Screening for novel enzymes

If a reaction is thermodynamically possible, it is likely that an enzyme exists which is capable of catalysing it. One of the major skills of enzyme companies and suitably funded academic laboratories is the rapid and cost-effective screening of microbial cultures for enzyme activities. Natural samples, usually soil or compost material found near high concentrations of likely substrates, are used as sources of cultures. It is not unusual at international congresses of enzyme technologists to see representatives of enzyme companies collecting samples of soil to be screened later when they return to their laboratories.

The first stage of the screening procedure for commercial enzymes is to screen ideas, i.e., to determine the potential commercial need for a new enzyme, to estimate the size of the market and to decide, approximately, how much potential users of the enzyme will be able to afford to pay for it. In some cases, the determination of the potential value of an enzyme is not easy, for instance when it might be used to produce an entirely novel substance. In others, for instance when the novel enzyme would be used to improve an existing process, its potential value can be costed very accurately. In either case, a cumulative cash flow must be estimated, balancing the initial screening and investment capital costs including interest, tax liability and depreciation, against the expected long term profits. Full account must be taken of inflation, projected variation in feedstock price and source, publicity and other costs. In addition, the probability of potential market competition and changes in political or legal factors must be considered. Usually the sensitivity of the project to changes in all of these factors must be estimated, by informed guesswork, in order to assess the risk factor involved. Financial re-appraisal must be frequently carried out during the development process to check that it still constitutes an efficient use of resources.

If agreement is reached, probably after discussions with potential users, that experimental work would be commercially justifiable, the next stage involves the location of a source of the required enzyme. Laboratory work is expensive in manpower so clearly it is worthwhile using all available databases to search for mention of the enzyme in the academic and patents literature. Cultures may then be sought from any sources so revealed. Some preparations of commercial enzymes are quite rich sources of enzymes other than the enzyme which is being offered for sale, revealing such preparations as potential inexpensive sources which are worth investigating.

If these first searches are unsuccessful, it is probably necessary to screen for new microbial strains capable of performing the transformation required. This should not be a 'blind' screen: there will usually be some source of microbes that could have been exposed for countless generations to the conditions that the new enzyme should withstand or to chemicals which it is required to modify. Hence, thermophiles are sought in hot springs, osmophiles in sugar factories, organisms capable of metabolising wood preservatives in timber yards and so on. A classic example of the detection of an enzyme by intelligent screening was the discovery of a commercially useful cyanide-degrading enzyme in the microbial pathogens of plants that contain cyanogenic glycosides.

The identification of a microbial source of an enzyme is by no means the end of the story. The properties of the enzyme must be determined; i.e., temperature for optimum productivity, temperature stability profile, pH optimum and stability, kinetic constants (K_m, V_max), whether there is substrate or product inhibition, and the ability to withstand components of the expected feedstock other than substrate. A team of scientists, engineers and accountants must then consider the next steps. If any of these parameters is unsatisfactory, the screen must continue until improved enzymes are located. Now that protein engineering (see Chapter 8) can be seriously contemplated, an enzyme with sufficient potential value could be improved 'by design' to overcome one or two shortcomings. However, this would take a long time, at the present level of knowledge and skill, so further screening of microbes from selected sources would probably be considered more worthwhile.

Once an enzyme with suitable properties has been located, various decisions must be made concerning the acceptability of the organism to the regulatory authorities, the productivity of the organism, and the way in which the enzyme is to be isolated, utilised (free or immobilised) and, if necessary, purified. If the organism is unacceptable from a regulatory viewpoint two options exist; to eliminate that organism altogether and continue the screening operation, or to clone the enzyme into an acceptable organism. The latter approach is becoming increasingly attractive especially as cloning could also be used to increase the productivity of the fermentation process. Cloning may also be attractive when the organism originally producing the enzyme is acceptable from the health and safety point of view but whose productivity is unacceptable (see Chapter 8). However, cloning is not yet routine and invariably successful so there is still an excellent case to be made for applying conventional mutation and isolation techniques for the selection of improved strains. It should be noted that although the technology for cloning glucose isomerase into 'routine' organisms is known, it has not yet been applied. Several of the glucose isomerase preparations used commercially consist of whole cells, or cell fragments, of the selected strains of species originally detected by screening.

The use of immobilised enzymes (see Chapter 3) is now familiar to industry and their advantages are well recognised so the practicality of using the new enzymes in an immobilised form will be determined early in the screening procedure. If the enzyme is produced intracellularly, the feasibility of using it without isolation and purification will be considered very seriously and strains selected for their amenability to use in this way.

It should be emphasised that there will be a constant dialogue between laboratory scientists and biochemical process engineers from the earliest stages of the screening process. Once the biochemical engineers are satisfied that their initial criteria of productivity, activity and stability can be met, the selected strain(s) of microbe will be grown in pilot plant conditions. It is only by applying the type of equipment used in full scale plants that accurate costing of processes can be achieved. Pilot studies will probably reveal imperfections, or at least areas of ignorance, that must be corrected at the laboratory scale. If this proves possible, the pilot plant will produce samples of the enzyme preparation to be used by customers who may well also be at the pilot plant stage in the development of the enzyme-utilizing process. The enzyme pilot plant also produces samples for safety and toxicological studies provided that the pilot process is exactly similar to the full scale operation.

Screening for new enzymes is expensive so that the intellectual property generated must be protected against copying by competitors. This is usually done by patenting the enzyme or its production method or, most usefully, the process in which it is to be used. Patenting will be initiated as soon as there is evidence that an innovative discovery has been made.