Affinity precipitation of enzymes

Lactate dehydrogenase has been purified by precipitation with a bis-ligand. The precipitating agent in this case was Bis-NAD. This approach of affinity precipitation is also applicable to other enzymes.     

Molecularly imprinted nanoreactors for regioselective huisgen 1,3-dipolar cycloaddition reaction

A molecularly imprinted polymer has been successfully utilized as nanoreactors for Huisgen 1,3-dipolar cycloaddition of azides and alkynes, leading to high product regioselectivity and kinetic acceleration. The MIP nanoreactors also showed remarkable selectivity toward the reactant structures, so that the "best fit" product was mostly amplified during the reaction. In contrast to previously reported regioselective MIPs, the present imprinted cavities bind reactants by means of only noncovalent molecular interactions, the same as that normally involved in biological systems. The results support the concept of drug "cloning" that further extends both the anti-idiotypic imprinting and in-cavity synthesis approaches into the modern drug discovery area.     

Sephadex-bound histamine in the catalysis of ester hydrolysis

Ester hydrolysis by Sephadex-bound catalysts was studied in a flow-through system. Three different immobilized preparations were synthesized and used: histamine-, coimmobilized histamine-octylamine-, and octylamine-Sephadex; octylamine-Sephadex was used as a reference. Immobilization was carried out using water-soluble carbodiimide, which gave amide linkages between carboxymethyl Sephadex and the groups attached. It was found that the coimmobilized histamine-octylamine preparation was three times more efficient than immobilized histamine alone in the hydrolysis of the esterp-nitrophenylcaproate, whereas hardly any difference was found in the hydrolysis of the less hydrophobic substratep-nitrophenylacetate. We attribute this enhancement of the hydrolysis ofp-nitrophenylcaproate to local enrichment of the substrate on the histamine-octylamine matrix caused by the presence of hydrophobic octyl groups.     

Enzyme thermistors for process monitoring and control

In today’s biotechnology there is an increasing demand for appropriate analytical systems for process control. At present the most widely used control systems are based on measurements of pH, pO₂, and pCO₂. Such systems do not allow the direct measurement of substrates and products. To overcome this drawback sensors such as enzyme thermistors and enzyme electrodes have been designed and their development into industrial useful sensors for monitoring and controlling is the subject of active research.     

Competitive amperometric morphine sensor based on an agarose immobilised molecularly imprinted polymer

A morphine-sensitive device was constructed based on a molecularly imprinted polymer. The imprinted polymer exhibited recognition properties previously. A method of detection based on competitive binding was used to measure morphine in the concentration range 0.1–10 μg/ml. A morphine concentration of 0.5 μg/ml gave a peak current (by oxidation) of 4 nA. The method of morphine detection involves two steps. In the first step, morphine binds selectively to the molecularly imprinted polymer in the sensor. In the second step, an electroinactive competitor (codeine) is added in excess, whence some of the bound morphine is released. The released morphine is detected by an amperometric method. The advantages of this type of sensor compared to biosensors based on antibodies, enzymes or cells are discussed. This sensor, based on an artificial recognition system, demonstrates autoclave compatibility, long-time stability and resistance to harsh chemical environments.     

Bayesian Error Propagation for a Kinetic Model of n‐Propylbenzene Oxidation in a Shock Tube

We apply a Bayesian parameter estimation technique to a chemical kinetic mechanism for n‐propylbenzene oxidation in a shock tube to propagate errors in experimental data to errors in Arrhenius parameters and predicted species concentrations. We find that, to apply the methodology successfully, conventional optimization is required as a preliminary step. This is carried out in two stages: First, a quasi‐random global search using a Sobol low‐discrepancy sequence is conducted, followed by a local optimization by means of a hybrid gradient‐descent/Newton iteration method. The concentrations of 37 species at a variety of temperatures, pressures, and equivalence ratios are optimized against a total of 2378 experimental observations. We then apply the Bayesian methodology to study the influence of uncertainties in the experimental measurements on some of the Arrhenius parameters in the model as well as some of the predicted species concentrations. Markov chain Monte Carlo algorithms are employed to sample from the posterior probability densities, making use of polynomial surrogates of higher order fitted to the model responses. We conclude that the methodology provides a useful tool for the analysis of distributions of model parameters and responses, in particular their uncertainties and correlations. Limitations of the method are discussed. For example, we find that using second‐order response surfaces and assuming normal distributions for propagated errors is largely adequate, but not always.     

Enzymic analysis of organophosphate insecticides using an enzyme thermistor

Quantitative analysis of water soluble insecticides using an enzyme thermistor was examined for organophosphates. A procedure based on an insecticide hydrolyzing enzyme was found suitable for continuous determination of insecticides at concentrations down to 30 µM. A more sensitive procedure, < 3.4µM, suitable for discontinuous monitoring was developed using reversible immobilization of acetylcholine esterase.