The research on synthesis and biological activitiy of alkyl 3,4-dihydroxybenzoates

酪氨酸酶(EC1.14.18.1)是一种含铜的氧化还原酶，广泛分布于微生物，动物，植物和人体中，是生物体生命活动的关键酶，其抑制剂的研究引起了广泛的关注。 本论文从酪氨酸酶抑制剂的构效关系出发，设计并合成了系列3，4-二羟基苯甲酸烷基酯，研究它们对酪氨酸酶的抑制效应及其抑制机理。以大肠杆菌，金黄色葡萄球菌等为作用对象，研究3，4-二羟基苯甲酸烷基酯的抑菌作用。以小菜蛾和菜青虫为实验对象，研究3，4-二羟基苯甲酸烷基酯的杀虫毒力及其对昆虫酪氨酸酶表达的影响。 本论文采用EDCHCl和DMAP为催化剂，通过酯化反应成功地合成了系列3，4-二羟基苯甲酸烷基酯（P1-P10），用质...Tyrosinase (EC 1.14.18.1), a copper containing enzyme, is ubiquitously distributed in microorganisms, animals, plants and human beings. It is the key enzyme in the vital function of biology. The inhibitors of tyrosinase have aroused the widespread interest. In this study, we synthetized a family of alkyl 3,4-dihydroxybenzoates, a new tyrosinsase inhibitor, from the structure activity relationship...学位：理学硕士院系专业：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：2172008115265

Study on the rheology of xanthan and synergistic interaction with konjac gum and other gum

研究前期筛选获得一株多糖胶质高产菌Xanthomonas axonopodis所产的黄原胶FJAT-10151-DTJZ的品质,为该黄原胶的开发应用提供基础数据及参考。通过分析浓度、剪切速率、p H、加热温度、时间、冻融处理等对FJAT-10151-DTJZ粘度的影响研究其流变性,并研究其与结冷胶、黄原胶、凝胶多糖、瓜尔豆胶、刺槐豆胶、魔芋胶、果胶和壳聚糖8种胶的协效性。实验结果表明,FJAT-10151-DTJZ溶液的粘度随浓度的升高而升高,且为非牛顿流体;当FJAT-10151-DTJZ的浓度为1%时,其粘度为343 m Pa·s;p H、冻融对FJAT-10151-DTJZ的粘度影响不大;FJAT-10151-DTJZ的最佳加热温度为75℃;粘度随加热时间先增大后减小,当加热温度为75℃,加热时间为150 min,1%浓度的FJAT-10151-DTJZ溶液的粘度为808 m Pa·s。FJAT-10151-DTJZ只与魔芋胶有强烈的协同增效作用,与壳聚糖、结冷胶、黄原胶、凝胶多糖、瓜尔豆胶、刺槐豆胶、果胶无协效性。A Xanthomonas axonopodis strain producing xanthan gum had been screened,and researched the quality of xanthan gum FJAT-10151-DTJZ in order to provide basic data and reference for development and application. The rheology properties of FJAT-10151-DTJZ were discussed according to the viscosity variation with different conditions including concentration,shearing,p H,heating temperature,heating time and freezingthawing. The synergistic interactions of FJAT-10151-DTJZ with chitosan,gellan gum,xanthan gum,curdlan,guar gum,locust bean gum and pectin were also investigated. The results showed that polysaccharide gum solution FJAT-10151-DTJZ was non-Newton fluid. It's viscosity rised with its concentration and reached to343 m Pa·s when the concentration was 1%. The p H change and freezing-thawing played negligible effects on its viscosity. Its viscosity was 808 m Pa·s when it was heated at 75 ℃ for 150 min. Besides,FJAT-10151-DTJZ had no synergistic interaction with chitosan,gellan gum,xanthan gum,curdlan,guar gum,locust bean gum and pectin except konjac gum.国家948项目(2014-Z48);; 福建省公益类科研院所专项(2015R1018-2);; 福建省农业科学院科技创新项目(2015CX-7

生物藕合技术的原理及其应用

生物藕合是指将两种或多种分子进行键合的过程,此过程形成一个新的联合体,该联合体拥有其所有组分所具有的各特性。生物藕合技术自产生以来其应用不断推广,几乎已经涉及到生命科学领域的各个方面,也极大地推动了生物、化学、医学、农业等学科的发展。本文结合生物藕合技术的原理、特点,探讨了其发展与应用的推广与问题

缩氨基硫脲化合物的合成及对菜青虫酚氧化酶抑制作用研究

菜青虫是危害农作物的主要害虫,酚氧化酶在昆虫的生长发育过程中起着重要的调节作用,高效的酚氧化酶抑制剂对于发展新型"环境友好"杀虫剂有着重要的意义.设计合成了4种缩氨基硫脲类化合物,采用红外光谱、质谱和核磁共振谱对其进行结构鉴定,进一步研究了它们对菜青虫酚氧化酶的抑制作用,结果表明,4种化合物均有显著的抑制作用,其半抑制率IC50分别为2.88,0.93,3.60和0.22μmol/L,有可能进一步开发成为以酚氧化酶为靶点的新型生物农药.同时,为基于缩氨基硫脲母核的酚氧化酶抑制剂的设计提供了数据基础

Cloning, prokaryotic expression and homology modeling analysis of midgut aminopeptidase gene PxAPN5 in Plutella xylostella (Lepidoptera:Plutellidae)

【目的】克隆和表达小菜蛾PluTEllA XylOSTEllA氨肽酶基因,并进行基因序列分析和同源建模分析。【方法】以小菜蛾中肠C dnA为模板克隆分析氨肽酶基因序列,原核表达氨肽酶蛋白并进行酶活性测定,应用配体印迹分析氨肽酶与Cry2Ab蛋白的结合,通过蛋白质建模对突变位点进行分析。【结果】从小菜蛾中肠C dnA扩增出氨肽酶基因,该基因全长2 853 bP,编码950个氨基酸,预测蛋白分子量为107.3871 k dA,等电点为5.24;进化树分析显示,克隆得到的氨肽酶基因属于APn家族5,将其命名为PX APn5(gEn bAnk登录号:kM034756)。PX APn5蛋白具有鳞翅目昆虫氨肽酶蛋白的保守性特征,即含有n-糖基化位点、O-糖基化位点和gPI锚定位点,具有“HEXXH“锌蛋白酶结构域和C端跨膜区域。在大肠杆菌ESCHErICHIA COlI中原核表达PX APn5,表达产物经SdS-PAgE电泳,在110 k dA附近出现特异性条带;酶活性测试显示菌体破碎上清液具有氨肽酶活性,比活力为1 047.2 u/g。配体印迹结果显示表达的PX APn5能与Cry2Ab蛋白特异性结合。多序列比对结果表明,与其他已报道的小菜蛾氨肽酶相比,PX APn5氨基酸序列有3个保守性位点发生了突变,并通过蛋白质建模的方式表征突变位点。【结论】本研究成功克隆和表达了具有氨肽酶活性的小菜蛾氨肽酶,并发现其能与Cry2Ab蛋白特异性结合;通过蛋白质建模对氨肽酶突变位点的特征及功能进行了预测。这些结果对小菜蛾氨肽酶的功能性研究提供了理论基础。【Aim】The aim of this study is to analyze the cloning,expression and homology modeling of midgut aminopeptidase gene Px APN5 in the diamondback moth,Plutella xylostella.【Methods 】 A aminopeptidase( APN) gene was cloned from the P.xylostella midgut,and bioinformatics analysis of the gene was performed.The APN protein was expressed using prokaryotic expression system,and its enzymatic activity was assayed.The interaction between APN and Cry2 Ab was determined by using ligand blot analysis.Homology modeling of APN gene was conducted for the prediction of characteristics and functions of mutation sites.【Results】The sequencing results showed that the cloned APN gene( NCBI accession no.: KM034756) is 2 853 bp in length and encodes 950 amino acids with the predicted molecular weight of 107.3871 k Da and isoelectric point of 5.24.Phylogenetic tree analysis indicated that this APN gene belongs to class 5 of APN family,and it was named as Px APN5.Px APN5 has the conservative features of the APN proteins in lepidopteran insects including N-glycosylation and Oglycosylation sites,GPI anchor point,C-transmembrane domains and zinc-metalloprotease domain(361HEXXH365).A 110 k Da specific protein band appeared when APN protein was inducibly expressed in Escherichia coli.Aminopeptidase activity assay showed that the supernatant of broken bacteria possessedaminopeptidase activity and its specific activity was 1 047.2 U / g.The ligand blot result indicated that Px APN5 could bind to Cry2 Ab specially.Multiple alignment of amino acid sequences demonstrated that there are three mutations in Px APN5 when compared to other known APN proteins from P.xylostella.【Conclusion】The Px APN5 protein with aminopeptidase activity was successfully cloned and expressed,and it could bind to Cry2 Ab.Prediction of characteristics and functions of mutation sites in Px APN5 was carried out by homology modeling.These results laid the foundation for the functional research of Px APN.国家自然科学基金项目(31371999;31370059

亚硫酸氢钠在马铃薯切片过程中防褐变作用机理的研究

多酚氧化酶(PPO)是蔬果酶促褐变的关键酶,抑制该酶活性是防止蔬果褐变的有效措施.在实验中发现NaHSO3对马铃薯PPO具有显著的抑制作用及在马铃薯切片护色中具有防褐变功能.实验结果表明:马铃薯PPO催化L-多巴形成的褐变物质在475 nm波长处有特征性的吸收峰,并且该吸收峰随着NaHSO3浓度的增加明显降低.NaHSO3的抑制作用表现为不可逆效应,既可以延长酶促反应的迟滞时间,也降低了稳态的酶活力.当NaHSO3浓度增至0.12 mmol/L时,酶促反应的迟滞时间从0延长至56 s,稳态的酶活力下降了35.5%.在马铃薯切片保鲜实验中,对照组在第3天褐变指数达到2,第6天已经完全褐变,而50 mmol/L NaHSO3处理的实验组在第6天褐变指数仅为0.6,有效遏制了马铃薯切片的酶促褐变,实验显示,NaHSO3通过抑制马铃薯PPO活力实现防褐变的效应

效应物对牛肠激酶活性的影响

肠激酶(EK,EC3.4.21.9)是一种在基因工程产品中广泛应用的工具酶.以小分子荧光物质甘氨酰-天冬氨酰-天冬氨酰-天冬氨酰-天冬氨酰-赖氨酰-β-萘胺(Gly-Asp-Asp-Asp-Asp-Lys-β-naphthylamide,GD4K-β-naphthylamide)为底物,采用荧光跟踪法研究了不同氨基酸、几种常用有机溶剂、EDTA、DTT等对牛肠激酶(BEK)活力的影响.结果表明:L-赖氨酸(L-Lys)、丙酮、EDTA、DTT对该酶的活性有较强的抑制作用,IC50分别约为25,50,50和120mmol/L.进一步研究了L-Lys与丙酮对BEK活力的抑制机理以及抑制类型,结果表明:L-Lys对该酶的抑制机理为可逆抑制,其抑制类型为竞争型抑制类型,其抑制常数KI为12.02mmol/L.丙酮对BEK的抑制类型表现为不可逆抑制

Effects of Initial p H Value for the Fermentation of Bacillus subtilis FJAT-14254 on the Generation of Metabolites Substances

为考察酸碱条件对枯草芽胞杆菌fJAT-14254代谢物产生的影响,采用气相色谱-质谱联用分析枯草芽胞杆菌fJAT-14254菌株的胞内成分。结果表明:通过谱库扫描得到32种高匹配率代谢物的初步鉴定结果,主要包括氨基酸类、酸类、烷烃类等,其中含量相对较高的有7种;枯草芽胞杆菌fJAT-14254代谢物的种类和含量与其培养环境的酸碱度变化呈一定的相关性,强酸性(P H=3)条件下培养的胞内代谢物具有特异性,而接近中性(P H=5、7)条件及碱性(P H=9、11)条件下培养胞内代谢物则相似。由此推断,不同P H条件影响发酵终点的P H,进而影响枯草芽胞杆菌fJAT-14254代谢物的产生。To study the relationship between p H of culture medium and intracellular metabolic of Bacillus subtilis FJAT-14254.Intracellular metabolites of B.subtilis FJAT-14254 were analyzed by GC-MS.564 metabolites were detected.31 metabolic markers including hexadecanoic acid, was obtained by data mining.The biochemical substances of B.subtilis FJAT-14254, produce in different p H conditions, had high specificity.The change of metabolites in different p H conditions was complex, not only linear relation.This research provided preliminary study of stress resistance of B.subtilis in metabolic level.公益性行业(农业)科研专项经费(No.201303094); 福建省农科院院青年创新基金(No.2013dqd-6); 福建省农科院杰出青年人才基金(No.2014JQ-2

Antimicrobial Activities of Cinnamic Acid and Its Derivatives to Microorganisms Isolated from Rotten Agaricus bisporus

双孢蘑菇(Agaricus bisporus(Lange)Sing)组织细嫩,营养价值高,采后呼吸代谢旺盛,为微生物的生长繁殖提供了有利条件,但容易引起一些不良的生理反应,加速组织的衰老和腐败,极大地降低了双孢蘑菇的商品价值.本文从腐烂的双孢蘑菇中分离鉴定得到6株细菌和8株真菌,分别为黏质沙雷氏菌(Serratia marcescens,2株)、产酸克雷伯菌(Klebsiella oxytoca,1株)、不动杆菌属(Acinetobacter guillouiae,1株)、沙雷氏菌属(Serratia sp.,1株)和阿氏肠杆菌(Enterobacter asouriae,1株)以及哈茨木霉(Trichoderma harzianum,5株)、多变根毛霉(Rhizomucor variabilis,1株)、绿色木霉(T.viride,1株)和米根霉(Rhizopus oryzae,1株).分别采用琼脂扩散法和生长速率法测定了肉桂酸及其衍生物对上述细菌和真菌的抑制作用,研究结果表明肉桂酸及衍生物对这些菌株有不同程度的抑制作用,抑制效果的强弱依次为4-甲基肉桂酸>4-甲氧基肉桂酸>肉桂酸>4-羟基肉桂酸.本研究结果可为双孢蘑菇的采后防腐提供依据.Agaricus bisporus has a vigorous respiration after harvesting,due to its delicate tissue and the high nutritional value,which provides favorable conditions for the growth of microbes,causing some bad physiological reaction,accelerating the aging and corruption,and greatly reducing the commodity value of A.bisporus.We identified six bacterial strains and eight fungal strains from rotten A.bisporus,which are Serratia marcescens(2),Klebsiella oxytoca(1),Acinetobacter guillouiae(1),Serratiasp.(1),Enterobacter asburiae(1),and Trichoderma harzianum(5),Rhizomucor variabilis(1),T.viride(1)and Rhizopus oryzae(1).Moreover,antimicrobial activities of cinnamic acid and its derivatives were investigated.The results showed that cinnamic acid and its derivatives exhibited different degrees of the growth inhibition on microbia.The strength of inhibition effects decreases following the sequence of 4-methyl cinnamic acid,4-methoxy cinnamic acid,cinnamic acid and 4-hydroxy cinnamic acid.These results may provide theoretical and practical basis in the application of antimicrobial rotting of postharvest A.bisporus in the future.国家自然科学基金(31271952);; 中国博士后科学基金(2015M570556

小菜蛾钙黏蛋白PxCR结构域的克隆、原核表达及功能分析

钙黏蛋白（cadherin）是一类跨膜糖蛋白,因其在苏云金芽胞杆菌（Bacillus thuringiensis,Bt）的杀虫过程中作为主要的受体而受到广泛研究.钙黏蛋白的特征之一是由若干钙黏蛋白重复单元（cadherin repeats,CR）组成的长链状蛋白,其中靠近细胞膜的CR结构域被认为是钙黏蛋白与Bt毒素发生互作的区域.小菜蛾（Plutella xylostella）钙黏蛋白由11个CR结构域和1个跨膜结构域组成,其中第10和11个CR结构域PxCR被认为是钙黏蛋白与Bt毒素的互作区域.本研究从小菜蛾中肠cDNA中克隆得到小菜蛾钙黏蛋白PxCR结构域的DNA片段,并通过原核表达系统对PxCR结构域进行表达.配体印迹结果表明PxCR可以特异性和Cry2Ab结合;杀虫实验结果表明PxCR能提高Cry2Ab对小菜蛾的毒力.此外,利用蛋白质同源建模和糖基化位点预测网站对PxCR蛋白质结构进行了分析.上述结果表明PxCR可能参与了Cry2Ab毒素对小菜蛾的毒杀过程,为后续研究小菜蛾钙黏蛋白和Cry2Ab的相互作用机制奠定了基础.国家自然科学基金(31371999；31370059)