Ralph A. Messing

ADSORPTION OF PROTEINS ON GLASS SURFACES AND PERTINENT PARAMETERS FOR THE IMMOBILIZATION OF ENZYMES IN THE PORES OF INORGANIC CARRIERS

Abstract The forces involved in the adsorption reactions between proteins and glass surfaces are primarily ionic amine-silanol bonding and hydrogen bonding. When protein solutions are exposed to porous glass surfaces, two rates of reaction are noted. The first appears to be dependent upon the relative number of amines on the protein surface and is very rapid. The second reaction is considerably slower than the first and is dependent upon the molecular weight of the protein. This second reaction, then, is governed by the ability of the protein to diffuse into the porous structure of the glass. The surface reaction of glass has been utilized to bind enzymes (biological catalysts) and other biologically active molecules. Enzymes, a precious material, can be immobilized on inorganic oxide surfaces and effectively employed, economically, in a manner similar to that of inorganic catalysts. The pore morphology of the carrier utilized to immobilize the enzyme offers a protected environment for extending the use of this unstable molecule.

Support-bound microbial cells

Index Entries: Immobilization; immobilized microbes; carriers; crosslinking; entrapment: biomass accumulation; encapsulation: continuous fermentation; covalent coupling; covalent binding; immobilized microbe technology; supports; continuous reactor; immobilization technology; adsorption; adsorptive forces; stationary phase: growth phase; sequential multi-enzyme reactions; synchronous growth: generation time; intracellular enzyme: enzyme; porous supports; polyacrylamide; collagen; carrageenan; multisequential enzyme reactions; microbe retention; periodicity.

Covalent coupling of alkaline bacillus subtilis protease to controlledpore silica with a new simplified coupling technique

SummaryA controlled-pore silica with an average pore diameter of 510 A° was used as the carrier for the immobilization of alkaline Bacillus subtilis protease. The coupling of the enzyme was accomplished by a simple two-step procedure which involved the aqueous reaction of a bifunctional diazonium salt with the surface of the silica followed by the coupling of the enzyme through the remaining available azo functional group.The stability of the coupled enzyme was compared to that of an adsorbed enzyme produced under similar conditions. Both immobilized enzymes were stored in water at room temperature and were repeatedly assayed at intervals up to 103 days. These protease preparations were assayed with 1% casein solutions at pH 7.8 at 37°C. Under these conditions, the adsorbed enzyme exhibited a half-life of approximately 48 days, while that of the coupled protease was somewhat greater than 80 days.

Patents and literature

Protein engineering and site-directed mutagenesis is becoming immensely important in both fundamental studies and commercial applications involving proteins and enzymes in biocatalysis. Protein engineering has become a powerful tool to help biochemists and molecular enzymologists elucidate structure-function relationships in enzymic active sites, to understand the intricacies of protein folding and denaturation, and to alter the selectivity of enzymatic catalysis. Commercial applications of engineered enzymes are being developed to increase protein stability, widen or narrow substrate specificity, and to develop novel approaches for use of enzymes in organic synthesis, drug design, and clinical applications. In addition to protein engineering, novel expression systems have been designed to prepare large quantities of genetically engineered proteins. Recent US patents and scientific literature on protein engineering, site-directed mutagenesis, and protein expression systems related to protein engineering are surveyed. Patent abstracts are summarized individually and a list of literature references are given.

Chapter 2 - Bioenergy Production and Pollution Control with Immobilized Microbes

Publisher Summary Anaerobic waste treatment has been primarily a batch process requiring residence times of approximately 30 days. These extended treatment periods are required because methanobacter exhibit very low growth rates. As contrasted to the aerobic treatment, the anaerobic process evolves relatively low cell mass. The anaerobic treatment of waste, although fuel efficient, did not represent a feasible approach to treating large volumes of waste solutions by continuous processing until the advent of the anaerobic filter, which is a vertical column containing rocks or other material for the retention of solids in the waste stream. But there are still three problems that are encountered in the application of the anaerobic filter. The first of these problems is that of achieving acceptable reductions of pollutants when very low pollutant concentrations are delivered to the system. The second problem is that of processing very high concentrations of pollutants without adversely affecting the system. The final problem is that of the additional costs of scrubbing the evolved gas to produce a satisfactory fuel. Three different modifications of the anaerobic filter appear to offer solutions to these three problems. This chapter describes three anaerobic systems for producing energy while dramatically reducing retention times. These systems appear to be competitive with aerobic waste treatment with respect to retention time and waste concentration. The positive aspect of energy production warrants both pilot plant and full-scale evaluation of these innovative systems as future high technology waste treatment approaches. If the performance of the scaled-up versions of these units are comparable to the laboratory models, major contributions will have been made to solve the increasing waste production from a rising population and for contributions to alternate energy.

Immobilized cells in anaerobic digestion for methane from poultry manure

The two-stage immobilized microbe waste processor designed for sewage treatment by Messing has been modified to process poultry manure.The Messing reactor of 120-mL volume was modified and scaled up to a 4-L volume.Three different carrier materials have been investigated. Temperatures for each of the two stages were examined, and residence time as well as feed concentration were explored.Analytical data has been computer analyzed using multiple variable correlations and the results of this analysis have indicated directions for optimization.

Rapid estimation of biochemical oxygen demand.

The feasibility of using columnar reactors containing immobilized microorganisms for the rapid estimation of BOD was demonstrated in this study. Dilutions of three types of industrial effluents were tested by the BOD5 test and by this experimental system. A high degree of correlation (r = 0.98) was observed between results of the two tests. The mean standard error of estimation of the experimental system was 11%.

Immobilized Glucose Oxidase and Catalase in Controlled Pore Titania

The immobilization of glucose oxidase and catalase by adsorption within the pores of controlled-pore titania has indicated that catalase acts as a stabilizer for glucose oxidase in this material. Flow rates effect the apparent activity of the immobilized enzyme system. Carrier parameters were varied to obtain optimum loading and stability information.

Aromatic amine coupling ofaspergillus niger lactase to controlled-pore silica witho-dianisidine

Lactose (β-galactosidase) derived fromAspergillus niger was immobilized on controlledpore silica with an average pore diameter of 425 Å. The coupling of this enzyme to the surface of the silica was accomplished by reacting the surface of the silica with o-dianisidine followed by the functionalization of the residual amine with glutaraldehyde or with nitrite to form the diazonium salt. The PH profiles of the immobilized enzymes were determined and compared. Continuous reactor studies of the glutaraldehyde-functionalized, immobilized enzyme indicated a half-life of 52 days at 50°C with a 5% lactose feed at pH 3.5.