Chimeric Proteins Combining Phosphatase and Cellulose-Binding Activities: Proof-of-Concept and Application in the Hydrolysis of Paraoxon

Title:Chimeric Proteins Combining Phosphatase and Cellulose-Binding Activities: Proof-of-Concept and Application in the Hydrolysis of Paraoxon

Volume: 21 Issue: 5

Author(s): Larissa M. Goncalves, Hernan Chaimovich, Iolanda M. Cuccovia and Sandro R. Marana

Affiliation:

Keywords: Carbohydrate-binding domain, chimeric protein, phosphatase, paraoxonase, organophosphate.

Abstract: Phosphatases for organophosphate degradation and carbohydrate-binding domains (CBMs) have potential biotechnological applications. As a proof-of-concept, a soluble chimeric protein that combines acid phosphatase (AppA) from Escherichia coli and a CBM from Xanthomonas axonopodis pv. citri (AppA-CBM) was produced in E.coli. AppACBM adsorbed in microcrystalline cellulose Avicel PH101 catalyzed the hydrolysis of p-nitrophenyl phosphate (PNPP). The binding to microcrystalline cellulose displayed saturation behavior with an apparent binding constant (K_b) of 22 ± 5 mg and a maximum binding (B_max) of 1.500 ± 0.001 enzyme units. Binding was highest at pH 2.5 and decreased above pH 6.5, as previously observed for family 2 CBMs. The K_m values for PNPP of AppA-CBM and native AppA were identical (2.7 mM). To demonstrate that this strategy for protein engineering has practical applications and is largely functional, even for phosphatases exhibiting diverse folds, a chimeric protein combining human paraoxonase 1 (hPON1) and the CBM was produced. Both PON1-CBM and hPON1 had identical K_m values for paraoxon (1.3 mM). Additionally, hPON1 bound to microcrystalline cellulose with a K_b of 27 ± 3 mg, the same as that observed for AppA-CBM. These data show that the phosphatase domains are as functional in both of the chimeric proteins as they are in the native enzymes and that the CBM domain maintains the same cellulose affinity. Therefore, the engineering of chimeric proteins combining domains of phosphatases and CBMs is fully feasible, resulting in chimeric enzymes that exhibit potential for OP detoxification.

Cite this article as:

Goncalves M. Larissa, Chaimovich Hernan, Cuccovia M. Iolanda and Marana R. Sandro, Chimeric Proteins Combining Phosphatase and Cellulose-Binding Activities: Proof-of-Concept and Application in the Hydrolysis of Paraoxon, Protein & Peptide Letters 2014; 21 (5) . https://dx.doi.org/10.2174/092986652105140218114537