William Cohen

Practical Processing with Cofactor -Requiring Enzymes

Abstract The use of cofactor requiring enzymes in practical processes is dependent on regeneration and multiple reuse of the cofactor. Various methods currently available for the accomplishment of this goal are evaluated. We conclude that, in most instances, enzymatic regeneration techniques are superior to chemical regeneration techniques. We also conclude that, except in very unusual cases, cofactor regeneration and reuse is most efficient when processing enzymes as well as cofactor regenerating enzymes are contained in the same reactor. A cofactor classification based on regeneration requirements is proposed. Methods for the retention of both modified (for example, macro molecular) and native cofactors within the process reactor are discussed, as well as various cofactor recovery techniques. Finally, a prototype enzyme reactor employing native cofactor without retention, and allowing high substrate to product conversion with an extremely high number of cofactor cycles, is proposed. Utilizing this react...

A continuously monitored spectrophotometric assay of glycosidases with nitrophenyl glycosides

Abstract A general method for a continuously monitored spectrophotometric assay of glycosidases at all values of pH using p -nitrophenyl glycosides is presented. The method is demonstrated specifically by the development of a routine assay for α-galactosidase from fig and Mortierella vinacea using p -nitrophenyl galactopyranoside (NPG) at pH 3.9 and 5.8, respectively, and also for jack bean meal β- N -acetylhexosaminidase using p -nitrophenyl-β-2-acetamido-2-deoxy- d -glucopyranoside (NPADG) at pH 5.0. A number of different wavelengths may be used for the assay depending upon the criterion of the user; maximum sensitivity at a selected pH, determination of enzyme pH optima with a pH-independent difference extinction coefficient, or the reduction of background absorbance for kinetic studies at high substrate concentrations.

An active-site titration method for immobilized trypsin

Abstract An active-site titration method for immobilized trypsin with p-nitrophenyl p′-guanidinobenzoate (NPGB) employing a recirculation reactor system is described. Trypsin, covalently linked to 35 and 150 μm diameter porous glass particles was titrated by this procedure, analyzed for protein content, and then compared with soluble trypsin. Analysis of the titration results indicates that the amount of p-nitrophenol produced by the burst is equal to the amount of active immobilized trypsin. This active site titration determines the absolute amount of active immobilized enzyme and is preferable to either total protein analysis or kinetic assays for characterization of immobilized enzymes.

Continuous Processing with Cofactor Requiring Enzymes: Coenzyme Retention and Regeneration

Until now continuous processing with enzymes has been limited to those enzymes that do not require cofactors. However, of the six classes of enzymes only two do not require cofactors. In order to widen the scope of enzyme engineering, the capability for continuous processing with cofactor requiring enzymes is highly desirable. Such processing necessitates cofactor reuse since continuous addition of expensive coenzyme is prohibitive. Regenerating the cofactor (reconverting it to its original state) is a necessary aspect of cofactor reuse. Another necessary aspect of cofactor reuse is coenzyme retention; the cofactor must either remain in the reactor, or a satisfactory method for separating the cofactor from the product stream must be developed.

High Turnover NAD Regeneration in the Coupled Dehydrogenase Conversion of Sorbitol to Fructose

Significant progress has recently been made in the commercial application of a limited number of enzyme processes. However, it is important to note that none of these applications involve enzymes with dissociable cofactors. Since four of the six classes of enzymes require cofactors, the absence of efficient and economic methods for cofactor-requiring enzymatic processes has presented a major obstacle to reaching the full potential of enzyme technology. Cofactor-requiring enzymatic processes of considerable interest include the transformation of glucose to a variety of chemicals, the conversion of lignin by-products to amino acids, enzymatic synthesis of pharmaceuticals such as chenodeoxycholate and bacitracin, the capture of solar energy through biophotolysis, stereo-specific steroid transformations, epoxidation of fatty acids and many others.

Removal of Heavy Metal Enzyme Inhibitors

Protection of immobilized enzyme activity by removal of heavy metal enzyme inhibitors from enzyme reactor feed streams has been studied with a recently developed heavy metal selective adsorbent. Enzyme protection from mercury-caused inhibition was demonstrated for the enzymes glucose isomerase, alcohol dehydrogenase and the lactases from Aspergillus niger and Saccharomyces fragilis. The adsorbent, which contains thiol groups covalently bound to a porous inorganic matrix, forms tight complexes with mercury. Mathematical simulation and experimental confirmation of adsorbent kinetics and column performance indicated that mercury adsorption on the adsorbent proceeds by a “progressive shell” mechanism. The simulation was used to project adsorbent performance for large-scale enzyme protection.

Membrane-Immobilized Liver Microsome Drug Detoxifier

The common occurrence of poisoning due to drug overdose has led to an increasing need for a safe, simple, effective, and inexpensive means of removing such drugs from the blood in the emergency treatment of drug-overdose victims. Various devices and techniques based on extracorporeal blood processing have been investigated. Physical techniques include hemodialysis (Kennedy et al., 1969), charcoal adsorption (Widdop et al.,1975; Chang et al., 1973), and ion-exchange adsorption (Rosenbaum et al., 1971; Medd et al., 1974). Biological techniques include liver perfusion (McDermott and Norman, 1971), liver slice perfusion (Koshino et al., 1975), perfusion over hepatic cell suspensions in direct contact with the blood (Eisman and Soyer, 1971; Soyer et al., 1973), and perfusion through hollow fibers containing cultured hepatic cells on the outer surface of the fibers (Wolf and Munkelt, 1975). It is well accepted that the biological system responsible for detoxification of a variety of drugs is located in the smooth endoplasmic reticulum of hepatic cells which can be isolated as microsomes by cell fractionation. The microsomes contain a complex mixture of enzymes, including cytochrome P-450, which requires the coenzyme NADPH and molecular oxygen for drug detoxification. In general these detoxification enzymes modify toxins by increasing their polarity and consequently their aqueous solubility, thus decreasing their permeation into tissues (Mandel, 1971) and promoting excretion. The chemical modifications catalyzed by the microsomal drug detoxification enzymes include, among others, hydroxylation, demethylation, and conjugation reactions. Several of the microsomal detoxification enzymes have been purified (Lu and Levin, 1974; Brunner, 1975; Ziegler and Mitchell, 1972) and at least two have been immobilized by covalent bonding to insoluble particles (Brunner, 1975; Parikh et al., 1976; Sofer et al.,1975). One approach for utilization of these enzymes in an extracorporeal drug detoxification system is to purify the individual enzymes and then reconstitute the multienzyme complex by binding to insoluble particles. An alternative approach, the one we have chosen, is to utilize the isolated microsomes themselves. This method has the advantage of simplicity and lower cost. More importantly, in contrast to the former approach, it assures that all the microsomal enzymes are present in proportions and molecular arrangement closely resembling the in vivo state. We have used an extracorporeal hollow fiber-based enzyme reactor in an attempt to develop a therapeutic technique suitable for treatment of drug and poison detoxification. Since all the protein components of the system are impermeable to the hollow fiber membranes, adverse immunological reactions will be avoided. In addition, the safety of blood processing using hollow fiber devices has been well established in recent years by extensive use in hemodialysis.

Characterization of Immobilized β-N-Acetylhexosaminidase1

Abstract The properties of fl-N -acetylhexosaminidase chemically bound to Sepharose 4B were determined and compared to those of the soluble enzyme. External diffusion effects on the kinetics of the immobilized enzyme were eliminated by assaying in a recirculation reactor with rapid flow rates. The immobilized 15-N-acety Hexosaminidase exhibited a broad pH optimum quite similar to that of the soluble enzyme. Compared to the soluble enzyme the immobilized enzyme demonstrated a markedly enhanced stability at each pH and temperature investigated. Immobilization caused an increase in both the apparent K and K., with rrm. A preliminaty account of this work was presented at the Annual Meeting of the Federation of American Societies for Experimental Biology, Atlantic City, N. J., April, 197 3.