The genes encoding four deoxynucleoside monophosphate kinase (dNMPkinase) enzymes, including ADK1 for deoxyadenylate monophosphate kinase (AK), GUK1 for deoxyguanylate monophosphate kinase (GK), URA6 for deoxycytidylate monophosphate kinase (CK), and CDC8 for deoxythymidylate monophosphate kinase (TK), were isolated from the genome of Saccharomyces cerevisiae ATCC 2610 strain and cloned into E. coli strain BL21(DE3). Four recombinant plasmids, pET17b-JB1 containing ADK1, pET17b-JB2 containing GUK1, pET17b-JB3 containingURA6, and pET17b-JB4 containing CDC8, were constructed and transformed into E. coli strain for overexpression of AK, GK, CK, and TK. The amino acid sequences of these enzymes were analyzed and a putative conserved peptide sequence for the ATP active site was proposed. The four deoxynucleoside diphosphates (dNDP) including deoxyadenosine diphosphate (dADP), deoxyguanosine diphosphate (dGDP), deoxycytidine diphosphate (dCDP), and deoxythymidine diphosphate (dTDP), were synthesized from the corresponding deoxynucleoside monophosphates (dNMP) using the purified AK, GK, CK, and TK, respectively. The effects of pH and magnesium ion concentration on the dNDP biosynthesis were found to be important. A kinetic model for the synthetic reactions of dNDP was developed based on the Bi–Bi random rapid equilibrium mechanism. The kinetic parameters including the maximum reaction velocity and Michaelis–Menten constants were experimentally determined. The study on dNDP biosynthesis reported in this article are important to the proposed bioprocess for production of deoxynucleoside triphosphates (dNTP) that are used as precursors for in vitro DNA synthesis. There is a significant advantage of using enzymatic biosyntheses of dNDP as compared to the chemical method that has been in commercial use.

The enzyme reaction mechanism and kinetics for biosyntheses of deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) from the corresponding deoxyadenosine diphosphate (dADP) and deoxyguanosine diphosphate (dGDP) catalyzed by pyruvate kinase were studied. A kinetic model for this synthetic reaction was developed based on a Bi-Bi random rapid equilibrium mechanism. Kinetic constants involved in this pyruvate kinase catalyzed phosphorylation reactions of deoxynucleoside diphosphates including the maximum reaction velocity, Michaelis-Menten constants, and inhibition constants for dATP and dGTP biosyntheses were experimentally determined. These kinetic constants for dATP and dGTP biosyntheses are of the same order of magnitude but significantly different between the two reactions. Kinetic constants involved in ATP and GTP biosyntheses as reported in literature are about one order of magnitude different from those involved in dATP and dGTP biosyntheses. This enzyme reaction requires Mg2+ ion and the optimal Mg2+ concentration was also determined. The experimental results showed a very good agreement with the simulation results obtained from the kinetic model developed. This kinetic model can be applied to the practical application of a pyruvate kinase reaction system for production of dATP and dGTP. There is a significant advantage of using enzymatic biosyntheses of dATP and dGTP as compared to the chemical method that has been in commercial use.

The enzyme reaction mechanism and kinetics for biosyntheses of deoxycytidine triphosphate (dCTP) and deoxythymidine triphosphate (dTTP) from the corresponding deoxycytidine diphosphate (dCDP) and deoxythymidine diphosphate (dTDP) catalyzed by pyruvate kinase were studied. The kinetic model for the two synthetic reactions was found to follow the Bi–Bi random rapid equilibrium mechanism similar to that of the biosynthesis of deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) from the corresponding deoxyadenosine diphosphate (dADP) and deoxyguanosine diphosphate (dGDP). Kinetic constants involved in the reactions including the maximum reaction velocity, the Michaelis–Menten constants, and the inhibition constants for dCTP and dTTP biosyntheses were experimentally determined. This enzyme reaction requires Mg2+ ion and the optimal Mg2+ concentration was also determined. The experimental results showed a good agreement with the simulation results obtained from the kinetic model developed. The kinetics of the four biosynthetic reactions for deoxynucleoside triphosphates (dNTP) including dATP, dGTP, dCTP, and dTTP from the corresponding deoxynucleoside diphosphates (dNDP) including dADP, dGDP, dCDP, and dTDP were analyzed. The results suggest that the binding kinetics of phosphoenolpyruvate (PEP) and pyruvate are similar for all four biosynthetic reactions. The affinity of the dNDP substrates to enzyme is of the same order of magnitude as the corresponding dNTP as inhibitors. The order of reactivity and substrate specificity for dNDP is dADP>dGDP>dCDP>dTDP in the pyruvate kinase (PK) reactions. The results obtained from this study can be applied to bioreactor design and production of dCTP and dTTP for biosynthesis of DNA at a significantly lower cost compared to the currently available chemical method.

The phospholipase D (PLD) gene encoding the mature PLD enzyme was cloned from the genomic DNA library of Streptoverticillium reticulum and its gene sequence was analyzed. The Stv. reticulum PLD gene sequence showed a high degree of similarity to that of Streptomyces spp. (66%), Streptomyces antibioticus (67%), Streptoverticillium cinnamoneum (70%), and Streptomyces somaliensis (71%). A highly conserved phospholipase superfamily-specific consensus sequence, HxKxxxxD (HKD), was also found in both N- and C-terminal regions of the peptides from Streptoverticillium reticulum. The effects of biochemical properties of phosphatidylcholines (PC) on Streptoverticillium reticulum PLD activity were studied. The biochemical properties of PC studied include the chain length, cis and trans conformation of PC, carboxyl and carbonyl bonds at position 1 and 2 of PC, hydrophobic chain of PC, and saturated and unsaturated acyl and alkyl groups. Also studied are the micelle sizes of PC and mixed TX-100-PC, the diacyl chain length, and the properties of various functional groups in the presence and absence of TX-100. The PLD activity was significantly affected by the biochemical properties of PC substrates used and the micelle size. A prospective reaction mechanism of this enzyme has been also suggested. The PLD activity was significantly affected by the biochemical properties of PC substrates used and the micelle size. A prospective reaction mechanism of this enzyme has been also suggested. The studies on the Stv. reticulum PLD gene analysis and the effect of PC substrates in PLD activity are important not only for better understanding of PLD function in cells but also for production of phospholipid derivatives used in pharmaceutical, food, and cosmetics industries.

In our previous works, the immobilized glucose oxidase (GO) plus manganese dioxide (MnO2) were prepared and efficiently used at 30 ◦C for production of calcium gluconate (CaG) in an external loop airlift. In the gel beads occurred the three reactions: (1) the GO-catalyzed air oxidation of glucose to produce gluconic acid (GA) and hydrogen peroxide (P) which inactivated GO, (2) the MnO2-catalyzed decomposition of P and (3) the neutralization of GA with calcium hydroxide added continuously resulting in accumulation of CaG at a constant pH. In this work, the stabilities of the immobilized GO and MnO2 activities to various operating conditions are observed and analyzed to develop an efficient prolonged use of GO plus MnO2 entrapped. The well-known first-order kinetics is assumed to analyze the deactivation of either GO or MnO2 since its stability is affected by a complicated interaction among the operating variables. The first-order deactivation rate constant for GO, kd,E is determined from the progressive decrease in the apparent effectiveness factor for GO, αapp based on the initial GO concentration CE,0 in the gel beads. This is because the decrease in the GO concentration, CE is too difficult to measure but approximately directly proportional to αapp. The kd,E value under any set of operating conditions is determined from the two values of αapp at the start and end of the 24 h reaction. To obtain the kd,E values for the prolonged times, the repeated 24 h reactions are carried out with the same gel beads. Examining the data on kd,E obtained under the various operating conditions gives an empirical correlation of kd,E with the pH value and the P concentration CP which is the time-averaged P concentration during every 24 h reaction. The other variables such as the glucose and gel beads concentrations and the superficial air velocity are found to exert almost no influence on the kd,E value. On the other hand, the deactivation rate constant for MnO2, kd,M is determined from the two values of the intrinsic P decomposition rate constant, kP in the same 24 h reaction as above since the kP value, which is proportional to the MnO2 concentration CM, can be calculated from its apparent value based on the effectiveness factor theory. The kd,M values are empirically correlated with the pH value only. The above two deactivation kinetic models are combined with our previous reaction and mass transfer model for the gel beads in the airlift to simulate the CaG production process in a prolonged batch bioreactor operation. A fair agreement obtained between the observed and calculated time courses of the process suggests the validity of the deactivation models developed. An application of the simulation model also predicts an optimal ratio of the amounts of GO and MnO2 immobilized in the gel beads.

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.

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.

The industrial catalytic-distillation process for the production of methyl tert-butyl ether (MTBE) from methanol and isobutylene was simulated by developing the process model as a user modular on Aspen plus platform. The model utilizes the Aspen plus system and retains the characteristics of the self-designed model, which has been verified in various scale-up processes. The experimentally determined reaction kinetics was applied in the model. NRTL and Redlick-Kwong-Soave equations were selected for the vapor-liquid equilibrium calculation. The NRTL binary interaction parameters were estimated from the experimental data of the two-component vapor-liquid equilibrium. Two typical industrial plants for the MTBE production, one using the loose-stack-type package technology and the other using the bale-type package technology in the catalytic-distillation column, were chosen as the sample processes to demonstrate the validity of the model. The flowsheet simulations of the two industrial plants were done on Aspen plus platform, in which the simulation of the catalytic-distillation column used the developed user modular. The results show that fair agreements between the calculated and operating data were obtained.

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 de3ned 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 ofthe proposed process was found to be comparable to that ofthe current batch fermentation process.

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 constantKI 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 constantKM. 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.