Characterization and functional properties of digestive proteases in two sparids; gilthead seabream (Sparus aurata) and common dentex (Dentex dentex)

Digestive proteases present in two sparids, seabream (Sparus aurata) and common dentex (Dentex dentex), have been characterized using both biochemical and electrophoretic techniques. Although optimum pH and temperature for maximum activity of both acid and alkaline proteases were similar in the two species, important differences in total activity, as well as in thermal and pH stability were found. Specific inhibitors and SDS-PAGE zymograms were used to clarify such differences. Evidences support the existence of a more active and complex protease set in dentex. Results are discussed from the perspective of their application to the formulation of feeds for each species.     

Characterization of Gelatinolytic Activity in Seminal Plasma of Some Teleost Fish

Gelatin-containing SDS-PAGE combined with the incubation of gels in buffers containing protease inhibitors was performed for the visualization and characterization of proteolytic activity in teleost fish seminal plasma. To demonstrate the class of detected enzymes we used serine protease inhibitor – benzamidine or EDTA which inhibits metalloproteases activity. Additionally the effects of calcium ions on protease activity were investigated. Multiple gelatinolytic activities in seminal plasma of 10 teleost fish species from three orders (Cypriniformes, Salmoniformes, Perciformes) were found. Most proteases were either stimulated by Ca²⁺ and inhibited by EDTA or inhibited by benzamidine. This suggests that metalloproteases and serine proteases are major gelatinolytic proteases of fish seminal plasma. In cyprinid species we found a common profile of two gelatinolytic activities; the first band (60–66 kDa) belonged to metalloproteases, and the second one (76–81 kDa) belonged to serine proteases. Other bands were also visible and they represented mostly serine protease activity. Species from the Salmoniformes order showed a similarity in metalloproteases with molecular weights of about 64 and 75 kDa. Salmonid species also had similar serine proteases with molecular weights of about 102 and 165 kDa. In European grayling seminal plasma we found metalloproteases with molecular weight of 51, 57, 64, 70 kDa and two serine proteases activities of 35 and 125 kDa. Percid species had metalloproteases activities of 53 and 63 kDa and serine protease activity of 100 kDa. Protease of other, presently unknown classes were also found in seminal plasma of asp, chub, European grayling and pikeperch. The physiological role of seminal plasma proteases is still unknown.     

芝麻种子中内源性蛋白酶的热稳定性及其运用

近年来,芝麻的深加工偏重于制油,而忽略了对芝麻蛋白的高值利用。为了实现芝麻蛋白与油脂的高值综合利用,该研究将湿法脱皮芝麻进行干燥,经压榨制取芝麻油和压榨粕后,再利用内源性蛋白酶对芝麻压榨粕中的蛋白质进行水解,制取蛋白水解物。在此过程中,考察了干燥温度对湿法脱皮芝麻压榨出油率和内源性蛋白酶活性的影响。结果显示,在80～180 ℃干燥1 h时,脱皮芝麻出油率上升至88.55%～90.98%（带皮芝麻为81.76%）;对于内源性蛋白酶而言,在130 ℃时,仍可保留约57%的活性,表明芝麻种子中内源性蛋白酶具有良好的热稳定性。同时,通过将低变性芝麻粕（干燥条件为50 ℃、4 h）制浆,在pH 值为4.5、50 ℃下孵育6 h,经3 000 g离心5 min后,分离得到透明清液（蛋白水解物）,其清液中蛋白质的分布率为66%,且主要由38%小肽（< 1500 Da）和37%游离氨基酸组成,除此之外,还存在多种生物活性物质。该研究为芝麻内源性蛋白酶的实际应用以及芝麻蛋白新产品的开发提供了一种新思路。     

文蛤肉酶法水解条件的优化及最适蛋白酶的选择

以文蛤肉为原料,探讨了胰蛋白酶的加酶量、水解时间、水解温度、pH值及液固比对文蛤肉水解液总氨基氮含量和水解得率的影响规律,确定文蛤肉酶解的最优条件为酶解温度50℃,加酶量4000U/g原料,pH值为（8．00±0．05）,水解时间5h,液固比3：1。再以文蛤肉水解液的水解度、水解得率及风味评分值为指标对该酶解优化条件下获得的产品与精制中性蛋白酶水解优化条件下获得的产品进行比较。结果表明,胰蛋白酶为酶法水解文蛤肉最适宜的蛋白酶,其酶解所得文蛤肉水解液的水解度、水解得率及风味评分值分别为42．44％、82．86％及205．92。     

大豆干酪的研制──豆乳凝乳条件的研究

通过几种蛋白酶和乳酸菌发酵对豆乳凝乳特性影响的研究，发现所选择的几种蛋白酶，均不适于大豆干酪的凝乳，而乳酸菌发酵则有利于凝乳粒得率的提高。进一步研究表明，乳酸菌发酵豆乳的最佳凝乳条件为：脂肪和蛋白质含量分别为３．０％和２．１％的豆乳，分别添加１．０％脱脂奶粉和乳清粉，接种７．０％的乳酸菌发酵剂，在４２℃下发酵９０ｍｉｎ。     

生姜蛋白酶和生姜汁对猪肉嫩化效果的比较

本文研究了生姜蛋白酶及生姜汁对猪肉的嫩化效果，对其用量、pH值、预处理温度进行了测试对比。结果表明：生姜蛋白酶及生姜汁对猪肉的嫩化效果十分显著(P≤O．01)，生姜蛋白酶最佳用量为O.01％，姜汁最佳用量5％，最佳pH都为7.O，最佳预处理温度生姜蛋白酶为300℃，生姜汁为40℃；并且生姜蛋白酶及生姜汁对猪肉的嫩化效果极为相似，可以证明，生姜汁对肉类的嫩化作用就是生姜汁中的生姜蛋白酶作用的结果。     

蛋白质的折叠调控与包涵体的形成

新合成的多肽链必须先经折叠和装配后形成特定的三维结构才有活性.分子伴侣和蛋白酶可有效地调控多肽链的正确折叠.然而,在多肽链的折叠过程中,往往也会产生一些折叠异常的蛋白,形成集聚体即包涵体.本文主要对蛋白质的折叠机制、分子伴侣和蛋白酶在折叠中的作用,以及集聚和包涵体的特性、形成机理等做一综述.     

毛霉产蛋白酶的特性及其应用研究

为探寻适合于制备大豆多肽的蛋白酶 ,对毛霉产蛋白酶的特性、毛霉蛋白酶的制备方法及其对大豆分离蛋白的降解进行了研究 ,并测定了酶解产物的相对分子质量 .结果表明 ,毛霉蛋白酶是诱导性很强的酶类 ,毛霉的产酶条件除与温度、培养时间、相对湿度等有关外 ,还与培养方式密切相关 ,以麦麸培养毛霉 ,当长有大量孢子时翻曲 1次 ,可大幅度提高酶活力 .毛霉蛋白酶在40℃ ,p H 7.3的条件下催化大豆分离蛋白水解 5 h,所产生的酶解产物的相对分子质量分布范围在 1～ 3 k D之间 ,酶解产物微咸 ,后味略鲜 ,无苦涩味     

Substrate-SDS-PAGE determination of protease activity through larval development in sea bream

Identification of alkaline proteases produced during larval stages of gilthead sea bream, Sparus aurata was achieved using SDS-PAGE and specific inhibitors. Such techniques were also applied to determine proteases existing in rotifers, Brachionus plicatilis, and Artemia nauplii, which are used as live food for these larvae, as well as proteases of adult fish. The results show a great prominence of trypsin-like proteases during the 4 weeks after hatching, but the number of enzyme species was reduced in adult fish. Alkaline proteases present in the rotifers and Artemia showed clear differences when compared with those of the larvae and were not detected in extracts obtained from fed larvae. The results obtained provide information about the role of exogenous enzymes in larval feeding of sea bream.     

Characterization of digestive enzymes protease and alpha‐amylase activities in the thick‐lipped grey mullet (Chelon labrosus,Risso 1827)

Activities of pepsin, trypsin, chymotrypsin, alpha‐amylase and lipase, as well as their optima and stability to pH and temperature, were determined in digestive extracts of thick‐lipped grey mullet, Chelon labrosus, of three different sizes: Group 1 (45.2 ± 3.0 g), Group 2 (180.9 ± 4.2 g), and Group 3 (328.5 ± 43.3 g). SDS‐PAGE zymograms were also used to assess the role of serine proteases in the digestive tract of C. labrosus. On the other hand, possible changes in the digestive enzyme profile of C. labrosus during development were observed, with a comparatively lower pepsin activity, higher activities of alkaline proteases and alpha‐amylase and no lipase activity recorded in pre‐adult specimens. It is suggested that these variations are linked to the changes in diet composition with age, moving from a partly carnivorous to a more herbivorous feeding habit.