PURIFICATION AND CHARACTERIZATION OF ALKALINE PROTEINASES FROM THE VISCERA OF ANCHOVY, ENGRAVLIS JAPONICA

Two electrophoretically homogeneous proteinases designated proteinase A and B were isolated from anchovy viscera. Purity was increased 17.7 and 24.6-fold with approximately 1.9 and 1.8% yield for proteinases A and B, respectively. The maximum caseinolytic activity was found to be at pH 9.4 for proteinase A and at pH 9.6 for proteinase B at the optimum temperature of 48°C. The molecular weights of proteinase A and B were determined to be 27, 300 and 25,100 D, respectively, using Sephadex G-100 gel filtration. The amino acid profiles of the enzymes were similar and relative proportion of amino acid residues was comparable to that in bovine pancreatic α-chymotrypsin. Proteinase A and B were identified as α-chymottypsin-like serine proteases by inhibitor and substrate specificity studies. Apparent K_m (K_m′) values of proteinase A and B for benzoyl-L-tyrosine ethyl ester were 4.6 × 10_?4 M and 1.2 × 10_?3 M, respectively.     

Autolysis and the endogenous proteinases characterised in beardless barb (Anematichthys apogon) muscle

Beardless barb is a common fish species used in fermentation of fish paste Ka-pi-plaa. Autolytic profile of beardless barb muscle showed the maximum autolysis was at 50 °C, at both acidic and alkaline pH values. With augmentation concentration of NaCl, autolytic activity slightly decreased. Endogenous proteinases isolated from fish muscle in crude extract forms were also characterised. The acidic proteinases had optimum activity at pH 3.0 and 50°C, and they showed higher proteolytic activity than the alkaline proteinases which were optimally active at pH 9.0 and 50 °C. Proteinases in peak at pH 3.0 were inhibited by pepstatin A, but those in peak at pH 9.0 were highly inhibited by PMSF, TLCK and soybean trypsin inhibitor, suggesting that both aspartic and serine proteinases were existed in beardless barb muscle. The proteinases were stable in pH range of 2.0-5.0 but unstable at the temperatures higher than 40 °C. NaCl suppressed the proteolytic activity, ATP activated the proteinase activity, while CaCl₂, MgCl₂ and CoCl₂ exhibited no influence on the activity. The results implied that cathepsin D is the predominant proteinase responsible for autolysis in beardless barb. The findings were useful to improve the processing and qualities of Ka-pi-plaa product using beardless barb as raw material.     

Acid Proteinases from Antarctic Krill, Euphausia superb: Partial Purification and Some Properties

The presence of neutral and alkaline proteinases in the extract from Antarctic krill, Euphausia superba was confirmed. In addition to these enzymes, acid proteinases were found in the extract from E. superba and the two major acid proteinases, A and B, were purified 80–140 fold. The molecular weights of acid proteinases A and B were estimated by gel filtration on Sephadex G-100 to be approximately 45,000 and 64,000, respectively. The optimal pH of enzymes A and B was 3.0 toward hemoglobin. They were partially inhibited by acid proteinase specific reagents: diazoacethyl-DL-norleucine methylester (DAN) and 1,2epoxy-3-(p-nitrophenoxy) propane (EPNP). However, enzyme B was less sensitive to pepstatin while enzyme A was inhibited by pepstatin like almost all other acid proteinases.     

PURIFICATION AND CHARACTERIZATION OF CHYMOTRYPSIN FROM PENAEUS VANNAMEI (CRUSTACEA: DECAPODA)

The purification and characterization of a chymotrypsin from the hepatopan-creas of the white shrimp Penaeus vannamei is described. Only one chymotrypsin was detected in contrast to other shrimp that have two major forms. P. vannamei chymotrypsin has a molecular mass of 33.2 kDa and a pI of 3.1. The molecular mass is high relative to other penaeid chymotrypsins. The proteinase is acid labile and exhibits optimum activity at pH 8. The enzyme is thermostable both at 25 and 37C. It is a serine proteinase. Phenylmethylsulphonyl fluoride and soybean trypsin inhibitor blocked the activity of the enzyme, and it was not affected by chymotrypsin inhibitors such as tosyl-PheCH₂Cl or benzyloxycarbonyl-Phe-CH₂Cl. Protein profiles of the hepatopancreas from two populations varied     

ACTIVITY OF THE VACUOLAR PROTEASES OF YEAST AND THE SIGNIFICANCE OF THE CYTOSOLIC PROTEASE INHIBITORS DURING THE POST-FERMENTATION DECLINE PHASE

The vacuolar proteases, proteinase A, proteinase B and carboxypeptidase Y, together with inhibitors for proteinase B and carboxypeptidase Y, have been identified in a brewing strain of Saccharomyces cerevisiae fermenting a malt extract wort at 30°C. The specific activity on a protein basis measured in freshly prepared extracts increased for all three enzymes during the post-fermentation decline phase but on a per cell basis the total activity, measured after removal of any inhibitor by autocatalysis, fell steadily. No evidence was found for the presence of proteinase A inhibitor at any point but the cells contained considerable amounts of proteinase B inhibitor especially during the growth and fermentation phases. Proteinase B only became detectable in freshly prepared extracts after a distinct loss in cell viability had become obvious. The situation for carboxypeptidase Y was intermediate between that for proteinase A and proteinase B. At high cell concentrations leakage of proteinase B and its inhibitor into the medium occurred but neither of the other two proteases were detectable outside the cells .     

CHARACTERIZATION OF TRYPSIN PURIFIED FROM THE PYLORIC CAECA OF THE SOUTHWEST ATLANTIC WHITE CROAKER Micropogonias furnieri (Sciaenidae)

A southwest Atlantic croaker protease was purified from the pyloric caeca by ammonium sulfate fractionation, acetone precipitation and affinity chromatography. The enzyme was classified as a trypsin on the basis of molecular weight, its ability to hydrolyze synthetic substrates N-α-benzoyl-arginine-p-nitroanilide (BAPA) and tosylarginine methyl ester (TAME), and its inhibition by known trypsin inhibitors. The isolated enzyme has a single band on SDS-PAGE with an estimated molecular mass of 24 kDa. Croaker trypsin activity was stable between pH 5 and pH 11 for 30 min at 0C, 10C and 25C and its maximal activity against BAPA was at pH 9.5. Thermostability was observed to about 55C for 30 min at pH 7.8 and its temperature optimum was 60C. Substrate turnover number was 850 BAPA units per μmol trypsin, and the K_mwas 0.081 mM at 25C. For the hydrolysis of TAME, V_maxwas 9273 units per μmol trypsin and the K_m was 0.155 mM at 25C. The catalytic efficiency V_max/K_mwas higher than that of trypsin from fish living in colder waters.     

PURIFICATION AND CHARACTERIZATION OF A SERINE PROTEINASE FROM THE TUNA PYLORIC CAECA

A 15.0 kDa serine proteinase with collagenase activity from pyloric caeca of tuna, Thunnus thynnus, was purified in four steps; acetone precipitation, gel filtration chromatography on a Sephadex G‐100, ion‐exchange chromatography on a DEAE‐Sephadex α‐50 and gel filtration chromatography on a Sephadex G‐75 column. The purification and yield were 30.5‐fold and 0.023%, respectively, as compared with those in the starting crude extract. The optimum pH and temperature for the purified collagenolytic enzyme were around pH 7.5 and 55C, respectively. The purified proteinase was strongly inhibited by metal ions (Hg²⁺ and Zn²⁺) and serine proteinase inhibitors (PMSF, TLCK and soybean trypsin inhibitor) suggesting it is a serine protease. The K_m and V_max of the purified enzyme for collagen type I were approximately 3.82 mM and 851.5 U, respectively.     

Degradation of Ochratoxin A by Proteases and by a Crude Enzyme of Aspergillus niger

Ochratoxin A is a mycotoxin present in food commodities as cereals, wine, coffee, figs, dried vine fruits or beer and in feeds for animals. The enhancement of its conversion into ochratoxin α is considered to be a way to reduce its presence in the body and, therefore, its toxicity. In this paper we report the ability of several commercial proteases to hydrolyze ochratoxin A into ochratoxin α in different amounts. After an incubation period of 25 h., a significant hydrolytic activity at pH 7.5 for Protease A (87.3%), and for Pancreatin (43.4%) was detected. At pH 3.0, a weak hydrolytic activity was detected for Prolyve PAC (3%). None of the other commercial enzymes tested were able to hydrolyze ochratoxin A in the tested conditions. Also, the isolation of an enzyme extract from an Aspergillus niger strain with very strong ochratoxin A hydrolytic activity at pH 7.5 (99.8%) is reported. This activity is similar to the activity detected in Protease A. Data about the inhibition effect of ethylenediaminetetraacetic acid and phenylmethanesulfonyl fluoride on the involved hydrolytic enzymes showed that enzymes involved in ochratoxin A hydrolysis are metalloproteins.     

Formation of oligosaccharides from whey UF-permeate by enzymatic hydrolysis — analysis of factors

Two-level fractional factorial experiments were designed to study effects of enzyme (0.05 and 0.1%) and initial lactose concentrations (L_o: 14 and 23%), pH (5.0 and 7.0) and temperature (35 and 45 °C) on enzymatic formation of oligosaccharides (OS) from whey UF-permeate in a batch reactor. β-d-galactosidase from Aspergillus oryzae (A), Kluyveromyces lactis (B) and K. fragilis (C) were compared. Hydrolysis with B and C gave comparable yields which were higher than that from A at identical conditions. L_o did not influence reaction time, but concentration of OS significantly increased by increasing L_o for all enzymes. Increasing L_o reduced the yield after hydrolysis with A and B, but improved the yield for C. L_o had negligible effect on degree of hydrolysis (DH) for A and C. However, increasing L_o significantly lowered DH for B. Increasing enzyme concentration significantly reduced reaction time for A, but it had no effect on that for B and C. DH significantly decreased after increasing concentration of A. Consequently, OS and yield were reduced. Applying B and C at higher concentration improved DH, OS and yield. Increasing temperature or pH reduced DH, OS and yield for A, but increased the responses for B and C.     

Breakdown of caseins by proteinases in bovine milks with high somatic cell counts arising from mastitis or infusion with bacterial endotoxin

Milk obtained from cows which were either infected by clinical mastitis or had been subjected to intramammary infusion of Escherichia coli endotoxin possessed high counts of somatic cells and very high levels of proteinase activity which hydrolysed the caseins almost completely in a few hours at 37 degrees C. The rate of hydrolysis of beta-casein was slightly greater than that of alpha S1-casein, but in both cases hydrolysis was enhanced by 6 cycles of freezing and thawing to disrupt somatic cell membranes. A study of the relationship between proteinase activity and cell count suggested that only some of the proteinase activity originated in the somatic cells and also that the identity of the cells making up the total cellular population was important. Maximum proteolysis occurred at 50-60 degrees C, but the temperature-activity curve was a broad peak. Likewise the pH versus activity plot was very broad and was almost flat over the pH range 6-9. Experiments with a number of inhibitors of proteinases failed to give a clear cut pattern of inhibition. All evidence obtained was consistent with the view that several different enzymes with different pH and temperature optima and different specificities contributed to the overall hydrolysis of caseins in these milks. From electrophoretic band patterns one of these enzymes was clearly plasmin, but in high cell count milks other proteinases also became significant.     

Factors affecting the caseinolytic activity of Lactobacillus casei and Lactobacillus plantarum

Whole cell suspensions of some strains of each Lactobacillus casei and Lactobacillus plantarum were assayed for their caseinolytic activity in 0.1 M NaH₂PO₄ buffer, pH 6.5, at 30 °C, using different assay methods. Azocasein was not as sensitive as casein (Hammarsten) as a substrate. Inclusion of glucose in the assay mixture reduced the released α-amino groups as evidenced by fluorescent labelling, but generally increased the amounts of excreted amino acids. Divalent cations, including calcium ions, played only a minor role in the activation of the cell-bound proteinase, whereas NaCl inhibited it markedly. Inhibitor studies suggest that the enzyme is a serine proteinase. The different assay methods used did not give identical results. Fluorescent labelling of the free α-amino groups at pH 6.0 appears, on the contrary, to be a more reliable method.     

Contamination of barley seeds with Fusarium species and their toxins in Spain: an integrated approach

Fusarium is a globally distributed fungal genus that includes different species pathogenic to cereals among others crops. Some of these Fusarium species can also produce toxic compounds towards animals and humans. In this work, the presence of the most important Fusarium toxins was determined in barley seeds from Spain, sampled according to European Union requirements. The results obtained were compared with the presence of mycotoxigenic species considered responsible for their synthesis by using species-specific polymerase chain reaction protocols. Fumonisins B₁ and B₂, zearalenone, trichothecenes type A (T-2 and HT-2) and trichothecenes type B (deoxynivalenol and nivalenol) were analysed by using high-performance liquid chromatography. Deoxynivalenol and zearalenone were detected in 72% and 38% of the barley samples, respectively, at levels below European Union limits in all cases. However, the co-occurrence of both toxins in 34% of the samples suggested that synergistic activity of these two mycotoxins should be evaluated. Nivalenol and HT-2/T-2 were detected at low levels in 17% and 10% of the samples, respectively. Fumonisins occurred in 34% of the samples at levels up to 300?µg/kg. This suggested that they might represent a risk in Spanish barley, and to our knowledge, this is the first report on the presence of fumonisins in barley in this country. The species-specific polymerase chain reaction assays to detect mycotoxin-producing Fusarium species showed a very consistent correlation between F. verticillioides detection and fumonisin contamination as well as F. graminearum presence and zearalenone, deoxynivalenol and nivalenol contamination in barley samples. The approach used in this study provided information of mycotoxin contamination of barley together with the identification of the fungal species responsible for their production. Detection of the species with the current polymerase chain reaction assay strategy may be considered predictive of the potential mycotoxin risk in this matrix.     

Characteristics of Metroxylon sagu Resistant Starch Type III as Prebiotic Substance

Resistant starch type III (RS₃) was produced from sago (Metroxylon sagu) and evaluated for its characteristics as a prebiotic. Two RS₃ samples designated sago RS and HCl‐sago RS contained 35.71% and 68.30% RS, respectively, were subjected to hydrolyses by gastric juice and digestive enzymes and to absorption. Both sago RS and HCl‐sago RS were resistant to 180 min hydrolysis by gastric acidity at pH 1 to 4 with less than 0.85% hydrolyzed. Both samples were also resistant toward hydrolysis by gastrointestinal tract enzymes and intestinal absorption with 96.75% and 98.69% of RS₃ were recovered respectively after 3.5 h digestion and overnight dialysis at 37 °C. Sago RS₃ supported the growth of both beneficial (lactobacilli and Bifidobacteria) and pathogenic microbes (Escherichia coli, Campylobacter coli, and Clostridium perfringens) in the range of 2.60 to 3.91 log₁₀ CFU/mL. Hence, prebiotic activity score was applied to describe the extent to which sago RS₃ supports selective growth of the lactobacilli and bifidobacteria strains over pathogenic bacteria. The highest scores were obtained from Bifidobacterium sp. FTDC8943 grown on sago RS (+0.26) and HCl‐sago RS (+0.24) followed by L. bulgaricus FTDC1511 grown on sago RS (+0.21). The findings had suggested that sago RS₃ has the prebiotic partial characteristics and it is suggested to further assess the suitability of sago RS₃ as a prebiotic material.     

Isolation and characterization of 1,4-β-glucan 4-glucanohydrolases (EC 3.2.1.4) from a technical Trichoderma viride cellulase

The following enzyme activities were detected in a Trichoderma viride cellulase (Röhm 2230 B): 1,4-β-d-glucan cellobiohydrolase (C₁), 1,4-β-d-glucan 4-glucanohydrolase (C_x), β-glucosidase, β-galactosidase, polygalacturonase, proteinase, xylanase, amylase, esterase and ‘polyphenoloxidase’. Isolation of cellulolytic enzymes was performed starting with adsorption chromatography on Avicel SF, leading to separation of more than 90% of non-cellulolytic enzymes and 96% of β-glucosidase activity (= fraction A). In fraction A, 30% of the C_x activity was determined whereas, in a separated fraction, B, the remaining C_x and the total C₁ activity was established. Further fractionation of B using ion-exchange and gel chromatography resulted in the separation of three purified enzyme fractions, PI to PIII, with endo C_x activities. Additionally, C₁ activity was found in PIII. PI-PIII were characterized by means of their pH optima, isoelectric points and molecular weights.     

Purification and some properties of a cysteine proteinase from sorghum malt variety SK5912

A cysteine proteinase from sorghum malt variety SK5912 was purified by a combination of 4 M sucrose fractionation, ion‐exchange chromatography on Q‐ and S‐Sepharose (fast flow), gel filtration chromatography on Sephadex G‐100 and hydrophobic interaction chromatography on Phenyl Sepharose CL‐4B. The enzyme was purified 8.4‐fold to give a 13.4% yield relative to the total activity in the crude extract and a final specific activity of 2057.1 U mg^?1 protein. SDS—PAGE revealed two migrating protein bands corresponding to apparent relative molecular masses of 55 and 62 kDa, respectively. The enzyme was optimally active at pH 6.0 and 50 °C, not influenced across a relatively broad pH range of 5.0–8.0 and retained over 60% activity at 70 °C after 30‐min incubation. It was highly significantly (P < 0.001) inhibited by Hg²⁺, appreciably (P < 0.01) inhibited by Ag⁺, Ba²⁺ and Pb²⁺ but highly significantly (P < 0.001) activated by Co²⁺, Mn²⁺ and Sr²⁺. The proteinase was equally highly significantly (P < 0.001) inhibited by both iodoacetate and p‐chloromercuribenzoate and hydrolysed casein to give the following kinetic constants: K_m = 0.33 mg ml^?1; V_max = 0.08 µmol ml^?1 min^?1. Copyright © 2004 Society of Chemical Industry     

Characteristics of an alkaline proteinase and exopeptidase from shrimp (Penaeus indicus) muscle

Activities of an alkaline proteinase and an exopeptidase were detected in the muscle of shrimp. Both enzymes could be extracted with unbuffered 0.5% KCI. The shrimp muscle alkaline proteinase, optimally active at pH 8.0 and 60°C, was partially purified and characterized. The heat stable enzyme had an apparent molecular weight of 250 KDa. Protein substrates such as casein, azocasein, azocoll and hemoglobin were hydrolyzed by the enzyme while it showed no action on bovine serum albumin and the synthetic substrates of proteinases. Active site directed inhibition experiments suggested that the enzyme was a metal-dependent serine proteinase. The shrimp exopeptidase cleaving amino acid naphthylamides at pH 6.8 and 40°C exhibited the characteristics of an aminopeptidase because of its susceptibility to bestatin, puromycin and a metal chelator.     

Optimization of peptic hydrolysis parameters for the production of angiotensin I-converting enzyme inhibitory hydrolysate from Acetes chinensis through Plackett-Burman and response surface methodological approaches

BACKGROUND: Angiotensin I‐converting enzyme (ACE) plays an important physiological role in regulating blood pressure. The elevation of blood pressure could be suppressed by inhibiting ACE. ACE inhibitory peptides derived from food proteins could exert antihypertensive effects without side effects. Acetes chinensis is a marine shrimp suitable for the production of ACE inhibitory peptides. The principal objective of this study was to screen for the significant variables, and further to optimize the levels of the selected variables, for the enzymatic production of ACE inhibitory peptides from Acetes chinensis. RESULTS: Plackett–Burman design and response surface methodology were employed to optimize the peptic hydrolysis parameters of Acetes chinensis to obtain a hydrolysate with potent ACE inhibitory activity. The peptic hydrolysis variables were subject to a Plackett–Burman design for screening the main factors. The selected significant parameters such as pH, hydrolysis temperature and enzyme/substrate (E/S) ratio were further optimized using a central composite design. The optimized conditions were: pH 2.5, hydrolysis temperature 45 °C, E/S ratio 17 800 U kg^?1 shrimp and substrate concentration 200 g L^?1. The results showed that 3–5 h hydrolysis could result in a hydrolysate with ACE inhibition IC₅₀ of 1.17 mg mL^?1 and a high DH of 25–27%. CONCLUSION: Plackett–Burman design and RSM performed well in the optimization of peptic hydrolysis parameters of Acetes chinensis to produce hydrolysate with ACE inhibitory activity. A hydrolysate with potent ACE inhibitory activity and high degree of hydrolysis was obtained, so that the yield of ACE inhibitory peptides in it was high. Copyright © 2011 Society of Chemical Industry     

Proteomic and peptidomic study of proteolysis in quarter milk after infusion with lipoteichoic acid from Staphylococcus aureus

Mastitic milk is associated with increased bovine protease activity, such as that from plasmin and somatic cell enzymes, which cause proteolysis of the caseins and may reduce cheese yield and quality. The aim of this work was to characterize the peptide profile resulting from proteolysis in a model mastitis system and to identify the proteases responsible. One quarter of each of 2 cows (A and B) was infused with lipoteichoic acid from Staphylococcus aureus. The somatic cell counts of the infused quarters reached a peak 6 h after infusion, whereas plasmin activity of those quarters also increased, reaching a peak after 48 and 12 h for cow A and B, respectively. Urea-polyacrylamide gel electrophoretograms of milk samples of cow A and B obtained at different time points after infusion and incubated for up to 7 d showed almost full hydrolysis of β- and α_S1-casein during incubation of milk samples at peak somatic cell counts, with that of β-casein being faster than that of α_S1-casein. Two-dimensional gel electrophoretograms of milk 6 h after infusion with the toxin confirmed hydrolysis of β- and α_S1-casein and the appearance of lower-molecular-weight products. Peptides were subsequently separated by reversed-phase HPLC and handmade nanoscale C₁₈ columns, and identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. Twenty different peptides were identified and shown to originate from α_s1- and β-casein. Plasmin, cathepsin B and D, elastase, and amino- and carboxypeptidases were suggested as possible responsible proteases based on the peptide cleavage sites. The presumptive activity of amino- and carboxypeptidases is surprising and may indicate the activity of cathepsin H, which has not been reported in milk previously.     

Polyphenoloxidase activity from strawberry fruit (Fragaria ananassa,Duch., cv Selva): characterisation and partial purification

In this work, polyphenoloxidase (PPO) from Selva strawberry fruit (Fragaria × ananassa, Duch) was extracted, characterised and partially purified. The activity of PPO was analysed in crude extracts obtained from either fresh fruits or acetone powder. The presence of NaCl and Triton X‐100 in the extraction buffer caused a marked increase in enzyme extractability. The enzyme showed an apparent K_m value of 11.2 mM with pyrocatechol as substrate. The maximum enzyme activity was observed at 50 °C and pH 5.3–6.0 without SDS and pH 7.2 in the presence of SDS. The presence of SDS increased PPO activity at pH 7.2 but diminished it at pH 6.0. The enzyme showed high thermal stability and maintained activities equal to or greater than 50% of its maximum activity in the 2.6–9.3 pH range. One polyphenoloxidase isoenzyme was detected in crude extracts of all ripening stages, showing an isoelectric point of 7.3. The specific activity of PPO decreased continuously through fruit ripening. Maximum specific activities were found at the ‘small green’ and ‘large green’ ripening stages. A total enzyme extract was partially purified by means of (NH₄)₂SO₄ precipitation and cationic exchange chromatography in an FPLC system. The purification grade achieved was near 25. The partially purified enzyme showed an isoelectric point equal to 7.3 and a molecular mass of 135 ± 4 kDa for the native protein. © 2000 Society of Chemical Industry