PURIFICATION AND CHARACTERIZATION OF PROTEASES FROM THE VISCERA OF MILKFISH (CHANOS CHANOS)

Proteases in acetone powder prepared from milkfish ( Chanos chanos ) viscera were extracted with deionized water and purified by ammonium sulfate fractionation, Sephadex G-75 gel filtration, repeated DEAE-Sephadex A-50 and CM-Sepharose CL-6B chromatography. Four fractions with caseinolytic activity, named A, B, C and D, were obtained from CM-Sepharose CL-6B and DEAE-Sephadex A-50 chromatography. The four proteases were purified to electrophoretic homogeneity. Substrate specificity studies indicated that proteases A and B were carboxypeptidase A-like and chymotrypsin-like enzymes, respectively; C and D were trypsin-like enzymes .

ABSTRACT

The optimal temperatures of proteases A, B, C and D for hydrolysis of casein were found to be 60, 60, 55 and 60°C, respectively. The optimal pH of protease A for hydrolysis of hippuryl-L-phenylalanine was 9.0, B for hydrolysis of acetyl-L-tyrosine ethyl ester was 8.0, C and D for hydrolysis of tosyl-L-arginine methyl ester was 8.0. The temperatures which inactivated 50% of enzymes in 5 min were 20°C for protease B; 51°C for protease C; 56°C for protease A; and 61°C for protease D. The molecular weights of proteases A, B, C, and D were 14,800, 16,800, 24,800 and 22,000 daltons, respectively.     

ISOLATION AND CHARACTERIZATION OF MICROORGANISMS INVOLVED IN THE FERMENTATION OF TRINIDAD'S CACAO BEANS

In order to isolate, identify and characterize the microflora of cacao beans before, during and after fermentation and locate possible sources contributing to microbial contamination, cacao beans from the Centeno and San Louis Estates in Trinidad were investigated. Prior to fermentation, the interior and exterior of the pods, hands of employees, utensils, dried pulp material of the sweatboxes and finally fruitflies ( Drosophila melanogaster ) were studied microbiologically. At Centeno Estate, beans were sampled at 5, 45 and 90 cm depths at 8-hr intervals for the first 72 hr and every 12 hr thereafter for 7 days. Sampling at San Louis Estate was carried out at 24 hr intervals for the same period. The changes in microbial population of the beans sampled at Centeno Estate ranged from 1.48 × 10⁵/g at 0 hr to 4.1 × 10⁵/g at the completion of the fermentation, whereas, at San Louis Estate they ranged from 6.8 × 10⁵/g to 9.2 × 10⁵/g during the same period. Taxonomical studies of isolates obtained during the fermentation period revealed the identification of 44 microorganisms at both Estates. Yeasts Zymomonas mobilis and several species of lactic acid organisms dominated the flora during the early stages of fermentation. As the fermentation progressed, these and other isolates were taken over by several species of genus Bacillus. Microbiological examination of dried and polished beans resulted in the identification of 22 organisms at Centeno Estate and 15 organisms at San Louis Estate     

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.     

PURIFICATION AND CHARACTERIZATION OF ACIDIC PROTEASES FROM THE STOMACH OF THE DEEPWATER FINFISH ORANGE ROUGHY (HOPLOSTETHUS ATLANTICUS)

Acidic proteases were extracted and purified from the stomach of orange roughy (Hoplostethus atlanticus). Protease I and II were glycoproteins with molecular weights of 33.5 and 34.5 KDa, respectively. Protease I had an isoelectric point of 5.30. The two forms of protease II (a and b) had isoelectric points of 4.35 and 4.40, respectively, and N-terminal sequence identity for 12 amino acids. The proteases exhibited optimal temperature activity at 37C. They had high activity at low temperatures and low thermal stability compared to mammalian pepsins. They were stable in the pH range of 2–4.5 and unstable above pH 6.5. Protease I and II had pH optima of 2.5 and 3.5, respectively, and K _m’values for the hydrolysis of hemoglobin (pH 3.0, 37C) of 124 μM and 517 μM, respectively. Enzyme activities were inhibited by pepstatin A and high NaCl concentrations, and were slightly stimulated by Ca²⁺ and Cu²⁺.     

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.     

HEAT-INDUCED GELATION OF MYOSIN IN THE PRESENCE OF ACTIN

ABSTRACT The rabbit muscle contractile proteins, myosin, actin and reconstituted actomyosin were mixed in 0.1–1.0 M KCl, 20 mM buffers, pH 5.0–8.0, and were tested quantitatively for thermally induced gelation properties by measuring the rigidity (shear modulus) of the system at 20–70°. Scanning electronmicroscopy (SEM) was also used to study the structure of the gels formed by gelation of myosin in the presence of F-actin. Under the standard condition, i.e. at 0.6 M KCl, pH 6.0 and 65°, decrease of the myosin/actin mole ratio to about 1.5–2.0 in the reconstituted acto-myosin system resulted in substantial augmentation of the rigidity of the gel formed. Further decreases in the myosin ratio relative to F-actin reduced the rigidity value of the gel to close to the level of myosin alone. Gel-formability of the reconstituted actomyosin was maximal at pH 5.5–6.0 and between 0.5 and 0.8 M KCl and decreased considerably at other pH values and KCl concentrations. The SEM studies revealed progressive changes in three dimensional ordering as actin concentration in the actomyosin varied. These were in concordance with the results of gel strength.     

REFINEMENTS OF THE CASEIN TURBIDITY METHOD FOR MEASURING PROTEASES IN BEER

The casein turbidity method for determining papain has been modified to allow it to be used for both normal and strong beers. Problems of instant casein coagulation which occur when the original reagents are mixed with strong beers were due to inadequate control of pH, and not to high alcohol levels. Methods are given for preparing convenient substrate and activator solutions that are stable for at least 8 days at 4°C. By carrying out estimations at 60°, rather than 50° as originally proposed, incubation times are roughly halved. It is shown that weak activators, like potassium metabisulphite, can reduce enzyme activities when added to a reaction mixture which also contains cysteine. The mixing sequence of beer, activator solution and substrate solution, as well as buffer strengths, influence the observed activities of papain. Further changes which might improve the method further have been proposed.     

CHARACTERIZATION OF THE SWINE SEX ODOR (SSO) COMPONENTS IN BOAR FAT VOLATILES

SUMMARY —Investigations were undertaken to characterize the volatiles collected from heated boar fat, particularly those components responsible for swine sex odor (SSO). Boar fat volatiles were collected by heating (to 150°C) the ground carcass fat in an all glass distillation apparatus with a liquid nitrogen trap attached. The condensed moisture and liquid nitrogen trap contents were combined and continuously extracted for 48 hr with diethyl ether. The ether was evaporated on a steam bath and the residue subjected to olfactory-gas chromatographic analysis by using a heated collection vent and a flame ionization detector. Using this analytical technique, a perspiration-like odor was detected resembling the SSO that normally emanates from heated boar fat. Further analysis using a gas chromatograph-mass spectrometer unit gave a molecular ion of 272 for this peak and major fragmentation ions identical to a 5a-androst-16-en-3-one standard. This standard was obtained by chromic acid oxidation of 3a-hydroxy-5a-androst-16-ene. Other types of odors were also noted in the boar fat volatile mixture, including those resembling perfume, wood, onion, musk and "Ivory" soap. This research demonstrated the presence of a unique smelling steroid accounting for the perspiration-like odor of heated boar fat. Furthermore, it has shown that the SSO component can be isolated directly from boar adipose tissue at normal cooking temperatures and atmospheric pressure.     

EFFECTS OF FEED DIETS ON DIGESTIVE PROTEASES FROM THE HEPATOPANCREAS OF WHITE SHRIMP (Penaeus vannamei)

Protease activities in the hepatopancreas extract (HP) from white shrimp (Penaeus vannamei) fed one of seven test diets for 30 days were evaluated by several methods. The test diet contained 85% of a reference ration for shrimp and 15% of either anchovy meal, tuna waste meal, deboned white fish meal, langostilla meal, soybean meal, and two menhaden meals as a protein replacer. One of the menhaden fish meals tested (B) had the lowest quality as a shrimp feed based on amino acid analysis. SDS-PAGE zymograms of HP from each of the seven diet groups showed similar proteins activity patterns with casein as substrate. The degree of hydrolysis of casein, measured by pH-stat, was also the same for HP from the seven diet groups (P > 0.05). However, total protease activity measured by azocasein hydrolysis (units/g HP) was higher for the diet group fed the test ration containing tuna waste as a replacer (P > 0.05). Trypsin and chymotrypsin activities measured with synthetic substrates (units/mg protein; units/g HP) from animals reared on the diet with menhaden meal B replacer were greater than in the other diet groups (P < 0.05). This study shows that a relatively small amount (15%) of a specific protein replacer in white shrimp rations can influence the protease activity of shrimp HP. Given that digestive proteases such as trypsin can leach into the muscle of postharvest shrimp and thereby cause softening of the meat, the impact of the rearing diet on postharvest shelf-life should be considered along with the standard measures of feed quality that are used by the fish farmer, i.e. animal growth and heatlh.     

IDENTIFICATION AND CHARACTERIZATION OF THE MICROFLORA AND SPOILAGE BACTERIA IN FRESHWATER CRAYFISH Procambarus clarkii (Girard)

Crayfish tails were collected from two commercial peeling plants in South Louisiana and stored at 0° and 5°C. Initially and after various periods of storage, bacteria were isolated from the peeled tails, identified generically and subsequently classified on the basis of their ability to produce "spoilage" in sterile crayfish tail flesh. Micrococcus, Staphylococcus, Alcaligenes and Achromobacter were the predominant genera in the fresh samples of tails. Achromobacter predominated during early storage and Pseudomonas and Achromobacter were the dominating organisms in the spoiled tails. Of the 280 isolated bacteria, 22.1% were classified as "rapid spoilers," 16.4% were "slow spoilers" and 61.5% were "nonspoilers." Most of the spoilers were Pseudomonas , while an appreciable number belonged to Achromobacter.     

THE ADDITION OF PROTEASES TO THE FERMENTER TO CONTROL CHILL-HAZE FORMATION

The addition of papain and proteases isolated from various yeast strains to fermentation to reduce chill-haze formation is discussed. Particular attention is paid to the behaviour of the yeast throughout fermentation and to the character of the final beers. The results suggest that fermentation in the presence of papain and a protease preparation prepared from Candida olea 148 progress normally and in the case of C. olea 148 protease-treated fermentation no change in beer flavour was detected.     

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.     

CHARACTERIZATION OF SURFACE AUXIN RESIDUE IN GREENHOUSE TOMATOES (LYCOPERSICON ESCULENTUM)

Greenhouse tomato samples (n = 20) was analyzed before and after peel removal in order to determine surface auxin residue. Mean 4‐CPA residue levels of greenhouse tomatoes with and without peels were 0.383 ± 0.123 mg kg^?1 and 0.241 ±0.085 mg kg^?1, respectively. This difference (36 ±13%) was statistically significant. The frequency distribution curve of tomatoes with peel had a peak point at 4‐CPA reside interval of 0.4‐ < 0.5 mg kg^?1 tomato, and shifted back to 4‐CPA residue interval of 0.2‐ < 0.3 mg kg^?1 for tomatoes without peel. Percentage of samples having 4‐CPA level lower than the critical concentration of 0.5 mg kg^?1 was 80% before peel removal, but increased to 100% upon being peeled. The mean 4‐CPA residue of peels was roughly estimated to be 3.449 mg kg^?1 peel based on peeled versus nonpeeled fruit residue.     

PARAMETERS OF TEXTURE CHANGE IN PROCESSED FISH: MYOSIN DENATURATION

Abstract The white muscle of the Sacramento blackfish ( Orthodon microlepidotus ) was processed by freezing, dehydration, and cooking. Myosin was extracted immediately afterwards or following a period of storage in order to examine evidence for denaturation. The tests used were the solubility of whole muscle protein and the intrinsic viscosity, isoelectric point, ATPase activity, ultra-violet absorption spectrum, and optical rotatory dispersion of purified myosin extract. Almost all measures used showed that denaturation increased in the order: fresh < frozen < frozen-stored < dehydrated < dehydrated-stored < cooked.     

玉米蛋白粉制备功能性短肽的研究

玉米功能短肽的制备对推动玉米蛋白资源的深加工和产业化进程有重要意义。在多次小试实验的基础上,利用酶工程技术制备玉米短肽,并对其抗氧化性进行了研究;结果表明:酶解100kg玉米蛋白粉可得玉米短肽干粉53kg,得率在50%以上,经测定,所得玉米短肽在浓度为1.67mg/mL时,其抗氧化抑制率为48.52%。