蓝圆鲹骨骼肌脯氨酸内肽酶的分离纯化及其对胶原肽的作用

以蓝圆鲹为研究对象,探讨脯氨酸内肽酶(Prolyl endopeptidase,PEP)对鱼类肌肉胶原蛋白的作用及其机理。通过硫酸铵分级盐析,DEAE-Sephacel阴离子交换,Phenyl-Sepharose疏水层析和Q-Sepharose阴离子交换,从蓝圆鲹骨骼肌中分离纯化得到一种脯氨酸内肽酶。SDS-PAGE结果显示,PEP的分子量为82 ku,肽质量指纹图谱分析得到16个肽片段,共169个氨基酸残基。片段序列与墨西哥鲷鱼(Neolamprologus brichardi)PEP的同源性达98.8%,证明纯化得到的酶是PEP。PEP的最适温度为35℃,但热稳定性较差;最适p H为6.0,在p H 5.0~7.5之间有较好的稳定性。将PEP与合成的鱼胶原蛋白小肽反应,利用反相高效液相色谱对产物进行分离,电喷雾质谱分析结果显示PEP的水解位点在脯氨酸残基的羧基端。以上结果表明,鱼类肌肉中的PEP能够协同金属蛋白酶,通过切割脯氨酸残基进一步降解胶原小肽从而参与到鱼肌肉胶原蛋白的新陈代谢中,是鱼死后参与胶原蛋白降解的重要酶类。     

Serine Protease Inhibitors as Good Predictors of Meat Tenderness: Which Are They and What Are Their Functions?

Since years, serine proteases and their inhibitors were an enigma to meat scientists. They were indeed considered to be extracellular and to play no role in postmortem muscle proteolysis. In the 1990's, we observed that protease inhibitors levels in muscles are a better predictor of meat tenderness than their target enzymes. From a practical point of view, we therefore choose to look for serine protease inhibitors rather than their target enzymes, i.e. serine proteases and the purpose of this report was to overview the findings obtained. Fractionation of a muscle crude extract by gel filtration revealed three major trypsin inhibitory fractions designed as F1 (Mr:50–70 kDa), F2 (Mr:40–60 kDa) and F3 (Mr:10–15kD) which were analyzed separately. Besides antithrombin III, an heparin dependent thrombin inhibitor, F1 and F2 comprised a large set of closely related trypsin inhibitors encoded by at least 8 genes bovSERPINA3-1 to A3-8 and able to inhibit also strongly initiator and effector caspases. They all belong to the serpin superfamily, known to form covalent complexes with their target enzymes, were located within muscle cells and found in all tissues and fluids examined irrespective of the animal species. Potential biological functions in living and postmortem muscle were proposed for all of them. In contrast to F1 and F2 which have been more extensively investigated only preliminary findings were provided for F3. Taken together, these results tend to ascertain the onset of apoptosis in postmortem muscle. However, the exact mechanisms driving the cell towards apoptosis and how apoptosis, an energy dependent process, can be completed postmortem remain still unclear.     

合浦珠母贝肉酶解液中抗氧化肽的分离及活性研究

合浦珠母贝肉酶解液经膜分离后,采用葡聚糖凝胶G-25进行分离纯化,对分离组分的总抗氧化性、DPPH自由基清除能力、羟自由基清除能力和在化妆品中的应用效果进行测定,结果表明:经分离得到10个有效组分F1-F10,其中F4组分的总抗氧化性最强,DPPH自由基清除能力44.1±0.3%,羟自由基清除率76.0±1.5%,将合浦珠母贝抗氧化肽(F4组分)添加到营养霜中,当添加量为1.0%时,抗氧化活性A700nm为1.599±0.004,能有效提高营养霜的抗氧化性,且营养霜的性状没有发生改变。该研究为合浦珠母贝肉抗氧化肽的结构和在化妆品中的应用研究奠定基础。     

SDS-PAGE法检测动物肌肉蛋白质加热终点温度的研究

为了探索检测动物组织蛋白质热变性程度的方法,分别对猪、牛、羊、鸡、鱼五种新鲜动物肌肉组织进行不同温度的热处理（新鲜未加热、50、60、70、80、90、100℃）,对处理后的样品提取蛋白质进行十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析。结果显示,同种动物组织经不同温度热处理,所得电泳图谱具有较大差异,电泳条带的数量随温度的升高逐渐减少,猪、牛、羊三种动物肌肉组织加热到80℃后,电泳图谱不再变化,达到加热终点温度;鸡和鱼的肌肉组织加热到70℃后,电泳图谱不再变化,达到加热终点温度。不同种动物组织经相同温度热处理所得电泳图谱显示：猪、牛、羊三种动物组织蛋白质的电泳图谱相似,与鸡和鱼组织蛋白质的电泳图谱差异显著。     

野生与池养刀鲚肌肉品质特性及抗氧化性的比较分析

比较研究了野生和池养刀鲚（Coilia nasus）肌肉营养成分组成、质构特性和抗氧化特性。结果显示,野生刀鲚肌肉中水分、羟脯氨酸、胶原蛋白含量以及胶原蛋白/总蛋白比值均显著低于池养型,而脂肪含量则显著提高;两者的蛋白质含量、pH均无显著性差异;池养刀鲚肌肉弹性、硬度、咀嚼度、胶着性相较于野生型呈上升趋势,但无统计学差异。野生刀鲚肌肉中SOD、CAT、T-AOC、MDA、GSH-PX、iNOS、GSH/GSSG均显著高于池养型。表明野生和池养刀鲚营养品质存在差异,建议在人工养殖刀鲚中应该注意饵料的种类和丰度,其养殖模式有待进一步改善。     

Characterisation of proteolytic enzymes from muscle and hepatopancreas of fresh water prawn (Macrobrachium rosenbergii)

BACKGROUND: Fresh water prawn in Thailand is widely consumed due to its delicacy. During postmortem handling and storage, prawn meat becomes soft and mushy, probably as a result of indigenous proteases. Therefore, an understanding of prawn proteases associated with the degradation of muscle proteins from fresh water prawn could pave the way for prevention of such a phenomenon during extended storage. RESULTS: Proteolytic enzymes in the crude extract (CE) from muscle and hepatopancreas of fresh water prawn (Macrobrachium rosenbergii) were characterised. CE from muscle exhibited the highest hydrolytic activities towards haemoglobin at pH 5 and 50 °C, while that from hepatopancreas had the highest activity on casein at pH 7 and 60 °C. Based on inhibitor study, cysteine protease and serine protease were dominant in CE from muscle and hepatopancreas, respectively. CE from muscle rarely hydrolysed natural actomyosin (NAM), but could not degrade pepsin‐soluble collagen (PSC). Conversely, NAM and PSC were susceptible to hydrolysis by CE from hepatopancreas as evidenced by the marked decreases in band intensity. Activity staining using haemoglobin, casein and gelatin as substrates revealed that no proteolytic or gelatinolytic activity was observed in CE from prawn muscle, while CE from hepatopancreas exhibited pronounced hydrolytic activities towards all substrates. CE from muscle showed calpain and cathepsin L activities but CE from hepatopancreas mainly exhibited tryptic and chymotryptic activities. CONCLUSION: Serine proteases, mainly trypsin‐like or chymotrypsin‐like, from hepatopancreas were probably responsible for the softening of prawn meat during postmortem storage via the degradation of both muscle and connective tissues. Copyright © 2010 Society of Chemical Industry     

Antiproliferative activity of peptides prepared from enzymatic hydrolysates of tuna dark muscle on human breast cancer cell line MCF-7

To produce and identify antiproliferative peptides, two commercial enzymes, papain (PA) and Protease XXIII (PR) were used to hydrolyse tuna dark muscle byproduct, and the protein hydrolysates were purified, before being evaluated for antiproliferative activities against human breast cancer cell line MCF-7. The results showed that the peptide fractions with the molecular weight ranging from 390 to 1400 Da possessed the greatest antiproliferative activity. The amino acid sequences of the two antiproliferative peptides isolated from PA and PR hydrolysates were Leu-Pro-His-Val-Leu-Thr-Pro-Glu-Ala-Gly-Ala-Thr (1206 Da) and Pro-Thr-Ala-Glu-Gly-Gly-Val-Tyr-Met-Val-Thr (1124 Da), whilst they show the dose-dependent inhibition effect of the MCF-7 cells with IC₅₀ values of 8.1 and 8.8 μM, respectively. We thus conclude that antiproliferative hydrolysates from tuna dark muscle byproduct may be useful ingredients in food and nutraceutical applications.     

Gelatinolytic activities in muscle of Atlantic cod (Gadus morhua), spotted wolffish (Anarhichas minor) and Atlantic salmon (Salmo salar)

The gelatinolytic activity in muscle from Atlantic cod (Gadus morhua), spotted wolffish (Anarhichas minor) and Atlantic salmon (Salmo salar) was studied using gelatin SDS‐PAGE, gelatin affinity chromatography and enzyme inhibitors. These fish species are known to differ markedly in fillet softening and gaping post mortem. Atlantic cod, which is a promising species for cold water marine aquaculture, often shows such negative properties, particularly after being well fed. Gelatinolytic activity bands were present in all three species. Using gelatin chromatography and enzyme inhibitors, both serine proteinases and metalloproteinases were detected in wolffish and cod muscle, while only the latter were found in salmon muscle. Activation of the metalloproteinases by p‐aminophenylmercuric acetate (APMA) resulted in a shift in activity from higher to lower molecular weight, as is known for mammalian matrix metalloproteinases. In all three species the molecular weight of the metalloproteinases was lowered from approximately 80 to about 70 kDa by activating with APMA. Copyright © 2003 Society of Chemical Industry     

沙光鱼肌肉营养成分分析及营养学评价

测定连云港特产鱼类——沙光鱼的肌肉营养成分并对其进行营养学评价。沙光鱼的肌肉(鲜样)中水分、粗脂肪、粗蛋白和粗灰分分别为76.32%、1.52%、18.06% 和1.23%。沙光鱼肌肉共检测出18 种氨基酸,其中,谷氨酸含量最高,达到2.27%,必需氨基酸占总氨基酸的比值为46.79%,必需氨基酸和非必需氨基酸的比值为87.93%。沙光鱼的必需氨基酸评分和化学评分表明其第一限制性氨基酸为色氨酸。必需氨基酸指数为75.95。单不饱和脂肪酸(MUFA)、多不饱和脂肪酸(PUFA)、二十碳五烯酸(EPA)+ 二十二碳六烯酸(DHA)在总脂肪中含量分别为37.97%、22.20% 和10.20%。以上结果表明沙光鱼营养价值较高、并具有保健作用,具有较好的开发前景。