Concanavalin a immobilized affinity adsorbents for reversible use in yeast invertase adsorption

Concanavalin A (Con A) immobilized poly(2-hydroxyethyl methacrylate) (PHEMA) beads were investigated for specific adsorption of yeast invertase from aqueous solutions. PHEMA beads were prepared by a suspension polymerization technique with an average size of 150-200 microm, and activated by epichlorohydrin. Con A was then immobilized by covalent binding onto these beads. The maximum Con A immobilization was found to be 10 mg/g. The invertase-loading capability of the PHEMA/Con A beads was 107 mg/g. The maximum invertase adsorption capacity on the PHEMA/Con A adsorbents was observed at pH 5.0. The values of the Michaelis constant K(m) of invertase were significantly larger upon adsorption, indicating decreased affinity by the enzyme for its substrate, whereas V(max) was smaller for the adsorbed invertase. Adsorption improved the pH stability of the enzyme as well as its temperature stability. Thermal stability was found to increase with adsorption. The adsorbed enzyme activity was found to be quite stable in repeated experiments. Storage stability of adsorbed invertase.     

A new enzyme model for enantioselective esterases based on molecularly imprinted polymers

An efficient enzyme model exhibiting enantioselective esterase activity was prepared by using molecular imprinting techniques. The enantiomerically pure phosphonic monoesters 4 L and 5 L were synthesized as stable transition-state analogues. They were used as templates connected by stoichiometric noncovalent interactions to two equivalents of the amidinium binding site monomer 1. After polymerization and removal of the template, the polymers were efficient catalysts for the hydrolysis of certain nonactivated amino acid phenylesters (2 L, 2 D, 3 L, 3 D) depending on the template used. Imprinted catalyst IP4 (imprinted with 4 L) enhanced the hydrolysis of the corresponding substrate 2 L by a factor of 325 relative to that of a buffered solution. Relative to a control polymer containing the same functionalities, prepared without template 4 L, the enhancement was still about 80-fold, showing the highest imprinting effect up to now. In cross-selectivity experiments a strong substrate selectivity of higher than three was found despite small differences in the structure of the substrate and template. Plots of initial velocities of the hydrolysis versus substrate concentration showed typical Michaelis-Menten kinetics with saturation behavior. From these curves, the Michaelis constant K(M) and the catalytic constant k(cat) can be calculated. The enantioselectivity shown in these values is most interesting. The ratio of the catalytic efficiency k(cat)/K(M), between the hydrolysis of 2 L- and 2 D-substrate with IP4, is 1.65. This enantioselectivity derives from both selective binding of the substrate (K(M)L/K(M)D=0.82), and from selective formation of the transition state (k(cat)L/k(cat)D=1.36). Thus, these catalysts give good catalysis as well as high imprinting and substrate selectivity. Strong competitive inhibition is caused by the template used in imprinting. This behavior is also quite similar to the behavior of natural enzymes, for which these catalysts are good models.     

Flow Injection Chemiluminescence for the Determination of Estriol via a Noncompetitive Enzyme Immunoassay

A novel approach to the detection of estriol using a flow injection system coupled to enhanced chemiluminescent immunoassay was developed based on noncompetitive immunoassay formats. A conjugated estriol-ovalbumin immobilized immunoaffinity column was inserted into the flow system to trap the unbound horseradish peroxidase (HRP)-labeled antibody after an off-line incubation of estriol and HRP-labeled anti-estriol antibody. The trapped enzyme conjugate was detected by the injection of chemiluminescent substrates to produce enhanced chemiluminescence. The linear range for the determination of estriol is 10.0 to 400 ng · mL⁻¹ with a correlation coefficient of 0.996 and a detection limit of 5.0 ng · mL⁻¹. The total time for sampling and chemiluminescent detection of one sample is 400 seconds after 30 min of pre-incubation. The results for pregnancy serum samples obtained by this method are in good agreement with those obtained using ELISA.     

Mechanism and potential applications of bio-ligninolytic systems in a CELSS

A large amount of inedible plant material, generated as a result of plant growth in a Controlled Ecological Life Support System (CELSS), should be pretreated and converted into forms that can be recycled on earth as well as in space. The main portion of the inedible biomass is lignocellulosic material. Enzymatic hydrolysis of this cellulose would provide sugars for many other uses by recycling carbon, hydrogen, oxygen, and nitrogen through formation of carbon dioxide, heat, and sugars, which are potential foodstuffs. To obtain monosaccharides from cellulose, the protective effect of lignin should be removed. White-rot fungi degrade lignin more extensively and rapidly than other microorganisms.Pleurotus ostreatus degrades lignin effectively, and produces edible and flavorful mushrooms that increase the quality and nutritional value of the diet. This mushroom is also capable of metabolizing hemicellulose, thereby providing a food use of this pentose containing polysaccharide. This study presents the current knowledge of physiology and biochemistry of primary and secondary metabolisms of basidiomycetes, and degradation mechanism of lignin. A better understanding of the ligninolytic activity of white-rot fungi will impact the CELSS Program by providing insights on how edible fungi might be used to recycle the inedible portions of the crops.     

合成天然产物曲古抑菌素A的新方法

报道了一条组蛋白去乙酰化酶抑制剂曲古抑菌素A的有效合成路线. 通过L-脯氨酸催化对硝基苯甲醛与丙醛的羟醛缩合反应, 高立体选择性地构建了目标分子的手性中心, 羟醛缩合产物的ee值大于99%, anti∶syn＝16∶1. 随后的合成过程中无消旋化现象, 合成的曲古抑菌素A是单一R型异构体, ee值大于99%.     

Utility of copper(II) oxide as a packed reactor in flow injection assembly for rapid analysis of some angiotensin converting enzyme inhibitors

A new simple, sensitive, rapid and precise flow injection (FI) procedure based on the formation of copper complexes with some angiotensin converting enzyme (ACE) inhibitors has been developed and evaluated for the analysis of lisinopril (LN), enalapril maleate (EP), ramipril (RP) and perindopril tert-butylamine (PD). In this method, samples were injected into a flowing stream of distilled-deionized water, carried through the packed reactor of CuO for derivatization followed by ultraviolet (UV) detection. The flow rate was 1.5 ml min⁻¹ and column temperature was ambient (25 °C). Lisinopril was injected directly into the flowing stream and the detector response was measured at 262 nm. The hydrolysis products of enalapril maleate, ramipril and perindopril tert-butylamine in 0.2N NaOH were injected after neutralization with 1N HCl and the detector response was measured at 272, 265 and 252 nm, respectively. The developed method was successfully applied to the determination of tested drugs in pharmaceutical preparations at a sampling rate of 60 samples h⁻¹ and a recovery near 100% for all compounds.     

Binding N2, N2H2, N2H4, and NH3 to transition-metal sulfur sites: modeling potential intermediates of biological N2 fixation

In the quest for low-molecular-weight metal sulfur complexes that bind nitrogenase-relevant small molecules and can serve as model complexes for nitrogenase, compounds with the [Ru(PiPr(3))('N(2)Me(2)S(2)')] fragment were found ('N(2)Me(2)S(2)'(2-)=1,2-ethanediamine-N,N'-dimethyl-N,N'-bis(2-benzenethiolate)(2-)). This fragment enabled the synthesis of a first series of chiral metal sulfur complexes, [Ru(L)(PiPr(3))('N(2)Me(2)S(2)')] with L=N(2), N(2)H(2), N(2)H(4), and NH(3), that meet the biological constraint of forming under mild conditions. The reaction of [Ru(NCCH(3))(PiPr(3))('N(2)Me(2)S(2)')] (1) with NH(3) gave the ammonia complex [Ru(NH(3))(PiPr(3))('N(2)Me(2)S(2)')] (4), which readily exchanged NH(3) for N(2) to yield the mononuclear dinitrogen complex [Ru(N(2))(PiPr(3))('N(2)Me(2)S(2)')] (2) in almost quantitative yield. Complex 2, obtained by this new efficient synthesis, was the starting material for the synthesis of dinuclear (R,R)- and (S,S)-[micro-N(2)[Ru(PiPr(3))('N(2)Me(2)S(2)')](2)] ((R,R)-/(S,S)-3). (Both 2 and 3 have been reported previously.) The as-yet inexplicable behavior of complex 3 to form also the R,S isomer in solution has been revealed by DFT calculations and (2)D NMR spectroscopy studies. The reaction of 1 or 2 with anhydrous hydrazine yielded the hydrazine complex [Ru(N(2)H(4))(PiPr(3))('N(2)Me(2)S(2)')] (6), which is a highly reactive intermediate. Disproportionation of 6 resulted in the formation of mononuclear diazene complexes, the ammonia complex 4, and finally the dinuclear diazene complex [micro-N(2)H(2)[Ru(PiPr(3))('N(2)Me(2)S(2)')](2)] (5). Dinuclear complex 5 could also be obtained directly in an independent synthesis from 1 and N(2)H(2), which was generated in situ by acidolysis of K(2)N(2)(CO(2))(2). Treatment of 6 with CH(2)Cl(2), however, formed a chloromethylated diazene species [[Ru(PiPr(3))('N(2)Me(2)S(2)')]-micro-N(2)H(2)[Ru(Cl)('N(2)Me(2)S(2)CH(2)Cl')]] (9) ('N(2)Me(2)S(2)CH(2)Cl'(2-) =1,2-ethanediamine-N,N'-dimethyl-N-(2-benzenethiolate)(1-)-N'-(2-benzenechloromethylthioether)(1-)]. The molecular structures of 4, 5, and 9 were determined by X-ray crystal structure analysis, and the labile N(2)H(4) complex 6 was characterized by NMR spectroscopy.     

链酚胺型配体锌(Ⅱ)配合物模拟碳酸酐酶研究

合成了酚胺型链状配体,N,N'-二(2-羟基苄基)丙二胺(H~2L)及其Zn(Ⅱ)配合物(ZnL),通过元素分析、IR和^1HNRM等手段进行了表征。采用pH电位滴定法,在25℃±0.1℃,I=0.1mol·dm^-^3(KNO~3)条件下,测定了配体的质子化常数以及配体与金属离子Zn(Ⅱ)配位反应平衡常数。讨论了配体与金属离子Zn(Ⅱ)的配位情况,得到了配位酚羟基的解离常数。运用分光光度法,在25℃±0.1℃,I=0.1mol·dm^-^3(KNO~3)条件下,在pH=5.5～9.0(50mmol·dm^-^3缓冲溶液)范围内,研究了配合物作为碳酸酐酶模拟物催化对-硝基苯酚乙酸酯(NA)水解动力学,得到了NA酯水解的配合物催化速率常数k~N~P。实验结果表明,ZnHL^+的配位酚羟基的解离常数pK~a为6.83;催化速率常数k~N~P与pH之间不存在Sigmoidal型曲线关系,而是在pH值中性附近有最大值,ZnHL^+对NA酯水解有很好的催化效果,并且采取双重催化机理,是碳酸酐酶很好的模拟物。     

Ternary β-Cyclodextrin Complexes as Models of Allosteric Effects

Two guests reacting with cyclodextrins (CDs) may form ternary complexes, in addition to the common competition of 1:1 complexes. One of the guests can really be included into the cavity of the CD, while the second guest molecule is either inserted close to the first one or attached to the outer surface of the supramolecule by H-bonding. There is a further possibility when the included guest bears a substituent outside the cavity and the second guest can interact with it. The properties of the ternary species formed are highly influenced by the solely (or primarily) included guest. The changes are attributed to the altered properties of the hydrophilic domain of the CD. The phenomena can be proved by NMR data obtained for some binary systems of -CD inclusion complexes and acetic acid and by the stability constants of the ternary complexes formed. Allosteric effects as well as coenzyme/apoenzyme/substrate interactions could be well modelled by these types of CD complexes.     

MMI作为铜的盐酸酸洗缓蚀剂作用机理的研究

用失重法研究了N-甲基-2-巯基咪唑（MMI）在5％盐酸中对铜的酸洗缓蚀性能.探讨了温度和MMI浓度对缓蚀效果的影响，从中得出了MMI在铜表面的吸附等温式，计算了吸附热及MMI的加入对铜在盐酸中腐蚀反应活化能的影响，进而探讨了MMI对铜缓蚀作用的机理.结果表明， 30 ℃下，在5％盐酸中，当MMI的浓度在3 mmol•L－1和8 mmol•L－1之间时,缓蚀率随MMI浓度的增加而迅速增加，当浓度达到8 mmol•L－1时，缓蚀率趋于定值，而当浓度小于3 mmol•L－1时， MMI的加入会加速铜的腐蚀；吸附在铜表面的MMI分子间的作用力整体表现为引力； MMI在铜表面的吸附是吸热反应； MMI的加入降低了铜的腐蚀反应活化能.