Adopting Selected Hydrogen Bonding and Ionic Interactions fromAspergillus fumigatusPhytase Structure Improves the Thermostability ofAspergillus nigerPhyA Phytase

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 (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 min 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.Phytase catalyzes the hydrolysis of phytate (myo-inositol hexakisphosphate), the major storage form of phosphorus in plant seeds (20), to phosphate and myo-inositol. The enzyme has been effectively used as an animal feed supplement to improve the bioavailability of phytate phosphorus to simplestomached swine and poultry (4, 13). Because temperatures in the practical feed pelleting process can reach as high as 70 to 90°C (18), phytase enzymes with sufficiently high thermal stability are desirable but rare among the naturally occurring sources (14). Therefore, different strategies have been used to enhance the thermal stability of phytase (22).Aspergillus fumigatus phytase (Afp) (19) is a well-known heat-resilient phytase; it retains 90% of its initial activity after being heated at 100°C for 20 min (21). In comparison, Aspergillus niger PhyA phytase (30) displays much less heat resistance, despite a higher specific activity and a better pH profile than those of Afp (17,29,33,34). More intriguingly, these two phytases have 66% sequence homology and very similar overall crystal structures (9,15,35). Both enzymes contain a small ␣ domain and a large ␣/␤ domain. The small ␣ domain is composed of a long ␣ helix and seven short ␣ helices, and the large ␣/␤ domain contains a six-stranded ␤ sheet surrounded by two long ␣ helices at one side and several short ␣ helices at the other side. Nevertheless, detailed structure comparisons between these two enzymes indicate that three amino acid residues in Afp-E35, R168, and R248-may be critical...

Improving the Thermostability and Catalytic Efficiency of Bacillus deramificans Pullulanase by Site-Directed Mutagenesis

“…It is known that both hydrogen bonding and hydrophobic interactions play important roles in the thermal stability of proteins. Previously, a number of studies have shown that the introduction of these interactions into thermophilic proteins is an efficient approach for further enhancing their stability (37,38). In the present study, analysis of the structure models of the mutants also showed that new hydrogen bonds and/or hydrophobic interactions were introduced to the disorder loop of Phe490-Asp513 (Fig.…”

Expression, purification and characterization of a phyA m -phyCs fusion phytase

“…Thermostability is a particularly important issue since feed pelleting is commonly performed at temperatures between 65 °C and 95 °C. About 51% to 63% of the original activity of fusion phytase remained after incubation at 75 °C to 95 °C for 10 min without a protective agent, which was more thermostable than phyA m phytase (Fig.6c) and phyA mutant which retained 20% activity after being heated at 80 °C for 10 min (Zhang et al, 2007). The expressed phyA m , phyCs and fusion phyA m -phyCs phytases showed a broad and diffuse band in SDS-PAGE due to the heavy glycosylation (Fig.5) as described by Markus et al(1999) and Zou et al(2006).…”

“…All purification steps were carried out at 4 °C unless otherwise stated (Janne et al, 1998;Zou et al, 2006;Zhang et al, 2007). The culture supernatant obtained by centrifugation at 5000×g for 10 min was supplemented with CaCl 2 to a final concentration of 2 mmol/L.…”

Current Progresses in Phytase Research: Three‐Dimensional Structure and Protein Engineering

“…The A. fumigatus phytase (Afp) which shares high sequence identity to AnPhyA (sequence identity, 60.5 %) but exhibits exceptional heat resilience was chosen as a template for AnPhyA modification [26,49]. According to the sequence alignment of two Aspergillus phytases and analysis of the Afp 3D structure, four Afp-unique residues that involve in hydrogen bond and salt bridge formations were identified [50,51]. The AnPhyA mutant harboring the four residues in the corresponding positions displayed 20 % higher residual activity at 100°C [51].…”

“…According to the sequence alignment of two Aspergillus phytases and analysis of the Afp 3D structure, four Afp-unique residues that involve in hydrogen bond and salt bridge formations were identified [50,51]. The AnPhyA mutant harboring the four residues in the corresponding positions displayed 20 % higher residual activity at 100°C [51]. Notably, the mutant residues A35E and P42S are located within the fungal HAP unique N-terminal region and may stabilize the local structure through forming a hydrogen bond to each other (Fig.…”