Kimitoshi Fukunaga

A kinetic study on air oxidation of glucose catalyzed by immobilized glucose oxidase for production of calcium gluconate

The kinetic studies on air oxidation of glucose catalyzed by free and immobilized glucose oxidase were carried out in the gluconate buffer solution prepared to develop an efficient production of calcium gluconate crystals. The optimal pH, temperature and gluconate concentration as well as the kinetic parameters in the Michaelis–Menten rate expression were determined for the free enzyme reaction in an airtight batch reactor. The fine manganese dioxide particles were entrapped together with glucose oxidase within the calcium alginate gel beads to decompose hydrogen peroxide produced in the oxidation. The various gel beads containing different concentrations of the enzyme and/or manganese dioxide particles were prepared. The intrinsic kinetics for the immobilized glucose oxidase with no peroxide inhibition were assumed to be the same as those for the free enzyme. The liquid–solid mass transfer around the gel beads suspended in the airtight reactor as well as the competitive inhibition effect of hydrogen peroxide were taken into account for the gel beads. A method to determine the effectiveness factor α and hydrogen peroxide inhibition constant KI for the immobilized glucose oxidase was proposed based on the observed time course of dissolved oxygen concentration. The values of the constant KI were found to be almost equal to those of the Michaelis constant KM. The rate constant for hydrogen peroxide decomposition kP as well as the corresponding effectiveness factor α′ were also determined from the time course of hydrogen peroxide concentration in a separate batch reactor. Both determinations proposed were verified by comparing the observed prolonged time courses of the dissolved oxygen and hydrogen peroxide concentrations in the reactor with those calculated by solving simultaneously the kinetic and mass transfer equations with the parameters obtained.

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.

Immobilization of organic solvent-soluble lipase in nonaqueous conditions and properties of the immobilized enzymes

Lipid-coated Pseudomonas lipase was successfully immobilized by entrapping with hydrophobic, photo-crosslinkable, resin prepolymers under non-aqueous conditions. We studied the feasibility of using this new type of biocatalyst, which is an immobilized organic solvent-soluble enzyme, for enzymatic synthesis in organic solvent. Esterification of lauric acid with benzyl alcohol in benzene and transesterification between racemic sulcatol and isopropenyl acetate in different organic solvents were carried out with the immobilized lipid-coated lipase as the model reactions. In both reaction systems, the immobilized organic solvent-soluble enzyme was observed to have excellent activity and stability.

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.

Removal of mixtures of acetaldehyde and propionaldehyde from waste gas in packed column with immobilized activated sludge gel beads

The removal of mixed acetaldehyde and propionaldehyde as a model of the binary contaminants in waste gas was studied in the packed column containing the immobilized activated sludge gel beads together with the hollow plastic balls developed for the removal of a single aldehyde in the previous work. The rate of each aldehyde biodegradation by the gel beads in the aldehydes mixture was expressed by the Michaelis-Menten type rate equation with an inhibitory term due to the other coexistent aldehyde. The kinetic parameters involved were found to be the same as those determined previously for biodegradation of a single aldehyde. A model for prediction of removal of each aldehyde in the packed column was developed assuming that each aldehyde dissolved in the aqueous phase within the gel bead was biodegraded according to the above rate equation with no mass transfer effect. The packed column was stable and efficient for removal of the binary aldehydes mixture with a very low pressure drop for gas flow due to a reduced gel beads bed compaction by the hollow plastic balls. Removal of each aldehyde decreased with increasing the inlet aldehyde concentrations since each biodegradation rate itself approached asymptotically the maximum one with increase in each aldehyde concentration. The observed removals for each aldehyde in the aldehydes mixture agreed well with those calculated from the design equations developed. The contact efficiency of gel beads with the waste gas stream was estimated to be the same value of 0.24 as in the previous work, supporting that the efficiency was specific to the geometrical and physical properties of the packed column used.

Glucose oxidation catalyzed by liposomal glucose oxidase in the presence of catalase-containing liposomes.

A catalase‐containing liposome (CAL) was prepared and characterized in terms of stability during storage and catalysis of the decomposition of hydrogen peroxide (H2O2) that was initially added or produced in the oxidation of glucose catalyzed by the glucose oxidase‐containing liposomes (GOL). The reactors used were a test tube and an external loop airlift bubble column as the static liquid and circulating liquid flow systems, respectively. The free catalase (CA) at low concentrations was unstable during storage at 4 °C as a result of dissociation of the tetrameric CA subunits. On the other hand, the deactivation of the CA activity in the CAL was depressed because of the high CA concentration in the CAL liposome. The CAL effectively catalyzed the repeated decompositions at 25 °C with 10 mM H2O2 added initially, whereas the free CA was significantly deactivated during the repeated reactions. The high stability of the CAL was attributed to the moderately depressed reactivity, which was essentially derived from the diffusion limitation of the CAL membrane to H2O2 in the liquid bulk. In the GOL‐catalyzed prolonged oxidation of 10 mM glucose at 40 °C in the static liquid in a test tube, both the free CA and CAL could continuously catalyze the decomposition of H2O2 produced. This was because the glucose oxidation rate was small due to the limited reactivity of the GOL to glucose with its low permeability through the GOL membrane. In the glucose oxidation catalyzed by the GOL with the free CA or the CAL in the airlift, much larger oxidation rates were observed compared to those in the test tube because the permeability of the GOL membrane to glucose was increased in the gas‐liquid two phase flow in the airlift. The GOL/CAL system in the airlift operated in an acidic condition, which was preferable to the GO activity, gave the largest oxidation rate with negligible accumulation of the H2O2 produced. On the other hand, the GOL/free CA system gave an oxidation rate smaller than that of the GOL/CAL system even under the acidic condition due to an unfavorable interaction of the free CA molecules with the GOL membranes leading to the decreased reactivity of the GOL.

Preparation of detergent-lipase complexes utilizing water-soluble amphiphiles in single aqueous phase and catalysis of transesterifications in homogeneous organic solvents.

A novel method of preparing detergent-enzyme complexes that can be employed in organic media was developed utilizing newly synthesized water-soluble nonionic gemini-type detergents, N,N-bis(3-D-gluconamidopropyl)-3-(dialkyl-L-glutamatecarbonyl)propanamides (BIG2CnCA: n = 10,12,14,16,18) and N,N-bis(3-D-lactonamidopropyl)-3-(dialkyl-L-glutamatecarbonyl)propanamides (BIL2CnCA: n = 16,18), and nonionic twin-headed detergents, N,N-bis(3-D-gluconamidopropyl)alkanamides (BIG1Cn: n = 12,14,16,18,delta9). This method simply entails mixing a selected enzyme with an appropriate detergent in an aqueous solution followed by lyophilization, and it offers the advantages of enhanced enzymatic activity in organic solvents and eliminates both enzyme loss and the necessity for an organic solvent in the preparation stage. Using various modified lipases originating from Aspergillus niger (Lipase A), Candida rugosa (Lipase C), Pseudomonas cepacia (Lipase P), and porcine pancreas (PPL), prepared using the novel method and detergents, including conventional synthesized nonionic detergents such as dialkyl N-D-glucona-L-glutamates (2CnGE: n = 12,18delta9) and octanoyl-N-methylglucamide (MEGA-8), enantioselective transesterifications of 6-methyl-5-hepten-2-ol (sulcatol) and 2,2-dimethyl-1,3-dioxolane-4-methanol (solketal) with a vinyl or isopropenyl carboxylate were carried out in an organic solvent. The modified lipase activity was influenced by both the lipases and the structure of the detergents. The value for the hydrophile-lipophile balance (HLB) of the detergent provided a means of correlating the structure and the obtained modified lipase activity. For detergents of the same class with a HLB value of approximately 9 and 12, the highest activity was obtained for Lipase A and Lipase P, and Lipase C and PPL, respectively. Among detergents of the same HLB value tested, the gemini-type detergents possessing the most bulky head and tail were most effective as a modifier for lipases of all types. The preparation and reaction conditions for these novel gemini-type detergent-modified lipases were optimized using BIG2C12CA (HLB = 9.4) by studying the effect of the detergent/lipase ratio and the nature of organic solvents on the complex formation. The high enzymatic activities of the BIG2C12CA-modified lipases were independent of the solubility of the lipases in organic solvents, unlike in the case of 2CnGE-modified lipases prepared using the conventional suspension system.

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.