饲料中苏氨酸含量对建鲤肉质及组织蛋白酶B、L的影响

该试验测定了饲料中3组苏氨酸含量,即7. 4 g/kg(缺乏组)、15. 7 g/kg(适宜组)、25. 2 g/kg(过量组),对建鲤肉质及组织蛋白酶B、L的影响。通过利用显微镜图像分析各处理组肌纤维特征、鱼肉品质指标,荧光合成肽底物法测定组织蛋白酶B(cathepsin B,CTS B)、组织蛋白酶L(Cathepsin L,CTS L)的活性,免疫组化法测定蛋白表达量,研究发现,饲喂90 d后,适宜组的肌纤维密度最高,肌纤维平均直径(22. 12μm)最小;肌原纤维耐折力(0. 116 3μm)相对较高,而其断裂指数(A_(540)=59. 70)最低;剪切力(0. 168 4 kg·f)最高,失水率(21. 02%)最低,p H值(6. 91)最低;组织蛋白酶B、L的酶活性及表达量最低。综上,饲料中添加适宜水平苏氨酸能够改善鱼肉肌纤维特性及硬度品质,为通过营养素水平调节而提高建鲤鱼肉品质提供一定理论依据。     

饲料中添加适宜水平铁对建鲤肉质、鱼糜稳定性及组织蛋白酶B/L的影响

探讨饲料中添加适量铁对建鲤肉质、鱼糜品质及其肌肉中组织蛋白酶B/L的影响。以添加适宜水平铁的饲料饲喂90 d的建鲤（适宜组,铁实测质量分数146.1 mg/kg）为研究对象,同时以未添加铁组（缺乏组,铁实测质量分数53.9 mg/kg）作为对照。采样后测定鱼肉品质指标及肌肉中组织蛋白酶B/L活性,并采用免疫组化法进行蛋白表达定量;而后将2组鱼肉加工为鱼糜,并测定其稳定性指标。结果表明,与缺乏组相比,适宜组肌原纤维耐折力、断裂指数分别稍有上升及下降,但差异不显著（P>0.05）;剪切力极显著增加31.80%（P<0.01）,可溶胶原蛋白含量、不溶胶原蛋白含量和总胶原蛋白含量均分别极显著增加305.45%、180.98%、219.35%（P<0.01）。适宜组鱼肉pH及失水率略呈下降趋势,差异无统计学意义（P>0.05）。适宜组组织蛋白酶B/L活性分别极显著（P<0.01）及显著（0.01相似文献   

鱿鱼肝脏蛋白酶的鉴定及组织蛋白酶L的分离纯化

鱿鱼肝脏含有丰富的蛋白酶,为利用其内源蛋白酶进行可控的酶解,本研究以鲤鱼肌原纤维蛋白为底物对鱿鱼肝脏内源蛋白酶的种类和性质进行了研究。反应体系中添加E-64、1,10-菲啰啉和苯甲基磺酰氟(phenylmethylsulfonyl?fluoride,PMSF)后,肌球蛋白重链(myosin?heavy?chain,MHC)的降解得到了显著抑制,确定了鱿鱼肝脏含有金属类、半胱氨酸类、丝氨酸类3类蛋白酶。半胱氨酸类蛋白酶热稳定性最好,在50℃以上仍然具有较大活性,可将肌原纤维蛋白酶解成小分子质量的降解产物。利用特异性底物对半胱氨酸蛋白酶种类进行鉴定发现,该酶只酶解Z-Phe-Arg-MCA,添加亮抑酶肽后相对酶活性为0%,添加E-64相对酶活性仅存0.6%,初步确定鱿鱼肝脏中的半胱氨酸蛋白酶主要为组织蛋白酶L。最后,通过硫酸铵沉淀、离子交换层析、凝胶过滤对组织蛋白酶L进行分离纯化,在电泳上得到了分子质量约为25?kD单一条带。     

杂色鲍组织蛋白酶L的分离纯化与性质研究

通过硫酸铵分级沉淀、SP-Sepharose阳离子交换层析、Sephacryl S-200HR凝胶过滤和Hydroxyapatite羟基磷灰石层析,从杂色鲍（Haliotis diversicolor）内脏中分离纯化了组织蛋白酶L。SDS-PAGE结果显示,纯化蛋白的分子量约为28ku。该酶最适温度37℃,最适pH5.5。当温度低于40℃,pH在5.0～6.5范围内该酶较稳定。底物特异性实验与抑制剂实验结果表明,该酶属于半胱氨酸蛋白酶。金属离子Mn2＋、Ba2＋和Mg2＋对该酶有不同程度的激活作用,而Co2＋、Cu2＋、Zn2＋、Fe2＋和Ca2＋等对该酶起抑制作用。     

饲料中适宜含量铁对建鲤鱼糜冻藏品质变化及组织蛋白酶B/L的影响

对经过不同铁含量的饲料(缺乏组53.9 mg/kg,适宜组146.1 mg/kg)饲喂90 d后的建鲤,取鱼肉加工为鱼糜,于-20℃条件下进行冻藏实验,取样点为0、1、2、4、6周,测定鱼糜冻藏品质指标的变化,包括Ca~(2+)-ATPase活性、总巯基、二硫键、表面疏水性、TBA值;鱼糜冻藏期间组织蛋白酶B、组织蛋白酶L活性采用荧光合成肽底物法进行测定。结果表明,适宜含量铁能极显著提高鱼糜肌动球蛋白初始Ca~(2+)-ATPase活性、总巯基含量(p0.01),降低二硫键含量(0.01p0.05)、表面疏水性(p0.01)及TBA值(p0.01),且可延缓冻藏期间Ca~(2+)-ATPase活性、总巯基含量的下降,以及表面疏水性、二硫键含量的增大;冻藏6周后,缺乏组和适宜组TBA值分别上升264%、143%;适宜组组织蛋白酶B/L活性均低于缺乏组,且对组织蛋白酶L活性下降有一定促进作用,而对组织蛋白酶B作用不明显。综上所述,饲料中添加适宜含量铁能够减缓冻藏期间建鲤鱼糜蛋白质的变性,降低组织蛋白酶B/L活性残留,对提高鱼糜冻藏品质稳定性具有潜在的促进作用。     

牛胰脏组织蛋白酶L的纯化和酶学性质

新鲜牛胰脏匀浆物经酸化粗提后,再通过盐析、柱层析等步骤纯化,制备了0.58 mg纯酶,纯化倍数达530.54。经凝胶电泳分析,该酶有2 个亚基,分子质量为29.1 kD和18.9 kD。牛胰脏组织蛋白酶L的最适反应温度为50 ℃,最适反应pH值为6.5。巯基还原剂二硫苏糖醇、L-半胱氨酸均明显激活了该酶活性,10 μmol/L的N-（反式-环氧丁二酰基）-L-亮氨酸-4-胍基丁基酰胺（E-64）可完全抑制其活性。1 mmol/L的Zn2＋对酶活性有明显抑制作用。该纯化酶可水解苄氧羰基-苯丙氨酰-精氨酰-甲基香豆素（Z-Phe-Arg-MCA）,其Km值为3.52 μmol/L。     

鲤鱼组织蛋白酶L的分离纯化与酶学性质研究

对鲤鱼鱼背部白肌中的组织蛋白酶L进行分离纯化并研究了其酶学性质。结果发现,鲤鱼组织蛋白酶L提取液经酸处理、硫酸铵分级沉淀、DEAE-Sepharose F.F.离子交换层析和SephacrylS-100凝胶过滤层析等步骤后得到部分纯化,纯化倍数为16.39。鲤鱼组织蛋白酶L的最适反应温度和pH值分别为40℃和5.0,在20～50℃和pH 4.5～5.5范围内具有较好的稳定性。     

鲤鱼组织蛋白酶L活性的影响因素研究

主要研究了鲤鱼组织蛋白酶L活性的影响因素。结果表明,鲤鱼组织蛋白酶L具有较强的稳定性,在-20℃冻藏、20⁵0℃加热处理、不同酸性环境中均保留了一定程度的活性;鱼肉中组织蛋白酶L在漂洗工艺中并不能完全去除,漂洗后仍残存了45.65%活性。此外,鲤鱼组织蛋白酶L在豆科类蛋白、大米蛋白、鸡蛋清蛋白、马铃薯蛋白、茶多酚和大豆异黄酮等各种食品组分作用下,其活性发生明显的下降,且呈现良好的量效关系。      

烟草半胱氨酸蛋白酶抑制剂（CPI）基因家族的克隆及组织表达谱分析

运用生物信息学方法,结合RT-PCR和SMART RACE技术从烟草（Nicotiana tabacum）中克隆了4个CPI基因的全长cDNA序列,分别命名为NtCPI1、NtCPI2、NtCPI3和NtCPI4, GenBank登陆号分别为KF057988、KF057989、KF057990和KF057991。基因序列分析表明4个基因分别编码98、98、120和123个氨基酸残基的蛋白质,都具有CPI反应位点的保守基序GG、QXVXQ和A/PW,同时具有植物CPI所特有的LARFAV基序,其中NtCPI3和NtCPI4的N端还包含一段27个氨基酸残基组成的信号肽。实时荧光定量PCR试验表明,4个基因的组织表达谱很广,在根、茎、叶和芽组织中都有表达。这些结果为进一步研究半胱氨酸蛋白酶抑制剂在植物中的生理功能奠定了基础。      

荧光素酶编码基因luc在大肠杆菌中的克隆、表达及纯化

以荧光虫荧光素酶报告载体pXP2 DNA为模板,扩增得到编码萤火虫荧光素酶(LUC)的基因(luc),构建了诱导型表达载体pQE30-luc.将载体导入 E.coli M15获得高效诱导表达萤火虫荧光素酶基因的重组菌M15/pQE30-luc.SDS-PAGE电泳分析显示:重组蛋白的相对分子质量为 60 000 ,表达量占全菌胞内可溶性蛋白质的50.9 %. 利用表达载体pQE30上的His*Tag标记选用Ni柱纯化表达具有活性的萤火虫荧光素酶(LUC),纯化后比酶活达到1.43×10 9 RFU/ mg,纯化倍数达10.6倍,回收率为25.3%.     

胍基乙酸对建鲤生产性能、体成分及肌肉能量代谢关键酶的影响

研究胍基乙酸(guanidinoacetic acid,GAA)对建鲤生产性能、体成分及肌肉能量代谢的影响。选取600尾平均体重为(23.23±0.14)g的建鲤随机分为4组,每组5个重复,每个重复30尾,分别投喂基础饲料(对照组)和在基础饲料中添加250、500和1 000 mg/kg GAA的试验饲料,试验为期42 d。结果表明:与对照组相比,添加250、500 mg/kg GAA可显著降低饵料系数(P0.05),但对体重、特定生长率、增重率、平均日采食量和成活率均无显著影响(P0.05)。添加GAA可显著提高脏体指数(P0.05),对肝体指数无显著影响(P0.05)。GAA对肌肉中粗脂肪、粗蛋白和粗灰分含量无显著影响(P0.05)。250 mg/kg GAA组可显著降低肌肉中丙酮酸激酶活性(P0.05);250、500 mg/kg GAA组显著降低琥珀酸脱氢酶活性(P0.05);1 000 mg/kg GAA组显著降低肌酸激酶活性(P0.05);GAA组均显著提高肌糖原含量(P0.05)。结果表明,饲料中添加胍基乙酸可降低肌肉中能量代谢关键酶的活性来改善其能量代谢,提高饲料转化率,以添加250 mg/kg GAA效果较好。     

Immunolocalization of Jian Carp (Cyprinus Carpio Var. Jian) Cathepsin B: Cloning,Expression, Characterization,and Antibody Preparation

Cathepsin B (CatB) cDNA of 759 bp from Jian carp (Cyprinus carpio var. Jian) with amino acid similarity of 99.6% to common carp was cloned. The mature CatB was expressed in Escherichia coli BL21 transferred with vector CatB‐pET‐30a. It was purified and identified as a single band (29 kDa) on SDS‐PAGE. Optimum CatB activity was observed at 40 °C and pH 5.5. Mouse anti‐CatB polyclonal antibody with a high titer of 1:256000 was prepared successfully and shown to specifically recognize the antigen both in prokaryotic cells and in the tissues of Jian carp according to western blotting and immunohistochemistry results. Immunolocation analysis showed that CatB distribution at protein level varied among the tested tissues. The results presented in this study may provide a significant reference for future research on the inherent relationship between CatB and the quality of fish or fish products at both the gene and protein levels.     

Purification and characterization of parvalbumins from silver carp (Hypophthalmichthy molitrix)

BACKGROUND: As the largest producer and consumer of freshwater fish in the world, many people suffer from allergy for consuming freshwater fish in China. However, the allergen profiles of freshwater fish are rarely known. RESULTS: Parvalbumins (PVs) from the white muscle of silver carp (Hypophthalmichthy molitrix) were purified by ammonium sulfate fractionation and column chromatography including DEAE‐Sepharose and Superdex 75. Three PV isoforms—PV‐I, PV‐II, and PV‐III—were obtained and their molecular masses as estimated by tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis were 12, 11, and 14 kDa, respectively. All the PVs could be detected by anti‐frog PV monoclonal antibody. PV‐I and PV‐II were quite possibly glycoproteins, while PV‐III was not glycosylated, as analyzed by periodic acid–Schiff (PAS) staining. Thermal stability revealed that PV‐I and PV‐II easily formed polymers, while these proteins were stable in a pH range of 4.0–10.0. A PV gene encoding 110 amino acid residues was cloned and it revealed high identity with PVs from other species of fish. CONCLUSION: Three isotypes of PV were purified to homogeneity and one distinct PV gene was cloned in silver carp white muscle. Copyright © 2010 Society of Chemical Industry     

Lipid peroxidation,protein oxidant and antioxidant status of muscle,intestine and hepatopancreas for juvenile Jian carp (Cyprinus carpio var. Jian) fed graded levels of myo-inositol

Lipid peroxidation, protein oxidant and antioxidant status of muscle, intestine and hepatopancreas in juvenile Jian carp (Cyprinus carpio var. Jian) fed graded levels of myo-inositol (MI) (163.5, 232.7, 384.2, 535.8, 687.3, 838.8 and 990.3 mg/kg diet) for 60 days were investigated. Total tissue malondialdehyde (MDA) and protein carbonyl (PC) content showed a downward trend to a point (P < 0.05). Conversely, total tissue anti-hydroxyl radical (AHR), catalase (CAT), glutathione-S-transferase (GST), glutathione peroxidase (GPx), glutathione reducase (GR) activities and glutathione (GSH) content were generally higher in MI-supplemented diets than MI-unsupplemented diet (P < 0.05). Muscle and intestinal superoxide dismutase (SOD), and intestinal anti-superoxide anion (ASA) were increased by MI supplementation (P < 0.05), whereas these parameters in the other tissue showed no alterations (P > 0.05). These results indicated that antioxidant status was improved, and lipid peroxidation and protein oxidant were depressed in muscle, intestine and hepatopancreas by MI.     

Physicochemical,micro-structural,and textural properties of different parts from farmed common carp (Cyprinus carpio)

The fish body of cultured common carp (Cyprinus carpio) was divided into six sections, including the upper back, lower back, jaw, chest, belly, and tail. Differences in the physicochemical, micro-structural, and textural properties of different muscle tissues were investigated. The upper and lower back, with high content of protein, low content of fat, high water-holding capacity, and desirable textural properties, was proven to be the most valuable part of common carp from both a nutritional point-of-view and an organoleptic perspective. This could provide a theoretic basis for the comprehensive utilization of freshwater fish.     

黄棒菜超氧化物歧化酶(SOD)分离纯化的技术

以植物新品种黄棒菜为原料,研究料液比,浸提温度及pH,热变性温度及时间、丙酮加入量等因素对黄棒菜超氧化物歧化酶(SOD)提取效果的影响,利用正交试验与单因素试验优化提取工艺。结果表明,提取SOD的最佳条件为:料液比1︰3(g/mL),浸提温度0℃,pH 7.0,50℃热变性20 min,1.5倍酶液体积的丙酮,此时SOD纯化倍数可达3.17。经层析纯化及真空冷冻干燥,获得SOD成品。最佳条件下处理新鲜黄棒菜叶茎、叶片组织,测得SOD最终比活力分别为7662.23 U/mg,4897.39 U/mg,纯化倍数达17.55、23.08,叶茎更适于提取SOD。经电泳检测,黄棒菜SOD相对分子质量约80 kDa,据文献鉴定为Mn-SOD。试验结果为黄棒菜SOD的分离纯化及资源开发提供技术参考。