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1.
An alkaline phosphatase was purified from the gut of sea cucumber Stichopus japonicus by n-butyl alcohol extract, ammonium sulfate precipitation, ion exchange chromatography with diethylaminoethyl cellulose, gel filtration chromatography with Sephacryl S-200 and preparative electrophoresis with polyacrylamide gel electrophoresis. The native enzyme was estimated to be 166 ± 9 kDa and produced a single predominant band corresponding to active enzyme on nondenaturing electrophoresis, but showed 2 bands of 97 and 35 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the native enzyme is composed of two dissimilar subunits. The enzyme displayed maximum activity at pH 11 and 40 °C, showing narrow pH stability (pH 10–12) and thermal instability at temperature higher than 30 °C. The activity of the purified alkaline phosphatase was enhanced by Mg2+, whereas inhibited by Zn2+, Ca2+ and EDTA at 1 and 10 mM, suggesting its activity is in a magnesium ion-dependent manner. The product-analog WO4 2? and product HPO4 2? showed strong inhibitory effects on the enzyme activity. Using p-nitrophenyl phosphate as substrate, the V max and K m values were 24.45 μmol/L min and 5.76 mM, respectively.  相似文献   

2.
Quantification of proteases activities and their class structure have been studied in a cladoceran, Daphnia carinata. Protease activity ranged from 0.28 to 0.55 Unit mg−1 protein min−1 with an average value of 0.42±0.06 Unit mg−1 protein min−1. Chymotrypsin activity was more than twofold higher (0.49±0.09 Unit mg−1 protein min−1) than the trypsin activity (0.21±0.02 Unit mg−1 protein min−1). Protease activity and reduction of activity in bands of samples treated with specific inhibitors were documented in photometric assay and substrate SDS–PAGE. Proteinase activity against azocasein was inhibited (91.4±1.5%) with SBTI. PMSF reduced the enzyme activity by 53.1±6.5%, and the azocasein hydrolysis was reduced up to 64.6±3.8% by the specific inhibitor of trypsin, TLCK. In the present investigation, the molecular weight of various activity bands ranged from 16.3 to 51.1 kDa. The molecular weights of several protein bands are similar to protease activity zones. The knowledge of digestive enzyme profiles of fish food organisms generated in the present study may assist in the formulation of age-specific feed.  相似文献   

3.
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   

4.
Lipases were purified from delipidated pyloric ceca powder of two New Zealand-sourced fish, Chinook salmon (Oncorhynchus tshawytscha) and hoki (Macruronus novaezelandiae), by fractional precipitation with polyethylene glycol 1000, followed by affinity chromatography using cholate-Affi-Gel 102, and gel filtration on Sephacryl S-300 HR. For the first time, in-polyacrylamide gel activity of purified fish lipases against 4-methylumbelliferyl butyrate has been demonstrated. Calcium ions and sodium cholate were absolutely necessary both for lipase stability in the gel and for optimum activity against caprate and palmitate esters of p-nitrophenol. A single protein band was present in native polyacrylamide gels for both salmon and hoki final enzyme preparations. Under denaturing conditions, electrophoretic analysis revealed two bands of 79.6 and 54.9 kDa for salmon lipase. It is proposed that these bands correspond to an uncleaved and a final form of the enzyme. One band of 44.6 kDa was seen for hoki lipase. pI values of 5.8 ± 0.1 and 5.7 ± 0.1 were obtained for the two salmon lipase forms. The hoki lipase had a pI of 5.8 ± 0.1. Both lipases had the highest activity at 35°C, were thermally labile, had a pH optimum of 8–8.5, and were more acid stable compared to other fish lipases studied to date. Both enzymes were inhibited by the organophosphate paraoxon. Chinook salmon and hoki lipases showed good stability in several water-immiscible solvents. The enzymes had very similar amino acid composition to mammalian carboxyl ester lipases and one other fish digestive lipase. The salmon enzyme was an overall better catalyst based on its higher turnover number (3.7 ± 0.3 vs. 0.71 ± 0.05 s−1 for the hoki enzyme) and lower activation energy (2.0 ± 0.4 vs. 7.6 ± 0.8 kcal/mol for the hoki enzyme) for the hydrolysis of p-nitrophenyl caprate. The salmon and hoki enzymes are homologous with mammalian carboxyl ester lipases.  相似文献   

5.
Precipitation techniques play a vital role in the industrial extraction of enzymes. The present study aimed to extract the proteases from the viscera of Labeo rohita (commonly called Rohu) and to compare the precipitation techniques for the isolation and purification of the enzyme. The enzyme is usually discarded as tons of waste during processing. Hence, a trial has been carried out to isolate the protease enzymes from viscera of the freshwater fish Labeo rohita. The proteases were precipitated with ammonium sulfate, ethanol, and acetone. Acetone precipitation was found to be the best option for the recovery of enzymes (54%) from the viscera of Rohu, and two caseinolytic protease bands were shown in the zymogram. The precipitates with highest proteolytic activity were further subjected to dialysis, and their molecular weight was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).  相似文献   

6.
The aim of this study was to examine proteinases and peptidases from the hepatopancreas of two sub‐adult stages of Farfantepenaeus subtilis: SAS6 (5.93 ± 0.69 g wet weight) and SAS13 (13.26 ± 0.60 g wet weight). Trypsin and chymotrypsin activity was higher in the extract from the SAS6 individuals (P < 0.05). The highest activity among aminoacyl‐β‐naphthylamide substrates was found using alanine‐, arginine‐, leucine‐ and lysine‐β‐naphthylamide. There was a positive correlation between the recommended concentration of essential amino acids in penaeid shrimp feed and aminopeptidase activity in both sub‐adult stages. Proteolytic activity of F. subtilis was strongly inhibited by specific trypsin inhibitors. The optimal temperature for trypsin, chymotrypsin and leucine aminopeptidase activity was between 45 and 55 °C. Six and seven bands were found in caseinolytic zymograms for SAS6 and SAS13 respectively. All bands were inhibited by phenylmethylsulfonyl fluoride in both sub‐adult stages. The use of tosyl‐lysine‐chloromethyl‐ketone and benzamidine caused strong inhibition of the proteolytic bands. Trypsin and chymotrypsin activity was the main difference observed between the protease pattern of SAS6 and SAS13F. subtilis.  相似文献   

7.
An acetylcholinesterase was purified from the gut of sea cucumber Stichopus japonicus by anion exchange chromatography followed by gel filtration chromatography. The enzyme was purified 35.49-fold with a total yield of 7.73 %. The molecular mass of purified acetylcholinesterase was 68 kDa as revealed on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme displayed maximum activity at pH 7.5 and 35 °C with acetylthiocholine iodide as substrate. The enzyme activity appeared to be stable over pH 6.0–8.0 and up to 40 °C. It displayed an apparent Michaelis–Menten behavior in the concentration range from 0.1 to 0.8 mM with K m values of 0.62 mM for acetylthiocholine iodide and 2.53 mM for butyrylthiocholine iodide. More than 95 % of acetylcholinesterase activity was inhibited by 1 mM eserine or 1,5-bis(4-allyldimethylammonium phenyl)-pentan-3-one dibromide (BW284C51), but only 19.1 % of the activity was inhibited by tetraisopropylpyrophosphoramide (iso-OMPA) at the same concentration. On the basis of the substrate and inhibitor specificities, the purified enzyme appeared to be a true acetylcholinesterase. Nevertheless, the purified acetylcholinesterase exhibited insensitivity to substrate inhibition phenomenon. Its biochemical properties were compared with those reported for different species.  相似文献   

8.
Aminopeptidases play important roles in turnover of proteins, metabolism of hormones and neurotransmission, cell maturation and immunological regulations. In the present study, an aminopeptidase was purified to homogeneity from the skeletal muscle of grass carp by ammonium sulfate fractionation and sequential chromatographic steps, including DEAE-Sephacel, Sephacryl S-200, hydroxyapatite and Phenyl-Sepharose. The purified enzyme revealed a molecular mass of approximately 105 kDa both on SDS–PAGE and on gel filtration of Superdex 200. The enzymatic activity toward synthetic substrates was optimal at 40°C and pH 7.0–7.5. Metal-chelating agents such as EDTA and EGTA effectively inhibited the enzyme activity while inhibitors to serine, asparatic and cysteine proteinases did not show much effect, suggesting its belonging to metalloproteinase family. A specific aminopeptidase inhibitor bestatin was most effective in suppressing the enzymatic activity and performed in a competitive fashion. The enzymatic activity was slightly enhanced by metal ions of Mg2+ and Mn2+ while inhibited to different extents by Co2+, Cu2+, Zn2+ and Ca2+. Sulfhydryl reagent was necessary to maintain its activity. Purified enzyme demonstrated amidolytic activity most effectively against synthetic aminopeptidase substrate Leu-methylcoumarylamide (MCA) while N-terminal-blocked substrates and myofibrillar proteins were not hydrolyzed. The enzyme purified in the present study was quite possibly a leucine aminopeptidase (LAP) and functions during muscular protein metabolism.  相似文献   

9.
Proteases from the midgut gland of the Farfantepenaeus paulensis juveniles were assessed. Enzyme activity was determined using protease substrates and inhibitors. The effect of pH, temperature and calcium on proteolytic activity was assayed. Caseinolytic activity was analysed in substrate-sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Trypsin, chymotrypsin and leucine aminopeptidase activity was detected. Proteolytic activity was strongly inhibited by the specific trypsin inhibitors. Tosyl-phenylalanine chloromethyl ketone inhibited 59.3% of chymotrypsin activity. The greatest trypsin-like activity occurred at pH 8.0 and 45 °C. Chymotrypsin-like activity reached maximal values at alkaline pH (7.2–9.0) and 55 °C. CaCl2 did not increase trypsin-like activity, but rather inhibited it at concentrations of 30 (20%), 50 (30%) and 100 mM (50%). The substrate-SDS-PAGE zymogram revealed eight proteinase bands. Two possibly thermal-resistant (85 °C, 30 min) chymotrypsin isoforms were found, which were inhibited by phenyl-methyl-sulphonyl-fluoride. Aminopeptidase activity of enzyme extracts (Arg, Leu, Lys, Phe and Val) and the recommended concentrations of these essential amino acids in penaeid shrimp diets were positively correlated ( P <0.05). Beause protein digestion involves the combined action of different enzymes, adequate knowledge of shrimp digestion and enzyme characteristics is required for the assessment of the digestive potential of different feed sources and development of in vitro digestibility protocols.  相似文献   

10.
Atypical Aeromonas salmonicida (AAS) causes generalized lethal infections in farmed Arctic charr, Salvelinus alpinus (L.), and European grayling, Thymallus thymallus (L.), and is thus a serious threat for culture of these fish species. Virulence factors were studied among isolates of AAS from Arctic charr (n = 20), European grayling (n = 19) and other fish species (n = 20), of which 48 were of Finnish and 11 of Swedish origin. All isolates produced an A-layer. Extracellular products (ECP) of the AAS isolates did not produce detectable gelatinase and caseinase activity in test assays. Analysis of the same ECP preparations with substrate sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed weak proteolytic activity, indicating the different sensitivity of the detection methods used. The ECP from AAS isolates showed low cytotoxic activity against cultured cells. However, the ECP did not induce mortality in challenged Arctic charr. The results suggest that toxic components, like ECP, secreted by the bacterium may not be the major virulence factor in AAS-infection in Arctic charr and European grayling, and hence the pathogenesis also differs from the pathogenesis of AAS-infection in Atlantic salmon, Salmo salar L.  相似文献   

11.
Abstract. A proteinase produced by the bivalve pathogen Vibrio alginolyticus NCMB 1339 was purified by preparative isoelectric focusing and gel filtration. Proteinase activity was associated with two components of molecular weights 43000 and 41000 and was toxic to larval Ostrea edulis . Production of this enzyme was maximal during the late exponential phase of growth and it remained stable throughout the stationary phase of growth. At 37°C, activity against azocasein was optimal at pH 7–5. The enzyme was inhibited by mercuric chloride, dithiothreitol and ethylene diamine tetra-acetic acid, but not by pepstatin-A, phenylmethylsulphonyl-fluoride or tosyl-L-lysine chloromethylketone.  相似文献   

12.
经热变性,硫酸铵二步分级沉淀,再经Sephadex G-100分子筛和DEAE-Sepharose离子交换层析,从口虾蛄肝胰脏中分离纯化得到超氧化物歧化酶。试验获得的酶紫外吸收峰在268 nm处,比活力为983.8 U/mg,提纯倍数为378.4。该酶分子量约31.5 ku,是由2个分子量均为15.6 ku的亚基组成的二聚体。该酶在30~60℃稳定性较好,最适pH值为6.7。Li+、K+、Ca2+、Zn2+对该酶活性的影响总趋势是抑制作用。紫外灯照射使酶活性呈现递减的变化趋势。  相似文献   

13.
Skin secretions of fishes constitute a rich source of proteins with a broad spectrum of antimicrobial properties. We report here the characterization of proteases from skin mucus of tambacu, an economically important Neotropical hybrid fish. The effects of pH on the proteolytic activities of the mucus acting on various substracts – hemoglobin, casein, bovine serum albumin (BSA) and ovalbumin (OVA) – were tested. Optimal pH values for protease activity on hemoglobin were 4.5 and 8.5, on casein, 8.5, on BSA, 5.0 and 7.5, and on ovalbumin, 4.5 and 6.5. The proteolytic activity was inhibited on all of these substrates in the presence of specific inhibitors: caseinolytic activity was inhibited by inhibitors of serine and metalloproteases; hemoglobinolytic activity was inhibited by serine, aspartic and metalloproteases inhibitors; albuminolytic activity on BSA was inhibited by serine and aspartic proteases inhibitors, and on ovalbumin, by cysteine and aspartic proteases inhibitors. Gelatin zymography revealed that the skin mucus of tambacu consisted primarily of serine and metalloproteases. Hemoglobin zymography showed one proteolytic band inhibited by EDTA, whereas casein zymography showed two proteases inhibited by serine proteases inhibitors. We were able to identify all classes of proteases in the mucus from the skin of tambacu. These, and these results suggest that the proteolytic activities of the skin mucus of fish may play an important role in the defense against microorganisms and ectoparasites.  相似文献   

14.
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   

15.
ABSTRACT:   A growth hormone (GH)-like substance was extracted from the rotifer Brachionus plicatilis and subsequently purified by gel filtration and ion exchange chromatography. The GH-like substance had a molecular weight of approximately 28 kDa and had cross-reactivity with salmon GH antibody. In vivo bioassay showed a higher intrinsic rate of increase and net reproduction rate of B. plicatilis treated with the GH-like substance.  相似文献   

16.
Biochemical assays and substrate SDS-PAGE were conducted to partially characterize and identify various types of proteases present in the digestive tract of PL15 giant freshwater prawn ( Macrobrachium rosenbergii ). Casein hydrolytic assay of the enzyme extracts showed major proteolytic activities at pH 3.0, 6.0 and 9.0, while assay of preincubated enzyme extracts with phenylmethylsulphonyl fluoride (PMSF), a serine protease inhibitor produced a 33.17% reduction in alkaline protease activity. When specific inhibitors tosyl-lysine chloromethyl ketone and tosyl-phenylalanine chloromethyl ketone were used, they resulted in a reduction in activity of proteases in the enzyme extracts by 82.41% and 55.03%, respectively, confirming the presence of trypsin and chymotrypsin, while ethylenediamine tetraacetic acid produced protease activity reduction in 33.92% showing the presence of metalloproteases in the digestive tract of the prawn. Further characterization of the alkaline proteases using SDS-PAGE technique, after incubating the extract in the presence or absence of specific inhibitors, produced six bands corresponding to molecular masses of between 13.48 and 136.1 kDa; two trypsin bands of 13.48 and 36.4 kDa, three chymotrypsin bands in the range of 23.0–73.4 kDa and one for metalloprotease of 136.1 kDa, all of which were identified from a zymogram. This study suggests that protein digestion in M. rosenbergii is initiated by an acid protease followed by a combination of action of alkaline proteases: trypsin, chymotrypsin and metalloproteases.  相似文献   

17.
Proteolytic activity in the different parts of the digestive tract of the turbot (Scophthalmus maximus L.) were studied in this work. One pure protease was isolated from turbot stomach and its behavior was studied. Results showed the optimum pH for proteases in the different parts of the digestive tract of the turbot were pH 2.0 for the stomach, pH 8.0 for the pylorus cecum, pH 8.0 for the foregut, pH 8.5 for the midgut, and pH 8.0 for the hindgut. The activity of proteases in the different parts of the digestive tract were in the sequence pylorus cecum protease > stomach protease > foregut protease > midgut protease > hindgut protease. The stomach protease was purified by ammonium sulfate precipitation and column chromatography on DEAE-Sepharose F.F. and Sephadex G-100. The purified enzyme gave a single band in SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Its molecular weight was found to be approximately 42,000 Da. The enzyme is stable at pH 1.0–9.0 and at temperatures below 40°C. Its activity was maximum at pH 2.0 and 40°C. When reaction time was prolonged the optimum temperature of the enzyme tended to decline. The enzyme was activated by Mn2+ and Cu2+ and inactivated by Fe3+. It was fully inhibited by pepstatin and partially inhibited by PMSF, TPCK, PCMB, and NBS. These results imply the enzyme is a pepsin.  相似文献   

18.
Abstract. Cultures of Aeromonas salmonicida , subspecies salmonicida and achromogenes , produced considerable quantities of inhibitors that affected extracellular bacterial proteases (endopeplidases), as well as some animal trypsins (from pig, salmon and trout) included in this study. Electrophoretic separation of these inhibitors revealed a complex of three to four single factors which were similar for the two subspecies. One of the factors only inhibited protease from the homologus subspecies, while another only affected enzyme from the other subspecies (cross-wise inhibition). Both subspecies produced a factor which inhibited animal trypsins only. Subspecies salmonicida also possessed a factor which inhibited most of the examined proteases unspecifically. In young cultures (2–3 days at 15°C), the inhibitors were demonstrable both in the disintegrated cell material and in cell-free culture filtrate. The activity of the factor which only inhibited the protease from subspecies salmonicida could be increased considerably by moderate heating of the material. The effect of inhibitors produced by other relevant Gram-negative bacteria on the proteases of the A. salmonicida subspecies included was more limited. Considerable quantities of inhibitors against proteases of the subspecies salmonicida and achromogenes were demonstrated in cell-free filtrates of two commercially available vaccines against furunculosis in fish.  相似文献   

19.
The monoclonal antibody (MAb-001), which was produced against a surface membrane glycoprotein on C. salmositica , significantly inhibited the activities of the intracellular proteases of the parasite. The total activity in the partially purified metallo-protease, and about 80% of activity in the partially purified cysteine protease, were inhibited by the antibody (at 10 μg protein ml–1). The inhibitory effects of the antibody on the proteases were also demonstrated using haemoglobin (substrate)-SDS-PAGE. The activities of the metallo-protease band (200 kDa) and the three cysteine protease bands (66, 70 and 97 kDa) were inhibited by MAb-001, but the activity of the fourth cysteine protease band (49 kDa) was not affected. Similar inhibitory effects of the antibody were also found in the crude protease extract (parasite lysate), except that more antibody was required to obtain the same level of inhibition. The metallo-protease band was more sensitive than the cysteine protease bands to the antibody.  相似文献   

20.
The effect of different dietary concentrations of shrimp protein hydrolysate (SPH) on digestive enzyme activity of Nile tilapia juveniles was evaluated. SPH concentrations in diets were 0, 15, 30 and 60 g kg?1 (treatments SPH0, SPH15, SPH30 and SPH60, respectively). Hemoglobin, azocasein, BApNA (Nα-benzoyl-dl-arginine-p-nitroanilide), SApNA (Suc-Ala-Ala-Pro-Phe p-nitroanilide), aminoacyl of β-naphthylamide and starch were used as substrates for enzyme activity determinations. The activity of total alkaline protease was significantly higher (P < 0.05) in fish under SPH15 and SPH60 treatments than in the control (SPH0). However, the effect was not dose-dependent. Substrate-SDS-PAGE was also performed to evaluate changes in the profile of Nile tilapia digestive proteases caused by SPH. Substrate-SDS-PAGE revealed 12 active proteolytic bands, eight of which responded to SPH dietary incorporation. Inhibition substrate-SDS-PAGE indicated a decrease in the activity of three enzymes, with trypsin activity decreasing with the increase of SPH concentration, whereas the opposite occurred for two aminopeptidases. Distinct protease profiles were also found for each treatment, suggesting adaptability of digestive proteases from Nile tilapia to the different diets.  相似文献   

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