Bacterial Cell Surface Display of Organophosphorus Hydrolase for Selective Screening of Improved Hydrolysis of Organophosphate Nerve Agents

ABSTRACT Organophosphorus hydrolase (OPH) is a bacterial enzyme that has been shown to degrade a wide range of neurotoxic organophosphate nerve agents. However, the effectiveness of degradation varies dramatically, ranging from highly efficient with paraoxon to relatively slow with methyl parathion. Sequential cycles of DNA shuffling and screening were used to fine-tune and enhance the activity of OPH towards poorly degraded substrates. Because of the inaccessibility of these pesticides across the cell membrane, OPH variants were displayed on the surface of Escherichia coli using the truncated ice nucleation protein in order to isolate novel enzymes with truly improved substrate specificities. A solid-phase top agar method based on the detection of the yellow product p-nitrophenol was developed for the rapid prescreening of potential variants with improved hydrolysis of methyl parathion. Two rounds of DNA shuffling and screening were carried out, and several improved variants were isolated. One variant in particular, 22A11, hydrolyzes methyl parathion 25-fold faster than does the wild type. Because of the success that we achieved with directed evolution of OPH for improved hydrolysis of methyl parathion, we believe that we can easily extend this method in creating other OPH variants with improved activity against poorly degraded pesticides such as diazinon and chlorpyrifos and nerve agents such as sarin and soman.

[1]  Hydrolysis of organophosphate insecticides by an immobilized‐enzyme system , 1979, Biotechnology and bioengineering.

[2]  C. Serdar,et al.  Enzymatic Hydrolysis of Organophosphates: Cloning and Expression of a Parathion Hydrolase Gene from Pseudomonas diminuta , 1985, Bio/Technology.

[3]  W. Mulbry,et al.  Identification of a plasmid-borne parathion hydrolase gene from Flavobacterium sp. by southern hybridization with opd from Pseudomonas diminuta , 1986, Applied and environmental microbiology.

[4]  D. Goeddel,et al.  A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction , 1989 .

[5]  F. Raushel,et al.  Purification and properties of the phosphotriesterase from Pseudomonas diminuta. , 1989, The Journal of biological chemistry.

[6]  A. Sharma,et al.  Degradation of methyl parathion by a mixed bacterial culture and a Bacillus sp. isolated from different soils , 1989 .

[7]  M. Jobling,et al.  Construction of vectors with the p15a replicon, kanamycin resistance, inducible lacZ alpha and pUC18 or pUC19 multiple cloning sites. , 1990, Nucleic acids research.

[8]  F. Raushel,et al.  Inactivation of organophosphorus nerve agents by the phosphotriesterase from Pseudomonas diminuta. , 1990, Archives of biochemistry and biophysics.

[9]  G. Chaudhry,et al.  Microbiological and biotechnological aspects of metabolism of carbamates and organophosphates. , 1992, Critical reviews in biotechnology.

[10]  W. Stemmer.  DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[11]  K. Lai,et al.  Bimetallic binding motifs in organophosphorus hydrolase are important for catalysis and structural organization. , 1994, The Journal of biological chemistry.

[12]  F. Raushel,et al.  Three-dimensional structure of the binuclear metal center of phosphotriesterase. , 1995, Biochemistry.

[13]  S. Harvey,et al.  Rational Enzyme Design: Computer Modeling and Site-directed Mutagenesis for the Modification of Catalytic Specificity in Organophosphorus Hydrolase , 1996, CHIMIA.

[14]  F. Raushel,et al.  Metal-substrate interactions facilitate the catalytic activity of the bacterial phosphotriesterase. , 1996, Biochemistry.

[15]  F. Raushel,et al.  Three-dimensional structure of the zinc-containing phosphotriesterase with the bound substrate analog diethyl 4-methylbenzylphosphonate. , 1996, Biochemistry.

[16]  Ashok Mulchandani,et al.  Biodegradation of organophosphorus pesticides by surface-expressed organophosphorus hydrolase , 1997, Nature Biotechnology.

[17]  F. Raushel,et al.  Augmented Hydrolysis of Diisopropyl Fluorophosphate in Engineered Mutants of Phosphotriesterase* , 1997, The Journal of Biological Chemistry.

[18]  F. Raushel,et al.  Stereochemical constraints on the substrate specificity of phosphotriesterase. , 1999, Biochemistry.

[19]  K. Lai,et al.  Rational design of organophosphorus hydrolase for altered substrate specificities. , 1999, Chemico-biological interactions.

[20]  F. Raushel,et al.  Rationally Engineered Mutants of Phosphotriesterase for Preparative Scale Isolation of Chiral Organophosphates , 2000 .

[21]  F. Raushel,et al.  The Binding of Substrate Analogs to Phosphotriesterase* , 2000, The Journal of Biological Chemistry.

[22]  F. Arnold,et al.  Designed evolution of enzymatic properties. , 2000, Current opinion in biotechnology.

[23]  A Mulchandani,et al.  Cell Surface Display of Organophosphorus Hydrolase Using Ice Nucleation Protein , 2001, Biotechnology progress.