James T. Hsu

Effects of ligand concentration on protein adsorption in dye-ligand adsorbents

Abstract The effect of ligand concentration on both equilibrium and fixed-bed adsorption has been studied for three model proteins on dye-ligand adsorbents. Adsorption equilibrium data corresponded well to the Langmuir isotherm. It was found that binding capacity was approximately proportional to immobilized dye concentration. Also it was observed that the more highly substituted adsorbents exhibited higher affinity for proteins. Breakthrough curves were measured for protein adsorption on fixed beds. These data were analyzed by a simple model to determine rate constants for the adsorption process. In general it was found that the rate constants decreased with increasing ligand concentration. The contributions of mass transfer and intrinsic adsorption kinetics to the overall rate were estimated, and it has been found that the adsorption kinetics are rate-limiting. The implication of these results on development of a dye-ligand process, as well as the validity and utility of this simple model for scale-up and design procedures, were discussed.

Correlation of protein partitioning in aqueous polymer two-phase systems

A correlation is presented for the partitioning of proteins in aqueous two-phase systems. The correlation relates the natural logarithm of the protein partition coefficient to the polyethylene glycol (PEG) concentration difference between the phases, (w1″ — w1′), by the equation, 1n K = A(w1″ — w1′) + b(w1″ — w1′)2. This relationship was shown to fit protein partitioning data obtained from eight two-phase sytems at pH 7.0, four consisting of PEG 8000—Dextran T-500—water systems and four consisting of PEG 3400—potassium phosphate—water systems.Seven different proteins, ribonuclease, lysozyme, trypsin, rhodanese, transferrin, hexokinase and invertase, with a molecular weight range of 10 000–270 000, were utilized in this work. Each of the proteins partitioned with different values of the empirical constants A and b.

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.

Phase diagrams for dextran-PEG aqueous two-phase systems at 22°C

We have determined phase diagrams at 22°C for the aqueous two-phase systems composed of dextran, polyethylene glycol, and water. The effects of polyethylene glycol and dextran molecular weight on phase separation are reported. These phase diagrams provide more complete data for dextran/PEG/water system, and will be needed for the correlation of biomolecule partitioning.

ADSORPTION EQUILIBRIUM OF PROTEINS ON A DYE-LIGAND ADSORBENT

The effects of pH and ionic strength on adsorption of lysozyme and bovine serum albumin to Blue Sepharose have been studied. Isotherms for both proteins obey the Freundlich model. Lysozyme binding involves both hydrophobic and cation exchange interactions with the adsorbent, while binding of albumin is due primarily to cation exchange. Protein properties will be discussed in relation to the binding patterns.

Novel sulfonic acid-modified starburst dendrimer used as a pseudostationary phase in electrokinetic chromatography

Abstract Starburst dendrimer macromolecules modified with sulfonic acid terminal moieties were used as a pseudostationary phase in electrokinetic chromatography to separate positional isomers of neutral phenol molecules in an aqueous mobile phase by capillary electrophoresis. Using these dendrimers optimal separation was achieved at a pH below the p K a values of the phenols and with better performance than with micellar electrokinetic chromatography using sodium dodecyl sulfate micelles.

Reversing the amino acid sequence of a dipeptide changes its partition in an aqueous two-phase system

Reversing the amino acid sequence of a dipeptide produces distinct differences in how they partition between a polyethylene glycol/potassium phosphate aqueous two-phase system. The partition coefficients for twenty-three dipeptides in this biphasic system are recorded.

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

Solubility of pure taxol in supercritical carbon dioxide

Abstract The solubility of taxol in supercritical carbon dioxide was measured using a high-pressure ultraviolet–visible-light-transmission static cell at pressures from 3000 psia to 7000 psia and temperatures 35°C to 45°C. The solubility of taxol increased by a factor of seven from 0.7×10 −7 to 5.0×10 −7 mole fraction from 3000 psia to 7000 psia. The experimental data were modeled using the Peng–Robinson equation of state and the Chrastil method. The Peng–Robinson equation of state method utilizing k 12 optimum did not provide an adequate fit. The Chrastil method provided a good fit with average absolute relative deviations of 4.6%, 3.6%, and 2.8% for 35°C, 40°C, and 45°C, respectively.

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.