Author
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Ullah, Abul |
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Mullaney, Edward |
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Submitted to: Biochemical and Biophysical Research Communications
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 8/26/1996 Publication Date: N/A Citation: N/A Interpretive Summary: Soybean, cotton seed and other legume seeds contain phytic acid that is considered an antinutrient. Phytic acid binds essential minerals and proteins, thus interfering with the bioavailability of metals and proteins. One fungal enzyme that can degrade phytic acid is phytase. When soybean meals supplemented with phytase are fed to broilers and hogs, it results in better utilization of the meal. Phosphates that are liberated from the soybean meals are utilized by the poultry and hogs, as a result no additional phosphates are needed in the feed. Moreover, the manure contains less phosphate, resulting in less phosphate entering the environment. A heat tolerant phytase in needed for supplementation of the soybean meal. Unfortunately the native phytase in not stable at 70 deg C. But the enzyme can function at 58 deg C. The present study indicated that some biochemical feature called disulfide bonds gives strength to the structure of the enzyme. By recombinant DNA method, we can engineer additional disulfide bonds in phytase to make them more heat tolerant. If heat tolerant phytase were used in the diets of all poultry and hogs reared in the U.S. it would release phosphorus that has a value of $168 million and also precludes 82.3 million Kg of phosphates from entering the environment. Technical Abstract: The function of disulfide bonds in Aspergillus ficuum phytase was elucidated by unfolding studies, using guanidinium hydrochloride (Gu.HCl) as denaturant. Although the enzyme is totally inactivated by 0.8 M Gu.HCl, at pH 5.0, the active conformation is instantaneously restored by 0.6 M Gu.HCl, at pH 5.0. Conditions which would permit refolding of phytase are completely negated by 10 mM beta-mercaptoethanol and causes its catalytic demise at pH 7.5. Assay of free thiols using Ellman's reagent indicates that none of the thiols in the ten cysteines in phytase are free; five disulfide bonds were predicted for the enzyme. Sequence comparison of mold phytases and yeast acid phosphatases indicates four conserved cysteines. Thus, disulfide bonds play an important role in the folding of fungal phytase; any perturbation of the process of its formation causes an altered three-dimensional structure that is inconsistent with catalytic activity. |
