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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

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

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Author(s):
Goncalves, Larissa M. [1] ; Chaimovich, Hernan [1] ; Cuccovia, Iolanda M. [1] ; Marana, Sandro R. [1]
Total Authors: 4
Affiliation:
[1] Univ Sao Paulo, Dept Bioquim, Inst Quim, BR-05513970 Sao Paulo - Brazil
Total Affiliations: 1
Document type: Journal article
Source: PROTEIN AND PEPTIDE LETTERS; v. 21, n. 5, p. 468-475, MAY 2014.
Web of Science Citations: 1
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. (AU)

FAPESP's process: 08/55914-9 - Development of beta-glycosidases designed to improve the efficiency of noncomplexed cellulase systems
Grantee:Sandro Roberto Marana
Support Opportunities: Program for Research on Bioenergy (BIOEN) - Thematic Grants