大豆种皮过氧化物酶的制备及其在ELISA中的应用

对大豆种皮过氧化物酶(SBP)进行了部分纯化,并对其标记的抗体的效价和稳定性进行了初步测定。大豆种皮用自来水提取,提取液经pH4.5沉淀去杂蛋白、DEAE-cellulose离子交换柱层析以及SephadexG-75分子筛柱层析,最后冻干得SBP制品。用SBP冻干品标记羊抗人IgG,于-20℃和室温保存2周,前者稀释8000倍、后者稀释2000倍可产生较强的信号。结果表明SBP可以用于酶联免疫检测。     

大豆种皮过氧化物酶的制备及其在ELISA中的应用

对大豆种皮过氧化物酶(SBP)进行了部分纯化,并对其标记的抗体的效价和稳定性进行了初步测定。大豆种皮用自来水提取,提取液经pH4.5沉淀去杂蛋白、DEAE-cellulose离子交换柱层析以及SephadexG-75分子筛柱层析,最后冻干得SBP制品。用SBP冻干品标记羊抗人IgG,于-20℃和室温保存2周,前者稀释8000倍、后者稀释2000倍可产生较强的信号。结果表明SBP可以用于酶联免疫检测。     

氯过氧化物酶稳定化技术及其应用

氯过氧化物酶因其独特的活性腔结构而具有多种多样的催化活性,因此它可广泛应用于有机合成、生物转化、医药工业和石油提炼等领域.但游离氯过氧化物酶在极端条件下稳定性差、易失活,导致其至今无法大量运用到工业中.因此近年来通过固定化、非共价修饰、蛋白质工程和离子液体等手段来开发氯过氧化物酶巨大的应用潜能已成为一个研究热点.该文根据近五年来的有关文献,介绍氯过氧化物酶稳定化技术的研究进展及其应用,并对其未来的发展趋势进行展望.     

过氧化物酶和PeroxiBase过氧化物酶数据库

过氧化物酶是存在于生物体中的一大类以过氧化物为电子受体催化底物氧化的酶,在生物体的生命活动中发挥着重要作用.过氧化物酶由多态性丰富的多基因家族所编码,其结构和功能具有多样性.近年来,随着研究和应用的深入,过氧化物酶领域迫切需要建立一个专业的信息交流平台.PeroxiBase数据库收集了大量的过氧化物酶数据,并进行生物信息学加工,为生命科学研究人员免费共享、最大化利用与综合开发过氧化物酶资源搭建了信息平台,在过氧化物酶的研究与应用中发挥着重要作用.     

Class Ⅲ过氧化物酶在植物中的作用及其研究进展

过氧化物酶广泛存在于各种有机体中,根据其结构和功能可分为不同的类型,其中ClassⅢ过氧化物酶是植物体内特有的一个多基因家族.ClassⅢ过氧化物酶的功能多样,能够参与生长素的代谢、细胞壁的延伸和加厚、活性氧和活性氮的代谢以及植物的抗病作用等各种生理活动.目前对ClassⅢ过氧化物酶的940个物种中的6 000条序列都已经进行了注释,包括其存在的物种、组织类型以及细胞中的定位等.该文对国内外近年来有关ClassⅢ过氧化物酶的结构特征及其在植物体内的功能等进行综述.     

植物谷胱甘肽过氧化物酶研究进展

氧化胁迫可诱导植物多种防御酶的产生,其中包括超氧化物歧化酶(SOD,EC1.15.L1)、抗坏血酸过氧化物酶(APX,EC1.11.1.11)、过氧化氢酶(CAT,E.C.1.11.1.6)和谷胱甘肽过氧化物酶(GPXs,EC1.11.1.9).它们在清除活性氧过程中起着不同的作用.GPXs是动物体内清除氧自由基的主要酶类,但它在植物中的功能报道甚少.最近几年研究表明,植物体内也存在类似于哺乳动物的GPXs家族,并对其功能研究已初见端倪.本文综述了有关GPXs的结构以及植物GPXs功能的研究进展.     

不同溶液中乳过氧化物酶系统对Pg和Fn的影响

比较唾液和缓冲液对含硫氰酸根离子(SCN-)或I-的乳过氧化物酶(LP)抗菌系统抑制牙龈卟啉单胞菌(Porphyromonasgingivalis,Pg)和具核梭杆菌(Fusobacteriumnucleatum,Fn)生长的影响。以Pg(6.0×108/mL)为例,根据所含底物不同,将实验分2大组:第1大组(含SCN-),分3小组:Saliva(对照组),Saliva (LPSCN-)组,Buffer (LPSCN-)组;第2大组(含I-)分3小组:Saliva(对照组),Saliva (LPI-)组,Buffer (LPI-)组;各组均含H2O2(对照组除外),Fn(1.0×108/mL)分组同Pg。将2大组分别在37℃震荡水浴培养30min和60min,5μLDTT终止反应,10倍浓度系列稀释,接种于BHIS琼脂培养基厌氧培养4d并记数CFU。反应至30min时,实验组抑制Pg和Fn生长的作用明显高于对照组(P<0.05),Buffer (LPI-)组抑制Pg和Fn生长的作用明显高于Saliva (LPI-)组,且持续到60min(P<0.05);反应60min时,Buffer (LPSCN-)组抑制Pg生长的作用明显高于Saliva (LPSCN-)组(P<0.05)。LP系统在唾液和缓冲液中均能有效抑制Pg和Fn的生长,但是唾液的存在可削弱LP抗菌系统的抑菌作用。     

大豆过氧化物酶－过氧化氢－碘化钾体系杀菌作用

【目的】研究豆壳过氧化物酶(SBP)-H2O2-KI三元体系杀菌作用及其可能的杀菌机制。【方法】以细菌生长的浊度(OD600)为指标检测SBP-H2O2-KI体系的抑菌作用;以活细胞计数(CFU)为指标检测SBP-H2O2-KI体系的杀菌作用;以最低抑菌浓度(MIC)为指标检测亚致死剂量的SBP-H2O2-KI体系作用下连续传代细菌的敏感性变化;以物理和化学方法检测SBP-H2O2-KI体系中活性氧基团等功能基团的生成与否,以期解释SBP-H2O2-KI体系的杀菌机制。【结果】SBP-H2O2-KI体系对多种细菌有高效、快速杀菌作用,作用时间仅为几分钟。在亚致死剂量浓度下连续培养的细菌悬液对体系的耐受能力(MIC)没有显著性变化,从中也不能分离到抗性/耐性突变株。物理和化学方法检测结果表明SBP-H2O2-KI反应过程中无羟基自由基产生;化学方法检测结果表明SBP-H2O2-KI反应过程中无超氧阴离子自由基产生;而有单线态氧和单质碘的产生。【结论】根据实验结果可以推测:SBP-H2O2-KI体系的杀菌力可能主要来源于单线态氧和碘的活性中间态,而不是羟基自由基和超氧阴离子。此外,SBP-H2O2-KI体系所具备的高效、快速的杀菌作用,以及细菌对其不易产生抗性的特点,预示此体系在医疗以及植保等方面有广泛的应用前景。     

辣根过氧化物酶同功酶C基因克隆及其在大肠杆菌中的表达

无论从应用还是从理论研究角度,辣根过氧化物酶（ＨＲＰ）是一种非常重要的酶．ＨＲＰ基因克隆与表达将有利于更深入研究ＨＲＰ的结构与功能．利用反转录ＰＣＲ从天然植物辣根中分离和克隆编码辣根过氧化物酶同功酶Ｃ（ＨＲＰ－Ｃ）一个ｃＤＮＡ,并测定其序列．结果发现,从基因推导出的氨基酸序列与Ｗｅｌｉｎｄｅｒ报道的辣根过氧化物酶序列有９０．６％的同源性．将该基因连接到表达载体ｐＥＴ－２４ｂ上,利用抗ＨＲＰ多克隆抗体进行Ｗｅｓｔｅｒｎｂｌｏｔ,检测有少量目标产物表达．在诱导表达过程中,没有发现细菌生长受抑制或受明显的毒害     

Lactoperoxidase: structural insights into the function,ligand binding and inhibition

Lactoperoxidase (LPO) is a member of a large group of mammalian heme peroxidases that include myeloperoxidase (MPO), eosinophil peroxidase (EPO) and thyroid peroxidase (TPO). The LPO is found in exocrine secretions including milk. It is responsible for the inactivation of a wide range of micro-organisms and hence, is an important component of defense mechanism in the body. With the help of hydrogen peroxide, it catalyzes the oxidation of halides, pseudohalides and organic aromatic molecules. Historically, LPO was isolated in 1943, nearly seventy years ago but its three-dimensional crystal structure has been elucidated only recently. This review provides various details of this protein from its discovery to understanding its structure, function and applications. In order to highlight species dependent variations in the structure and function of LPO, a detailed comparison of sequence, structure and function of LPO from various species have been made. The structural basis of ligand binding and distinctions in the modes of binding of substrates and inhibitors have been analyzed extensively.     

Inhibition of Lactoperoxidase by Its Own Catalytic Product: Crystal Structure of the Hypothiocyanate-Inhibited Bovine Lactoperoxidase at 2.3-Å Resolution

To the best of our knowledge, this is the first report on the structure of product-inhibited mammalian peroxidase. Lactoperoxidase is a heme containing an enzyme that catalyzes the inactivation of a wide range of microorganisms. In the presence of hydrogen peroxide, it preferentially converts thiocyanate ion into a toxic hypothiocyanate ion. Samples of bovine lactoperoxidase containing thiocyanate (SCN⁻) and hypothiocyanate (OSCN⁻) ions were purified and crystallized. The structure was determined at 2.3-Å resolution and refined to R_cryst and R_free factors of 0.184 and 0.221, respectively. The determination of structure revealed the presence of an OSCN⁻ ion at the distal heme cavity. The presence of OSCN⁻ ions in crystal samples was also confirmed by chemical and spectroscopic analysis. The OSCN⁻ ion interacts with the heme iron, Gln-105 N^ɛ1, His-109 N^ɛ2, and a water molecule W96. The sulfur atom of the OSCN⁻ ion forms a hypervalent bond with a nitrogen atom of the pyrrole ring D of the heme moiety at an S–N distance of 2.8 Å. The heme group is covalently bound to the protein through two ester linkages involving carboxylic groups of Glu-258 and Asp-108 and the modified methyl groups of pyrrole rings A and C, respectively. The heme moiety is significantly distorted from planarity, whereas pyrrole rings A, B, C, and D are essentially planar. The iron atom is displaced by ≈0.2 Å from the plane of the heme group toward the proximal site. The substrate channel resembles a long tunnel whose inner walls contain predominantly aromatic residues such as Phe-113, Phe-239, Phe-254, Phe-380, Phe-381, Phe-422, and Pro-424. A phosphorylated Ser-198 was evident at the surface, in the proximity of the calcium-binding channel.     

动物线粒体遗传系统理论与应用研究进展

随着线粒体与人类疾病之间关系研究的不断深入,线粒体DNA及其编码的13个多肽的功能和进化引起了众多研究者的关注。作为有氧呼吸的后生动物主要的产能系统——线粒体电子传递系统(ETS)由线粒体和核基因组共同编码,自然选择被认为偏爱那些增强ETS功能的突变,此类突变可发生在线粒体或核编码的ETS蛋白中并引起积极的基因组间的相互作用,即"共适应"。线粒体DNA进化通常被认为遵循一种稳定的突变速率平衡中性模型,但有证据表明该假设可能并不可靠。对线粒体遗传系统研究的最新进展进行了综述,这对科学和合理地使用线粒体DNA分析技术具重要意义。     

Antibacterial Activity of the Lactoperoxidase System in Milk Against Pseudomonads and Other Gram-Negative Bacteria

Products of thiocyanate oxidation by lactoperoxidase inhibit gram-positive bacteria that produce peroxide. We found these products to be bactericidal for such gram-negative bacteria as Pseudomonas species and Escherichia coli, provided peroxide is supplied exogenously by glucose oxidase and glucose. By the use of immobilized glucose oxidase the bactericidal agent was shown to be dialyzable, destroyed by heat and counteracted, or destroyed by reducing agents. Because the system is active against a number of gram-negative bacteria isolated from milk, it may possibly be exploited to increase the keeping quality of raw milk.     

南瓜属植物再生体系的建立及其应用

对南瓜属植物再生体系的研究及其应用作了介绍,并对存在问题和改进措施进行了分析讨论。     

漆酶及其应用的研究进展

漆酶是一种含铜的多酚氧化酶,白腐真菌普遍能分泌该酶.有氧条件下,漆酶能催化多种高分子化合物降解,利用该性质漆酶已经广泛应用于生物制浆、污染物生物降解、生物漂白等领域.介绍了目前漆酶在农业和工业各个领域中的应用.     

Role of Porins in Sensitivity of Escherichia coli to Antibacterial Activity of the Lactoperoxidase Enzyme System

Lactoperoxidase is an enzyme that contributes to the antimicrobial defense in secretory fluids and that has attracted interest as a potential biopreservative for foods and other perishable products. Its antimicrobial activity is based on the formation of hypothiocyanate (OSCN⁻) from thiocyanate (SCN⁻), using H₂O₂ as an oxidant. To gain insight into the antibacterial mode of action of the lactoperoxidase enzyme system, we generated random transposon insertion mutations in Escherichia coli MG1655 and screened the resultant mutants for an altered tolerance of bacteriostatic concentrations of this enzyme system. Out of the ca. 5,000 mutants screened, 4 showed significantly increased tolerance, and 2 of these had an insertion, one in the waaQ gene and one in the waaO gene, whose products are involved in the synthesis of the core oligosaccharide moiety of lipopolysaccharides. Besides producing truncated lipopolysaccharides and displaying hypersensitivity to novobiocin and sodium dodecyl sulfate (SDS), these mutants were also shown by urea-SDS-polyacrylamide gel electrophoresis analysis to have reduced amounts of porins in their outer membranes. Moreover, they showed a reduced degradation of p-nitrophenyl phosphate and an increased resistance to ampicillin, two indications of a decrease in outer membrane permeability for small hydrophilic solutes. Additionally, ompC and ompF knockout mutants displayed levels of tolerance to the lactoperoxidase system similar to those displayed by the waa mutants. These results suggest that mutations which reduce the porin-mediated outer membrane permeability for small hydrophilic molecules lead to increased tolerance to the lactoperoxidase enzyme system because of a reduced uptake of OSCN⁻.     

SELEX技术在神经系统疾病研究中的应用

配体指数级富集系统进化（systematic evolution of ligands by exponential enrichment,SELEX）技术是一种组合化学技术,可经过反复筛选扩增得到针对靶分子的高亲和力和高特异性的适配子.适配子通过识别、结合特定靶分子并对其进行功能调控从而达到对疾病诊断和治疗的目的 .近年来SELEX技术在神经系统功能和疾病研究中的应用越来越多.现已经筛选出针对朊蛋白、肌腱蛋白-C、β-淀粉样肽、乙酰胆碱受体的自身抗体等靶标的适配子,促进了对朊病毒病、脑肿瘤、阿茨海默病、重症肌无力等神经系统疾病的诊断和治疗研究,为这些疾病的诊治提供了新的研究工具.     

草酸脱羧酶及其应用

草酸脱羧酶是一种含锰的酶,在白腐菌中广泛存在,少数低等真菌和细菌中也能产生。目前,至少10多种草酸脱羧酶得到了分离和纯化。该酶是一种氨基酸残基在379个左右的单体酶,一般都为酸性糖蛋白,含有2个锰离子,形成2个活性区域;表面一些氨基酸被不同程度地糖基化。晶体结构和其它一些波谱学研究解释了其空间结构和可能的电子传递机制。运用PCR技术和cDNA文库技术,越来越多的草酸脱羧酶基因被克隆。已研究的该酶基因中都含有17个左右的内含子,这些内含子在活性域位置上有比较高的保守性。一些特殊氨基酸序列的存在决定了该酶的表达形式为诱导型,菌株的基因调控序列中含有一段受草酸化合物作用的序列。该酶在一些酵母和植物等异源表达系统中有成功表达的报道。该酶的应用主要集中在以下几方面：造纸废水中的草酸盐降解;食品中的草酸降解;草酸生物检测（如,临床诊断）等。