Chymosin Hydrolysis Research Articles

Influence of Chymosin on Physicochemical and Hydrolysis Characteristics of Casein Micelles and Individual Caseins.

The effects of chymosin on the physicochemical and hydrolysis characteristics of casein micelles and individual caseins were investigated. Adding 0.03 units of chymosin/mL led to the casein micelles in skim milk coagulating after a 3 h incubation period at 30 °C. SDS–PAGE investigation showed that β-CN, κ-CN, αs-CN, and a portion of β-lactoglobulin (β-LG) in the milk supernatant fraction (MSF) were precipitated into the milk pellet fraction (MPF). The mean particle size of the MSF with chymosin decreased from 254.4 nm to 179.2 nm after a 3 h incubation period. Mass spectrometry and SDS–PAGE analysis suggested that chymosin hydrolyzed individual β-CN, κ-CN, and αs-CN, but not β-LG. Chymosin hydrolysis led to a decrease in the molecular weights of the hydrolyzed β-CN, κ-CN, and αs-CN. Particle size analysis indicated that there was no difference in the particle size distribution of hydrolyzed β-CN and αs-CN. Moreover, our outcomes demonstrated that the hydrolysis of κ-CN by chymosin occurs before that of β-CN and αs-CN.

Investigation of the mechanism of bivalent iron binding by casein phosphopeptides

The article presents the results of investigations of mechanism of binding (chelation) of iron by casein phosphopeptides (CPPs). Molecular mass distribution of peptides in the form of the aqueous solutions of casein phosphopeptides, obtained by enzymatic hydrolysis of sodium caseinate by various proteinases has been studied. The greatest number of low-molecular peptides with molecular weight less than 2.8 kDa has been detected in the trypsin hydrolysate. Peptic and chymotryptic hydrolysates have similar qualitative composition and contain peptides with higher molecular weight. Under chymosin hydrolysis of sodium caseinate, mainly peptides with high molecular weight were formed, and fragments with molecular weight less than 2.8 kDa were not detected. In the study of chelating ability of CPPs it has been found that the maximum amount of iron was bound by peptide fraction of trypsin hydrolysate. Iron content in peptide complexes of trypsin hydrolysate was 18.8 mg/g of protein, and in those of himotrypsin hydrolysate and pepsin hydrolysate - 13.2 and 11.3 mg/g protein, respectively. It was noted that with increasing doses of iron, the degree of chelation of this micronutrient in all hydrolysates reduced. Chromatographic studies have shown that more than 70% of chelated iron in pepsin and trypsin hydrolysates, was found in peptide fractions with a molecular weight of 0.5-1.4 kDa and only 30% in fractions with a molecular weight of 1.4-4.5 kDa. Amino acid profiles of CPPs peptides have been studied in trypsin hydrolysate by using mass-spectroscopy. It was found that after the interaction of amino acids with iron the value of molecular ion peaks on the plots -Val-Ser-Ser-Glu-Glu-, and -Ala-Glu-Ser-Ser-Ser-Glu- Glu- increased by 15-25%, indicating the binding of these amino acids with the metal. The data obtained showed that most of the iron was associated with low molecular weight fraction of relatively short peptides of molecular weight from 1.4 to 4.5 kDa. It was noted that enzymatic hydrolysis of sodium caseinate by trypsin produced the greatest amount of low molecular weight peptides that bond iron and increased its bioavailability. It should be noted that chelated complexes are easy to penetrate into the cells, freeing the metal exactly where it is needed, protecting it from oxidation and interaction with other elements in the gastrointestinal tract. The results obtained allowed to optimize the nutrient medium and to provide high chelation of iron.

Camel and Bovine Chymosin Hydrolysis of Bovine αS1- and β-Caseins Studied by Comparative Peptide Mapping

In many cheese varieties, the general proteolytic activity of the coagulant is of great importance to the development of flavor and texture during ripening. This study used capillary electrophoresis and LC-MS/MS to compare the in vitro proteolytic behavior of camel and bovine chymosin (CC/BC) on bovine α(S1)- and β-casein (CN) at pH 6.5 and 30 °C. β-CN hydrolysis was also studied at pH 5.2 and in the presence of 0, 2, and 5% (w/v) NaCl. A total of 25 α(S1)- and 80 β-CN peptides were identified, and initial rates of early peptide formation were determined. The modes of proteolytic action of CC and BC shared a high degree of similarity generally. However, except for a few peptide bonds, CC was markedly less active, the magnitude of which varied widely with cleavage site. Preferential α(S1)-CN (Phe23-Phe24) and β-CN (Leu192-Tyr193) hydrolysis by CC proceeded at an estimated 36 and 7% of the initial rate of BC, respectively. The latter rate difference was largely pH and NaCl independent. Several cleavage sites appeared to be unique to CC and especially BC action, but qualitative differences were often predetermined by quantitative effects. In particular, negligible CC affinity to α(S1)-CN₁₆₄/₁₆₅ and β-CN₁₈₉/₁₉₀ prevented further exposure of the N-terminal products. β-CN hydrolysis by either enzyme was always stimulated at the lower pH, yet either inhibited or stimulated by the presence of NaCl, depending mainly on the predominating type of molecular substrate interactions involved at the specific site of cleavage. The potential impact of this proteolytic behavior on cheese quality is discussed.

Comparative proteomic analysis of casein and whey as prepared by chymosin-induced separation, isoelectric precipitation or ultracentrifugation

Fractionation of bovine milk was performed using chymosin-induced separation, isoelectric precipitation or ultracentrifugation as separation techniques prior to gel-based proteomic analysis. This approach allowed for comparative display and identification of proteins partitioned into casein and whey, respectively. Initially, three different staining methods (silver staining, colloidal Coomassie Blue G-250 or fluorescent Flamingo Pink staining) for two-dimensional gel electrophoresis (2-DGE) analysis were compared for their suitability as staining agent, especially in relation to their suitability to reveal differences in the casein fractions. Fluorescent staining proved to be the most appropriate for this purpose, giving a high sensitivity, and using this staining method, characteristic 2-DGE fingerprints were obtained for each casein and whey fraction from each separation method. A number of protein spots in both casein and whey fractions varied with separation method and these spots were subsequently identified using tandem mass spectrometry (MS). In rennet casein, proteolytic fragmentation of caseins (α(s1)-, α(s2),-, β- and κ-) was identified as a result of chymosin hydrolysis, whereas the 2-DGE profile of acid and ultracentrifuged casein was dominated by the presence of multiple isoforms of κ-caseins. Furthermore, casein remnants were identified in milk serum after ultracentrifugation. This study shows that gel-based proteomic analysis is suitable for characterisation of subtle variations in protein composition of milk fractions that occur as a consequence of different milk fractionation strategies.

Study of chymosin hydrolysis of casein micelles under ultra high pressure: Effect on re-association upon pressure release

The re-association of disintegrated casein micelles was investigated using a combined high pressure and enzymatic treatment with chymosin at 20 °C and 150 or 200 MPa. The amphiphilic, growth-limiting character of κ-casein was altered in the pressure-disintegrated state of the casein micelle. With increasing amounts of κ-casein being hydrolysed, less micellar surface can be covered with intact κ-casein after high pressure treatment. Therefore, larger particles with shifted surface-to-volume ratio were expected to occur with increasing degree of hydrolysis (DH) of κ-casein. Higher DH values led to larger reformed casein micelles. As compared with a control sample, the relative particle diameter showed a strong correlation to DH. This could be attributed to the re-association of caseins or micellar fragments forming micelles similar in structure as compared with native ones. Gelling similar to that in standard renneting, only occurred at DH levels above 0.80.

Bulk and interfacial behaviour of caseinoglycomacropeptide (GMP)

Caseinoglycomacropeptide (GMP) is a hydrophilic glycopeptide released from milk κ-casein by chymosin hydrolysis during cheese making. GMP is thought to be a potential ingredient for specific dietary applications with several health benefits. In this study GMP was characterized at the air–water interface and its behaviour was related with the self-assembly of GMP in solution as affected by pH. This GMP self-assembly was investigated by dynamic light scattering and the interfacial properties were determined by tensiometry and surface dilatational measurements at pH 4, 5 and 7. At pH 5 GMP exhibited higher surface pressure at equilibrium than at pH 7. At pH 4 the behaviour was more complex due to self-assembly close to GMP pI. Dynamic measurement showed that the adsorption/penetration rate constant ( K ads) is facilitated at higher GMP bulk concentrations, while the rate constant of rearrangement ( K r) decreased at higher GMP concentrations which could be attributed to the existence of a steric restriction due to the higher GMP load at the interface. K r was higher at pH 5 because of lower electrostatic interactions close to the pI. The viscoelastic properties showed a complex behaviour due to the existence of protein–protein interactions depending on the GMP concentration, on the pH of the bulk and on the rates of diffusion, adsorption and rearrangement of GMP at the air–water interface.

Sequence analysis of UTR and coding region of kappa-casein gene of Indian riverine buffalo (Bubalus bubalis)

In this study, complete nucleotide as well as derived amino acid sequence characterization of water buffalo (Bubalus bubalis) kappa-casein gene has been presented. Kappa-casein cDNA clones were identified and isolated from a buffalo lactating mammary gland cDNA library. Sequence analysis of kappa-casein cDNA revealed 850 nucleotides with an open reading frame (ORF) of 573 nucleotides, encoding mature peptide of 169 amino acids. The 5' untranslated region (UTR) comprised 71 nucleotides, while 3' UTR was of 206 nucleotides. A total of 11 nucleotide and seven amino acid changes were observed in, buffalo (Bubalus bubalis) as compared to cattle (Bos taurus), sheep (Ovis aries) and goat (Capra hircus). Among these nucleotide changes, eight were unique in buffalo as they were fully conserved in cattle, sheep and goat. Majority of the nucleotide changes and all the amino acid changes; 14 (Asp-Glu), 19(Asp/Ser-Asn), 96(Ala-Thr), 126(Ala-Val), 128(Ala/Gly-Val), 156(Ala/Pro-Val) and 168(Ala/Glu-Val) were limited to exon IV. Three glycosylation sites, Thr 131, Thr 133 and Thr 142 reported in cattle and goat kappa-casein gene were also conserved in buffalo, however, in sheep Thr 142 was replaced by Ala. Chymosin hydrolysis site, between amino acids Phe 105 and Met 106, important for rennet coagulation process, were found to be conserved across four bovid species. Buffalo kappa-casein with the presence of amino acids Thr 136 and Ala 148 seems to be an intermediate of "A" and "B" variants of cattle. Comparison with other livestock species revealed buffalo kappa-casein sharing maximum nucleotide (95.5%) and amino acid (92.6%) similarity with cattle, whereas with pig it showed least sequence similarity of 76.0% and 53.2%, respectively. Phylogenetic analysis based on both nucleotide and amino acid sequence indicated buffalo kappa-casein grouping with cattle, while sheep and goat forming a separate cluster close to them. The non-ruminant species viz. camel, horse and pig were distantly placed, in separate lineages.

Susceptibility of equine κ- and β-caseins to hydrolysis by chymosin

Abstract Equine whole casein was hydrolysed by chymosin and some peptides generated were characterised by microsequencing after reversed-phase high performance liquid chromatography or sodium dodecyl sulphate polyacrylamide gel electrophoresis. β-Casein was readily hydrolysed into amino- and carboxy-terminal fragments after cleavage of the Leu190–Tyr191 bond. These two fragments seemed to be resistant to further chymosin hydrolysis on incubation for up to 24 h. Equine κ-casein was purified by affinity chromatography on immobilised wheat germ agglutinin. O-Glycosylated κ-casein was found to represent less than 6.8% of the equine casein components. Equine κ-casein was also hydrolysed and para-κ-casein and glycomacropeptide were generated after cleavage of the Phe97–Ile98 bond.

Detection of the Cholera Toxin-binding Activity of κ-Casein Macropeptide and Optimization of Its Production by the Response Surface Methodology.

The cholera toxin (CT)-binding activity of purified κ-casein macropeptide (CMP) from bovine κ-casein was detected. In addition, a statistical model was developed to optimize the production of CMP. CMP was prepared by chymosin hydrolysis of κ-casein and a subsequent 3% trichloroacetic acid treatment. CMP was further fractionated in an ion-exchange column by FPLC. CT binding activity was eluted at 0.18 M NaCl and was a single 8.9 kDa peptide without tyrosine and arginine residues. The CT binding activity was rapidly lost by a carbohydrase treatment. The conditions for CMP production with chymosin were optimized by using the response surface methodology (RSM). The estimated optimum levels of the factors were as follows: reaction temperature, 38.5°C; pH, 6.44; and time, 35.9 min. A validation experiment was performed in which CMP was prepared under the predicted parameters, and it was ascertained that the estimated optimum conditions gave better production of CMP than any other conditions.

Profiling of genetic variants of bovine κ-casein macropeptide by electrophoretic and chromatographic techniques

Abstract Bovine κ-caseins of single variant phenotype (AA and BB) and mixed phenotype (AA, BB and AB) were purified from milk and the corresponding κ-casein macropeptides prepared by chymosin hydrolysis. The macropeptides were characterized by PAGE, Mono Q- and reverse-phase HPLC. Chromatographic profiles showed marked differences between the monovariant macropeptides, attributable to differences in glycosylation. The B variant macropeptide was found to be more highly glycosylated than the A variant with an apparently greater number of oligo-saccharide chains per peptide unit and a higher level of sialylation. The analytical profiles for the mixed variant sample were a composite of those for the individual variants, all components being accounted for by one or other variant. It was concluded that while the overall extent of glycosylation may vary, there are consistent patterns of glycosylation for each variant.

Comparative study of methods for the isolation and purification of bovine κ-casein and its hydrolysis by chymosin

kappa-Casein was purified from a single batch of whole acid casein (kappa-A variant) using different methods in order to compare their merits in producing a purified material with a carbohydrate and phosphate heterogeneity representative of the whole kappa-casein complement in milk. Ion-exchange methods of purification gave products of higher purity than precipitation techniques involving final purification by ethanol fractionation, but all methods resulted in kappa-caseins of apparently similar heterogeneity and chemical composition. The purified kappa-caseins were hydrolysed with chymosin and the derived macropeptides isolated. These were all virtually identical as determined by reversed-phase chromatography and gel electrophoresis. Some observations on chymosin hydrolysis of kappa-casein were made. In addition to formation of the major para-kappa-casein (Glu1-Phe105) and macropeptide (Met106-Val169), chymosin hydrolysis at pH 6.6 also resulted in two minor para-kappa-caseins with N-termini corresponding to Phe18 and Ser33 of kappa-casein. At pH 5.5 and 4.5 para-kappa-casein was rapidly hydrolysed into at least six fragments, one of which had an N-terminus corresponding to Trp76 of kappa-casein. At pH 6.6, 5.5 and 4.5 the kappa-casein macropeptide was stable to chymosin, but at pH 2.3 it was hydrolysed by chymosin into fragments with N-termini corresponding to Met106, Ile125, Ala138, Val139, Thr145 and Glu147 of kappa-casein.

Influence of pH on Chymosin Action in Solutions of Different .kappa.-Casein Variants

Chymosin hydrolysis of κ-casein variants A, B, or AB or heated (85 o C×10 min) mixtures of κ-casein variants plus β-lactoglobulin A, B, or AB in buffers at pH 6.7, 6.4, 6.0, 5.6, or 5.3 were monitored spectrophotometrically at 420 nm by reaction with 2,4,6-trinitrobenzenesulfonic acid. The genetic type of κ-casein and pH of the medium influenced the rate and extent of hydrolysis. At pH 5.3 and 5.6 κ-casein B was most extensively hydrolyzed, but at pH 6.0-6.7 the hydrolyses of the three variants were identical. κ-Casein A was most susceptible to hydrolysis (at pH 5.3) in heated solutions containing different genetic combinations of κ-casein plus β-lactoglobulin

Effect of Ferric Chloride on Chymosin Hydrolysis and Rennet Clotting Time of Milk

The influence of FeCl3 on the renneting properties of milk in the preparation of iron-fortified cheeses were evaluated. The FeCl3 (0 to .86mM) was added to milk at various stages of its thermal history: 1) raw milk not cooled below 30°C and FeCl3 added at 30°C; 2) raw milk cooled to 4°C and FeCl3 added at 4°C; 3) FeCl3 added to raw cooled milk at 4°C and pasteurized at 64°C for 30min; and 4) raw cooled milk pasteurized at 64°C for 30min, cooled to 30°C, and FeCl3 added at 30°C. The pH of milk decreased with FeCl3 addition. Rate of chymosin hydrolysis of micellar κ-casein, as assessed by release of macropeptide, and Formagraph rennet clotting time were determined at 30°C without pH correction. Addition of FeCl3 to raw uncooled milk increased the rate of chymosin hydrolysis but did not affect the enzymic reaction when it was added either to raw cooled milk or to milk before or after pasteurization. Ferric chloride decreased rennet clotting time when it was added to raw uncooled milk and to milk before pasteurization and increased rennet clotting time when it was added to raw cooled milk and to milk after pasteurization. Ferric chloride at concentrations (about .2mM) normally employed in iron fortification of Cheddar cheese did not affect the rennet clotting time of whole milk whether it was added to raw cooled milk or to milk before or after pasteurization. However, at high concentrations (>.43mM), addition of FeCl3 to milk before pasteurization is beneficial when making iron-fortified cheese.

Proteolytic Activity of Some Milk-Clotting Enzymes on κ-Casein

Proteolytic activity of some milk-clotting enzymes [calf rennet (chymosin), Mucor miehei rennet, and Cryphonectria parasitica rennet] on κ-casein was determined with reverse-phase HPLC. All enzymes were standardized to the same milk-clotting activity and incubated with κ-casein at 37°C for 1, 5, 15, 30, and 60min. Protein hydrolysis was stopped by addition of 10–5M pepstatin in 8 M urea. Chromatograms of hydrolysis products revealed that the enzymes hydrolyzed κ-casein differently. Chymosin hydrolysis was limited to formation of κ-macropeptide and para-κ-casein; the microbial rennets, particularly that from M. miehei, effected extensive nonspecific hydrolysis of both κ-casein and para-κ-casein.

Interaction of .beta.-lactoglobulin with .kappa.-casein in micelles as assessed by chymosin hydrolysis. Effects of added reagents

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTInteraction of .beta.-lactoglobulin with .kappa.-casein in micelles as assessed by chymosin hydrolysis. Effects of added reagentsI. Mohan Reddy and John E. KinsellaCite this: J. Agric. Food Chem. 1990, 38, 2, 366–372Publication Date (Print):February 1, 1990Publication History Published online1 May 2002Published inissue 1 February 1990https://doi.org/10.1021/jf00092a005RIGHTS & PERMISSIONSArticle Views89Altmetric-Citations4LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (916 KB) Get e-Alerts Get e-Alerts

Interaction of .beta.-lactoglobulin with .kappa.-casein in micelles as assessed by chymosin hydrolysis: effect of temperature, heating time, .beta.-lactoglobulin concentration, and pH

Les interactions a chaud entre les 2 proteines sont etudiees en suivant la cinetique d'hydrolyse par la chymosine. Discussions des resultats en fonction des changements dans la conformation des lactoglobulines et leur teneur en groupements SH et S.S, a differents pH et temperatures

The involvement of one of the three histidine residues of cow κ-casein in the chymosin-initiated milk clotting process

Cow κ-casein has been modified by photo-oxidation in the presence of rose bengal and by the chemical reagents diethyl pyrocarbonate, 2-hydroxy-5-nitro-benzyl bromide and iodoacetic acid. Photo-oxidation resulted in the destruction of histidine and tryptophan residues and all of the histidines could be ethoxyformylated by treatment with diethyl pyrocarbonate. Both procedures caused a loss in the susceptibility of the Phe-Met linkage of κ-casein to chymosin hydrolysis. Treatment of κ-casein with 2-hydroxy-5-nitrobenzyl bromide and iodoacetic acid caused the loss of tryptophan and methionine residues respectively but, in both cases, the susceptibility of the modified protein to chymosin hydrolysis remained unaffected. Of the amino acids examined it is concluded that only the histidine residues of cow κ-casein are important for the hydrolytic action of chymosin and, furthermore, the treatment with diethyl pyrocarbonate suggests that only one of the three histidines plays an essential role.