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1.
Three trypsin isoforms A, B and C were purified to homogeneity from the viscera of sardinelle (Sardinella aurita). Purification was achieved by ammonium sulfate precipitation (20–70% (w/v)), Sephadex G-100 gel filtration and Mono Q-Sepharose anion-exchange chromatography. The molecular weights of these purified enzymes were estimated to be 28.8 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Based on the native PAGE and casein-zymography, each purified trypsin appeared as a single band. Trypsins A and C exhibited the maximal activity at 55°C, while trypsin B at 50°C. All isoforms showed the same optimal pH (pH 9.0) using Nα-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as a substrate. The three trypsins were stable at temperatures below 40°C and over a broad pH range (7.0–11.0). The activities of the three isoforms were strongly inhibited by soybean trypsin inhibitor and phenylmethylsulfonyl fluoride, a serine protease inhibitor, and partially inhibited by ethylenediaminetetraacetic acid, a metalloenzyme inhibitor. Kinetic constants of trypsins A, B and C for BAPNA were evaluated at 25°C and pH 9.0. The values of K m and k cat were 0.125, 0.083 and 0.10 mM, and 2.24, 1.21 and 5.76 s−1, respectively. The N-terminal sequences of the first 10 amino acids were “I V G G Y E C Q K Y” for trypsin A and “I V G G Y E A Q S Y” for trypsins B and C. These sequences showed highly homology to other fish trypsins.  相似文献   

2.
Trypsin from the pyloric ceca of masu salmon (Oncorhynchus masou) cultured in fresh water was purified by a series of chromatographies including Sephacryl S-200, Sephadex G-50 and diethylaminoethyl cellulose to obtain a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and native PAGE. The molecular mass of the purified trypsin was estimated to be approximately 24,000 Da by SDS–PAGE. The enzyme activity was strongly inhibited by phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, and N α -p-tosyl-l-lysine chloromethyl ketone. Masu salmon trypsin was stabilized by calcium ion. The optimum pH of the masu salmon trypsin was around pH 8.5, and the trypsin was unstable below pH 5.0. The optimum temperature of the masu salmon trypsin was around 60°C, and the trypsin was stable below 50°C, like temperate-zone and tropical-zone fish trypsins. The N-terminal 20 amino acid sequence of the masu salmon trypsin was IVGGYECKAYSQPHQVSLNS, and its charged amino acid content was lower than those of trypsins from frigid-zone fish and similar to those of trypsins from temperate-zone and tropical-zone fish. In the phylogenetic tree, the masu salmon trypsin was classified into the group of the temperate-zone fish trypsin.  相似文献   

3.
Trypsin from viscera of Indian mackerel (Rastralliger kanagurta) was purified by ammonium sulphate precipitation and chromatographic techniques such as size exclusion, ion exchange, and affinity chromatography, with a 14.4-fold increase in specific activity and 18.7% recovery. The molecular weight of the trypsin was estimated to be approximately 26 kDa using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purified trypsin showed amidase-specific activity which was determined using benzoyl-dl-arginine-p-nitroanilide (BAPNA). The optimum pH and temperature for isolated trypsin activity were 9.0 and 50°C, respectively. The purified trypsin was strongly inhibited by soybean trypsin inhibitor (SBTI) and N-p-tosyl-1-lysine chloromethyl ketone (TLCK). Purified trypsin showed almost 40% recovery at high NaCl concentration (30%). The N-terminal amino acid sequence of the first 10 amino acids of purified trypsin was IVGGYESQPH. The Michaelis-Menten constant (Km) and catalytic constant (Kcat) of purified trypsin were 0.430 mM and 0.77 s?1, respectively, determined using BAPNA as a substrate. Purified trypsin showed digestion of casein similar to bovine trypsin by the fluorometric method.  相似文献   

4.
Trypsin from the pyloric ceca of orange-spotted grouper, Epinephelus coioides, was purified by fractionation with ammonium sulfate, ionic exchange, and affinity chromatography. The protein was purified 161.85-fold with a yield of 4%. Purified trypsin had an apparent molecular weight of 24 kDa according to an SDS-PAGE analysis. Optimal profiles of temperature and pH of the enzyme were 50°C and 8–10, respectively, using Nα-benzoyl-l-arginine ethyl ester as the substrate. The results of thermal and pH stability assays showed that the enzyme was stable at temperatures of up to 50°C and in the pH range of 6–8. Trypsin activity decreased with an increasing NaCl concentration (0–0.6 M). The activity of purified trypsin was effectively inhibited by a soybean trypsin inhibitor and N-p-tosyl-l-lysine chloromethyl ketone, and was slightly inhibited by iodoacetic acid, ethylenediaminetetraacetic acid, 1-(l-trans-epoxysuccinyl-leucylamino)-4-guanidinobutane, and pepstatin A. Protein identification of the purified protease showed that the sequences of two peptides, LGEHNI and NLDNDIML, were highly homologous to other fish trypsins. The measurement of trypsin activity in different tissues showed that the highest activity was detected in pyloric ceca, followed by anterior intestine, middle intestine, hind intestine and spleen, but very low activities were found in other tissues. An inverse relationship between the trypsin activity in four tissues of pyloric ceca, anterior intestine, middle intestine and hind intestine and fish body weight as a result of increased pepsin in stomach indicated grouper growth status was increased.  相似文献   

5.
Two cystatins (cst-I and cst-II) were purified from crucian carp eggs by acidification and subsequent ion exchange and molecular sieve chromatography. The molecular masses of cst-I and cst-II analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis were 11.9 and 14.4 kDa, respectively, under reducing conditions and 13.5 and 12.7 kDa, respectively, under non-reducing conditions. The cst-I and cst-II molecules were stable after 30 min of incubation at 60 and 50°C, respectively. There was no significant loss in the inhibitory activity of either cst in the pH range 4–11. These two cystatins were able to affect the proteolysis of papain, cathepsin L, and bromelain, but they were unable to inhibit cathepsin B and trypsin. The partial N-terminal amino acid sequences of both cst inhibitors were homologous and that of cst-I was recognized as NH2-AGIPGGLVDADINDADVQ. This latter fragment shared 88.9% identity to common carp cystatin and 44.4–55.6% to cystatins of other aquatic animals. Based on these results, we conclude that the two cst inhibitors are members of family II cystatin.  相似文献   

6.
ABSTRACT:   We purified cathepsins B1 and B2 from the ordinary muscle of carp Cyprinus carpio . The N-terminal amino acid sequences (12 residues) of 29 kDa bands of cathepsins B1 and B2 are the same and showed high homology of 75% and 83%, respectively, with the heavy chain of rat and human cathepsins B. Based on conserved sequences of other cathepsins B and the N-terminal amino acid sequences of 29 kDa bands, we cloned carp cathepsin B cDNA. The nucleotide sequence of carp cathepsin B cDNA consists of 1470 bp including a 993 bp open reading frame, encoding a deduced protein of 330 amino acids. The deduced amino acid sequence of carp cathepsin B has similarity of 80% to rainbow trout cathepsin B and of 76–78% to other vertebrate cathepsins B. The sequence of its isoform was also determined during molecular cloning, which has 94.8% similarity with first cloned cathepsin B. They are completely same in N-terminal amino acid sequence of heavy chain, active site and potential N-glycosylation site. This indicates there are at least two kinds of cathepsin B functioning in vivo in carp.  相似文献   

7.
SUMMARY: Cathepsin S was purified from carp hepatopancreas to homogeneity up to 300-fold. The amino acid sequence of its NH2-terminus was determined to be V-P-D-A-M-D-W-Y-N-K-G-Y-V-T-D-V-K-N-Q. On the contrary, that of purified cathepsin L from carp hepatopancreas was to be V-P-N-S-L-D-W-R-E-K-G. Purified cathepsin S consisted of a single chain with 37 kDa estimated by sodium dodecylsulfate–polyacrylamide gel electrophoresis. The enzyme had strong hydrolytic activity toward Z-Phe-Arg-MCA with the pH optimum of 7.0, but this lacked the ability to hydrolyze most of the other MCA substrates. The optimum pH of cathepsin S for protein substrate (carp myosin heavy chain) was also to be pH 7.0. These properties of purified cathepsin S obviously differ from cathepsins B and L. The enzyme activity was totally inhibited by E-64, leupeptin, 5–5'-dithiobis (2-nitro-benzoic acid) and p -tosyl-lys chloromethylketone as well.  相似文献   

8.
ABSTRACT:   Lysozyme was purified from purple washington clam Saxidomus purpurata by sequential procedures using Chitopearl Basic BL-01 affinity and TSKgel ODS-120T column chromatographies. Molecular mass of the purified enzyme was estimated to be 12 kDa by SDS-PAGE. Optimum pH of the enzyme was 5.2 toward Micrococcus lysodeikticus cells. The optimum temperature was 50°C. The enzyme was stable in the range of pH 4.8–6.8 and 20–90°C. Further, the N-terminal amino acid sequence of the enzyme showed similarity to lysozymes from invertebrates. However, the specific activity of the enzyme toward M. lysodeikticus cells and p -nitrophenyl penta- N -acetyl- β -chitopentaoside was 143 times and 12 times higher than that of hen egg white lysozyme, respectively.  相似文献   

9.
Anionic trypsin from Pacific saury (Cololabis saira) pyloric ceca was purified to homogeneity by ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration chromatography. It was purified to 53.7-fold with a yield of 6.1%. The apparent molecular weight of the enzyme was about 24 kDa, as determined by size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). On native-PAGE, trypsin showed a single band. The purified anionic trypsin displayed optimal activity at pH 8.5 and 55°C. The enzyme was stable at neutral and alkaline pH and in the temperature range of 20–50°C. The stability was affected by the calcium ion. The activity of purified anionic trypsin was completely inhibited by soybean trypsin inhibitor and N-p-tosyl-L-lysine chloromethyl ketone (TLCK) and partially inhibited by ethylenediaminetetraacetic acid (EDTA). NaCl (0–30%) decreased the activity in a concentration-dependent manner. The kinetic trypsin constants Km and Kcat were 0.19 mM and 210 s?1, respectively, while the catalytic efficiency (Kcat/Km) was 1105.26 s?1 mM?1. The N-terminal amino acid sequences of anionic trypsin, IVGGYECQAH, were found and were homologous to those of trypsin from other fish species.  相似文献   

10.
Trypsin, with molecular weight of 28 kDa from the intestine of genetically improved Nile tilapia (Oreochromis niloticus), was purified by ammonium sulfate precipitation, gel filtration, and anion-exchange chromatography. Purified trypsin had maximal activity at pH 8.0 and 60°C for hydrolysis of N α-p-tosyl-L-arginine methyl ester. The enzyme was stable at temperatures up to 50°C and pH range of 6.0–11.0. Its activity was strongly inhibited by metal ions such as Pb2+ and Fe3+ and protease inhibitors including soybean trypsin inhibitor and phenylmethylsulfonyl fluoride. Also, the ion Ca2+ slightly inhibited this activity. The Michaelis-Menten constant (K m) and catalytic constant (K cat) of purified trypsin were 0.036 mM and 152 s?1, respectively. Furthermore, trypsin contained low amounts of hydrophobic and aromatic amino acids as well as β-sheet (20.2%) and β-turn (25.0%).  相似文献   

11.
Two extracellular metalloproteases were purified from a culture filtrate derived from Aeromonas salmonicida ssp. salmonicida . One enzyme, leucine aminopeptidase (LAP), which had a molecular mass 37 kDa, hydrolysed aminoterminal l -leucine and l -phenylalanine. The activity was inhibited by 1,10-o-phenanthroline, but not by EDTA. The addition of excess Zn2+ to an o-phenanthroline-inhibited enzyme restored most of its activity. The peptidase was temperature stable, and had an optimum temperature and pH of 60 °C and 8, respectively. The other enzyme, metalloprotease 3 (MP3), which had a molecular mass 20 kDa, was an endoprotease, and hydrolysed azocoll and hide powder-azure, but not gelatine. The MP3 enzyme had an optimum temperature and pH of ≈40 °C and 7.5, respectively, and a cationic isoelectrical point.  相似文献   

12.
Trypsin from the intestine of common kilka (Clupeonella cultriventris caspia) was purified using ammonium sulfate precipitation (30–50% saturation), Sephadex G-75, and DEAE-cellulose chromatography with the purity of 30-fold and the yield of 12%. The molecular weight of trypsin was estimated to be 23.2 kDa based on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The trypsin had optimal activity at pH 8.0 and 60°C using N-α-benzoyl-DL-arginine-ρ-nitroanilide hydrochloride (BAPNA) as a substrate and showed high stability in the pH range of 7.0–10.0. It was stable up to 50°C. Soybean trypsin inhibitor (SBTI) and N-ρ-tosyl-L-lysine-chloromethylketone (TLCK) significantly inhibited trypsin activity (p < 0.05). Protein hydrolysate from common kilka muscle with different degrees of hydrolysis (DHs; 20, 30, and 40%) was prepared using the purified trypsin, and antioxidative activities were determined. 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activities, ferric reducing antioxidant power, and ferrous chelating activity of hydrolysate increased with increasing DH up to 40% (p < 0.05). Therefore, trypsin from intestine of common kilka could be used as a processing aid for production of fish protein hydrolysate with antioxidative activity.  相似文献   

13.
ABSTRACT:   A glycerolipid acyl-hydrolase was purified 19-fold with a yield of 11% from the prostaglandin-producing red alga Gracilaria vermiculophylla by ammonium sulfate precipitation, anion-exchange chromatoraphy and gel filtration chromatography. Sodium dodecylsulfate– polyacrylamide gel electrophoresis of the final preparation showed a single band corresponding to a molecular mass of 20 kDa, but Superdex 200 fast protein liquid chromatography exhibited a molecular mass of 40 kDa. Accordingly, it was suggested that the purified enzyme was a homodimer of a 20 kDa subunit. The optimal temperature and pH were 37°C and 7–8, respectively. The purified enzyme catalyzed hydrolysis of the acyl groups of both glycoglycerolipids and phospholipids, especially monogalactosyldiacylglycerol and phosphatidylcholine. These results suggest that the enzyme hydrolyze the membrane lipids of the alga to release various saturated and unsaturated fatty acids, including arachidonic acid as substrate for prostaglandin synthesis.  相似文献   

14.
Abstract. The lysine requirements of fingerling carp, Cyprinus carpio L., for optimum growth and efficiency of food utilization were determined in two experiments by the addition of graded supplements of lysine to a basal diet deficient in this amino acid.
The lysine requirement was estimated to be around 14.5 g/kg dry matter at an ambient temperature of 20°C. At 25°C continuing growth responses occurred up to a dietary lysine concentration of 22.4 g/kg DM. However, when these responses are considered in relation to daily lysine intake, the gross efficiency of utilization of dietary lysine for growth appeared to be unaffected by environmental temperature.
Dietary lysine concentrations exerted no consistent effect upon carcass composition of carp. However, fat deposition tended to increase as food intakes increased in response to lysine additions or to elevated ambient temperature.  相似文献   

15.
Carboxypeptidase B (CPB) from zebra blenny (Salaria basilisca) viscera was purified using ammonium sulphate precipitation and Sephadex G-100 gel filtration, with a 28-fold increase in specific activity and 21.72% recovery. The molecular weight of the enzyme was estimated to be 34.5 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature for the enzyme activity were around pH 8.0 and 60°C, respectively, using Hippuryl-l-Arg as a substrate. The enzyme was unstable above 50°C and below pH 5.0. The enzyme was activated by Co2+ and Zn2+ and inhibited by ethylenediaminetetraacetic acid (EDTA). The N-terminal amino acid sequence of the enzyme was determined as S P S Y T K Y N T. The CPB kinetic constants, Km and kcat for Hippuryl-l-Arg, were 0.32 mM and 36.23 s?1, respectively.  相似文献   

16.
Abstract. The ultimate upper lethal temperature (UULT) was 43.0 or 46.0°C for common carp and 43.5 or 46.5°C for silver carp, depending on the method of calculation. The influence of fish age on lethal temperature values was low. Using the relationship between the UULT and the optimum growth temperature the latter was calculated as being 38°C for common carp and 39°C for silver carp. No significant differences were found in survival of fish reared at lower and higher temperatures when proper food was used.  相似文献   

17.
Trypsin from the viscera of Bogue (Boops boops) was purified to homogeneity by precipitation with ammonium sulphate, Sephadex G-100 gel filtration and Mono Q-Sepharose anion exchange chromatography, with an 8.5-fold increase in specific activity and 36% recovery. The molecular weight of the purified enzyme was estimated to be 23 kDa by SDS–PAGE and size exclusion chromatography. The purified trypsin appeared as a single band on native-PAGE and zymography staining. The purified enzyme showed esterase-specific activity on N-α-benzoyl-l-arginine ethyl ester (BAEE) and amidase activity on N-α-benzoyl-dl-arginine-p-nitroanilide (BAPNA). The optimum pH and temperature for the enzyme activity, after 10 min incubation, were pH 9.0 and 55°C, respectively, using BAPNA as a substrate. The trypsin kinetic constants K m and k cat on BAPNA were 0.13 mM and 1.56 s−1, respectively, while the catalytic efficiency k cat /K m was 12 s−1 mM−1. Biochemical characterisation of B. boops trypsin showed that this enzyme can be used as a possible biotechnological tool in the fish processing and food industries.  相似文献   

18.
ABSTRACT: Transglutaminase seems to be related to the setting phenomenon of fish meat paste that occurs at temperatures below 40°C. In many reports on the relationship between transglutaminase and setting phenomenon, the enzyme activity has been measured at 25°C. However, it is known that the setting phenomenon is complicated and the effect of calcium and the 3-D structure of myofibrils, which are sensitive to temperature, play important roles in the reaction. In the present study, total activities of transglutaminase of threadfin bream, white croaker, red sea bream and carp meats were measured at various temperatures. Total transglutaminase activities at 25°C of tested fish meats are arranged in order as follows; white croaker, red sea bream, carp, threadfin bream. In contrast, optimal temperature of carp meat is 30°C, red sea bream 40°C, threadfin bream 50°C and white croaker 50–55°C. In carp meat, the enzyme activity at optimal temperature (30°C) became 8.5-fold higher than at 25°C. The data of the total activity at various temperatures are useful in order to comprehend the details of the reaction.  相似文献   

19.
Vitellogenin (VTG), the egg yolk precursor protein, was purified from plasma of estradiol-3-benzoate (E2B)-treated male shorthead redhorse (Moxostoma macrolepidotum) and immature copper redhorse (Moxostoma hubbsi) by a two-step chromatographic procedure without precipitation. Intact VTGs appeared as dimers with apparent molecular masses, determined by gel filtration, of ∼425 kDa (copper redhorse) and ∼450 kDa (shorthead redhorse). In native polyacrylamide gel electrophoresis (PAGE), dimeric redhorse VTGs appeared as a 520 kDa band. Both VTGs were reduced to a single monomer of ∼150 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and nonreducing conditions, indicating that monomers are not linked by disulfide bonds in the dimer form. The purified proteins were characterized as phospholipoglycoproteins. Isoelectric focusing of both VTGs revealed components with isoelectric points ranging from 5.3 to 6.0, suggesting charge heterogeneity. The amino acid composition of both VTGs contains a high proportion of nonpolar amino acids and was similar to those of other teleosts. An antibody developed against carp (Cyprinus carpio) VTG showed cross-reactivity with VTG from both redhorse species. Using this antibody, VTG was detected in plasma and surface mucus of E2B-treated redhorse. This is the most extensive report on purification and characterization of vitellogenin from catostomidid species.  相似文献   

20.
Histidine decarboxylase (HDC) from Staphylococcus epidermidis TYH1, a halotolerant histamine-producing bacterium isolated from Japanese fermented fish-miso, was purified to homogeneity for the first time. The enzyme was purified 182-fold from cell-free extracts by ammonium sulfate precipitation, anion exchange chromatography and gel filtration chromatography. The N-terminal amino acid sequences of two polypeptide chains of 27–30 and 7–9 kDa were highly homologous with those of α- and β-chains of other staphylococcal HDCs. The optimum pH and temperature for the enzyme were 6.0 and 60 °C, respectively. This enzyme did not decarboxylate lysine, arginine, tyrosine, tryptophan or ornithine. The enzyme activity decreased with the addition of NaCl. At pH 4.8, the V max and K m values were 45.5 μmol histamine min?1 mg?1 and 1.10 mmol/L, respectively. Moreover, this enzyme was resistant to heat treatment (80 °C for 15 min) and was stable upon freezing at ?30 °C for 7 days. The very similar physiological properties of this enzyme and the almost identical N-terminal amino acid sequence to that of the HDC from S. capitis indicated that this enzyme may be evolutionally highly conserved in the genus Staphylococcus. The biophysical properties of staphylococcal HDC were elucidated using native purified enzyme.  相似文献   

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