七种常见养殖鱼血清胰酶抑制活性的比较

α2巨球蛋白能结合并抑制蛋白酶的活性,但不抑制该蛋白酶对人工合成的小分子底物的酶活。利用这一特性,选择胰酶的一种有色小分子底物BAPNA,建立BAPNA比色检测法,对7种常见养殖鱼:草鱼、鲫、鲢、胡子鲇、鳙、加州鲈和黄鳝的血清胰酶抑制活性进行了比较,胰酶抑制活性由高到低依次为:草鱼>鲫>黄鳝>鳙>鲢>胡子鲇>加州鲈;α2巨球蛋白活性由高到低依次为:黄鳝>草鱼>胡子鲇>加州鲈>鲢>鳙>鲫;黄鳝和草鱼的α2巨球蛋白活性明显高于其他几种鱼的α2巨球蛋白活性。     

几种肉食性鱼类在太湖渔业中生态地位初析

调查表明，乌鳢、鳜鱼、沙塘鳢、黄颡鱼对太湖经济鱼类危害不大，且能抑制小杂鱼的过量繁殖，而红鳍鱼白大量掠食鲤鲫鱼苗与鱼卵，且对小杂鱼抑制作用不明显，因此提出了适当保护和增殖乌鳢、鳜鱼、沙塘鳢和黄颡鱼，抑制红鳍鱼白的主张。     

WSSV蛋白酶性质的研究

从感染白斑综合症病毒(Whitespotsyndromevirus,WSSV)的对虾中提取和纯化WSSV,对其蛋白酶活力进行分析。结果表明,WSSV蛋白酶具广泛的pH稳定性,当pH达7.5时,蛋白酶活性最大;pH高于10 0时,酶活力很低,其蛋白酶偏碱性。丝氨酸蛋白酶抑制剂苯甲基磺酰氟(PMSF)对酶活性有抑制作用。Ca2 、Mn2 、Fe2 和Cu2 可降低WSSV蛋白酶活性,但Mg2 有轻微的激活作用。胰蛋白酶抑制剂在浓度为12.5～25.0mg/L时,对WSSV蛋白酶活性无影响。Leupeptin使蛋白酶活性降低12.29%。Chymostatin在质量浓度为12.5～25.0mg/L时,对WSSV蛋白酶活性有强烈的抑制作用,表明WSSV蛋白酶类属胰凝乳蛋白酶。蛋白质修饰剂对WSSV蛋白酶活性影响的研究结果表明,组氨酸残基为WSSV蛋白酶活性基团,而巯基为非必需基团,说明WSSV蛋白酶为非巯基依赖型的蛋白酶。     

草鱼酶解技术研究

应用对比试验和正交试验方法对草鱼进行酶解技术研究。试验结果表明，胃蛋白酶酶解草鱼酶用量为（E／S）0．6％，温度55℃，pH2．0，酶解5h氨基态氮含量较高，为0．0190g／L；枯草杆菌蛋白酶酶解草鱼酶用量为（E／S）0．01％，温度45℃，pH6．5，酶解4h氨基态氮含量较高，为0．0417g/L；胰蛋白酶酶解草鱼酶用量为（E／S）0．64％，温度50℃，pH8．0，酶解5h氨基态氮含量较高，为0．0472g／L。通过对3种蛋白酶在最优条件下酶解草鱼肉的比较可知，枯草杆菌蛋白酶和胰蛋白酶酶解效果较好。为草鱼深加工提供了一定的科学依据。     

鱼类胰弹性蛋白酶的研究进展

本文综述了国内外鱼类胰弹性蛋白酶的分离纯化、特性研究和系统发育分析。阐明了鱼类胰弹性蛋白酶结构与功能的关系,有助于了解鱼类的消化生理特点,为水产饲料的研究提供理论依据。     

草鱼消化道碱性蛋白酶的分析鉴定

试验结果表明,草鱼肠道粗提取物在pH 10.3有最高酶活力。SDS-底物-PAGE电泳表明,草鱼肠道粗酶中有4种碱性蛋白酶,其中3种是丝氨酸蛋白酶(胰蛋白酶),1种是非丝氨酸蛋白酶,它们的相对分子质量分别为:26 400、30 750、43 000、约105 000。     

乌鳢消化道蛋白酶脂肪酶的研究

采用Folin-酚法测定了乌鳢消化道胃、肠及幽门盲囊中的蛋白酶、脂肪酶活性,蛋白酶最适pH。结果表明,蛋白酶活性由高到低为：胃〉肠〉幽门盲囊,最适pH依次为2．2、8．2、7．8。脂肪酶的测定通过检测新鲜牛乳中脂肪的降解作用产生的脂肪酸含量变化来进行,结果表明,脂肪酶活性由高到低为：肠〉胃〉幽门盲囊。     

杂交鳢及其亲本仔鱼耐饥饿能力的比较研究

利用人工繁殖孵化得到的杂交鳢、乌鳢、斑鳢仔鱼,在水温26±1℃下,进行三种仔鱼饥饿致死时间的试验。结果表明,三种仔鱼50%饥饿致死时间分别为14d、16-17d、10d;100%饥饿致死时间分别为19d、21d、15d。杂交鳢、乌鳢、斑鳢三种仔鱼对饥饿的耐受力强弱顺序依次为:乌鳢>杂交鳢>斑鳢。     

不同发育时期月鳢消化酶活性的研究

对月鳢(Channa asiatica)稚鱼、幼鱼和成鱼的主要消化酶活性及分布进行了研究。结果表明,随着鱼体的发育和生长,月鳢消化系统各器官组织的各种消化酶活性逐渐增强,尤其是蛋白酶和脂肪酶活性的增强更为明显。月鳢幼鱼蛋白酶活性以肠组织最大、肝胰脏次之、胃组织最小,且肠组织和肝胰脏中该酶活性显著高于胃组织;胰蛋白酶活性以肝胰脏最大,胃组织和肠组织均表现较小;淀粉酶和脂肪酶活性均以胃组织最大,且显著大于肠组织和肝胰脏。月鳢成鱼蛋白酶活性以中肠粘膜组织和后肠粘膜组织最高,前肠粘膜和肝胰脏次之,胃粘膜最低;胰蛋白酶活性以中肠粘膜组织和前肠粘膜最高,胃粘膜组织次之,肝胰脏小于胃粘膜,后肠粘膜组织的胰蛋白酶活性最小;淀粉酶活性以后肠粘膜组织和中肠粘膜组织最大,肝胰脏次之,胃粘膜组织和前肠粘膜组织最小;脂肪酶活性以胃粘膜组织最大,其它器官组织该酶活性均表现较小。     

鱼降压肽的酶法制备工艺及其理化性质研究

采用高压液相色谱测定降血压肽的血管紧张素转化酶抑制活性，对碱性蛋白酶水解草鱼蛋白制备鱼降压肽进行研究。结果表明，碱性蛋白酶水解草鱼蛋白制备鱼降压肽的较佳工艺条件为：pH9.0、温度50℃、酶与底物比48AU.kg-1、水解度34.52%。鱼降压肽成分分析结果显示，鱼降压肽中可溶性氮含量为81.26%，肽含量为72.81%，脂肪为0.12%，水分为3.54%，灰分为9.47%。鱼降压肽的血管紧张素转化酶抑制活性为70.35%；体积排阻色谱测定鱼降压肽的相对分子质量在124～10581之间，主要集中在124～1062之间；溶解度的测定结果表明，鱼降压肽在pH3.0～11.0时溶解度为96.0%左右，可广泛应用于食品中。 关键词：草鱼； 降压肽；碱性蛋白酶；高压液相色谱；相对分子质量；体积排阻色谱     

鳜鱼、青鱼、草鱼、鲤、鲫、鲢消化酶活性的研究

鳜鱼、青鱼、草鱼、鲤、鲫、鲢肝脏蛋白酶活性低于肠蛋白酶。肠蛋白酶活性，鳜鱼最高，其余依次为青鱼、鲤、鲢、草鱼、鲫。鳜鱼、鲢的肠蛋白酶活性由前肠向后肠递减；而青鱼、鲤、草鱼的则由前肠向后肠递增；鲫则中肠活性最低。六种鱼不同组织的脂肪酶活性因鱼而异。青鱼、鳜鱼、鲤、鲢肝脏中脂肪酶活性低于肠脂肪酶活性（Ｐ＜０．０１）；而草鱼和鲫肝脏中脂肪酶活性与肠脂肪酶活性差异不明显（Ｐ＞０．０５）。六种鱼肝脏中脂肪酶活性由高到低依次为鳜鱼＞鲢＞鲫＞草鱼＞青鱼＞鲤；肠脂肪酶活性顺序为鳜鱼＞鲢＞青鱼＞鲤＞草鱼＞鲫。脂肪酶活性与鱼类食性无明显相关性。不同组织间淀粉酶活性存在差异。鳜鱼的肝脏和肠均有较高淀粉酶活性，青鱼和鲫肝脏中淀粉酶活性低于肠中的；草鱼、鲤和鲢肝脏中淀粉酶活性高于肠中的，但差异不显著。六种鱼中鳜鱼的肝脏淀粉酶活性明显高于其它五种无胃鱼，它们的淀粉酶活性顺序依次为鳜鱼＞鲢＞鲤＞草鱼＞青鱼＞鲫；肠淀粉酶活性顺序为鳜鱼＞鲫＞鲤＞青鱼＞鲢＞草鱼。     

Partial characterization and activity distribution of proteases along the intestine of grass carp, Ctenopharyngodon idella (Val.)

A series of studies based on biochemical assays and electrophoretical observations has been conducted in order to investigate activity distributions and partially characterize various types of proteinases in the digestive tract of grass carp, Ctenopharyngodon idella (Val.). The casein digestion assays revealed that the presence of acidic proteinase had the highest activity at pH 2.5–3.0 and the alkaline proteinases at pH 10.0. The acidic proteinase activity distribution was found to decrease gradually from the oesophagus to the anus. Pepstatin A and EDTA inhibited the acidic proteinases activity. The SDS‐substrate‐PAGE showed that crude extraction of grass carp intestine contained an acidic proteinase active component with molecular mass of 28.5 ku. The substrate‐PAGE at neutral pH condition showed the presence of two acidic proteinase active components. The activity distribution of alkaline proteinase was found to slightly fluctuate along the intestine. And the whole intestine had very high activity. The inhibition assays and substrate specificity assays showed that trypsin was the main active component of the alkaline proteinases. The SDS‐substrate‐PAGE further showed that the crude extraction of grass carp intestine had four types of alkaline proteinase with molecular mass of 26.4, 30.8, 43.0 and 105.0 ku respectively. They were characterized to be trypsin (26.4, 30.8 and 43.0 ku) and un‐serine proteinase (105.0 ku) respectively. No chymotrypsin was detected.     

鲤肌肉肌原纤维结合型丝氨酸蛋白酶的分子克隆

肌原纤维结合型丝氨酸蛋白酶(myofibril-bound serine proteinase,MBSP)是最近发现的一种蛋白酶。该酶参与肌原纤维蛋白的降解及鱼糜制品弹性的下降。但是,对该酶一级结构的研究,迄今为止,未有报道。本文根据已测定的鲤MBSP N-末端氨基酸序列以及丝氨酸蛋白酶活性中心保守序列设计兼并引物,结合RT-PCR技术实现了MBSP基因片段的扩增。再根据克隆到的MBSP片段序列设计基因特异引物,用于MBSP基因的5′和3′末端快速扩增。综合以上结果,鲤MBSP的全长被确定。序列分析表明,MBSP cDNA含有一732 bp的开放阅读框,编码243个氨基酸残基,其中信号肽长度为21个氨基酸残基。组成丝氨酸蛋白酶活性中心的氨基酸残基(His61,Asp107和Ser197)在MBSP中保守存在。成熟MBSP含有222个氨基酸残基,分子量为24.5 ku,比其天然蛋白的分子量30 ku略小。成熟MBSP的等电点为10.43。鲤MBSP与鲫MBSP,猪胰蛋白酶,牛胰蛋白酶,美洲鲽胰蛋白酶的同源性分别为80.6%,55.8%,55.3%和53.9%。而与仓鼠肌肉中具有胰凝乳蛋白酶性质的蛋白酶的同源性为39.2%。MBSP有高含量的赖氨酸残基(11.93%),此特性可能与该酶的肌原纤维结合特性有关。     

Characterization of protease inhibitors of seminal plasma of cyprinids

Anti-proteinase activity was demonstrated in the seminal plasma of cyprinid fish species (bream, chub, ide, dace, asp, goldfish, roach, common carp) using electrophoretic techniques combined with a detection method based on inhibition of bovine trypsin. We found species-specific protease inhibitors in the seminal plasma of cyprinids. At least three bands of protease inhibitors with different migration rates could be identified by native PAGE. Higher variability was characterized for bands with slower migration rates. Visualization of inhibitors after SDS-PAGE under non-reducing conditions allowed estimation of their molecular weights. Apparent molecular weights were within the range of 51–59 and 47–54 kDa for the bands with slower and moderate migration rates, respectively. The molecular weight of fast migration bands for roach and common carp were estimated to 23 and 30 kDa, respectively. Inhibitors of common carp seminal plasma differed in their affinity toward serine proteases. Three inhibitors in common carp seminal plasma could be visualized using cod and bovine trypsin, but only two inhibitors (of high molecular weight) were recognized with chymotrypsin. There were differences in anti-proteinase activity and seminal plasma protein concentration in relation to the origin of common carp seminal plasma (breeding lines) and time of milt collection (spawning vs. post-spawning season).     

鱼类消化酶活性与其体长、体重和水质的相关性研究

对主养草鱼(Ctenopharyngodon idellus)池塘中4种鱼类消化器官的胃蛋白酶、胰蛋白酶、脂肪酶和淀粉酶的活性与鱼体长、体重及主要水质指标的相关性进行了研究。结果显示,草鱼肠道和肝胰脏的胃蛋白酶与水温具有显著正相关(P<0.05),而草鱼肠道胰蛋白酶、肠道和肝胰脏的脂肪酶以及鲢(Hypophthalmichthys molitrix)肝胰脏中淀粉酶与水温呈负相关(P>0.05)。4种鱼的消化酶活性与总氮和总磷呈正相关。匙吻鲟(Polyodon spathula)的脂肪酶和淀粉酶活性与化学耗氧量呈负相关;除鲢外,其他3种鱼的胃蛋白酶活性均与水体硬度和碱度呈正相关,而胰蛋白酶、脂肪酶和淀粉酶均与水体硬度和碱度呈负相关。草鱼和鳙(Aristichthys nobilis)肠道胰蛋白酶、脂肪酶和淀粉酶以及匙吻鲟消化道和肝脏胰蛋白酶与鱼的体长、体重均具有显著的正相关。     

Digestive proteases of three carps Catla catla, Labeo rohita and Hypophthalmichthys molitrix: partial characterization and protein hydrolysis efficiency

Characteristics and functional efficacy of digestive proteases of Catla catla, catla, Labeo rohita, rohu and Hypophthalmichthys molitrix, silver carp were studied. Total protease activity was significantly (P < 0.05) higher in rohu (1.219 ± 0.059 U mg protein⁻¹ min⁻¹) followed by silver carp (1.084 ± 0.061 U mg  protein⁻¹ min⁻¹), and catla (0.193 ± 0.006 U mg  protein⁻¹ min⁻¹). Trypsin activity of silver carp and rohu was 89–91% higher than catla. Chymotrypsin activity was significantly (P < 0.05) higher in silver carp compared with rohu and catla. The protease activity of rohu and silver carp displayed bell‐shaped curves with maximum activity at pH 9; whereas in catla, maximum activity was found between pH 8 and 11. Inhibition of protease activity with soybean trypsin inhibitor (SBTI), phenylmethylsulfonyl fluoride (PMSF) revealed the presence of serine proteases and inhibition of activity with N‐α‐p‐tosyl‐L‐lysine‐chloromethyl ketone (TLCK) and N‐tosyl‐L‐phenylalanychloromethane (TPCK) indicated the presence of trypsin‐like and chymotrypsin‐like enzymes in all these three carps. SDS‐PAGE showed the presence of several protein bands ranging from 15.3 to 121.9 kDa in enzyme extracts of catla, rohu and silver carp. The substrate SDS‐PAGE evidenced the presence of various protease activity bands ranging from 21.6–93.7, 21.6–63.8 and 26.7–98.5 kDa for catla, rohu and silver carp respectively. In pH‐stat hydrolysis of Chilean fishmeal showed significantly (P < 0.05) higher degree of hydrolysis compared with soybean meal, silver cup (a commercial fish feed of Mexico) and wheat flour, with enzyme preparations of three fishes. The rate of hydrolysis was significantly (P < 0.05) higher in silver carp compared with others.     

Identification and partial characterization of selected proteolytic enzymes in the digestive system of giant freshwater Prawn Macrobrachium rosenbergii (De Man) Postlarvae

Biochemical assays and substrate SDS-PAGE were conducted to partially characterize and identify various types of proteases present in the digestive tract of PL₁₅ giant freshwater prawn ( Macrobrachium rosenbergii ). Casein hydrolytic assay of the enzyme extracts showed major proteolytic activities at pH 3.0, 6.0 and 9.0, while assay of preincubated enzyme extracts with phenylmethylsulphonyl fluoride (PMSF), a serine protease inhibitor produced a 33.17% reduction in alkaline protease activity. When specific inhibitors tosyl-lysine chloromethyl ketone and tosyl-phenylalanine chloromethyl ketone were used, they resulted in a reduction in activity of proteases in the enzyme extracts by 82.41% and 55.03%, respectively, confirming the presence of trypsin and chymotrypsin, while ethylenediamine tetraacetic acid produced protease activity reduction in 33.92% showing the presence of metalloproteases in the digestive tract of the prawn. Further characterization of the alkaline proteases using SDS-PAGE technique, after incubating the extract in the presence or absence of specific inhibitors, produced six bands corresponding to molecular masses of between 13.48 and 136.1 kDa; two trypsin bands of 13.48 and 36.4 kDa, three chymotrypsin bands in the range of 23.0–73.4 kDa and one for metalloprotease of 136.1 kDa, all of which were identified from a zymogram. This study suggests that protein digestion in M. rosenbergii is initiated by an acid protease followed by a combination of action of alkaline proteases: trypsin, chymotrypsin and metalloproteases.     

三角帆蚌钩介幼虫在5种寄主鱼体寄生效果的比较

选用黄颡鱼、草鱼、鲢、鳙和鲫作为三角帆蚌(Hyriopsis cumingii)的寄主鱼,分析了寄苗悬液钩介幼虫密度和寄苗时间对5种鱼初始寄苗量的影响,并对寄生在这5种鱼上的钩介幼虫发育情况进行了观察。结果表明:无论高密度寄苗还是低密度寄苗,长时间寄苗还是短时间寄苗,黄颡鱼和草鱼的寄苗量都明显高于鲢、鳙和鲫。提高寄苗悬液钩介幼虫密度,寄主鱼的寄苗量显著增加,但增加寄苗时间,寄主鱼的寄苗量未见显著提高。黄颡鱼和草鱼的稚蚌脱苗率显著高于鲢、鳙和鲫,寄生在鲢、鳙和鲫鳃上的钩介幼虫约80%未发育至稚蚌便提前脱落。不论在寄苗量方面,还是在稚蚌脱苗率方面,黄颡鱼和草鱼均是三角帆蚌钩介幼虫的最佳寄主。     

天然滁州鲫氨基酸成分分析与营养价值评价

应用生化分析方法对滁州鲫鱼中氨基酸成分进行了定量分析,并对氨基酸含量进行营养分析评价。结果显示：滁州鲫鱼中氨基酸总量为86.35%（占干重）,其中8种必需氨基酸和2种半必需氨基酸总量为36.76%,4种呈味氨基酸含量为29.29%,必须氨基酸总量和呈味氨基酸含量均高于草鱼、鲢、鳙鱼等鱼类。表明滁州鲫较之其他鱼类是一种营养价值高,肉质鲜美的鱼类。