5.2 Cloning strategy for Enzymes
The modification of enzymes to improve/alter their catalytic properties has been carried out for several decades. In the past, this was achieved by random multinational programmes, but in recent years advanced technology has brought about major changes in the field. Table 5.5 gives some of the main objectives to which this research has been directed.
Protein engineering or 'molecular surgery' has been used to alter the performance of enzyme molecules. Protein engineering of enzymes involves the creation of a three-dimensional graphical model of the purified enzyme obtained from X-ray crystallographic data. Changes to the enzyme structure can then be considered which might result in increased stability to, for example, pH and temperature and the requisite molecular changes made in the gene coding for the enzyme.
Table 5.5. Objectives for the preparation of modified enzymes
To enhance the activity of the enzyme
To improve the stability
To permit the enzyme to function in a changed environment
To change pH or temperature optima
To change the specificity of an enzyme so that it catalyses the conversion of a different substrate
To change the reaction catalysed
To enhance the efficiency of a process
Adapted from Gray (1990).
Two main avenues of research have been pursued in order to alter the performance of enzymes. In one approach, mutagenesis of the cloned-gene product — amino acid residues at defined positions in the structure of the enzyme — can be replaced by other suitably coded amino acid residues. The altered gene is then transformed into a suitable host organism and the mutant enzyme subsequently produced with the requisite changes in position. This process is known as 'site-directed mutagenesis'. The second method used involves the isolation ofthe natural enzyme and modifications to its structure carried out by chemical or enzymatic means — sometimes referred to as 'chemical' mutation. A successful example of protein engineering is that of the enzyme phospho- lipase A2, which was modified structurally to resist higher concentrations of acid. This enzyme is widely used as a food emulsifier. Clearly, genetic engineering and protein engineering will have dramatic impacts on the enzyme industry in its many forms. Genetic engineering will ensure better product economy, production ofenzymes from rare microorganisms, faster development programmes, etc. Also, extensive tests of the enzymes now used have shown no harmful effects on the environment. 5.4 The technology of enzyme productionAlthough many useful enzymes have been derived from plant and animal sources, it is clear that most future developments in enzyme technology will rely on enzymes of microbial origin. Even in the malting process of brewing, where the amylases of germinated barley which hydrolyse the starch are relatively inexpensive and around which existing brewing technology has developed, there are now some competitive processes involving microbial enzymes.
The use of microorganisms as a source material for enzyme production has developed for several important reasons:
There is normally a high specific activity per unit dry weight of product.
Seasonal fluctuations of raw materials and possible shortages due to climatic change or political upheavals do not occur.
In microbes, a wide spectrum of enzyme characteristics, such as pH range and high temperature resistance, is available for selection.
Industrial genetics has greatly increased the possibilities for optimising enzyme yield and type through strain selection, mutation, induction and selection of growth conditions and, more recently, by using the innovative powers of gene transfer technology and protein engineering.
Novel enzymes from unusual sources can now be produced by cloning the relevant gene into a well-characterised and easily grown microorganism such as Aspergilus oryzae.
The rationale for selection between different microorganisms is complex and involves many ill-defined factors such as economics of cultivation, whether the enzyme is secreted in the culture broth or retained in the cell, and the presence of harmful enzymes. Depending on source material, enzymes differ greatly in their stability to temperature and to extremes of pH. Thus, Bacillus subtilis proteases are relatively heat-stable and active under alkaline conditions and have been most suitable as soap-powder additives. In contrast, fungal amylases, because of their greater sensitivity to heat, have been more useful in the baking industry.
When selecting for enzyme production, the industrial geneticist must seek to optimise desired properties (high enzyme yield, stability, independence of inducers, good recovery, etc.) while also attempting to remove or suppress undesirable properties (harmful accompanying metabolites, odour, colour, etc.). Sophisticated genetic techniques have not yet been widely practised, most manufacturers relying mainly on mutagenisation combined with good selection methods. A common feature of most industrial producer organisms is that their genetics is little understood. However, gene transfer technology, together with protein engineering, will alter this and present new horizons to enzyme technology.
The raw materials for industrial enzyme fermentations have normally been limited to substances which are readily available in large quantities at low cost, and which are nutritionally safe. Some of the most commonly used substrates are starch hydrolysate, molasses, corn-steep liquor, whey and many cereals. Solid-substrate methods of producing fungal enzymes have long historical applications, particularly in Japan and other Far East countries. In practice, this method uses moist wheat or rice bran with added nutrient salts as substrates. The growing environment is usually rectangular or circular trays held in constant-temperature rooms. Commercial enzymes of importance produced in this way include fungal amylases, proteases, pectinases and cellulases.
Since microbial enzymes are mostly low-volume, medium-cost products, the production methods using submerged liquid systems have generally relied on bioreactors that are similar in design and function to those used in antibiotic production processes (Fig. 5.3).
Fig. 5.3 The stages in the production of a liquid enzyme preparation
The choice of fermentation medium is important since it supplies the energy needs as well as carbon and nitrogen sources. Raw material costs will be related closely to the value of the final product.
Enzyme synthesis in microorganisms is often repressed, i.e. the enzyme will only be produced in the presence of an inducer molecule — most often the substrate. The inducer functions by interfering with the controlling repressor, as exemplified by starch for amylase production and sucrose for invertase production. Feedback repression can occur in the biosynthesis of small molecules in which usually the first enzyme in the chain of production is inhibited by the final product. In some cases excess of specific nutrients, such as carbon nitrogen, etc., can shut down or repress the production of enzymes involved in related or unrelated compounds — catabolic repression.
The use of inducers for industrial enzyme production can often be difficult and the most common solution is to produce regulatory mutants in which inducer dependence has been eliminated by creating constituent mutants. For catabolic repression, mutants resistant to this phenomenon have been developed while it is also possible to control the effect of these substrates by feeding them into the bioreactor by a fed-batch regime.
A typical enzyme-producing bioreactor is constructed from stainless steel and has a capacity of 10—50 m3 (Fig. 5.4). In most cases enzymes are produced in batch fermentations lasting from 30 to 150 hours; continuous- cultivation. processes have found little application in industrial enzyme production. Sterility of the bioreactor system is essential throughout production.
At the completion of the fermentation, the enzyme may be present within the microorganism or excreted into the liquid or solid medium. Commercial enzyme preparations for sale will be either in a solid or a liquid form, crude or highly purified. The concentration and purification of an enzyme is shown in Fig. 5.5.
