钙激活酶激活蛋白的研究进展

钙激活酶（calpain)是促进宰后肌肉嫩化的主要的内源酶，钙激活酶激活蛋白（calpain activator)是钙激活酶系统的成员之一，是区别于钙激活酶抑制蛋白（calpastatin)的钙激活酶的正调节蛋白，目前已对其激活特性，氨基酸序列及cDNA序列实行了研究，但其结构和激活机理还不清楚。     

钙蛋白酶系统及其对肉嫩度的影响

钙蛋白酶系统作为影响肉嫩度的重要因素引起了科研人员的广泛关注。钙蛋白酶系统是高度可调的复杂系统，包括钙激活酶、钙激活酶抑制蛋白和钙激活酶激活蛋白，三者相互作用，共同调控宰后肉的嫩化。本文综述了钙蛋白酶系统的结构及功能，并在此基础上阐述了其影响肉嫩度的作用机理。     

钙蛋白酶的活性调节及其与肉嫩度的关系

钙蛋白酶(calpain)是与肉嫩度相关的重要内源性蛋白酶之一,广泛存在于细胞质中,且需要一定量的Ca2 激活。研究表明,钙蛋白酶在Ca2 的激活下能降解肌原纤维蛋白,从而改善肉品嫩度。但是,由于钙蛋白酶抑制蛋白(calpastatin)等因素对钙蛋白酶活性的抑制作用,钙蛋白酶的活性调节成为一系列高度可调的复杂过程。在综述钙蛋白酶结构与功能关系的基础上,进一步阐述了钙蛋白酶的活性调节如何影响肉品嫩度。     

黑曲霉 G-1125生淀粉糖化酶基因的克隆与序列分析

从黑曲霉Aspergillus niger G-1125克隆到了生淀粉糖化酶菌基因的DNA及cDNA序列。序列分析表明,该生淀粉糖化酶基因组DNA 序列编码区长2 172 bp,cDNA编码区长1 923 bp,该基因含有4个内含子,共编码640个氨基酸,前18个氨基酸为信号肽序列,该氨基酸序列中共含有2个潜在的糖基化位点,软件预测出该酶的分子质量约为68 ku,等电点为pH值4.07。     

畜禽宰后肌肉嫩化相关酶研究进展

目前普遍认为参与宰后肌肉嫩化的相关蛋白酶主要有溶酶体组织蛋白酶、蛋白酶体、钙激活蛋白酶、钙激活酶抑制蛋白和半胱天冬酶5 种,然而对于成熟过程中肉的嫩化程度及机理颇具有争议。大量的研究表明,上述5 种嫩化酶在参与肉嫩化过程中其自身生理生化特性在宰后也发生变化。本文综述5 种参与宰后肉嫩化酶的分子质量、存在部位、作用底物、作用位点、激活条件、最适pH值以及对其活性具有一定影响的抑制剂,并就其影响宰后肉嫩度的作用机理及其自身状态的变化进行分析与阐述,旨在为后续研究嫩化酶在宰后改善肉嫩度方面的应用条件提供参考。     

钙蛋白酶系统与肌肉嫩度的关系

钙蛋白酶系统主要由钙蛋白酶(μ-calpain,m-calpain)及钙蛋白酶抑制蛋白(calpastatin)组成,calpain是存在于细胞质中的依赖于Ca2+的中性蛋白酶,calpastatin是钙蛋白酶的内源抑制蛋白。本文综述了钙蛋白酶系统各种酶的结构、作用、活性调节机能及其与肉质嫩度的关系。     

内源蛋白酶在肉嫩化中的作用(综述)

本文阐述了与肉成熟有关的两类蛋白酶,即钙激活蛋白酶和溶酶体组织蛋白酶,并分别就两类酶的分类、影响酶活性的因素、在肉嫩化中的作用、分离提纯及活性测定方法等作了介绍.     

内源蛋白酶与肉的嫩化

肉在低温成熟时,钙激活酶对嫩化起关键作用,此时组织蛋白酶存在于溶酶体内。高温成熟使肉ｐＨ位下降迅速,将促使溶酶体裂解、组织蛋白酶释放,降解肌纤维蛋白,从而加速嫩化,此时组织蛋白酶也是导致肌肉嫩化的主要因素,Ｃａ￣（２＋）注射及加速溶酶体裂解的处理将加快嫩化进程。目前,由于对溶酶体裂解检测方法的缺乏,对其稳定性与嫩化关系的研究还未展开。     

黑曲霉木聚糖酶（xynZF-2）基因克隆、表达及酶学性质分析

利用RT-PCR技术从黑曲霉（Aspergillusniger）XZ-3S中成功克隆出木聚糖酶基因（xynZF-2）的cDNA序列（Genbank登录号为：JQ700382）。该基因核苷酸序列阅读框全长678bp,编码225个氨基酸,其中成熟肽为107个氨基酸。以重组质粒pUCm-T—xynZF-2为模板,扩增得到编码xynZF－2成熟肽的cDNA片段（624bp）。将其与表达载体pET－28a连接,构建重组表达载体pET－28a—xynZF－2,并转化大肠杆菌（Escherichiacoh）BL21（DE3）,获得重组工程菌BL21／xynZF-2。经IPTG诱导表达,木聚糖酶的的比酶活最高可达42．33U／mg。重组表达的木聚糖酶最适反应温度为40℃,最适反应pH为5．0,在碱性条件下具有良好的稳定性。Fe^3＋对酶的激活作用最为明显。     

西兰花(Brassica oleracea var.italica)硫代葡萄糖苷酶基因 cDNA分子克隆及其特征分析

目的:研究西兰花硫代葡萄糖苷酶(简称硫苷酶)cDNA的结构特征,为硫苷酶异源表达打下基础.方法:以西兰花幼苗为试材.根据同源序列法PCR扩增硫苷酶特异cDNA片段,再用3＇RACE和5＇RACE分别扩增cDNA 3＇端序列和5＇端序列,经序列拼接获得西兰花硫苷酶全长cDNA序列.利用在线蛋白质分析系统对其编码的氨基酸序列进行结构特征分析.在NCBI CDD数据库中查找保守结构域,在SWISS-MODEL上进行在线蛋白同源模建,用MEGA3.1构建NJ系统树.结果:该基因cDNA全长1830bp,含有一个1641bp的ORF及31bp的5＇端非翻译区和158bp的3＇端非翻译区,命名为BoMyr2(GenBank登录号为EU004075);编码氨基酸序列含有20个氨基酸长度的信号肽及9个天冬酰胺N末端糖基化位点,编码的蛋白分子质量(Mw)约为62.2 kD,等电点(pI)为8.71;BoMyT2推测氨基酸序列舍有糖基水解酶家族1特有的糖基水解酶结构域,其编码蛋白与PDB库中白芥硫苷酶有类似的三维结构,具有明显的β/α(TIM)桶结构;BoMyr2应归为硫苷酶基因家族的MB亚族.     

钙激活酶激活蛋白初探

<正> 近年来,许多科学家对钙激活酶的研究产生了浓厚的兴趣,主要是因为细胞内蛋白质的降解,被认为是与细胞生长和萎缩密切相关。细胞内大部分蛋白质的降解是由非溶菌酶系统所引起的,calpain就是此系统中的一个重要成员。     

Changes in calpain and calpastatin activities of osmotically dehydrated bovine muscle during storage after treatment with calcium

Calpain and calpastatin activities were investigated in calcium-treated beef after osmotic dehydration. Dehydrated beef was soaked in 150 mM calcium chloride solution for 3 h, and then stored for 48 h at 3-4 °C. The untreated sample (control) was soaked in deionized water for 3 h instead of calcium chloride solution, after osmotic dehydration. The increase and decrease in the relative activity of crude calpain were observed in the untreated and the calcium-treated meat, respectively, during the storage. When the crude calpains were subjected to DEAE-Sephacel column chromatography, it was found that μ-calpain activity decreased rapidly during the storage in the untreated meat, whereas there was almost no change in the activity of m-calpain during the storage. The decrease of calpastatin activity was moderate compared with the decrease of μ-calpain activity. In the calcium chloride-treated meat, however, no μ-calpain nor calpastatin activities was detectable after 48 h at cold-room temperature, and m-calpain activity after 48 h had decreased to 6.1% of its activity immediately after thawing. It was concluded that 150 mM calcium chloride treatment after osmotic dehydration was sufficient to introduce calcium ions into the meat. In the presence of sufficient calcium, autolysis of calpains and proteolytic degradation of calpastatin, which eventually related to the rate of decrease in calpain and calpastatin activities, clearly seem to be related to a decrease in meat toughness.     

Contribution of postmortem muscle biochemistry to the delivery of consistent meat quality with particular focus on the calpain system

Tenderness has been repeatedly reported as the most important quality aspect of meat. However, a number of studies have shown that a significant portion of retail meat can be considered tough. As a consequence, a significant consumer segment is willing to pay a premium for guaranteed tender meat. However, apart from measuring the shear force, there is no reliable method to predict tenderness. Most of the branded meat programs therefore attempt to ensure eating quality by controlling some of the factors that affect tenderness. Meat tenderness is determined by the amount and solubility of connective tissue, sarcomere shortening during rigor development, and postmortem proteolysis of myofibrillar and myofibrillar-associated proteins. Given the effect of postmortem proteolysis on the muscle ultrastructure, titin and desmin are likely key substrates that determine meat tenderness. A large number of studies have shown that the calpain proteolytic system plays a central role in postmortem proteolysis and tenderization. In skeletal muscle, the calpain system consists of at least three proteases, μ-calpain, m-calpain and calpain 3, and an inhibitor of μ- and m-calpain, calpastatin. When activated by calcium, the calpains not only degrade subtrates, but also autolyze, leading to loss of activity. m-Calpain does not autolyze in postmortem muscle and is therefore not involved in postmortem tenderization. Results from a number of studies, including a study on calpain 3 knockout mice, have shown that calpain 3 is also not involved in postmortem proteolysis. However, a large number of studies, including a study on μ-calpain knockout mice, have shown that μ-calpain is largely, if not solely, responsible for postmortem tenderization. Research efforts in this area should, therefore, focus on elucidation of regulation of μ-calpain activity in postmortem muscle. Discovering the mechanisms of μ-calpain activity regulation and methods to promote μ-calpain activity should have a dramatic effect on the ability of researchers to develop reliable methods to predict meat tenderness and on the meat industry to produce a consistently tender product.     

Calpain3的初步提取和对其自动降解性的研究

骨骼肌特异性的Calpain3(又称Ncl-1、p94、CAPN3)是除μ-Calpain和m-Calpain以外,另一个可能与肉类嫩化有关的Calpains成员。由于Calpain3具有很强的自动降解性,使得对其的提取纯化和依钙性的研究很难进行。本实验从牛肉背最长肌中提取到了具有一定活性的Calpain3粗酶液,通过蛋白质免疫印迹法,对Calpain3的随时间的降解变化进行了研究,并对不同Ca2+浓度下Calpain3自动降解的变化进行了比较系统的探讨,研究发现Calpian3的自动降解分为四个连续性的过程:首先降解为89、68和58kDa的中间片断,然后进一步降解为55kDa的片断并在一定时间内保持不变,其降解的速度与Ca2+浓度和反应时间成正比。     

Calpain and cathepsin activities in post mortem fish and meat muscles

Post mortem tenderization is one of the most unfavourable quality changes in fish muscle and this contrasts with muscle of mammalian meats. The tenderization can be partly attributed to the acid lysosomal cathepsins and cytosolic neutral calcium-activated calpains. In this study, these proteases from fish and bovine muscles were quantified and compared. The cathepsin B and L activities were in more important amounts in sea bass white muscle than in bovine muscle. On the other hand, cathepsin D activity was 1.4 times higher in meat that in fish muscle, while cathepsin H was negligible in both muscles. Calpain activities were similar in both types of muscle. Moreover, calpastatin (calpain endogenous inhibitor) level is 3.9 times higher in sea bass white muscle. These differential activities are considered in relation to their probable involvement in post mortem degradation of muscle.     

不同年龄牦牛宰后钙激活酶活性及嫩度指标变化

本研究对不同年龄牦牛宰后成熟过程中钙激活酶活性和嫩度指标（肌纤维直径、肌原纤维小片化指数（myofibrillar fragmentation index,MFI）、肌原纤维超微结构）进行测定。结果表明：成熟过程中,不同年龄牦牛肉肌纤维直径、MFI都发生显著变化（P＜0.05）,肌原纤维在成熟过程中Z线断裂,框架结构被完全破坏;钙激活酶活性在宰后的前2 d均显著下降（P＜0.05）,且成熟过程中不同年龄公牦牛的钙激活酶活性均高于母牦牛;成熟时间与钙激活酶活性、嫩度指标均成极显著相关性（P＜0.01）;牦牛宰后经过7 d的成熟,可以降低牦牛因屠宰年龄不同而产生的差异,同时也可以改善肉的嫩度;与公牦牛相比母牦牛宰后成熟时间可以适当的延长。     

白羽肉鸡胸腿肉的氧化对食用品质和钙蛋白酶活性的影响

为了研究氧化对禽类肌肉的影响程度,为鸡肉品质调控的研究提供参考,本实验以白羽肉鸡为研究对象,选取鸡的胸、腿肉,测定氧化对肌肉持水性能、营养素含量以及对胸肉钙蛋白酶活力的影响。结果显示：氧化可造成肉鸡胸肉和腿肉持水力下降,蒸煮损失率增加,产品得率降低;且过氧化氢（H2O2）溶液浓度对蛋白氧化程度和脂肪氧化程度有影响,随着H2O2溶液浓度的增大,肌肉蛋白质和脂质等营养素水平呈显著下降趋势。而在氧化对钙蛋白酶的活性研究中发现钙蛋白酶活力随H2O2浓度增大而降低,从而推测钙蛋白酶活力水平变化与白羽肉鸡肉质改变的关系密切。