The active site motif of proteins belonging to "Histidine Acid Phosphatase" (HAP) contains a hepta-peptide region, RHGXRXP. A close comparison among fungal and yeast HAPs revealed the fourth residue of the h...The active site motif of proteins belonging to "Histidine Acid Phosphatase" (HAP) contains a hepta-peptide region, RHGXRXP. A close comparison among fungal and yeast HAPs revealed the fourth residue of the hepta-peptide to be E instead of A, which is the case with A. niger PhyA phytase. However, another phytase, PhyB, from the same microorganism has a higher turnover number and it shows E in this position. We mutated A69 residue to E in the fungal PhyA phytase. The mutant phytase shows a myriad of new kinetic properties. The pH profile shifted 0.5 pH unit in both 5.0 and 2.5 bi-hump peaks. The optimum temperature shifted down from 58℃ to 55℃. However, the greatest difference was observed in the mutant protein's reaction to GuCl at a concentration of 0.1 to 0.2 M. The activity of the mutant phytase jumped 100% while the wild type protein showed no activity enhancement in the same concentration range of GuCl. The kinetics performed at higher concentration of GuCl also contrasted the difference between the wild type and mutant phytase. While Km was least affected, the Vmax increased for the mutant and decreased for the wild type. The sensitivity towards myo-inositol hexasulfate, a potent inhibitor, was decreased by the mutation. All in all, A69E mutation has affected a multitude of enzymatic properties of the protein even though the residue was thought to be non-critical for phytase's catalytic function notwithstanding its location in the conserved hepta-peptide region of the biocatalyst.展开更多
Despite yeast having its own native phytase, the high levels of phytate found in DDGS, a byproduct of ethanol (ETOH) fermentation, suggest that its activity is diminished in the presence of ETOH. Ethanol, a product of...Despite yeast having its own native phytase, the high levels of phytate found in DDGS, a byproduct of ethanol (ETOH) fermentation, suggest that its activity is diminished in the presence of ETOH. Ethanol, a product of grain fermentation, is known to inactivate several hydrolytic enzymes but its effect on phytases is relatively unknown. In this study, two phytases, Aspergillus niger (PhyA) and Escherichia coli (AppA2), were tested for ETOH tolerance. The E. coli phytase displayed greater ethanol tolerance over fungal phytase in the 5% to 10% range. However, ETOH inactivation was found to be reversible for both the enzymes. These differences in ETOH tolerance do suggest that there is a potential to achieve higher ETOH tolerance in phytases by 'structure-function' studies to lower phytic acid levels in DDGS and for other applications.展开更多
文摘The active site motif of proteins belonging to "Histidine Acid Phosphatase" (HAP) contains a hepta-peptide region, RHGXRXP. A close comparison among fungal and yeast HAPs revealed the fourth residue of the hepta-peptide to be E instead of A, which is the case with A. niger PhyA phytase. However, another phytase, PhyB, from the same microorganism has a higher turnover number and it shows E in this position. We mutated A69 residue to E in the fungal PhyA phytase. The mutant phytase shows a myriad of new kinetic properties. The pH profile shifted 0.5 pH unit in both 5.0 and 2.5 bi-hump peaks. The optimum temperature shifted down from 58℃ to 55℃. However, the greatest difference was observed in the mutant protein's reaction to GuCl at a concentration of 0.1 to 0.2 M. The activity of the mutant phytase jumped 100% while the wild type protein showed no activity enhancement in the same concentration range of GuCl. The kinetics performed at higher concentration of GuCl also contrasted the difference between the wild type and mutant phytase. While Km was least affected, the Vmax increased for the mutant and decreased for the wild type. The sensitivity towards myo-inositol hexasulfate, a potent inhibitor, was decreased by the mutation. All in all, A69E mutation has affected a multitude of enzymatic properties of the protein even though the residue was thought to be non-critical for phytase's catalytic function notwithstanding its location in the conserved hepta-peptide region of the biocatalyst.
文摘Despite yeast having its own native phytase, the high levels of phytate found in DDGS, a byproduct of ethanol (ETOH) fermentation, suggest that its activity is diminished in the presence of ETOH. Ethanol, a product of grain fermentation, is known to inactivate several hydrolytic enzymes but its effect on phytases is relatively unknown. In this study, two phytases, Aspergillus niger (PhyA) and Escherichia coli (AppA2), were tested for ETOH tolerance. The E. coli phytase displayed greater ethanol tolerance over fungal phytase in the 5% to 10% range. However, ETOH inactivation was found to be reversible for both the enzymes. These differences in ETOH tolerance do suggest that there is a potential to achieve higher ETOH tolerance in phytases by 'structure-function' studies to lower phytic acid levels in DDGS and for other applications.
