Kinetic study of the action of bovine chymosin and pepsin A on bovine kappa-casein

A study was carried out to determine the Michaelian parameters relative to the action of chymosin and pepsin A on bond Phe105-Met106 of bovine kappa0-casein (carbohydrate-free fraction in micellar state). The reaction was performed in citrate buffer, pH 6.2, at 30 degrees C. The reaction mixture was analysed by reverse phase HPLC. Dosages of peptide 106-169 (caseino macropeptide) at different reaction times from recordings of its absorbance at 220 nm gave the initial rates of reaction at each substrate concentration. From these values the following parameters were determined: kcat = 68.5 s-1, Km = 0.048 mM, kcat/Km = 1,413 mM-1 s-1 for chymosin, and kcat = 45 s-1, Km = 0.018 mM, kcat/Km = 2,439 mM-1 s-1 for pepsin A. For chymosin they are similar to those obtained previously in dimethyl glutarate buffer, pH 6.6, at 30 degrees C, using fragment 98-111 of kappa-casein as substrate. It can thus be concluded that neither the micellar state nor the presence of the whole peptide chain of kappa-casein (our conditions) significantly affect the action of chymosin on fragment 98-111, which seems to contain all information that makes bond 105-106 highly sensitive to chymosin. For pepsin A, only the information contained in fragment 103-108 appears to be required.     

Hydrolysis of beta-casein by gastric proteases. I. Comparison of proteolytic action of bovine chymosin and pepsin A

Hydrolysis of beta A2-casein by bovine chymosin and pepsin A was performed in order to compare the hydrolysis of the two enzymes on this protein. Different conditions have been tested: pH 5.5 for 116h and pH 3.5 for 7 h [E/S = 1/100 (w/w)] for chymosin. pH 3.0 for 24 h [E/S = 1/1000 (w/w)] for pepsin A. Under these conditions 17 peptides were obtained after the action of chymosin and 23 after the action of pepsin A. They corresponded respectively to the cleavage of 14 and 15 peptide bonds for chymosin and pepsin A. However, six of the peptide bonds were only hydrolyzed by chymosin and seven other bonds only by pepsin A. Our results showed a preferential splitting at the Leu-X, Ser-X, and Trp-X bonds for chymosin and Leu-X, Met-X, and Thr-X, for pepsin A. Some of the identified peptides contained sequences with possible physiological roles.     

Isolation and properties of human kappa-casein

Human kappa-casein was isolated from human whole casein by gel filtration with Sephadex G-200 and hydroxylapatite chromatography in the presence of sodium dodecyl sulfate (SDS). The kappa-casein was calcium-insensitive and did stabilize human beta-casein and bovine alpha s1-casein against precipitation by calcium ions. Formation of micelles from human beta- and kappa-caseins, and calcium ions was confirmed by electron microscopic observation. On SDS-polyacrylamide gel electrophoresis (SDS-PAGE), a single band was obtained. The formation of para-kappa-caseins by chymosin was confirmed by SDS-PAGE. Two para-kappa-caseins with apparent molecular weights of 13,000 and 11,000 appeared. The molecular weight of intact human kappa-casein was estimated to be approximately 33,000. The human kappa-casein contained about 40% carbohydrate (15% galactose, 3% fucose, 15% hexosamines, and 5% sialic acid) and 0.10% (1 mol/mol) phosphorus. Its amino acid composition was similar to that of bovine kappa-casein except for serine, glutamic acid, and lysine contents.     

Detection of bovine αs1-casein genomic variants using the allele-specific polymerase chain reaction

A method was developed to distinguish between genotypic variants B and C of bovine alpha s1-casein, using the allele-specific polymerase chain reaction (ASPCR). The alpha s1-casein genotype determined for 17 Jersey cows by the ASPCR method was confirmed by typing the alpha s1-casein milk proteins on isoelectric focusing gels. Using the ASPCR method described, rapid analysis of the alpha s1-casein genotype of bulls is now possible. In addition, kappa-casein genotypes can be determined from the same PCR reaction.     

Direct Effect of Sodium Nitrite on Extracted Histones

Irpex lacteus milk-clotting enzyme hydrolyzed the Phe(105)-Met(106) bond of κ-casein, causing the precipitation of para-κ-casein along with other casein fractions in the presence of calcium ions, with a mechanism similar to other milk-clotting enzymes. Furhtermore, Irpex enzyme hydrolyzed at the positions Leu(79)-Ser(80) and Tyr(30)-Val(31) of para-κ-casein.

Degradation patterns of β-casein by Irpex and Mucor miehei enzymes were almost the same by polyacrylamide gel electrophoresis, but Endothia parasitica enzyme showed a different degradation pattern. Under the conditions employed, β-casein appeared to be scarcely hydrolyzed by chymosin.

Comparing the specificity of Irpex enzyme on β-casein with that of chymosin, the common cleaving points were Leu(165)-Ser(166), Ala(189)-Phe(190), and Tyr(192)-Glu(193). The difference in the specificity between the enzymes was exhibited in the cleavage at the Leu(139)-Leu(140) bond by chymosin and of the Ser(142)-Trp(143) bond by Irpex enzyme. Although the cleaving points of β-casein by both enzymes resembled each other, each enzyme exhibited different degradation patterns of β-casein because of thier different order of cleavage.     

Electrophoretic study of the caseinolytic activity of a pepsin from the dogfish Scyliorhinus canicula

Electrophoretic patterns of casein and casein subfractions were studied following proteolysis by dogfish pepsin II or calf chymosin. Both enzymes hydrolyze the kappa casein subfraction with the production of kappa paracasein peptide. alpha S1 and beta subfractions hydrolysis is stronger with dogfish enzyme than with chymosin. It is concluded that, despite a broader specificity, the activity spectrum of dogfish enzyme is, in many respects, similar to that of calf chymosin.     

Comparative Study of Action of Cell Wall Proteinases from Various Strains of Streptococcus cremoris on Bovine alpha(s1)-, beta-, and kappa-Casein

Partially purified cell wall proteinases of eight strains of Streptococcus cremoris were compared in their action on bovine alpha(s1)-, beta-, and kappa-casein, as visualized by starch gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and thin-layer chromatography. Characteristic degradation profiles could be distinguished, from which the occurrence of two proteinases, represented by strain HP and strain AM(1), was concluded. The action of the HP-type proteinase P(1) (also detectable in strains Wg(2), C(13), and TR) was established by electrophoretic methods to be directed preferentially towards beta-casein. The AM(1)-type proteinase P(III) (also detectable in strain SK(11)) was also able to degrade beta-casein, but at the same time split alpha(s1)- and kappa-casein more extensively than did P(I). Strain FD(27) exhibited mainly P(I) activity but also detectable P(III) degradation characteristics. The cell wall proteinase preparation of strain E(8) showed low P(I) as well as low P(III) activity. All proteinase preparations produced from kappa-casein positively charged degradation products with electrophoretic mobilities similar to those of degradation products released by the action of the milk-clotting enzyme chymosin. The differences between P(I) and P(III) in mode of action, as detected by gel electrophoresis and thin-layer chromatography, were reflected by the courses of the initial degradation of methyl-C-labeled beta-casein and by the effect of alpha(s1)- plus kappa-casein on these degradations. The results are discussed in the light of previous comparative studies of cell wall proteinases in strains of S. cremoris and with respect to the growth of this organism in milk.     

Characterization of the particles of purified kappa-casein: trypsin as a probe of surface-accessible residues

-Casein as purified from bovine milk exhibits a rather unique disulfide bonding pattern as revealed by SDS–PAGE. The disulfide-bonded caseins present range from dimer to octamer and above and preparations contain about 10% monomer. All of these heterogeneous polymers, however, self-associate into nearly spherical particles with an average diameter of 13 nm at pH 8.0, as revealed by negatively stained transmission electron micrographs and dynamic light scattering. The weight-average molecular weight of the aggregates at pH 8.0, as judged by analytical ultracentrifugation, is 648,000. Trypsin digestion at pH 8.0 was used to probe the surface groups of the -casein A polymers. The reaction with trypsin was rapid and the peptides liberated were identified by separation with reverse-phase HPLC, amino acid analysis, and protein sequencing. The most rapidly released peptides (t _1/2 < 30 sec) were from cleavage at Arg 97 and Lys residues 111 and 112. These results suggest a surface orientation for these residues, and the data are in accord with earlier proposed 3D predictive models for -casein. It is speculated that Arg 97, together with adjacent His residues (98 and 100) and Lys residues 111 and 112, form two positively charged clusters on the surface of the otherwise negatively charged casein. These clusters bracket the neutral chymosin cleavage site (whose hydrolysis triggers a well-known digestive process) and so these clusters may facilitate docking of the substrate caseins with chymosin.     

Comparison of conformations of κ-casein,para-κ-casein and glycomacropeptide

The conformation of κ-casein was compared with those of para-κ-casein and glycomacropeptide formed by the cleavage of κ-casein with chymosin. Para-κ-casein is insoluble in water at room temperature, but is slightly soluble in 0.07 M NaCl (pH 7.0) at 3°C. The secondary structure of κ-casein, para-κ-casein and glycomacropeptide was estimated from CD spectra measured at 3°C by the method of Yada and that of Provencher and Glockner. The surface hydrophobicity of these molecules was estimated by the fluorometric method. It was concluded that the secondary structures of para-κ-casein and glycomacropeptide segments were changed slightly by cleavage with chymosin. Para-κ-casein was estimated to have more β-sheet structure than glycomacropeptide. Para-κ-casein had larger hydrophobic regions on the molecular surface compared with the corresponding part in κ-casein.     

Evolution of the casein multigene family: conserved sequences in the 5'' flanking and exon regions.

The rat alpha- and bovine alpha s1-casein genes have been isolated and their 5' sequences determined. The rat alpha-, beta-, gamma- and bovine alpha s1-casein genes contain similar 5' exon arrangements in which the 5' noncoding, signal peptide and casein kinase phosphorylation sequences are each encoded by separate exons. These findings support the hypothesis that during evolution, the family of casein genes arose by a process involving exon recruitment followed by intragenic and intergenic duplication of a primordial gene. Several highly conserved regions in the first 200 base pairs of the 5' flanking DNA have been identified. Additional sequence homology extending up to 550 base pairs upstream of the CAP site has been found between the rat alpha- and bovine alpha s1-casein sequences. Unexpectedly, the 5' flanking promoter regions are conserved to a greater extent than both the entire mature coding and intron regions of these genes. These conserved 5' flanking sequences may contain potential cis regulatory elements which are responsible for the coordinate expression of the functionally-related casein genes during mammary gland development.     

Amyloid fibril formation by bovine milk alpha s2-casein occurs under physiological conditions yet is prevented by its natural counterpart, alpha s1-casein

The calcified proteinaceous deposits, or corpora amylacea, of bovine mammary tissue often comprise a network of amyloid fibrils, the origins of which have not been fully elucidated. Here, we demonstrate by transmission electron microscopy, dye binding assays, and X-ray fiber diffraction that bovine milk alpha s2-casein, a protein synthesized and secreted by mammary epithelial cells, readily forms fibrils in vitro. As a component of whole alpha s-casein, alpha s2-casein was separated from alpha s1-casein under nonreducing conditions via cation-exchange chromatography. Upon incubation at neutral pH and 37 degrees C, the spherical particles typical of alpha s2-casein rapidly converted to twisted, ribbon-like fibrils approximately 12 nm in diameter, which occasionally formed loop structures. Despite their irregular morphology, these fibrils possessed a beta-sheet core structure and the ability to bind amyloidophilic dyes such as thioflavin T. Fibril formation was optimal at pH 6.5-6.7 and was promoted by higher incubation temperatures. Interestingly, the protein appeared to be less prone to fibril formation upon disulfide bond reduction with dithiothreitol. Thus, alpha s2-casein is particularly susceptible to fibril formation under physiological conditions. However, our findings indicate that alpha s2-casein fibril formation is potently inhibited by its natural counterpart, alpha s1-casein, while is only partially inhibited by beta-casein. These findings highlight the inherent propensity of casein proteins to form amyloid fibrils and the importance of casein-casein interactions in preventing such fibril formation in vivo.     

Comparative Study of Action of Cell Wall Proteinases from Various Strains of Streptococcus cremoris on Bovine αs1-, β-, and κ-Casein

Partially purified cell wall proteinases of eight strains of Streptococcus cremoris were compared in their action on bovine α_s1-, β-, and κ-casein, as visualized by starch gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and thin-layer chromatography. Characteristic degradation profiles could be distinguished, from which the occurrence of two proteinases, represented by strain HP and strain AM₁, was concluded. The action of the HP-type proteinase P₁ (also detectable in strains Wg₂, C₁₃, and TR) was established by electrophoretic methods to be directed preferentially towards β-casein. The AM₁-type proteinase P_III (also detectable in strain SK₁₁) was also able to degrade β-casein, but at the same time split α_s1- and κ-casein more extensively than did P_I. Strain FD₂₇ exhibited mainly P_I activity but also detectable P_III degradation characteristics. The cell wall proteinase preparation of strain E₈ showed low P_I as well as low P_III activity. All proteinase preparations produced from κ-casein positively charged degradation products with electrophoretic mobilities similar to those of degradation products released by the action of the milk-clotting enzyme chymosin. The differences between P_I and P_III in mode of action, as detected by gel electrophoresis and thin-layer chromatography, were reflected by the courses of the initial degradation of methyl-¹⁴C-labeled β-casein and by the effect of α_s1- plus κ-casein on these degradations. The results are discussed in the light of previous comparative studies of cell wall proteinases in strains of S. cremoris and with respect to the growth of this organism in milk.     

Mechanistic study of the urocanase reaction using deuterated substrates and 1H-NMR spectroscopy

1. Samples of (alpha-2H1, 5-2H1) and (alpha-2H1, beta-2H1) urocanic acid were prepared by a combination of chemical and enzymic methods. 2. The enzymic conversion of unlabelled urocanate was followed by 1H-NMR spectroscopy at 500 MHz in deuterium oxide. It was found (a) that urocanase promotes the exchange of the 5-hydrogen atom of the substrate faster than it catalyses the overall reaction, (b) that the product is an equilibrium mixture of racemic beta-(5-oxoimidazol-4-yl)propionate and beta-(5-hydroxyimidazol-4-yl)propionate and (c) that beta-(5-oxoimidazol-4-yl)-propionate is spontaneously hydrolysed under physiological conditions to N-formylisoglutamine. The rate of this hydrolysis is considerably diminished at +8 degrees C. 3. It was shown by ultraviolet and 1H-NMR spectroscopic measurements that beta-(5-hydroxyimidazol-4-yl)-propionate (gamma max approximately equal to 234 nm) exists in protonated from at low pH (less than 1) whereas pH (approximately equal to 7.5) it exists in equilibrium with beta-(5-oxoimidazol-4-yl)propionate (gamma max approximately equal to 269 nm). 4. (alpha-2H1, beta-2H1)Urocanate was reacted with urocanase in deuterium oxide. 1H-NMR spectroscopy at 500 MHz showed a slight incorporation of protium into the side-chain of the product. The incorporated protium corresponded roughly to the protium contamination of the solvent and was equally distributed between the alpha and beta positions. No transfer of the 5-hydrogen atom to the side-chain was detected. 5. Kinetic deuterium isotope effects of between 2 and 3 were measured when the urocanase reaction was conducted in deuterium oxide at different p2H values. 6. Implications of these findings for the mechanism of action of urocanase are discussed.     

Anomalous behavior of bovine alpha s1- and beta-caseins on gel electrophoresis in sodium dodecyl sulfate buffers

Electrophoresis in the presence of sodium dodecyl sulfate (SDS) provides a relatively simple means of determining molecular weights of proteins. This technique relies on the validity of a correlation between some function of Mr and the mobility of the protein through the gel matrix. However, bovine caseins (especially alpha s1-casein) have lower mobilities than expected on the basis of their known Mr. The binding of SDS to both alpha s1-casein (Mr 23,600) and beta-casein (Mr 24,000) reached a maximum at the slightly low value of 1.3 g SDS/g protein. Gel-filtration chromatography showed, however, that the alpha s1-casein:SDS complex was larger than the beta-casein:SDS complex at pH 6.8 or 7.0, but that they were similar in size at pH 2.9 or 3.0. Circular dichroism spectra indicated that the low helical structure content of both alpha s1- and beta-casein increased with the addition of SDS and/or decreasing the pH to 1.5. 13C NMR results showed that SDS bound to alpha s1- and beta-casein in the same way as it did to bovine serum albumin. Either esterification or dephosphorylation followed by amidation of alpha s1-casein increased its mobility in SDS-gel electrophoresis, but neither modification affected beta-casein mobility. These and other results indicate that the low electrophoretic velocity of alpha s1-casein in SDS-gel electrophoresis results from its unexpectedly large hydrodynamic size. This is caused by localized high negative charges on certain segments of alpha s1-casein, which would induce a considerable amount of inter- and intrasegmental electrostatic repulsion, leading to an expanded or extended structure for portions of the alpha s1-casein molecule in the presence of SDS. It is clear that the conformation, and hence the equivalent radius, of an SDS:protein complex is determined by the sequence of amino acids in the protein and that, a priori, it cannot be anticipated that the electrophoretic mobility of such a complex will bear more than a casual relationship to the Mr of the protein.     

The secondary structure of peptides derived from caseins: a circular dichroism study

Three peptides have been formed by proteolytic digestion of individual casein proteins and their secondary structures characterised by far-UV circular dichroism (CD). Peptide alpha s1(1-23), residues 1-23 of alpha s1-casein, was generated by treatment of the parent protein with chymosin. Peptides beta(1-28) and beta(1-52), residues 1-28 and 1-52 of beta-casein, were plasmin- and chymotrypsin-generated fragments, respectively. Analysis of the CD spectra revealed that in aqueous solution all three peptides have secondary structures composed exclusively of beta-sheet and random coil. A limited amount of alpha-helix was formed in two of the three peptides upon treatment with high concentrations (greater than 40% (v/v] of 2,2,2-trifluoroethanol. Partial dephosphorylation (60%) of beta(1-28) and beta(1-52) by treatment with alkaline phosphatase resulted in homogeneous preparations, as judged by polyacrylamide gel electrophoresis, which exhibited increased hydrophobicity. This reduction in the level of phosphorylation of serine residues 15, 17, 18 and 19 led to increased propensity for helix formation in the peptides in the presence of 2,2,2-trifluoroethanol, but no alpha-helical structures were detected in the dephosphorylated peptides in the absence of 2,2,2-trifluoroethanol.     

Codon optimization of the calf prochymosin gene and its expression in <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis>

Chymosin as an important industrial enzyme widely used in cheese manufacture. The yeast Kluyveromyces lactis is a promising host strain for expression of the chymosin gene. However, low yields (80 U/ml in shake flask cultures) were obtained when the K. lactis strain GG799 was used to express chymosin. We hypothesized that the codon-usage bias of the host may have resulted in inefficient translation and chymosin production. To improve expression efficiency of recombinant calf chymosin in K. lactis strain GG799, we designed and synthesized a DNA sequence encoding calf prochymosin using optimized codons, while keeping the G + C content relatively low. We altered 333 nucleotides to optimize codons encoding 315 amino acids. In shaking flask culture, chymosin activity was 575 U/ml in the strain expressing the optimized gene, a sevenfold higher expression level compared with the non-optimized control. SDS–PAGE analysis revealed that the purified recombinant calf chymosin had a molecular mass of 35.6 kDa, the same as the molecular weight of native calf chymosin. Alpha-casein, beta-casein, and kappa-casein were incubated with the recombinant calf chymosin from K. lactis strain GG799 or chymosin from calf stomach and the breakdown products were analyzed by SDS–PAGE. Both the recombinant calf chymosin and the native calf chymosin specifically hydrolyzed kappa-casein. Our results show that codon optimization of the calf chymosin gene improves expression in K. lactis strain GG799. Genetic manipulation to optimize codon usage has important applications for industrial chymosin production.     

Restriction fragment length polymorphism identification of goat αs1-casein alleles: a potential tool in selection of individuals carrying alleles associated with a high level protein synthesis

The extensive polymorphism of caprine alpha s1-casein, which is controlled by at least seven autosomal alleles segregating in a Mendelian fashion, was investigated by RFLP analysis. Genomic DNA from 77 lactating goats, whose genotypes had been previously determined by electrophoretic analysis of milk proteins, was digested with 11 restriction endonucleases and Southern blots were probed with a radiolabelled ovine alpha s1-casein cDNA. Three enzymes, PstI, TaqI and Rsa I, allowed the unambiguous identification of known alleles alpha s1-CnA, E and O and of the allelic pairs [alpha s1-CnD and F] and [alpha s1-CnB and C]. Evidence for a second null allele, termed alpha s1-CnO', and for an additional allele, designated alpha s1-CnF', was provided, which leads to the identification of nine alleles at the alpha s1-Cn locus, in this species. Although only 15 out of the 45 expected genotypes could be fully ascertained, this procedure allows the identification at birth of animals carrying the alpha s1-CnA, B or C alleles associated with a high alpha s1- and whole-casein content.     

Diversity in specificity of the extracellular proteinases in Lactobacillus helveticus and Lactobacillus delbrueckii subsp. bulgaricus

AIMS: To investigate the diversity in specificity of cell-bound extracellular proteinases in Lactobacillus helveticus and Lactobacillus delbrueckii subsp. bulgaricus. METHODS AND RESULTS: HPLC analysis of whole-cell preparations of 14 Lact. delbrueckii subsp. bulgaricus and eight Lact. helveticus strains incubated with alpha (s1)-casein (f 1-23) detected at least six distinct proteolytic patterns. Differences between groups were found in both the primary and secondary specificity toward alpha(s1)-casein (f 1-23) and its breakdown products. No correlation was found between the o-phthaldialdehyde (OPA) general proteolysis analysis and alpha(s1)-casein (f 1-23) cleavage profiles. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF STUDY: Using the alpha(s1)-CN (f 1-23) method, six patterns of proteolysis were found in the dairy lactobacilli tested. Understanding the influence of Lactobacillus proteinase specificity on casein degradation should facilitate efforts to develop starter cultures that predictably improve the functional properties of Mozzarella cheese.