定点突变提高N-酰基高丝氨酸内酯酶酶活和温度稳定性

【目的】提高N-酰基高丝氨酸内酯酶(N-acylhomoserine lactonase,AiiA)酶活及温度稳定性。【方法】本研究基于AiiA同源蛋白的三维结构对AiiA进行定点突变,分析野生型AiiA及其突变蛋白酶活和温度稳定性。【结果】野生型AiiA较不稳定,在45℃下温浴30 min,或4℃储存5 d后均失去降解N-酰基高丝氨酸内酯(N-acylhomoserine lactone,AHL)的活性。但是突变AiiA蛋白(N65K,T195R和A206E)的酶活力较野生型AiiA均提高了20%以上,且4℃储存时间延长到7 d。此外,突变株N65K比野生型AiiA对高温具有更强的耐受性,在45℃温浴后剩余酶活力达到45%以上,55℃温浴30 min后仍保留5.0%的酶活力。【结论】通过定点突变改造AiiA蛋白结构,提升了AiiA蛋白的酶活和温度稳定性。     

枯草芽孢杆菌SS6N-酰基高丝氨酸内酯酶基因的克隆表达及其酶学特性

摘要:【目的】从一株具有细菌群体感应(Quorum Sensing,QS)信号分子淬灭活性的枯草芽孢杆菌(Bacillus subtilis) SS6中扩增N-酰基高丝氨酸内酯酶(N-acylhomoserine lactonase,AiiA)基因aiiASS6并异源表达,研究此信号降解酶的酶学特性。【方法】设计特异性引物,从B.subtilis SS6中克隆N-酰基高丝氨酸内酯酶基因aiiASS6,测序并进行生物信息学分析;将此基因克隆到表达载体pET28(a),构建重组菌株并提纯目的蛋白AiiASS6;然后用高效液相色谱(high performance liquid chromatography,HPLC)分析目的蛋白AiiASS6降解QS信号分子N-(3-Oxooctanoyl)-L-homoserine lactone (OOHL)的酶学特性。【结果】克隆得到基因片段,命名为 aiiASS6 (GenBank: KP125494),其编码一条含有297氨基酸残基的多肽,用pET28(a)成功构建重组质粒pET28-aiiASS6。生物信息学分析表明,AiiASS6的氨基酸序列含有N-酰基高丝氨酸内酯酶典型的“HXHXDH”基序和194 位的Tyr残基。在Escherichia coli BL21(DE3)中异源表达AiiASS6,用Ni柱纯化后,AiiASS6含量达2.76 mg/mL。HPLC检测结果表明AiiASS6对OOHL具有很强的催化活性及耐热性,Km和Vmax分别为0.998 mmol/L和22.3 U/mg,最适pH为7.6,最适温度范围为50－90℃;此酶在4℃保存3个月后其残余活性仍达到86%,表现出较强的稳定性。【结论】从B．subtilis SS6中获得的QS淬灭酶AiiASS6表现出降解QS信号分子的高活性,其酶学特性表明它具有作为微生物制剂防控植物或水产养殖中基于QS调控的病原菌毒力的应用潜力。     

细菌群体感应信号分子N-酰基高丝氨酸内酯的检测

群体感应是细菌生长到一定密度时相互感应,并进行基因表达及调控产生的独特、多样的群体行为现象。N-酰基高丝氨酸内酯(AHL)类化合物是革兰阴性菌群体感应中最重要的一类信号分子,调控许多生理特性基因的表达。快速、简便、有效地检测细菌能否产生AHL或产生何种信号分子,成为深入研究和了解细菌群体感应的重要手段。我们就细菌群体感应信号分子AHL检测的基本原理和方法及国内外研究进展进行了总结。     

细菌信号分子N-酰基高丝氨酸内酯调控植物抗性的研究进展

自然界中植物与细菌长期共存,共同进化,二者之间形成由不同信号分子介导的复杂的相互作用网络。N-酰基高丝氨酸内酯（AHLs）是革兰氏阴性细菌胞间通讯的信号分子,可被植物感知,并能调控植物多种生理行为,包括植物的天然免疫、生长发育、耐逆性等。本文综述近年来的相关研究进展,有助于全面了解植物与细菌间的信息交流机制,并对农业生产提供理论指导。     

红球菌来源QsdA型N-酰基高丝氨酸内酯酶水产养殖饲用潜力分析

通过研究QsdA型N-酰基高丝氨酸内酯酶酶学性质来评估其饲用潜力。研究通过提取红球菌(Rhodococcus erythropolis)BLJF-1的基因组, 利用 PCR 技术克隆得到N-酰基高丝氨酸内酯酶基因qsdA-rh5。构建重组表达载体pET28a/qsdA-rh5转化大肠杆菌BL21(DE3), 筛选得到具有N-酰基高丝氨酸内酯酶活性的转化子即为重组菌株, 随后经Ni-NTA柱纯化得到的重组蛋白QsdA-RH5进行补充酶学性质的研究。结果表明, 克隆得到972 bp的目的基因。构建重组载体, 筛选得到重组菌株经诱导表达后得到具有N-酰基高丝氨酸内酯酶活性的目的蛋白即QsdA-RH5, 经分析表明, 该蛋白的理论分子量为36 kD, 属于金属依赖性水解酶PET超家族。酶学性质研究表明: 其最适作用 pH 为 8.0, 作用温度为 35℃, 在 pH 611内能够稳定的存在, 在1040℃, 酶活性能够维持在 80% 以上, 且该酶对多种金属离子、化合物具有很好的抗性。该融合蛋白具有较为专一的底物特异性, 只对没有取代基团的底物具有水解作用, 以C7-HSL 为底物时的Km值为0. 0125 mmol/L。实验经酶学性质研究表明, 该酶具有较为专一的底物特异性, 因此可具有针对性的控制外源性病原菌毒性效应对维护畜禽(水产)消化道健康方面具有一定的应用前景。       

N-酰基高丝氨酸内酯调控植物生长发育的研究进展

植物与细菌之间存在着复杂的相互作用关系。N-酰基高丝氨酸内酯（AHLs）是革兰氏阴性细菌进行胞间通讯的信号分子,也是介导植物与细菌互作的重要信号分子,在调控植物生长发育方面起着重要作用。本文对近年来的相关研究进展作一综述,这将有助于全面了解植物与细菌间的信息交流机制,并对实际农业生产具有指导意义。     

一株降解N-酰基高丝氨酸内酯酵母菌菌株的分离鉴定及其降解特性

利用N酰基高丝氨酸内酯(N-acyl-homoserine lactone,简称AHL)为唯一碳源和能源,筛选得到一株能够降解AHL的菌株R1。常规鉴定和18S rDNA序列分析表明,菌株R1属于红冬孢酵母菌(Rhodosporidium toruloides),定名为R.toruloidesR1。结果显示R.toruloidesR1能利用所测试的3种AHL作为唯一碳源和能源生长,具有降解AHL的能力,其对AHL依赖型胡萝卜欧文氏软腐病菌(Erwinia carotovora subsp.carotovora)的致病有一定的抑制作用。     

细菌群体感应信号分子——酰基高丝氨酸内酯的检测

革兰氏阴性菌根据信号分子N-酰基高丝氨酸内酯(AHLs)的浓度可以监测周围环境中自身或其他细菌的数量变化,当信号分子达到一定浓度阈值时,能启动相关基因的表达来适应环境的变化,这一调控系统被称为细菌的群体感应(quorumsensing,QS)系统。快速简便而有效地检测细菌是否以及产生何种信号分子成为深入研究和了解细菌群体感应的重要手段。现对信号分子AHLs敏感的用于检测不同的信号分子AHLs的微生物传感菌进行综述,并对其检测能力进行了讨论。     

高效表达N-乙酰高丝氨酸内酯酶-木聚糖酶融合蛋白及其酶学性质

摘要: 【目的】构建N-乙酰高丝氨酸内酯酶-木聚糖酶双酶活性表达毕氏酵母重组菌株,并对经纯化的重组蛋白SL2B 进行N-乙酰高丝氨酸内酯酶及木聚糖酶酶学性质的研究。【方法】利用PCR 拼接技术得到N-乙酰高丝氨酸内酯酶基因aiiA-B546 和木聚糖酶基因xynAS27cd 融合而成的基因Sl2b。构建重组表达载体pPIC9 / Sl2b 转化毕氏酵母,筛选得到同时具有木聚糖酶和N-乙酰高丝氨酸内酯酶活性的重组子,随后对经硫酸铵沉淀、分子筛纯化后得到的重组蛋白SL2B 进行N-乙酰高丝氨酸内酯酶及木聚糖酶     

芽孢杆菌酰基高丝氨酸内酯酶基因的克隆及表达

N-酰基高丝氨酸内酯(N—acyl—homoserine hctones．AHLs)作为细菌群体应答系统(Quorum—sensing)中的关键信号分子,其浓度是决定许多动、植物病原菌致病基因的表达的关键因子,酰基高丝氨酸内酯酶基因可以水解AHk丹子的内酯键,使．MILs失去生物活性,从而减弱致病菌的危害．该研究旨在从芽孢杆菌中克隆酰基高丝氨酸内酯酶基因并获得纯化蛋白。根据已知酰基高丝氨酸内酯酶基因的保守序列设计引物,利用PCR方法从2株芽孢杆菌的基因组DNA中克隆出阿个基因SS1和SS10。利用在基因库中进行同源比对．结果表明SS1和SS10编码的蛋白产物SS1和SS10均为酰基高丝氨酸内酯酶。将两个基因在大肠杆菌中诱导表达,通过亲和层析获得了纯化蛋白。     

大黄酸和大黄素的热分析及其动力学研究

本文采用热重法(TG)和差热分析法(DTA)测定了大黄酸和大黄素的DTA,TG-DTG曲线。两者的DTA曲线中皆有两个较为明显的吸热峰,第一个在熔化过程中出现,第二个发生在热分解过程中并伴随有明显的失重现象。TG曲线均有一个失重平台,失重率在90%以上。用TG-DTG法对两者在非等温条件下进行热分解动力学研究,把从TG-DTG曲线中取得的数据和31个不同的方程采用Achar微分法和Madhusudanan-Krishnan-Ni-nan(MKN)积分法对其进行非等温分解动力学研究,得到动力学参数活化能(E和指前因子A)和分解动力学机理及方程。得出结论:大黄酸和大黄素的动力学方程为dα/dt=Aexp(-E/RT)3/2(1-α)4/3[1/(1-α)1/3-1]-1和dα/dt=Aexp(-E/RT)3/2(1-α)2/3[1/(1-α)1/3]-1,其分解等合3D抗理。二者的活化能E(kJ/mol)分别为117.6和86.79,lnA/s-1分别是36.72和27.44。     

草鱼CNBP的分子特征及其进化分析

从草鱼(Ctenopharyngodon idella)肝肾cDNA文库中克隆到细胞核酸结合蛋白基因CNBP的完整开放阅读框序列.分析表明草鱼CNBP由163个氨基酸残基组成,含有7个保守CCHC型锌指结构、核定位信号区和RGG框,与其他鱼类的同源性很高.与人及其他脊椎动物的相比,草鱼细胞核酸结合蛋白在第3个锌指中的第5个氨基酸残基由Gly变成His,另外在第1锌指和第2锌指结构间,缺失6～14个氨基酸残基.虽然如此,适应性进化分析显示细胞核酸结合蛋白没有经历正达尔文选择(ω≤1),即这种结构的差异还不足以产生新的功能.这表明CNBP处于中性进化中.     

低分子肝素的绿色荧光蛋白荧光标记及其活性检测

增强型绿色荧光蛋白（EGFP）是生物领域常用的标记物. 本实验首先利用高碘酸法活化低分子肝素（LMWH）,与EGFP连接得到LMWH EGFP.然后用Sephacryl S-200 HR对其进行初步分离,再用Sephadex G-10 HR进行脱盐纯化. 采用Sephacryl S 200 HR检测LMWH EGFP的纯度,为单一对称峰. 经检测,LMWH-EGFP具有良好的热稳定性和耐碱性. 通过荧光分光检测器检测LMWH-EGFP的λEx为488 nm,λEm为509 nm. 通过抗凝实验发现LMWH EGFP仍具有抗凝活性. 本实验建立了LWMH荧光标记的方法,为多糖的荧光标记提供了新的选择.     

Novel roles of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> to control plant diseases

Bacillus thuringiensis is well known as an effective bio-insecticidal bacterium. However, the roles of B. thuringiensis to control plant diseases are not paid great attention to. In recent years, many new functions in protecting plants from pathogen infection have been discovered. For example, acyl homoserine lactone lactonase produced by B. thuringiensis can open the lactone ring of N-acyl homoserine lactone, a signal molecule in the bacterial quorum-sensing system. This in turn, significantly silences bacterial virulence. This finding resulted in the development of a new strategy against plant bacterial diseases by quenching bacterial quorum sensing. Another new discovery about B. thuringiensis function is zwittermicin A, a linear aminopolyol antibiotic with high activity against the Oomycetes and their relatives, as well as some gram-negative bacteria. This paper summarized the relative progresses of B. thuringiensis in plant disease control and its favorable application prospects.     

“False” thymine—1H-enol guanine base pair. Low misinsertion rate by DNA polymerase explained by computational chemistry consideration

Formation of correct TA and GC and “false” thymine—1H-enol guanine (TGenol) base pairs is here considered to control nucleotide insertion into DNA via low substrate concentration Michaelis-Menten controlled kinetics. Contributions of base pairing to formation of Gibbs free energies in water solution, ΔΔG, are calculated for the correct and false base pairs with the semi-empiric MNDO/PM3 method for base pairing energies in vacuum and the BEM method for hydration effects. The results for ΔΔG indicate equal insertion rates for correct base pairing and a 10⁻³−10⁻⁴ error probability for false insertion controlled by the TGenol false pair. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 3, pp. 402–406.     

Peptide Modulation of Class I Major Histocompatibility Complex Protein Molecular Flexibility and the Implications for Immune Recognition

T cells use the αβ T cell receptor (TCR) to recognize antigenic peptides presented by class I major histocompatibility complex proteins (pMHCs) on the surfaces of antigen-presenting cells. Flexibility in both TCRs and peptides plays an important role in antigen recognition and discrimination. Less clear is the role of flexibility in the MHC protein; although recent observations have indicated that mobility in the MHC can impact TCR recognition in a peptide-dependent fashion, the extent of this behavior is unknown. Here, using hydrogen/deuterium exchange, fluorescence anisotropy, and structural analyses, we show that the flexibility of the peptide binding groove of the class I MHC protein HLA-A*0201 varies significantly with different peptides. The variations extend throughout the binding groove, impacting regions contacted by TCRs as well as other activating and inhibitory receptors of the immune system. Our results are consistent with statistical mechanical models of protein structure and dynamics, in which the binding of different peptides alters the populations and exchange kinetics of substates in the MHC conformational ensemble. Altered MHC flexibility will influence receptor engagement, impacting conformational adaptations, entropic penalties associated with receptor recognition, and the populations of binding-competent states. Our results highlight a previously unrecognized aspect of the “altered self” mechanism of immune recognition and have implications for specificity, cross-reactivity, and antigenicity in cellular immunity.     

1株产多糖芽胞杆菌的分子鉴定和诱变选育

以中国农业科学院北京畜牧兽医研究所鸡舍附近土壤为材料,采用稀释平板法对其中的菌株进行分离与纯化培养,并对其进行形态学鉴定和粗多糖提取量的检测,同时对分离得到的菌株进行紫外线和亚硝基胍的诱变。从土壤中分离得到1株产多糖的芽胞杆菌(编号P-30),结合形态学鉴定、16S rRNA序列分析和系统发育树分析结果,确定该菌株为短短芽胞杆菌(Bacillus brevis)。其16S rRNA GenBank登录号为HM185814。经过诱变选育后,获得3株(N-05、N-11、U-01)多糖产量为P-30的1.44、1.44和1.29倍的诱变菌株,具有良好的开发前景。     

Molecular Characterization of Caveolin-induced Membrane Curvature

The generation of caveolae involves insertion of the cholesterol-binding integral membrane protein caveolin-1 (Cav1) into the membrane, however, the precise molecular mechanisms are as yet unknown. We have speculated that insertion of the caveolin scaffolding domain (CSD), a conserved amphipathic region implicated in interactions with signaling proteins, is crucial for caveola formation. We now define the core membrane-juxtaposed region of Cav1 and show that the oligomerization domain and CSD are protected by tight association with the membrane in both mature mammalian caveolae and a model prokaryotic system for caveola biogenesis. Cryoelectron tomography reveals the core membrane-juxtaposed domain to be sufficient to maintain oligomerization as defined by polyhedral distortion of the caveolar membrane. Through mutagenesis we demonstrate the importance of the membrane association of the oligomerization domain/CSD for defined caveola biogenesis and furthermore, highlight the functional significance of the intramembrane domain and the CSD for defined caveolin-induced membrane deformation. Finally, we define the core structural domain of Cav1, constituting only 66 amino acids and of great potential to nanoengineering applications, which is required for caveolin-induced vesicle formation in a bacterial system. These results have significant implications for understanding the role of Cav1 in caveola formation and in regulating cellular signaling events.