Study on immobilization of lipase B from Candida Antarctica and its application

摘要 南极假丝酵母脂肪酶B（CandidaantarcticalipaseB，CALB）是工业上广泛应用的脂肪酶。维生素A棕榈酸酯是具有良好市场前景的维生素A衍生物。利用南极假丝酵母脂肪酶B催化合成维生素A棕榈酸酯具有条件温和，反应选择性高，副反应少，产品的稳定性好，绿色环保等优点。 本文首先培养重组毕赤酵母获得高浓度CALB的发酵上清液。发酵144h后，游离酶酶活可达108U/mL，目标蛋白占总蛋白含量的百分比为63.4%。通过对游离酶固定化方法的比较，发现与共价法相比，吸附法操作简单，同时对酶活破坏小。采用大孔吸附树脂HPD826吸附固定脂肪酶，其催化月桂酸酯化的活性可达9200PLU...Abstract Candida antarctica lipase B (CALB) has been widely used in industry and vitamin A palmitate has great market prospects. The synthesis of vitamin A palmitate catalyzed by Candida antarctica lipase B has many advantages including mild conditions, high reaction selectivity, less side effects, good product stability, environmental friendly etc. Firstly, the high concentration of CALB were o...学位：工学硕士院系专业：化学化工学院_化学工程学号：2062013115148

Immobilization of Oxidoreductase Based on Two-Dimensional Graphene Materials

氧化还原酶（Oxidoreductase）是重要的生物催化剂，其催化的氧化还原反应具有反应条件温和、立体选择性强等优点，但依赖NADH（NADPH）的氧化还原酶需要昂贵的辅酶参与反应。甲酸脱氢酶（FormateDehydrogenase，FDH）在辅酶酶法再生中具有明显优势，甲酸/甲酸脱氢酶再生体系的明显优势在于其唯一副产物CO2对酶的活性没有影响，且易于从反应体系中逃逸出来。苯丙氨酸脱氢酶（PhenylananineDehydrogenase，PheDH），在临床上可用于苯丙酮尿症的检测，且可与甲酸脱氢酶耦联合成苯丙氨酸。 氧化石墨烯（GrapheneOxide，GO）具有独特的片层结构，...Oxidoreductase is one of the most important biocatalysts, which can catalyze reactions with high effienciency and stereoselectivity in mind conditions, but the expensive cofactor is an indispensable part of the reactions. Formate dehydrogenase (FDH) is suitable for the regeneration of cofactor NADH. It has many advantages, namely, favorable thermodynamic equilibrium, the inertness of the substrate...学位：工学硕士院系专业：化学化工学院_生物化工学号：2062014115140

Preparation and Characteristic Research of Immobilized Acetylcholinesterase

本研究以期研制出能重复使用的固定化乙酰胆碱酯酶(ACHE),为天然产物复杂体系中ACHE抑制剂筛选新方法的发展奠定基础.以氨基化硅胶(APS-SI)微球为载体,戊二醛为交联剂对乙酰胆碱酯酶进行交联固定化,并研究了酶的最佳固定化条件和固定化酶的性质.结果表明,0.05 g氨基化硅胶微球载体,用戊二醛溶液活化6 H后,在给酶量5 u,28℃固定16 H条件下,得到固定化酶的活性最大.固定化酶在常温(20~40℃),以及较宽PH范围内(PH 6~10)均具有较高的活性,并且具有良好的保存稳定性和可重复利用率,为基于固定化靶酶亲和-色谱质谱联用分析快速筛选乙酰胆碱酯酶抑制剂新方法的发展奠定了基础.A new method for preparation of a reusable immobilized acetylcholinesterase(AchE) was developed in order to lay a foundation for screening AchE inhibitors from natural products.AchE was immobilized on aminated silica gel microspheres with glutaraldehyde as cross-linking reagent.The optimum conditions and properties of immobilized enzyme were investigated.The optimal immobilization process was as follows: 0.05 g of aminated silica gel microspheres was activated by glutaraldehyde solution for 6 h,and mixed with AChE solution with the activity of 5 U.Then,the mixture was shook for 16 h at 28 ℃.The immobilized enzyme produced by this method had the highest enzyme activity,and it could remain better activity with a wide range of pH(pH 6～10) at room temperature(20～40 ℃).The immobilized AChE showed higher storage stability and could be used repeatedly.国家自然科学基金(No.20905017);国家海洋局青年基金(No.2010140);海洋一所基本科研业务专项(No.2010G25)资助项目---

Preparation of Immobilized Nucleoside Phosphorylase and Research on Its Property

目的优化核苷磷酸化酶的固定化工艺,并对固定化酶的性质进行研究。方法通过正交试验优化核苷磷酸化酶(包括嘌呤和嘧啶核苷磷酸化酶)固定化条件,并进一步对固定化核苷磷酸化酶的最适P H、温度及其稳定性进行研究。结果固定化酶条件为P H=8.0,温度为25℃,蛋白载量为20 Mg/g载体,固定化时间为6 H;固定化核苷磷酸化酶的最适P H=7.5,最适温度为40℃,固酶连续使用20批次酶活力无明显损失。结论固定化核苷磷酸化酶稳定可靠,适合应用于工业化生产。Objective To optimize the immobilization process of the nucleoside phosphorylase and research on its property.Methods The immobilization process of the nucleoside phosphorylase(including purine and pyrimidine nucleoside phosphorylase)was optimized by orthogonal test,and took further study of the finest p H,temperature and stability of immobilized nucleoside phosphorylase.Results The finest condition of immobilized enzyme was p H = 8.0,25 ℃,20 mg/g protein loads and immobilize for 6 h; the finest condition for the immobilized nucleoside phosphorylase to catalyze was p H = 7.5 and 40 ℃.There was no significant loss of activity of the enzyme after use for catalyzing 20 batches consecutively.Conclusion The immobilized nucleoside phosphorylase is stable and reliable,which is suitable to application in industrial productio

Enzymology, Gene Cloning and Proteomics of Aspergillus glaucus XC9

本研究从灰绿曲霉发酵液中分离纯化出一种外切葡聚糖酶(CBH)和一种内切葡聚糖酶(EG)。SephadexG-100凝胶柱层析和SDS-PAGE测得CBH全酶分子量为71kDa，由两个35kDa亚基组成；EG为单体蛋白，全酶分子量为32kDa。CBH催化pNPC的最适pH为6.0，最适温度为55oC，Km值为1.4mmol/L，酶活在pH5.0~8.0区间和温度低于55oC时稳定；EG催化CMC-Na的最适pH为4.0，最适温度为50oC，Km值为5.0mg/mL，酶活在pH3.5~7.5区间和温度低于65oC时稳定。Na+、K+、Ba2+、Mg2+以及NO3-和SO42-对CBH和EG酶活均无...A cellobiohydrolase (CBH) and an endoglucanase (EG) were purified from fermentation liquor of Aspergillus glaucus by following procedures: ammonium sulfate precipitation, gel filtration on Sephadex G-100, ion-exchange chromatography on DEAE Sepharose Fast Flow and hydrophobic chromatography on Phenyl Sepharose Fast Flow. By the methods of gel column chromatography and SDS-PAGE, the molecular weigh...学位：理学博士院系专业：生命科学学院生物化学与生物技术系_生物化学与分子生物学学号：2162007015382

The Research Survey of the Degrading-bacteria and Degrading-enzyme of Synthetic Pyrethroid Insecticides

通讯作者：Tel: 0592-2186195; : [email protected][中文文摘]拟除虫菊酯类农药目前已成为我国出口的蔬菜、水果中主要的3类农药残留之一,引起急慢性中毒事件也越来越多,对人类、水生生物和自然环境造成很大危险。而农药生物降解作为去除农药污染的有效手段,逐渐成为环境科学研究的热点。重点综述了拟除虫菊酯类农药降解菌的分离、降解酶的提取、纯化、降解机理、固定化研究,并对以后要解决的问题进行了展望。[英文文摘]Synthetic pyrethroid insecticides have become one of the three major kinds of pesticide residues in the exported vegetabes and fruits in China nowadays. They also caused more and more acute or chronic poisoning affair and do great damage to human beings, aquatic biology and natural environment. As an efficient way to remove pesticides pollution, the biodegradation of pesticides has gradually become the hot issue in the field of environmental sciences. The study on the separation of degrading-bacteria and the extraction, purification, mechanism and immobilization of the degrading-enzyme of pyrethroids is reviewed in this paper. And the problems under resolved in the future work are also prospected.福建省青年科技人才创新项目(No.2007F3094);厦门大学引进人才科研启动费项目(No.0000-X071C3);厦门大学近海海洋环境科学国家重点实验室开放基金(No.MEL0603);国家海洋局近岸海域生态环境重点实验室开放基金(No.200702);国家海洋局第三海洋研究所海洋生物遗传资源重点实验室开放研究基金(No.HY200601

固定化双酶耦联体系制备手性胺

胺脱氢酶(amine dehydrogenase,Am DH)能够在辅酶的作用下不对称还原前手性酮制备手性胺,并与甲酸脱氢酶(formate dehydrogenase,FDH)构建耦联双酶反应体系,通过酶活检测与高效液相色谱法得出,Am DH和FDH浓度比为4∶1、底物浓度为10 mmol/L、辅酶浓度为0. 025 mmol/L时,是游离双酶耦联体系最优反应条件.为提高双酶耦联体系的稳定性,将聚乙烯亚胺-双酶复合物作为模板和催化剂,诱导钛前驱体Ti-BALDH(titanium(IV) bis-(ammoniumlactato)-dihydroxide)缩聚,形成固定化耦联体系.与游离酶体系相比,辅酶可在固定化酶体系的微环境中高效循环再生,大大提高了多酶耦联体系的催化效率

Breeding the strain 2A00015 (Candida parapsilosis) from Deep Sea and Preliminary Study of the Preparation of Microbio-diesel

微生物油脂(microbialoils)又称单细胞油脂(singlecelloil，SCO)，即微生物以碳水化合物、碳氢化合物和普通油脂为碳源、氮源、辅以无机盐生产的油脂和另一些有商业价值的脂质。随着人口的增长、社会的进步使油脂需求量与自然资源短缺的矛盾日益加剧，环境污染日趋严重，开发新型油脂资源及清洁能源是当务之急。微生物生产油脂具有原料来源丰富、可再生、成本低、周期短、环境友好等优点，已引起人们的高度关注。开展微生物油脂的研究对解决人类面临的资源短缺、环境恶化等现实问题具有重要意义。 本文针对当前所关注的有关深海微生物、生物能源、废物综合利用3个热点问题，旨在寻找一株可以高效利用廉价碳源...Microbial oils (or single cell oil) refers to the oil produced by microorganisms with carbohydrate source (carbohydrates, hydrocarbons and ordinary oil), nitrogen sources and inorganic chemicals. With the population and social developing. New types of cleaning energy resource is key to solve problems among oil needs, shortage of natural resource and the serious environmental pollution. Now, many ...学位：理学硕士院系专业：生命科学学院_生物化学与分子生物学学号：2162010115230

Co-immobilization of Glycerol dehydrogenase and coenzymeⅠ

烟酰胺腺嘌呤二核苷酸NAD/NADH作为一类重要的辅酶，协助多种氧化还原酶催化细胞内包括糖酵解、细胞呼吸等途径的重要反应。NAD(H)价格昂贵，易溶于水，不稳定，且回收难度大，成为限制氧化还原酶大规模工业化应用的“瓶颈”。本文尝试将共固定化技术与辅酶酶法再生结合，拓展氧化还原酶的工业应用。 本课题以辅酶和甘油脱氢酶（GDH）为研究对象，通过海藻酸修饰、环氧修饰、戊二醛交联、羰二亚胺活化等方法将辅酶固定在壳聚糖、纳米二氧化硅或碳纳米管载体上，选取较优载体及方法进行固定化条件优化；利用NAD+与GDH的亲和吸附，尝试NAD+与GDH的共固定化，并对共固定化条件进行优化；探讨辅酶、甘油与甘油脱氢酶...Nicotinamide adenine dinucleotide,as an important class of coenzyme, plays an important role in variety of cellular oxidoreductase catalytic reaction, Including glycolysis, cellular respiration and other reaction pathways. NAD(H) is expensive, easy to dissolve in water, leading to the difficulty of its recovery, which is the "bottleneck" factor of the large-scale industrial application with oxi...学位：工学硕士院系专业：化学化工学院化学工程与生物工程系_生物化工学号：2062008115161

Study on NaCS Preparation and Immobilized Cultivation of Dictyostelium discoideum in NaCS-PDMDAAC Microcapsules

本文的研究目的是利用纤维素硫酸钠-聚二甲基二烯丙基氯化铵（NaCS-PDMDAAC）生物微胶囊系统固定化培养重组盘基网柄菌，以提高其细胞密度并高通量表达目的蛋白。由于纤维素硫酸钠（NaCS）尚未商品化，故本文先对纤维素硫酸钠的制备工艺进行了研究。在成功制备出纤维素硫酸钠后，对NaCS-PDMDAAC微胶囊进行了一些表征，然后将其应用到细胞培养中。 首先利用硫酸和正丙醇作为反应液制备纤维素硫酸钠。为了减小纤维素硫酸钠的制备成本，用无水乙醇代替了反应液中的正丙醇。利用响应面实验设计，在100mL反应液体系下，以制备胶囊（2%NaCS溶液滴加到6%PDMDAAC溶液中，反应10min）的机械强度为...In this work, NaCS (sodium cellulose sulphate)-PDMDAAC (ploy dimethyl diallyal ammonium chlorid) microcapsule immobilization system was applied to cultivate recombinant Dictyostelium discoideum (D. discoideum), in order to raise its cell density and increase the product concentration. However, the NaCS has not been commercialized, thus its preparation process, characterization of NaCS-PDMDAAC micr...学位：工学硕士院系专业：化学化工学院化学工程与生物工程系_生物化工学号：2062006115204