Purification and characterization of a β–glucan binding protein from the haemolymph of freshwater prawn Macrobrachium rosenbergii

β‐glucan binding protein (βGBP), a pattern recognition protein was purified from the haemolymph of freshwater prawn Macrobrachium rosenbergii by heparin affinity chromatography that showed a single band in native gradient PAGE. The β‐glucan binding property of the purified protein was confirmed in a phenoloxidase (PO) assay, where addition of βGBP along with β‐glucan increased the specific PO activity compared with that of β‐glucan alone. The molecular weight of the βGBP was found to be ~316 kDa on gel filtration chromatography. In SDS‐PAGE, βGBP molecule was reduced to one polypeptide chain of molecular weight ~113 kDa. Thus the βGBP in M. rosenbergii is possibly a homotrimeric molecule. The purified sample run on unreduced condition in SDS‐PAGE also revealed a similar size band (~113 kDa) and hence, the polypeptide chains of βGBP are held by non‐covalent interactions. The purified βGBP samples run in native PAGE was stained positively with alcian blue for carbohydrates and Sudan black for lipids indicating the βGBP to be a glycolipoprotein. With rabbit polyclonal anti‐βGBP serum developed, an indirect ELISA was standardized and the normal βGBP concentration in adult M. rosenbergii serum was quantified to be ~2 mg mL^?1. Furthermore, the applicability of the developed ELISA is discussed.     

Purification of vitellogenin from the air breathing catfish, Clarias gariepinus

Vitellogenin (Vtg) is a female specific glycophospholipoprotein which can be induced both in male and female with estradiol and xeno-estrogens. The basic theme behind the purification of vitellogenin from the fish is to understand the evolutionary relationship and for the purification and characterization of the Vtg receptor. The male catfish, Clarias gariepinus was administrated with estradiol over a period of time for the synthesis of Vtg and the serum was collected. The Vtg was purified from the serum using a two step chromatographic technique. The serum was passed on to DEAE-ion exchange column and the protein was eluted using a salt gradient. The fractions containing the Vtg were pooled and passed onto a gel permeation chromatography column and the pure protein was obtained. The molecular weight is around 200 kDa on the SDS-PAGE and around 520 kDa on the native gel electrophoresis.     

Isolation and partial characterization of Asian sea bass (Lates calcarifer) Vitellogenin

A study was conducted to isolate, partial characterize Asian sea bass (Lates calcarifer) vitellogenin (vtg). Two-year-old juvenile L. calcarifer (n = 10) were given three intraperitoneal injections of 17-β estradiol (E₂) at a dose of 2 mg/kg body weight to induce vitellogenesis. Blood was collected 3 days after the last injection, and plasma was purified through gel filtration chromatography. A broad single symmetrical peak consisting of vtg molecule was produced. Protein concentration was 0.059 mg/ml as determined by Bradfrod assay using bovine serum albumin as a standard. The protein appeared as one circulating form in Native PAGE considering the dimeric form of putative vtg with molecular weight of 545 kDa. In SDS-PAGE under reducing conditions, two major bands appeared at 232.86 and 118.80 kDa and minor bands at 100.60, 85.80 and 39.92 kDa, respectively. The purified vtg was used to generate a polyclonal antibody, and the specificity of antibody was assessed by Western blot analysis. Two major bands were immunoreacted, but no cross-reactivity was observed with plasma from non-induced males. The protein was characterized as phosphoglycolipoprotein as it positively stained for the presence of lipid, phosphorus and carbohydrate using Sudan Black B, methyl green and periodic acid/Schiff reagent solution, respectively. The amino acid composition was analyzed by high sensitivity amino acid analysis that showed high percentage of non-polar amino acids (~48 %). The results suggest the potential utilization of vtg as a basis tool to further study about reproductive physiology of this important economical species.     

尼罗罗非鱼卵黄脂磷蛋白的分离纯化与性质鉴定

采用Sephacryl S-300过滤层析和DEAE-Sepharose Fast Flow离子交换层析相结合的方法从尼罗罗非鱼(Oreochromis niloticus)成熟卵子匀浆液中分离纯化出了一种高分子量的蛋白。该蛋白能被Schiff试剂、甲基绿和苏丹黑B着色,Western blot显示能被金鱼卵黄脂磷蛋白(lipovitellin,Lv)多克隆抗血清特异性识别,在非变性条件下分子量约为560 k D,在SDS变性条件下分子量约为112 k D,结果表明分离纯化的蛋白是一种含有糖、磷、脂基团的蛋白,符合鱼类Lv的性质,且与金鱼Lv有免疫交叉反应,从蛋白的性质和免疫原性以及分子量大小等角度判断,本研究获得的高纯度蛋白为尼罗罗非鱼卵黄脂磷蛋白;纯化的罗非鱼Lv在反复冻融、37℃及60℃处理条件下均未出现降解,表明罗非鱼Lv比鱼类卵黄原蛋白(Vitellogenin,Vtg)更为稳定。研究结果为罗非鱼Lv抗体的制备奠定了基础。     

Vitellogenin of the Chilean flounder <Emphasis Type="Italic">Paralichthys adspersus</Emphasis> as a biomarker of endocrine disruption along the marine coast of the South Pacific. Part I: induction,purification, and identification

We sought to provide a useful indicator of the presence of endocrine-disrupting contaminants along the marine coast of the South Pacific using Chilean flounder (Paralichthys adspersus). In light of the lack of information on vitellogenin for this species, we induced, purified, and identified the plasma vitellogenin of Chilean flounder inhabiting the Chilean coast. Vitellogenin (Vg) from Chilean flounder was purified by size exclusion and ion-exchange chromatography using plasma from juvenile males induced by injecting 17β-estradiol. The Vg was detected by SDS–PAGE and Western blot analyses using an antibody against turbot (Scophthalmus maximus) vitellogenin. These analyses revealed a protein band of 205 kDa and three minor bands of 120, 90, and 68 kDa. These proteins were identified as Vg by means of mass spectrometry (LCQ Duo ESI-IT-MS), matching sequences of tryptic peptides to known sequences for several other fish species. The matches showed the presence of vitellogenin (VgI, VgII, Vg A and Vg B) in Chilean flounder, similar to species such as mummichog (Fundulus heteroclitus), Japanese medaka (Oryzias latipes), and white perch (Morone americana). These results are discussed in terms of identifying Vg in Paralichthys adspersus with the antibody to turbot Vg. Moreover, we compare the molecular size of Vg from Chilean flounder (large) with that of other flatfish species. Finally, we discuss the potential use of this molecule as a biomarker for the presence of xeno-estrogenic compounds along the Chilean coastline.     

Purification and partial characterization of vitellogenin from shorthead redhorse (<Emphasis Type="Italic">Moxostoma macrolepidotum</Emphasis>) and copper redhorse (<Emphasis Type="Italic">Moxostoma hubbsi</Emphasis>) and detection in plasma and mucus with a heterologous antibody

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.     

Purification and characterization of lipovitellin from Pacific saury Cololabis saira

ABSTRACT:   Lipovitellin (Lv), the major yolk protein derived from vitellogenin (Vg), was purified from vitellogenic ovaries of Pacific saury Cololabis saira using hydroxylapatite column chromatography followed by gel filtration. The apparent native mass of purified Lv was approximately 420 kDa, while the tertiary structure of Lv revealed by sodium dodecylsulfate–polyacrylamide gel electrophoresis was typical of teleost Lvs, consisting of a heavy chain (∼99 kDa) and a light chain (∼34 kDa). Western blot analysis using rabbit antiserum raised against Pacific saury Lv revealed a specific reaction with a polypeptide (∼194 kDa) that is present in serum from female Pacific saury but not in male serum, suggesting the approximately 194-kDa polypeptide to be the Vg monomer. This study describes the first step toward the development of specific immunoassays for Pacific saury Vg, which will be an effective tool for monitoring the reproductive development of this species.     

Effect of size‐fractionated fish protein hydrolysate on growth and feed utilization of turbot (Scophthalmus maximus L.)

Four experimental diets were fed to turbot to examine the effect of fish hydrolysate and ultra‐filtered fish hydrolysate on growth performance, feed utilization and non‐specific immune response. Fish hydrolysate was produced by enzymatic treatment and size fractionated using ultra‐filtration (UF). The permeate (molecular weight <1000 Da) after UF and the non‐ultra‐filtered fish hydrolysate (NUF) were tested as feed ingredients. Diets UF1, UF2 contained 3.7%, 1.2% ultra‐filtered fish hydrolysate to replace fish meal protein respectively. The diets UF1, NUF were identical in composition except that the molecular weight of fish hydrolysate in the diet. Fish meal was used in the control diet. All diets were made equal in protein, lipid and energy. Each experimental diet was fed to juvenile turbot (27.87 ± 0.04 g) in triplicate for 8 weeks. Results of this study indicate that the best overall growth and feed utilization of turbot juveniles were obtained with a diet containing higher dose of the small molecular weight compounds in fish hydrolysate. Acid phosphatase, alkaline phosphatase, lysozyme and superoxide dismutase activity in serum were not affected by diet. Total antioxidant capacity was improved with increasing level of low molecule weight fish hydrolysate (UF1).     

Identification, purification and classification of multiple forms of vitellogenin from white perch (Morone americana)

Three forms of female-specific plasma protein (FSPP 1-3) were purified from blood plasma of estrogen-treated white perch (Morone americana) by combining several types of ion-exchange chromatography including a novel, fast flow, strong anion exchanger (POROS media), followed by gel filtration. Native FSPP 1, FSPP 2 and FSPP 3 had molecular masses of 532 kDa, 532 kDa and 426 kDa, respectively. The apparent mass of purified FSPP 1 and FSPP 2 after SDS-PAGE under reducing conditions was ∼ 180 kDa, while FSPP 3 appeared as a major ∼ 148 kDa band. All of the FSPPs resembled one another with respect to amino acid composition but each appeared to be immunologically distinct. In double immunodiffusion using anti-total FSPP (antiserum raised against vitellogenic female plasma pre-absorbed by male plasma), each FSPP formed one precipitin line that crossed those produced by both others. A rabbit antiserum was raised against each FSPP and absorbed with combinations of the other two FSPPs to ensure specificity. Using the antisera, each FSPP was detected by immuno-electrophoresis in plasma from vitellogenic females or estrogen-treated male or immature fish, but no FSPP was detected in normal male plasma. Endoprotease (Asp-N) digests of the FSPPs were subjected to HPLC separation for N-terminal sequencing and mapping of isolated peptides to published vitellogenin (Vg) sequences. Results of these analyses indicate that white perch FSPP 1, FSPP 2, and FSPP 3 can be classified into three Vg groups identified in previous studies: VgA, VgB, and VgC-like protein, respectively. This is the first report, of which we are aware, on isolation of more than two Vg proteins from any species of vertebrate except the chicken. This revised version was published online in July 2006 with corrections to the Cover Date.     

Purification and partial characterization of GtHs (cfLH and cfFSH) from Indian walking catfish (Clarias batrachus) (L.) and development of a homologous ELISA for cfLH

Two gonadotropins (GtH; Qa and Qb) were purified by gel filtration and ion exchange chromatography from the pituitaries of Indian walking catfish (Clarias batrachus). The presence of GtH during purification was assessed by in vitro oocyte maturation and in vivo steroidogenic activity, and their identities were determined by elution profiles, molecular weight, biological activities and yield. The molecular weights of Qa and Qb were 37 and 42 kDa, respectively, and composed of distinct subunits (Qa: 20 and 14 kDa and Qb: 26 and 18 kDa). Polyclonal antibodies raised against Qa immunostained Qa, Qb and pituitary GtH cells. A competitive Qa‐ELISA was developed whose sensitivity was 6.25 ng mL^?1 (1.25 ng well^?1) with intra‐ (3.5%) and inter‐ (12.4%) assay coefficients of variation. Displacement curves parallel to the standard were obtained with plasma and pituitary extracts of catfish, Qb and carp GtHII. The assay was validated by measuring the plasma Qa levels after LHRH treatment and in relation to ovarian growth in the female catfish during different reproductive phases. Based on the results, Qa and Qb corresponded to fish LH and FSH respectively. The findings will increase the knowledge of the mechanisms controlling fish reproduction and identification of sensitive phases in fish in captivity for hormonal manipulation.     

17β—雌二醇对雄性金鱼卵黄原蛋白的诱导作用

采用腹腔注射17β-雌二醇的方法诱导雄性金鱼卵黄原蛋白产生,注射浓度为0.05mg·g-1BW,诱导2周后取尾静脉血,离心分离血浆,进行血浆常规聚丙烯酰胺凝胶电泳,通过对卵黄原蛋白特性基团磷、脂和糖蛋白的染色,确定了卵黄原蛋白在电泳图谱上的位置,开发了一种简便、高效的定性卵黄原蛋白的聚丙烯酰胺凝胶电泳法。电泳结果表明,在0.05mg·g-1BW的注射浓度下,2周后17β-雌二醇诱导了雄性金鱼卵黄原蛋白产生,并通过ELISA检测卵黄原蛋白的平均含量为690.2ng·mL-1,与对照组雄性金鱼平均含量为10.7ng·mL-1的差异极显著(P<0.01),比雌性对照组检出量285.5ng·mL-1高1倍多;17β-雌二醇诱导组雄鱼血浆钙离子和血总蛋白含量明显增加。     

Effects of dietary linolenic acid on growth,fatty acid composition,immune function and antioxidant status of juvenile blunt snout bream,Megalobrama amblycephala

A nine‐week feeding trial was performed to determine the dietary linolenic acid (LNA; 18:3n–3) requirements of juvenile blunt snout bream. Six iso‐nitrogenous, semi‐purified diets were prepared with different concentrations of LNA (0–25 g/kg). Dietary LNA had no significant effects on survival rate. However, final fish weight, weight gain (WG), specific growth rate (SGR) and feed efficiency ratio (FER) increased with increasing dietary LNA concentrations up to 20 g/kg. Dietary LNA increased muscle LNA and total n‐3 polyunsaturated fatty acid (PUFA) contents, but decreased total saturated fatty acid content. Fish fed 20 g/kg LNA had the highest plasma alkaline phosphatase activity, total protein, albumin and white blood cell count levels. Additionally, fish fed 20 g/kg LNA had higher triglyceride levels than control fish. Plasma glucose increased with increasing dietary LNA concentrations. Superoxide dismutase and glutathione peroxidase activities significantly increased with increasing dietary LNA concentrations up to 15 g/kg. Based on SGR and FER, the optimal dietary LNA requirements of juvenile blunt snout bream were 17.5 and 15.6 g/kg respectively.     

Effects of dietary selenium on growth performance and oxidative stress in juvenile grass carp Ctenopharyngodon idellus

An 8‐week feeding trial was conducted to investigate the effect of dietary selenium (Se) on feed intake, weight gain and antioxidant activity in juvenile grass carp (11.2 ± 0.03 g). Six Se levels (0.13, 0.41, 0.56, 1.12, 2.18 and 4.31 mg/kg) of semi‐purified diets were assayed in triplicate. The maximum weight gain, specific growth rate and feed intake were obtained in fish fed with 1.12 mg Se/kg diet. Hepatic glutathione peroxidase activity was markedly increased when dietary Se ≤1.12 mg/kg diet and reached a plateau when dietary Se ≥1.12 mg/kg diet. Hepatic superoxide dismutase and serum catalase activities in juvenile grass carp fed with 0.56, 1.12 and 2.18 mg Se/kg diets were all significantly higher than those in the other groups. The malondialdehyde content in liver and serum was firstly decreased and then increased with increasing dietary Se content, and the lowest content was observed in fish fed with 1.12 mg Se/kg diet. With the increase in Se level, the activities of serum alanine aminotransferase and aspartate aminotransferase were reduced. In addition, serum alkaline phosphatase activity and albumin content were highest in fish fed with 1.12 mg Se/kg diet. This study indicated that both the Se deficiency and excess of Se caused negative effect on the oxidative stress in juvenile grass carp and suggested that the health‐giving concentration of dietary inorganic Se was 1.12 mg/kg diet. Moreover, based on the broken‐line regression analysis of weight gain, the optimal concentration of dietary inorganic Se was 0.83 mg/kg for juvenile grass carp.     

Response of juvenile pacu (Piaractus mesopotamicus Holmberg, 1887) to balanced digestible protein

This 45‐day work aimed to determine the response of juvenile pacu (Piaractus mesopotamicus) to balanced digestible protein (BDP) and to use these responses to determine whether the optimum economic levels of BDP would differ depending on the form in which the fish is sold. Six isoenergetic diets containing 163, 201, 238, 272, 315 and 348 g/kg BDP (dry‐matter basis) were prepared through the serial dilutions of a high‐protein diet with the low‐protein diet. Fish (initial average body weight, 10.82 ± 0.14 g) were fed with respective experimental diets three times a day until apparent satiation. The optimum biological level of BDP was calculated as 326 g/kg (dry‐matter basis) by the quadratic regression model for maximum body weight gain of juvenile pacu. To maximize economic returns (US$/kg) for different end products, the optimum economic levels of BDP were calculated as 311, 317 and 319 g/kg (dry‐matter basis) by an economic model for whole‐body, eviscerated and sliced juvenile pacu respectively. This finding revealed that obtaining maximum biological performance of fish in the case of high feed costs or low prices of the end product in consumer market would substantially reduce the economic returns.     

Digestive proteases of three carps Catla catla, Labeo rohita and Hypophthalmichthys molitrix: partial characterization and protein hydrolysis efficiency

Characteristics and functional efficacy of digestive proteases of Catla catla, catla, Labeo rohita, rohu and Hypophthalmichthys molitrix, silver carp were studied. Total protease activity was significantly (P < 0.05) higher in rohu (1.219 ± 0.059 U mg protein⁻¹ min⁻¹) followed by silver carp (1.084 ± 0.061 U mg  protein⁻¹ min⁻¹), and catla (0.193 ± 0.006 U mg  protein⁻¹ min⁻¹). Trypsin activity of silver carp and rohu was 89–91% higher than catla. Chymotrypsin activity was significantly (P < 0.05) higher in silver carp compared with rohu and catla. The protease activity of rohu and silver carp displayed bell‐shaped curves with maximum activity at pH 9; whereas in catla, maximum activity was found between pH 8 and 11. Inhibition of protease activity with soybean trypsin inhibitor (SBTI), phenylmethylsulfonyl fluoride (PMSF) revealed the presence of serine proteases and inhibition of activity with N‐α‐p‐tosyl‐L‐lysine‐chloromethyl ketone (TLCK) and N‐tosyl‐L‐phenylalanychloromethane (TPCK) indicated the presence of trypsin‐like and chymotrypsin‐like enzymes in all these three carps. SDS‐PAGE showed the presence of several protein bands ranging from 15.3 to 121.9 kDa in enzyme extracts of catla, rohu and silver carp. The substrate SDS‐PAGE evidenced the presence of various protease activity bands ranging from 21.6–93.7, 21.6–63.8 and 26.7–98.5 kDa for catla, rohu and silver carp respectively. In pH‐stat hydrolysis of Chilean fishmeal showed significantly (P < 0.05) higher degree of hydrolysis compared with soybean meal, silver cup (a commercial fish feed of Mexico) and wheat flour, with enzyme preparations of three fishes. The rate of hydrolysis was significantly (P < 0.05) higher in silver carp compared with others.     

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.     

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.     

Effects of Vibrio harveyi on plasma clotting protein (coagulogen) of white shrimp Litopenaeus vannamei

Blood clotting exhibits various important functions, including the prevention of body fluid loss and invasion of pathogens in shrimp. The effects of pathogenic Vibrio harveyi on plasma of white shrimp (Litopenaeus vannamei) in vitro and in vivo were investigated in this study. The clotting protein (coagulogen) in plasma of white shrimp pre‐incubated with extracellular products (ECP) of V. harveyi was found apparently decreased and fast‐migrated in crossed immunoelectrophoresis (CIE) gels. In addition, the coagulogen had been degraded to many low molecular‐weight protein bands in plasma pre‐incubated with ECP on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) gels. When pre‐challenged with bacterial cells and ECP of V. harveyi, the white shrimp began to die at about 30 and 16 h respectively. Moreover, plasma coagulogen was decreased more obvious in shrimp challenged with ECP than that with bacterial cells as visualized in CIE gels, and total plasma protein in both group of shrimp were all decreased. Haemolymph withdrawn from moribund shrimp pre‐challenged with V. harveyi or its ECP was observed unclottable. However, the addition of clotting factors (transglutaminase and/or Ca²⁺) to these unclottable plasma could apparently promote their re‐clotting ability as jelly‐like solid observed in microtubes. The recovery of clotting ability of plasma from moribund shrimp was due to the reformation of coagulogen (200 kDa) after adding the two clotting factors as shown on CIE and SDS‐PAGE gels. The present results suggest that the infection of V. harveyi in white shrimp may not only degrade coagulogen but also influence the presence of transglutaminase and Ca²⁺ ion.     

Evaluation of pea proteins and poultry protein as fish meal alternatives in the diets for juvenile black sea bream,Acanthopagrus schlegelii

The potential of three different protein resources (pea protein isolate, PPI; pea protein concentrate, PPC; enzyme treated poultry protein, ETPP) as fish meal (FM) alternative protein in diets for juvenile black sea bream, Acanthopagrus schlegelii. (initial average weight 7.90 ± 0.13 g) was evaluated. Seven isonitrogenous and isoenergetic diets were formulated to replace FM at 0% (T0, control diet), 8% (designated as T1‐T3) and 16% (designated as T4‐T6) using PPI, PPC and ETPP respectively. Each diet was randomly assigned to triplicate groups of 25 juvenile fish for 8 weeks. At the end of the feeding period, survival rate was not significantly affected by dietary treatments. Growth performance in T6 (16% ETPP) group was significantly inferior to T0 group, however, weight gain and specific growth rate in other treatments showed no significant differences (P > 0.05). Mean feed intake, feed efficiency ratio and protein efficiency ratio were also poorer in fish fed in T6 than those of fish fed with the control diet respectively. Apparent digestibility coefficients (ADCs) of dry matter and crude protein for fish fed ETPP diets were significant lower than those of fish fed with the control diet, whereas ADCs of lipid were unaffected by dietary treatments. ADC's of dietary Leu, Ile, His and Lys was also significantly influenced. There were no marked variations in proximate compositions of dorsal muscle. With regard to plasma characteristics, significant difference was observed in triacylglycerol content. Ammonia concentration in plasma tended to increase in alternative protein diets as substitution level increased. There were significant differences in aspartate aminotransferase activities among groups, but alanine aminotransferase levels were unaffected by treatments. In conclusion, the present study demonstrated that PPI and PPC were potential protein sources for using in juvenile black sea bream diet. However, the substitution level of FM by ETPP should be limited within 16%.     

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.