实验室海月水母大规模解体的初步研究

本实验针对两次突发的饲养海月水母(Aureliaaurita)大规模烂洞解体状况,通过对病灶处进行细菌培养,从其中每次发病水母中均各分离到2种优势病原菌菌株,利用PCR扩增和DNA测序技术,获得优势菌株16SrRNA基因序列,通过生物信息比对分析显示,导致两次海月水母发病的病原菌菌株分别为2种嗜冷杆菌(Psychrobacter sp.)和2种弧菌(Vibrio splendidusVibrio neptunius)。通过利用福林酚法进行此四种优势菌株的细菌发酵培养液蛋白酶活性的测定,结果显示,此4种细菌均具有较强的蛋白酶活性。根据实验结果推测,分离所得的2种嗜冷杆菌菌株(Paa1Paa2)和2种弧菌菌株(Vaa1Vaa2)作为此次发病海月水母的病原菌,有可能均是通过分泌胞外蛋白酶侵蚀水母伞体,从而导致伞体烂洞解体。通过本次实验分析,推测自然条件下水母暴发后的快速消亡是由水母自身免疫下降与细菌侵染的共同作用导致。     

环境因子对鳗弧菌生长和胞外蛋白酶表达的影响

鳗弧菌(Vibrio anguillarum)是一种水产动物的重要致病菌,鳗弧菌分泌的胞外蛋白酶是该菌的致病因子之一.研究了环境因子对鳗弧菌W-1生长及胞外蛋白酶表达的影响.结果表明,鳗弧菌的最适生长温度为28℃,菌体生长量在21 h达到最高,胞外蛋白酶的活力在24 h最高.在37℃培养时,菌体生长和酶活力显著降低;添加葡萄糖、蔗糖、麦芽糖等碳源物质均能显著促进菌体的生长,但对于胞外蛋白酶表达有明显抑制作用;1 mmol/L EGTA对茵体的生长和胞外蛋白酶表达没有明显影响,而1 mmol/L的EDTA强烈抑制鳗弧菌的生长及其胞外蛋白酶的表达.添加不同浓度的氨苄青霉素对菌体的生长及其胞外蛋白酶表达有明显的抑制作用.     

海洋生物蛋白的酶解及酶解产物的抗氧化活性

应用4种蛋白酶：实验室保藏菌株Bacillus sp．SM98011和Bacillus sp．SM97010所产的中性蛋白酶SM98011和SM97010以及购自诺维信公司的碱性蛋白酶和风味蛋白酶分别酶解鲨鱼、牡蛎、扇贝和毛虾,分析了酶解液中蛋白质含量、氨基酸组成及肽的含量,并测定了酶解液的抗氧化能力。结果表明,酶解液中必需氨基酸含量丰富、均衡,营养价值较高;SM98011蛋白酶、SM97010蛋白酶和碱性蛋白酶的酶解液中肽的比例较高;而风味蛋白酶的酶解产物主要以游离的风味氨基酸为主,肽的含量较低。SM97010和SM98011蛋白酶的酶解液具有较高的抗氧化能力,预示在功能食品开发方面具有较好的前景。     

PROPERTIES OF PROTEOLYTIC TOXIN OF VIBRIO ANGUILLARUM FROM DISEASED FLOUNDER

Extracellular products(ECP)produced by Vibrio anguillarum strain M3 originally isolated from diseased flounder(Paralichthys olivaceus)were prepared.ECP of M3 showed gelatinase,casinase,amylase and haemolytic activity on agarose plates.High protease activity against azocasin was detected.Bacterium M3 showed highest growth and protease activity at 25℃。The Protease present in ECP showed maximal activity at pH 8 and 55℃；was completely inactivated by application of 80℃ heat for 30 min;was completely inhibited by EDTA and HgCl2,and was partially inhibited by PMSF,SDS,MnCl2 and iodoacetic acid;but not inhibited by CaCl2 and MgCl2.The ECP was toxic to flounder fish at LD50 values of 3.1 μg protein/g body weight.The addition of HgCl2 and application of heat at 50℃ decreased the lethal toxicity of ECP.When heated at 100℃,ECP lethality to flounder was completely in-hibited.After intramuscular injection of ECP into flounder,it showed evident histopathological changes including necrosis of muscle,extensive deposition of haemosiderin in the spleen,dilated bolld vessels congested with numerous lymphocytes in the liver.These results showed that ECP protease was a lethal factor produced by the baterium V.anguillarum M3.     

舟山海域一株产碱性蛋白酶海洋放线菌的鉴定、选育及发酵条件的初步研究

从舟山海域潮间带海泥筛选到产碱性蛋白酶的海洋放线菌,利用Folin-酚法进行酶活测定,选取酶活最高的菌株进行鉴定,绘制系统进化树。再通过紫外线和DES诱变从而得到高产碱性蛋白酶的海洋放线菌菌株,并进行初步的发酵条件研究。结果表明:分离筛选得到2株菌株,在脱脂奶筛选平板上能产生较大的水解透明圈,通过Folin-酚法进行酶活测定,挑选出酶活较高的菌株A20(初始酶活为104.7U/mL),生理生化试验和16S rDNA试验结果显示该菌株为Streptomyces roseus。对菌株A20进行紫外线和硫酸二乙酯(DES)诱变,最终得到高产碱性蛋白酶的菌株,酶活为227.5U/mL,酶活提高117.3%,传代试验显示该菌株具有较好的遗传稳定性。单因素试验显示最适发酵温度为50°C、pH值为9.0、培养时间为72h。     

Alkaline protease production by a strain of marine yeasts

Yeast strain 10 with high yield of protease was isolated from sediments of saltern near Qingdao, China. The protease had the highest activity at pH 9.0 and 45℃. The optimal medium for the maximum alkaline protease production of strain 10 was 2.5 g soluble starch and 2.0 g NaNO3 in 100 mL seawater with initial pH6.0. The optimal cultivation conditions for the maximum protease production were temperature 24.5 ℃, aeration rate 8.0 L min^- 1 and agitation speed 150 r min^-1 . Under the optimal conditions, 623.1 Umg^-1 protein of alkaline protease was reached in the culture within 30 h of fermentation.