Antioxidative Activitiy of Protein Hydrolysate from the Muscle of Common Kilka (Clupeonella cultriventris caspia) Prepared Using the Purified Trypsin from Common Kilka Intestine

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.     

Oxidative Stability of Common Kilka (Clupeonella cultriventris caspia) Oil Supplemented with Microwave Extracted Ghure (Unripe Grape) Marc Extract

The objective of this study was to evaluate the effect of microwave extracted Ghure (unripe grape) marc extract on common kilka (Clupeonella cultriventris caspia) oil oxidation during accelerated storage. The antioxidant activity of Ghure marc extract was compared with those of α-tocopherol and BHT. The Ghure marc extract significantly reduced the peroxide and p-anisidine value of kilka oil. Even though the effect of Ghure marc extract on reducing the oxidation of kilka oil was similar to the effect of BHT, it was functionally more effective than α-tocopherol. Generally, Ghure marc extract could be considered as a potential source of natural antioxidants, with inexpensive costs, for improving the oxidative stability of kilka oil.

Abbreviations: AA, Antioxidant activity; AOP, Antioxidant power; AV, p-Anisidine value; CUPRAC, Cupric ion reducing antioxidant capacity; DHA, Docosahexaenoic acid; EPA, Eicosapentaenoic acid; FIC, Ferrous ion chelating; GME, Ghure marc extract; FRAP, Ferrous ion reducing antioxidant power; KO, Kilka oil; IP, Induction period; MAE, Microwave-assisted extraction; MUFA, Monounsaturated fatty acid; PUFA, Polyunsaturated fatty acid; PV, peroxide value; PF, Protection factor; RSA, Radical scavenging activity; SFA, Saturated fatty acids; TFC, Total flavonoid content; TPC, total phenolic content TV, totox value.     

Anionic Trypsin from the Pyloric Ceca of Pacific Saury (Cololabis saira): Purification and Biochemical Characteristics

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 K_m and K_cat were 0.19 mM and 210 s^?1, respectively, while the catalytic efficiency (K_cat/K_m) 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.     

Isolation,Purification, and Biochemical Characterization of Trypsin from Indian Mackerel (Rastralliger kanagurta)

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 (K_m) and catalytic constant (K_cat) 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.     

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。     

鳙鳃凝集素的分离纯化及理化性质的研究

利用DEAE-Sepharose FF离子交换和Sephacryl S-200 HR及Superdex 200 10/300 GL分子筛层析技术,从鳙鳃组织中分离纯化到一种岩藻糖专一的凝集素,命名为GANL.在还原SDS-PAGE电泳上显示单一蛋白染色带,其亚基相对分子质量为37 kD.经Superdex 200凝胶过滤层析测得其天然相对分子质量为220 kD.GANL的中性糖含量为13.4%.因此,GANL作为一种由相同亚基组成多亚基糖蛋白.GANL对兔红细胞有专一的凝集活性,其凝集活性不依赖Ca~(2+).在被测的单糖、双糖及糖蛋白中,仅岩藻糖能抑制其凝集活性.氨基酸分析表明GANL的Asp、Glu、Leu、Val、Lys的含量较高,Cys-S的含量为0.81%.GANL的最适pH为8～9;具有很高的热稳定性,最适温度为50 ℃.     

Isolation and Characteristics of Carboxypeptidase B from Zebra Blenny (Salaria basilisca) Viscera

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, K_m and k_cat for Hippuryl-l-Arg, were 0.32 mM and 36.23 s^?1, respectively.     

皱纹盘鲍脏器多糖的分离纯化及鉴定

利用碱性蛋白酶从皱纹盘鲍脏器中提取多糖,采用胃蛋白酶和Sevag相结合的方法脱除蛋白质;经Sephadex G-100凝胶柱层析和DEAE-cellu lose 52离子交换层析分离纯化得到AHP-2;AHP-2经高效液相色谱和比旋光度法鉴定其为均一性多糖;紫外扫描无蛋白质的特征吸收峰;红外光谱分析,AHP-2具有典型的多糖吸收峰和硫酸基吸收峰;凝胶色谱法测定其分子量为10 000~15 000;化学组成分析表明,AHP-2是硫酸酯多糖,硫酸根含量11.55%;气相色谱测定单糖组成为鼠李糖、岩藻糖、木糖、半乳糖和葡萄糖,其摩尔比Rha∶Fuc∶Xyl∶Gal∶G lu=6.7∶2.0∶3.9∶7.4∶1.0。     

口虾蛄肝胰脏超氧化物歧化酶的分离纯化及部分性质的研究

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。     

鲫卵黄脂磷蛋白的纯化鉴定及其夹心ELISA的建立

鲫(Carassius carassius)卵黄原蛋白(vitellogenin,Vtg)是检测水体环境雌激素活性常用的生物标志物。本研究利用凝胶过滤结合离子交换层析与选择性沉淀结合离子交换层析2种方法,从鲫卵巢匀浆液中纯化得到了卵黄脂磷蛋白(lipovitellin,Lv),经鉴定该蛋白含有糖、磷、脂基团,天然分子量约为521 kD,SDS变性电泳显示分子量为117 kD和103 kD的2个亚基。Western blot结果显示,金鱼(Carassius auratus)Lv抗体和斑马鱼(Danio rerio)Lv抗体都能与鲫Lv发生很好的交叉反应。利用纯化的鲫Lv与金鱼Lv抗体和斑马鱼Lv抗体建立了2种夹心ELISA,发现金鱼Lv和鲫Lv的结合曲线基本重合,并且利用鲫Lv与金鱼Lv抗体建立的夹心ELISA工作范围为15.6~1000 ng/mL,检出限约为6.8 ng/mL,显著低于利用斑马鱼Lv抗体建立的夹心ELISA,结合此前研究者建立的鲫Vtg竞争ELISA,为鲫Vtg指标的测定提供了可靠方法。     

黄颡鱼肝脏胞质中NADP依赖性的异柠檬酸脱氢酶(IDPc)的分离纯化和酶学性质

采用Sephadex G-100凝胶过滤、DEAE Sepharose离子交换、Sephadex G-25凝胶过滤等方法对黄颡鱼肝脏胞质中NADP依赖性的异柠檬酸脱氢酶(IDPc)进行纯化,并研究其相关的酶学性质。结果显示,IDPc的比活力为7.94 U/mg,亚基分子量为36.7 ku。IDPc活性最大时的pH和温度分别为8.0和65℃,活化能为81.33 kJ/mol,底物米氏常数KmNADP和KmIC分别为0.056和0.175 mmol/L,底物最大反应速率VmNADP和VmIC分别为9.04和10.51U/mg,催化效率KcatNADP和KcatIC分别为0.16和0.06 min/mg,产物NADPH对IDPc表现为竞争性抑制,抑制常数KiNADPH为0.034 mmol/L。IDPc的催化作用强烈依赖于金属离子Mn2+、Mg2+,没有金属离子存在时反应几乎不进行,二价金属离子激活IDPc的顺序为Mn2+Mg2+Zn2+Ca2+Cu2+,Cu2+几乎不能激活IDPc的活性。Ca2+和Zn2+既是激活剂也是抑制剂,与其作用浓度有关,Cu2+基本上对其无影响。通过对IDPc系统的酶学性质探讨,能够为深入研究IDPc催化与调节机制奠定基础。     

贻贝胰蛋白酶抑制剂的纯化,结晶和生化特性的研究

本文首次报道用0.05mol/L pH7.8磷酸缓冲液抽提,热变性,饱和硫酸铵盐析和Sephadexg—50凝胶柱层析。从软体动物殆贝(Mytilus)中纯化出仅对胰蛋白酶专一性抑制的蛋白酶制剂结晶。通过SDS聚丙烯酰胺凝胶电泳,N—末端分析及氨基酸组成的测定表明贻贝胰蛋白酶抑制剂的分子量约为32100,是以甘氨酸、赖氨酸为末端,由253个氨基酸组成的二条多肽链或亚基聚成的一组蛋白。     

Purification and characterization of a new reovirus from the Chinese mitten crab, Eriocheir sinensis

A new reovirus was recently isolated from a freshwater crab, the Chinese mitten crab, Eriocheir sinensis, in China. The complete viral particles are 55 nm in diameter, icosahedral, non-enveloped and have a mean buoyant density of 1.39 g cm(-3) in CsCl gradient. The viral genome is composed of 12 pieces of dsRNA with an electrophoretic pattern of 3/4/2/3. This virus infects connective tissue of the gills, gut and hepatopancreas. Partial cDNA cloning and sequence analysis showed that the RNA-dependent RNA polymerase is located in the first RNA segment. From its biochemical, ultrastructural and physicochemical properties, this virus is quite different from the genus Aquareovirus (Reoviridae). It may represent a new genus of Reoviridae, different from the other crab reoviruses, P and W(2).     

凡纳滨对虾精氨酸激酶的分离纯化及性质研究

经过CM-纤维素批量层析、Separdex G-100柱层析、DEAE_纤维素柱层析等步骤,从凡纳滨对虾肌肉组织分离得到精氨酸激酶,经SDS-PAGE检测达到电泳纯,分子量约为40kDa.对该酶的性质进行分析结果表明,精氨酸激酶的最适作用温度为55℃,当温度高于65℃时,酶活力显著下降;pH 8时酶活力较高,低浓度的精氨酸对酶活力有促进作用,高浓度时表现抑制作用,而底物类似物精胺和氨基胍则对酶促反应表现出完全的抑制.NaCl,KCl对酶的活力具有促进作用,低浓度(10 mmol·L-1)MgCl2对酶活力表现出激活作用,而CuCl2与MnCl2则表现出完全抑制酶活力,CaCl2与ZnCl2在低浓度时对酶活力无明显影响,但是随着浓度升高,对酶具有抑制作用.     

Purification and characterization of a nucleoplasmin-like protein from carp (Cyprinus carpio) eggs

ABSTRACT: Nucleoplasmin, first isolated from Xenopus laevis eggs, promotes nucleosome assembly. Hereby, we have purified a nucleoplasmin-like protein from carp ( Cyprinus carpio ) eggs using ion exchange and subsequent gel filtration columns. The protein was recognized by a polyclonal antiserum against Xenopus laevis nucleoplasmin and had an amino acid composition similar to other member of the nucleoplasmin family proteins. Partial amino acid sequences from the cyanogen bromide (CNBr)-cleaved fragments showed high homology with Xenopus nucleoplasmin. The protein was also found to form an oligomeric complex and to be phosphorylated. Moreover, this protein promoted sperm nuclear decondensation as well as that of nucleoplasmin from Xenopus laevis eggs. These results suggest that the fish protein isolated here is a member of nucleoplasmin family.     

Characterization of cellulase-producing bacteria from the digestive tract of tilapia, Oreochromis mossambica (Peters) and grass carp, Ctenopharyngodon idella (Valenciennes)

Isolation and characterization of cellulase‐producing aeorobic bacterial flora in the intestine of omnivorous tilapia (Oreochromis mossambica) and phytophagous Chinese grass carp (Ctenopharyngodon idella) have been carried out using selective carboxymethylcellulose‐agar (CMC‐agar) medium. The cellulolytic activity was measured both qualitatively and quantitatively. It was found that the ability of different strains in degrading cellulose varies within a wide range. Among the strains isolated from the gut of each test fish, TM1 and CI3 isolated from O. mossambica and C. idella, respectively exhibited maximum cellulolytic activity (67.02 and 35.8 U mL^?1 respectively). Pure cultures of these strains were selected for morphological, physiological and biochemical characterization. On the basis of these tests, the isolated strains were identified as Bacillus circulans (TM1) and Bacillus megaterium (CI3). Both the strains are rod‐shaped, motile and show better temperature (15–42°C) and pH (5–11) tolerance. The selected strains were further quantitatively assayed for amylase and protease activities. Maximum amylase and protease activities were exhibited by TM1 and CI3 respectively. Information generated from the present study might contribute towards better‐feed formulation incorporating plant ingredients.     

草鱼Stefin克隆表达、纯化及活性特征鉴定

文章克隆了草鱼(Ctenopharyngodon idellus)Stefin c DNA全长序列,全长294 bp,编码97个氨基酸,无二硫键,N端存在高度保守的Gly(3、4)残基及QXVXG(45~49)序列,比对结果显示其氨基酸序列与Burton's mouthbrooder(Haplochromis burtoni)Stefin A1一致性最高,为47.5%。进化树分析表明草鱼Stefin A与Burton's mouthbrooder(Haplochromis burtoni)、southern platyfish(Xiphophorus maculatus)、Colisa chuna(Trichogaster chuna)、lamprologini(Neolamprologus brichard)、elephant shark(Callorhinchus milii)及bicolor damselfish(Stegastes partitus)Stefin A聚为一类。将构建的原核表达载体Stefin-Pet30a转入E.coli BL21,以1 mol·L-1IPTG诱导表达重组Stefin蛋白,而后经梯度尿素洗涤和镍亲和层析纯化,并分别利用SDS-PAGE和TSK-GEL G2000SWxl高效液相色谱检测诱导及纯化效果,SDS-PAGE结果显示重组Stefin蛋白得到高度纯化,最终呈现相对分子量11.4 k D的单一条带;其在高效液相上保留时间25.98 min处亦呈单一活性峰,纯度为96.28%。以荧光合成肽底物(Z-Phe-Arg-MCA)测活法鉴定重组草鱼Stefin对鲤鱼组织蛋白酶B、L的抑制活性,发现该重组蛋白对二者均体现了明显的抑制活性。     

鲢鱼骨骼肌过敏原小清蛋白的分离纯化及多克隆抗体制备与应用

小清蛋白是鱼类的主要过敏原,对该蛋白的研究不仅有利于过敏原检测方法的建立也可为低致敏性水产品的开发提供理论依据。通过组织捣碎、冷冻离心、热处理、Superdex75凝胶过滤等方法从鲢白色肉中纯化得到过敏原小清蛋白。Tricine-SDS-PAGE显示,在非还原条件下,该蛋白呈分子量分别为12ku、14ku、24ku的3个条带。而在还原条件下,仅有分子量为12ku的条带。Western-blotting分析表明,分子量为12ku、14ku和24ku的这3个条带都与小鼠抗蛙小清蛋白单克隆抗体(PARV-19)发生特异性反应,提示它们均为小清蛋白的不同形态。用纯化的小清蛋白制备多克隆抗体,经Protein A Sepharose亲和层析纯化得到高纯度的免疫球蛋白G(IgG)。Dot-blot检测发现,抗体稀释至1/51200时仍能与纯化的小清蛋白有显色反应。用制备的多克隆抗体进行Western-blotting分析,能特异地检测4种鱼(鲤、鲢、鲫、黄鳍鲷)中的小清蛋白。     

建鲤组织蛋白酶L 的原核表达、纯化鉴定及多克隆抗体的制备

对原核表达的重组建鲤组织蛋白酶L(Cathepsin L,CAT L)蛋白进行尿素洗涤和Ni-NTA亲和层析纯化,该目的蛋白经300 mmol/L咪唑洗脱为单一峰,SDS-PAGE结合TSK-GEL G2000SWxl凝胶过滤高效液相色谱分析表明重组CAT L获得了高度纯化,分子量约28 k D,纯度超过95%。Z-Phe-Arg-MCA底物测活法显示该重组CAT L表现为半胱氨酸蛋白酶活性,能与其内源抑制因子Cystatin以1︰1的摩尔比结合,具有生物学活性。以纯化的重组CAT L蛋白免疫Balb/C小鼠获得抗血清,经ELISA法检测获得的CAT L抗血清效价高于1︰512000;Western blotting鉴定结果表明该抗体具有良好的特异性,能够识别原核表达的重组CAT L蛋白。免疫组织化学分析结果表明,该抗体还能识别建鲤小肠、肝胰脏、脾、背肌和心肌组织表达的内源性CAT L蛋白。因此可利用该抗体从蛋白水平检测CAT L在鱼类不同组织中的表达和分布情况。     

Isolation and Characterization of Pepsin-Soluble Collagen from the Skin of Peru Squid (Dosidicus gigas)

Pepsin-soluble collagen (PSC) was isolated from Peru squid (Dosidicus gigas) skin and physicochemical properties of the PSC were determined. The PSC exhibited a maximum absorbance at 220 nm. Sodium dodecyl sulfate polyacrylamide gel electrophoresis suggested the collagen containing α1 and α2 chain was classified as type I collagen. Amino acid composition indicated that the collagen had lower amino acid content than that of mammalian collagen. Denaturation temperature (T_d) of the PSC was 26.8°C. The PSC had relatively high solubility in alkaline condition or NaCl concentrations below 2%. Fourier transform infrared spectroscopy investigations showed the existence of helical arrangements of collagen. The lyophilized collagen had a uniform and regular network structure. These results suggested that Peru squid skin was a potential source of collagen for further research and application.