Chimeric Proteins Combining Phosphatase and Cellulose-Binding Activities: Proof-of-Concept and Application in the Hydrolysis of Paraoxon
Larissa M. Goncalves, Hernan Chaimovich, Iolanda M. Cuccovia and Sandro R. Marana
Affiliation: Departamento de Bioquimica, Instituto de Quimica, Universidade de Sao Paulo, CP 26077, Sao Paulo, 05513-970, SP, Brasil.
Keywords: Carbohydrate-binding domain, chimeric protein, phosphatase, paraoxonase, organophosphate.
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 (Kb) of 22 ± 5
mg and a maximum binding (Bmax) 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 Km 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 Km values for paraoxon (1.3 mM). Additionally, hPON1
bound to microcrystalline cellulose with a Kb 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.
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