. Camel and bovine chymosin share the same overall fold, except for the antiparallel central ß-sheet that connects the N-terminal and C-terminal domains. In bovine chymosin the N-terminus forms one of the strands which is lacking in camel chymosin. This difference leads to an increase in the flexibility of the relative orientation of the two domains in the camel enzyme. Variations in the amino acids delineating the substrate-binding cleft suggest a greater flexibility in the ability to accommodate the substrate in camel chymosin. Both enzymes possess local positively charged patches on their surface that can play a role in interactions with the overall negatively charged C-terminus of κ-casein. Camel chymosin contains two additional positive patches that favour interaction with the substrate. The improved electrostatic interactions arising from variation in the surface charges and the greater malleability both in domain movements and substrate binding contribute to the better milk-clotting activity of camel chymosin towards bovine milk.

Monitoring the aggregation of single casein micelles using fluorescence microscopy.

The aggregation of casein micelles (CMs) induced by milk-clotting enzymes is a process of fundamental importance in the dairy industry for cheese production; however, it is not well characterized on the nanoscale. Here we enabled the monitoring of the kinetics of aggregation between single CMs (30-600 nm in diameter) by immobilizing them on a glass substrate at low densities and subsequently imaging them with fluorescence microscopy. We validated the new method by a quantitative comparison to ensemble measurements of aggregation. Single-particle statistics allowed us to observe for the first time several heterogeneities in CM aggregation. We observed two types of CM growth: a slow increase in the size of CMs and a stepwise increase attributed to interactions between aggregates preformed in solution. Both types of growth exhibit a lag phase that was very heterogeneous between different CMs, suggesting significant differences in their composition or structure. Detailed size histograms of CMs during aggregation also revealed the presence of two distinct subpopulations with different growth amplitudes and kinetics. The dependence of these distinct nanoscale processes/parameters on aggregation conditions is not accessible to bulk measurements that report only ensemble-average values and may prove important to an in-depth understanding of CM aggregation.

Bovine chymosin: a computational study of recognition and binding of bovine kappa-casein.

Bovine chymosin is an aspartic protease that selectively cleaves the milk protein kappa-casein. The enzyme is widely used to promote milk clotting in cheese manufacturing. We have developed models of residues 97-112 of bovine kappa-casein complexed with bovine chymosin, using ligand docking, conformational search algorithms, and molecular dynamics simulations. In agreement with limited experimental evidence, the model suggests that the substrate binds in an extended conformation with charged residues on either side of the scissile bond playing an important role in stabilizing the binding pose. Lys111 and Lys112 are observed to bind to the N-terminal domain of chymosin displacing a conserved water molecule. A cluster of histidine and proline residues (His98-Pro99-His100-Pro101-His102) in kappa-casein binds to the C-terminal domain of the protein, where a neighboring conserved arginine residue (Arg97) is found to be important for stabilizing the binding pose. The catalytic site (including the catalytic water molecule) is stable in the starting conformation of the previously proposed general acid/base catalytic mechanism for 18 ns of molecular dynamics simulations.

Initial stage of cheese production: a molecular modeling study of bovine and camel chymosin complexed with peptides from the chymosin-sensitive region of κ-casein.

Bovine chymosin has long been the preferred enzyme used to coagulate cow's milk, in the initial stage of cheese production, during which it cleaves a specific bond in the milk protein κ-casein. Recently, camel chymosin has been shown to have a 70% higher clotting activity toward cow's milk and, moreover, to cleave κ-casein more selectively. Bovine chymosin, on the other hand, is a poor clotting agent toward camel's milk. This paper reports a molecular modeling study aimed at understanding this disparity, based on homology modeling and molecular dynamics simulations using peptide fragments of κ-casein from cow and camel in both bovine and camel chymosin. The results show that the complex between bovine chymosin and the fragment of camel κ-casein is indeed less stable in the binding pocket. The results also indicate that this in part may be due to charge repulsion between a lysine residue in bovine chymosin and an arginine residue in the P4 position of camel κ-casein.

Biophysical consequences of remodeling the neutral side chains of rhamnogalacturonan I in tubers of transgenic potatoes.

Two lines of transgenic potato (Solanum tuberosum L.) plants modified in their cell wall structure were characterized and compared to wild type with regard to biomechanical properties in order to assign functional roles to the particular cell wall polysaccharides that were targeted by the genetic changes. The targeted polymer was rhamnogalacturonan I (RG-I), a complex pectic polysaccharide comprised of mainly neutral oligosaccharide side chains attached to a backbone of alternating rhamnosyl and galacturonosyl units. Tuber rhamnogalacturonan I molecules from the two transformed lines are reduced in linear galactans and branched arabinans, respectively. The transformed tuber tissues were found to be more brittle when subjected to uniaxial compression and the side-chain truncation was found to be correlated with the physical properties of the tissue. Interpretation of the force-deflection curves was aided by a mathematical model that describes the contribution of the cellulose microfibrils, and the results lead to the proposition that the pectic matrix plays a role in transmitting stresses to the load-bearing cellulose microfibrils and that even small changes to the rheological properties of the matrix have consequences for the biophysical properties of the wall.