the choice of separation techniques as some are difficult to operate on a large scale.
All microbial enzyme products that will be used in foods or medically related aspects are required to meet strict specifications with regard to toxicity (Table 5.6). At present, only a small number of microorganisms are used for enzyme production. Responsibility for the safety of an enzyme product remains with the manufacturer. In practice, a safe enzyme product should have low allergenic potential and be free of toxic materials and harmful microor-ganisms. Enzymes from animal and plant sources do not require toxicological studies to be performed. When enzymes are derived from microorganisms that are traditionally used in food or food processing, no testing is required (Table 5.6). Enzymes from other microorganisms may require extensive test-ing and also analysis for toxic metabolites such as exo- and endotoxins and mycotoxins. All bulk enzymes are supplied with a detailed Material Safety Data Sheet which covers potential dangers and also handling procedures for using the enzyme.
5.5 Immobilised enzymes
Almost 95% of all commercial enzymes are purchased in a soluble form, with the majority being used directly on a single-use basis in the areas listed in Table 5.1. The use of enzymes in a soluble or free form must be considered as very wasteful because the enzyme generally cannot be recovered at the end of the reaction. A new and valuable area of enzyme technology is that concerned with the immobilisation of enzymes on insoluble polymers, such as membranes and particles, which act as supports or carriers for the enzyme activity. The enzymes are physically confined during a continuous catalytic process and may be recovered from the reaction mixture and re-used over and over again, thus improving the economy of the process; this is merely a return to the natural immobilised state of most enzymes in living systems. Some enzymes that are rapidly inactivated by heat when in cell-free form can be stabilised by attachment to inert polymeric supports, while in other examples such insolubilised enzymes can be used in non-aqueous environments. Whole microbial cells can also be immobilised inside polyacrylamide beads and used for a wide range of catalytic functions. The variety of new enzymes and whole-organism systems that are likely to become cheaply available presents exciting possibilities for the future, especially in the pharmaceutical and diagnostic fields.
Table 5.6. Safety testing of food enzymes based on the Association of Microbial Food Enzyme Producers’ classification
Group (a) Microorganisms
X, test to be performed.
aOnly to be performed under exceptional conditions.
From Godfrey and Reichelt (1983).
5.5 Immobilised enzymes 95
Fig. 5.6 Techniques of enzyme / cell immobilisation.
Present applications of immobilised catalysts are mainly confined to indus-trial processes, e.g. production of l-amino acids, organic acids and fructose syrup. The future potential for immobilised biocatalysts lies in novel applica-tions and the development of new products rather than as an alternative to existing processes using non-immobilised biocatalysts.
Immobilised enzymes are normally more stable than their soluble counter-parts and are able to be re-used in the purified semi-purified or whole-cell form.
Catalytic properties of immobilised enzymes can often be altered favourably to allow operation under broader or more rigorous reaction conditions; for example, immobilised glucose isomerase can be used continuously for over 1000 hours at temperatures of 60–65◦C.
How are enzymes immobilised? In practice, both physical and chemical methods are routinely used for enzyme immobilisation. Physically, enzymes may be absorbed onto an insoluble matrix, entrapped within a gel, or encapsu-lated within a microcapsule or behind a semi-permeable membrane (Fig. 5.6).
Chemically, enzymes may be covalently attached to solid supports or cross linked.
A large number of chemical reactions have been used for the covalent bind-ing of enzymes, by way of their non-essential functional groups, to inorganic carriers such as ceramics, glass, iron, zirconium and titanium, to natural poly-mers such as sepharose and cellulose, and to synthetic polypoly-mers such as nylon, polyacrylamide and other vinyl polymers and co-polymers possessing reactive chemical groups. In many of these procedures the covalent binding of enzymes to the carriers is non-specific, i.e. the binding of the enzyme to the carrier by way of the enzyme’s chemically active groups distributed at random. More
Table 5.7. Limitations of immobilised enzyme techniques
Method Advantages Disadvantages
Covalent attachment Not affected by pH, ionic strength of the medium or substrate concentration.
Active site may be modified;
costly process.
Covalent crosslinking Enzyme strongly bound, thus unlikely to be lost.
Loss of enzyme activity during preparation; not effective for macromolecular substrates; regeneration of carrier not possible.
Adsorption Simple with no modification of enzyme; regeneration of
Entrapment No chemical modification of enzyme.
Diffusion of substrate to, and product from, the active site; preparation difficult and often results in enzyme inactivation; continuous loss of enzyme due to distribution of pore size; not effective for macromolecular substrates; enzyme not subject to microbial or proteolytic action.
recent studies have attempted to develop techniques of enzyme immobilisa-tion in which the enzyme binds to a carrier with high activity without affecting its catalytic activity. The limitations of immobilised enzyme techniques are shown in Table 5.7.
The entrapment of enzymes in gel matrices is achieved by carrying out the polymerisation or precipitation/coagulation reactions in the presence of the enzyme. Polyacrylamides, collagen, silica gel, etc., have all proved to be suitable matrices, but the entrapment process is relatively difficult and results in low enzyme activity.
Immobilised whole microbial cells are becoming increasingly utilised and tend to eliminate the tedious, time-consuming and expensive enzyme-purification steps. Immobilisation of whole cells is normally achieved by the
5.5 Immobilised enzymes 97
Table 5.8. The advantages of immobilised biocatalysts
(1) Permits the re-use of the component enzyme(s) (2) Ideal for continuous operation
(3) Product is enzyme-free
(4) Permits more accurate control of catalytic processes (5) Improves stability of enzymes
(6) Allows development of a multi-enzyme reaction system (7) Offers considerable potential in industrial and medical use (8) Reduces effluent disposal problems
same methods as for cell-free enzymes. The greatest potential for immobilised cell systems lies in replacing complex fermentations such as secondary prod-uct formation (i.e. semi-synthetic antibiotics) in the continuous monitoring of chemical processes (via enzyme electrodes), water analysis and waste treat-ment, continuous malting processes, nitrogen fixation, synthesis of steroids and other valuable medical products. The advantages of using immobilised biocatalysts are summarised in Table 5.8.
As a consequence of successful immobilisation techniques, in the form of enzyme capsules, enzyme beads, enzyme columns and enzyme membranes, many types of bioreactors have been developed on a laboratory scale and, to a lesser extent, on an industrial scale. These include batch-stirred tank bioreactors, continuous packed-bed bioreactors and continuous fluidised-bed bioreactors (Fig. 5.7). In industrial practice the catalytic properties of iso-lated enzymes, immobilised enzymes or immobilised whole cells are gener-ally utilised within the confines of bioreactor vessels. Bioreactor systems can have many forms, depending on the type of reactions and stability of the enzyme.
In Europe, immobilised penicillin acylase is used to prepare 6-amino-penicillic acid (6-APA) from naturally produced penicillin G or V (Fig. 5.8).
This compound is an important intermediate in the synthesis of semi-synthetic penicillins, which are so essential in our fight against bacterial diseases. Two types of penicillins are produced by industrial fermentation: penicillin G (phenyl acetyl-6-APA) and penicillin V (phenoxy acetyl-6-APA), each con-taining a nucleus of 6-APA and a side-chain. The antibiotic activity of the penicillin molecule is governed by the side-chain and, when removed and replaced with another, it can profoundly alter the antibiotic spectrum and other properties. Many pharmaceutical companies now operate immobilised enzyme processes for the production of 6-APA on an industrial scale.
Batch-stirred tank reactor
Immobilised microbial cells/enzymes
Immobilised microbial cells/enzymes Stirred tank
bioreactor
Continuous packed-bed reactor Substrate
Substrate
Substrate Substrate
Substrate Product
Product Product
Product
Product
Immobilised microbial cells/enzyme column
Continuous fluidised-bed
reactor Fluidised bed
of immobilised microbial cells/enzymes
Recycle Upward flow
Downward flow
Fig. 5.7 Immobilised cell / enzyme bioreactors.
Fig. 5.8 Formation of 6-APA by hydrolysis of penicillin.
5.5 Immobilised enzymes 99
Table 5.9. Some industrial applications of immobilised enzymes
Method of
Industry Enzyme immobilisation Process
Food Glucose isomerase AE-cellulose Conversion of glucose to fructose
Chemical Nitrilase Polyacrylamide Production of
acrylamide from acrylonitrile
At least 3500 tonnes of 6-APA are produced each year, requiring the pro-duction of about 30 tonnes of the enzyme (Table 5.9).
Immobilised glucose isomerase is used in the USA, Japan and Europe for the industrial production of high-fructose syrups by partial isomerisation of glucose derived from starch. Thousands of tonnes of high-fructose syrup are produced annually by this enzyme process, and it is undoubtedly the most widely used of all the immobilised enzymes. The industrial and commercial success of this process is due to the following facts: glucose derived from starch is relatively cheap; fructose is sweeter than glucose; the high-fructose syrup contains approximately equivalent amounts of glucose and fructose, and, from a nutritional aspect, is similar to sucrose. The overall production of fructose from starch is shown in Fig. 5.9.
Fig. 5.9 Production of fructose from starch.
Platinum anode
Teflon membrane Electrolyte
‘O’-ring Enzyme
Substrate (analyte) Cathode
Product Semi-permeable membrane
Fig. 5.10 A simple biosensor combining an electrochemical electrode and an enzyme immobilised onto a semi-permeable membrane (from Wymer, NCBE Newsletter).
5.5 Immobilised enzymes 101
Another important use of immobilised enzymes is aminoacylase production of amino acids. Aminoacylase columns are used in Japan to produce thousands of kilograms of L-methionine, L-phenylalanine, L-tryptophan and L-valine.
Enzyme polymer conjugates are now being used extensively in analytical and clinical chemistry. Immobilised enzyme columns or tubes can be used repeatedly as specific catalysts in assays of substrates. Enzyme electrodes are a new type of detector or biosensor designed for the potentiometric or amperometric assay of substrates such as urea, amino acids, glucose, alcohol and lactic acid.
In design, the electrode is composed of a given electrochemical sensor in close contact with a thin, permeable enzyme membrane that is capable of reacting specifically with the given substrates. The embedded enzymes in the membrane produce O2, hydrogen ions, ammonium ions, CO2or other small molecules, depending on the enzymatic reactions occurring, which are readily detected by the specific sensor; the magnitude of the response determines the concentration of the substrate (Fig. 5.10). While the biological component in a biosensor may more often be an enzyme or multi-enzyme system, it can also be an antibody, an organelle, a microbial cell or whole slices of tissue.
The application of enzyme technology to existing processes, e.g. brew-ing, food processbrew-ing, medicine, pharmaceuticals, chemical industry and waste treatment, has enormous potential and is examined in later chapters.
Looking to the future, it seems reasonable to expect that the production and application of enzymes will continue to expand. The growing world concern about the environment and natural resources, in particular the rising prices of oil and other raw materials, is promoting new avenues of research and there is little doubt that enzymes will play a major role in solving some of these problems.