Purification and characterization of heat-stable alkaline proteinase from bigeye snapper (Priacanthus macracanthus) muscle

Heat-stable alkaline proteinase was purified from bigeye snapper (Priacanthus macracanthus) ordinary muscle by heat-treatment and a series of chromatographies including Phenyl-Sepharose 6 Fast Flow, Source 15Q and Superose 12 HR 10/30. It was purified to 5180-fold with a yield of 0.8%. The molecular weight of purified proteinase was estimated to be 72 kDa by gel filtration. The proteinase appeared as two proteinase activity bands with molecular weights of 66 and 13.7 kDa on non-reducing SDS-substrate gel. Accordingly, it was found to consist of two different subunits. The optimum pH and temperature for casein hydrolysis were 8.5 and 60 °C, respectively. The proteolytic activity was strongly inhibited by soybean trypsin inhibitor and partially inhibited by ethylenediaminetetraacetic acid, while pepstatin A and E-64 showed no inhibition. Purified proteinase was able to hydrolyze Boc-Phe-Ser-Arg-MCA, but rarely hydrolyzed Z-Phe-Arg-MCA and Z-Arg-Arg-MCA. In addition, it mainly degraded myosin heavy chain, not actin. These results suggest that purified proteinase was serine proteinase, which is probably involved in gel weakening of bigeye snapper surimi.     

Presettlement schooling behaviour of a priacanthid,the Purplespotted Bigeye Priacanthus tayenus (Priacanthidae: Teleostei)

We report in situ behavioural observations of presettlement schooling in Priacanthus tayenus off Coral Bay, Western Australia collected using pelagic Baited Remote Underwater stereo-Video systems. Two groups of fish (8 and 9 individuals) were observed that aggregated into a single school. Mean total length was 24.1 mm (12.5–30.2 mm). The fish swam at a mean speed of 8.5 cm s^?1 in a group spacing themselves more or less evenly at a distance of around one body length from the nearest neighbour within the school. P. tayenus appeared to be sometimes associated with juveniles of other species. The results presented here add to the limited, but growing body of literature on the schooling behaviour of the early pelagic stages of demersal fishes.     

Effect of adding insoluble solids from surimi wash water on the functional and mechanical properties of pacific whiting grade A surimi

Surimi processors are seeking means to improve the utilization of seafood resources to increase productivity and also in response to the strong public pressure on this industry to reduce the organic matter in processing water discharged into the environment. Insoluble solids (IS) can be recovered from surimi wash water (SWW) by centrifugation. The quality implications of adding 0 (control), 1%, 3% and 5% of solids (SWW-IS) into surimi paste and gels were evaluated by determining their mechanical properties, moisture retention and color. This study showed that adding 1% SWW-IS improved the mechanical properties of Pacific whiting surimi with a minimal effect on color. Higher additions resulted in quality deterioration in mechanical properties and color.     

Autoxidation of myoglobin from bigeye tuna fish (Thunnus obesus)

Native oxymyoglobin (MbO2) was isolated directly from the skeletal muscle of bigeye tuna (Thunnus obesus) with complete separation from metmyoglobin (metMb) on a CM-cellulose column. It was examined for its stability properties over a wide range of pH values (pH 5-12) in 0.1 M buffer at 25 degrees C. When compared with sperm whale MbO2 as a reference, the tuna MbO2 was found to be much more susceptible to autoxidation. Kinetic analysis has revealed that the rate constant for a nucleophilic displacement of O2- from MbO2 by an entering water molecule is 10-times higher than the corresponding value for sperm whale MbO2. The magnitude of the circular dichroism of bigeye tuna myoglobin at 222 nm was comparable to that of sperm whale myoglobin, but its hydropathy profile revealed the region corresponding to the distal side of the heme iron to be apparently less hydrophobic. The kinetic simulation also demonstrated that accessibility of the solvent water molecule to the heme pocket is clearly a key factor in the stability properties of the bound dioxygen.     

Isolation and characterization of microsatellites in lane snapper (Lutjanus synagris), mutton snapper (Lutjanus analis), and yellowtail snapper (Ocyurus chrysurus)

Polymerase chain reaction primer pairs for a total of 25 nuclear‐encoded microsatellites (loci) were developed from genomic DNA libraries of lane snapper (Lutjanus synagris), mutton snapper (Lutjanus analis), and yellowtail snapper (Ocyurus chrysurus). The microsatellites include 24 perfect (21 dinucleotide and three trinucleotide) and one imperfect (combination tetranucleotide/tetranucleotide) repeat motifs. A total of 32 individuals of each species were assayed for allelic variation at all 25 microsatellites; reliable amplification products were generated for lane snapper (25 loci), mutton snapper (21 loci), and yellowtail snapper (24 loci). Significant deviations from Hardy–Weinberg expectations, following Bonferroni corrections, were found for one microsatellite in lane and yellowtail snappers, and for three microsatellites in mutton snapper. All pairwise comparisons of microsatellites (all three species) did not deviate significantly from genotypic equilibrium.     

Effect of pH on the oxidation-reduction properties of thioredoxins

Oxidation-reduction midpoint potential (E(m)) versus pH profiles were measured for wild-type thioredoxins from Escherichia coli and from the green alga Chlamydomonas reinhardtii and for a number of site-directed mutants of these two thioredoxins. These profiles all exhibit slopes of approximately -59 mV per pH unit, characteristic of the uptake of two protons per reduction of an active-site thioredoxin disulfide, at acidic, neutral, and moderately alkaline pH values. At higher pH values, these profiles exhibit slopes of either -29.5 mV per pH unit, characteristic of the uptake of one proton per disulfide reduced, or are pH-independent, indicating that neither proton uptake nor proton release is associated with reduction of the active-site disulfide. Reduction of the two wild-type thioredoxins is accompanied by the uptake of two protons even at pH values where the more acidic cysteine thiol group of the reduced proteins would be expected to be completely unprotonated. The effect of site-directed mutagenesis of two highly conserved aspartate residues that play important structural and/or catalytic roles in both thioredoxins, and which could in principle play a role in proton transfer, on the pK(a) values of redox-linked acid dissociations (deduced from changes in slope of the E(m) versus pH profiles) has also been determined for both E. coli thioredoxin and C. reinhardtii thioredoxin h.     

Properties and microstructure of protein-based film from round scad (Decapterus maruadsi) muscle as affected by palm oil and chitosan incorporation

The properties of protein-based film prepared from round scad (Decapterus maruadsi) muscle in the absence and the presence of palm oil and/or chitosan were investigated. Films added with 25% palm oil (as glycerol substitiution) had the slight decrease in water vapor permeability (WVP) and elongation at break (EAB) (p < 0.05). WVP and tensile strength (TS) of films increased but EAB decreased when 10–40% chitosan (as protein substitution) was incorporated (p < 0.05). Hydrophobic interactions and hydrogen bonds, together with disulfide and non-disulfide covalent bonds, played an important role in stabilizing the film matrix. The a* and b*-values increased with increasing chitosan levels (p < 0.05). Films added with chitosan were less transparent and had the lowered transmission in the visible range. The incorporation of 25% palm oil and 40% chitosan yielded the films with the improved TS but decreased water vapor barrier property. Apart from film strengthening effect, chitosan inconjunction with Tween-20 most likely functioned as the emulsifier/stabilizer in film forming solution containing palm oil.     

Effect of pH on the Kinetic Parameters of NADP-Malic Enzyme from a C(4)Flaveria (Asteraceae) Species

We have used the pH variation in the kinetic parameters with respect to malate of NADP-malic enzyme purified from the C₄ species, Flaveria trinervia, to compare the pK values of its functional groups with those for the pigeon liver NADP-malic enzyme (MI Schimerlik, WW Cleland [1977] Biochemistry 16: 576-583) and the plant NAD-malic enzyme (KO Willeford, RT Wedding [1987] Plant Physiol 84: 1084-1087). Like the other enzymes, the C₄ enzyme has a group with a pK of about 6.0 (6.6 for the C₄ enzyme), as indicated from plots of the log V_max/K_m (V_max = maximum rate of catalysis) versus pH, which must lose a proton for malate binding and subsequent catalysis. The optimum ionization for the C₄ enzyme-NADP-Mg²⁺ complex occurs at pH 7.1 to 7.5. From pH 7.5 to 8.4, the K_m increases, but V_max remains constant. The log V_max/K_m plot in this pH range indicates a group with a pK of about 7.7. The other malic enzymes exhibit a similar pK. Above pH 8.4, deprotonation leads to a marked increase in K_m and a decrease in V_max for the C₄ enzyme. As in the case of the animal enzyme, the log V_max/K_m plot for the C₄ enzyme appears to approach a slope of two. The curve suggests an average pK of 8.4 for the groups involved, while the animal enzyme exhibits an average pK of 9.0. The NAD-malic enzyme does not exhibit any pK values at these high pK values. We hypothesize that the putative groups with the high pK values may be at least partially responsible for the ability of the C₄ NADP-malic enzyme to maintain high activity at pH 8.0 in illuminated chloroplasts.     

Isolation and characterization of microsatellite DNA loci from Russell's snapper (Lutjanus russellii)

A total of 37 microsatellite loci from the Russell's snapper, Lutjanus russellii, were successfully isolated and characterized. Thirty‐four loci were polymorphic in L. russellii samples. Twenty of the 37 markers did not differ significantly from Hardy–Weinberg expected genotype proportions. Significant linkage disequilibrium was detected in one pairwise comparison. The numbers of alleles and observed heterozygosities in polymorphic loci ranged from two to 16 and from 0.41 to 0.95, respectively. These markers will be useful for studying the population genetic structure of this species.     

红笛鲷仔鱼体内特异性母源抗体降解动力学研究

应用山羊IgG免疫红笛鲷亲鱼,人工催产孵化出仔鱼后,用双抗体夹心法检测仔鱼特异性抗体的降解过程,发现抗体水平随时间变化呈下降趋势且可以持续7d,每尾仔鱼特异性抗体降解量约0.001μg,最低检测限为0.482μg/mL。采用饱和硫酸铵分步盐析与亲和层析等方法纯化红笛鲷仔鱼特异性抗体,结果显示,盐析仔鱼特异性抗体最理想的硫酸铵饱和度为40%－50%;亲和层析分离到两个明显独立的层析峰。SDS-PAGE证明纯化的抗体具有极高纯度,其L、H链分子量分别为29 kD和77.5 kD,抗体分子量约为852 kD。该抗体不仅能够与山羊IgG产生特异性免疫反应,而且能与兔抗红笛鲷IgM抗血清产生免疫反应,因而认为红笛鲷母源抗体可以垂直转移至仔鱼。     

First record of Randall's snapper Randallichthys filamentosus (Perciformes: Lujanidae) from the eastern Indian Ocean (north-western Australia)

The rare Randall's snapper Randallichthys filamentosus is recorded here for the first time in the eastern Indian Ocean from Scott Reef, north-western Australia. A single specimen of R. filamentosus , measuring 495 mm fork length, was captured on 9 July 2006 at a depth of 211 m on the outer reef slopes of Scott Reef. The capture of this specimen indicates that this deepwater fish is more widely distributed than was previously acknowledged and further implies that this species has a dispersal capacity that extends across ocean boundaries.     

Effect of pH on conformation and kinetic properties of phosphorylase from bovine skeletal muscles

Interaction of phosphorylase with 8-anilino-1-naphthalene-sulfonate (ANS) results in the formation of an ANS-protein complex. The microenvironment of the protein-bound dye changes depending on pH. Using fluorimetric titration, the dissociation constants for the complex (Kd = 23 and 57 microM for pH 6.2 and 6.8, respectively) were determined. The mode of the enzyme inhibition by ANS also changes depending on pH. At pH 6.8, ANS competitively inhibits the enzyme with respect to AMP, but does not compete with the nucleotide at pH 6.2; the corresponding Ki values are equal to 160 and 26 microM. The protective effect of ligands from the inhibiting effect of ANS was studied. It was shown that at pH 6.2, the enzyme is protected from the inhibition only by the substrate, glucose-1-phosphate, whereas at pH 6.8--by the allosteric inhibitor, glucose-6-phosphate. These findings suggest that at pH 6.2 the conformation of the enzyme molecule is induced by the substrate, while at pH 6.8--by the allosteric inhibitor. ANS binding in the vicinity of the active or allosteric centers is due to the pH-dependent conformational transition. The data obtained suggest that the pH changes within the range of 6.2-6.8 are essential for the regulation of enzyme activity.     

Isolation and characterization of polymorphic microsatellite loci from black snapper Sebastodes fuscescens (Houttuyn)

Black snapper Sebastodes fuscescens (Houttuyn) is an economocally important species. Fourty-five microsatellite loci were isolated from an enriched genomic library of S. fuscescens. Ten of these loci were polymorphic in a test population with alleles per locus ranging from 2 to 5, and observed and expected heterozygosities per locus from 0.21 to 1.00 and from 0.19 to 0.86, respectively. Three loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction and no significant linkage disequilibrium was found between pairs of loci. These polymorphic microsatellite loci would be useful for investigating genetic diversity of S. fuscescens and other related species.     

Improved film properties of radiation-treated medium-chain-length poly(hydroxyalkanoates)

Medium-chain-length poly(hydroxyalkanoates) (mcl-PHAs) were synthesized from coconut oil (PHA-C), tallow (PHA-T), and soybean oil (PHA-S) by bacterial fermentation using Pseudomonas resinovorans as the producer strain. Films were solution-cast and subjected to 50 kGy of -irradiation. This resulted in crosslink formation based on the number of olefinic groups present in the polymer side-chains. In each case, radiation improved the tensile strength (104% and 63%), percent elongation (49% and 13%), and Young's modulus (30% and 76%) of PHA-C and PHA-T films, respectively. The greatest effect was on PHA-S, which was converted from an amorphous, liquid-like material to a solid elastomeric film. © Rapid Science Ltd. 1998     

Age,growth, mortality and population structure of silver croaker Pennahia argentata (Houttuyn, 1782) and red bigeye Priacanthus macracanthus Cuvier, 1829 in the north‐central Taiwan Strait

The main objectives of this study were to estimate the age, growth, mortality and population structure of silver croaker Pennahia argentata (Houttuyn, 1782) and red bigeye Priacanthus macracanthus Cuvier, 1829 and to explore the factors causing the fluctuation of the fish populations and the ecological parameters. The two species were randomly sampled, with 80~120 individuals of each species captured every month from a single trawler fishery (mesh size 250 mm), March to November 2006, in the north‐central Taiwan Strait. A total of 994 speciments of silver croaker and 851 speciments of red bigeye were collected. Compared with previous studies, the mean length and weight, mean age, the minimum size at first sexual maturity, the asymptotic length (L_∞) and weight (W_∞) of the two species has decreased in recent decades, indicating that the populations were younger, smaller and earlier in sexual maturity. Meanwhile, the growth coefficient (K) increased, the mortality coefficients (Z, F, M) were higher, and the exploitation rate (E) indicated overfishing. Fishing is a key driving force that can cause abundant fluctuations and changes in ecological characteristics in these two demersal fishes in this area. Accordingly, the traditional fishery management approach, such as the reduction in fishing effort, coupled with the Ecosystem‐based Fishery Management (EBFM) should be implemented to achieve sustainable demersal fisheries.     

Effect of pH on the physico-chemical and immunologic properties of rabbit antibodies

The effects of an incubation at low pH (during 20 h at 37 degrees) on the antibody activity and anticomplementary activity of rabbit IgG have been studied. Modifications have also been examined by physicochemical methods. The properties of rabbit anti-sheep red cell IgG are not modified by incubation at pH values between 7.4 and 4.0 during 20 h at 37 degrees. Below pH 4 a decrease of hemolytic activity is apparent concomitant with an important increase of the agglutinating activity. This phenomenon is due to the formation of polymers from native IgG. At pH values below 3.8 the anticomplementary activity of a nonspecific IgG decreases rapidly. One observes an increase of optical rotation, a finding which is compatible with the appearance of heavier compounds with sedimentation coefficients of 9.5 and 11.5 S, probably dimers of native IgG. The increase of optical rotation is partially reversible when the pH is readjusted to 7.4. The use of starch-gel and immunoelectrophoresis has shown the appearance of compounds with higher mobility which are closely related to a peptide (PEP III') which was isolated from a peptic hydrolysate of rabbit IgG. The decrease of anticomplementary activity of nonspecific IgG seems to be closely related to the liberation of PEP III'.     

Effect of brief treatment at alkaline pH on the properties of UDP-glucuronosyltransferase

The kinetic properties of UDP-glucuronosyltransferase were measured after brief treatment of liver microsomes at alkaline pH, followed by assay with p-nitro-phenol as aglycone, at pH 7.5. Enzyme activity increased in a graded fashion as the pH of pretreatment was increased above 8.0, with apparent maximal activation of eight-fold for a pretreatment pH of 11.1. The pH for half maximal activation was 10.6. Brief treatment at alkaline pH prior to assay at pH 7.5 was associated too with a graded conversion of the kinetics of the enzyme from non-Michaelis-Menten to Michaelis-Menten at pH 11.7. Sensitivity to the allosteric modulator, UDP-N-acetylglucosamine decreased as the pH increased. A fifty percent loss of sensitivity to UDP-N-acetylglucosamine-induced activation occurred at pH 10.6. Thus, pretreatment at alkaline pH had irreversible effects on the properties of UDP-glucuronosyltransferase in microsomes. In order to establish the cause for the irreversibility of the changes induced by alkaline pH, microsomes were treated at pH 11.6 prior to purifying UDP-glucuronosyltransferase. Enzyme purified from alkali-treated and untreated microsomes had approximately the same specific activity. More importantly, responses to activation by lipids, and regeneration of allosteric properties were the same for both purified enzymes (from alkali-treated and control microsomes). Pure enzyme was not activated by pretreatment at alkaline pH. We interpret these data to mean that the irreversible effects of alkaline pH on the properties of UDP-glucuronosyltransferase in microsomes were not due to direct effects on the enzyme, but to how the enzyme interacted normally with molecules within the plane of the membrane.     

Effect of phenolic compounds on protein cross-linking and properties of film from fish myofibrillar protein

The effects of several phenolic ocmpounds (caffeic acid, catechin, ferullic acid and tannic acid) at various concentrations (1, 3 and 5% based on protein) on cross-linking and properties of film from myofibrillar proteins of bigeye snapper (Priacanthus tayenus) were investigated. Among all phenolic compounds used, tannic acid exhibited the highest cross-linking ability on myofibrillar protein as evidenced by higher decrease in free amino groups with coincidentally lower band intensity of myosin heavy chain (MHC). In addition, the extent of protein cross-linking increased with increasing concentration of phenolic compounds. Addition of phenolic compounds could enhance mechanical properties of the resulting films. As phenolic compounds content increased, Young's modulus (E) and tensile strength (TS) of the films increased, while their elongation at break (EAB) decreased (P<0.05), suggesting stronger and stiffer film structure. At the same concentration used, tannic acid rendered the film with higher mechanical properties, compared to others. Phenolic compounds decreased film transparency and affected color of the films differently, depending on types and concentrations used. Films from myofibrillar proteins with and without polyphenol generally had the excellent barrier properties to UV light at the wavelength of 200-800nm. Therefore, it could potentially be used as inner packaging material for high-fat foods to prevent the lipid oxidation and thus prolonging the shelf-life of foods during storage.