A. Constantinides

Characterization of glucose oxidase immobilized on collagen

SummaryThe enzyme glucose oxidase (E.C. 1.1.3.4) was immobilized on collagen — a proteinaceous material found in biological systems as a structural material for a wide variety of cells and membranes. The novel technique of electrocodeposition, which utilizes the principles of electrophoresis, was used to deposit the enzyme-collagen complex on stainless steel helical supports. This technique has been developed in our laboratory. The mechanism of complex formation between collagen and enzyme involves multiple salt linkages, hydrogen bonds and van der Waals interactions.As a first step toward examining its feasible technical use, the kinetic behavior of the collagen-supported glucose oxidase was studied in a batch recycle type reactor and was compared with that for the soluble form. A novel reactor configuration consisting of multiple concentric electrocodeposited helical coils was used. The reactor was found to attain a stable level of activity which was maintained for several months under cyclic testing. The optimum levels of pH and temperature for the immobilized form of the enzyme were the same as those of the soluble enzyme, but the immobilized enzyme was more active than the soluble form at higher temperatures and pH. The values of the Michaelis-Menten parameters indicate that the overall reaction rate of the immobilized enzyme may be partially restricted by bulk and matrix diffusion.

Synthesis of Organic Acids and Modification of Steroids by Immobilized Whole Microbial Cells

As part of our continued efforts to develop a technology based on collagen immobilized enzymes and whole microbial cells, we have investigated a number of reaction schemes catalyzed by collagen-whole cell complexes. We initiated our studies on immobilized whole cell systems with simple systems such as glucose isomerization which involve only a single enzymatic reaction. The encouraging results obtained in this case (production of high fructose syrup by immobilized Streptomyces venezuelae) (1,2) prompted us to examine other systems listed in Table 1.

Design and Analysis of Immobilized-Enzyme Flow Reactors

Publisher Summary The overall efficiency of an enzyme reactor design would be determined by myriad factors. Although some general analyses and design procedures for enzyme reactors are known, the dearth of experimental information—particularly on a pilot-plant scale—renders the task of designing and scaling-up an enzyme reactor difficult. A designer, contemplating the design of a given enzymic reaction system, might be posed with several alternative approaches—none of them having a clear advantage or design precedence. If comparative information was available on different alternative design schemes, it would facilitate the choice of a particular system. This chapter presents the comparison between the relative efficiencies of several reactor configurations, based on research done in the laboratory on free and immobilized enzymes. The data was obtained under conditions that permit their meaningful comparison. The most important factors governing the overall reactor efficiency include the enzyme loading factor, carrier loading factor, operational stability of enzyme, external and internal diffusional efficiency, and residence time distribution.

Scaleup and Optimization of Oxygen Transfer in Fermentors

The basic function of scaleup is to determine the operating conditions in equipment of different size and mass transfer characteristics so as to achieve the same process yield. A typical problem is the determination of operating conditions in existing large-scale equipment that will provide similar oxygen transfer as observed in laboratoryscale fermentors. In most fermentations of industrial importance, the critical substrate is oxygen due to its low solubility in water. For good process productivity, it is important to supply sufficient oxygen to satisfy the needs of the microorganism. However, supplying excessive oxygen is wasteful of power and may even lead to poor performance due to oxygen toxicity in some unusual cases. One way of ensuring adequate oxygen availability is to determine operating conditions that keep the dissolved oxygen concentrations above predetermined critical levels in all parts of the fermentor. In order to minimize power consumption, these operating conditions should be such that the dissolved oxygen levels are not kept higher than necessary. The work here will be restricted to geometrically similar stirred-tank fermentors. These units are common in the industry because of their versatility and somewhat standardized design. The main operating conditions that must be optimized are the agitator speed and the aeration rate. Suitable methods for the design of equipment for both Newtonian as well as nowNewtonian fermentation broths will be discussed. On-line techniques will be developed for the continuous optimization of oxygen transfer efficiency.

Modeling of cell viability and specific alcohol productivity.

A model has been proposed to correlate two viability tests and specific alcohol productivity of a yeast culture. Two viable populations with different activity are assumed to coexist. The difference between two populations is the ability to reproduce. Satisfactory agreement between the model and the experimental data has been observed.

Sugar transport in enzymatically active proteinaceous membranes and the application of a sorption theory

Abstract The transport of sucrose through lightly crosslinked, reconstituted biopolymeric (i.e., collagen) membranes, blank and enzyme-bound, was investigated. Transient models of the transport mechanism are presented for the following cases: no solute immobilization, and partial solute immobilization, with and without enzymatic reaction. Enzyme-bound collagen membranes were studied through permeation experiments conducted at a system temperature of 25°C so as to examine the effect of reaction on the diffusive transport. The imposed reaction was the enzymatic hydrolysis of sucrose by invertase. An understanding of the sucrose transport mechanism was necessary prior to the analysis of the enzyme membrane permeation data. Through time lag and sorption data collected from blank, unreactive collagen membranes, this mechanism was identified. Upon the elimination of the extramembrane film resistances, a site saturation effect for these membranes was observed. The apparent effective diffusivity was determined from the data collected near system saturation. This value was estimated to be 1.01 × 10−6 cm2/s ± 5%. Thus, a transport pathway on the structural protein is found to control the sugar permeability. The enzyme membrane data reflected a prolonged transient and a significant reduction in the emergent substrate flux. These effects could be important in modeling delays or lags in physiological responses to changes in substrate concentration(s). With the transport mechanism described, the kinetic parameters characterizing these membranes were extractable from these same data.

Complexation of Enzymes or Whole Cells with Collagen

Usually the most important criterion in selecting the carrier material and process for enzyme immobilization is that of economy. Collagen was selected with this idea in mind, because hide pulp is priced in the range of

Steroid transformation at high substrate concentrations using immobilized Corynebacterium simplex cells

0.50 per lb. Employing this material, we have been investigating different approaches to achieve enzyme immobilization on collagen with the goal of developing simple and economical processes. We have reported the processes of impregnation of enzyme (1–7) into a preformed collagen membrane and electrocodeposition of enzyme codispersed with collagen to form a membrane complex on an electrode (8). As shown on the left side of Fig. 1, the process to be introduced here is a newly developed method of immobilization through direct macromolecular complexation.