中华绒螯蟹免疫因子—溶菌酶的初步研究

研究了在自然状态下中华绒螯蟹成蟹血淋巴和大眼幼体组织匀浆上清液中溶菌酶的活性。结果表明,在中华绒螯蟹成蟹血淋巴中均检测出溶菌酶的活性,为(0.0286±0.0081)活力单位(n=30),而在中华绒螯蟹大眼幼体组织匀浆上清液中亦检测到溶菌酶的活性,为(0.0318±0.0073)活力单位(n=4)。     

pH胁迫对中华绒螯蟹5种非特异性免疫指标的影响

测定了不同pH(4.5,6.0,7.5,9.0,10.5)胁迫1、4、7、10d后中华绒螯蟹(Eriocheir sinensis)血细胞呼吸爆发(RP)、血细胞破碎物上清液(HLS)酚氧化酶(PO)、血清溶菌酶(LSZ)、酸性磷酸酶(ACP)及碱性磷酸酶(ALP)活性的变化.结果表明:在pH胁迫后第1天,pH4.5组...     

玉米蛋白粉替代鱼粉对暗纹东方鲀溶菌酶活性及c型溶菌酶mRNA表达的影响

以初重为(41.26±1.09)g的暗纹东方鲀为实验鱼,配制5种玉米蛋白粉使用量为0%、5%、10%、15%、20%的实验饲料,玉米蛋白粉对饲料中鱼粉的替代水平分别为0%、7.4%、14.8%、22.2%和29.6%。用实验饲料饲养暗纹东方鲀60d,研究玉米蛋白粉替代鱼粉对暗纹东方鲀肝胰脏、头肾和脾脏组织的溶菌酶活性及其c-型溶菌酶mRNA相对表达量的影响。结果表明,(1)暗纹东方鲀肝胰脏、头肾和脾脏的溶菌酶比活力分别为9.14、42.12和40.49U/mgprot,头肾和脾脏组织中c-型溶菌酶mRNA相对表达量分别是肝胰脏的3.76和3.24倍。(2)玉米蛋白粉替代鱼粉显著影响暗纹东方鲀溶菌酶比活力。5%和10%组的肝胰脏和头肾组织溶菌酶比活力显著高于对照组(P<0.05),而15%和20%组肝胰脏和头肾组织溶菌酶比活力则显著低于对照组(P<0.05);5%和10%组的脾脏组织溶菌酶活力与对照组无显著性差异(P>0.05),而15%和20%组显著低于对照组和5%组(P<0.05)。(3)玉米蛋白粉对暗纹东方鲀c型溶菌酶基因mRNA相对表达量有显著影响(P<0.05)。肝胰脏组织中5%和10%组表达量显著高...     

水产动物溶菌酶研究概况

溶菌酶，全称为1，4-β-N-溶菌酶，又称粘肽N-乙酰基胞壁酰水解酶，属于α-乳白蛋白家族。人们对溶菌酶的研究始于上世纪初，英国细菌学家Fleming发现人的唾液、眼泪中存在有溶解细菌细胞壁的酶，因其具有溶菌作用，故命名为溶菌酶；此后人们在微生物、植物、无脊椎动物和高等动物中也发现了溶菌酶的存在。本文对水产动物溶菌酶的分型、特性及在饲料中的潜在应用价值进行了总结。     

史氏鲟及小体鲟不同组织中溶菌酶水平的比较

对史氏鲟及小体鲟血清和几种主要组织的溶菌酶含量进行了测定和比较后发现,同小体鲟相比,史氏鲟的血清、鳃、肝、胃、前肠及后肠的溶菌酶量均较高,血清之间有极显著的差异,鳃、肝及后肠均有显著性的差别,胃及前肠之间无差异;小体鲟的粘液、幽门垂及脾中的溶菌酶含量较史氏鲟高,但无显著性差异,由此可见,虽然同为鲟科鲟属,溶菌酶在两种鱼之间的差别还是很显著的。     

海洋低温溶菌酶抗肿瘤活性的初步研究

通过观察海洋低温溶菌酶对体外培养细胞株增殖的影响、对角膜诱生血管的影响、对小鼠肉瘤S180（实体型）及对小鼠肝癌HAC（实体型）的抑制作用，探讨海洋低温溶菌酶抑制肿瘤生长活性。结果表明，该酶与蛋清溶菌酶在抑瘤作用机制上有很大区别，海洋低温溶菌酶具有血管生成抑制因子的活性，故对肿瘤生长具有明显的抑制作用。     

pH、温度和盐度对单环刺螠消化酶和溶菌酶活力的影响

采用L25(56)正交试验法测定了不同pH(6、7、8、9、10)、温度(10、15、20、25、30℃)和盐度(15、20、25、30、35)对体质量为(71.2±8.5)g的单环刺螠肠蛋白酶、淀粉酶、纤维素酶和脂肪酶活力的影响,以及不同盐度对单环刺螠血液溶菌酶活力的影响。试验结果表明,单环刺螠肠消化酶活力的最适环境条件分别为,蛋白酶:pH 8、温度30℃、盐度35;淀粉酶:pH 9、温度25℃、盐度25;纤维素酶:pH6、温度30℃、盐度35;脂肪酶:pH 6、温度25℃、盐度30。盐度15和20两组单环刺螠血液溶菌酶活力均先降后升再降,而盐度30和35两组则先升后降。至处理第4d,各试验组溶菌酶活力依次为:盐度3025352015,盐度15和20两组溶菌酶活力显著低于盐度较高试验组(P0.05)。盐度25~35、pH 6~9为单环刺螠适宜的环境条件,高温(25~30℃)下其消化酶活力较高,而低盐度(15~20)下其消化酶活力和免疫能力明显降低。     

甲基盐霉素对鲤白细胞吞噬活性和溶菌酶活力的影响

选用60 g左右健康正常的鲤540尾,随机平均分为6组,每组设3个重复。试验设6种处理,即基础饲料中分别添加甲基盐霉素0(对照组)、10、20、40、80、160 mg/kg。研究了甲基盐霉素对鲤白细胞吞噬活性和溶菌酶活性的影响。试验结果表明,饲料中添加10 mg/kg甲基盐霉素对鲤白细胞吞噬活性和溶菌酶活性影响不显著,而添加20~160 mg/kg甲基盐霉素对鲤白细胞吞噬活性和溶菌酶活性有一个从促进到抑制的转变,剂量越高,这个转变越快,并且随饲喂时间的延长,其抑制作用越来越强。     

魁蚶各组织溶菌酶活性对鳗弧菌侵染的响应

为了有效防治细菌及其他病原微生物对魁蚶等贝类的危害,本实验研究了魁蚶各组织中溶菌酶活性对鳗弧菌侵染的响应过程,以期探讨魁蚶体内溶菌酶的免疫功能。本实验采用注射活菌的方法侵染20月龄魁蚶个体,随机选取16只个体,在每只个体的斧足处注射1 mL(约1×10~9个)鳗弧菌菌悬液作为感染组;随机选取16只个体不注射鳗弧菌作为对照组。两组魁蚶分别于洁净海水中暂养4、12、24和48 h后,每组随机取4只魁蚶个体的血液、外套膜、鳃、斧足、肝胰腺和闭壳肌等组织,采用ELISA试剂盒测定其溶菌酶含量变化。结果显示,对于鳗弧菌的侵入,魁蚶血液中溶菌酶含量由正常低值迅速增高并一直维持较高的含量,说明血液是魁蚶机体防御病原菌的主要免疫组织之一;魁蚶外套膜在无感染的情况下,对外界水环境的干扰始终保持较高的溶菌酶含量;鳃、斧足的溶菌酶含量均在注射细菌24 h之后明显高于正常值,说明外套膜、鳃、斧足作为魁蚶机体与外界接触的第一道屏障也能应对病原菌入侵,但反应较血液延迟;肝胰腺和闭壳肌的溶菌酶含量变化不明显,推测肝胰腺和闭壳肌不是魁蚶的重要免疫组织或器官。本实验结果可为魁蚶抗病选育及免疫机理方面的研究提供相关的参数。     

海洋微生物溶菌酶的抑菌作用及抑菌机理初步研究

以一些革兰氏阴性菌、革兰氏阳性菌和真菌对海洋微生物溶菌酶的敏感性进行研究。结果表明,海洋微生物溶菌酶对受试菌株均有不同程度的抑制或杀灭作用,尤其对临床分离的致病菌及养殖致病菌效果显著。海洋微生物溶菌酶对试验菌株的生长繁殖和代谢均有影响,抑制作用主要发生在指数期初期。通过透射电镜观察发现,海洋微生物溶菌酶对大肠埃希菌和金黄色葡萄球菌的作用效果不同,推测其作用机理与二者细胞壁结构不同有关。     

金乌贼肌肉中三甲胺脱甲基酶的分离纯化及酶学性质

为了提取纯化金乌贼肌肉中的三甲胺脱甲基酶(TMAOase),本研究采用含有0.1 mol/L NaCl、pH 7.0、浓度为20 mmol/L的三羟甲基氨基甲烷(Tris)-醋酸缓冲液提取粗酶液,经透析、浓缩处理后,通过DEAE-52阴离子交换柱层析和Sephacryl S-300柱层析得到了纯化的TMAOase,并对其酶学性质进行了研究。结果显示,经Sephacryl S-300柱层析的TMAOase相比粗酶纯化了209.54倍;粗酶和纯化酶的最适温度分别为55和50°C,当温度高于最适温度时,酶活性开始出现显著下降,粗酶在80°C仍残留21.9%的活性;而纯化酶在80°C时,几乎检测不到酶活;粗酶和纯化酶的最适p H均为7.0,中性条件下表现稳定,在酸性和碱性条件下稳定性下降,p H为9.0时,粗酶残留60.7%的活性,而纯化酶的活性仅为20.5%。以双倒数作图法(Lineweaver-Burk法)测得纯化的TMAOase的Km值为22.8 mmol/L;经SDS-PAGE电泳分析,测得其分子量为21.3 ku;在化学物质中,柠檬酸和CaCl_2对酶活性具有显著的促进作用,H_2O_2和Na_2S对TMAOase活性有显著抑制作用。     

海洋侧孢短芽孢杆菌S-12-86溶菌酶的分离纯化与冻干技术研究

海洋微生物溶菌酶发酵液经低温离心、超滤浓缩、乙醇提取、Superdex 75 10/300凝胶层析和反相高效液相色谱层析纯化得到电泳纯海洋溶菌酶,分子量为17.1 kD,比活达到3 987.7 U/mg,纯度提高41.98倍,活力回收率为21.7%。对该酶冷冻干燥过程的研究结果表明,海藻糖、蔗糖和麦芽糖对该酶均有一定的冻干保护作用。其中,以海藻糖的保护效果最佳。0.5 mol/L海藻糖与20 mg/ml吐温80复合作为冻干保护剂,使冻干后的溶菌酶活性维持在95%以上,为该酶进一步研究和应用提供了稳定的酶制剂。     

温度、pH对克氏原螯虾血清酚氧化酶活力及稳定性的影响

以克氏原螯虾(Procam barus clarkii)为材料,研究温度、pH对其血清酚氧化酶活力及稳定性的影响。实验以L-dopa为反应底物,在试验设计的温度范围(20,30,40,50,60,70℃)内,测得酚氧化酶活力的最适温度为20℃,随温度升高酶活力迅速下降,该酶在20~40℃范围内表现出较高的热稳定性,而30℃时最稳定;该酶的最适pH 7.0,在pH 6.0和7.0的缓冲系统中表现出较强的稳定性。     

Purification and physicochemical properties of deoxyribonuclease from pyloric caeca of Atlantic cod (Gadus morhuaL.)

A deoxyribonuclease (DNase) of pancreatic origin has been purified from extracts of the pyloric caeca from Atlantic cod (Gadus morhua L.). The crude extract was prepared by mincing frozen caeca tissue in equal volumes of buffer. The enzyme was isolated from the supernatant after streptomycin sulfate precipitation and centrifugation. The purification scheme further included chromatography on Q-Sepharose Fast Flow and hydroxyapatite columns. Affinity adsorption chromatography of the hydroxyapatite fraction on 8-(6-aminohexyl)-amino-5′-AMP-Sepharose, revealed an apparently homogeneous protein with molecular weight of 35,000 Da as judged by NaDodSO₄-PAGE. In sum a 644-fold enzymatic enrichment and 3.5% total enzyme recovery was achieved. The cod enzyme resembles DNase I-type enzymes with an alkaline pH activity optimum and shows dependency for Mg²⁺. The pI of the enzyme is 6.5 as determined by isoelectric focusing and DNase-zymography. Our findings suggest that the nuclease is a member of the cod's digestive enzymes secreted from the connective tissue surrounding the caeca.     

产海洋细菌MP-2酯酶菌株的鉴定及酯酶理化性质的研究

从渤海海泥样品中分离获得1株新型酯酶菌株,经鉴定为地衣芽孢杆菌Bacillus licheniformis。所得的MP-2酯酶的最适作用温度范围50～70℃,在60℃表现出了最高活性,属于耐热酶;最适作用pH为10,属于碱性酶,其pH值作用范围比较窄;具有良好的热稳定性;金属离子Co2＋,Li＋对酶具有激活作用,Ca2＋对酯酶的活力没有显著影响,化学试剂SDS、EDTA及Tween-20对酯酶的抑制效果显著,对常见有机溶剂具有良好的耐受力;该酯酶对碳链长短不同的底物表现出不同的酶活。     

Lysozyme in the ovary of tilapia (Oreochromis mossambicus): its purification and some biological properties

Lysozyme was purified from the ovary of tilapia, Oreochromis mossambicus, with two steps, chitin coated-cellulose and Sephadex G-100, and its biological properties were investigated. Purified lysozyme had a molecular mass of 15kDa on SDS-PAGE under reducing condition. Analyses with antibody (a-EL) against the purified lysozyme revealed that serum and egg extract reacted with a-EL and the precipitin lines fused completely. The enzyme activities in serum and egg extract were inhibited by adding serially diluted a-EL. Therefore, egg extract and serum lysozyme was immunologically identical. Immunohistochemically, lysozyme was observed in the ooplasm of the oocytes laden with yolk but not in the follicle layers, egg envelope or immature oocytes (the peri-nucleolus stage). In addition, the enzyme activity in the large oocytes was higher than that in the small ones. These results suggest that lysozyme detected in the oocytes is derived from extra-ovarian tissue and transfers from the maternal circulation. Lysozyme activity in the serum of female tilapia increased with oocyte development, suggesting that the change in the enzyme level may be partially related to the reproductive events (especially vitellogenesis) of the female fish.     

Purification and characterization of lysozyme from purple washington clam Saxidomus purpurata

ABSTRACT:   Lysozyme was purified from purple washington clam Saxidomus purpurata by sequential procedures using Chitopearl Basic BL-01 affinity and TSKgel ODS-120T column chromatographies. Molecular mass of the purified enzyme was estimated to be 12 kDa by SDS-PAGE. Optimum pH of the enzyme was 5.2 toward Micrococcus lysodeikticus cells. The optimum temperature was 50°C. The enzyme was stable in the range of pH 4.8–6.8 and 20–90°C. Further, the N-terminal amino acid sequence of the enzyme showed similarity to lysozymes from invertebrates. However, the specific activity of the enzyme toward M. lysodeikticus cells and p -nitrophenyl penta- N -acetyl- β -chitopentaoside was 143 times and 12 times higher than that of hen egg white lysozyme, respectively.     

Purification and characterization of cathepsin S from hepatopancreas of carp Cyprinus carpio

SUMMARY: Cathepsin S was purified from carp hepatopancreas to homogeneity up to 300-fold. The amino acid sequence of its NH₂-terminus was determined to be V-P-D-A-M-D-W-Y-N-K-G-Y-V-T-D-V-K-N-Q. On the contrary, that of purified cathepsin L from carp hepatopancreas was to be V-P-N-S-L-D-W-R-E-K-G. Purified cathepsin S consisted of a single chain with 37 kDa estimated by sodium dodecylsulfate–polyacrylamide gel electrophoresis. The enzyme had strong hydrolytic activity toward Z-Phe-Arg-MCA with the pH optimum of 7.0, but this lacked the ability to hydrolyze most of the other MCA substrates. The optimum pH of cathepsin S for protein substrate (carp myosin heavy chain) was also to be pH 7.0. These properties of purified cathepsin S obviously differ from cathepsins B and L. The enzyme activity was totally inhibited by E-64, leupeptin, 5–5'-dithiobis (2-nitro-benzoic acid) and p -tosyl-lys chloromethylketone as well.     

紫外诱变原生质体选育溶菌酶高产菌株的研究

以1株产海洋溶菌酶活性较高的菌株（S_12-86）为原始菌株，对其原生质体的制备、再生及紫外诱变育种进行了研究。实验所确定的原生质体的最佳制备条件为：菌株S-12—86培养18h，溶菌酶浓度为1．0mg／ml，在35℃下，酶解30min，原生质体形成率为97．6％，再生率为23．6％。同时实验所确定的原生质体诱变的适宜条件为：30w紫外灯下80cm照射120S。对大量再生突变株进行筛选，最终获得了1株遗传性能稳定的菌株R—J—i01，其产酶活力达到1808U／mg，比原始菌株（1290U／mg）提高了40％。