Enzyme Technology
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 (Km,
Vmax), 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.
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This page was established in 2004 and last updated by Martin
Chaplin on
6 August, 2014
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