Relationship between the Conformation and Enzyme Activity of Commercial and Purified Glucose Oxidase

Glucoseoxidase(5-D-glucosefoxygenoxidoreductase,ECl.l.3.4,abbreviationGOD)isaglucoproteincontainingl6%(w/w)carbohydrate.H.M.Kaliszetal.1havereportedthatthecontaminatesincludingcarbohydratecanhinderthecrystallizationprocess.Inthisexperiment,fastproteinliquidchromatography(FPLC)togetherwithMonoQcolumnhavebeenusedtopurifyGOD,andtheconformationofcommercialGODandpurifiedGODhasbeenstudiedbyrr-Acurves,LBtechniqueandCDspectrum.PurificationwasperformedwithaPharmaciaFPLCunit(PharmaciaLKB,…     

Esterification Activity of Novel Fungal and Yeast Lipases

The main objective of this work was the isolation and screening of microorganisms with potential for producing lipases for the synthesis of fatty esters as well as evaluating the specificity of the enzymes produced, using different alcohols (methanol, ethanol, n-propanol, and butanol) and fatty acids (oleic and lauric acids) as substrates. Promising biocatalysts for organic synthesis were obtained in this work. The isolated strains 69F and 161Y showed ability to efficiently catalyze the reaction for production of n-propyl oleate. Other strains can also be considered of potential interest, as 74F, 111Y, and 186Y. The future development of production using different substrates could result in cheap crude lipase of high importance to industrial applicability.     

Assessment of Activities and Conformation of Lipases Treated with Sub- and Supercritical Carbon Dioxide

In order to illustrate the underlining mechanism of the effect of high pressure on lipases from different resources, the influence of compressed carbon dioxide treatment on the esterification activities and conformation of the three lipases Candida rugosa lipase (CRL), Pseudomonas fluorescens lipase, and Rhizopus oryzae lipase was investigated in the present work. The results showed that the lipases activities were significantly enhanced in most of high-pressure treatments, except the pressure had a negative effect on CRL activity in supercritical condition. Mild depressurization rate could remain the lipase’s activity by protecting its rigid structure under supercritical fluid. Conformational analysis by Fourier transform-infrared spectrometry and fluorescence emission spectra revealed that the variances of lipase activity after high-pressure treatment were correlated with the changes of its α-helix content and fluorescence intensity. Additionally, transesterification catalyzed by three lipases in supercritical carbon dioxide were conducted, and 87.2 % biodiesel conversion was obtained by CRL after 3 h, resulting in a great reduction of reaction time.     

十二烷基磺酸钠对大豆过氧化物酶活性和构象的影响

在不同的pH值体系中, 利用酶活测量、圆二色谱、荧光谱和电子吸收谱研究了十二烷基磺酸钠(SDS)对大豆过氧化物酶(SBP)活性与构象的影响情况. 在pH 2.6和4.2 的体系中, 少量的SDS分子可通过静电作用与SBP结合, 进而与SBP分子中的His169残基结合, 降低其与铁卟啉的配位能力, 使其Soret吸收带蓝移, 二级结构发生轻微的变动, 活性永久丧失. 在pH 5.2体系中, SDS和SBP分子都带负电, 由于静电排斥作用, SDS无法进入SBP的分子内部, 失去与SBP分子中His169残基结合的能力, 对SBP分子的二级结构没有影响, 仅对SBP分子的三级结构有所影响. 当SDS的浓度大于临界胶束浓度时, 由于胶束与SBP的静电排斥作用增强, 限制了铁卟啉中乙烯基的运动, 乙烯基与卟啉环的共轭程度增大, Soret 吸收带红移. 由于SBP活性可完全恢复, 此变化是可逆的.     

Water Activity Dependence of Lipases in Non-aqueous Biocatalysis

Eleven lipases are tested and it was found that lipases can be divided into three types according to water activity dependence. The first type is lipase that has low water activity dependence and works in a low water activity, its performance changes little with the change of water activity. The optimum water activity is 0.19 and Newlase F (Rhizopus niveus), lipase FAP-15 (Rhizopus oryzae) belong to this type. The second type is lipase that has medium water activity dependence and its performance changes with the change of water activity. Most lipases belong to this type and the optimum water activity in this type is about 0.60. The third type is lipase that has a high water activity dependence and works only in a high water activity (a _w  > 0.75). WGL (wheat germ) belongs to this type and the optimum water activity is 0.90. The relationship between enantioselectivity and water activity is also discussed and the enantioselectivity seems to be independent of water activity. And we also compared the two control methods of water activity, it was found that the method which add solid salt hydrates to the reaction mixture (method II) is more stable and effective throughout the reaction than the method that pre-equilibrate via the vapor phase (method I). The addition concentration of salt hydrates is also investigated and the optimum concentration is 1 g/l.     

The Effect of Trifluoroethanol on Tyrosinase Activity and Conformation: Inhibition Kinetics and Computational Simulations

We studied the inhibitory effects of trifluoroethanol (TFE) on the activity and conformation of tyrosinase. TFE increased the degree of secondary structure of tyrosinase, which directly resulted in enzyme inactivation. A reciprocal study showed that TFE inhibited tyrosinase in a slope-parabolic mixed-type inhibition manner (K _I = 0.5 ± 0.096 M). Time-interval kinetic studies showed that the inhibition was best described as first order with biphasic processes. Intrinsic and 1-anilinonaphthalene-8-sulfonate-binding fluorescences were also measured to gain more insight into the supposed structural changes; these showed that TFE induced a conspicuous tertiary structural change in tyrosinase by exposing hydrophobic surfaces. We also predicted the tertiary structure of tyrosinase and simulated its docking with TFE. The docking simulation was successful with significant scores (binding energy for Autodock4 = −4.75 kcal/mol; for Dock6 = −23.07 kcal/mol) and suggested that the TRP173 residue was mainly responsible for the interaction with TFE. Our results provide insight into the structure of tyrosinase and allow us to describe a new inhibition strategy that works by inducing conformational changes rather than targeting the active site of the protein.     

Influence of N-Methylation and Conformation on Almiramide Anti-Leishmanial Activity

The almiramide N-methylated lipopeptides exhibit promising activity against trypanosomatid parasites. A structure–activity relationship study has been performed to examine the influences of N-methylation and conformation on activity against various strains of leishmaniasis protozoan and on cytotoxicity. The synthesis and biological analysis of twenty-five analogs demonstrated that derivatives with a single methyl group on either the first or fifth residue amide nitrogen exhibited greater activity than the permethylated peptides and relatively high potency against resistant strains. Replacement of amino amide residues in the peptide, by turn inducing α amino γ lactam (Agl) and N-aminoimidazalone (Nai) counterparts, reduced typically anti-parasitic activity; however, peptide amides possessing Agl residues at the second residue retained significant potency in the unmethylated and permethylated series. Systematic study of the effects of methylation and turn geometry on anti-parasitic activity indicated the relevance of an extended conformer about the central residues, and conformational mobility by tertiary amide isomerization and turn geometry at the extremities of the active peptides.     

Conformation and Biological Activity of Cyclic Peptides

Cyclic peptides containing biologically active hormone sequences are suitable models for studying conformation-activity relationships. In such models the usual flexibility of peptide chains is reduced by their cyclic arrangement. However, conformational analysis of such systems by experimental means is possible only if a single conformer predominates at equilibrium, and criteria for this are put forward. NMR spectroscopic methods, including many recent advances, are discussed in relation to their ability to contribute to peptide conformational analysis.     

Collagen-Immobilized Lipases Show Good Activity and Reusability for Butyl Butyrate Synthesis

Candida rugosa lipases were immobilized onto collagen fibers through glutaraldehyde cross-linking method. The immobilization process has been optimized. Under the optimal immobilization conditions, the activity of the collagen-immobilized lipase reached 340 U/g. The activity was recovered of 28.3 % by immobilization. The operational stability of the obtained collagen-immobilized lipase for hydrolysis of olive oil emulsion was determined. The collagen-immobilized lipase showed good tolerance to temperature and pH variations in comparison to free lipase. The collagen-immobilized lipase was also applied as biocatalyst for synthesis of butyl butyrate from butyric acid and 1-butanol in n-hexane. The conversion yield was 94 % at the optimal conditions. Of its initial activity, 64 % was retained after 5 cycles for synthesizing butyl butyrate in n-hexane.     

Effect of the Pretreatment of Lipase with Organic Solvents on its Conformation and Activity in Reverse Micelles

The activity and conformation of Chromobacterium viscosum lipase-pretreated with various organic solvents were investigated. The pretreatment of lipase led to a substantial increase of enzyme activity in AOT (sodium bis [2-ethyl -1-hexyl] sulfosuccinate)/isooctane/water reverse micelles. Among the organic solvents used, n-hexane was found to be most effective. It was observed that higher hexane content with shorter agitation time and vice versa had almost the same effect on the initial activity of lipase. The kinetic study showed that the Michaelis constant (K _m) and the substrate adsorption equilibrium constant (K _ad) were reduced by the pretreatment of lipase with hexane, whereas the change in the maximum reaction rate (V _max) was insignificant. The two spectroscopic techniques (Fluorescence spectra of lipase encapsulated in RMs and Fourier transform infrared [FTIR] spectra of lipase powders) were performed to detect possible conformational changes in the enzyme caused by the pretreatment. A correlation between the maximum fluorescence intensity and the activity of treated lipase was found as a function of agitation time. The FTIR spectrum of lipase showed a new shape peak corresponding to 1,500 cm⁻¹ as a result of pretreatment with organic solvents.     

Changes in the Activity of Lipases in the Presence of Synthetic and Natural Polymers

Moscow University Chemistry Bulletin - Abstract—Both synthetic and natural polymers are promising carriers for the immobilization of enzymes, including lipases from different sources. This...     

Influence of Side Chain Conformation on the Activity of Glycosidase Inhibitors

Substrate side chain conformation impacts reactivity during glycosylation and glycoside hydrolysis and is restricted by many glycosidases and glycosyltransferases during catalysis. We show that the side chains of gluco and manno iminosugars can be restricted to predominant conformations by strategic installation of a methyl group. Glycosidase inhibition studies reveal that iminosugars with the gauche,gauche side chain conformations are 6- to 10-fold more potent than isosteric compounds with the gauche,trans conformation; a manno-configured iminosugar with the gauche,gauche conformation is a 27-fold better inhibitor than 1-deoxymannojirimycin. The results are discussed in terms of the energetic benefits of preorganization, particularly when in synergy with favorable hydrophobic interactions. The demonstration that inhibitor side chain preorganization can favorably impact glycosidase inhibition paves the way for improved inhibitor design through conformational preorganization.     

单核铁(Ⅲ)双卟啉配合物在溶液中的构象平衡及其对环己烷羟化反应的催化活性的影响

合成及表征了一系列以柔韧碳氢链相连不同长度的ｐ／ｐ型单核铁（Ⅲ）双卟啉配合物，以可见光谱首次观察到该系列配合物在氯仿溶液中开放式及闭合式构象的平衡，发现６８０ｎｍ处吸收峰强度与这种构象平衡有关，烷氧链越长，该吸收峰强度越大．利用该类配合物模拟了细胞色素Ｐ４５０单加氧酶对环己烷的羟化作用，催化结果表明，在以分子氧为氧源及还原剂存在的温和条件下，该类配合物的催化活性显著高于单核铁卟啉（ＦｅＴＰＰＣｌ），随着柔韧碳氢链长度的增加，双卟啉的催化活性依次增加．高的催化活性与双卟啉配合物在溶液中的构象平衡，即闭合式构象引起的立体位阻及电子转移有关．     

Trifluoroethanol-Induced Changes in Activity and Conformation of Manganese-Containing Superoxide Dismutase

Superoxide dismutase (SOD, EC 1.15.1.1) plays an important role in antioxidant defense in organisms exposed to oxygen. However, there is a lack of research into the regulation of SOD activity and structural changes during folding, especially for SOD originating from extremophiles. We studied the inhibitory effects of trifluoroethanol (TFE) on the activity and conformation of manganese-containing SOD (Mn-SOD) from Thermus thermophilus. TFE decreased the degree of secondary structure of Mn-SOD, which directly resulted in enzyme inactivation and disrupted the tertiary structure of Mn-SOD. The kinetic studies showed that TFE-induced inactivation of Mn-SOD is a first-order reaction and that the regional Mn-contained active site is very stable compared to the overall structure. We further simulated the docking between Mn-SOD and TFE (binding energy for Dock 6.3, −9.68 kcal/mol) and predicted that the LEU9, TYR13, and HIS29 residues outside of the active site interact with TFE. Our results provide insight into the inactivation of Mn-SOD during unfolding in the presence of TFE and allow us to describe ligand binding via inhibition kinetics combined with computational predictions.     

Study on the Conformation of Cyclodecapeptide Loloatin C with Obvious Antibiotic Activity

The conformation of cyclodecapeptide loloatin C with obvious antibiotic activity has been investigated in 2,2,2-trifluoroethanol/sodium acetate buffer solution and then characterized by Fr-IR, CD and NMR spectrum. The results of FT-IR show that there exists β-strand or β-tum secondary structure in the molecule. According to the CD spectrum, the helical turn is dominant but the β-turn structure also exists. Conformation of the whole molecule is probably a helical β-turn.The chemical shifts and coupling constants prove the existence of a β-structure in the regions of Val,Orn2 and Leu3. NOESY data and temperature gradients of amide protons suggest that the molecular conformation is a dumbbell-like structure with the waist located between ornithyl (position 2) and D-phenylalanyl (position 7) and β-turn on both ends.     

核糖核酸酶A在DAB-环乙烷溶液中的活性和构象

The activity and conformation of ribonuclerse A (RNaseA) solubilized in cyclohexane via dodecylammonium butyrate(DAB) reverse micelles were investigated. The activity of RNaseA was studied using the cytidine 2’,3’ -phosphate as the substrate, and it was found that kcat increases significantly with respect to that in water attended by an increased Km•FT-IR spectra of RNaseA in reverse micellar solution were investigated as a function of w0(= [H2O]/ [DAB]), and it was noted that the structure of RNaseA became losser in reverse micelles campared to that in aqueous solution. The relation between activity and conformation was discussed.     

溶液组成对乌头酸梅构象热稳定性的影响

乌头酸酶（ＥＣ４．２．１．３）又名乌头酸水合酶或柠檬酸（异柠檬酸）水解酶 ,从被发现至今已有６０年的研究利用历史［１］．然而 ,仅仅约１０年前才弄清其一级结构［２］和晶体结构［３］,但它的热稳定性研究至今未见报导．它是一个四结构域蛋白 ,在第３结构域内的铁 硫簇是酶促立体有择反应的催化中心．在大多数活细胞中心代谢途径的核心———Ｋｒｅｂｓ三羧酸循环的第２及第３步中 ,将柠檬酸经过顺 乌头酸最终转化为异柠檬酸．因此 ,它是活细胞中心代谢途径中不可缺少的酶．显然 ,认识该酶构象的稳定性及影响因素 ,有助于控制细胞…     

Ce4+对过氧化氢酶构象的影响

为了探讨Ce4+对过氧化氢酶(CAT)构象的影响, 以离体的牛肝过氧化氢酶(CAT)为实验材料, 用紫外-可见吸收光谱、荧光光谱、 CD谱、红外光谱等方法分析Ce4+作用后CAT构象的变化, 发现CAT的构象与Ce4+的浓度相关, Ce4+的浓度较低时, CAT的α-螺旋含量增加, β-折叠含量减小;Ce4+的浓度较高时, 结果相反. 结果表明, 不同浓度的Ce4+对CAT构象的影响不同.     

磁场对大分子构象的影响研究进展

对近年来有关磁场对大分子构象的影响研究进展进行了较全面综述.     

Lipases,liposomes and lipid-prodrugs

Colloidal and interfacial phenomena lie at the core of drug formulation, drug delivery, as well as drug binding and action at diseased sites, e.g., in cancer therapy. We review a class of liposome-based drug-delivery systems whose design and functional properties are intimately controlled by the stability of sub-micron structures, lipid-bilayer interfaces, and interfacially activated enzymes that can be exploited to target and deliver drugs. Moreover these drugs can themselves be special lipid molecules in the form of lipid prodrugs that both form the liposomal carrier as well as the substrate for endogenously upregulated lipases that turn the prodrugs into potent drugs precisely at the diseased site.