耐高温植酸酶研究进展2010
植物免疫学
植物免疫学第一章绪论Table of Contents1.1 抗病性利用与植物病害防治植物病害是作物生产的最大威胁之一▪1844—1846年，马铃薯晚疫病流行，造成爱尔兰饥荒▪1870年，咖啡锈病流行，斯里兰卡的咖啡生产全部毁产▪1942年，水稻胡麻斑病流行，造成孟加拉饥荒▪1950年，小麦条锈病大流行，我国损失小麦120亿斤▪1970年、1971年，玉米小斑病流行，美国玉米遭受重大损失1.1 抗病性利用与植物病害防治抗病性利用在植物病害防治中的作用▪利用抗病性来防治植物病害，是人类最早采用的防治植物病害的方法▪“综合治理”策略中，抗病性利用是最基本、最重要的措施▪经济、简便、易行，且不污染环境1.2 植物免疫学1.2.1 植物免疫学植物免疫学（plant immunology）是关于植物抗病性原理和应用的综合学科，以植物与病原物的相互作用为主线，探索植物免疫的本质，合理实行人为干预，以达到有效而持久控制植物病害的目的植物病理学的一门新兴分支学科系统研究植物的抗病性的类型、机制和遗传、变异规律及植物抗病性合理利用，使其在植物病害防治中发挥应有的作用1.2 植物免疫学1.2.2 植物免疫学的研究内容①植物抗病性的性质、类型、遗传特点和作用机制②植物病原物致病性的性质、类型、遗传特点和作用机制③植物与病原物的识别机制和抗病信号的传递途径④植物抗病性鉴定技术、抗病种质资源、抗病育种和抗病基因工程⑤病原物群体毒性演化规律、监测方法和延长品种抗病性持久度的途径和方法⑥人工诱导植物免疫的原理和方法1.2 植物免疫学1.2.3 植物免疫学与其它学科的关系▪以植物病理学、生物化学、遗传学和分子生物学为基础▪基础理论层面：与植物病原学、植物生理学、真菌生理学、细胞学、生物物理学等学科有密切关系▪应用层面：与植物育种学关系最密切，与植物保护学、作物栽培学、植物遗传工程、农业生物技术、田间试验与统计等学科有密切关系▪在植物病理学各分支学科中，植物免疫学与生理植物病理学、分子植物病理学最接近，内容有所重叠，但学科范畴和侧重点不同1.3 植物免疫学发展简史1.19世纪中期至20世纪初期阶段（萌芽阶段）•1380年，英国选种家J. Clark用马铃薯“早玫瑰”品种与“英国胜利”杂交育成抗晚疫病品种“马德波特∙沃皮特”•L. Liebig，1863发现增施磷肥可提高马铃薯对晚疫病的抗性，偏施氮肥可加重发病•1896年，J. Eriksson和E. Hening发现小麦对锈病的反应有严重感染、轻度感染和近乎完全抵抗3种类型，并建议在生产上应用近乎完全抵抗的品种•1879年和1894年，Shrodter和Eriksson先后发现醋梨锈病菌（Puccinia caricis）和禾谷类秆锈菌（Puccina graminis）有寄生专化现象1.3 植物免疫学发展简史2.20世纪30〜70年代（学科体系建立和完善的阶段）（一）开始建立了遗传学理论•1900年，G. J. Mendel的遗传定律被重新肯定，为植物抗病性的研究和利用提供了遗传学理论•1905年，R. H. Biffen用小麦抗条锈品种American Club与感锈品种Michigan Bronze杂交和用大麦抗白粉病品种与感病品种杂交证明，植物的抗病性不但可以遗传，而且是按照孟德尔的遗传定律遗传•1909年N. A. Orton用栽培种西瓜Eden与饲料西瓜Citon杂交，并按照孟德尔的遗传定律在子2代和子3代继续选择，选出了抗萎蔫病食用西瓜“胜利者”（二）发现病菌有生理分化现象▪1917年，E. C. Stakman和F. J. Piemeisel发现小麦秆锈菌内有生理小种的分化（三）开始研究病原菌致病性的遗传和变异▪1904年，Blackeslee发现毛霉菌有异宗配合现象▪1927年，G. H. Criegie发现秆锈菌有异宗配合现象▪1932年，A. F. Hansen和Smith还在半知菌中发现有异核性（四）提出了一些有关植物免疫机制的学说▪Ward，1902，毒素和抗毒学说▪Comes，1910，酸度学说▪Dougal，1910，渗透压学说▪Rivera，1913，膨压学说▪Κричевский，1916，抗体、拟抗体学说▪瓦维洛夫，1919，植物免疫发生学说▪瓦维洛夫，于1939年出版了“植物对侵染性病害的免疫学”专著20世纪中期阶段▪H. H. Flor，1942，提出“基因对基因”假说（gene-for-gene hypothesis）▪植物病原菌致病性的遗传和变异研究：病菌可以通过准性生殖（parasexualism）产生变异（Pontecorvo，1953）；▪在植物抗病性的遗传变异规律和寄主与病原物相互关系方面取得了较大进展▪开始物理、化学和人工免疫研究▪在植物抗病机制方面做了大量研究，提出了一些新的假说。
毕赤酵母表达知识归纳
毕赤酵母表达知识归纳1a.配制500×BIOTIN stock solution（0.02％）有这么3种方案：1、懒人是将Biotin直接溶在去离子水中，放过夜，基本就能溶；2、急性子是将溶液配成0.02N的NaOH，就很容易溶解了；3、水浴加热，温度不能高于50度。
D－生物素是具有生物活性的生物素，也就是vitaminH。
在毕赤酵母代谢过程中，作为多种酶的辅基起作用。
天然培养基中一般可以不单独添加，因为YNB中、酵母粉、蛋白胨中均含有一定量的生物素，但是做高密度发酵还是必须要添加的。
b.有几个比较迷惑的问题请教大家：(很典型的小问题）1、制感受态细胞，OD多少比较好？pyrimidine 战友的方法：取1mlGS115过夜培养物(OD约6-10) 分装到1.5ml EP管中。
说明书还有一些文献是说在1.3左右效率高，再高了效率会很低2、关于高效转化法，文献说用(LiAc)，而invitrogen的说明书说转化毕赤酵母用(LiAc)没用，要用LiCl。
Lithium acetate does not work with Pichia pastoris. Use only lithium chloride.3、YNB到底能高温灭么？有的说能有的说不能。
过滤灭菌的怎么操作？我是把滤器装好膜绑到瓶口用纱布盖上，报纸包上，瓶盖放烧杯里单灭。
然后把配好的溶液用注射器一点点推进去。
4、葡萄糖为什么在YPD里一起灭颜色很深，单灭则不会。
该115度还是121度灭？网上搜了下，都有人用！5、电转化参数用400欧还是200欧？有的用400，有的还专门说不是用400。
都是从园里看到的！电击参数：1.5KV，25uF，200欧姆（不是400）6、电转后，在MD平板上长的应该就是整合了目的基因的重组子了吧？如果不想筛高拷贝的，是否PCR验证一下即可？网友的回答：ynb最好不灭菌，我是0.22um过滤处理的。
invitrogen手册上可以灭菌的。
枯草芽孢杆菌实际应用与发展前景的研究Microsoft Office Word 97-2003 文档
枯草芽孢杆菌实际应用与发展前景的研究[摘要] 随着国家对农业的支持，微生物化肥有农业中的应用也越来越受到重视。
而枯草芽孢杆菌在农业方面和其它方面都有广泛的应用，因此，研究枯草芽孢杆菌的实际应用是非常必要的。
本文从工业酶生产、生物防治领域、微生物添加剂领域和医药卫生领域等方面对枯草芽孢杆菌的实际应用进行研究。
[关键词] 枯草芽孢杆菌实际应用发展前景Bacillus subtilis application and development prospect of[ Abstract ] along with the country the support to agriculture, microbial fertilizer has application in agriculture has been paid more and more attention. And Bacillus subtilis in agriculture and other aspects of a wide range of applications, therefore, research of Bacillus subtilis and practical application is very necessary.This article from the industrial enzyme production, biological control field, microbial additives and medical and health fields on Bacillus subtilis applied research.[ Key words] Bacillus subtilis; application; development prospects1.1 枯草芽孢杆菌简介枯草芽孢杆菌，是芽孢杆菌属的一种。
玉米种植调查报告
玉米种植调查报告玉米种植调查报告玉米种植调查报告1玉米产业现状及问题浅析玉米产业：围绕玉米生产形成的产业链，包括玉米生产前的种子、及产出后在产品消费过程中涉及的饲料工业、食品工业、化工工业、医药工业、生物燃料等。
一、玉米产业概况1.玉米生产概况在世界谷物总产量中，玉米居第2位，仅次于小麦。
在世界经济发展中，拉美、非洲把玉米生产放在首位；而亚洲则放在水稻、小麦后的第3位。
玉米是种植最广泛的谷类作物，全世界有70多个国家，包括53个发展中国家种植玉米。
未来10～20年，世界玉米种植面积基本稳定，平均产量持续提高，总产量不断增长；玉米的用途将更加广泛，加工更精细；饲用玉米的数量将占有更大的比例；玉米在世界经济发展中的地位日趋重要。
目前，美国、中国、巴西、墨西哥、阿根廷是世界上最主要的玉米生产国，这五国的产量之和达到世界玉米总产量的70%以上。
从数据来看，五国的总产量仍在不断的上升当中。
20xx/11年度（7月到次年6月）全球玉米产量预计为8.253亿吨。
其中，美国玉米产量为3.302亿吨，相比之下，早先的预测为3.363亿吨，上年为3.33亿吨。
预计中国20xx年玉米产量为1.68亿吨，较上年增长2.5%或403万吨。
欧盟27国玉米产量可能达到5560万吨，低于早先预测的5770万吨，上年为5580万吨。
美国和中国的玉米产量占世界玉米总产的60%以上。
预计到20xx/2013年度，世界玉米种植面积将稳定在1.38亿至1.39亿公顷之间，单产将由4.56吨/公顷稳定增加到5.12公顷。
因单产的增加，总产量也稳定增加到8.2亿吨。
年平均增长1.34%。
20xx 年我国玉米的种植面积达到2898万公顷，较20xx年增加18万公顷。
预计20xx年中国玉米播种面积为3,056万公顷，较上年增加10万公顷，增幅为0.3%。
2.玉米消费情况自1999年起全球玉米总需求一直保持在6亿吨以上，并且呈刚性增长态势。
世界玉米消费主要有三个方面。
高考生物一轮复习 第30讲 微生物的利用夯基提能作业本(含解析)-人教版高三全册生物试题
微生物的利用A组基础过关1.根据分离以尿素为氮源的微生物实验,回答下列问题:(1)写出脲酶降解尿素的反应式:。
(2)该实验分离细菌时用浓度为10-4和10-5土壤稀释液接种,更容易形成单菌落的是。
(3)A同学筛选出的菌落为150个,其他同学只筛选出50个,并且他在设计实验时并没有设置对照。
你认为A同学结果产生的原因可能有哪些?。
如何设计对照实验排除可能影响实验结果的因素:。
(4)涂布平板的所有操作怎样保证无菌?。
(5)有脲酶的细菌在生态稳态上起什么作用?。
答案(1)CO(NH2)2+H2O 2NH3+CO2(2)10-5土壤稀释液(3)可能是土样不同,也可能是培养基污染或操作失误方案有二:一是由其他同学用与A同学一样的土壤进行实验,如果结果与A同学一致,则证明无误,如果结果不同,则证明A同学存在操作失误或培养基配制有问题;另一种方案是将A同学配制的培养基在不加土样的情况下进行培养,作为空白对照,以证明培养基是否受到污染(4)应从操作的各个细节保证“无菌”,都应在火焰附近进行。
例如,酒精灯与培养皿的距离要合适、移液管头不要接触任何其他物质、移液管要在酒精灯火焰周围等(5)如果土壤中有许多尿素,在自然界较高温度下会被水解,水解后的氨使土壤碱化,氨还与其他阴离子物质如S、C、N等形成化合物,则会使土壤板结。
有脲酶的细菌使植物废弃物和动物尸体降解,并利用了所形成的氨,有利于自然界中氮的循环解析脲即尿素,是蛋白质降解的产物。
有一些细菌含有脲酶可以分解尿素,利用尿素作为其生长的氮源。
本实验使用涂布分离法分离以尿素为氮源的微生物,用浓度为10-4和10-5土壤稀释液接种,更容易形成单菌落的是10-5土壤稀释液。
A同学结果产生的原因可能是土样不同,也可能是培养基污染或操作失误,排除方案有二:一是由其他同学用与A同学一样的土壤进行实验,结果与A同学一致,则证明无误,如果结果不同,则证明A同学存在操作失误或培养基配制有问题;另一种方案是将A同学配制的培养基在不加土样的情况下进行培养,作为空白对照,以证明培养基是否受到污染。
酶制剂——植酸酶
酶制剂——植酸酶早在1915年，Anderson提出天然植酸磷利用率不同于化学分离纯化产品的一个可能原因是饲料成分中存在水解植酸磷为无机磷的酶——植酸酶，并对植酸酶的来源、理化特性及作用机理进行了研究，从而引起了许多学者的广泛关注。
近年来，随着发酵工程和生物技术的迅速发展以及人们环境保护意识的提高，采用DNA重组技术使微生物产生植酸酶活性大幅度提高，大大降低了植酸酶生产成本，从而使之得到广泛应用。
植酸酶现已成为饲料酶制剂研究的一个热点，尤其在一些畜禽饲养密度大、环境污染严重的国家如美国、加拿大、芬兰、荷兰、法国、瑞士等。
许多科学家对这一课题的研究很感兴趣，欧洲、北美和其它地区对此的兴趣也与日俱增。
1994年欧共体、美国、芬兰、丹麦、德国等国的生产企业均前后推出各种植酸酶制剂,并利用DNA重组技术获得生产植酸酶的工程菌,为广泛应用植酸酶提供了可能。
一、植酸酶结构及性质植酸酶，又称为肌醇六磷酸水解酶，是一种可使植酸磷复合物中的磷变成可利用磷的酸性磷酸酯酶。
植酸酶广泛存在于动植物组织中，也存在于微生物（细菌、真菌和酵母）。
目前分离出的植酸酶主要有两种：3-植酸酶（EC 3.1.3.8）和6-植酸酶（EC 3.1.3.26），前者最先水解的是肌醇3号碳原子位置的磷酸根，主要存在于动物和微生物；后者最先水解的是6号碳原子的磷酸根，主要存在于植物组织。
因此，动物胃肠道可能有三种来源的植酸酶，但主要来源于饲料本身以及来源于微生物合成。
大量高浓度的植酸酶主要存在于无花果曲霉和黑曲霉与小麦麸的培养物中。
因此饲料植酸酶的生产目前主要使用微生物曲霉菌株。
霉菌植酸酶分子量一般在60 ~ 100KDal之间，曲霉植酸酶分子量较大。
如土曲霉为214Kdal，无花果曲霉为85 ~ 100KDal，黑曲霉为200KDal。
细菌植酸酶分子量一般较小，如大肠杆菌为42Kdal，枯草杆菌为38KDal。
霉菌植酸酶通常有一个最适pH，在4.2 ~ 5.5范围内。
淀粉酶生产
淀粉酶生产淀粉酶类的生产淀粉酶属于水解酶类，是催化淀粉(包括糖原，糊精)中糖苷键水解的一类酶的统称。
它是研究较多，生产最早，产量最大和应用最广泛的一种酶。
几乎占整个总产量的50,以上。
根据淀粉酶对淀粉的作用方式不同，淀粉酶可分为四种主要类型，即a-淀粉酶，β-淀粉酶，葡萄糖淀粉酶和异淀粉酶。
此外，还有一些应用不是很广泛，生产量不大的淀粉酶，如环状糊精生成酶，及α-葡萄糖苷酶等。
表5—1 淀粉酶的分类常用名作用特性存在 E.C编号系统名称不规则地分解淀粉唾液，胰脏，麦芽，α-1，4葡聚糖- α-淀粉酶，液化霉菌，细菌 E.C. 4-葡聚糖水解酶酶，淀粉-1， 4-糖原类物质的α-13.2.1.1 糊精酶，内断型4糖苷键淀粉酶E.C. α-1，4葡聚糖- Β-淀粉酶，淀粉从非还原性末端甘薯，大豆，大3.2.1.2 4-麦芽糖水解酶 -1，4-麦芽糖苷以麦芽糖为单位麦，麦芽等高等酶，外断型淀粉顺次分解淀粉，植物以及细菌等酶糖原类物质的α微生物-1，4糖苷键E.C. α-1，4葡聚糖葡糖化型淀粉酶，从非还原性末端霉菌，细菌，酵3.2.1.3 萄糖水解酶糖化酶，葡萄糖以葡萄糖为单位母等淀粉酶，淀粉-1，顺次分解淀粉，4-葡萄糖苷酶，糖元类物质的α淀粉葡萄糖苷酶 -1，4糖苷键E.C. 支链淀粉6-葡聚异淀粉酶，淀粉分解支链淀粉，植物，酵母，细3.2.1.9 糖水解酶 -1，6-糊精酶，糖元类物质的α菌R-酶，茁酶多糖-1，6糖苷键酶，脱支酶淀粉酶的种类不同，对直链淀粉和支链淀粉的作用方式也不一样。
各种不同的淀粉酶对淀粉的作用有各自的专一性。
淀粉是自然界中分布极广的碳水化合物，它是由葡萄糖基相连接聚合而成的，根据连接方式不同一般可将其分为直链淀粉和支链淀粉两种。
直链淀粉的葡萄糖基几乎都是以α-1，4键相互连接成的直连，聚合度为100—6000个葡萄糖单位不等，最近研究认为直链淀粉分子中也有极少量的分枝结构存在。
2008 world journal of microbiology and biotechnology
ORIGINAL PAPERPurification and characterization of a thermostable uricase from Microbacterium sp.strain ZZJ4-1Lei Kai ÆXiao-Hang Ma ÆXue-Lai Zhou ÆXiao-Ming Jia ÆXia Li ÆKang-Ping GuoReceived:10April 2007/Accepted:30June 2007/Published online:25July 2007ÓSpringer Science+Business Media B.V.2007Abstract In order to study the properties of a thermo-stable uricase produced by Microbacterium sp.strain ZZJ4-1,the enzyme was purified by ammonium sulfate precipitation and DEAE-cellulose ion exchange,hydro-phobic and molecular sieve chromatography.The molec-ular mass of the purified enzyme was estimated to be 34kDa by SDS-PAGE.The enzyme was stable between pH 7.0and 10.00.The optimal reaction temperature of the enzyme was 30°C at pH 8.5.The K m and K cat of the enzyme were 0.31mM and 3.01s –1,respectively.Fe 3+could enhance the enzyme activity,whereas Ag +,Hg 2+,o -phenanthroline and SDS inhibited the activity of the enzyme considerably.After purification,the enzyme was purified 19.7-fold with 31%yield.As compared with uri-cases from other microbial sources,the purified enzyme showed excellent thermostability and other unique char-acteristics.The results of this work showed that strains of Microbacterium could be candidates for the production of a thermostable uricase,which has the potential clinical application in measurement of uric acid.Keywords Uricase ÁThermostability ÁMicrobacterium .ÁPurification ÁCharacterization ÁUric acidIntroductionUricases (urate oxidoreductase;EC 1.7.3.3),which catalyse the oxidative breakdown of uric acid,belong to a group ofenzymes in the purine degradation pathway found in ani-mals (Keilin 1959;Wallrath and Friedman 1991),plants (Montalbini et al.1997),fungi (Montalbini et al.1999),yeasts (Adamek et al.1990;Hongoh et al.2000;Koyama et al.1996)and bacteria (Yamamoto et al.1996).Since Bongaert et al.(Bongaert et al.1978)found that Bacillus fastidiosus could produce uricase and use uric acid as the only carbon source for growth in 1978,many reports on bacterial and yeast uricases,such as those from Arthrob-acter globiformis (Nobutoshi et al.2000),Bacillus subtilis (Hunag and Wu 2004),Candida utilis (Liu et al.1994)and Pseudomonas aeruginosa (Ishikawa et al.2004),have been published.In the human body,uric acid is a final product of purine catabolism and is excreted out of the body by the kidney.When the level of uric acid in blood increases over the normal value,it can lead to a group of diseases,such as gout (Nakagawa et al.2006),idiopathic calcium urate nephrolithiasis (Masseoud et al.2005)and renal failure (Capasso et al.2005),and it was also reported that a high level of uric acid was related to leukemia in children (Larsen &Loghman-Adham 1996).Consequently,uric acid concentration is an important parameter monitored in urine and blood samples in routine clinical examinations.At present,the colorimetric method that employs uricase and peroxidase is widely accepted as a simple,sensitive and highly specific test for uric acid examination.In this enzymatic system of uric acid analysis,uricase plays an important role:uric acid À!uricase5Àhydroxyisourate þH 2O 2þCO 2By determining the amount of peroxide formed in the reaction,the concentration of uric acid can be estimated.For the best result in this examination,the properties of theL.Kai ÁX.-H.Ma (&)ÁX.-L.Zhou ÁX.-M.Jia ÁX.Li ÁK.-P.GuoCollege of Life Sciences,Zhejiang University,Yuhangtang Road 388#,Hangzhou,Chinae-mail:maxiaohong@World J Microbiol Biotechnol (2008)24:401–406DOI 10.1007/s11274-007-9489-1uricase in this reaction system are important,especially its stability,which will determine the precision of the mea-surement.At present,most enzymes applied in the clinical test are used in solution,and most proteins,including this enzyme,are relatively unstable when dissolved in aqueous solution.Therefore,for the enzymatic examination appli-cation,research was undertaken to search for a thermostable enzyme(Guo et al.2006;Huang et al.1998;Zhou et al.2005).As mentioned above,there were many uricases that have been isolated from microorganisms,but the thermostability of the published uricases were relatively low(Suzuki et al. 2004).The most thermostable uricase was reported by Suzuki,but it would lose its activity after a short period of treatment at60°C(Suzuki et al.2004).This low stability is a disadvantage in clinical applications.In a previous study,we isolated a bacterium Micro-bacterium sp.strain ZZJ4-1that produced a thermostable uricase.The enzyme was stable at65°C and its solution retained its original activity even after storage at37°C for 40days(Zhou et al.2005).Considering that this enzyme has a potential value in practical application,the present study was undertaken to purify and study the properties of this new enzyme.Materials and methodsMaterials and chemicalsThe culture of Microbaterium sp.(strain ZZJ4-1)was maintained in our laboratory(Zhou et al.2005).The protein standards for SDS-PAGE were purchased from Invitrogen(Shanghai,China).All other chemicals used were of reagent or molecular biology grade and pur-chased from Hangzhou Huadong Medicine Group Co.,Ltd (Hangzhou,China).Cultivation conditionsThe fermentation medium consisted of3.0g of uric acid, 10.0g of maize milk,0.5g of MgSO4Á7H2O,0.5g of KH2PO4,2.0g of K2HPO4Á3H2O,0.1g of NaCl,1.0l of tap water and the pH was adjusted to7.5(Zhou et al. 2005).To cultivate the strain for production of the uricase, a loop of bacteria from a slant was inoculated into a500ml flask containing100ml liquid medium and incubated at 30°C for30h with a rotary shaker at120rev/min.Enzyme assay and protein measurementThe principle of enzyme measurement was as follows: uricase can catalyse the oxidation of uric acid to form 5-hydroxyisourate and H2O2,which is then measured using a reaction system containing4-aminoantipyrine,phenol and peroxidase as chromogens.In practical analysis, 0.10ml enzyme solution was incubated with a mixture of 0.6ml0.1M sodium borate buffer(pH8.5)containing 2mM uric acid,0.15ml of30mM4-aminoantipyrine, 0.1ml of1.5%phenol and0.05ml peroxidase(15U/ml) at37°C for20min(Masaru1981).The reaction was stopped by addition of1.0ml ethanol and the absorbance at540nm was read against the blank in a spectropho-tometer.One unit of enzyme was defined as the amount of enzyme that produces1.0l mole of H2O2per minute under the standard assay conditions.The protein was measured by the Folin-phenol method (Lowry et al.1951).Enzyme purificationUnless otherwise stated,all of the enzyme purification processes were performed with0.1M phosphate buffer at pH7.0(buffer A)at4°C.Cells from10.0l of media were harvested by centrifu-gation and washed twice with50mM phosphate buffer(pH 7.5),re-suspended in buffer A and disrupted by ultrasonic oscillation(120W oscillating for3s with6s intervals, repeated100times).After the cell debris had been sepa-rated by centrifugation,solid ammonium sulfate was added to the enzyme solution and the precipitate of the fractions from55%to80%saturation was collected by centrifuga-tion(8000rpm,40min,4°C).The enzyme was then dis-solved in a small amount of buffer(2:1,volume of the buffer/weight of the precipitate),dialyzed against0.01M phosphate buffer until the ammonium sulfate was removed.The dialyzed enzyme solution was put onto a DEAE-Cellulose column(5.5·50cm)previously equilibrated with buffer A and it was then eluted with2.0l of buffer A with a linear gradient of0–1.5M KCl(flow rate:2ml/ min).The fractions containing enzyme activity were col-lected.After the ammonium sulfate had been added to65% saturation,the enzyme solution was loaded onto a Toyo-pearl HW-65column(5.5·85cm)equilibrated previ-ously with buffer A containing65%ammonium sulfate. The enzyme was eluted with2.0l of the same buffer with a linear gradient of ammonium sulfate from65%to0%(flow rate:2ml/min).When the fractions containing enzyme activity had been collected,the ammonium sulfate was added to get85%saturation.The precipitate of the enzyme was then collected by centrifugation,dissolved in a small amount of buffer A,dialysed with the same buffer con-taining0.1M KCl and applied to a Sephadex G-75column (5.5·100cm)equilibrated with the same buffer.The enzyme was then eluted with2.0l of the same buffer(flow rate:2ml/min)and the fractions containing the highest specific activity were collected for further study.Characterization of enzymeTo study the effect of pH on the activity of uricase,the enzyme was assayed at different pHs in the range from4.0 to11.0with intervals of0.5.The following buffers were used:100mM citrate for pH4.0–6.0,100mM phosphate for pH6.0–8.5and100mM borate for pH8.5–11.The pH stability was studied by incubating the purified enzyme solution in the corresponding buffers in the range from4.0 to11.0at25°C for18h and measuring the residual activity.To study the effect of temperature on the uricase activity,the standard enzyme reaction solution was pre-incubated at temperatures of20–60°C with5°C intervals for5min and the enzyme solution was then added and incubated for20min at the same temperature to measure its activity.For thermostability testing,the purified uricase solution was incubated at temperatures in the range from 20°C to80°C for30min and the remaining activity was then measured.The apparent K m of the uricase was estimated by the double reciprocal plot method.At different concentrations of uric acid,the enzyme activity was assayed and the Km was calculated by the Lineweaver-Burk plotting according to the Michaelis-Menten equation.To study the effects of chemicals on the uricase activity, the enzyme solution was pre-incubated with the chemicals for30min at room temperature in phosphate buffer and the remaining uricase activity was assayed with the standard reaction system containing the corresponding chemical.The relative molecular mass of the enzyme was deter-mined by SDS-PAGE with10%polyacrylamide gels (Laemmli1970).The purity was determined by the specific activity of the enzyme and PAGE with10%polyacryl-amide gels.The protein was stained with0.1%Coomassie brilliant blue R250in4:1:5methanol/acetic acid/water (vol/vol/vol)solution and destained in the same solution without dye.ResultsPurification of the enzymeIn thefirst step of the purification,ammonium sulfate precipitation was applied.The amount of protein and the enzyme activity of each fraction were measured.The fractions with ammonium sulfate concentrations from65% to80%had the highest enzyme specific activity(0.43U/ mg),while fractions from35–55,55–65to80–85had the specific activity of0.02U/mg,0.09U/mg and0.07U/mg, respectively.The fractions with concentrations from65% to80%were collected,dialyzed and loaded onto a DEAE-cellulose column.The enzyme was eluted with the same buffer containing a linear concentration gradient of KCl from0M to1.5M.Enzyme activity was found in fractions from75to130and fractions from80to105were pooled, solid ammonium sulfate was added to65%saturation and the solution was loaded onto a Toyopearl HW65-C col-umn.The enzyme was then eluted with the same buffer with a decreasing linear concentration of ammonium sul-fate from65%to0%.The enzyme activity was found in the fractions from100to160and fractions from110to135 were pooled,concentrated and applied to the Sephadex G-75column.After being washed with buffer A containing 0.1M NaCl,the enzyme activity was observed in the fractions from30to80.The fractions from50to60were pooled as purified enzyme for further study.The purity of the enzyme after each purification step was examined by SDS-PAGE and is shown in Figure1. The results of the purification process are summarized in Table1.During the purification,the enzyme was purified 19.7-fold with a recovery of31%and the purified enzyme had a specific activity of5.32U mg-1.Molecular mass determinationThe purified enzyme showed a single protein band in SDS-PAGE and its molecular mass was estimated to be34kDa (Fig.2).Optimum reaction temperature and thermostability ofthe purified enzymeThe purified enzyme was stable at a relative high temper-ature.As shown in Fig.3,it was stable at65°C and it retained64%of its original activity even after beingtreated Fig.1SDS-PAGE pattern of uricase samples from different steps of purification.(A)crude extract;(B)ammonium sulfate precipitation;(C)DEAE-cellulose chromatography;(D)Toyopearl HW-65chro-matography;(E)Sephadex G-75chromatographyat70°C for30min.By comparison between the optimal reaction and stable temperature,it was shown that although the enzyme was stable at65°C,its optimum temperature was30°C,which was relatively low,and it only showed 21%relative activity at60°C.Optimum pH and the stability of the enzymeat different pHsThe activity of the enzyme was measured in different buffers with a pH range from4.0to11.0.The results showed that the enzyme had low activity at pH below5.5 or over10.5and had relatively high activity in the range from7.0to10.0,with the optimal reaction pH at8.5 (Fig.4).As shown in Fig.4,after being incubated in dif-ferent buffers at25°C for18h,the uricase was stable in the pH range from5.5to9.5.Kinetics and effect of chemicals on the activityof the enzymeThe K m of the purified enzyme for uric acid was estimated to be0.31mM,which was calculated from the slopes and intercepts of the regression lines of the Lineweaver-Burk plot by determining the enzyme activity at37°C and the K cat of the enzyme was estimated to be3.01s–1.The effects of different chemicals on the activity of the enzyme are summarized in Table2.It was shown that among the metal ions,Li+,Ag+and Hg+greatly inhibitedTable1Summary of the uricase purification process Purification steps Activity(U)Total protein(mg)Specific activity(U mg–1)Purification(fold)Yeild(%)Crude enzyme6012220400.271100 Ammoniumsulfate488595190.51 1.9081 DEAE-cellulose338540840.83 3.0756 Toyopearl HW-6528451530 1.86 6.8947 Sephdax G-751866351 5.3219.7031 Fig.2SDS-PAGE electrophoregram of uricase.M:Standard ProteinMarker;U:Uricasethe enzyme activity.The strongest suppression was ob-served in the case of Hg+,which suppressed almost all of the activity of this enzyme.The chelating reagents had different effects;EDTA had no inhibitory effect,whereas o-phenanthroline(OPT)could inhibit the activity of uricase considerably.DiscussionAt present,the uricases from many microorganisms have been studied and some gene sequences of uricases have been cloned and studied.It was shown that uricases belong to a group of enzymes that have the same catalytic char-acter,but a great diversity of molecular structures.Uricases from different sources may have different molecular mas-ses and amino acid sequences.In this study,the molecular mass of the uricase from strain ZZJ4-1was estimated to be 34kDa by SDS-PAGE,whereas uricases produced by Candida utilis(Koyama et al.1996)and Pseudomonas aeruginosa(Ishikawa et al.2004)had the molecular mas-ses of34and54kDa,respectively.The apparent K m value of this uricase was0.31mM,while uricases from Ar-throbacter,Bacillus sp.and Candida sp.had K m values of 75,75,46l M,respectively(Suzuki et al.2004).Some relatively thermostable uricases,such as those produced by Arthrobacter globiformis FERM BP-360, Bacillus sp.TB-90and Candida utili s,have been studied and these were stable at55or60°C(Suzuki et al.2004). In this work,the uricase produced by strain ZZJ4-1showed good thermostability.After incubation at65°C for30min, the enzyme from strain ZZJ4-1still retained98.9%of its original activity,and even after incubation at70°C for 30min,the remaining activity was64%.Additionally,the pH stability of the enzyme from strain ZZJ4-1was a little broader than that of the uricases from the above three species(Suzuki et al.2004).The effects of metal ions on the enzyme activity from strain ZZJ4-1were also compared with the above three enzymes(Suzuki et al.2004).Considering that some uri-cases require metal ion cofactors for activity(Wu et al. 1989;Chu et al.1996)and whether or not an enzyme is inhibited by certain metals is an important characteristic, experiments were carried out to examine the effect of metals on the activity of this enzyme.It was shown that the enzyme from strain ZZJ4-1was not inhibited by Cu2+,Fe3+ or Zn2+,while uricases from the other three strains were strongly inhibited by these ions.Ag+is a strong inhibitor of the uricases from strain ZZJ4-1,Candida utilis and Bacillus sp.TB-90,while it had no such effect on the uricase from Arthrobacter globiformis FERM BP-360. When EDTA,a chelating reagent,was added to the enzyme solution at afinal concentration of20mM,the activity of the uricase was only slightly inhibited.When the stronger metal-ion-chelating reagent o-phenanthroline was added to the enzyme solution,the activity of the enzyme was strongly inhibited.This phenomenon indicates that some yet unidentified metal ion is strongly bound in the enzyme and forms part of the uricase structure,which is very important to keep its catalytic activity.This property is also different from the uricase of Arthrobacter globiformis FERM BP-360(Suzuki et al.2004).It was shown that although the uricase from the strain ZZJ4-1was stable at65°C,the enzyme was at its optimal activity at30°C and it only showed21%of its maximal activity at60°C.This implied that the molecular structure of enzyme had a reversible change at a temperature be-tween30°and60°C,which had a negative effect on its catalysis activity.But considering that most clinical enzy-matic examinations are 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