Amylase is a digestive enzyme (present in saliva, for example) that breaks down long-chain carbohydrates (such as starch) as an initial step in the production of ethanol from grain starch.

Very broadly, in this type of production, corn or starchy grain is ground into flour (“meal”), which is then slurried with water to form a mash. Enzymes are added for the conversion of starch to sugar, the whole mash is processed in a high-temperature cooker and then transferred to fermenters where yeast is added and the conversion of sugar to ethanol and CO₂ begins.

The starch is usually heated at around 105°C or higher in the presence of thermostable alpha amylase, and then liquefied further at a lower temperature (around 90ºC). The high temperatures help reduce bacteria levels in the mash (high bacteria levels reduce yield).

Another key factor in the outcome of the process besides temperature is pH.

The pH is a measure of the acidity or alkalinity of a solution expressed on a scale of 1-14. Neutral is pH 7, pH 1-7 is acid, and pH 7-14 is alkaline. Yeast will grow only in a slightly acid solution (and the growth of harmful bacteria is further retarded by the slight acidity).

Conventional alpha-amylase enzymes, however, function best in a slightly alkaline environment.

As a result, ethanol producers spend time and material managing the pH of the mash to optimize the different stages of the process. And as a further result, producers have been seeking an enzyme that works well in a lower pH environment for years. As late as 2000, low pH-high temperature amylases were one of the top research requests from the industry to DOE and NREL.

The new Diversa/Valley research “Ultra-Thin” enzyme appears to meet that need. Capable of operating robustly at pH 4.5—the same pH of the production process—it reduces the cost associated with managing the mash pH.

To develop the enzyme, Innovase, a 50–50 venture between Diversa and Dow Chemical, used the process of directed evolution to screen DNA from a variety of unusual natural environments, including those with extremes of pH, temperature, and other physical parameters, for alpha-amylases with the desired characteristics. The DNA that Innovase screened was either obtained from available cultures of microorganisms (including some proprietary strains) that had been isolated from such environments, or obtained by screening the environments without an intervening step of actually isolating or cultivating the microorganisms.

From this DNA screening, Innovase selected three enzymes and then used a gene reassembly process to isolate a series of fragments from each of these three enzymes and then combine fragments to obtain the hybrid enzyme.

Resources:

• FDA GRAS (Generally Recognized as Safe) Response Letter, with description of the enzyme and process