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.     

INHIBITION OF LIPOXYGENASE AND BLOOD THINNING EFFECTS OF MACKEREL PROTEIN HYDROLYSATE

Mackerel (Scomber australasicus) muscle was homogenized with deionized water at a ratio of 35:70 (w/w) for 2 min, boiled for 10 mm, and then cooled. Protease N was added to hydrofyze the homogenate at 50C for 2 h, then heated at 90C for 10 mm to stop the reaction. The supernatant obtained after centrifugation and filtration was the mackerel protein hydrolysate (MPH), It showed inhibitory effects on both lipoxygenase (LOX)‐catalyzed ami hemoglobin (Hb)‐catalyzed oxidation of arachidonic acid and linoleic add. Similar inhibitory effects were found on LOX's of soybean, tilapia gill and grey mullet gill. Blood thinning effects were observed in‐vitro by mixing MPH with red blood cells. The addition of MPH resulted in a reduced fluid consistency from 0.03 to 0.02, and an increased flow behavior index from 0.67 to 0.87 indicative of a flow behavior becoming closer to Newtonian type and a thinning consistency.     

PURIFICATION AND CHARACTERIZATION OF LIPOXYGENASE ISOZYMES FROM CANOLA (Brassica napus cv, WESTAR) SEED

Four isozymes, I', II'a, II'b and III’ of lipoxygenase (EC 1.13.11.12) from Canola (Brassica napus, cv Westar) seed were purified by successive chromatography on ion-exchange and size-exclusion columns using a Fast Protein Liquid Chromatograph (FPLC). The homogeneity of each isozyme was demonstrated by a single protein band on SDS-polyacrylamide gel electrophoresis. The molecular weights of isozymes I', II'a, II'b and III’ were 72, 000, 106, 000, 78, 000 and 62, 000, respectively. The optimum pH for lipoxygenase activity was 6.5 for isozyme I’ and 6.0 for isozymes II'b, II'b and III'. Apparent K_m value for isozymes I', II'a, II'b and III’ were 5.5 × 10^?4 M, 3.4 × 10^?4 M, 4.0 × 10^?4 M and 3.8 × 10^?4 M, respectively. Isozyme I’ displayed preferential activity towards monolinoleate and dilinoleate, while isozyme II'a demonstrated preferential activity towards dilinoleate followed by mono- and trilinoleate. No enzymatic activity was observed with both isozymes I’ and II'a toward free linoleic acid. Isozyme II'b showed activity towards free linoleate as well as mono-, di- and trilinoleate. Isozyme III’ showed preferential activity towards free linoleate. The activity of isozymes I’ and II'a was inhibited completely by the addition of 10 mM and 4 mM KCN, respectively, while the addition of 3 mM and 10 mM KCN to isozymes II'b and III', respectively, increased activity by approximately 20%.     

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.     

IDENTIFICATION AND CHARACTERIZATION OF MATRIX METALLOPROTEINASES FROM THE SARCOPLASMIC FRACTION OF COMMON CARP (CYPRINUS CARPIO) DARK MUSCLE

Two gelatin hydrolyzing proteinases in the sarcoplasmic fraction of common carp muscle were detected using gelatin zymography. A comparative study shows that the gelatin hydrolyzing activity in dark muscle is obviously higher than that in white muscle. The enzymes can transform to their active forms after treated by aminophenylmercuric acetate, an activator of matrix metalloproteinases. Optimum pH and temperature of these enzymes were around 8.0 and 40C using gelatin as substrate. Metalloproteinase inhibitors (ethylenediaminetetraacetic acid and ethylene glycol-bis (2-aminoethylother)-N, N, N', N'-tetraacetic acid) completely suppressed the activities and 1,10-phenanthroline also showed great inhibitory effects. However, other proteinase inhibitors, such as soybean trypsin inhibitor, benzamidine, E-64 and pepstatin A, did not show any effect. Metal ions Ca^{2 +} and Zn^{2 +} are essential for these gelatinolytic activities. All these facts indicate that these proteinases are matrix metalloproteinases. Furthermore, these enzymes hydrolyze collagen effectively and maybe thus proposed to be responsible for the tenderization of fish muscle during the postmortem stage.     

PARTIAL PURIFICATION AND CHARACTERIZATION OF SWEET CORN GERM LIPOXYGENASE

Lipoxygenase (LPO) extracted from the germ fraction of sweet corn (Zea mays L. var. Jubilee) was purified by acetone powder preparation; extraction with 0.1 M Tris-HCl, pH 8; 40–60% fractionation with ammonium sulfate; and conventional column chromatography on Sephacryl S-300 HR and Fast Protein Liquid Chromatography (FPLC) on a Mono Q column. A 124-fold purification was achieved with 26.3% recovery. Further purification was achieved by FPLC chromatofocusing on a Mono P column and size exclusion chromatography on Superose-12, with a resultant 436-fold purification and 16.8% recovery. The apparent molecular weight and isoelectric point (pI) determined by FPLC on Superose-12 and Mono P columns were 90,500 and 5.06, respectively. Lipoxygenase-catalyzed formation of conjugated dienes was inhibited by both synthetic (BHA, BHT) and natural phenolic antioxidants (quercetin, chlorogenic acid) at a concentration of 0.2 mM with 57.2, 16.3, 61.4 and 32.3% inhibition, respectively. The activation energy for thermal inactivation of sweet corn germ lipoxygenase from the ammonium sulfate preparation at 55–70C was 56.3 kcal/mol.     

EFFECT OF SLAUGHTER METHOD ON DEGRADATION OF INTRAMUSCULAR TYPE V COLLAGEN DURING SHORT-TERM CHILLED STORAGE OF CHUB MACKEREL SCOMBER JAPONICUS

The present paper demonstrates that a nonstntggling slaughter method can delay degradation of type V collagen in meat of chub mackerel Scomber japonicus and softening of the meat during postharvest chilled storage. The fish were slaughtered by piercing a knife into nape (nonstruggling method) or by leaving on ground (struggling method) and then stored in an ice box. Sensory study revealed that the postharvest softening of the meat was moderated at 4 and 8 h by the non‐struggling slaughter method in comparison with the struggling method. On the basis of the specific solubilization of type V collagen and reduced tyrosine content in it, a cleavage of the nonhelical regions (telopeptides) of the type V collagen occurred during the chilled storage in the fish slaughtered by the struggling method. The degradation of type V collagen was also slower in the meat of the fish slaughtered by the nonstruggling method, which can be directly linked to the moderation of the postharvest softening.     

CHARACTERIZATION OF PROTEOLYTIC ACTIVITY IN OCTOPUS (Octopus vulgaris) ARM MUSCLE

A new proteolytic activity assay was devised to avoid the interference of paramyosin which causes gelling during the enzymatic assay. Extremely high autolytic activity was observed in octopus arm muscle, which was 40–500 fold higher than those of various other fish species. The proteinase was inhibited strongly by leupeptin and iodoacetic acid and, to a lesser degree, by transepoxysuccinyl-L-leucylamino (4-guanidono)butane (E-64), indicating the class as a thioi proteinase. The proteinase exhibited optimum activity at pH 2.5 and 40C, although it contained a sulfhydryl group in the active site. Myosin heavy chain was the primary myofibrillar protein which was hydrolyzed during the autolysis of octopus arm followed by paramyosin. Actin showed no signs of hydrolysis during the incubation of up to 8 h. Due to its high affinity for myosin, the enzyme activity should be controlled during processing octopus to ensure the functionality of myosin.     

SOLUBILITY OF THE PROTEINS OF MACKEREL LIGHT MUSCLE AT LOW IONIC STRENGTH

Over 90% of the proteins of mackerel light muscle were soluble in solutions of physiological ionic strength or less. To accomplish this solublization, it was necessary to extract certain proteins at moderate ionic strength and neutral pH before extracting the rest of the myofibrillar and cytoskeletal proteins in water. Six proteins were favorably solubilized by sodium chloride solutions of moderate ionic strength at neutral pH under conditions that allowed later dissolution of myofibrillar and cytoskeletal proteins in water. The possibility is suggested that three of these proteins were involved in preventing the solubilization in water of other myofibrillar and cytoskeletal proteins of mackerel light muscle. Based on molecular masses and relative abundance, these proteins could possibly be M-protein (166 kDa), α-actinin (95 kDa) and desmin (56 kDa).     

PURIFICATION AND CHARACTERIZATION OF A MYOFIBRIL-BOUND SERINE PROTEINASE FROM THE SKELETAL MUSCLE OF SILVER CARP

Recently, a myofibril‐bound serine proteinase (MBSP) in the skeletal muscle of silver carp was identified. MBSP could be dissociated from myofibrils by treatment at pH 4.0. Following ultrafiltration concentration and chromatography on Sephacryl S‐200, High Q ion‐exchange and affinity column of Arginine Sepharose‐4B, MBSP was partially purified. The enzyme with an estimated molecular weight of 28 kDa cleaves synthetic fluorogenic substrates specifically at the carboxyl sites of arginine and lysine residues. MBSP activity is suppressed by serine proteinase inhibitors such as Pefabloc SC, lima bean trypsin inhibitor and benzamidine; it is insensitive to Pepstatin, l ‐3‐carboxy‐trans‐2, 3‐epoxypropionyl‐l ‐leucine‐4‐guanidinobutylamide and ethylenediaminetetraacetic acid, suggesting MBSP is a trypsin‐like serine proteinase. Optimal profiles of pH and temperature of the enzyme are 8.5 and 55C, respectively. Hydrolysis of myofibrillar proteins such as myosin heavy chain, actin and tropomyosin by purified MBSP occurred especially at around 55C, consistent with our proposal that MBSP plays a significant role in the Modori phenomenon.     

APPLICATION OF ACTINIDIN FROM KIWIFRUIT TO MEAT TENDERIZATION AND CHARACTERIZATION OF BEEF MUSCLE PROTEIN HYDROLYSIS

The effect of actinidin on the tenderness of broiled bovine semitendinosus (ST) steaks was studied. An actinidin activity of 400 U/mL resulted in Kramer shear values and sensory tenderness scores equivalent to that produced by Adolph's papain-based meat tenderizer (18 U/mL). Both were significantly (P<0.05) more tender than steaks having no tenderizing treatment. Actinidin did not over-tenderize the steak surface as did Adolph's meat tenderizer. Hydrolysis of myofibrillar proteins in enzyme-treated steaks prior to broiling was considerably less for actinidin than for papain when using activities to attain equal Kramer shear values of broiled steaks.     

CHARACTERIZATION OF POLYPHENOLOXIDASE FROM WILD PEAR (PYRUS ELAEGRIFOLIA)

Wild pear polyphenoloxidase (PePPO) was extracted and purified using a Sepharose 4B- l -tyrosine- p -amino benzoic acid affinity column. Optimum conditions for pH, temperature and heat inactivation were determined. At the optimum pH and temperature, K _M and V _max values for PePPO with catechol and pyrogallol were determined. The V _max/ K _M showed that PePPO has the greatest activity toward catechol. Optimum pH for PePPO was pH 6.0 using catechol as substrate. Optimum temperatures of PePPO for pyragallol and catechol were 65 and 35C, respectively. Enzyme activity decreased because of heat denaturation with increasing temperature. Inhibition of PePPO was investigated using p -aminobenzoic acid, ethyleneglycol, l -cysteine, l -tyrosine, sodium azide, p -aminobenzenesulfonamide, β-mercaptoethanol and dithiothreitol and catechol as substrate. Competitive-type inhibition was obtained with ethyleneglycol, l -cysteine, l -tyrosine, p -aminobenzenesulfonamide and dithiothreitol. Uncompetitive inhibition was obtained with β-mercaptoethanol, sodium azide and p -aminobenzoic acid. These results show that the most effective inhibitor for PePPO was dithiothreitol and that the type of inhibition depended on the origin of PPO.

PRACTICAL APPLICATIONS

In this present work, the properties of polyphenoloxidase in Pyrus elaegrifolia , including optimum temperature, optimum pH, substrate specificity and response to inhibitors, were studied.     

RECOVERY OF FISH BONE FROM HOKI (JOHNIUS BELENGERI) FRAME USING A PROTEOLYTIC ENZYME ISOLATED FROM MACKEREL INTESTINE

A new enzymatic treatment procedure was devised to recover and further utilize fish bones from processing which are normally discarded. This procedure includes the use of an enzyme preparation isolated from raw mackerel intestine by acetone precipitation. The caseinolytic activity of mackerel intestine crude enzyme (MICE) was approximately 0.58 U/mg protein, which was comparable to those of trypsin and α‐chymotrypsin, or even higher than those of papain and Aspergillus saitoi protease. The optimum pH, temperature and enzyme/substrate ratio of MICE for hydrolysis of protein with hoki (Johnius belengeri) frames were determined to be pH 9.0, 40C and 1:100 (w/w). When the hoki frame was treated by MICE for 6 h under the optimum reaction conditions, the yield of bone recovery was approximately 90%. Compared to Alcalase, trypsin, α‐chymotrypsin and Neutrase, the yield of bone recovery using MICE was higher. These results suggest that MICE may be utilized for bone recovery from fish frame.     

CHARACTERIZATION OF PHYTOCHELATIN-LIKE COMPLEXES FROM FLAX (LINUM USITATISSIMUM) SEED

Proteins were extracted from dehulled and delipidated powder of flaxseed (cv. NorMan), and fractionated by DEAE‐Sephacel onion exchange chromatography. Ultraviolet absorbance scans of ion exchange fractions eluting in buffers at pH 8.6 containing 0.45 M and 0.50M NaCl revealed A₂₅₅ >A₂₈₀. Furthermore, the A₂₅₅ was reduced upon acidification and restored by adjusting to the original pH, confirming spectral characteristics typical of metal‐thiolate complexes. Characteristic circular dichroism spectra and reversible aggregation‐dissociation behavior under low and high salt conditions observed for these fractions are also typical of metal binding phytochelatins reported for other plants, while the presence of aromatic amino acids indicated by fluorescence spectra suggests some similarity to nonmetallothionein proteins. Further work is underway to investigate the prevalence of these and other complexes as well as the distribution of metals in different protein fractions of flaxseed, as a function of cultivar and geographical location.     

PURIFICATION AND CHARACTERIZATION OF PROTEASES FROM OYSTER (CRASSOTREA GIGAS)

Proteases in oyster (Crassotrea gigas) were extracted with 10 mM Tris-HCl buffer solution and purified by ammonium sulfate fractionation, Sephadex G-150 gel filtration, repeated DEAE-Sephadex A-50 and CM-Sepharose CL-6B chromatography. Three fractions with caseinolytic activity, named I, II and III, were obtained from CM-Sepharose CL-6B and DEAE-Sephadex A-50 chromatography. The three proteases were purified to electrophoretic homogeneity. Substrate specificity studies indicated that protease I was a carboxypeptidase A-like enzyme; II and III were trypsin-like enzymes. The optimal pH of protease I for hydrolysis of hippuryl-L-phenylalanine was 9.0, II and III for hydrolysis of p-toluenesulfonyl-L-arginine methyl ester (TAME) was 8.0. The temperatures which inactivated 50% of enzymes were 78°C for protease I in 30 min; 50 and 52°C for protease II and III, respectively, in 5 min. The molecular weights of proteases I, II and III were 23,000, 34, 400 and 31, 000, respectively.