米根霉富马酸酶三维结构的同源建模与分析

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fuma基因富集解读

fuma基因富集解读
FUMA（Functional Mapping and Annotation of Genome-Wide Association Studies）是一个用于解读基因组关联研究（GWAS）结果的工具。

它通过将GWAS结果与基因组中的各种功能元素（如编码蛋白质的基因、非编码RNA、启动子、增强子等）进行比对，以找出与特定表型或疾病相关的基因和功能元素。

FUMA的主要功能包括富集分析、功能注释、基因网络分析等。

首先，FUMA可以进行富集分析，即确定在GWAS结果中是否存在特定功能元素的富集。

这可以帮助研究人员理解与特定表型或疾病相关的生物学过程和通路。

通过比对GWAS结果和基因组功能元素的位置，FUMA可以识别那些可能与疾病或表型相关的功能元素。

其次，FUMA还提供功能注释，即对GWAS结果中的基因进行功能注释，包括编码蛋白质的基因和非编码RNA。

这有助于识别与特定表型或疾病相关的基因，并进一步理解这些基因在生物学过程中的作用。

此外，FUMA还可以进行基因网络分析，帮助研究人员理解与特定表型或疾病相关的基因之间的相互作用和调控关系。

通过构建基
因网络，研究人员可以发现潜在的关键基因和通路，从而深入探究疾病的发病机制。

总之，FUMA是一个强大的工具，可以帮助研究人员解读GWAS 结果，发现与特定表型或疾病相关的基因和功能元素，并深入理解它们在生物学过程中的作用。

通过FUMA的使用，研究人员可以更全面地理解复杂疾病的遗传基础，为疾病的预防、诊断和治疗提供重要参考。

生物化学笔记

生物化学（Biochemistry）刘国琴参考书介绍1. 王镜岩朱圣庚徐长法 (2002) 生物化学2. 张曼夫（2002）生物化学中国农业大学出版社3. Albert L. Lehninger (1993)Principles of Biochemistry4. Lubert Stryer (1998) Biochemistry什么是生物化学？生物化学是生命的化学。

研究生命体的化学组成与化学变化。

用化学术语解释生命的本质。

生命体共有特征新陈代谢遗传、进化、变异生长、发育、繁殖生物化学组成的同一性相对稳定的内环境严整的结构对环境的感应性和适应性生命--是物质运动的高级形式，它建立在物理、化学规律之上，但又不能完全归结为物理、化学规律。

生命是以蛋白质和核酸为主的高度有序的多分子体系，是能进行新陈代谢和自我繁殖的开放系统。

生物化学I （50学时）教学内容第一章生物化学导论第二章糖类第三章蛋白质I：蛋白质的组成第四章蛋白质II：蛋白质的结构与功能第五章蛋白质III：蛋白质的性质、分离与鉴定第六章酶第七章核酸化学第八章维生素与辅酶、辅基第九章脂类与生物膜第十章生物能学与生物氧化第一章生物化学导论一、生物化学学科的建立和发展二、生命物质特征及生物化学反应类型三、水—生命的溶剂一、生物化学学科的建立和发展（一）生物化学发展简史十八世纪下半叶法国化学家Lavoisier（1783）发现生物氧化过程十九世纪生物化学进展缓慢（德国领先）J.von Liebig (1803-1873) 新陈代谢（1842）Hoppe-Seyler (1825-1895) Biochemie（1877）二十世纪初德、美、英、法生物化学发展生物化学领域三大发现：酶、维生素、激素生物化学作为一个独立学科出现！二十世纪中叶生物化学快速发展原因：1. 生物化学研究方法的改进2. 跨学科的合作研究美国生物学家W atson , 英国物理学家Crick：1953 DNA双螺旋（1962获诺贝尔奖）英国化学家 Sanger : 牛胰岛素结构（1958）英国物理学家 Kendrew: 解析肌红蛋白结构（1962）美国生物化学家Nirenberg：破遗遗传密码（1969）美国生物化学家 Holly：解析 tRNA结构（1969）法国生物学家 Jocob, Monod：遗传信息流（1965）英国化学家 Sanger : DNA测序（1983）（二）现代生物化学不断取得新进展光合作用机理酶作用机理生物固氮机理核酸、蛋白质三维空间结构基因克隆基因表达基因调控最新生物学研究成果激动人心新概念新知识新技术人类基因组→后基因组→功能基因组→蛋白质组pseudogeneT-DNA mutantRNAiMALDI-TOF-MS（三）生物化学与其它学科的关系生物化学是生物学科的专业基础课！生物化学的现代进展为分子生物学奠定了基础，对发育生物学、分子进化、细胞生物学、遗传学产生了重要影响促使许多边缘学科诞生：量子生物学、结构生物学、生物信息学、生物系统学21世纪生命科学的特点（1）生命科学是21世纪自然科学的前沿学科–美国2000年对不同学科投入●生命科学49％●物质科学11.36%●环境科学8.06%●数学与计算机科学 5.77%●心理科学 4.57%●21世纪生命科学的特点（2）生物学与多学科发生交叉●21世纪生命科学的特点（3）分析与综合相结合大分子相互作用（四）我国科学家在生物化学领域中的贡献1965 生化所与有机化学所人工合成有功能的蛋白质－牛胰岛素1973 X－射线分析出猪胰岛素空间结构1983 酵母丙氨酸转移核糖核酸的人工全合成(tRNAAla)2002 水稻基因组测序●我国生物学研究相对滞后（1992-2001年SCI论文占2.15%，生物论文占0.89%）―中国人离诺贝尔奖仅一步之遥‖原创性分析问题、解决问题的能力基础知识（五）学好生化，勇攀高峰！二、生命物质特征及生物化学反应类型（一）生命体的化学组成大量元素 H O N C S P Cl Na K Ca微量元素Mg V Mn Fe Co Ni Cu Zn Se Mo I元素含量排序（％）人体H（63） O（25.5） C（9.5）N（1.4） Ca（0.31 P（0.22） Cl（0.08）K（0.06）海水H（66） O（33） Cl（0.33） Na（0.28） Mg（0.033） S（0.017） Ca（0.0062）K（0.0060） C（0.0014）地壳O（47） Si（28）Al（7.9） Fe（4.5） Ca（3.5） Na（2.5） K（2.5） Mg（2.2）生命来自海洋！化学元素—原始物质—生物分子Oparin(1922)生物起源假说：地球上的生命是由非生命物质通过长期的化学进化逐步演变而来的。

鲍曼不动杆菌UDP-葡萄糖4-差向异构酶Gne1的表征

JournalofChinaPharmaceuticalUniversity2021，52（6）：742-750

学报

鲍曼不动杆菌UDP-葡萄糖4-差向异构酶Gne1的表征张展1，冯雁1，李谦2*，崔莉1**

（1上海交通大学生命科学技术学院微生物代谢国家重点实验室，上海200240；2中国药科大学生命科学技术学院，南京210009）

摘要异源表达鲍曼不动杆菌AB0057的UDP-葡萄糖4-差向异构酶并表征其酶学性质以及分析其结构与功能。将异构酶基因构建到pET-28a表达载体并在大肠埃希菌BL21（DE3）中异源表达，使用高效液相色谱检测酶活力及表征酶学性质。系统发育分析、序列比对、同源建模与分子对接分析其结构与关键催化位点。结果显示，重组酶Gne1获得可溶性表达，质量约为38.9kD，催化最适温度为44℃，最适pH为6.0，米氏常数KM与催化常数kcat分别为（1.227±0.0824）mmol/L和（82.64±3.562）×10-3•min-1。该酶属于NADB_Rossmann超家族并分属于UDP_G4E_1_SDR_e亚组，分别具有典型GXGXXG基序与YXXXK基序。N端结构域与NAD结合，而C端结构域用来结合底物，催化关键位点为S125和Y150。本研究验证了Gne1的差向异构酶活性，阐释了其序列特点和结构特征，揭示了其与底物、辅因子的结合模式，分析了关键催化位点。为蛋

白质工程改造提高酶活力进而利用生物酶法合成稀有功能糖提供了理论依据。关键词差向异构酶；酶学性质表征；序列及结构分析；稀有功能糖合成中图分类号Q558+.1；Q71；Q814.1文献标志码A文章编号1000-5048（2021）06-0742-09doi：10.11665/j.issn.1000-5048.20210613

引用本文张展，冯雁，李谦，等.鲍曼不动杆菌UDP-葡萄糖4-差向异构酶Gne1的表征［J］.中国药科大学学报，2021，52（6）：742–750.Citethisarticleas：ZHANGZhan，FENGYan，LIQian，etal.CharacterizationofaUDP-glucose4-epimerasefromAcinetobacterBaumannii［J］.JChinaPharmUniv，2021，52（6）：742–750.

细胞生物学习题

翟中和细胞生物学(2000版)配套习题第一章：绪论填空题：细胞生物学是细胞整体、超微结构和分子水平上研究及其规律的科学。

名词解释：1、细胞学讲〔celltheory〕3、选择题：1、现今世界上最有碍事的学术期刊是。

a：Natuneb:Cellc:PNASd:Science2、自然界最小的细胞是〔a〕病毒〔b〕支原体〔c〕血小板〔d〕细菌4、是非题：1、现代细胞生物学的全然特征是把细胞的生命活动和亚细胞的分子结构变化联系起来。

…………………………〔〕5、咨询答题：1.当前细胞生物学研究的热点课题哪些？2.细胞学讲的全然要点是什么？细胞学讲在细胞学开展中有什么重大意义？3.细胞生物学的开展可划分为哪几个时期？各时期的要紧特点是什么？第二章：细胞全然知识概要1、名词解释：1.血影〔Ghost〕2.通道形成蛋白〔Porin〕3.纤维冠〔fibrouscorona〕2、选择题：1、立克次氏体是〔a〕一类病毒〔b〕一种细胞器〔c〕原核生物〔d〕真核生物2、原核细胞的呼吸酶定位在〔a〕细胞质中〔b〕质膜上〔c〕线粒体内膜上〔d〕类核区内3、最小的细胞是〔a〕细菌〔b〕类病毒〔c〕支原体〔d〕病毒4、在英国引起疯牛病的病原体是：〔a〕朊病毒〔prion〕〔b〕病毒〔Virus〕〔c〕立克次体〔rickettsia〕〔d〕支原体〔mycoplast〕5、逆转病毒〔retrovirus〕是一种〔a〕双链DNA病毒〔b〕单链DNA病毒〔c〕双链RNA病毒〔d〕单链RNA病毒6、英国疯牛病病原体是〔a〕DNA病毒〔b〕RNA病毒〔c〕类病毒〔d〕朊病毒7、线虫基因组的全序列测定目前已接近尾声，发觉其一共约有〔〕种的编码基因〔a〕6000〔b〕10000〔c〕20000〔d〕500008、原核细胞与真核细胞虽有许多不同，但根基上〔a〕核仁〔b〕核糖体〔c〕线粒体〔d〕内质网9、前病毒是〔a〕RNA病毒〔b〕逆转录RNA病毒RNA病毒〔c〕整合到宿主DNA中的逆转录DNA〔d〕整合到宿主DNA中的DNA病毒3、是非题：1.类病毒仅由裸露的DNA所构成，不能制造衣壳蛋白。

生物信息学实验报告

生物信息学实验报告班级：：学号：日期：实验一核酸和蛋白质序列数据的使用实验目的了解常用的序列数据库，掌握基本的序列数据信息的查询方法。

教学基本要求了解和熟悉NCBI 核酸和蛋白质序列数据库，可以使用BLAST进行序列搜索，解读BLAST 搜索结果，可以利用PHI-BLAST 等工具进行蛋白质序列的结构域搜索，解读蛋白质序列信息，可以在蛋白质三维数据库中查询相关结构信息并进行显示。

实验容提要在序列数据库中查找某条基因序列（BRCA1），通过相关一系列数据库的搜索、比对与结果解释，回答以下问题：1. 该基因的基本功能？2. 编码的蛋白质序列是怎样的？3. 该蛋白质有没有保守的功能结构域 (NCBI CD-search)？4. 该蛋白质的功能是怎样的？5. 该蛋白质的三级结构是什么？如果没有的话，和它最相似的同源物的结构是什么样子的？给出示意图。

实验结果及结论1. 该基因的基本功能？This gene encodes a nuclear phosphoprotein that plays a role in maintaining genomic stability, and it also acts as a tumor suppressor. The encoded protein combines with other tumor suppressors, DNA damagesensors, and signal transducers to form a large multi-subunit protein complex known as the BRCA1-associated genome surveillance complex (BASC). This gene product associates with RNA polymerase II, and through the C-terminal domain, also interacts with histone deacetylase complexes. This protein thus plays a role in transcription, DNA repair of double-stranded breaks, and recombination. Mutations in this gene are responsible for approximately 40% of inherited breast cancers and more than 80% of inherited breast and ovarian cancers. Alternative splicing plays a role in modulating the subcellular localization and physiological function of this gene. Many alternatively spliced transcript variants, some of which are disease-associated mutations, have been described for this gene, but the full-length natures of only some of these variants has been described. A related pseudogene, which is also located on chromosome 17, has been identified. [provided by RefSeq, May 2009]2. 编码的蛋白质序列是怎样的？[Homo sapiens]1 mdlsalrvee vqnvinamqk ilecpiclel ikepvstkcd hifckfcmlk llnqkkgpsq61 cplcknditk rslqestrfs qlveellkii cafqldtgle yansynfakk ennspehlkd121 evsiiqsmgy rnrakrllqs epenpslqet slsvqlsnlg tvrtlrtkqr iqpqktsvyi181 elgsdssedt vnkatycsvg dqellqitpq gtrdeislds akkaacefse tdvtntehhq241 psnndlntte kraaerhpek yqgssvsnlh vepcgtntha sslqhenssl lltkdrmnve301 kaefcnkskq pglarsqhnr wagsketcnd rrtpstekkv dlnadplcer kewnkqklpc361 senprdtedv pwitlnssiq kvnewfsrsd ellgsddshd gesesnakva dvldvlnevd421 eysgssekid llasdpheal ickservhsk svesniedki fgktyrkkas lpnlshvten481 liigafvtep qiiqerpltn klkrkrrpts glhpedfikk adlavqktpe minqgtnqte541 qngqvmnitn sghenktkgd siqneknpnp ieslekesaf ktkaepisss isnmelelni601 hnskapkknr lrrksstrhi halelvvsrn lsppnctelq idscssseei kkkkynqmpv661 rhsrnlqlme gkepatgakk snkpneqtsk rhdsdtfpel kltnapgsft kcsntselke721 fvnpslpree keekletvkv snnaedpkdl mlsgervlqt ersvesssis lvpgtdygtq781 esisllevst lgkaktepnk cvsqcaafen pkglihgcsk dnrndtegfk yplghevnhs 841 retsiemees eldaqylqnt fkvskrqsfa pfsnpgnaee ecatfsahsg slkkqspkvt 901 feceqkeenq gknesnikpv qtvnitagfp vvgqkdkpvd nakcsikggs rfclssqfrg 961 netglitpnk hgllqnpyri pplfpiksfv ktkckknlle enfeehsmsp eremgnenip 1021 stvstisrnn irenvfkeas ssninevgss tnevgssine igssdeniqa elgrnrgpkl 1081 namlrlgvlq pevykqslpg snckhpeikk qeyeevvqtv ntdfspylis dnleqpmgss 1141 hasqvcsetp ddllddgeik edtsfaendi kessavfsks vqkgelsrsp spfththlaq 1201 gyrrgakkle sseenlssed eelpcfqhll fgkvnnipsq strhstvate clsknteenl 1261 lslknslndc snqvilakas qehhlseetk csaslfssqc seledltant ntqdpfligs 1321 skqmrhqses qgvglsdkel vsddeergtg leennqeeqs mdsnlgeaas gcesetsvse 1381 dcsglssqsd ilttqqrdtm qhnliklqqe maeleavleq hgsqpsnsyp siisdssale 1441 dlrnpeqsts ekavltsqks seypisqnpe glsadkfevs adsstsknke pgversspsk 1501 cpslddrwym hscsgslqnr nypsqeelik vvdveeqqle esgphdltet sylprqdleg 1561 tpylesgisl fsddpesdps edrapesarv gnipsstsal kvpqlkvaes aqspaaahtt 1621 dtagynamee svsrekpelt astervnkrm smvvsgltpe efmlvykfar khhitltnli 1681 teetthvvmk tdaefvcert lkyflgiagg kwvvsyfwvt qsikerkmln ehdfevrgdv 1741 vngrnhqgpk raresqdrki frgleiccyg pftnmptdql ewmvqlcgas vvkelssftl 1801 gtgvhpivvv qpdawtedng fhaigqmcea pvvtrewvld svalyqcqel dtylipqiph 1861 shy3. 该蛋白质有没有保守的功能结构域 (NCBI CD-search)？有保守的供能结构域。

生物化学问答题(附答案)

生物化学解答题(一档在手万考不愁)整理:机密下载有淀粉酶制剂1g，用水溶解成1000ml酶液，测定其蛋白质含量和粉酶活力。

结果表明，该酶液的蛋白质浓度为0.1mg/ml；其1ml的酶液每5min 分解0.25g淀粉，计算该酶制剂所含的淀粉酶总活力单位数和比酶活（淀粉酶活力单位规定为：在最适条件下，每小时分解1克淀粉的酶量为一个活力单位）。

答案要点：①1ml的酶液的活力单位是×（2分）酶总活力单位数是3×1000=3000U（1分）②总蛋白是0.1×1000=100mg（1分）,比活力是（1分）。

请列举细胞内乙酰CoA的代谢去向。

（5分）答案要点：三羧酸循环；乙醛酸循环；从头合成脂肪酸；酮体代谢；合成胆固醇等。

（各1分）酿酒业是我国传统轻工业的重要产业之一，其生化机制是在酿酒酵母等微生物的作用下从葡萄糖代谢为乙醇的过程。

请写出在细胞内葡萄糖转化为乙醇的代谢途径。

答案要点：在某些酵母和某些微生物中，丙酮酸可以由丙酮酸脱羧酶催化脱羧变成乙醛，该酶需要硫胺素焦磷酸为辅酶。

乙醛继而在乙醇脱氢酶的催化下被NADH 还原形成乙醇。

葡萄糖+2Pi+2ADP+2H+生成2乙醇+2CO2+2ATP+2H2O（6分）脱氢反应的酶：3-磷酸甘油醛脱氢酶（NAD＋），醇脱氢酶（NADH+H+）（2分）底物水平磷酸化反应的酶：磷酸甘油酸激酶，丙酮酸激酶（Mg2+或K+）（2分）试述mRNA、tRNA和rRNA在蛋白质合成中的作用。

答案要点：①mRNA是遗传信息的传递者，是蛋白质生物合成过程中直接指令氨基酸掺入的模板。

（3分）②.tRNA在蛋白质合成中不但为每个三联体密码子译成氨基酸提供接合体，还为准确无误地将所需氨基酸运送到核糖体上提供运送载体。

(4分)③.rRNA与蛋白质结合组成的核糖体是蛋白质生物合成的场所（3分）。

物合成过程中直接指令氨基酸掺入的模板。

（3分）②.tRNA在蛋白质合成中不但为每个三联体密码子译成氨基酸提供接合体，还为准确无误地将所需氨基酸运送到核糖体上提供运送载体。

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 undertaken at25to37°C,this property will not affect its application in clinical exami-nation.ReferencesAdamek V,Suchova M,Demnerova K et al(1990)Fermentation of Candida utilis for uricase production.J Indu Microbiol6:85–90 Bongaerts GP,Uitzetter J,Brouns R et al(1978)Uricase of Bacillus fastidiosus.Properties and regulation of synthesis.Biochim Biophys Acta527:348–358Capasso G,Jaeger Ph,Robertson WG et al(2005)Uric acid and the kidney:urate transport,S tone disease and progressive renal failure.Curr Pharm Des11:4153–4159Table2Effects of chemicals on uricase activityChemicals Final Concentration Residual activity(%)Blank–100Fe3+1mM118.5Ca2+1mM107.7Ba2+1mM103.1Mn2+1mM101.5Zn2+1mM100.7Cu2+1mM98.9Li+1mM81.5Ag+1mM7.5Hg2+1mM 1.8NaN320mM101.2EDTA20mM99.8o-Phenanthroline2mM 1.2Tween200.10%(w/v)99.7Tween800.10%(w/v)80.5Triton X-1000.10%(w/v)99.7SDS0.50%(w/v)51.3Chu R,Lin Y,Usuda N,Rao MS et al(1996)Mutational analysis of the putative copperbinding site of rat urate oxidase.Ann NY Acad Sci804:777–780Guo K,Ma X,Sun G et al(2006)Expression and characterization of a thermostable sarcosine oxidase(SOX)from Bacillus sp.in Escherichia coli.Appl Microbiol Biotechnol73:559–566 Hongoh Y,Sasaki T,Ishikawa H(2000)Cloning,sequence analysis and expression in Escherichia coli of the gene encoding a uricase from the yeast-like symbiont of the brown planthopper, Nilaparvata lugens.Insect Biochem Mol Biol30:173–182 Huang HS,Kabashima T,Ito K et al(1998)Thermostable glycerol kinase from Thermusﬂavus:cloning,sequencing,and expression of the enzyme gene.Biochim Biophys Acta1382:186–190 Hunag S,Wu T(2004)Modiﬁed colorimetric assay for uricase activity and a screen for mutant Bacillus subtilis uricase genes following StEP mutagenesis.Eur J Biochem271:517–523 Ishikawa J,Yamashita A,Mikami Y et al(2004)The complete genomic sequence of Nocardia farcinica IFM10152.Proc Natl Acad Sci USA101:14925–14930Keilin J(1959)The biological signiﬁcance of uric acid and guanine excretion.Biol Rev34:265–296Koyama Y,Ichikawa T,Nakano E(1996)Cloning,sequence analysis, and expression in Escherichia coli of the gene encoding the Candida utilis urate oxidase(uricase).J Biochem(Tokyo), 120:969–973Laemmli UK(1970)Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature227:680–685 Larsen G,Loghman-Adham M(1996)Acute renal failure with hyperuricemia as initial presentation of leukemia in children.J Pediatr Hematol Oncol18:191–194Liu J,Li G,Liu H et al(1994)Puriﬁcation and properties of uricase from Candida sp.and its application in uric acid analysis in serum.Appl Biochem Biotechnol47:57–63Lowry OH,Rosebrough NJ,Farr AL et al(1951)Protein measure-ment with the Folin Phenol reagent.J Biol Chem193:265–275Masaru P(1981)Puriﬁcation and some properties of Sarcosine Oxidase from Corynebacterium sp.U-96.J Biochem89:599–607 Masseoud D,Rott K,Liu-Bryan R et al(2005)Overview of hyperuricaemia and gout.Curr Pharm Des11:4117–4124 Montalbini P,Aguilar M,Ineda M(1999)Isolation and character-ization of uricase from bean leaves and its comparison with uredospore enzyme.Plant Sci147:139–147Montalbini P,Redondo J,Caballero JL(1997)Uricase from leaves: its puriﬁcation and characterization from three different higher plants.Planta202:277–283Nakagawa T,Mazzali M,Kang DH et al(2006)Uric acid-a uremic toxin?Blood Purif24:67–70Nobutoshi K,Keisuke S,Takao M et al(2000)Determination of uric acid in plasma by closed-loopﬁa with a coimmobilized enzyme ﬂow cell.Anal Sci16:1203–1205Suzuki K,Sakasegawa SI,Misaki H et al(2004)Molecular cloning and expression of uricase gene from Arthrobacter globiformis in Escherichia coli and characterization of the gene product.J Biosci Bioeng98:153–158Wallrath LL,Friedman TB(1991)Species differences in the temporal pattern of Drosophila urate oxidase gene expression are attrib-uted to trans-acting regulatory changes.Proc Natl Acad Sci USA 88:5489–5493Wu XW,Lee CC,Muzny DM et al(1989)Urate oxidase:primary structure and evolutionary implications.Proc Natl Acad Sci USA 86:9412–9416Yamamoto K,Kojima Y,Kikuchi T et al(1996)Nucleotide sequence of the uricase gene from Bacillus sp.TB-90.J Biochem119:80–84Zhou XL,Ma XH,Sun GQ(2005)Isolation of a thermostable uricase-producing bacterium and study on its enzyme production conditions.Process Biochem40:3749–3753。

纤维素的微生物实验报告

一、实验目的1. 了解纤维素酶的来源及作用机理。

2. 探讨不同微生物对纤维素的降解效果。

3. 分析影响纤维素降解的因素。

二、实验材料1. 菌株：枯草芽孢杆菌、曲霉、黑曲霉、白腐真菌等。

2. 纤维素酶：纤维素酶原液、纤维素酶活力测定试剂盒。

3. 培养基：纤维素培养基、LB培养基、PDA培养基等。

4. 仪器：恒温培养箱、高压蒸汽灭菌器、离心机、显微镜等。

三、实验方法1. 菌株活化：将保存的菌株接种于LB培养基，37℃培养24小时，得到活化菌株。

2. 纤维素酶活力测定：采用酶活力测定试剂盒，按照说明书操作，测定不同菌株产生的纤维素酶活力。

3. 纤维素降解实验：将活化菌株接种于纤维素培养基，37℃培养24小时，观察菌株对纤维素的降解效果。

4. 影响因素分析：分别考察pH值、温度、接种量等因素对纤维素降解效果的影响。

四、实验结果与分析1. 纤维素酶活力测定结果表1 不同菌株纤维素酶活力测定结果菌株名称纤维素酶活力（U/g）枯草芽孢杆菌 0.20曲霉 0.30黑曲霉 0.35白腐真菌 0.40由表1可知，白腐真菌产生的纤维素酶活力最高，其次是黑曲霉、曲霉和枯草芽孢杆菌。

2. 纤维素降解实验结果表2 不同菌株纤维素降解效果菌株名称纤维素降解率（%）枯草芽孢杆菌 25.0曲霉 35.0黑曲霉 40.0白腐真菌 50.0由表2可知，白腐真菌对纤维素的降解效果最好，其次是黑曲霉、曲霉和枯草芽孢杆菌。

3. 影响因素分析（1）pH值：在pH值4.0-8.0范围内，纤维素降解率随pH值升高而增加，当pH 值超过8.0时，降解率逐渐降低。

（2）温度：在37℃时，纤维素降解效果最好，当温度超过50℃时，降解效果明显下降。

（3）接种量：接种量在1%时，纤维素降解率最高，超过1%时，降解率逐渐降低。

五、结论1. 白腐真菌对纤维素的降解效果最好，其次是黑曲霉、曲霉和枯草芽孢杆菌。

2. 纤维素降解效果受pH值、温度、接种量等因素的影响。

米氏方程

米氏方程（Michaelis-Menten Equation）或米曼氏动力学（Michaelis-Menten kinetics）是由Leonor Michaelis和Maud Menten在1913年提出，是酶学中极为重要的可以描述多种非变异构酶动力学现象、表示一个酶促反应的起始速度V （有些资料中也称为Vo）与底物浓度[S]关系的速度方的方程，米氏方程形式如下：
其中，Vmax表示酶被底物饱和时的反应速度，Km值称为米氏常数，是酶促反应速度V为最大酶促反应速度值一半时的底物浓度。

在酶促反应中，底物情况在低浓度下，反应相对于底物是一级反应（first order reaction）；而当底物浓度处于中间范围时，反应（相对于底物）是混合级反应（mixed order reaction）；当底物浓度增加时，反应由一级反应向零级反应（zero order reaction）过渡；当底物浓度[S]逐渐增大时，速度V相对于[S]的曲线为一双曲线。

下图为米氏方程的模拟作图：
酶促反应中的米氏常数的测定和Vmax的测定有多种方法。

比如固定反应中的酶浓度，然后测试几种不同底物浓度下的起始速度，即可获得Km和Vmax值。

但直接从起始速度对底物浓度的图中确定Km或Vmax值是很困难的，因为曲线接近Vmax时是个渐进过程。

因此，通常情况下，我们都是通过米氏方程的双倒数形式来测定，即Lineweaver-Burk plot，也可称为双倒数方程(double-reciprocal plot)：
将1/V 对1/[S]作图，即可得到一条直线，该直线在Y轴的截距即为1/Vmax，在X轴上的截距即为1/Km的绝对值。

示意图如下：。

植物炭疽病真菌金属硫蛋白的三级结构预测

１方法构建
距离几何算法【＿ｌ川是一种用原子间的相对距０
离表示三维结构信息的方法．它根据Ｂｍｅｔ定ｌｎｎｈ＇ａ理对原子间的最大距离矩阵——上限矩阵和最小距离矩阵——下限矩阵进行修饰，以排除那些违背三一讯联通系ｋ
． ቤተ መጻሕፍቲ ባይዱ
纪才金醵重人基和 ★
Ｔ：１６？２．，ｌＨｙｌ出Ｌｅ０ —・４Ｆ：＠。耐ｌｌ０２２３７ｔ收期：０－－，受日２１００稿日２１７０接期：０ —－００３０１３
Ｏ５，（３．．１；９５１９学科分类号
植物炭疽病真菌孢子发芽时在芽管顶端长出一种称为附着器的结构，粘着于寄主表皮，并于其下长出侵入丝，侵入寄主表皮细胞．对于这一过程的形态和生理方面的研究已经比较深入，然而其分子机理，特别是孢子发芽和分化过程中寄主植物信号分子的作用机制还不清楚．Ｈａｇ等从被鳄梨表ｗｎ层蜡质诱导的附着器中，筛选到仅在附着器形成过程中表达的三个ｅＮＡ．其中一个ｅＡ表达生成ＤＤＮ的，由２６个氨基酸组成的小蛋白质称为ＣＰ．Ａ３ＣＰ富含半胱氨酸，与粗糙麦孢菌的铜诱导ＭＴＡ３
（清华大学生物科学与技术系，生物膜与膜生物工程国家重点实验室，北京１０８）００４
摘耍根据实验钡定的Ｉ类金属硫蛋白（ｔｌｈｏｅ，）三级结构的实验数据，给出该类蛋白质的两种特ＩｍｅｌｔｌｎｌＭＴａｏｎ
征结构（Ｘ、ｃｘ－￣ＣＣｘｃ
０４ｌ．２ｒｎ的正四面体．Ｉ＇

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中图分类号：９９５Ｔ３１Ｑ３．。Ｐ９文献标识码：Ａ文章编号：６２５５２０）０ —９ —０１７ —５６（０ｒ一３ｒＩ７７５
Ｈｏｌｇｈｅ．ｉｅｓｏａｔｒｅ— ｄｍｎｉｎｌｓｒｃｕｅｍｏｅｉｆｆｎｕｒｓｆｏＲｈＴｕｒｚｅａｄｉｎｌｓｒｍｉ￣ｐｓｏｙａｎｓａａｙｉｔｓ
ｔａｆｒｅｆ￣ｈｚｐｓｒａｉｓｌｎｉｓｆｈｅｄｍｉ，ａｄｔ呲ｄｍｉｉｃ０ｅｆｅｒｒｌｌ一｝Ｘ．ｈｔｌ￣ｘｎＲｌｕｙｅｉｔｓｔｙｏｓｔｏｔｅｏａｓｎｅｏｍａｉｉｏｏｚｎｈＩｃｓｒｄｎｈｏａ０ｅｄｏ５ｎａｙｐａｅａ￣Ｌｎｓ蚰ｐｌａｌｓｅ
维普资讯
生物信息学
ＣｍｕｎｌｆｉｎｒａｃｈａｏｒｏｆｍｔｓＪａｏＢｉｏｉ
研究论文
米根霉富马酸酶三维结构的同源建模与分析
张昆，黄和，宁，刘高振，明杰，金韦萍
ｌ￣ｖｎｅｒｌｂｌｙｏｔｅｒｓｌａｒｖｄｈｏｉｎｃｉｅｓｅｆｌｒａｔｄｅａｅ［ｔｅｒｓｔ．Ｔｅｒｓｌｈｗｌ７７ａｄｔｅａｉｔｆｈｕｔｗｓｐｏｅ，ｔｅｄｍａｎａｄａｔｉｓｏｆｍａ￣ｅｗｓｓｉｄｂｓｄ０Ｉｈｕｓｅｈｉｉｅｓｖｔｉｕｅｌｈｅｕｔｓｏｓ
结构，并对二者的立体构象和催化活性位点进行了深入的研究【７４Ｊ＿。然而，针对ｆｒ立体结构方面的ｕｏｒ
题，微生物发酵法替代传统的化学法已大势所趋。而以米根霉（ｈｚｕｏＲ／ｐｓ珊）ｏ为代表的丝状真菌是当
前生产富马酸的热门微生物。在米根霉产生富马酸的代谢过程中，三羧酸循环还原途径为主要的产酸途径，中催化苹果酸生成富马酸的富马酸酶是该其过程的一个关键酶ｕ３。－］该酶与线粒体的富马酸酶同功酶都属于第二类富马酸酶４－。除了米根霉富马酸酶（ｆｒ表以ｕｏｒ表示）以及大肠杆菌ｆＣ基因编码的富马酸酶（ｕｍ以
（南京工业大学制药与生命科学学院，南京。１０）２０９０
摘要：利用Ｍ￣ｌｒｖｏｌ７７软件对米根霉（］ｕｏｅ富马酸酶（ｅＲｕｓｒ）￣ｐ￣ａ
）进行了三级结构的同源建模并对结果的空间和能量
上的合理性进行了验证，步对酶的结构域和催化活性位点进行了研究。结果表明：进一富马酸酶由三个结构域组成，中心区
ｔｅｃｈ五ｃｍｅｈｎｓｏｉｍａ￣ｅａｄＩａｔｅｓｅ一叩ｅｃｍｕａｅｅｌｔｒｖｈｒｄｃｉｎｏｆ￣ｃａｉｔｒ．ｃａｉｍｆｆｒｎｌｌｈｉｌＩｔｃｔｇｎｓｏｉｏｅｔｅｐｏｕｔｆｍｓｎｏｉｃｄｉｆｕｅｎｕ
Ｋｅｏｄ：，ｒｓ；Ｒｉｐｓｏｙａ；ｓｕｔ￣；ｈｍｏｇｄｌｇａｔｉｙＷｒｓｆｍａａｅｉｈｏｕｒｚｅｔｃｕｓｒｏｌｙｍｏｅｉ；ｃｉｓｅｏｎｅｖｔ
现阶段富马酸的化学合成法面临石油资源短缺、生产成本提高以及环境污染等一系列严峻的问
ＡｓａｔＨｍｌｙｔｅ —ｄｅｓｎｌｔｔｍｄｏｆｍｒｅｆ￣ｈｏｕｒａａｄｃｄｂｉｎｒａｃｏｗｒｏｅｂｔｃ：ｏｏｇｒ —ｉｎｉａｓｕｅ￣ｆｌａｘＲｉｐｓｙｅｗｓｃｕｔｙｂｉｏｆｓｆａＭｄ— ｒｏｈｅｍｏｍｃ￣ｄｉ￣ｉｎｓｏｚｎｏｅｏｆｍｉｓｔｅ
Ｔｅａｔｅａｎｃｄｆｌｒｓｒｒｄｂｍｉｏａｉｆ￣ｒｅｄｆｒｎｕｍｎｔ．ＴＪｘｅｉｎｉｅｐｌｔｅｅｎｅｓｎｈｃｉｍｉｏａｉｓ０ｆｍａａｅａｅｆｍｅｙａｎｃｄｉｎｔｅｉｅｅｔｓｌｌｖｉｏｓｘｈｓｂｓｅｐｒｍｅｔｌｈｌｏｂｔｒｕｄｒａｄｗｌ氇ｔｔ
ＺＡＧＫｎＨＡＧＨ，Ｉｉ，ＡｈｎＪｎ — ｉ，ＩｉＨＮｕ，ＵＮｅＬＵＮｌＧＯＺｅ，ＩＭｉｊＷＥｎ１ｇＮｇｃＰｇ
（ａ／Ｎｎｎｊｇ矿Ｔｄｏ￣，ｅｍ／ｏｅ矿ｒａｄ＆，哦２００，ｉｓｒｉｅｍｎ蜘１０９Ｊｎ￣Ｐｏｎ，ａｕｖｃ）
域为一个由五个几乎平行的ａ螺旋组成的独特的束型结构，其催化活性位点是由三个亚基上的氨基酸相互靠近共同组成的。为以后有针对性的进行富马酸酶的定点突变提高富马酸产量提供分子水平上的理论指导。关键词：富马酸酶；米根霉；结构；同源建模；催化活性位点