|Lei, Xin Gen|
Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/26/2007
Publication Date: 5/1/2007
Citation: Zhang, W., Mullaney, E.J., Lei, X. 2007. Adopting selected hydrogen bonding and ionic interactions from Aspergillus fumigatus phytase structure improves the thermostability of Aspergillus niger PhyA phytase. Applied and Environmental Microbiology. 73(9):3069-3076. Interpretive Summary: Fungal phytase is now a widely accepted feed additive for swine and poultry to lower phosphorus levels in their manure. Phytase, an enzyme, breaks down a compound, phytic acid, commonly found in soybean and other plants meals in this feed and transforms it into a form of phosphate animals can digest. However, in processing animal feed it is often necessary to heat the ingredients briefly to pelletize the mixture. This heating partially inactivates phytase and thus lowers its efficiency. To improve on the usefulness of this enzyme, its structure has been closely studied and compared with another phytase with higher heat tolerance, but lower overall activity. In this study certain essential components, amino acids, of the fungal phytase were identified that could be changed and produce a phytase with significant higher heat tolerance. By coupling a molecular modification technique, site-directed mutagenesis, and detailed molecular analysis of the phytase molecule, an improved phytase with both high activity and heat tolerance is now available for the animal feed industry. This new engineered phytase has a 20% increase in thermostability over the currently available phytase. The success of this approach supports further application of this technology for additional enhancement of phytase and other agricultural important enzymes.
Technical Abstract: Although it has been widely used as a feed supplement to reduce manure phosphorus pollution of swine and poultry, Aspergillus niger PhyA phytase is unable to withstand heat inactivation during feed pelleting. Crystal structure comparisons with its close homolog, the thermostable Aspergillus fumigatus phytase (Afp), suggest associations of thermostability with several key residues (E35, S42, R168, and R248) that form a hydrogen bond network in the E35-to-S42 region and ionic interactions between R168 and D161 and between R248 and D244. In this study, loss-of-function mutations (E35A, R168A, and R248A) were introduced singularly or in combination into seven mutants of Afp. All seven mutants displayed decreases in thermostability, with the highest loss (25% (P<0.05) in the triple mutant A(E35A R168A R248A). Subsequently, a set of corresponding substitutions were introduced into nine mutants of PhyA to strengthen the hydrogen bonding and ionic interactions. While four mutants showed improved thermostability, the best response came from the quadruple mutant (A58E P65S Q191R T271R), which retained 20% greater (P<0.05) activity after being heated at 80°C for 10 minutes and had a 7°C higher melting temperature than that of wild-type PhyA. This study demonstrates the functional importance of the hydrogen bond network and ionic interaction in supporting the high thermostability of Afp and the feasibility of adopting these structural units to improve the thermostability of a homologous PhyA phytase.