ENZYMATIC CHARACTERISTICS OF TRYPSIN FROM PYLORIC CECA OF SPOTTED MACKEREL (SCOMBER AUSTRALASICUS)

Trypsin was purified from the pyloric ceca of spotted mackerel (Scomber australasicus) by gel filtration on Sephacryl S‐200 and Sephadex G‐50. The purification and yield were 20‐fold and 81%, respectively, as compared to those in the starting crude extract. Final enzyme preparation was nearly homogeneous in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and the molecular weight of the enzyme was estimated to be 24,000 Da by SDS–PAGE. The trypsin was stable at pH 5–11 for 30 min at 30C, and its maximal activity against N^α‐p‐tosyl‐L‐arginine methyl ester was pH 8.0. Trypsin was heat‐stable up to about 50C for 15 min at pH 8.0. Optimum temperature of the trypsin enzyme was 60C. The enzyme was stabilized by calcium ion. The purified trypsin was strongly inhibited by serine protease inhibitors such as N‐p‐tosyl‐L‐lysine chloromethyl ketone and soybean trypsin inhibitor, suggesting that it is a trypsin‐like serine protease. N‐Terminal amino acid sequence of spotted mackerel trypsin was IVGGYECTAHSQPHQVSLNS.     

COMPARATIVE STUDY OF ENZYMATIC CHARACTERISTICS OF TRYPSINS FROM THE PYLORIC CECA OF YELLOW TAIL (SERIOLA QUINQUERADIATA) AND BROWN HAKELING (PHYSICULUS JAPONICUS)

Trypsins from the pyloric ceca of two fish species, yellow tail (Seriola quinqueradiata) and brown hakeling (Physiculus japonicus) were purified by a series of chromatographic separations. Purity increased 62‐ and 106‐fold with approximately 55 and 10% yield for yellow tail trypsin and brown hakeling trypsin, respectively. Final enzyme preparations were homogeneous in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), and the molecular weights of both enzymes were estimated to be 24 kDa by SDS‐PAGE. Yellow tail and brown hakeling trypsins had maximal activity at pH 8.0 for hydrolysis of N_α‐p‐tosyl‐L‐arginine methyl ester hydrochloride and was unstable at acidic pH. The optimum temperatures for yellow tail and brown hakeling trypsins were 60 and 50C, respectively. Yellow tail trypsin was stable up to 50C, whereas brown hakeling was stable up to 40C. Both trypsins were stabilized by calcium ions. The activities of both trypsins were strongly inhibited by soybean trypsin inhibitor and N_α‐p‐tosyl‐L‐lysine chloromethyl ketone hydrochloride, and were partially inhibited by ethylenediaminetetraacetic acid. The N‐terminal amino acid sequences of yellow tail trypsin and brown hakeling trypsin were determined as IVGGYECTPYSQPHQVSLNS and IVGGYECPKHSQPHQVSLNS, respectively.     

PURIFICATION AND CHARACTERIZATION OF TRYPSIN FROM PYLORIC CAECA OF BIGEYE SNAPPER (PRICANTHUS MACRACANTHUS)

Trypsin from the pyloric caeca of bigeye snapper was purified and characterized. Trypsin had an apparent molecular weight of 23.8 kDa when analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and substrate‐gel electrophoresis. The trypsin fraction consisted of three isoforms as evidenced by the appearance of three different bands on native‐PAGE. Optimal activity was observed at 55C and pH range of 8–11. The activity of trypsin fraction was completely inhibited by soybean trypsin inhibitor and was partially inhibited by E‐64 and ethylenediaminetetraacetic acid. CaCl₂ partially protected the trypsin fraction from activity loss at 40C, while NaCl (0–20%) decreased the activity in a concentration‐dependent manner. The apparent Michaelis–Menten constant (K_m) and catalytic constant (k_cat) were 0.312 mM and 1.06 s, respectively when Nα‐Benzoyl‐dl ‐arginine ρ‐nitroanilide was used as a substrate. Trypsin from the pyloric caeca of bigeye snapper generally showed similar characteristics to other fish trypsins.     

PURIFICATION AND CHARACTERIZATION OF PROTEASES FROM HEPATOPANCREAS OF CRAWFISH (PROCAMBARVS CLARKII)1

Four trypsin‐like enzymes (CP‐I, II, III and IV in order of elution on DEAE‐Sepharose chromatography), purified from the hepatopancreas of crawfish, were inhibited by protease inhibitors such as phenyl methyl suifonyl fluoride (PMSF), soybean trypsin inhibitor (SBTI), aprotinin and tosyl lysine chloromethyl ketone (TICK). The molecular weights of CP‐I, II, III and IV were determined to be 35.0, 41.2, 37.9 and 39.5 kDa, respectively, using sodium dodecyl sulfate polyacryl‐amide gel electrophoresis (SDS‐PAGE), These proteases had optimal esterase activity at pH 8.0–8.5 and showed the highest activity at 60–70C. Crawfish proteases were rich in acidic amino acids. Activation energies for hydrolysis of tosyl arginine methyl ester (TAME) by these proteases were 6.98 – 8.34 kcal/mole. Unlike other serine proteases, the activities of CP‐I and CP‐II were activated by mercury while CP‐HI and IV were inhibited.     

Quality changes of farmed cobia steaks held in cold stores (−18 °C)

Cobia (Rachycentron canadum) steaks held in cold store (?18 °C) were analysed aseptically in triplicates for the sensory, total aerobic bacterial count, proximate composition, pH, thiobarbituric acid‐reactive substance (TBA‐RS), formaldehyde, total volatile base nitrogen (TVB‐N), trimethyl amine‐nitrogen (TMA‐N), salt soluble nitrogen (SSN), nonprotein nitrogen (NPN), sodium dodecyl sulphate‐poly acrylamide gel electrophoresis (SDS‐PAGE) pattern. Steaks were sensorially acceptable up to 5 months of storage and the total bacterial counts did not exceed 6 log CFU counts. There were no significant changes in the pH values. TBA‐RS values increased significantly (P < 0.05) and reached 7.34 mg of malonaldehyde kg^?1 fat at the end. Formaldehyde content remained constant upto 4th month and later increased to 2.06 μg g^?1 (P < 0.05). TVB‐N and TMA‐N values did not exceed the acceptable limits. NPN contents showed no change, while SSN contents increased to 1.24% after 5 months. SDS‐PAGE pattern indicated no protein denaturation in the fish tissue. Results indicated that TBA‐RS value can only be considered as the valuable indicator in determining the quality of fish steaks held in cold stores.     

INFLUENCE OF HARVEST SEASON ON THE PROTEOLYTIC ACTIVITY OF HEPATOPANCREAS AND MANTLE TISSUES FROM JUMBO SQUID (DOSWICUS GIGAS)

Jumbo squid (Dosidicus gigas) size and protease activities were evaluated after harvest in April (A) and November (N). A were smaller (560±120 g vs 6040±1130 g) and hepatopancreas was a larger percent of body weight (8.3±2.5 vs 4.6±2.1). Mantle from A had lower water (73.9±l.l vs 79.1± 1.2) and higher protein (29.0±1.1 vs 23.1±0.8). Lipid and protein contents of N and A hepatopancreas tissues did not differ (P < 0.05). Azocaseinolytic, trypsin‐like, chymotrypsin‐like, aminopeptidase, and carboxypeptidase activities were detected in mantle (ME) and hepatopancreas extracts (HPE). HPE and ME from N had higher activity than A for all substrates (P < 0.05). With azocasein substrate, HPE activity from A had a pH optimum of 5 ‐ 6 and a temperature optimum of 70C, whereas HPE from Nhad highest activity at pH 9 and 40–70C. ME from A had maximum proteolytic activity at pH 6 and 60C, whereas that from N had maximum activity at pH 10 and 40C. SDS‐PAGE zymograms of HPE from A and N revealed different patterns of activity and 1 and 2 major protease zones, respectively.     

INFLUENCE OF TRANSGLUTAMINASE-INDUCED CROSS-LINKING ON IN VITRO DIGESTIBILITY OF SOY PROTEIN ISOLATE

The influence of covalent cross‐linking by microbial transglutaminase (MTGase) on the sequential in vitro pepsin and trypsin digestion process and the digestibility of soy protein isolate (SPI), was investigated by sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and nitrogen release analyses. Various subunits of β‐conglycinin and acidic subunits of glycinin were cross‐linked by MTGase to form high molecular weight (MW) biopolymers, while basic subunits of glycinin were unaffected. SDS‐PAGE analysis indicated that the cross‐linking mainly affected in vitro pepsin digestion pattern of various subunits of β‐conglycinin, while the trypsin digestion pattern of native SPI was nearly unaffected. Nitrogen release analysis showed that the in vitro pepsin or/and trypsin digestibility of native SPI (at the end of pepsin or trypsin ingestion) was significantly decreased (P ≤ 0.01) by the MTGase treatment (for more than 2 h). The cross‐linking by MTGase also significantly decreased the in vitro digestibility of preheated SPI. These results suggest that the cross‐linking by means of transglutaminase may negatively affect the nutritional properties of food proteins.     

THE PURIFICATION OF PROTEASE FROM COWSLIP (PRIMULA VERIS) AND ITS USE IN FOOD PROCESSING

Proteases perform very important functions and are used in different objectives as in vitro and in vivo. In recent years, proteases have been used for clinical, pharmaceutical and industrial applications. Most of these proteases are obtained from animal sources. News of diseases passing over from animals to human (severe acute respiratory syndrome [SARS], mad cow) has created suspicion of contamination in animal food and its product. Therefore, a new vegetal protease source for use in the food industry was studied. Ammonium sulfate fractionation and CM‐sephadex ion exchange chromatography were used in the purification of the enzyme. The purified enzyme has an optimum pH of 7.0 and its optimum temperature was 70C. The V_max and K_m values determined by Lineweaver–Burk graphics were 0.44 mg/mL.min and 40 mM, respectively. The purification degree was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE ). The native molecular weight of the enzyme was 46 kDa using a gel filtration chromatograph. SDS‐PAGE results show that the enzyme has two subunits, which have protease activity at 32 and 14 kD. It was investigated whether the purified and characterized protease enzyme would cause to congeal milk or could produce digestion of chicken and cow meat. The results showed that the protease enzyme can be used for industrial production.     

Isolation and Characterization of Collagen from the Body Wall of Sea Cucumber Stichopus monotuberculatus

To exploit a new collagen resource from the body wall of tropical sea cucumber, pepsin‐solubilized collagen of Stichopus monotuberculatus (PSC‐Sm) was isolated and characterized with UV‐vis spectra, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), amino acid composition, enzyme‐digested peptide maps, Fourier transform infrared spectroscopy (FTIR), maximum transition temperature (T_m), and solubilities. The maximum absorbance of PSC‐Sm was exhibited at 218 nm in UV‐vis spectra. The triple helical structure and activity of PSC‐Sm could be indicated by FTIR. SDS‐PAGE showed that the triple helix of PSC‐Sm was formed as (α₁)₃ by 3 α₁ chain homologous with molecular weight of 137 kDa. The T_m of PSC‐Sm and calf skin collagen (CSC) were 30.2 and 35.0 ºC, respectively, which consistent with the result of FTIR that CSC contained more stable triple‐helix than PSC‐Sm. Peptide maps were different between PSC‐Sm and CSC, indicating the differences in their amino acid compositions and sequences. The maximum and minimum solubilities of PSC‐Sm were observed at pH 2.0 and 4.0, respectively. A sharp decrease in solubility appeared when NaCl concentration was between 3% and 5%. These results showed that collagen from S. monotuberculatus had the type I collagen characteristics and good thermal stability, and therefore, it could be used as an alternative resource of collagen.     

PARTIAL PURIFICATION AND CHARACTERIZATION OF TROPOMYOSIN-BOUND SERINE PROTEINASE FROM THE SKELETAL MUSCLE OF YELLOW CROAKER (PSEUDOSCIAENA CROCEA)

A myofibril‐bound serine proteinase (MBSP) in the skeletal muscle of yellow croaker (Pseudosciaena crocea) was isolated from myofibril by heat treatment and chromatographies on Sephacryl S‐200, fast performance liquid chromatography (FPLC) on Mono Q column and high performance liquid chromatography (HPLC) using a Bio‐Sil SEC‐125 column. A single protein band with a molecular weight of 34 kDa was observed on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Optimum temperature and pH of the purified protein was 55C and 8.0. Inhibitor susceptibility analysis indicated that the enzymatic activity was effectively suppressed by serine proteinase inhibitors such as Pefabloc 4‐(2‐aminoethyl)‐benzenesulfonyl fluoride hydrochloride and aprotinin, while inhibitors for other proteinases (namely cysteine, asparatic and metallo) did not show any inhibitory effect. At the last purification stage, the electrophoretic study revealed that the proteinase associated with tropomyosin, as the N‐terminal amino acid sequence of the 34 kDa main band protein, exhibited high sequence homology (90%) to α‐tropomyosin from white croaker, human and rat. This result suggested that yellow croaker MBSP is an α‐tropomyosin‐binding proteinase.     

Kinetic Analysis of the Digestion of Bovine Type I Collagen Telopeptides with Porcine Pepsin

Collagen is frequently digested using pepsin in industries to produce a triple helical collagen without the N‐ and C‐terminal telopeptides. However, kinetic analysis of this reaction is difficult because several Lys residues in the N‐ and in the C‐terminal telopeptides form covalent bonds, leading to multiple substrates species, and pepsin cleaves collagen at various sites in the N‐terminal and in the C‐terminal telopeptides, yielding different products. Here we performed kinetic analysis of the digestion of bovine type I collagen with porcine pepsin. The reaction could be monitored by SDS‐PAGE by measuring the intensity of the protein bands corresponding to the variant β11 chain. We obtained kinetic parameters relative to the decrease in the variant β11 chain upon digestion. At pH 4.0, the K_m and k_cat values increased with increasing temperature (30 to 65 °C), although the k_cat/K_m values were stable. Additional cleavage at the helical region was detected at 45 to 65 °C. At 37 °C, the K_m and k_cat values increased with decreasing pH, and the k_cat/K_m values at pH 2.1 to 4.5 were stable and higher than those at pH 5.0 and 5.5. No additional cleavage was detected at the examined pH. Thus, the optimal pH and temperatures for selective digestion of collagen telopeptides with pepsin are 2.1 to 4.5 and 30 to 40 °C, respectively. These results suggest that the method might be useful for the kinetic analysis of the digestion of collagen telopeptides with pepsin.     

PURIFICATION AND PARTIAL CHARACTERIZATION OF AN ENDO-POLYGALACTURONASE FROM ASPERGILLUS NIGER

A pectinase was identified and isolated from a commercial Aspergillus niger pectinase preparation. The crude enzyme preparation, which was prepared by precipitation of the water extract of the culture of A. niger with ammonium sulfate, was further fractionated by three steps of chromatography, i. e., cation exchange, hydrophobic interaction and onion exchange, to obtain an electrophoretically homogeneous pectinase. The molecular weight of the purified enzyme was estimated by SDS‐PAGE to be about 40.4 kDa under both nonreducing and reducing conditions, with the optimum pH at 5.0 and the optimum temperature at 36C. The enzyme was stable at temperatures below 35C. The partial N‐terminal ammo acid sequence data analysis of the first 19 amina acids of the obtained pectinase revealed 94.7% and 89.5% homology with two reported pectinases from A. niger.     

PURIFICATION AND CHARACTERIZATION OF A MALATE DEHYDROGENASE FROM PHASEOLUS MUNGO

A malate dehydrogenase (MDH) was identified and isolated from the seeds of the mung bean (Phaseolus mungo). The procedure entailed extraction, ammonium sulfate precipitation, ion exchange chromatography on CM‐Sephadex and high performance liquid chromatography on POROS HS‐20. The purified protein exhibited a molecular mass of 38 kDa in SDS‐polyacrylamide gel electrophoresis under both nonreduced and reduced conditions. The pI was 9.7 by isoelectric focusing. The specific activity of the MDH was estimated to be 199 U/mg. The enzyme expressed its optimum activity at pH 7.2, 35C, and showed stable activity below 40C. The Km for oxaloacetate was 112 µM. The partial N‐terminal amino acid sequence data analysis of the first 20 amino acids of the mung bean MDH revealed 95 and 80% homology with two reported MDH from soya bean (Glycine max) and potato (Solanum tuberosum), respectively.     

Secretagogue and bacteriostatic active fractions derived from a peptic hydro‐ lysate of alfalfa RuBisCO small purified subunit

For the first time a purification process for small RuBisCO (ribulose‐1,5‐bisphosphate carboxylase/oxygenase) subunit (SRS) was developed from an industrial by‐product of alfalfa, taking advantage of its solubility at low pH. Only one protein strip (14 kDa) was clearly detected in the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) profile of the supernatant at pH 2. The recovery of SRS was 48% by this method, with a purity estimated as 98% by densitometry and reverse phase high‐performance liquid chromatography (RP‐HPLC). Moreover, most polyphenolic compounds were discarded, as confirmed by spectrophotometry and RP‐HPLC. SRS hydrolysis was performed for 20 h at 37 °C using pepsin in ammonia/formic acid buffer at pH 3. The hydrolysate was fractionated on a Sephadex G25 column equilibrated with ethanolamine/HCl buffer. Biological activities were found in two fractions. The first fraction showed slight bacteriostatic properties against two pathogenic bacteria, Salmonella arizonae and Shigella sonnei. The second fraction, tested by radioimmunoassay (RIA), presented a secretagogue activity comparable to that of gastrin. Copyright © 2006 Society of Chemical Industry     

Purification,characterisation and kinetics of a trypsin inhibitor from black gram (Vigna mungo)

A black gram (Vigna mungo) trypsin inhibitor (BGTI) was purified to homogeneity by ammonium sulphate precipitation, DEAE-cellulose chromatography and affinity chromatography. The homogeneity of the inhibitor was identified by polyacrylamide gel electrophoresis (PAGE), SDS–PAGE, immunodiffusion and immunoelectrophoresis. The isoelectric point of BGTI was 8.32. The molecular weight of the BGTI was found to be about 12500 by both gel filtration and SDS–PAGE. One mole of the BGTI was found to contain 75 amino acid residues, and it was identified as a glycoprotein, comprising about 18.2% carbohydrate. The BGTI was an uncompetitive inhibitor, stable in the pH range 2–10 and in the temperature range 30–80°C for 30 min.     

Effect of extrusion cooking on structure and functional properties of pea and kidney bean proteins

Pea (Pisum sativum L cv Ballet) and kidney bean (Phaseolus vulgaris L cv Pinto) seeds were extruded at 148 and 156 °C respectively. Protein solubility at various pH values and in various solvents was determined and analysis of protein fractions was carried out by SDS‐PAGE. Also, sulphhydryl and disulphide groups, water‐holding capacity (WHC), water solubility index (WSI) and oil absorption capacity (OAC) were determined. No changes in total nitrogen content of pea and kidney bean seeds occurred as a result of thermal treatment. Protein solubility from raw and extruded legumes was significantly higher in saline solutions than in water in the pH range 2–10. The solubility of proteins from extruded pea and kidney bean flours was greatly decreased with respect to native flours when extraction was in buffer (pH 7.0) alone. Extraction with buffer containing 2‐mercaptoethanol (2‐ME) or sodium dodecyl sulphate (SDS), alone or in combination, greatly increased protein extractability. As a result, the relative solubility was nearly 100% in buffer with SDS and 2‐ME for both raw and extruded samples. Total and free sulphhydryl group and disulphide contents decreased significantly (P < 0.05) after extrusion cooking. Moreover, extrusion treatment caused major changes in the band patterns of the albumin and globulin fractions obtained by SDS‐PAGE. WHC and WSI of extrudates increased significantly in both peas and kidney beans. A significant reduction in OAC was observed in extruded kidney bean flour. © 2000 Society of Chemical Industry     

Purification of diamine oxidase and its properties in germinated fava bean (Vicia faba L.)

Background: γ‐Aminobutyric acid (GABA) is a non‐protein amino acid with bioactive functions for human health. Diamine oxidase (DAO, EC 1.4.3.6) is one of the key enzymes for GABA formation. In the present study, this enzyme was purified from 5 day germinated fava bean and its properties were investigated in vitro. Results: The molecular mass of the enzyme estimated by Sephadex G‐100 gel filtration was 121 kDa. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) displayed a single band at a molecular mass of 52 kDa. The enzyme had optimal activity at 40 °C and retained its activity after being incubated at 30 °C for 30 min. It showed higher activity at pH 6.5 than at other pH values. The enzyme was significantly inhibited by Mg²⁺, Cu²⁺, Fe³⁺, aminoguanidine, ethylene glycol tetraacetic acid (EGTA), ethylene diamine tetraacetic acid disodium salt (EDTA‐Na₂), L ‐cysteine and β‐mercaptoethanol. The K_m value of DAO was 0.23 mmol L^?1 for putrescine and 0.96 mmol L^?1 for spermidine. However, the enzyme did not degrade spermine. Conclusion: DAO from germinated fava bean was purified. The optimal reaction temperature and pH of the enzyme were mild. The enzyme had higher affinity to putrescine than to spermidine and spermine. Copyright © 2011 Society of Chemical Industry     

ALBUMINS FROM THE BEAN PHASEOLUS VULGARIS: EFFECTS OF HEAT TREATMENT

Bean albumins heated at different temperature and time intervals (60C to 135C, 1 to 30 min) suffered a reduction of in vitro digestibility, proportional to the intensity of treatment. The electrophoretic (SDS‐PAGE) and chromatographic (gel filtration) profiles showed the formation of high molecular weight protein aggregates, involving disulfide bonds. More intense heat treatments caused formation of bonds not broken by β‐mercaptoethanol or SDS, resulting in aggregates resistant toproteolysis. Determination of available lysine showed that these residues were not implied in possible crosslinking reactions, and heated albumins showed the same amino acid profile as the control. The decrease of SH groups upon heating followed a first order kinetics, with an activation energy of 76.9kJ/mol, and an increasing k value ranging from 0.0012 at 60C to 0.267 at 135C. DSC thermograms of albumins showed two peaks, with maximum denaturation temperature of 87C and 98C, and total enthalpy of denaturation of 7.7 J/g.     

PURIFICATION AND CHARACTERIZATION OF β-GALACTOSIDASE FROM MUCOR PUSILLUS

The β-galactosidase from Mucor pusillus was purified by acetone precipitation, gel filtration and ion-exchange chromatography. Its molecular weight was 129 kDa on SDS PAGE, and its pI was 4.55. Optimum activity was observed at pH 4 and at 65C. Thermal denaturation at temperatures above 60C was essentially first order with an activation energy of 26.4 KJ/mole. Activity was not affected by metal ions or EDTA but was inhibited by galactose and galactono 1–4 lactone. The K_m for lactose at 37C was 22 mM. The enzyme was devoid of cysteine/cystine and stained positive for carbohydrate. Overall the enzyme is similar in structural and kinetic properties to the β-galactosidase from Aspergillus niger.     

Purification and characterization of extracellular proteinase produced by Brevibacterium linens ATCC 9172

The micro-organism Brevibacterium linens ATCC 9172 produced five extracellular proteinases, as shown by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) with copolymerized gelatine. One of these was purified to homogeneity by ion-exchange chromatography and native preparative PAGE. The optimum pH and temperature for the proteinase were 8.0 and 50°C, respectively. The enzyme remained stable over a pH range from 6 to 10. The molecular weight estimated by SDS–PAGE was 56 kDa. Serine proteinase inhibitors 3,4-dichloroisocoumarin (3,4-DCI) and phenylmethylsulphonylfluoride (PMSF) inhibited, while Mg²⁺ and Ca²⁺ ions activated the proteinase.