Engineering the residual side chains of HAP phytases to improve their pepsin resistance and catalytic efficiency

Canfang Niu,Huoqing Huang,Peilong Yang,Yaru Wang,Huiying Luo,Bin Yao

doi:10.1038/srep42133

Canfang Niu, Huoqing Huang + Show 4 more

Open Access

https://doi.org/10.1038/srep42133

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Abstract

Strong resistance to proteolytic attack is important for feed enzymes. Here, we selected three predicted pepsin cleavage sites, L99, L162, and E230 (numbering from the initiator M of premature proteins), in pepsin-sensitive HAP phytases YkAPPA from Yersinia kristensenii and YeAPPA from Y. enterocolitica, which corresponded to L99, V162, and D230 in pepsin-resistant YrAPPA from Y. rohdei. We constructed mutants with different side chain structures at these sites using site-directed mutagenesis and produced all enzymes in Escherichia coli for catalytic and biochemical characterization. The substitutions E230G/A/P/R/S/T/D, L162G/A/V, L99A, L99A/L162G, and L99A/L162G/E230G improved the pepsin resistance. Moreover, E230G/A and L162G/V conferred enhanced pepsin resistance on YkAPPA and YeAPPA, increased their catalytic efficiency 1.3–2.4-fold, improved their stability at 60 °C and pH 1.0–2.0 and alleviated inhibition by metal ions. In addition, E230G increased the ability of YkAPPA and YeAPPA to hydrolyze phytate from corn meal at a high pepsin concentration and low pH, which indicated that optimization of the pepsin cleavage site side chains may enhance the pepsin resistance, improve the stability at acidic pH, and increase the catalytic activity. This study proposes an efficient approach to improve enzyme performance in monogastric animals fed feed with a high phytate content.

Highlights

Strong resistance to proteolytic attack is important for feed enzymes
Phytases are grouped into four major classes, histidine acid phosphatase (HAP), cysteine phosphatase (CP), purple acid phosphatase (PAP), and β-propeller phytase (BP), based on their catalytic characteristics[12,13]
Modeled structures of Yersinia phytases (Fig. 1) showed three theoretical pepsin cleavage sites: L162 and E230 in YeAPPA and YkAPPA, corresponding to V and D in YrAPPA, respectively, and L99 was conserved in the three phytases

Summary

Introduction

Strong resistance to proteolytic attack is important for feed enzymes. Here, we selected three predicted pepsin cleavage sites, L99, L162, and E230 (numbering from the initiator M of premature proteins), in pepsin-sensitive HAP phytases YkAPPA from Yersinia kristensenii and YeAPPA from Y. enterocolitica, which corresponded to L99, V162, and D230 in pepsin-resistant YrAPPA from Y. rohdei. E230G/A and L162G/V conferred enhanced pepsin resistance on YkAPPA and YeAPPA, increased their catalytic efficiency 1.3–2.4-fold, improved their stability at 60 °C and pH 1.0–2.0 and alleviated inhibition by metal ions. E230G increased the ability of YkAPPA and YeAPPA to hydrolyze phytate from corn meal at a high pepsin concentration and low pH, which indicated that optimization of the pepsin cleavage site side chains may enhance the pepsin resistance, improve the stability at acidic pH, and increase the catalytic activity. The optimal activity is usually between pH 1.3–5.518,19 and 45–70 °C19–21, the substrate specificity is diverse[22] and HAP family enzymes display different tolerance to acidity, heat, and proteolytic digestion[23,24,25,26]. The proteinase K cleavage site of bovine www.nature.com/scientificreports/

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