应用于肉品嫩化的组织蛋白酶的研究进展

大量研究表明,成熟嫩化过程是肌肉内源蛋白水解酶的作用所致。组织蛋白酶是其中一个重要的内源蛋白,可以水解肌原纤维。本文简要介绍了溶酶体中的组织蛋白酶,以及其稳定性和在骨骼肌中的表达特性。并且就组织蛋白酶的分类、影响其活性的因素、在肉嫩化中的作用、分离提纯、活性测定方法及应用前景等作了介绍。     

蓝圆鲹肌肉组织蛋白酶B的纯化与性质分析

在鱼糜制品生产过程中,凝胶劣化现象是引起鱼糜品质下降的重要原因。研究表明,溶酶体中的内源性组织蛋白酶B会促进肌原纤维蛋白的降解,进而导致鱼糜凝胶劣化。迄今为止,多种鱼体肌肉中的组织蛋白酶B已有研究,但海水经济低值鱼蓝圆鲹中该酶的情况却尚未报道。本研究采用硫酸铵沉淀和柱层析相结合的方法,从蓝圆鲹肌肉中分离纯化得到分子量约为27ku的组织蛋白酶B。酶学性质结果显示,该酶最适温度和最适pH分别为55℃和5.5,半胱氨酸蛋白酶抑制剂E-64能有效抑制其活性。对肌原纤维蛋白的降解实验表明,在最适条件下,蓝圆鲹组织蛋白酶B对肌原纤维蛋白有一定的降解作用。因此,组织蛋白酶B参与肌原纤维蛋白的降解,更可能参与在低pH条件下鱼糜凝胶劣化。     

腌干鲢鱼组织蛋白酶B、L活力变化的响应面法预测研究

以鲢鱼为原料,进行腌制和热泵干燥加工,研究内源组织蛋白酶B、L活性在加工过程中的变化。选取加工过程对鲢鱼(Hypophthalmichthys molitrix)组织蛋白酶B、L活力影响较大的3个工艺参数(温度、pH值、盐质量分数)作为试验因素,并根据酶学特性和干燥过程中各理化指标的测定结果确定其试验水平。采用响应面法考察此3因素对组织蛋白酶B、L活性的影响,并根据回归方程对腊干鲢鱼加工过程中的组织蛋白酶活力进行预测。结果表明：回归方程可用于组织蛋白酶B、L实际活力预测;两种酶的潜在活力在加工过程中均呈下降趋势,加工结束时组织蛋白酶B和L的潜在酶活力分别为初始的43.18%和26.08%;通过回归方程对组织蛋白酶B、L在腌制前、腌制后、干燥2d、干燥5d、干燥7d实际活力进行预测,组织蛋白酶B实际酶活力分别为潜在酶活力的36.7%、39.9%、46.5%、46.5%和53.5%,而组织蛋白酶L则为6.6%、5.6%、10.4%、10%和12.6%。     

巴西研究牛肉中的钙和钾含量对内洛尔牛嫩度的影响

<正>钙和钾是动物营养中的必需营养素,由于它们在细胞中的作用,其含量对肉的嫩度有一定影响。研究表明,由钙蛋白酶和钙蛋白酶抑制酶组成的蛋白水解系统需要钙离子的参与,是宰后骨骼肌嫩度的主要影响因子。而钾的含量与肌肉收缩有关,也会影响肉的嫩度。巴西的科学家通过对老化14 d的肉进行测试发现钾含量对WarnerBratzler剪切力具有正效应,这也就意味着钾的含量越高肉的嫩度就越低。另外,编码     

内源蛋白酶对肉类食品风味的影响

内源蛋白酶是引起蛋白质水解的重要因素。在肉类食品中,对肉类食品品质具有作用的内源蛋白酶主要有组织蛋白酶、钙中性蛋白酶、氨肽酶和二肽酶,在这些酶的作用下,可产生对风味具有明显作用的游离氨基酸和小肽,使其具有自身独特的风味,同时这些降解产物也可以参与香味形成反应如美拉德反应贡献于肉品香味。该文对内源蛋白酶在肉类食品风味形成中的作用进行了阐述,以期为肉类食品风味的形成提供理论参考。     

内源性蛋白酶在宰后猪肉成熟过程中的作用

本文分别阐述了两类内源性蛋白酶,即钙蛋白酶和溶酶体组织蛋白酶的结构、性质、影响酶活性的因素及其在猪肉成熟过程中的作用,认为在猪肉宰后成熟过程中,钙蛋白酶发挥的作用更显著。     

鱼肉内源性蛋白酶对其贮藏期品质影响的研究进展

鱼肉贮藏期间会发生一系列物理和化学性质的改变，进而引起鱼肉品质劣变。鱼肉品质劣变的影响因素众多，内源性蛋白酶在这一过程中发挥重要作用。本文重点围绕与贮藏期鱼肉品质下降相关的基质金属蛋白酶、钙蛋白酶和组织蛋白酶三种内源性蛋白酶展开介绍，梳理这三种酶对鱼肉蛋白作用的理论基础，并对其在鱼肉的风味、保水性、颜色及质地等品质变化中的作用机理加以阐述，以表征内源性蛋白酶在鱼肉品质劣变中的作用。同时论述了现今鱼肉内源蛋白酶抑制的几种技术方法，旨在为鱼肉及其制品的品质检测及控制技术开发提供一定参考。     

酵母双杂交法筛选组织蛋白酶B相互作用蛋白质

组织蛋白酶B是肿瘤侵袭和转移密切相关的一种蛋白酶,研究其作用网络对于开发基于组织蛋白酶B的抗肿瘤药物非常重要。采用酵母双杂交技术构建相关文库,以人组织蛋白酶B作为诱饵蛋白质,寻找其在肿瘤细胞中的直接相互作用蛋白质。在84个初始阳性克隆中,共有15个能通过3个报告基因的检测。BLAST分析发现,在GenBank数据库中可以匹配7个基因,编码7种已知的蛋白质:BCL2-associated athanogene 6 (BAG6), filaminA,alpha(FLNA),ras homolog family member G (RHOG),metallothionein 2A (MT2A), ZXD family zinc finger C(ZXDC),DEP domain containing 7(DEPDC7),A kinase(PRKA) interacting protein 1(AKIP1)。     

内源性蛋白酶对鱼糜凝胶的影响及其抑制方法研究进展

鱼肉中的内源性蛋白酶是造成鱼糜凝胶劣化,影响鱼糜质构特性的主要内在原因。添加内源性蛋白酶抑制剂是控制鱼糜凝胶劣化常用的方法。本文综述了鱼肉中三种主要内源性蛋白酶(钙蛋白酶、组织蛋白酶和肌原纤维结合型丝氨酸蛋白酶)的性质及其对鱼糜凝胶特性的影响,并总结抑制内源性蛋白酶引起的鱼糜凝胶劣化和其他改善鱼糜凝胶特性的方法,旨在为控制鱼糜凝胶劣化及改善鱼糜凝胶特性提供理论依据。     

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.     

骨骼肌中组织蛋白酶(英文)

<正>Introduction Although cathepsin and its main functions have beenfound in animal muscle tissue,since man foundthat calcium-activated enzyme and calcium alone candirectly induce the degradation of     

The similarities between the activities of the calcium activated sarcoplasmic factor and the kinase activating factor from rabbit skeletal muscle

The similarity, that has been suggested by other investigators, between the kinase activating factor (KAF) and the calcium activated sarcoplasmic factor (CASF) of rabbit skeletal muscle is extended through a comparison of enzyme activities. Proteolytic activity on casein, the ability to activate skeletal muscle glycogen phosphorylase b kinase, inhibition by bovine heart KAF inhibitor and effects on myofibrillar integrity were examined using these two skeletal muscle proteases and a typical bacterial protease isolated from Pseudomonas perolens (ATCC 10757). The study demonstrated that the skeletal muscle proteases have more in common with each other than either protease has with the bacterial protease. The ability to influence the integrity of the myofibril was demonstrated by phase-contrast microscopy and SDS polyacrylamide gel electrophoresis. The KAF and CASF demonstrated similar effects that again differed from the bacterial protease effects.     

PROPERTIES OF AN ALKALINE PROTEASE FROM THE SKELETAL MUSCLE OF ATLANTIC CROAKER1

An alkaline protease was partially purified from the skeletal muscle of Atlantic croaker. The protease is a cytoplasmic enzyme and heat stable. The enzyme preparation was shown to degrade fish actomyosin in vitro between 50–60°C. The enzyme is a sulfhydryl protease and does not require Ca⁺⁺ ions for its activity. Preparations of the enzyme do not hydrolyze TAME, BTEE or denatured hemoglobin. Column chromatographic analyses suggest an apparent molecular weight of 80,000 ± 4,000 and the isoelectric point is 6.0 ± 0.2.     

ROLE OF MUSCLE PROTEINASES IN MAINTENANCE OF MUSCLE INTEGRITY AND MASS

Current evidence indicates that, of the thirteen known lysosomal peptide hydrolases, only seven, cathepsins A, B, C, D, H, L, and lysosomal carboxypeptidase B are located inside skeletal muscle cells. Only one of the reported neutral and alkaline proteases is located inside skeletal muscle cells', this neutral protease is the Ca²⁺-dependent proteinase, CAF. With the possible exception of cathepsin N, which can degrade collagen, it seems probable that any protease that contributes to postmortem tenderization needs to be located inside muscle cells. Because very little degradation of myosin or actin occurs in postmortem muscle, most of the small amount of proteolytic degradation of the myofibrillar proteins that occurs during postmortem storage must be due to CAF, which is unique in being unable to degrade myosin and actin. It is not certain that postmortem proteolysis by CAF causes increased tenderness; some recently discovered actin-fragmenting proteins could be involved.     

HYDROLYSIS OF Z-DISC ACTIN BY Ca2+-ACTTVATED PROTEASE1

A 42,000 dalton protein has been identified in chicken breast muscle which differs from actin in that it is soluble at low ionic strength and insoluble in 1 MKI solutions. This protein is readily hydrolysed by Ca⁺⁺-activated neutral protease from chicken breast muscle, and is not present in Ca⁺⁺-activated neutral protease treated myofibrils. This protein may be involved in the integrity of the Z—disc in skeletal muscle.     

Investigation into Potential Sources of Heat-Stable Alkaline Protease in Mechanically Separated Atlantic Croaker (Micropogon undulatus)

Alkaline protease in croaker exists not only in the sarcoplasmic fraction of skeletal muscle but also in the skin and internal organs. As characterized by optimal pH, thermal stability, and column chromatography, the alkaline proteases obtained from different fish tissues such as muscle, skin, kidney, and alimentary canal exhibit similar enzymatic properties. An experiment using chloramphenicol to inhibit bacterial growth suggests that the heat-stable alkaline protease present in the minced (mechanically separated) croaker is likely not of bacterial origin. The high specific activity of alkaline protease from kidney, liver, and visceral tissue in comparison to that of skin and muscle suggests that inclusion of residual tissue in even small amounts from the former sources could contribute greatly to the total activity measured in fish mince.     

Contribution of Retained Organ Tissues to the Alkaline Protease Content of Mechanically Separated Atlantic Croaker (Micropogon undulatus)

The greater texture breakdown observed in gel-type products prepared from mechanically separated tissue as opposed to manually separated tissue can to some extent be attributed to the significantly higher protease activity measured in mechanically separated tissue. Failure to thoroughly wash eviscerated fish prior to mincing appears to result in the retention of tissue from internal organs as evidenced by a concommitant increase in protease activity in the minced tissue. Alkaline protease activity in croaker kidney and liver is several thousand fold higher than in skeletal muscle. Addition of kidney and liver tissue to deboned tissue from thoroughly washed fish results in increased protease activity and degradation of the fish tissue upon comminuting and cooking at 60°C. Isoelectric focusing provided evidence that the increased alkaline protease activity observed in mechanically separated fish tissue resulted to some degree from retention of kidney tissue in the fish mince. Isolation of Ca⁺⁺-dependent protease fractions from liver also implicated contamination from this source as an additional contribution to the total protease activity of minced fish.     

CONTINUATION OF SYMPOSIUM: FUNDAMENTAL PROPERTIES OF MUSCLE PROTEINS IMPORTANT IN MEAT SCIENCE: ROLE OF NEW CYTOSKELETAL ELEMENTS IN MAINTENANCE OF MUSCLE INTEGRITY

Evidence suggests that desmin, titin and nebulin, three recently discovered proteins, have cytoskeletal roles in muscle cells. The three proteins have been purified from mature skeletal muscle and partially characterized. Properties of the three proteins are described, with special regard to their probable roles and importance in maintaining muscle cell integrity. Results will be shown that demonstrate ability of purified desmin to self-assemble into synthetic 10-nm (intermediate) diameter filaments. Taken together with immunoelectron microscope results (Richardson et al. 1981), it is evident that desmin is the major component of 10-nm filaments of mature skeletal muscle cells and that the desmin filaments link adjacent myofibrils at their Z-line levels and seemingly tie the myofibrils into the cell cyto-skeleton. Desmin is degraded at about the same rate as is the highly susceptible troponin-T in bovine semitendinosus muscle postmortem. Alterations in desmin and other recently discovered cytoskeletal proteins would be expected to disrupt muscle cell integrity and to have marked effects on properties of muscle important to its use as food.     

Proteolytic activity of isolated lamb calpains on myofibrils under the conditions of pH, Ca2+ concentration and temperature existing in postmortem muscle.

The two calcium-activated neutral proteinases (calpains I and II) and their specific inhibitor were isolated by ion exchange chromatography in DEAE-Sephacel from lamb skeletal muscle (longissimus dorsi). Their proteolytic activities were then determined using myofibrils as substrate. The Ca2+ requirements were different for each form of the enzyme: calpain I needed only 50 mumol Ca2+ for half-maximal activity, while the other isoenzyme, calpain II, needed 1,000 mumol Ca2+ for reaching 50% of its maximum activity. Both calpains showed a relevant activity in the pH range 5.5-6.5 (over 40% of maximum activity found at pH 7.5). With regard to the effect of temperature, both isoenzymes retained about 25% of their activity at 25 degrees C with a temperature reduction down to 4 degrees C. It is concluded that calpain I is an active protease under conditions similar to that prevalent in lamb meat during postmortem storage.     

Comparison of the relative expression of caspase isoforms in different porcine skeletal muscles

The present study investigated the relationship between muscle type and components of the caspase protease system in porcine trapezius (TZ), psoas (PS), longissimus dorsi (LD) and semitendinosus (ST) muscles. Muscles were classified according to slow and fast myosin heavy chain (MHC) content determined by western blotting. MHC slow, but not MHC fast protein expression was significantly different between muscles (p<0.001). Protein levels of caspases 3, 8 and 12 and the caspase inhibitor apoptosis repressor with caspase recruitment domain (ARC) were determined. In addition the level of caspase 3 mRNA and activity levels of caspase 3/7 were determined. There was a significant difference in protein levels and activity between muscles (p<0.01), although no difference was observed in mRNA abundance. The data show that multiple components of the caspase system are expressed in porcine skeletal muscle and that their levels are variable, but there is not a distinct association of expression with a particular muscle.