Enzymes, specialized proteins that act as very specific catalysts to speed chemical reactions, have been used to make foods and beverages since ancient times. However, the modern era of industrial enzymes began in the 1890s when Jokichi Takamine patented a fungal amylase-an enzyme that breaks down starches-for producing potable alcohol.

Since then, enzymes have been shown to reduce costs through more efficient processing, the replacement of harsh or toxic chemicals, and the reduction of energy and water use. This realization has dramatically increased the use of enzymes for processing raw materials from plants and animals (see Table 1) and has led to new or improved products.



Enzymes that work under mild reaction conditions can certainly be a benefit to manufacturing, but what about industrial processes, like pulp and paper production or vegetable oil processing? These processes operate under extreme temperatures and pH levels that aren't compatible with typical enzyme functions.

Robust specialty enzymes optimized to perform under industrial process conditions are needed in these cases. Some products have already been commercialized that do just that. As an example, Diversa Corporation's trademarked Ultra-Thin alpha-amylase used for producing fuel ethanol from starch, can more effectively reduce viscosity of high dry-solid corn mashes, and performs effectively under the extreme temperatures and pH levels found in the dry-mill process.

Where are the Enzymes for Biofuels?
The alpha-amylase contained in Ultra-Thin is actually a chimera of three alpha-amylase proteins. The genes encoding the three parent alpha-amylase were recovered from samples containing hyperthermophilic bacteria, including samples harvested close to a hydrothermal vent or black smoker two miles beneath the Atlantic Ocean by the unmanned submersible Alvin. More than a dozen novel alpha-amylase genes were discovered, and the properties of the best three were combined using a proprietary technology to produce a robust alpha-amylase with an effective mode of action that functions from pH 4 to 6 at temperatures up to 107 degrees Celsius (224 degrees Fahrenheit). This enzyme enables great flexibility in use in the ethanol dry-mill.

Broad access to microbial diversity was utilized in a similar fashion to discover the active component of Diversa's trademarked Purifine product in a soil sample collected from Texas farmland. The Purifine enzyme product was designed to increase oil yield and reduce chemical usage when refining vegetable oils for biodiesel and food applications.

Oil Degumming for Biodiesel
Degumming is considered essential for purifying vegetable oils and producing a quality feedstock for biodiesel. Degumming removes contaminated phospholipids (phosphorus-containing lipids composed of diacylglycerol-a phosphate group-and a simple organic molecule) contained in crude vegetable oils and lowers the phosphorus content of the oil to less than 10 parts per million (ppm). The names and chemical structures of these phospholipids are shown in Figure 1.



In current degumming processes, the phospholipids are removed from crude oil by efficiently mixing a small volume of water with the oil. The majority of the phospholipids form a gummy mass that is removed by centrifugation. Phosphoric acid or citric acid is added to the oil to help remove residual phospholipids that aren't removed in water alone.

A New Approach to Enzymatic Degumming
The Purifine enzyme product provides a novel approach for removing oil phospholipids and also improves the efficiency of oil recovery without requiring major changes to conventional processing conditions.

The active component of the Purifine product is a phospholipase C (PLC) enzyme. Purifine enzymes operate in a low-water environment to quickly hydrolyze up to 70 percent of the phospholipids contained in crude vegetable oil and releases a product-1,2-diacylglycerol oil-that is much like the neutral oil or triacylglycerol that makes up the bulk of the oil. This product of the Purifine enzyme reaction remains in the oil, contributing to an increase in oil yield during subsequent refining. The other product of the Purifine enzyme reaction, the phosphorus-containing component, goes into the water (gum fraction) and is removed by centrifugation.

Phospholipases, and in particular phospholipase A enzymes, have been used in vegetable oil refining previously as processing aids. However, the novel aspect of the Purifine enzyme degumming reaction is the hydrolysis of the major phospholipids and the creation of a product that boosts oil yield. Since conventional degumming only removes intact phospholipids, which act as emulsifiers and entrain neutral oil in the gum, additional oil losses result. Because the Purifine enzyme reduces the concentration of these emulsifiers (Figure 3), oil yield is enhanced both by reducing entrainment of oil in the gum and by producing 1,2-diacylglycerol in the oil.



Optimal Degumming Conditions Boost Yield
Purifine enzymes are used to increase yield during the refining of many plant oils, including canola and rapeseed oils. However, Purifine enzymes provide the greatest benefits when used during the degumming of soybean oil because of the high levels of phospholipids present in soy oil that can be hydrolyzed by the enzyme. Crude soybean oil typically contains 1 percent to 2.4 percent phospholipids (400-1,000 ppm). Purifine enzymes hydrolyze up to 70 percent of the total phospholipids, resulting in an increase in total oil yield ranging from 0.90 percent (400 ppm crude) to 2.1 percent (1,000 ppm crude). These yield gains can be obtained with minimal process changes to current chemical refining, as shown in Figure 2. Following enzymatic hydrolysis of the major phospholipids, the oil may be further processed using the standard chemical refining process to remove the residual phospholipids and the free fatty acids.



Assuming an average 1.5 percent yield increase for a typical soybean oil processing facility operating at 500 metric tons of soybean oil per day, the use of Purifine enzymes can result in a total yield increase of over 2,500 metric tons per year (or approximately $1.4 million, assuming an oil price of $550 per metric ton). Minimal changes are needed to implement Purifine enzymes in a conventional refining process. In a typical soybean processing facility, a yield increase worth more than $1 million per year should be feasible.

Broad access to biodiversity combined with the use of powerful protein optimization techniques have led to the development of robust specialty enzymes that perform efficiently under the harsh conditions often present in many industrial processes. Specialty enzymes can improve the economics of traditional processes by increasing throughput, improving overall yield and reducing costly waste streams. In addition, continuing advances in our ability to tailor the specificity and performance of enzymes will lead to the development of new processes-for example the efficient and economical conversion of cellulosic biomass to liquid fuel and other valuable products.

Nelson Barton, PhD., is director of product development at Diversa Corporation. Reach him at (800) 523-2990 or nbarton@diversa.com.