Purification and characterization of stomach protease from the turbot (Scophthalmus maximus L.)

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.     

Purification and characterization of three trypsin isoforms from viscera of sardinelle (<Emphasis Type="Italic">Sardinella aurita</Emphasis>)

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.     

Effects of dietary protein levels on the activity of the digestive enzyme of albino and normal Apostichopus japonicus (Selenka)

We evaluated the effects of different proportions of dietary protein (5%, 10%, 15%, 20%, 25% and 30% protein) on the activity of digestive enzymes of normal and albino Apostichopus japonicus. The experimental diets were fed for 60 days, the optimal conditions for digestive enzyme activity in sea cucumbers were studied. The optimal temperature for protease was 29.3°C and the optimal pH was 1.8. The optimal temperature for amylase was 34.3°C and the optimal pH was 6.7. The optimal temperature for cellulase was 56°C and the optimal pH was 5.9. The activity of intestinal protease increased at first and then decreased as the proportion of dietary protein increased, reaching the maximum when the proportion of protein was 19.7%. The activity of protease in the intestine of normal sea cucumber was significantly lower than that of albino sea cucumber, and the activity of amylase was significantly higher than that of albino sea cucumber. This study is expected to provide a basis for further explaining the ecological difference of albino and normal A. japonicus.     

Characterization of acetylcholinesterase from the gut of sea cucumber Stichopus japonicus

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.     

Ontogeny of protease, amylase and lipase in the alimentary tract of hybrid Juvenile tilapia (Oreochromis niloticus × Oreochromis aureus)

Juvenile tilapias (Oreochromis niloticus × O. aureus), with average body weights of groups I, II and III of 55.14, 122.82, and 225.68 g respectively were used to study changes in the activity of protease, amylase, and lipase in different organs and sections of the alimentary tract. Additionally, tract containing digesta, which is closer to physiological conditions, may help reveal the mechanisms of whole chemical digestion process. Another group of tilapias (mean body weight of 111.81 ± 0.14 g) was used to analyze changes in activity of protease, lipase, and amylase in response to varying pH and temperatures. The activity of enzymes in all digestive organs increased when body weight increased from 55 to 122 g, and the activity further increased when the weights increased from 122 to 225 g. The rates of increase in digestive enzyme activity were significantly greater than the rates of growth of the fish. When the activity was compared in different sections of the tract, a similar regularity was found for the three enzymes in all fish. The highest amounts of activity were seen in the foregut and the lowest in the rectum, with the middle amount of activity in the hindgut. The pH ranges for the maximum activity of tilapia protease in the stomach and intestine were 1.6–3.3 and 9.5–10.5 respectively and the optimal temperature in these organs was 55°C. The pH ranges for the maximum amylase activity in the intestine and hepatopancreas were 6–7 and 7.5 respectively, and high amylase activity in these organs was found at 25–35°C. The activity of lipase in the intestine remained unchanged at pH 6.0–9.0 and with temperatures between 25 and 35°C. These results suggest that the ability of tilapia to utilize feed varies at different growth stages and remains relatively stable with changes in environmental pH and temperature, which should be taken into account when designing feed formulas for tilapia.     

Purification and biochemical characterization of a cellulase from the digestive organs of the short-spined sea urchin Strongylocentrotus intermedius

We isolated a cellulase from the digestive organs of the short-spined sea urchin Strogylocentrotus intermedius using a combination of ion-exchange chromatography and gel filtration together with an assay for carboxymethylcellulase activity. The isolated cellulase was stained as a single band by Congo red. The molecular weight of the isolated cellulase, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, was 59?kDa. The isolated cellulase exhibited hydrolytic activity toward carboxymethyl cellulose, with an optimum temperature and pH of 30?°C and pH 8.0, respectively. The thermal stability of the enzyme was characterized by determining the temperature at which activity decreased by 50?% with treatment for 30?min at pH 7.0 and found to be 32?°C. Cellulase activity remained at a high level at 5?C20?°C, which is the growth temperature of the short-spined sea urchin. These results confirm that the short-spined sea urchin should preferably be reared at a water temperature of <20?°C.     

Trypsin from the viscera of Bogue (<Emphasis Type="Italic">Boops boops</Emphasis>): isolation and characterisation

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.     

Purification and characteristics of trypsin from masu salmon (<Emphasis Type="Italic">Oncorhynchus masou</Emphasis>) cultured in fresh-water

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.     

AMP-deaminase from goldfish white muscle: regulatory properties and redistribution under exposure to high environmental oxygen level

AMP-deaminase was partially purified from white skeletal muscle of goldfish, Carassius auratus. The enzyme was highly stable, showing virtually no change in activity at 1 month following the purification process when stored in 1 M KCl at 2–4°C. The specific activity of the purified enzyme was 130–150 U/mg protein, with a pH optimum of about pH 6.5. AMP-aminohydrolase (AMPD) showed non-Michaelis–Menten kinetics, with a S_0.5 (half saturation by the substrate) for AMP of 0.73 ± 0.03 mM, a Hill coefficient of 2.01 ± 0.26, and a V _max (maximum velocity) of 176 ± 46 U/mg protein. Both sodium and potassium ions activated goldfish AMPD at low concentrations, with maximal activation at about 80 mM of each chloride salt, whereas higher concentrations became inhibitory. Magnesium and calcium ions also inhibited goldfish muscle AMPD, as did phosphate and fluoride; at a concentration of 8 mM, each anion reduced activity by about 66%. ADP and ATP were strong activators and both demonstrated concentration-dependent activation, with maximal effects at 0.5–1.5 mM. Fish exposure to a high concentration of oxygen (18–20 mg/l against 5–6 mg/l in the control) and recovery to the initial level induced a redistribution of AMPD between free and bound forms in goldfish white muscle and brain in a tissue-dependent manner. A spatial–temporal redistribution may be among the mechanisms regulating enzyme operation in vivo. Possible regulatory mechanisms of AMP-deaminase function in fish muscle are discussed.     

Purification and characterization of alkaline phosphatase from the gut of sea cucumber Stichopus japonicus

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.     

Purification and characterization of hatching enzyme from flounder Paralichthys olivaceus

Using chorion of Paralichthys as a specific substrate, hatching enzyme (HE) from Paralichthys olivaceus (PHE) was purified by gel-filtration and ion-exchange chromatography, and characterized in terms of its molecular weight and enzymatic properties in this study. It was found that the molecular size of PHE is about 34.8 kDa in SDS-PAGE. The PHE had obvious choriolytic activity, which was optimal at pH 7.0 and temperature of 35 °C, respectively. The Km value of the PHE for casein was 4.28 mg ml⁻¹. The PHE was very sensitive to trypsin-specific inhibitors, especially serine protease-specific inhibitors, such as LBTI, SBTI, bestatin and p-APMSF, leupeptin, ovomucoid, PMSF, pepstatin A and TLCK, indicates that it is a trypsin-type serine protease. The PHE was also extremely sensitive to Cu²⁺ and Ca²⁺, combined with the results that it was inhibited by EDTA in a dose-dependent manner, indicates this PHE is also a kind of metalloprotease.     

Physicochemical and kinetic characteristics of rhodanese from the liver of African catfish <Emphasis Type="Italic">Clarias gariepinus</Emphasis> Burchell in Asejire lake

Two forms of rhodanese were purified from the liver of Clarias gariepinus Burchell, designated catfish rhodanese I (cRHD I) and rhodanese II (cRHD II), by ion-exchange chromatography on a CM-Sepharose CL-6B column and gel filtration through a Sephadex G-75 column. The apparent molecular weight obtained for cRHD I and cRHD II was 34,500 ± 707 and 36,800 ± 283 Da, respectively. The subunit molecular weight determined by sodium dodecyl sulphate–polyacrylamide gel electrophoresis was 33,200 ± 283 and 35,100 ± 141 Da for cRHD I and cRHD II, respectively. Atomic absorption spectrophotometric analysis revealed that cRHD II contained a high level of iron (Fe), which presumably was responsible for the brownish colour of the preparation. In contrast, no Fe was identified in cRHD I, and its preparation was colourless. Further characterization of cRHD II gave true Michaelis–Menten constant (K _m) values of 25.40 ± 1.70 and 18.60 ± 1.68 mM for KCN and Na₂S₂O₃, respectively, an optimum pH of 6.5 and an optimum temperature of 40°C. The Arrhenius plot of the effects of temperature on the reaction rate consisted of two linear segments with a break occurring at 40°C. The apparent activation energy values from these slopes were 7.3 and 72.9 kcal/mol. Inhibition studies on the cRHD II enzyme showed that the activity of the enzyme was not affected by Mn²⁺, Co²⁺, Sn²⁺, Ni²⁺ and NH₄ ⁺, but Zn²⁺ inhibited the enzyme considerably.     

Identification of an aminopeptidase from the skeletal muscle of grass carp (<Emphasis Type="Italic">Ctenopharyngodon idellus</Emphasis>)

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.     

Effects of stocking density,temperature, and salinity on larval survival and growth of the red race of the sea cucumber <Emphasis Type="Italic">Apostichopus japonicus</Emphasis> (Selenka)

The red race of the sea cucumber Apostichopus japonicus was introduced into China from Japan for large-scale seed production because of its economic value. This paper reports the effects of stocking density, temperature, and salinity on survival and growth of early larvae before and after feeding, in order to establish conditions for optimal larval growth and production. To maximize the yield per unit of space, densities of 0.5–1 larvae/ml are recommended for non-feeding larvae, while 0.1–0.2 larvae/ml are best for feeding larvae. Higher survival and growth values were obtained for both non-feeding and feeding larvae at temperature ranges from 21 to 24°C. Larvae reared at a salinity of 30‰ always showed maximum growth and survival. Based on results of this study, a temperature range from 21 to 24°C and a salinity of 30 are considered optimal for early development of the red A. japonicus.     

Purification and properties of digestive lipases from Chinook salmon (<Emphasis Type="Italic">Oncorhynchus tshawytscha</Emphasis>) and New Zealand hoki (<Emphasis Type="Italic">Macruronus novaezelandiae</Emphasis>)

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.     

High water-temperature tolerance in photosynthetic activity of <Emphasis Type="Italic">Zostera marina</Emphasis> seedlings from Ise Bay,Mie Prefecture,central Japan

Photosynthetic activities of seedlings of Zostera marina were successively measured using a gas volumeter for 6 days at seven light (0–400 μmol photons/m² per s) and 11 water temperature conditions (5–35°C). The seedlings were collected from mature plants (Ise Bay, central Japan), and stored and cultured in incubators accurately controlled at each test temperature. The maximum gross photosynthesis (P _maxg) was recorded at an optimal water temperature of 29°C after 0 days. After 6 days, P _maxg appeared at 25°C and most plants cultured at 29–30°C bleached and withered after the drastic increase of light compensation point (I _c). On the contrary, at 5–28°C, the photosynthetic activities either changed little (5–25°C) or recovered after a temporal reduction (26–28°C); seedlings survived and looked healthy after being cultured for 6 days. The recovery was thought to be an acclimation to tolerate higher water temperature. As a result, the critical upper water temperature for Z. marina seedlings was proposed as 28°C. The temperature was consistent with the previously reported maximum water temperature in habitats around the southern boundary of Z. marina in the northern hemisphere.     

Effect of ration size on growth,body composition,and energy and protein maintenance requirement of fingerling Indian major carp, <Emphasis Type="Italic">Labeo rohita</Emphasis> (Hamilton)

An eight-week feeding trial has been conducted to determine the optimum ration for Indian major carp, Labeo rohita, fingerling (4.10 ± 0.30 cm, 0.55 ± 0.16 g) by feeding a purified diet (40% CP; 3.61 kcal g⁻¹ GE) at six levels, 2, 4, 6, 8, 10, and 12% of body weight per day, at 0800 and 1600 h, in triplicate, to 20 fish per trough fitted with a water flow-through system. Highest weight gain, best feed conversion ratio (FCR), best specific growth rate (SGR%), and highest protein efficiency ratio (PER) were evident for rations of 6–8% body weight. Second-degree polynomial regression analysis for FCR, PER, protein, and energy retention data indicated the break-points occurred at 6.55, 6.75, 6.80, and 6.95% bw per day, respectively. Significant (P < 0.05) differences between body composition were observed for fish fed different rations. Maximum body protein content was recorded for 6% and 8% rations. A linear increase in body fat content was evident with increasing ration. Body moisture and ash content remained non-significantly (P > 0.05) low for higher rations, however. On the basis of these results it is recommended that feeding in the range 6.5–7.0% bw per day corresponding to 2.6–2.8 g protein and 23.49–25.31 kcal energy per 100 g of the diet per day is optimum for growth and efficient feed utilization of Labeo rohita. Results for 2–4% rations (0.8–1.6 g protein and 7.23–14.46 kcal energy) suggest these amounts approximate to the maintenance requirement of this fish.     

Characterization of gelatinolytic enzymes in the skeletal muscle of red sea bream Pagrus major

Gelatinolytic enzymes were partially purified from the skeletal muscle of red sea bream Pagrus major and characterized to obtain information on post mortem tenderization of fish muscle. Four gelatinolytic activities, G1 (90 kDa), G2 (65 kDa), G3 (60 kDa), and G4 (100 kDa), were detected in the Q Sepharose column. G1, the major gelatinolytic enzyme, and G4 were identified as serine proteinases from results of inhibitor spectrum and substrate specificity. By contrast, G2 and G3 were found to be metalloproteinases since these were inhibited by ethylenediamine tetraacetic acid and o-phenanthroline, and activated by 4-aminophenylmercuric acetate. The optimum pH and temperature of these enzymes were in the ranges of 7–9 and 20–40°C, respectively.     

Degradation of myofibrils in cultured yellowtail <Emphasis Type="Italic">Seriola quinqueradiata</Emphasis> burnt meat: effects of a myofibril-bound EDTA-sensitive protease

“Burnt meat” is a term used to describe the white (pale, grainy, exudative) muscle of yellowtail or tuna. It (with lightness parameter, L* ≥ 55) was observed after 2 h storage in the suffocate in air (SA) 29°C group and after 4 h storage in the spinal cord destruction (SCD) 29°C group. In the SA 17°C group, burnt meat was also observed after 4 h storage. In contrast, the meat in the SCD 17°C group was normal until after 12 h storage. The myosin heavy chain (MHC) was more degraded than the other myofibrillar proteins, and some protein bands increased in the burnt meat. The protease that leads to the degradation of MHC was investigated using myofibrils from the meat. EDTA completely suppressed the degradation, indicating that a myofibril-bound EDTA-sensitive protease (MBESP) may exist in yellowtail muscle and this caused the degradation of MHC. The optimum pH and temperature of MBESP in yellowtail were 5.0 and 50–60°C, respectively.     

刺参养殖池塘中新敌害生物澳洲异尾涡虫的鉴定及其危害

为了对导致辽宁大连、山东东营的2家养殖场池塘养殖刺参大量化皮死亡的新的敌害生物进行鉴定并确定其对养殖刺参的危害。本实验通过形态学观察、分子鉴定及系统发育分析确定了涡虫的分类地位,通过生态学方法确定了其生态适应条件,通过切割后培养的方法观测了其再生能力,通过与刺参苗种的共培养实验测试了该物种对刺参的危害及其危害方式。形态学观察结果显示,该涡虫体长0.96～3.26 mm,体宽0.49～1.93 mm,外观黄色或黄褐色,头部钝圆,具一对暗红色棒状眼点,尾部具两条并列的尾垂;显微镜镜检发现其表皮下分布密集的虫黄藻,体表周生纤毛,雌雄同体,口后具有两个生殖孔;对该物种COⅠ及18S r DNA基因片段扩增测序结果进行分析,并构建基于18S rDNA基因的系统发育树,结果显示该生物与澳洲异尾涡虫序列同源性达99.64%,根据其形态学特征,并结合18S rDNA分子鉴定结果,将该生物鉴定为澳洲异尾涡虫;进一步对其生活习性进行了研究,结果显示,该生物具有避光性,其适宜温度为18～24°C,适宜pH为5.5～8.0,适宜盐度为20～40;再生实验表明,该物种具有很强的前后轴极性再生能力;该生物与刺参的共培养实验表明,澳洲异尾涡虫对刺参体表表现出很强的趋向性,可以吸附在刺参体表导致刺参苗种溃疡、化皮甚至死亡,但刺参的体腔、肠道、呼吸树内均未发现虫体寄生。研究表明,澳洲异尾涡虫是营自由生活的池塘养殖刺参的一种新的敌害生物,在养殖过程中需要密切关注并防范该敌害生物。