Large quantities of glucose-containing syrups are manufactured by the enzymatic hydrolysis of corn starch. The first step of this process is usually accomplished by treating a starch-water mixture with an alpha-amylase enzyme at temperatures near the boiling point of water. A thermostable alpha-amylase is most efficient at these temperatures. For this reason, there is a great incentive to obtain a low-cost source of such a thermostable enzyme.
The alpha-amylases used commercially are produced by various microorganisms. It is known that these microorganisms contain genetic material which codes for the production of the enzymes by the organism. This genetic material is present in the form of deoxyribonucleic acid, hereafter referred to as DNA, within the cell.
Most genetic material in a bacterium exists as giant DNA molecules which are present as the chromosome of the cell. However, a certain amount of the genetic material may also be present in the form of smaller closed circular molecules of DNA, known as plasmids.
By techniques referred to as genetic engineering, it is possible to transfer a portion of the DNA from one organism to another. Various workers have attempted to use these techniques to develop microorganisms which are superior alpha-amylase producers.
One technique that has been used is to remove the total DNA, i.e., chromosomal plus plasmid DNA, from a microorganism known to produce an amylase. Fragments of this DNA are then linked with the DNA of a bacteriophage. The bacteriophage containing this combined DNA is used to infect another microorganism whereby it transfers this genetic material onto the chromosomal DNA of the microorganism. The cells of the infected organism that receive DNA containing an amylase gene in an active form become amylase producers. These cells can be selected and grown for further use.
An additional genetic engineering technique that has been used with thermostable alpha-amylases involves extracting the total DNA from a microorganism known to produce such amylases. Some fragments of the DNA are transformed into a new host microorganism whereby they are incorporated into the chromosome of the host microorganism. Any cells into which the DNA containing active amylase-coding genetic material has been introduced are then selected and grown.
It has now been found that DNA coding for a thermostable alpha-amylase can exist in a naturally-occurring plasmid. Furthermore, it has been found that such naturally-occurring genetic material containing a thermostable alpha-amylase coding gene can be combined directly with another plasmid, known as a plasmid vector, to give a synthetic plasmid, hereafter called a chimeric plasmid. This chimeric plasmid can be inserted into and multiplied in a new host microorganism. This permits the development of microorganisms that are superior alpha-amylase producers which can be grown readily on a commercial scale. It is to the use of the newly-discovered, naturally-occurring plasmids containing alpha-amylase genes, the formation of chimeric plasmids containing their genetic material, and the development of new microorganisms containing these chimeric plasmids, that the present invention is directed.