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.     

辣根过氧化物酶模拟酶研究进展

辣根过氧化物酶 (HRP)是一种常用的工具酶 ,对其模拟酶的研究是近年来生物化学和有机化学的重要课题 ,具有重要的理论意义和应用价值。本文评述了近十年来HRP模拟酶的研究进展。     

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。     

植物抗坏血酸过氧化物酶

植物抗坏血酸过氧化物酶沈文飚黄丽琴徐朗莱（南京农业大学理学院应用化学系,南京２１００９５）关键词抗坏血酸过氧化物酶植物抗坏血酸过氧化物酶（ＡＰＸ,ＥＣ１．１１．１．１１）的发现至今已有２０多年了。Ｆｏｙｅｒ和Ｈａｌｉｗｅｌ［１］首先于１９７６年发现以...     

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.     

Potent Inhibitory Effects of Some Phenolic Acids on Lactoperoxidase

Lactoperoxidase (LPO) plays a key role in immune response against pathogens. In this study, we examined the effects of some phenolic acids on LPO. For this purpose, bovine milk LPO was purified 380.85‐fold with a specific activity of 26.66 EU/mg and overall yield of 73.33% by using Amberlite CG‐50 H⁺ resin and CNBr‐activated Sepharose‐4B‐l ‐tyrosine‐sulfanilamide affinity chromatography. After purification, the in vitro effects of phenolic acids (tannic acid, 3,4‐dihydroxybenzoic acid, 3,5‐ dihydroxybenzoic acid, chlorogenic acid, sinapic acid, 4‐hydroxybenzoic acid, vanillic acid, salicylic acid, and 3‐hydroxybenzoic acid) were investigated on LPO. These phenolic acids showed potent inhibitory effect on LPO. K_i values for these phenolic acids were found as 0.0129 nM, 0.132 μM, 0.225 μM, 0.286 μM, 0.333 μM, 2.33 μM, 10.82 μM, 0.076 mM, and 0.405 mM, respectively. Sinapic acid and 4‐hydroxybenzoic acid exhibited noncompetitive inhibition; 3,4‐dihydroxybenzoic acid showed uncompetitive inhibition, and other phenolic acids showed competitive inhibition.     

双杂合系统及其应用

蛋白质与蛋白质的相互作用是生物体内包括复制、转录、分泌、信号传导、代谢等生命活动得以进行的物质基础．以基因工程技术为基础的双杂合系统可以在体内检测蛋白质与蛋白质的相互作用,并推广应用到寻找同某已知蛋白质相互作用的未知蛋白质,直接克隆未知蛋白质的基因,鉴别同已知蛋白质相互作用的关键氨基酸残基,绘制蛋白质联系图谱等．双杂合系统在药物设计中的应用使其具有更重大的实用价值．     

The Lactoperoxidase System: a Natural Biochemical Biocontrol Agent for Pre‐ and Postharvest Applications

Controlling pests in pre‐ and postharvest crops using natural and low‐impact products is a major challenge. The lactoperoxidase system is an enzymatic system that exists in all external secretions in mammals and is part of the non‐immune system. We tested its efficacy in in vitro microplates on Phytophthora infestans, Penicillium digitatum, Penicillium italicum, Penicillium expansum and Botrytis cinerea to determine the most suitable concentrations for use. Then, we verified its efficacy in planta under controlled conditions. Solutions prepared with 5.4 mm iodide and 1.2 mm thiocyanate and diluted threefold inhibited pathogen growth in vitro by 63–100%. Twofold‐diluted solutions protected potato plants against P. infestans by 60–74% under controlled conditions. Undiluted solution inhibited orange's and apple's postharvest pathogens in curative application with efficacy levels ranging between 84 and 95% in orange and between 63 and 74% in apple. 1.5‐fold concentrated solutions inhibited postharvest pathogens of apple in curative application with efficacy levels ranging between 84 and 92%. Our results also show that the oxidative stress response of fruit following wounding could interfere with ion efficiency. Our tests demonstrate for the first time that this biochemical method is as efficient as a conventional synthetic chemical method under controlled conditions.     

CRISPR-Cas9系统在疾病模型中的应用

规律成簇间隔短回文重复（CRISPR）及相关核酸内切酶（Cas）系统是最近发现的一种关于RNA指导核酸内切酶的基因编辑技术,这一技术的发现促进了生物学和医学研究的发展。CRISPR-Cas9系统的简便性使其广泛应用于细胞基因组编辑、动物模型的构建及疾病模型的基因治疗。现就CRISPR-Cas9系统的结构特点、作用机制及应用进行了综述。     

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.     

微生物适冷酶及其应用研究新进展

生物圈的80%是由低温环境构成,大约90%的海水平均温度为5℃或者更低,这里孕育着极为丰富的微生物。根据微生物对环境温度的耐受性和其生长温度上限/下限的不同,将微生物分为嗜冷微生物和适冷微生物两大类。由这些微生物产生的适冷酶在低温下具有较高的催化效率和特异性,因而在生物技术领域具有巨大的应用潜力和开发价值。总结了近5年适冷酶新酶的筛选、发现及适冷酶稳定性的改造和低温表达系统等方面的最新研究进展,并对该领域的研究方向进行了展望。     

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.     

CRISPR/Cas9技术及其在转基因动物中的应用

CRISPR/Cas9技术是一种新型的基因组定点编辑技术,具有设计简单、特异性强、效率高及可以在目标位点产生多种类型的编辑结果等特点,适用于在多种细胞中进行大规模的基因编辑。综述了CRISPR/Cas9技术的研究背景、基本原理和研究进展,从靶基因敲除(knock-out)、外源基因整合(knock-in)和目标基因转录沉默(knock-down)等方面总结了CRISPR/Cas9在转基因动物中的应用概况,并对现有的三种基因组定点编辑技术进行了比较。CRISPR/Cas9技术在转基因动物中具有明显的应用优势和良好前景。