Submitted to: Advances in Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 12, 2012
Publication Date: November 30, 2012
Citation: Mullaney, E., Sethumadhavan, K., Boone, S., Lei, X.G., Ullah, A.H.J. 2012. Elimination of a disulfide bridge in Aspergillus niger NRRL 3135 Phytase (PhyA) enhances heat tolerance and optimizes its temperature versus activity profile. Advances in Biological Chemistry. 2(4):372-378. Interpretive Summary: Today, much of the animal feed produced for swine, poultry and other animals with simple stomachs contain grains and other plant material rich in phytate. Phytate is common in plants and is high in phosphorus, which is an essential nutrient. However these animals lack an enzyme in their digestive tract, a phytase, that would allow them to break the phytate down and to access the phosphorus bound up in phytate. To overcome this problem, a phytase from a mold is produced and marketed as a feed additive. It allows the animals to use the phytin phosphorus and also as a bonus lowers the phosphorus levels in their manure to protect the environment. The enzyme has been widely accepted and attempts are now underway to improve on the enzyme and to make it better. In this study, molecular technology was used to modify the gene for this enzyme by removing one structural feature of the enzyme. This made the structure of the enzyme less rigid and when this new version of phytase was tested it was shown to have an optimum temperature nearer the body temperature of the animal and to have more resistance to high temperature. Both of these are important. The lower optimum temperature means the phytase works more efficiently at the body temperature of animals and the high heat resistance allows more of the enzyme activity to survive the brief period of exposure to elevated temperatures, which is often necessary during the manufacturing of the animal feed. These findings have potentially broad applications to be incorporated along with other desirable features to engineer a phytase with superior attributes for the animal feed applications.
Technical Abstract: The utilization of microbial phytases in animal feed, rich in phytate, and intended for animals with simple stomachs is now widely accepted. The commercial phytases currently available are all histidine acid phosphatases (HAP) and have been termed histidine acid phytases (HAPhy). The HAPhy enables swine, poultry, and other monogastric animals to hydrolyze phytate and thus utilizes the phytin phosphorus for nutrition and also lowers the phosphate level of manure. The optimum temperature for the wild type HAPhys is substantially higher than the body temperature of all these animals and thus the activity of the enzyme is not at its optimum. A significant activity loss also occurs when the enzyme is subjected to elevated temperatures for a brief period, which is necessary for the processing of the animal feed. In this study the optimum temperature was lowered while the residual phytase activity after heating to 70°C was raised in a widely utilized phytase, Aspergillus niger NRRL 3135 PhyA. This was accomplished by site-directed mutagenesis of the cysteines that are involved in the formation of a single disulfide bridge (DB). When compared to wild type (WT), three of the four mutant phytases displayed a lower optimum temperature, 42°C, and up to a four-fold increase in activity after heating. These findings have a potentially broad application to be incorporated along with other desirable features to engineer a phytase with superior attributes for animal feed applications.