鱿鱼肝脏自水解过程中内源蛋白酶的作用研究

对鱿鱼肝脏蛋白酶的酶学性质及其在鱿鱼肝脏自水解过程中的作用进行研究。结果表明：以偶氮酪蛋白为底物,TCA可溶性肽为评价指标,测得鱿鱼肝脏蛋白酶粗酶的最适反应温度为40℃,最适反应pH值为5.0;该粗酶在pH3.0～8.0较为稳定。在鱿鱼肝脏自水解过程中,内源性半胱氨酸蛋白酶发挥主要作用。此外,丝氨酸蛋白酶和金属蛋白酶也有一定作用。     

凡纳滨对虾组织蛋白酶L性质分析及其对肌肉蛋白的降解

本研究从凡纳滨对虾肝胰腺中纯化获得一种组织蛋白酶L,其分子质量约为31 k D,肽质量指纹图谱分析得到8个片段共112个氨基酸残基,与报道的凡纳滨对虾组织蛋白酶L序列完全一致。该酶的最适温度和最适pH值分别为35℃和5.5,且在0~40℃以及pH 5.5~6.5之间酶活力稳定。该酶仅对底物Z-Phe-Arg-MCA特异分解。半胱氨酸蛋白酶抑制剂E-64和Leupeptin对其有明显的抑制作用,而金属蛋白酶抑制剂乙二胺四乙酸和乙二醇二乙醚二胺四乙酸对其有少量的激活作用。扫描电子显微镜观察结果显示,随着低温贮藏时间的延长,对虾肌肉纤维的断裂程度不断增加。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析显示,组织蛋白酶L可使肌肉蛋白发生分解,推测其可能参与对虾低温贮藏过程中肌肉的降解。     

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。      

利用鱿鱼下脚料生产鱿鱼粉的工艺研究

利用鱿鱼皮和鱿鱼内脏两种鱿鱼下脚料生产鱿鱼粉。经实验确定了酶解最佳工艺为:鱿鱼皮酶解添加风味蛋白酶1‰、复合蛋白酶1‰,酶解时间为3.5 h;鱿鱼内脏酶解添加风味蛋白酶1‰、复合蛋白酶1‰,酶解时间为4.5 h。美拉德反应最佳工艺为:鱿鱼皮酶解液添加量20%、木糖添加量0.6%、鱿鱼内脏酶解液添加量25%、美拉德反应温度110℃、反应时间40 min。喷雾干燥工艺中变性淀粉的最佳添加量为3%。按照上述工艺制作的鱿鱼粉香气浓郁,风味纯正,颜色自然浓郁,颗粒细腻,流动性强,水溶性好,可以广泛用于膨化食品、方便食品、香精香料中,对鱿鱼资源的再利用和提高附加值有重要意义。     

葵花籽粗油体中内源性蛋白酶系的性质探究以及鉴定

通过电泳探究葵花籽粗油体中内源性蛋白酶系的水解性质,以及用液相色谱-串联质谱（LC-MS/MS）鉴定内源性蛋白酶的种类。结果表明,油质蛋白在pH 4~8水解速度相对较快,22 kDa球蛋白在pH 3~5水解速度相对较快,35 kDa球蛋白在pH 4~8水解速度相对较快。葵花籽粗油体中内源性蛋白酶系在pH 4、50 ℃水解速度相对最快。通过抑制剂实验发现,在pH 4下,葵花籽粗油体中有金属蛋白酶类、天冬氨酸蛋白酶类、丝氨酸蛋白酶类的酶活表征,几乎无半胱氨酸蛋白酶酶活的存在。通过LC-MS/MS分析发现,葵花籽粗油体中存在金属蛋白酶类、天冬氨酸蛋白酶类和丝氨酸蛋白酶类和天冬氨酸蛋白酶类抑制剂、丝氨酸蛋白酶类抑制剂以及半胱氨酸蛋白酶类抑制剂。研究结果为利用内源性蛋白酶类进行水相加工提供了理论基础。     

金鲳鱼内脏酸性蛋白酶的分离纯化及酶学性质研究

以金鲳鱼内脏为原料,依次采用p H7.0的磷酸钠缓冲液浸提,0~55%硫酸铵沉淀,Sephadex G-100和Sephadex G-50凝胶过滤法得到纯化的酸性蛋白酶,并研究了其酶学性质。结果表明,纯化酶的分子量为18.5 ku,最适p H为4.0,最适温度为35℃,具有较好的酸稳定性和耐盐能力;以血红蛋白为底物时,该蛋白酶的米氏常数Km为10.13 g/L;胃蛋白酶抑制剂(pepstatin A)对该酶活性有抑制作用,而乙二胺四乙酸(EDTA)、反-环氧丁二酰基-L-亮氨酰胺基(4-胍基)丁烷(E-64)和苯甲基磺酰氟(PMSF)对此酶活性基本无影响,据此推断该蛋白酶是一种天冬氨酸蛋白酶;此酶对罗非鱼鱼皮明胶的酶解能力强于猪胃蛋白酶,与木瓜蛋白酶和碱性蛋白酶的能力相近。     

牡蛎中HIV-1蛋白酶抑制肽的制备及其抑制率研究

牡蛎HIV-1蛋白酶抑制肽的制备是以其对HIV-1蛋白酶的抑制率及IC50值为指标,以中性蛋白酶为工具酶对牡蛎进行酶解时间单因素和加酶量单因素实验,确定最佳酶解条件。结果表明:最佳酶解条件为温度37℃、时间5 h、酶加量20%、底物浓度37.5%。把酶解液经过凝胶柱和HPLC分离纯化。结果表明:粗提取物中含有很多肽,对HIV-1蛋白酶的抑制率最高的肽在浓度为1 000μg/m L时达到81.95%,IC50值为68.66μg/m L。     

鱿鱼肝脏蛋白中α-葡萄糖苷酶抑制肽的研究

通过酶解技术和凝胶过滤分离等技术对鱿鱼肝脏蛋白水解液中活性肽进行研究,为鱿鱼副产物的高效利用奠定了理论基础。实验得出:6种蛋白酶(酸性蛋白酶、木瓜蛋白酶、中性蛋白酶、碱性蛋白酶、胰蛋白酶、胃蛋白酶)中,胃蛋白酶为鱿鱼肝脏蛋白水解的最佳酶类,同时以水解度和α-葡萄糖苷酶(AG)抑制活性为指标,得出胃蛋白酶水解的最佳条件:底物浓度0.4%,酶与底物的质量比3.0%,体系p H3.0,温度37℃,反应时间12 h。经过上述条件处理后的水解液用Sephadex LH-20凝胶层析柱进行分离,得到6个组分,其中组分Ⅲ的AG抑制活性最高,其半抑制浓度(IC50)达到0.215 mg/m L。同时对组分Ⅲ的稳定性和抑制动力学进行研究,得出组分Ⅲ对酸碱、热及消化道酶系统具有较高的稳定性。对AG为典型的非竞争性抑制,其米氏常数为50 mmol/L。     

金鲳鱼内脏酸性蛋白酶的分离纯化及酶学性质研究

以金鲳鱼内脏为原料,依次采用p H7.0的磷酸钠缓冲液浸提,0⁵5%硫酸铵沉淀,Sephadex G-100和Sephadex G-50凝胶过滤法得到纯化的酸性蛋白酶,并研究了其酶学性质。结果表明,纯化酶的分子量为18.5 ku,最适p H为4.0,最适温度为35℃,具有较好的酸稳定性和耐盐能力;以血红蛋白为底物时,该蛋白酶的米氏常数Km为10.13 g/L;胃蛋白酶抑制剂（pepstatin A）对该酶活性有抑制作用,而乙二胺四乙酸（EDTA）、反-环氧丁二酰基-L-亮氨酰胺基（4-胍基）丁烷（E-64）和苯甲基磺酰氟（PMSF）对此酶活性基本无影响,据此推断该蛋白酶是一种天冬氨酸蛋白酶;此酶对罗非鱼鱼皮明胶的酶解能力强于猪胃蛋白酶,与木瓜蛋白酶和碱性蛋白酶的能力相近。      

米曲霉3．042种曲蛋白酶特性研究

蛋白酶活性中心对米曲霉育种、制曲等研究至关重要,本研究选择四种专一性蛋白酶抑制剂AEBSF、E-64、Pepstatin A和EDTA,研究其对制曲时间为72 h的种曲蛋白酶酶活的影响,并确定三类蛋白酶的活性中心.研究发现酸性蛋白酶的活性中心主要由丝氨酸和天冬氨酸构成,中性蛋白酶的活性中心主要是天冬氨酸,碱性蛋白酶的活性中心最为复杂,含有四大类酶的活性中心.     

Identification of a cysteine proteinase from Jumbo squid (Dosidicus gigas) hepatopancreas as cathepsin L

A cysteine proteinase from Jumbo squid (Dosidicus gigas) hepatopancreas was partially purified by a two step procedure involving ammonium sulfate precipitation and gel filtration chromatography and further by SDS–PAGE. The molecular weight of the proteinase was 24 kDa determined by SDS–PAGE and 23.7 kDa with mass spectrometry. The activity had an optimum pH of 4.5 and optimum temperature of 55 °C under the assay for cathepsin L specific synthetic substrate Z-PAAFC. The cathepsin B and H specific synthetic substrates Z-AAAFC and H-AMC did not show any hydrolysis with the partially purified enzyme. Peptide mapping of trypsin digests of the 24 kDa band from SDS–PAGE showed the squid cysteine proteinase was homologous to cathepsin L from different animal sources. The activity of the partially purified fraction with the cathepsin L specific substrate Z-PAAFC was inhibited 75–89% by enzyme inhibitors specific for cysteine proteinases but was also significantly inhibited by serine and aspartate proteinase inhibitors.     

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。     

Participation of cysteine protease cathepsin L in the gel disintegration of red bulleye (Priacanthus macracanthus) surimi gel paste

BACKGROUND: Endogenous proteases, among them cysteine‐type proteases, are reported to contribute to gel disintegration, resulting in kamaboko of poor quality. Severe gel disintegration occurs in red bulleye surimi gel paste. The objective of this study was to clarify the participation of cysteine protease cathepsin L in the gel disintegration of red bulleye surimi. The surimi was made into kamaboko with and without cathepsin L inhibitors. To confirm its hydrolysis action, crude cathepsin L was also extracted and added to the surimi to make kamaboko. RESULTS: The gel strength of kamaboko obtained by both one‐step (50 °C, 2 h) and two‐step (50 °C, 2 h + 80 °C, 20 min) heating was very low in the absence of inhibitors. Protease inhibitors E‐64 and leupeptin were found to enhance the gel strength considerably. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the hydrolysis of kamaboko was promoted by crude cathepsin L and inhibited by E‐64 and leupeptin. The gel strength of two‐step heated kamaboko was increased from 12 to 110 and 130 g cm^?2 by E‐64 and leupeptin respectively at a concentration of 0.2 g kg^?1 surimi. CONCLUSION: Endogenous cathepsin L of red bulleye surimi participates in gel disintegration during kamaboko processing. It does so by degrading the myosin heavy chain of actomyosin and consequently hindering the gelation of red bulleye surimi. Copyright © 2009 Society of Chemical Industry     

Cysteine protease activity in bovine milk

Proteolytic activity of native cysteine proteases was studied in bovine milk. Five fractions (fI–fV) with cysteine protease activity were separated from acid whey prepared from raw bovine milk by ion-exchange chromatography on Q-Sepharose. The hydrolytic action of the most active fractions (fIII and fV), after further purification using gel permeation chromatography on Superdex S75, was studied against individual caseins. The two fractions contained different cysteine protease activities capable of hydrolyzing both α_s1- and β-casein. Studies of the effects of different reagents on the activity of partially purified fIII showed that the activity in this fraction was unaffected by aprotinin, slightly inhibited by p-chloromercuribenzoate and o-phenanthroline and completely inhibited by -trans-epoxysuccinyl- -leucylamido (4-guanidino) butane, consistent with identification as a cysteine protease. Phenylmethylsulphonyl fluoride and pepstatin A reduced activity of fIII by 40% and 50%, respectively. The partially purified fIII retained 20% of its cysteine protease activity after heating at 55°C, 60°C, 65°C and 72°C for 40 min, 20 min, 10 min and 30 s, respectively. Immunoblotting of fIII with antibodies to the bovine lysosomal cysteine protease, cathepsin B, clearly indicated the presence of immunoreactive cathepsin B in this fraction. This study presents strong evidence for the presence of a heterogeneous group of cysteine proteases in bovine milk, with one of these enzymes probably being cathepsin B.     

Characterization of proteolysis in muscle tissues of sea cucumber <Emphasis Type="Italic">Stichopus japonicus</Emphasis>

The proteolysis in muscle tissues of sea cucumber Stichopus japonicus (sjMTs) was characterized. The proteins from sjMTs were primarily myosin heavy chains (MHCs), paramyosin (Pm), and actin (Ac) having a molecular mass of approximately 200, 98, and 42 kDa, respectively. Based on SDS-PAGE analysis and quantification of trichloroacetic acid (TCA)-soluble peptides released, degradation of muscle proteins from sjMTs was favorable at pH 5 and 50°C. Proteolysis of MHCs was mostly inhibited by cysteine protease inhibitors, including trans-epoxysuccinyl-L-leucyl-amido (4-guanidino) butane (E-64) and antipain (AP). E-64 and AP completely inhibited the degradation of Pm and Ac, while iodoacetic acid showed a partially inhibitory effect. These results indicated that the proteolysis of sjMTs was mainly attributed to cysteine proteases. Avoidance of setting the tissues at 40–50°C and slightly acidic condition and inhibition of cysteine proteases are helpful for decreasing sea cucumber autolysis.     

Heat-activated proteolysis in lizardfish (Saurida tumbil) muscle

Autolysis of lizardfish mince and washed mince during heating at elevated temperatures was studied. Higher degradation of myosin heavy chain (MHC) was generally observed in mince, compared to washed mince. The highest autolysis was observed at 65 and 60 °C for mince and washed mince, respectively. Autolysis was extended as the incubation time increased by the result of the decrease in MHC band intensity on SDS-PAGE and the increase in TCA-soluble peptides. Autolysis of washed mince was markedly inhibited by E-64 and soybean trypsin inhibitor, suggesting that myofibril-associated proteinases were both cysteine and serine proteinases. Sarcoplasmic proteinase was characterized to be heat-activated alkaline proteinase, which had the optimal pH and temperature of 8.0 and 65 °C, respectively. The preteinase was inhibited by E-64 and activated by reducing agents, which was one of the characteristics of cysteine proteinase. Therefore, endogenous sarcoplasmic and myofibril-associated proteinases play an important role in degradation of myofibrillar proteins of lizardfish during heat-induced gelation, which results in gel weakening.     

CHARACTERIZATION OF THE PROTEASES INVOLVED IN HYDROLYZING PADDLEFISH (POLYODON SPATHULA) MYOSIN

An extract from paddlefish surimi possessed activities of B, L, and H‐like cathepsins. The optimal pH was around 5.0 for cathepsins B and L, and was between 6.0–6.5 for the H‐like cathepsin. The enzyme activities were not impaired by heating at 40Cfor 20 min. However, the protease extract lost about 20% of its cathepsin B, 50% B+L, and 90% H‐like cathepsin activities after heating at 50C for 20 min. The activity of H‐like cathepsin was not inhibited by E‐64, suggesting that it did not belong to ike known cysteme protease group. The protease extract was capable ofhydrolyzing myosin heavy chain, producing a major fragments) around 140 kDa. Degradation of myosin by the protease extract was substantially reduced by protease inhibitors including E‐64, a protease inhibitor mixture, and bovine plasma powder.     

Broad‐spectrum inhibition of proteolytic enzymes by allicin and application in mitigating textural deterioration of ice‐stored grass carp (Ctenopharyngodon idella) fillets

The inhibitory effect of allicin on proteolytic enzymes and textural deterioration of ice‐stored grass carp (Ctenopharyngodon idella) fillets was investigated. The results of in vitro study showed that allicin inhibited the activity of cathepsin B, L and D, calpain and collagenase in crude extract of grass carp muscle. Among endogenous enzymes, cathepsin B, L and collagenase were the most susceptible to allicin. Proteolysis of myofibrillar proteins by either crude enzyme or cathepsin B and L was almost prevented by allicin when employed at a concentration higher than 100 mm . After storage of 21 days, shear force of fillets treated with allicin at 10–100 mm was 39–51% higher than that of control. Myofibrillar proteins of fillets during storage were well protected against degradation when allicin concentration increased to 100 mm , as evidenced by SDS‐PAGE. Therefore, allicin could be a potential broad‐spectrum inhibitor to retard softening of fish fillets via mitigating myofibrillar proteolysis by endogenous enzymes especially cathepsin B and L during ice‐storage.