Characterisation of the citrate synthase reaction with propionyl-CoA

Experiments with propionyl-CoA stereoselectively deuteriated in the propionyl moiety demonstrate that the formation of (2S,3S)-methylcitric acid (1) catalysed by citrate (si)-synthase occurs with inversion of configuration in the propionyl moiety; the absolute configurations of the methylcitric acids 1 and 2 indicate a si attack on oxaloacetate. Deuterium in the pro-S position is exchanged for protium 60 times faster than deuterium in the pro-R position. Experiments with (R,S)-(2-2H1)propionyl-CoA allowed the determination of isotope effects. For the enzymatic formation of 1, a primary deuterium isotope effect kH/kD = 1.8 and a secondary alpha-deuterium isotope effect kH/kD = 0.99 were calculated; both are effects on Vmax/KM.     

Investigation of enzyme-substrate complexes by affinity chromatography. Application to pig heart citrate synthase

The relative affinities of Sepharose gels, to which coenzyme A (CoA-SH) and CoA-SH analogues were bound through a well defined site, for citrate synthase were determined. The relative eluting power of coenzyme derivatives for the CoA-SH-gel and the Matrex Gel Blue-bound enzyme was measured, and the influence of oxaloacetate on the binding of the enzyme investigated. From the results, the contributions of different parts of the coenzyme to its binding in the active site and kinetic concepts are derived and found to be in complete agreement with corresponding data for citrate synthase obtained from kinetic measurements reported in the literature. It is demonstrated for some other CoA-SH-specific enzymes that affinity chromatography is of value as an additional tool for the comparative investigation of binding sites of enzymes which depend on the same coenzyme.     

Immobilized biocatalysts

Recent work on immobilized biocatalysts at Helsinki University of Technology, Finland, is described, with starch processing, Β-galactosidase, glucose isomerase, invertase, and the immobilization of live cells as special examples.     

High-level QM/MM modelling predicts an arginine as the acid in the condensation reaction catalysed by citrate synthase

High-level ab initio quantum mechanical/molecular mechanical (QM/MM) modelling of citryl-CoA formation in citrate synthase reveals that an arginine residue acts as the proton donor; this proposed new mechanism helps to explain how chemical and large scale conformational changes are coupled in this paradigmatic enzyme.     

Immobilized plant cells

Plant cells have been immobilized in alginate, where they have been shown to retain their biological activity. Such systems can be utilized for bioconversions.     

Immobilized carboxypeptidase N

Carboxypeptidase N-Sepharose was prepared by covalent immobilization of purified human plasma carboxypeptidase N. More than 98% of the carboxypeptidase N was immobilized; 42% of the applied activity can be detected on the support. The column has excellent capabilities to quantitatively remove carboxy-terminal basic amino acids from peptides, as is demonstrated using the synthetic peptide substrate hippuryl-L-arginine and the nonapeptide bradykinin, and remains stable for several months. In contrast with apocarboxypeptidase B-Sepharose, apocarboxypeptidase N-Sepharose poorly binds its substrates.     

Cryoscopic studies of aqueous solutions of tartaric acid,sodium hydrogen tartrate,potassium tartrate,sodium dihydrogen citrate,potassium dihydrogen citrate,disodium hydrogen citrate,sodium citrate and potassium citrate

Freezing temperature lowerings of aqueous solutions of tartaric acid, sodium hydrogen tartrate, sodium dihydrogen citrate, potassium dihydrogen citrate, disodium hydrogen citrate, sodium citrate and potassium citrate were determined. These values and those taken from the literature for potassium tartrate were used in the determination of the osmotic and activity coefficients in the studied systems, via the numerical integration of the Gibbs–Duhem equation.     

Enzyme:nanoparticle bioconjugates with two sequential enzymes: stoichiometry and activity of malate dehydrogenase and citrate synthase on Au nanoparticles

We report the synthesis and characterization of bioconjugates in which the enzymes malate dehydrogenase (MDH) and/or citrate synthase (CS) were adsorbed to 30 nm diameter Au nanoparticles. Enzyme:Au stoichiometry and kinetic parameters (specific activity, k(cat), K(M), and activity per particle) were determined for MDH:Au, CS:Au, and three types of dual-activity MDH/CS:Au bioconjugates. For single-activity bioconjugates (MDH:Au and CS:Au), the number of enzyme molecules adsorbed per particle was dependent upon the enzyme concentration in solution, with multilayers forming at high enzyme:Au solution ratios. The specific activity of adsorbed enzyme increased with increasing number adsorbed per particle for CS:Au, but was less sensitive to stoichiometry for MDH:Au. Dual activity bioconjugates were prepared in three ways: (1) by adsorption of MDH followed by CS, (2) by adsorption of CS followed by MDH, and (3) by coadsorption of both enzymes from the same solution. The resulting bioconjugates differed substantially in the number of enzyme molecules adsorbed per particle, the specific activity of the adsorbed enzymes, and also the enzymatic activity per particle. Bioconjugates formed by adding CS to the Au nanoparticles before MDH was added exhibited higher specific activities for both enzymes than those formed by adding the enzymes in the reverse order. These bioconjugates also had 3-fold higher per-particle sequential activity for conversion of malate to citrate, despite substantially fewer copies of both enzymes present.     

微胶囊固定化过氧化氢酶

以乙基纤维素为膜材，用液中干燥法将过氧化氢酶微胶囊化，研究了温度和ｐＨ值对酶活性的影响及过氧化氢和尿素对固定化酶的抑制作用，由于乙基纤维素膜的保护，明胶对ｐＨ值的缓冲作用及明胶与尿素的反应，明显地扩大了酶对温度和ｐＨ值的适应性，降低了酶活性抑制剂的影响。     

Immobilized microfluidic enzymatic reactors

The use of enzymes for cleavage, synthesis or chemical modification represents one of the most common processes used in biochemical and molecular biology laboratories. The continuing progress in medical research, genomics, proteomics, and related emerging biotechnology fields leads to exponential growth of the applications of enzymes and the development of modified or new enzymes with specific activities. Concurrently, new technologies are being developed to improve reaction rates and specificity or perform the reaction in a specific environment. Besides large-scale industrial applications, where typically a large processing capacity is required, there are other, much lower-scale applications, benefiting form the new developments in enzymology. One such technology is microfluidics with the potential to revolutionize analytical instrumentation for the analyses of very small sample amounts, single cells or even subcellular assemblies. This article aims at reviewing the current status of the development of the immobilized microfluidic enzymatic reactors (IMERs) technology.     

固定化多酶级联反应器

多酶级联反应在生命活动过程中发挥着重要作用。固定化多酶级联反应器是将不同功能的酶通过物理化学或生物手段固定于特定载体上,以之模拟生物体内多种酶协同作用方式促使底物发生降解和转化等反应的新型仿生催化技术。该技术具有固定化酶的稳定性、可重复利用以及酶级联的高效协同催化等优点,近年来在生物传感、模拟生物学以及生物转化等领域得到越来越多的关注。本文从多酶级联反应原理、反应器制备、级联反应的影响因素及应用等方面对近年来固定化多酶级联反应器的进展进行详细评述,并展望其发展前景。     

Immobilized Proteins and Peptides

In recent years a great deal has been learned about the structure and function of proteins and about the roles they play in the complex process of a living organism. The classical approach of the biochemist in studying a given protein is to free it from the cellular species. The properties and structure of the protein in solution are then examined and analyzed. While this approach is logical and has yielded much information about proteins, one should always keep in mind that in natureagreat many proteins function not in solution, but at an interface or within solid state assemblages in cells. The protein removed from a surface is thus often not in its natural environment and can display an altered reactivity and stability.     

Stereochemistry of the citrate anion in compounds containing guanidinium and citrate ions

Journal of Structural Chemistry - An analysis is presented for the results obtained in the studies initiated by A. E. Shvelashivili, Corresponding Member, Academy of Sciences of Georgia, and...     

Electrospray ionisation with selected reaction monitoring for the determination of Mn‐citrate,Fe‐citrate,Cu‐citrate and Zn‐citrate

Citrate complexes of Mn and Fe, and potentially those of Cu and Zn, are considered as important low molecular mass species in human serum and cerebrospinal fluid (CSF). For example, Mn is supposed to enter the brain under excess exposure as Mn‐citrate leading to neurotoxic effects. Mn‐citrate has been characterised in human CSF using chromatography and electrophoresis online with inductively coupled plasma mass spectrometry, but not yet with molecular mass spectrometry. Therefore, this study explores the potential of electrospray ionisation (ESI) with selected reaction monitoring (SRM) for the detection of metal‐citrate complexes, in particular Mn‐citrate. The collision‐induced dissociation of precursor ions with various metal:citrate stoichiometries was studied for Mn‐citrate, Fe‐citrate, Cu‐citrate and Zn‐citrate. High selectivity was achieved for Mn(II)‐citrate even in respect to Fe(III)‐citrate which forms isobaric precursor ions. The limit of detection for Mn‐citrate was estimated to be around 250 µg L^?1 (referring to the total Mn content in the standard) using flow injection. The sensitivity was sufficient for the determination of Mn‐citrate in standard solutions and in an extract of an Mn‐citrate‐containing supplement. An improved ESI source design is expected to reduce the limits of detection significantly. The developed ESI‐SRM method has the potential to provide complementary data for the quality control of current separation methods for metal citrates using element‐selective detection, with application to biomedical samples and further matrices. Copyright © 2009 John Wiley & Sons, Ltd.     

Immobilized enzymes in chemical analysis

Immobilization of enzymes has proved to be useful in stabilizing them, in retrieving and reusing them, and in producing unstable or expensive reagents. Specific technologies have been developed for analytical applications of immobilized enzymes, including probes (e.g., enzyme electrodes) and reactors. The characteristics of these devices and their advantages and disadvantages will be presented.     

Immobilized Nanomaterials for Environmental Applications

Nanomaterials (NMs) have been extensively used in several environmental applications; however, their widespread dissemination at full scale is hindered by difficulties keeping them active in engineered systems. Thus, several strategies to immobilize NMs for their environmental utilization have been established and are described in the present review, emphasizing their role in the production of renewable energies, the removal of priority pollutants, as well as greenhouse gases, from industrial streams, by both biological and physicochemical processes. The challenges to optimize the application of immobilized NMs and the relevant research topics to consider in future research are also presented to encourage the scientific community to respond to current needs.     

Lipase Immobilized in Organic-Inorganic Matrices

Enzyme lipase was immobilized with ferrite powder and deposited in layers on glass slides from lipase to a solution of silicone alkoxides. The highest hydrolytical activity was observed with the magnetic lipase prepared by mixing the paste of ferrite powder and lipase with tetramethoxysilane, 3-aminopropyltriethoxysilane and propyltrimethoxysilane. In a mixed reactor, the particles of the magnetic lipase were desintegrated by mechanical stirring which caused loosing the lipase linked to magnetic material and resulted in a significant drop of activity after magnetic separation. Transparent layers were prepared by dip- or spin-coating from partially hydrolyzed tetraethoxysilane and solutions containing methyltriethoxysilane with 3-aminopropyltriethoxysilane or tetraethoxysilane with 3-mercaptopropyltriethoxysilane. The lipases immobilized in films with magnetic particles were active in tests with 4-nitrophenyl butyrate and were not inhibited by 0,0-dimethyl-0-(2,2-dichlor-vinyl)-phosphate.     

Immobilized enzymes as analytical reagents

Immobilized enzymes are becoming increasingly popular as analytical reagents because of their reusability, stability, and sensitivity to many inhibitors that would seriously interfere in assays using soluble enzymes. In this article, some of the kinetic and catalytic effects of immobilized enzymes in analysis will be discussed. The shift of the activity-pH profile curves on immobilization, the changes in temperature dependence. the inhibitor constants (K₁). Michaelis constants (K _m ), and the maximum velocity (V_max). plus others, will be discussed. Finally, the use of these immobilized enzymes in fluorometric and electrochemical monitoring systems will be shown, and the future of these reagents in various areas will be discussed. A survey of enzyme electrodes will be presented as an example of the use of immobilized enzymes. Application of immobilized enzyme technology to the assay of BUN, glucose, uric acid, amino acids, ethanol. and other metabolites will be discussed.