Pancreatic hydrolysis of bovine casein: Peptide release and time-dependent reaction behavior

Pancreatic hydrolysis of bovine casein was carried out at pH 8.0 in a batch stirred tank reactor. The resulting peptides were identified by liquid chromatography–tandem mass spectrometry and their release kinetics were tracked. During 24 h of digestion, a total of 297 peptides consisting of 109, 48, 126, and 14 peptides, from α_s1-, α_s2-, β-, and κ-caseins (α_s1-, α_s2-, β-, and κ-CN), respectively, were identified by LC–MS–MS. We found that pancreatin attacked different caseins with varying hydrolysis rates (β-CN > α_s1-CN > α_s2-CN > κ-CN). Based on our analysis of the peptide fragments produced, we determined that pancreatin preferentially cleaved the N-terminal phosphorylated region, the C-terminal hydrophobic region and the middle region of β-casein, the terminal regions of α_s1-casein, and both the terminals and the middle region of α_s2-casein. The hydrolysis of κ-casein occurred at the slowest rate, releasing peptides only after 30 min of hydrolysis.     

Expression of recombinant Thermomonospora fusca xylanase A in Pichia pastoris and xylooligosaccharides released from xylans by it

The mature peptide of Thermomonospora fusca xylanase A (TfxA) was successfully expressed in Pichia pastoris under the control of AOX1 promoter. The activity of recombinant T. fusca xylanase A (reTfxA) in culture supernatant was 117.3 ± 2.4 U/mg, which is 3 times higher than that of the native TfxA. The optimal temperature and pH for reTfxA were 60 °C and 6.0, respectively. When treated at 70 °C and pH 6.0 for 2 min, the residual activity of the reTfxA was 70%. The reTfxA was very stable over a wide pH range (5.0–9.0). After incubation over pH 5.0–9.0 at 25 °C for 1 h, all the residual activity of reTfxA was over 80%. The K_m and k_cat values for reTfxA were 2.45 mg/ml and 139 s⁻¹, respectively. HPLC analysis revealed that xylobiose (X2) was the main hydrolysis product released from birchwood xylan and wheat bran insoluble xylan by reTfxA. Hydrolysis results of xylooligosaccharides showed that reTfxA was an endo-acting xylanase and xylobiose, xylotriose (X3), xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6) could be hydrolysed. This is the first report on the expression of reTfxA in yeast and on the determining and quantifying of the hydrolysis products released from xylans and xylooligosaccharides by reTfxA.     

Short communication: Effect of kefir grains on proteolysis of major milk proteins

The effect of kefir grains on the proteolysis of major milk proteins in milk kefir and in a culture of kefir grains in pasteurized cheese whey was followed by reverse phase-HPLC analysis. The reduction of κ-, α-, and β-caseins (CN), α-lactalbumin (α-LA), and β-lactoglobulin (β-LG) contents during 48 and 90 h of incubation of pasteurized milk (100 mL) and respective cheese whey with kefir grains (6 and 12 g) at 20°C was monitored. Significant proteolysis of α-LA and κ-, α-, and β-caseins was observed. The effect of kefir amount (6 and 12 g/100 mL) was significant for α-LA and α- and β-CN. α-Lactalbumin and β-CN were more easily hydrolyzed than α-CN. No significant reduction was observed with respect to β-LG concentration for 6 and 12 g of kefir in 100 mL of milk over 48 h, indicating that no significant proteolysis was carried out. Similar results were observed when the experiment was conducted over 90 h. Regarding the cheese whey kefir samples, similar behavior was observed for the proteolysis of α-LA and β-LG: α-LA was hydrolyzed between 60 and 90% after 12 h (for 6 and 12 g of kefir) and no significant β-LG proteolysis occurred. The proteolytic activity of lactic acid bacteria and yeasts in kefir community was evaluated. Kefir milk prepared under normal conditions contained peptides from proteolysis of α-LA and κ-, α-, and β-caseins. Hydrolysis is dependent on the kefir:milk ratio and incubation time. β-Lactoglobulin is not hydrolyzed even when higher hydrolysis time is used. Kefir grains are not appropriate as adjunct cultures to increase β-LG digestibility in whey-based or whey-containing foods.     

Value-added utilization of yak milk casein for the production of angiotensin-I-converting enzyme inhibitory peptides

Yak (Bos grunniens) milk casein derived from Qula, a kind of acid curd cheese from northwestern China, was hydrolysed with alcalase. The hydrolysates collected at different hydrolysis times (0 min, 60 min, 120 min, 180 min, 240 min, 300 min, 360 min) were assayed for the inhibitory activity of angiotensin-I-converting enzyme (ACE), and the one obtained at 240 min hydrolysis showed the highest ACE inhibitory activity. The active hydrolysate was further consecutively separated by ultrafiltration with 10 kDa and then with 6 kDa molecular weight cut-off membranes into different parts, and the 6 kDa permeate showed the highest ACE-inhibiting activity. This active fraction was further purified to yield two novel ACE-inhibiting peptides, whose amino acid sequences were Pro–Pro–Glu–Ile–Asn (PPEIN)(κ-CN; f156–160) and Pro–Leu–Pro–Leu–Leu (PLPLL) (β-CN; f136–140), respectively. The molecular weight and IC₅₀ value of the peptides were 550 Da and 566.4 Da, and 0.29 ± 0.01 mg/ml and 0.25 ± 0.01 mg/ml, respectively.     

A β-galactosidase from chick pea (Cicer arietinum) seeds: Its purification,biochemical properties and industrial applications

A β-galactosidase from Cicer arietinum seeds has been purified to apparent electrophoretic homogeneity using a combination of various fractionation and chromatographic techniques, giving a final specific activity of 220 units mg⁻¹, with approximately 1840 fold purification. Analysis of the protein by SDS–PAGE revealed two subunits with molecular masses of 48 and 38 kDa, respectively. These bands were further confirmed with LC–MS/MS, indicating that Chick pea β-galactosidase (CpGAL) is a heterodimer. Molecular mass was determined to be 85 kDa by Superose-12 FPLC column, which is in agreement with the molecular mass suggested by mass spectroscopy to be 83 kDa. The optimum pH of the enzyme was 2.8 and it hydrolysed o-nitrophenyl β-d galactopyranoside (ONPG) with a K_m value of 1.73 mM at 37 °C. The energy of activation (E_a) calculated in the range of 35 to 60 °C, using Arrhenius equation, was determined to be 11.32 kcal mol⁻¹. The enzyme could also hydrolyse lactose, with an optimum pH of 4.0 at 40 °C. K_m and E_a for lactose hydrolysis was found to be 10 mM and 10.57 kcal mol⁻¹, respectively. The enzyme was found to be comparatively thermostable showing maximum activity at 60 °C for both ONPG and lactose. Galactose was found to be the competitive inhibitor. β-Galactosidase also exhibited glycoproteineous properties when applied on Con-A Sepharose column. The enzyme was localised in germinated seeds with X-gal activity staining and shown to be expressed prominently at grown radical tip and seed coat. Sequence alignment of CpGAL with other known plant β-galactosidase showed high amino acid sequence homology.     

Whey protein isolate polydispersity affects enzymatic hydrolysis outcomes

The effects of heat-induced denaturation of whey protein isolate (WPI) on the enzymatic breakdown of α-La, caseinomacropeptide (CMP), β-Lg A and β-Lg B were observed as hydrolysis proceeded to a 5% degree of hydrolysis (DH) in both unheated and heat-treated (80 °C, 10 min) WPI dispersions (100 g L⁻¹). Hydrolysis of denatured WPI favoured the generation of higher levels of free essential amino acids; lysine, phenylalanine and arginine compared to the unheated substrate. LC–MS/MS identified 23 distinct peptides which were identified in the denatured WPI hydrolysate – the majority of which were derived from β-Lg. The mapping of the detected regions in α-La, β-Lg, and CMP enabled specific cleavage points to be associated with certain serine endo-protease activities. The outcomes of the study emphasise how a combined approach of substrate heat pre-treatment and enzymology may be used to influence proteolysis with attendant opportunities for targeting unique peptide production and amino acid release.     

Protein hydrolysates from meriga (Cirrhinus mrigala) egg and evaluation of their functional properties

Protein hydrolysates from underutilised meriga (Cirrhinus mrigala) fish egg were prepared by using commercial Alcalase and papain enzymes. The degree of hydrolysis was 62% for Alcalase and 17.1% for papain, after 90 min digestion at 50–55 and 60–65 °C, respectively. The protein content of Alcalase-produced hydrolysate was higher (85%) than that of papain hydrolysate (70%) (p < 0.05). Hydrolysis by both enzymes increased protein solubility of fish egg protein hydrolysates to above 72.4% over a wide pH range (2–12). Results showed that the hydrolysates had good fat absorption capacity (0.9 and 1.0 g/g sample), foam capacity (70% and 25%) and emulsifying capacity (4.25 and 5.98 ml/g hydrolysate), respectively for Alcalase and papain protein hydrolysates. Gel filtration chromatograms and SDS–PAGE analysis indicated the distribution of smaller peptides. These results suggested that fish egg protein hydrolysates could be useful in the food industry.     

Influence of buffer type and concentration on the peptide composition of trypsin hydrolysates of β-lactoglobulin

Proteins acquire conformation in aqueous media due to the influence of the buffer salt composition and concentration. Such influence may have impact on the enzyme–substrate interaction and somehow steer the enzyme attack properties, leading to release of dissimilar products. Our group has sought to investigate the influence of the hydrolysis environment on the trypsinolysis of a model protein, β-lactoglobulin (β-Lg). This work was aimed at investigating the effect of different buffers and their concentrations on the trypsinolysis patterns of β-Lg. The traditional NaOH-buffered water, in comparison to Tris–HCl and potassium-phosphate buffer at 62.5 mM–1.0 M were used at pH 8.5 for the pH drop and pH 7.8 for the hydrolysis. Bovine trypsin (EC 3.4.21.4) was used at an enzyme-to-substrate ratio of 1%. The samples were analysed for mass composition, using LC-ESI-TOF/MS and MALDI-TOF/MS for monitoring time-dependence of peptide evolution. In all buffer types and concentrations, peptides f(1–8), f(15–40, f(125–138) and f(142–148) were detected, implicating ease of hydrolysis of the terminal regions of β-Lg. A peptide from f(9–14), with sequence Gly-Leu-Asp-Ile-Gln-Lys, was detected at >0.5 M Tris–HCl only, while peptide f(71–75) was unique to <125 mM Tris–HCl and >250 mM potassium-phosphate buffer. Hydrolysis under buffer produced trypsin-specific peptides, numerous chymotrypsin-like non-specific peptides but no disulphide-linked peptides. Trypsinolysis shifted to the N-terminal region of lysine under some conditions. Hydrolysis under buffer holds potential for the avoidance of some peptides with undesirable characteristics while preserving a diversity of different peptides with possible bioactive properties.     

Biochemical characterisation of MX-4, a plant cysteine protease of broad specificity and high stability

A new proteinase, mexicain-IV (MX-4), has been purified to homogeneity from the latex fruits of Jacaratia mexicana (formely Pileus mexicanus), a plant member of the Caricaceae family. MX-4 shows a Mr of 23.7 kDa, pI of 9.3 and maximum proteolytic activity on casein and BAPNA at pH 8.0–8.5 and pH 7.0–7.5, respectively. The amino acid sequence of MX-4 and its reversible inhibition by HgCl₂ show that the proteinase belongs to the family of cysteine proteinases. This enzyme exhibits rather broad substrate specificity, although there seems to be a slight preference for cleavage of peptides having certain hydrophobic residues in the P2 position. Biochemical and circular dichroism studies revealed that this enzyme belongs to the α + β class of proteins, in agreement with the results obtained by X-ray crystallographic structure determination, and showed that MX-4 has a higher pH and thermal stability than other members of the Caricaceae family, including papain. These properties make this novel protease a suitable novel analytical tool for the proteomic analysis of peptide fragments of great potential interest in the food industry and other industries.     

Genetic analysis of detailed milk protein composition and coagulation properties in Simmental cattle

The objective of this study was to estimate genetic parameters for milk protein fraction contents, milk protein composition, and milk coagulation properties (MCP). Contents of α_S1-, α_S2-, β-, γ-, and κ-casein (CN), β-lactoglobulin (β-LG), and α-lactalbumin (α-LA) were measured by reversed-phase HPLC in individual milk samples of 2,167 Simmental cows. Milk protein composition was measured as percentage of each CN fraction in CN (α_S1-CN%, α_S2-CN%, β-CN%, γ-CN%, and κ-CN%) and as percentage of β-LG in whey protein (β-LG%). Rennet clotting time (RCT) and curd firmness (a₃₀) were measured by a computerized renneting meter. Heritabilities for contents of milk proteins ranged from 0.11 (α-LA) to 0.52 (κ-CN). Heritabilities for α_S1-CN%, κ-CN%, and β-CN% were similar and ranged from 0.63 to 0.69, whereas heritability of α_S2-CN%, γ-CN%, and β-LG% were 0.28, 0.18, and 0.34, respectively. Effects of CSN2-CSN3 haplotype and BLG genotype accounted for more than 80% of the genetic variance of α_S1-CN%, β-CN%, and κ-CN% and 50% of the genetic variance of β-LG%. The genetic correlations among the contents of CN fractions and between CN and whey protein fractions contents were generally low. When the data were adjusted for milk protein gene effects, the magnitude of the genetic correlations among the contents of milk protein fractions markedly increased, indicating that they undergo a common regulation. The proportion of β-CN in CN correlated negatively with κ-CN% (r = −0.44). The genetic relationships between CN and whey protein composition were trivial. Low milk pH correlated with favorable MCP. Genetically, contents and proportions of α_S1- and α_S2-CN in CN were positively correlated with RCT. The relative proportion of β-CN in CN exhibited a genetic correlation with RCT of −0.26. Both the content and the relative proportion of κ-CN in CN did not correlate with RCT. Weak curds were genetically associated with increased proportions in CN of α_S1- and α_S2-CN, decreased contents of β-CN and κ-CN, and decreased proportion of κ-CN in CN. Negligible effects on the estimated correlations between a₃₀ and κ-CN contents or proportion in CN were observed when the model accounted for milk protein gene effects. Increasing β-CN and κ-CN contents and relative proportions in CN and decreasing the content and proportions of α_S1-CN and α_S2-CN and milk pH through selective breeding exert favorable effects on MCP.     

Hydrolysis of β-lactoglobulin by trypsin under acidic pH and analysis of the hydrolysates with MALDI–TOF–MS/MS

Trypsin (EC 3.4.21.4) hydrolysis of food proteins are done at the optimum pH (7.8) and temperature (37 °C). Little information is available on the effect of sub-optimal conditions on hydrolysis. Bovine β-lactoglobulin (β-Lg) was hydrolysed by trypsin under acidic pH (pH 4–7) between 20 and 60 °C and the substrate concentration from 2.5% to 15% (w/v) and compared with hydrolysis at pH 7.8 and 37 °C. Aliquots were taken at different times (t = 0 up to 10 min). Samples were analysed using matrix-assisted laser desorption/ionisation time-of-flight tandem mass spectrometry (MALDI–TOF–MS/MS) with α-cyano-4-hydroxycinnamic acid (HCCA) and 2,5-dihydroxyacetophenone (DHAP) matrices. Hydrolysis patterns of β-Lg were generally similar at pH 7.8, 7, 6 and 5 while at pH 4 fewer peptides were detected except a unique fragment f(136–141). The different cleavage sites of β-Lg showed low resistance to trypsin at optimum conditions and pH 7 while being random and simultaneous. At lower pH, some cleavage sites showed increased resistance, while hydrolysis was relatively slow and ordered. Initial attack by trypsin occurred at Arg⁴⁰–Val⁴¹, Lys¹⁴¹–Ala¹⁴² and Arg¹⁴⁸–Leu¹⁴⁹ resistance was at Lys⁶⁰–Trp⁶¹, Arg¹²⁴–Thr¹²⁵ and Lys¹³⁵–Phe¹³⁶. Five domains were identified based on β-Lg resistance to trypsin in the order f(1–40) < f(41–75) < f(76–91) > f(92–138) > f(139–162). Results suggest that hydrolysis away from trypsin optimum offer better hydrolysis process control and different peptides. This strategy may be used to protect target bioactive or precursor peptides, or avoid the production of unwanted peptides.     

Characterisation of a thermostable family 42 β-galactosidase (BgalC) family from Thermotoga maritima showing efficient lactose hydrolysis

A β-galactosidase gene (TM_1195) of Thermotoga maritima was cloned and expressed in Escherichia coli. The recombinant β-galactosidase (BgalC), belonging to glycosyl hydrolase (GH) family 42, was purified to homogeneity with 23.4-fold purification and a recovery of 36.6%. Its molecular mass was estimated to be 78 kDa by SDS–PAGE. BgalC exhibited maximum activity at an optimal pH of 5.5 and an optimum temperature of 80 °C. The enzyme displayed important properties, such as stability over a broad pH range of 5.0–9.0 and thermostability up to 75 °C. K_m values of BgalC for p-nitrophenyl-β-galactopyranoside (pNPGal), o-nitrophenyl-β-galactopyranoside (oNPGal) and lactose were 1.21, 7.31 and 6.5 mM, respectively. BgalC was efficient in complete removal of lactose from milk. BgalC is significantly one of the few β-galactosidases from family 42 displaying significant hydrolysis of lactose. These properties make BgalC an ideal candidate for commercial use, in the production of lactose-free milk.     

Preparation and antihypertensive activity of peptides from Porphyra yezoensis

This research was to develop a antihypertensive peptide, an efficient angiotensin converting enzyme (ACE) inhibitor (ACEI), from Porphyra yezoensis. Seven commercial enzymes were screened and then enzymatic hydrolysis conditions were optimised. The results showed that alcalase was the most effective for hydrolysis and its optimum conditions for achieving the highest antihypertensive activity of peptide were 1.5% substrate, 5% alcalase, pH 9.0, and temperature of 50 °C at a hydrolysis time of 60 min. The antihypertensive peptide produced under the optimum conditions had a high ACE inhibition rate of 55.0% and a low IC₅₀ value of 1.6 g/l and remained at high stability at temperatures of 4, 25, and 37 °C, pH values of 2.0 and 8.0, and after pepsin and trypsin treatments. Major proteins from P. yezoensis were glutelin, albumin, and gliadin. The albumin and glutelin had higher hydrolysis rates than the gliadin, but the IC₅₀ value of glutelin was the lowest, which indicated that the antihypertensive peptide from glutelin was more functional.     

An extra-cellular alkaline metallolipase from Bacillus licheniformisMTCC 6824: Purification and biochemical characterization

An extra-cellular lipase produced by Bacillus licheniformis MTCC 6824 was purified to homogeneity by ammonium sulphate fractionation, ethanol/ether precipitation, dialysis, followed by anion-exchange chromatography on Amberlite IRA 410 (Cl⁻ form) and gel exclusion chromatography on Sephadex G 100 using Tris–HCl buffer (pH 8.0). The crude lipase extract had an activity of 41.7 LU/ml of culture medium when the bacterium was cultured for 48 h at 37 °C and pH 8.0 with nutrient broth supplemented with sardine oil as carbon source. The enzyme was purified 208-fold with 8.36% recovery and a specific activity of 520 LU/mg after gel exclusion chromatography. The pure enzyme is a monomeric protein and has an apparent molecular mass of 74.8 kDa. The lipase had a V_max and K_m of 0.64 mM/mg/min and 29 mM, respectively, with 4-nitro phenylpalmitate as a substrate, as calculated from the Lineweaver–Burk plot. The lipase exhibited optimum activity at 45 °C and pH 8.0, respectively. The enzyme had half-lives (T_1/2) of 82 min at 45 °C, and 48 min at 55 °C. The catalytic activity was enhanced by Ca²⁺ (18%) and Mg²⁺ (12%) at 30 mM. The lipase was inhibited by Co²⁺, Cu²⁺, Zn²⁺, Fe² even at low concentration (10 mM). EDTA, at 70 mM concentration, significantly inhibited the activity of lipase. Phenyl methyl sulfonyl fluoride (PMSF, 70 mM) completely inactivated the original lipase. A combination of Ca²⁺ and sorbitol induced a synergistic effect on the activity of lipase with a significantly high residual activity (100%), even after 45 min, as compared to 91.5% when incubated with Ca²⁺ alone. The lipase was found to be hydrolytically resistant toward triacylglycerols with more double bonds.     

Caseinophosphopeptide enrichment and identification

Caseinophosphopeptides (CPPs) were generated via tryptic hydrolysis of sodium caseinate followed by heat treatment (75 °C) at pH 6.0, 7.0 and 8.0. Calcium aggregation, ethanol precipitation and gallium immobilised metal affinity chromatography (IMAC) were applied to enrich the CPPs. Nano‐liquid chromatography electrospray ionisation‐quadrupole time‐of‐flight tandem mass spectrometry was used for phosphopeptide separation and identification. The combination of gallium IMAC isolation with calcium and ethanol precipitation appears to be a useful tool in characterising the fate of CPPs in CN hydrolysates during thermal processing. CPPs appeared to be less susceptible to thermal modification when heat‐treated at pH 7.0 than at pH 6.0 and 8.0. Different extents of methionine oxidation occurred in the CPPs depending on heat‐treatment pH. The results herein may have important implications for the manufacture of CPPs and for the quality control of CPP products.     

Characterisation of a thermostable family 42 β-galactosidase from Thermotoga maritima

A β-galactosidase gene (TM_0310) of Thermotoga maritima MSB8 was expressed in Escherichia coli. The recombinant β-galactosidase (designated BgalB) was purified to homogeneity by heat treatment and Ni-NTA affinity chromatography. BgalB belongs to the glycoside hydrolase family 42. Its molecular mass was estimated to be 78 kDa and 76 kDa by SDS–PAGE and gel filtration, respectively. The enzyme was optimum at pH 5.5, and it was quite stable over the pH range 5.0–11.4 at 70 °C. It was optimally active at 80 °C and was stable up to 75 °C. Besides, BgalB exhibited broad substrate specificity with a preference for p-nitrophenyl-β-galactopyranoside (pNPGal). K_m values of the purified enzyme for pNPGal, o-nitrophenyl-β-galactopyranoside (oNPGal) and pNP-β-fucopyranoside were 2.7 mM, 12.5 mM and 1.4 mM, respectively. These properties make this enzyme an interesting candidate for biotechnological applications. This is the first report of the family 42 β-galactosidases from T. maritima.     

Effects of milk protein variants on the protein composition of bovine milk

The effects of β-lactoglobulin (β-LG), β-casein (β-CN), and κ-CN variants and β-κ-CN haplotypes on the relative concentrations of the major milk proteins α-lactalbumin (α-LA), β-LG, α_S1-CN, α_S2-CN, β-CN, and κ-CN and milk production traits were estimated in the milk of 1,912 Dutch Holstein-Friesian cows. We show that in the Dutch Holstein-Friesian population, the allele frequencies have changed in the past 16 years. In addition, genetic variants and casein haplotypes have a major impact on the protein composition of milk and explain a considerable part of the genetic variation in milk protein composition. The β-LG genotype was associated with the relative concentrations of β-LG (A » B) and of α-LA, α_S1-CN, α_S2-CN, β-CN, and κ-CN (B > A) but not with any milk production trait. The β-CN genotype was associated with the relative concentrations of β-CN and α_S2-CN (A² > A¹) and of α_S1-CN and κ-CN (A¹ > A²) and with protein yield (A² > A¹). The κ-CN genotype was associated with the relative concentrations of κ-CN (B > E > A), α_S2-CN (B > A), α-LA, and α_S1-CN (A > B) and with protein percentage (B > A). Comparing the effects of casein haplotypes with the effects of single casein variants can provide better insight into what really underlies the effect of a variant on protein composition. We conclude that selection for both the β-LG genotype B and the β-κ-CN haplotype A²B will result in cows that produce milk that is more suitable for cheese production.     

Distinct composition of bovine milk from Jersey and Holstein-Friesian cows with good,poor, or noncoagulation properties as reflected in protein genetic variants and isoforms

The objective of this study was to examine variation in overall milk, protein, and mineral composition of bovine milk in relation to rennet-induced coagulation, with the aim of elucidating the underlying causes of milk with impaired coagulation abilities. On the basis of an initial screening of 892 milk samples from 42 herds with Danish Jersey and Holstein-Friesian cows, a subset of 102 samples was selected to represent milk with good, poor, or noncoagulating properties (i.e., samples that within each breed represented the most extremes in regard to coagulation properties). Milk with good coagulation characteristics was defined as milk forming a strong coagulum based on oscillatory rheology, as indicated by high values for maximum coagulum strength (G′_max) and curd firming rate (CFR) and a short rennet coagulation time. Poorly coagulating milk formed a weak coagulum, with a low G′_max and CFR and a long rennet coagulation time. Noncoagulating milk was defined as milk that failed to form a coagulum, having G′_max and CFR values of zero at measurements taken within 1 h after addition of rennet. For both breeds, a lower content of total protein, total casein (CN) and κ-CN, and lower levels of minerals (Ca, P, Mg) were identified in poorly coagulating and noncoagulating milk in comparison with milk with good coagulation properties. Liquid chromatography/electrospray ionization-mass spectrometry revealed the presence of a great variety of genetic variants of the major milk proteins, namely, α_S1-CN (variants B and C), α_S2-CN (A), β-CN (A¹, A², B, I, and F), κ-CN (A, B, and E), α-lactalbumin (B), and β-lactoglobulin (A, B, and C). In poorly coagulating and noncoagulating milk samples of both breeds, the predominant composite genotype of α_S1-, β-, and κ-CN was BB-A²A²-AA, which confirmed a genetic contribution to impaired milk coagulation. Interestingly, subtle variations in posttranslational modification of CN were observed between the coagulation classes in both breeds. Poorly coagulating and noncoagulating milk contained a lower fraction of the least phosphorylated α_S1-CN form, α_S1-CN 8P, relative to total α_S1-CN, along with a lower fraction of glycosylated κ-CN relative to total κ-CN. Thus, apparent variation was observed in the milk and protein composition, in the genetic makeup of the major milk proteins, and in the posttranslational modification level of CN between milk samples with either good or impaired coagulation ability, whereas the composition of poorly coagulating and noncoagulating milk was similar.     

Characterisation of thermostable trypsin and determination of trypsin isozymes from intestine of Nile tilapia (Oreochromis niloticus L.)

Trypsin from intestinal extracts of Nile tilapia (Oreochromis niloticus L.) was characterised. Three-step purification – by ammonium sulphate precipitation, Sephadex G-100, and Q Sepharose – was applied to isolate trypsin, and resulted in 3.77% recovery with a 5.34-fold increase in specific activity. At least 6 isoforms of trypsin were found in different ages. Only one major trypsin isozyme was isolated with high purity, as assessed by SDS-PAGE and native-PAGE zymogram, appearing as a single band of approximately 22.39 kDa protein. The purified trypsin was stable, with activity over a wide pH range of 6.0–11.0 and an optimal temperature of approximately 55–60 °C. The relative activity of the purified enzyme was dramatically increased in the presence of commercially used detergents, alkylbenzene sulphonate or alcohol ethoxylate, at 1% (v/v). The observed Michaelis–Menten constant (K_m) and catalytic constant (K_cat) of the purified trypsin for BAPNA were 0.16 mM and 23.8 s⁻¹, respectively. The catalytic efficiency (K_cat/K_m) was 238 s⁻¹ mM⁻¹.     

PURIFICATION AND CHARACTERIZATION OF AN ENDOPEPTIDASE FROM PSEUDOMONAS FLUORESCENS ATCC 948

Intracellular, ca 55 kDa monomeric endopeptidase (PsPepO) from Pseudomonas fluorescens ATCC 948 was purified to homogeneity by chromatography on Q-Sepharose, Sephacryl 200, Phenyl-Superose and Mono Q. It was strongly inhibited by the metalloproteinase inhibitor, 1,10 phenanthroline and by dithiothreitol, but less strongly by EDTA; it was stimulated by Co²⁺. Activity was optimal at pH 7.0 and 40–45C, with considerable activity at pH 5.0 and 12C. The enzyme was relatively heat-stable with a D_80Cvalue of 1.2 min. It did not hydrolyze α_s1-, β-or κ-casein (CN) and peptides with less than 5 amino acids but readily hydrolyzed α_s1-CNfl-23, α_s1-CN f157-164, α_s1-CN f165-199 and various β-CN fragments and peptide hormones. On α_s1-CN fl-23, α_s1-CN f165-199, insulin B-chain and bradykinin, it mainly catalyzed the hydrolysis of peptide bonds in which a hydrophobic amino acid (most commonly Phe or Leu) residue occupied the P'₁position. β-CN f193- or 194-209, which are the source of bitter peptides in cheese ripening, were hydrolyzed slowly or not at all. β-CN fragments from the sequence 58-70 were degraded or did not inhibit the endopeptidase activity as well as β-CN f193-209. The characteristics of the endopeptidase of Ps. fluorescens ATCC 948 were compared with those of lactococcal endopeptidases.