Robert G. Luo

Kinetic Study on the Enzymatic Resolution of Homophenylalanine Ester Using Ionic Liquids

Two ionic liquids (ILs) were investigated as novel media for the enzymatic resolution of amino acid ester to obtain enantiomeric amino acid homophenylalanine. The effects of solvent nature, polarity, and concentration on the kinetic resolution were investigated. With change in solvent concentration, a systematic study shows that an improved enzyme activity can be obtained by adjusting these solvent parameters.

Preparation and characterization of three room-temperature ionic liquids

Three important room-temperature ionic liquids (RTILs) were prepared using straightforward precipitation reactions, 1-ethyl-3-methylimidazolium tetrafluoroborate ([EMIM] + [BF 4 ] - ), N-ethyl pyridinium tetrafluoroborate ([EtPy] + [BF 4 ] - ) and N-ethyl pyridinium trifluoroacetate ([EtPy]+[CF 3 OO] - ). They were characterized by density measurements and IR spectroscopy.

Chromatographic removal of endotoxin from hemoglobin preparations. Effects of solution conditions on endotoxin removal efficiency and protein recovery

In this work, affinity chromatography was used to remove endotoxin from human hemoglobin preparations with a Sterogene Acticlean Etox column. The effects of solution conditions on endotoxin removal efficiency and protein recovery have been investigated. It has been found that cations Na(+) or Ca(2+) reduced endotoxin removal efficiency from 73% (sample prepared with endotoxin-free water) to 31% (sample prepared with 0.15 M NaCl, ionic strength, I = 0.15 M), and from 73% sample prepared with endotoxin-free water) to 9% (sample prepared with 0.05 M CaCl2, ionic strength, I = 0.15 M). It has also been found that the protein recovery was increased from 90% to 99%, respectively.

Use of Ca+2 to re-aggregate lipopolysaccharide (LPS) in hemoglobin solutions and the subsequent removal of endotoxin by ultrafiltration

Divalent cations such as Ca+2 can act as “bridges” between lipopolysaccharide subunits causing large vesicles to form. During a two-stage ultrafiltration Ca+2 re-aggregated lipopolysaccharide subunits in protein solutions into large vesicles that could not pass a 300,000 nominal molecular weight cut-off ultrafiltration membrane. Such re-aggregation occurred at concentration ratio of hemoglobin Ao / lipopolysaccharide equal to or less than 17.0, or at concentration ratio of Ca+2 / hemoglobin Ao / lipopolysaccharide equal to or greater than 8.5.

Concise Synthesis and Enzymatic Resolution of L-(+)-Homophenylalanine Hydrochloride

The N-acetyl-homophenylalanine ethyl ester was synthesized by a simple three-step-reaction strategy. L-(+)-homophenylalanine hydrochloride with 98% ee was obtained through a kinetic resolution process using industrial enzyme alcalase. Compared with other methods, this strategy has the advantage of economical and simple procedure giving high product optical purity.

Protein concentration effect on protein-lipopolysaccharide (LPS) binding and endotoxin removal

It is known some proteins can disaggregate endotoxins and form complexes with lipopolysaccharide (LPS). Nevertheless, how protein concentration affects protein-LPS binding and endotoxin removal is unknown. In this study, protein samples at various concentrations were incubated with endotoxin samples at a fixed concentration. The mixtures were filtered by ultrafiltration membranes. As protein concentration increased, the amount of endotoxin detected in the filtrates increased too. This result indicates protein concentration has significant effect on protein-LPS binding and the amount of endotoxin disaggregated.

A New Integrated Membrane Filtration and Chromatographic Device

To improve protein separation, a novel integrated device combining membrane filtration and chromatography has been developed. The device basically consists of a hollow fiber filtration module whose shell side is filled with chromatographic resin beads. However, there is an essentially impermeable coated zone near the hollow fiber module outlet. The integrated device enjoys the advantages of both membrane filtration and chromatography; it also allows one to load the chromatographic media directly from the fermentation broth or lysate and separate the adsorbed proteins through the subsequent elution step in a cyclic process. Interfacial polymerization was carried out to coat the bottom section of the hollow fiber membrane; the rest of the hollow fiber membrane remained unaffected. Myoglobin (Mb) and α‐lactalbumin (α‐LA) were primarily used as model proteins in a binary mixture; binary mixtures of Mb and bovine serum albumin (BSA) were also investigated. Separation behaviors of binary protein mixtures were studied in devices having either an ultrafiltration (UF) or a microfiltration (MF) membrane. Experimental results show that the breakthrough time and the protein loading capacities were dramatically improved after introducing the impermeable coating in both UF and MF modules. For a synthetic yeast fermentation broth feed, four loading‐washing‐elution‐reequilibration‐based cyclic runs for separation of Mb and α‐LA were performed in the device using a MF membrane with a coated zone without cleaning in between. The Mb and α‐LA elution profiles for the four consecutive runs were almost superimposable. Due to lower transmembrane flux in this device plus the periodical washing‐elution during the chromatographic separation, fouling was not a problem, unlike in conventional microfiltration.

Effects of ionic strength and pH on endotoxin removal efficiency and protein recovery in an affinity chromatography

Abstract In this study endotoxin was removed from hemoglobin preparations using an affinity chromatographic column. The effects of ionic strength and pH on endotoxin removal efficiency and protein recovery have been investigated. Both ionic strength and pH had significant effects on endotoxin removal from hemoglobin solutions. When the ionic strength was increased from 0 to 0.25 M, endotoxin removal efficiency was reduced from 71 to 38% (samples prepared with NaCl solutions), and from 71 to 37% (samples prepared with CaCl 2 solutions). When pH was varied from 4.5 to 9.0, endotoxin removal efficiency was reduced from 92 to 43%. However, both ionic strength and pH had very limited effects on protein recovery in the process for hemoglobin-LPS feeds as well as pure hemoglobin feeds. Hemoglobin recovery was above 94% in all cases.

Quantitative Determination of Ca2+ Effects on Endotoxin Removal and Protein Yield in a Two-Stage Ultrafiltration Process

Endotoxin subunits normally aggregate into vesicles with a molecular mass around 1,000,000. Some proteins can bind and disaggregate endotoxin to form protein–endotoxin complexes, which makes endotoxin removal from protein solution more complicated. It is also known that divalent cations such as Ca2+ can act as “bridges” between lipopolysaccharide subunits, causing large vesicles to form. Such a property has potential utility in endotoxin removal from biological solutions. A two-stage ultrafiltration was used in this study during which Ca2+ reaggregated lipopolysaccharide subunits in protein solutions into large vesicles that hence were retained by a 300,000 nominal molecular weight cutoff ultrafiltration membrane. After the reaggregation of lipopolysaccharide subunits, the endotoxin removal efficiency was in the range of 54.78 to 73.10%, meanwhile protein yield was in the range of 71.57 to 89.54% at various protein/endotoxin concentration ratios.

An Integrated Process for Biomolecule Isolation and Purification

Biomolecule isolation and purification from a fermentation broth usually involve centrifugation, filtration, adsorption, and chromatography steps. Each step contributes to the product cost and product loss. In this research, a cyclic process integrating commercially available ultrafiltration membranes and chromatographic resin beads was developed to achieve the same goal in one device. The device consisted of ion exchange beads on the shell side of a hollow fiber ultrafiltration module. Loading of proteins on the stationary phase on the shell side was carried out for a period of 5-20 min from the permeate on the shell side produced from tube-side feed in ultrafiltration. The eluent was then introduced either from the shell-side inlet or tube-side inlet; the chromatographic fractions were collected from the shell-side outlet. The column was regenerated/washed next to start a new cycle. Systems studied in this cyclic process include the following binary mixtures: myoglobin and beta-lactoglobulin; hemoglobin and bovine serum albumin; and myoglobin and alpha-lactalbumin. Excellent resolutions of the proteins were obtained. A yeast-based cellular suspension containing a mixture of myoglobin and alpha-lactalbumin was also applied to this device. The target proteins were recovered and purified successfully. The cyclic process-based device integrates clarification, concentration, and chromatographic purification of biomolecules and is suitable for both extracellular and intracellular products.