Little is known about the substrate specificities of most of the enzymes as compared with common reagents used in organic synthesis. The velocity of the enzymatic reactions is much affected by the neighboring substituents: it cannot be predicted whether an unnatural chemical can be a substrate or not with most of the enzymes. Therefore, we screen microorganisms for an enzyme when (i) no information is available for the desirable reaction, (ii) nothing is known about the specific reaction but a homologous reaction is known to occur, (iii) an enzyme is known only in another biological source, (iv) insufficient amount of the enzyme is produced by a certain organism for a practical transformation, (v) increased stability of the enzyme for practical use is required, etc.
Enrichment culture is a technique to isolate microorganisms having special growth characteristics.3 Some microorganisms grow faster than others in media with limited nutrients, high temperature, extreme pH values, etc. Microorganisms that grow faster than other species become dominant after several transfers of the culture. The isolation method utilizing the difference of growth velocity gives us only limited group of microorganisms, since only a few kinds of fast-growing microorganisms usually appear on the plate containing a starting material in the successive transfer of microorganisms in the enrichment culture. The isolates are classified according to their biochemical and morphological characteristics. At the same time, microorganisms deposited among the culture collections are tested for the activity, dependent on or independent of the isolation experiments. During such studies, there may be unexpected findings and discovery.
An acclimation technique3 is applied when a toxic or unnatural compound is used as a substrate and usually run long term to isolate microorganisms which are not easily isolated by the enrichment culture. An adaptation to a synthetic medium containing a target compound sometimes results in isolation of microorganisms having a desirable new enzyme. Genetic changes in the microorganisms may be expected. Since fast-growing microorganisms do not always possess the desired enzyme activity and suppress the growth of other microorganisms, one may look for microorganisms growing rather slowly on certain compounds by methods such as spreading diluted soil sample directly to the medium allowing an isolation of microorganisms having an ability to convert the compound.
If the product is located in the downstream of the starting material in the metabolism, enzymes converting such compounds could be screened. In some cases, a new metabolic pathway of the compound may be discovered during the screening. Many industrial microorganisms have been isolated in such ways. However, when one wants to synthesize compounds located at the end of anabolism, such as polysaccharides, whose synthesis requires multistep enzyme reactions and energy, the strategy cannot be applied, since a compound with higher energy could be synthesized by coupling of an energy-yielding reaction. Oxidoreductases utilizing NAD+ or NADP+ as cofactors are also useful in the oxidation or reduction reactions because of their reversible nature.
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