BIOTECHNOLOGY - V. G. Herasymenko - 2006

Part II. Special Biotechnologies

CHAPTER 9. APPLICATION OF IMMOBILIZED ENZYMES IN ANALYTICAL PRACTICE

9.1. ANALYTICAL FLOW REACTORS WITH IMMOBILIZED ENZYMES

The USE OF IMMOBILIZED Enzymes has paved the way for The Development of flow analyzers essential for bioprocess monitoring and control.

Various types of immobilized enzyme reactors have been proposed, including columns, tubes, and hollow fibers. Column packing Materials typically utilized enzymes covalently linked to various Supports, such as amino-functionalized Glass, acrylic polymers, agarose or Sepharose, nylon powder, silica gel, and Silochrom.

The primary requirements for these supports include the capacity to bind a high amount of enzyme (high loading capacity), ensure rapid flow of the analyzed mixture through the Reactor, and minimize non-specific sorption of sample components, as this can lead to diminished enzymatic activity and increased backpressure in the column.

One of the finest supports for column reactors is Cyanogen bromide-activated Sepharose. It has been successfully used to immobilize not only individual enzymes but also multienzyme systems. Tryptophan, an essential amino acid, was the first analyte determined using such a reactor.

R. Sundaram (1977) utilized nylon tubing as enzyme reactors. The inner surface of the tubing was partially acid-hydrolyzed, after which the enzyme was covalently immobilized onto the nylon. These tubes were coupled to Technicon automated flow analyzers. Such reactors were employed to assay various metabolites and drugs in Blood serum, including urea, uric acid, Amino Acids, glucose, lactose, maltose, and penicillin.

An original design of an enzyme reactor was proposed by Italian chemists (Marconi V. et al., 1975). They developed a method for entrapping enzymes inside Cellulose triacetate hollow fibers during their formation—specifically, during the spinning of the fiber from solution. The enzyme became entrapped within the interior cavity, accessible only to low-molecular-weight substrates. These fibers were wound into coils, placed inside a glass casing, and the test mixture was passed through the enzyme reactor. Today, this method is used to determine penicillin, urea, glucose, and other substances.