Makoto Yoshimoto

Enzymatic reactions in confined environments

Within each biological cell, surface- and volume-confined enzymes control a highly complex network of chemical reactions. These reactions are efficient, timely, and spatially defined. Efforts to transfer such appealing features to in vitro systems have led to several successful examples of chemical reactions catalysed by isolated and immobilized enzymes. In most cases, these enzymes are either bound or adsorbed to an insoluble support, physically trapped in a macromolecular network, or encapsulated within compartments. Advanced applications of enzymatic cascade reactions with immobilized enzymes include enzymatic fuel cells and enzymatic nanoreactors, both for in vitro and possible in vivo applications. In this Review, we discuss some of the general principles of enzymatic reactions confined on surfaces, at interfaces, and inside small volumes. We also highlight the similarities and differences between the in vivo and in vitro cases and attempt to critically evaluate some of the necessary future steps to improve our fundamental understanding of these systems.

Competitive inhibition by hydrogen peroxide produced in glucose oxidation catalyzed by glucose oxidase

Abstract In our previous paper, the glucose oxidation catalyzed by the immobilized glucose oxidase was found to be competitively inhibited by hydrogen peroxide produced, and the inhibition constant KI was determined to be almost equal to the apparent Michaelis constant with respect to oxygen KM. In the present study, the value of KI has been determined in the late stage of the free glucose oxidase catalyzed reaction with an appreciable amount of hydrogen peroxide produced. The KI value has been found also to be almost equal to the KM value, which agrees with the result of the previous paper. Furthermore, the kinetic data obtained with too high concentrations of hydrogen peroxide added initially have been shown to give the same values of KM and KI as determined above. Finally, such a finding as an approximate equality of KI to KM has suggested that the reduced form of glucose oxidase has almost the same affinity to hydrogen peroxide as that to oxygen.

Enhanced enzyme activity and enantioselectivity of lipases in organic solvents by crown ethers and cyclodextrins.

Lipases from Candida rugosa (CRL) and Pseudomonas cepacia (PCL) were co-lyophilized with cyclic oligoethers including four crown ethers and nine cyclodextrins (CyDs), and their transesterification activity and enantioselectivity in organic solvents were evaluated. The PCL co-lyophilized with each additive showed simultaneously enhanced enzyme activity and enantioselectivity when compared to the native lipase lyophilized from buffer alone; in contrast, such enhancement was not observed for the co-lyophilized CRL. Among the cyclic oligoethers examined, permethylated betaCyD (Me1.78betaCyD), as the most suitable additive, was used for the optimization of both the co-lyophilized PCL preparation and reaction conditions by determining the effects of varying the additive/lipase ratio, aqueous pH, the nature of organic solvents, and temperature. The initial rate determined for the transesterification between racemic 2,2-dimethyl-1,3-dioxolane-4-methanol and vinyl butyrate in diisopropyl ether at 30 degrees C increased by up to 17-fold and the enantioselectivity represented by E could be doubled. While there was an inverse correlation between temperature and enantioselectivity, with the Me1.78betaCyD-PCL co-lyophilizate, the reaction rate even at 0 degrees C was much higher than that at higher temperatures in the native PCL-catalyzed reaction. Hence, this method seems to be of practical use for the large-scale production of optically active compounds.

Modification of lipases with poly(ethylene glycol) and poly(oxyethylene) detergents and their catalytic activities in organic solvents

The alpha-chymotrypsin-poly(ethylene glycol) complex, which was prepared by lyophilizing an aqueous solution, was found to have high catalytic activity in organic media even when the molar ratio of polymer/enzyme in its preparation stage is unity. In this study, we obtained freeze-dried complexes of lipases and poly(ethylene glycol) or poly(oxyethylene) detergents including newly synthesized gemini-type detergents, and their transesterification activity in organic solvents was evaluated. The freeze-dried lipase from Pseudomonas cepacia prepared by using each modifier showed enhanced transesterification activity, exhibiting a similar dependence on the concentration of the modifier in the preparation stage to that of the alpha-chymotrypsin-poly(ethylene glycol) complex; in contrasts, the one from Candida rugosa did not do so.

Optimal operation of an integrated bioreaction–crystallization process for continuous production of calcium gluconate using external loop airlift columns

Abstract A kinetic model was proposed to optimize the integrated bioreaction–crystallization process newly developed for production of calcium gluconate crystals using external loop airlift columns. The optimal operating conditions in the bioreactor were determined using an objective function defined to maximize the productivity as well as to minimize biocatalyst loss. The optimization of the crystallizer was carried out by matching the crystallization rate to the optimal production rate in the bioreactor because the bioreaction was found to be the rate controlling process. The calcium gluconate productivity under the optimal conditions of the integrated process was obtained by the simulation based on the process model. The productivity of the proposed process was found to be comparable to that of the current batch fermentation process.

Gas holdup, liquid circulating velocity and mass transfer properties in a mini-scale external loop airlift bubble column

The external loop airlift bubble column has been regarded as a promising type of gas–liquid or gas–liquid–solid biooreactor because of the liquid circulating flow between the riser and downcomer. A mini-scale column is useful and efficient in the process research and development for highly specialized materials such as fine chemicals, advanced bioproducts and biocatalysts utilized in two or three phase system. In this work, a mini-scale glass column of in volume was designed and characterized. The gas holdup eG in the riser was obtained by measuring the volume expansion through photographs taken with a digital camera. The liquid circulating velocity UL was measured by observing the time required for a tracer particle to travel a fixed distance in the downcomer through analysis of the images taken by a video camera. The gas–liquid volumetric oxygen transfer coefficient kLa and liquid–solid oxygen transfer coefficient kS were determined by our previous method in which the air oxidation of glucose was catalysed by the immobilized glucose oxidase gel beads suspended in the column to obtain a pseudo steady state concentration of the dissolved oxygen and the corresponding constant rate of glucose consumption. It was shown that even such a mini-scale external loop bubble column could be characterized in terms of gas holdup, liquid circulating velocity and mass transfer properties according to our previous correlations proposed for the bench to pilot scale column.

Rapid conversion of sorbitol to isosorbide in hydrophobic ionic liquids under microwave irradiation.

Sorbitol was effectively converted to isosorbide by treatment with [TMPA][NTf2 ] in the presence of catalytic amounts of TsOH under microwave heating at 180 °C. The reaction completed within 10 min and isosorbide was isolated to about 60%. Ionic liquids were readily recovered by an extraction treatment and reused several times.

A kinetic study on crystallization of calcium gluconate in external loop airlift column and stirred tank for an immobilized glucose oxidase reaction with crystallization

The batch crystallization of calcium gluconate from the reaction solution of the immobilized glucose oxidase catalyzed oxidation of glucose was kinetically studied not only in an external loop airlift column but also in a small scale stirred tank. The solubility of calcium gluconate under a wide range of the glucose oxidation conditions was measured and correlated. The crystallization was started by charging successively a saturated calcium gluconate solution prepared at 30 °C and a known amount of its seed crystals in either crystallizer being thermostated at a constant temperature between 5 and 15 °C. The time courses of the dissolved calcium gluconate concentration during crystallization were observed to be unaffected either by superficial gas velocity in the airlift column or by impeller speed in the stirred tank. The time courses in the column were found to be identical with those in the tank under the same operating conditions of pH, temperature, and glucose and seed crystals concentrations. Almost no decrease in the dissolved calcium gluconate concentration was observed during the crystallization without any seed crystals, while negligible change in the crystal size distribution was microscopically found during the crystallization with seed crystals. These results obtained suggested that the crystallization proceeded under the surface reaction control and no secondary nucleation occurred in both crystallizers. A crystal growth model was proposed to reproduce the time courses of the dissolved calcium gluconate concentration during crystallization under the various operating conditions. The order of the crystal growth rate with respect to the calcium gluconate supersaturation was found to be 3.2. The calculated time courses of dissolved calcium gluconate concentration agreed well with the observed ones in both airlift and stirred tank crystallizers.

Evaluation of interaction between liposome membranes induced by stimuli responsive polymer and protein

Immobilized liposome chromatography was utilized as a novel method for the quantitative evaluation of the interaction between liposome membranes. The capacity factors evaluated from the elution profile showed that interaction between 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposome membranes occurred in the presence of a stimuli responsive polymer and protein under specific stimulus conditions. The occurrence of such interaction was supported by experimental results for POPC liposome membrane fusion under corresponding stimuli conditions.

Direct conversion of polyamides to ω-hydroxyalkanoic acid derivatives by using supercritical MeOH

We examined the decomposition of polyamides such as nylon-6 and nylon-12 by using supercritical MeOH as the reaction media. The treatment of waste nylon-6 with supercritical MeOH resulted in smooth depolymerization, forming caprolactam as the first product which was then converted to a mixture of methyl 6-hydroxycapronate and methyl 5-hexenoate in a ratio of approximately 1 : 1. The reaction progress was traced using gas chromatography (GC) analyses, and precise product distribution was estimated. During the decomposition of nylon-6, N-methylcaprolactam and methyl 6-(N,N-dimethylamino)capronate were detected as intermediates. The sum of the all detectable products and intermediates exceeded 80%. In addition, we examined the decomposition reaction initiating from caprolactam, N-methylcaprolactam, and methylN,N-dimethylcapronate under similar reaction conditions, and observed that the final two products were formed in similar yields and ratios. Kinetic analyses by using a simulation study based on the experimental data were performed, and kinetic parameters for each step were estimated. Nylon-12 underwent similar conversion to produce methyl 12-hydroxydodecanoate in good yield. Because methyl ω-hydroxyalkanoate is known to be an important intermediate in the chemical industry, the present method has the potential for producing valuable compounds from waste material. Thus, the first upgrade in the chemical recycling of plastics was accomplished.