Peculiarities of substrate hydrolysis by endopeptidases from hepatopancreas of king crab

Kinetic parameters of hydrolysis of peptide and protein substrates by psychrophilic endopeptidases from hepatopancreas of the king crab Paralithodes camtschaticus (PC), in particular, by trypsin, collagenolytic protease, and metalloprotease, were measured at different temperatures. The PC trypsin was shown to hydrolyze Bz-Arg-pNA in the temperature range studied (4–37°C) 19 times more effectively than bovine trypsin. The rate constants of hydrolysis of Glp-Ala-Ala-Leu-pNA by the PC collagenolytic protease increased approximately by one order of magnitude along with temperature decrease, while K _m decreased by 3.5 times. The effective values of K _m for the hydrolysis of azocasein by the metalloprotease insignificantly depend on temperature. We proposed that electrostatic interactions of negative charges around the cavity of active site are critical for the effective hydrolysis of substrates by endopeptidases of the PC hepatopancreas.     

Glycolytic activity of enzyme preparation from the red king crab (Paralithodes camtschaticus) hepatopancreas

Enzyme preparation exhibiting glycolytic activity yielding chitooligosaccharides along with N-acetyl-D-glucosamine was obtained from the red king crab (Paralithodes camtschaticus) hepatopancreas. The results of the analysis confirmed the presence of endo- and exochitinase activities in the preparation. HPLC showed that the hydrolysis products of chitin and chitosan did not contain D(+)-glucosamine, which is indicative of the absence of deacetylase and, apparently, exochitosanase activities. A comparison of the dependence of the enzyme preparation activity on temperature and pH of the incubation medium suggests that chitinase and protease activities are exhibited by different enzymes.     

Characteristics of substrate hydrolysis by endopeptidases from the hepatopancreas of the king crab

Kinetic parameters of hydrolysis of peptide and protein substrates by psychrophilic endopeptidases from hepatopancreas of the king crab Paralithodes camtschaticus (PC), in particular, by trypsin, collagenolytic protease, and metalloprotease, were measured at different temperatures. The PC trypsin was shown to hydrolyze Bz-Arg-pNA in the temperature range studied (4-37 degrees C) 19 times more effectively than bovine trypsin. The rate constants of hydrolysis of Glp-Ala-Ala-Leu-pNA by the PC collagenolytic protease increased approximately by one order of magnitude along with temperature decrease, while Km decreased by 3.5 times. The effective values of Km for the hydrolysis of azocasein by the metalloprotease insignificantly depend on temperature. We proposed that electrostatic interactions of negative charges around the cavity of active site are critical for the effective hydrolysis of substrates by endopeptidases of the PC hepatopancreas.     

Hydrolysis of chitosan sulfate by an enzyme complex from Streptomyces kurssanovii

The possibility of enzymatic hydrolysis of chitosan was shown. The optimum conditions for the process are: sodium acetate buffer pH 6.0, 37 degrees C, 24 h, and the chitosan sulfate-protein volume ratio of 500:1 in the enzyme preparation. During hydrolysis, the intrinsic viscosity of chitosan sulfate solution decreased by a factor of 2.7.     

Hydrolysis of chitozan with an enzyme complex from Myceliophthora sp.

Samples of low-molecular weight chitosan with molecular masses of 8–24 kDa, identical deacetylation degrees (85%), and polydispersity indexes soluble at pH 5–7 were obtained by enzymatic hydrolysis using an enzyme complex from the mycelial fungi Myceliophthora fergusii with yields of 50–80%. The optimal conditions for hydrolysis were found (pH 5.6, 37°C, an enzyme/substrate ratio 1/800, 15–60 min).     

Glycolytic activity of enzyme preparation from the red king crab (<Emphasis Type="Italic">Paralithodes camtschaticus</Emphasis>) hepatopancreas

Enzyme preparation exhibiting glycolytic activity yielding chitooligosaccharides along with N-acetyl-D-glucosamine was obtained from the red king crab (Paralithodes camtschaticus) hepatopancreas. The results of the analysis confirmed the presence of endo- and exochitinase activities in the preparation. HPLC showed that the hydrolysis products of chitin and chitosan did not contain D(+)-glucosamine, which is indicative of the absence of deacetylase and, apparently, exochitosanase activities. A comparison of the dependence of the enzyme preparation activity on temperature and pH of the incubation medium suggests that chitinase and protease activities are exhibited by different enzymes.     

Hydrolysis of a phosphonate ester catalyzed by an enzyme from Dictyostelium discoideum.

An activity capable of hydrolyzing the phosphonate ester, 4-nitrophenyl phenylphosphonate has been detected in the eucaryotic microorganism Dictyostelium discoideum. This activity (4-nitrophenyl phenylphosphonate-esterase) was found in both membrane-enriched and soluble fractions. Two other activities present in this fraction, namely cyclic AMP-diesterase and an ATP-hydrolase, did not copurify with the 4-nitrophenyl phenylphosphonate-esterase. Also while ATP and cAMP inhibited the hydrolysis of 4-nitrophenyl phenlyphosphonate, they did so in a noncompetitive manner and, furthermore, saturating concentrations of 4-nitrophenyl phenylphosphonate had no effect on the hydrolysis of either cyclic AMP or ATP. The partially purified preparation was more active at pH 7 than at either pH 5 or 9, more active with acetate than chloride, and more active at 22 °C than at either 12 or 30 °C. The activity is inhibited by AMP in a competitive manner, but is not affected by levamisole, an inhibitor specific for alkaline phosphatase activity.     

Seasonal and geographical variations of sterol composition in snow crab hepatopancreas and pelagic fish viscera from Eastern Quebec

Sterol composition was determined in snow crab hepatopancreas and mackerel and herring viscera for various locations and collection periods. A simple and valuable method, using direct saponification and extraction with water-cyclohexane has been optimized to recover total sterol. They were identified and quantified as trimethylsilyl ether derivatives by GC-MS analysis. Method validation indicated excellent sensitivity (limit of quantification: 1.25 mg/100 g wet basis for cholesterol and desmosterol; 0.03-0.05 mg/100 g for other sterols), good reproducibility (CV%: 1.5-6.8) and accuracy (recovery%: 94-107). In crab hepatopancreas, cholesterol was the main sterol (67-76%), followed by desmosterol (19-24%). Phytosterols and molluscan sterols were also present in low quantity. A lower total sterol content with different composition was found in crabs from Magdalen Islands compared to those from Gaspé Peninsula or North Shore of the St-Lawrence Gulf. No seasonal variation was observed between collection periods, which were probably too close. Mackerel and herring viscera contained the same sterols as crab except for campesterol and sitosterol, but the cholesterol proportion was higher (93-98%). The higher abundance of sterols in herring caught in September vs. May would be related to an increase of the body lipid content during the summer.     

真江蓠(Gracilaria verrucosa)对网箱养殖海区的生态修复及生态养殖匹配模式

2006年8～9月,在浙江象山港花鲈(Lateolabrax japonicus)养殖网箱中吊养真江蓠(Gracilaria verrucosa)对网箱养殖造成的水体富营养化进行生态修复研究.通过45d内的平面监测、定点跟踪监测和断面监测,结果表明:该网箱养殖区水体呈严重富营养化状态,营养状态指数(E)为32.00,其营养盐分布由高浓度的中心区向周围150m非养殖水域扩散;真江蓠对养殖区的富营养化海水具有较好的修复效果:江蓠生态修复区及其相邻网箱中水体PO4-P、NO2-N、NH4-N和NO3-N含量显著低于非修复区(P＜0.01),修复区海水PO4-P、NO2-N、NH4-N和NO3-N浓度比非修复区分别降低22%～58%、24%～48%、22%～61%和24%～47%.养殖真江蓠45d后,修复区水体DO浓度和透明度显著高于非修复区(P＜0.05),DO平均提高28%,透明度平均提高30%;而修复区水体Chl-a浓度显著低于非修复区(P＜0.05),平均降低49%.通过建立基于N平衡的鱼藻生态养殖模式,每收获1kg花鲈至少需要匹配江蓠4.7 kg wet wt才可实现对鱼类排放N的完全吸收.因此网箱内栽培江蓠的混合生态养殖模式,可平衡因经济动物养殖所带来的额外营养负荷,有利于实现动物养殖环境的自我修复.     

Core protein I as an artifact in complex III.

SDS electrophoresis gels of complex III from yeast mitochondria were run after incubation of the enzyme at several different temperatures. It was found that the intensity of the more slowly moving core protein band was substantially affected by the incubation temperature. Four low molecular weight polypeptides were eluted from gels electrophoresed after preincubation of the enzyme at 15° C for 12 hours. These polypeptides were then incubated for 5 minutes at 100° followed by SDS gel electrophoresis. A polypeptide with the same molecular weight as the anomalous core protein was resolved.     

Enzymic hydrolysis of sphingolipids: Hydrolysis of ceramide lactoside by an enzyme from rat brain

Ceramide lactoside [1-O-(galactosido-4-β-glucosido)-2-N-acyl-sphingosine] was hydrolysed to ceramide glucoside and galactose by β-galactosidase of rat brain. The reaction was not reversible, required cholate or taurocholate, had optimum pH5·0 and K_m 2·2×10⁻⁵m. It was inhibited by γ-galactonolactone and galactose as well as by ceramide, sphingosine and fatty acid. Ceramide lactoside could be degraded to ceramide, galactose and glucose by mixtures of rat-brain β-galactosidase and ox-brain β-glucosidase.     

Enzymic hydrolysis of sphingolipids: Hydrolysis of ceramide glucoside by an enzyme from ox brain

Ceramide glucoside (1-O-glucosido-2-N-acyl-sphingosine) was hydrolysed to ceramide (N-acyl-sphingosine) and glucose by β-glucosidase from ox brain. The reaction was stimulated by the non-ionic detergent, Triton X-100, or by the anionic detergents, cholate or taurocholate. It was not reversible, had optimum pH5·0 (with acetate buffer) or 5·6 (with pyridine buffer), had K_m 1·8×10⁻⁴m and was inhibited by δ-gluconolactone and sphingosine, but not by ceramide or palmitic acid.     

Anticoagulant and anticomplement effects of an endogenous inhibitor from hepatopancreas of red king crab (<Emphasis Type="Italic">Paralithosed camtschaticus</Emphasis>) on human blood

Serpins are the superfamily of serine and cysteine protease inhibitors (SERine Protease Inhibitors) acting by an irreversible suicide mechanism. A novel serpin from hepatopancreas of red king crab (Paralithosed camtschaticus) was isolated and its effect on the process of human blood plasma coagulation was investigated. The investigated serpin exhibited a significant anticoagulant effect, which dramatically increased in the combination with heparin. The study of the crab serpin on C1s (C1 esterase) revealed its competition with the C1 inhibitor from blood plasma. Although the inhibitor weakly influenced thrombin activity, inhibition constant for C1s was (2.02 ± 0.71) 10⁻⁷ M. Unlike the C1 inhibitor the novel red king crab serpin does not inhibit fibrinolysis but inhibits blood coagulation. This creates certain clinical perspectives.     

Functional,antioxidant and antibacterial properties of protein hydrolysates prepared from fish meat fermented by Bacillus subtilis A26

Composition, functional properties and in vitro antioxidant and antibacterial activities of protein hydrolysates prepared with a proteolytic bacterium, Bacillus subtilis A26, through fermentation of fish proteins were investigated. Fermented fish meat protein hydrolysates (FPHs) were prepared from sardinelle (SPH), zebra blenny (ZPH) goby (GPH) and ray (RPH). The protein content of freeze-dried FPHs ranged from 74.3% to 81%. All fermented hydrolysates had an excellent solubility and possessed interfacial properties. The antioxidant activities of FPHs were evaluated by different methods, including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical method, reducing power assay, β-carotene bleaching and DNA nicking assay. All hydrolysates showed dose-dependent antioxidant activities. Further, FPHs exhibited antibacterial activity and SPH was the most effective, particularly against Gram positive bacteria.     

Efficient identification of proteins from ovaries and hepatopancreas of the unsequenced edible crab,Cancer pagurus,by mass spectrometry and homology-based,cross-species searching

Proteome maps of hepatopancreas (midgut gland) and ovarian tissues of the crustacean, Cancer pagurus (Decapoda; edible crab) have been produced by 2D-PAGE and identification of proteins, following trypsin proteolysis, by electrospray MS/MS and database searching. Owing to the lack of sequence information on proteins and fully sequenced genomes amongst the decapod crustaceans and given the evolutionary distance to the nearest full genome database (Daphnia), it was necessary to adopt a non-conventional identification approach. Thus, a strategy was developed for effective identification of decapod proteins by sequence similarity, homology-based cross-species database searching, using various algorithms and a combination of NCBI Crustacea and Arthropoda databases, together with the Arthropoda PartiGene database (Blaxter, University of Edinburgh). In both hepatopancreas and ovary tissues, the largest group of proteins identified were a variety of enzymes, followed by a smaller number of storage/transport proteins [including vitellogenin (yolk protein), several subunits of hemocyanin, cryptocyanin, ferritin and calreticulin], with fewer structural proteins (actin, tubulin) and heat-shock proteins, in addition to a number of proteins of miscellaneous functions. Such protein identifications allow the development of tools, such as antibodies and RNA/DNA probes, to investigate the functions of the proteins in specific tissues during development.