Satish C. Mohapatra

Optimizing lipase activity, enantioselectivity, and stability with medium engineering and immobilization for β‐blocker synthesis

Lipase from Pseudomonas cepacia showed poor activity and moderate enantioselectivity (E) in pure aqueous systems for hydrolysis of a racemic mixture (+/-)-1-chloro-2-acetoxy-3-(1-naphthyloxy)-propane, which is a potential intermediate for beta-blocker synthesis. However, addition of polar organic solvents to the reaction can change both the activity and the enantioselectivity for this chiral reaction significantly. It was observed, in general, that the activity increases and the enantioselectivity decreases with the increase in the polarity of the organic solvent added to the medium. Among the six solvents chosen (i.e., dimethylsulfoxide [DMSO], 1, 4-dioxane, dimethylformamide [DMF], acetone, 1-propanol, and tetrahydrofuran [THF]), maximum activity and minimum enantioselectivity was obtained with DMSO, whereas minimum activity and maximum enantioselectivity was obtained with THF as the cosolvents. In the subsequent studies, native or polyethylene glycol (PEG)-modified lipase was immobilized by entrapping in Caalginate gel beads. In a fixed-bed continuous reactor containing these catalyst beads, the enzyme was found to be at least three times more enantioselective than the native form in a batch reactor. This fixed-bed reactor with the beads could be operated with high concentration of acetone (33% v/v) for about 1 month without a significant loss of enzyme activity and enantioselectivity.

Immobilization of α-chymotrypsin for use in batch and continuous reactors

α-Chymotrypsin from bovine pancrease (EC 3.4.21.1) was entrapped in Ca-alginate gel particles to carry out hydrolysis of N-acetyl-L-phenylalanine methyl ester (APME) in batch as well as continuous fixed bed reactor. The enzyme was covalently modified with homo-bifunctional polyethylene glycol derivatives in order to reduce its leakage from the beads; 85% modification of the ∈-NH2 groups of lysine residues caused reduction in the enzyme activity by 50%. However, this modification was helpful in a long run because it reduced both enzyme leakage and deactivation. Effective diffusivities and the distribution coefficients of the substrate and the product were determined experimentally, and later used in simulation of a batch experiment employing the beads. A continuous fixed bed reactor with the gel beads was operated to study the deactivation of the enzyme. During a 15-day period, the enzyme showed about 15% loss in the conversion which occurred only during the first 5 days. After that the enzyme did not deactivate further which demonstrates that this method can be applied for continuous reactions. © 2000 Society of Chemical Industry

Polymerase chain reaction engineering.

A mathematical model for polymerase chain reaction (PCR) is developed, taking into account the three steps in this process: melting of DNA; primer annealing; and DNA synthesis (polymerization). Activity and deactivation of the polymerase enzyme as a function of temperature is incorporated in the kinetic model to get a better understanding of the amplification of DNA. Computer simulation of the model is carried out to determine the effects of various parameters, such as the cycle number, initial DNA concentration (copynumber), initial enzyme concentration, extension time, temperature ramp, and enzyme deactivation on the DNA generation. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 359-366, 1997.

Lipase kinetics in organic-water solvent with amphipathic substrate for chiral reaction.

Lipase from Pseudomonas cepacia was used for asymmetric hydrolysis of the substrate (+/-)1-chloro-2-acetoxy-3-(1-naphthyloxy)-propane, which is a precursor for (S)-(-)-beta-blocker synthesis. Because this substrate is insoluble in water and partially soluble in hydrophobic solvents such as hexane and octane, a mixture of hydrophilic organic solvents and aqueous buffer was used to study the initial reaction rates. Because of the amphipathic nature of the substrate, it can remain in three different forms: (1) monomeric (solution); (2) micellar; and (3) emulsion, depending on the acetone and substrate concentrations in the medium. This behavior is presented in a phase diagram. The enzyme was found to be active with micelle as well as emulsion form of the substrate, whereas it showed negligible activity with the monomeric form. Michaelis-Menten constants were determined experimentally for the emulsion and micellar part of the substrate. The initial rate of hydrolysis (v(0)) goes through a maximum with respect to the acetone content of the mixture. It is due to the combined effect of various factors occurring simultaneously with the increase in acetone content in the solvent. These phenomena are discussed based on the interfacial activation of lipase, deactivation of the enzyme at very high acetone concentration, and increase in critical micelle concentration (CMC) and critical emulsion concentration (CEC) with the increase in acetone content in the solvent. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 399-407, 1997.

Studies on primer-dimer formation in polymerase chain reaction (PCR)

A viable mechanism for primer-dimer formation in polymerase chain reaction (PCR) process has been proposed based on experimental results. This mechanism results in a kinetic description of the primer-dimer formation process with the Taq DNA polymerase enzyme, the two primers and the dNTPs as the starting materials. The possible impact of this primer-dimer formation on the selectivity and yield of PCR process is discussed.

Time dependent behaviour of the cross-linking reaction of α-chymotrypsin with glutaraldehyde

Immobilization of α-chymotrypsin was done by cross-linking the enzyme with glutaraldehyde. Studies were done in a batch reactor to find out the effects of the concentrations of α-chymotrypsin and glutaraldehyde on the rate of particle formation, the final yield of the crosslinked particles, and the residual activity of the cross-linked enzyme. Both intra- and inter-molecular cross-linking play important role at different time course of the reaction. At the begining of the cross-linking reaction, the large drop in the activity is because of the intra-molecular cross-linking and later on the large increase in the particle formation is because of the inter-molecular cross-linking.

Kinetics of deactivation for thermostable DNA polymerase enzymes

The kinetics of thermal deactivation for thermostable DNA polymerase enzymes were investigated by using the experimental data published elsewhere (Nielson et al. 1996. Strategies. 9, 7–8). The order of deactivation (a) and the deactivation rate constants (kd) were determined for different Taq DNA polymerase enzymes and were found to be of first order.

STUDIES ON GRADIENT ELUTION CHROMATOGRAPHY FOR SEPARATION OF DNA AND OLIGONUCLEOTIDES

Rate parameters and gradient correlations for nucleic acids are very important to engineering investigations of the separation of DNA products by gradient elution chromatography. Before carrying out computer simulations for gradient process, these rate parameters and gradient correlation must be determined from the experimental data. In this work, moment method was used to calculate the equilibrium adsorption constants (Ki ) as a function of the ionic strength (I) of the mobile phase for different sizes of oligonucleotides from the isocratic elution data and later, these coefficient values were used in the computer simulation to predict the elution behavior. This analysis can eliminate extensive experimental trial and error and hence suitable for separation of oligonucleotides and DNA in a more effective manner.