John L. Gainer

Esterification of fatty acids using nylon-immobilized lipase in n-hexane: kinetic parameters and chain-length effects

The esterification of long-chain fatty acids in n-hexane catalyzed by nylon-immobilized lipase from Candida rugosa has been investigated. Butyl oleate (22 carbon atoms), oleyl butyrate (22 carbon atoms) and oleyl oleate (36 carbon atoms) were produced at maximum reaction rates of approximately equal to 60 mmol h(-1) g(-1) immobilized enzyme when the substrates were present in equimolar proportions at an initial concentration of 0.6 mol l(-1). The observed kinetic behavior of all the esterification reactions is found to follow a ping-pong bi-bi mechanism with competitive inhibition by both substrates. The effect of the chain-length of the fatty acids and the alcohols could be correlated to some mechanistic models, in accordance with the calculated kinetic parameters.

Hypoxia and atherosclerosis: re-evaluation of an old hypothesis.

It was hypothesized over 40 years ago that a decrease in the oxygenation of the vascular wall might somehow be involved in atherogenesis. Recent studies concerning the mechanism of oxygenation indicate that diffusion through plasma is of major importance, meaning that a moderate, prolonged hypoxia could be induced by small decreases in the oxygen diffusivity. Combining all of this leads to a comprehensive theory of atherosclerotic change, indicating how aging, smoking, diabetes, hypercholesterolemia and hypertension are correlated with the disease, and suggesting methods of prevention (or reversal).

OXYGEN DIFFUSION AND ATHEROSCLEROSIS

Abstract The intramuscular injection of the carotenoid compound, Cocetin, in rabbits fed an atherosclerosis-producing diet, resulted in greatly reduced severity of the atherosclerosis. In addition, serum cholesterol levels were reduced by 50%. Crocetin also causes increased oxygen diffusion through the blood plasma. Thus, these results would appear to add evidence that hypoxia, due to reduced diffusion of oxygen at the blood-tissue interface, plays an important and perhaps an initiating part in the pathogenesis of atherosclerosis.

Synthesis of esters using a nylon-immobilized lipase in batch and continuous reactors

Direct esterifications using a nylon-immobilized lipase from Candida cylindracea were carried out in batch and continuous-flow reactors. The immobilized enzyme was effective in catalyzing the synthesis of ethylpropionate, isoamylpropionate, and isoamylbutyrate. With ethanol dissolved in hexane as a substrate, the maximum initial esterification rate was 0.02 mole/(h x g of immobilized protein), but the enzyme was stable only when the substrate concentrations were lower than 0.2 M. With isoamyl alcohol in hexane as a substrate, esterification rates as high as 0.085 mole/(h x g of immobilized protein) were observed and the immobilized enzyme was stable over a much broader concentration range. However, in this case, the use of a solvent, such as hexane, was not necessary for esterification, and the enzyme could be employed in equimolar acid/alcohol mixtures. A packed-bed reactor was operated successfully for the continuous synthesis of esters. The reactor was stable for long periods of time, and the steady-state performance could be accurately predicted on the basis of batch reaction experiments.

Asymmetric Ketone Reduction with Immobilized Yeast in Hexane: Biocatalyst Deactivation and Regeneration

There is a need to develop methods for producing enantiomerically pure pharmaceuticals because the racemic mixtures made today will probably not be allowed in the future. Synthetic chiral catalysts are being developed for this purpose, as well as new product separation techniques. Another possible option is to use biocatalysts, such as purified enzymes or whole microbial cells, since these can result in the production of mostly a single enantiomer. This study emphasizes the use of alginate‐entrapped yeast cells to catalyze the reduction of ketones as a model system. The emphasis is on the factors that might limit the reactivity of such cells, such as equilibrium conditions, substrate or product inhibition, solvent toxicity, loss of cell viability, or the degradation of intracellular levels of enzymes or cofactors.It was found that there was a progressive loss of catalytic activity of the immobilized yeast cells, which appeared to be mainly associated with a loss of cell viability and a decline of intracellular NAD(H) levels during the reaction. The other factors investigated did not have a large effect. A regeneration scheme was developed in order to replenish the intracellular NAD(H) lost during the reaction, which involved removing the biocatalyst from the reaction and supplying the cells with a nutrient source. This resulted in an increase in the NAD(H) to initial levels and also resulted in a maintenance of the ketone reduction rate over time.

Asymmetric Reduction of Acetophenone with Calcium-Alginate-Entrapped Baker's Yeast in Organic Solvents

Bakers yeast cells entrapped in alginate beads are shown to catalyze reactions in organic solvents when a cofactor regeneration scheme is implemented. This study focused on the reduction of acetophenone to 1‐phenylethanol, using bakers yeast as well as a cosubstrate to regenerate the cofactor. The product is a chiral alcohol, and it was desired to maintain a high enantiomeric excess. The effects of parameters such as the addition of a cosubstrate, water content, fermentation time, buffer pH, and bead diameter have been investigated. Such a general process may be quite useful when single enantiomers are needed, as well as for the production of other chemicals.

Predicting partition coefficients in polyethylene glycol-potassium phosphate aqueous two-phase systems

Abstract Partition coefficients in polyethylene glycol-potassium phosphate aqueous two-phase systems are predicted using a previously developed mathematical model. The model is based on a simplification of equations which arise from an osmotic pressure virial expansion and relates the partition coefficient to the concentration difference between phases of one of the phase-forming components and to the solute hydrophobicity. The predicted partition coefficients are compared to experimental values for several different solutes in this phase system over the range of pH of 5.5 to 9.2. The predictions are generally good for uncharged solutes, but show disagreement with experimental values for charged solutes.

In situ extraction versus the use of an external column in fermentation

SummaryOrganic extraction of 2,3-butanediol produced by Klebsiella oxytoca fermentation was studied to determine if the use of an external column offered advantages over in situ extractive fermentation. Dodecanol was chosen from 24 tested solvents, 11 of which were non-toxic to K. oxytoca. Although growth occurred in all shakeflask experiments containing dodecanol, growth was never observed when dodecanol was used in an in situ arrangement in a fermentor. Using dodecanol in an external column, however, resulted in a cell yield of 0.10 g/g d-xylose, and a 2,3-butanediol yield of 0.32 g/g d-xylose, similar to results obtained in control (solventless) experiments. Although the partitioning of 2,3-butanediol in the organic phase was low, this study suggests that external columns, with recycling to the fermentor, can offer substantial advantages over in situ extraction.

PLASMA PROTEINS, OXYGEN TRANSPORT AND ATHEROSCLEROSIS

Abstract In summary, we have presented evidence to support the hypoxic theory for the formation of atherosclerosis. More importantly, though, we have presented the following etiology for such hypoxia: 1. (1) Oxygen transport is affected by plasma protein concentrations and in most humans probably decreases with age; 2. (2) The result of hypoxia at the aortic lining is a degeneration of surface features which results in increased cellular permeability; and 3. (3) The interior structure of the vessel is further disorganized due to the influx of lipids and other plasmatic matter. This in turn accelerates oxygen demand and augments hypoxia. These ideas concerning the effects of proteins on oxygen transport, as well as the importance of the diffusion resistance of blood plasma, provide strong indications of a comprehensive mechanism for the occurrence of atherosclerosis and other vascular changes associated with aging.

A novel fluid resuscitation therapy for hemorrhagic shock

Fluid resuscitation is the usual therapy for hemorrhagic shock, and frequently consists of the infusion of large volumes of electrolyte solutions. However, to be successful, this therapy should be implemented soon after injury. A new treatment method in which the infusion could be delayed might result in a greater survival rate. Reducing the volume of fluid needed is also important. Both of these aspects of fluid resuscitation therapy were addressed in this study by supplementing the electrolyte solution with trans-sodium crocetinate (TSC). Rats were subjected to a severe hemorrhage, with 55% (or greater) of the estimated blood volume being removed over a period of approximately 10 min. There were five animals in each treatment group, and two types of experiments were done. In one, a bolus injection of TSC (or saline control) was given immediately after hemorrhage, followed 30 min later with an infusion of isotonic saline. In the other experiments, reduced infusion volumes of a TSC-saline infusion fluid were used. In both cases, TSC resulted in the survival of the animals while the controls all died. Whole-body oxygen consumption also increased with TSC, reaching 75% of the normal resting value after about 15 min. This correlates well with the increased survival rates seen, since mortality after hemorrhagic shock is associated with decreased oxygen consumption. These results suggest that the use of TSC could allow for later implementation of fluid resuscitation therapy as well as reducing the volume needed.