t edia ion & em B r i o o i d B e f g o r a Journal of l d a Makkar et al., J Bioremed Biodeg 2013, 4:3 a n r t i o u n o DOI: 10.4172/2155-6199.1000182 J ISSN: 2155-6199 Bior emediation & Biodegradation Research Article OpOpenen Access Access Enzyme-Mediated Bioremediation of Organophosphates Using Stable Yeast Biocatalysts Randhir S. Makkar1, Augustine A. DiNovo1, Caroline Westwater2 and David A. Schofield1* 1Guild Associates, Inc., Charleston, South Carolina, 29407, USA 2Department of Craniofacial Biology, Medical University of South Carolina, South Carolina, 29425, USA Abstract Organophosphates are extremely toxic compounds, which pose a threat to the environment and public due to their widespread use as common pesticides or due to their deliberate release as chemical weapons. The bacterial enzyme organophosphorus hydrolase (OPH, EC. 3.1.8.1) can hydrolyze, and thereby detoxify a broad range of organophosphate nerve agents. This enzyme therefore offers the opportunity for the development of naturally occurring newer bioremediation strategies. The aim of this research was to generate a stable yeast biocatalyst that was capable of hydrolyzing the poorly hydrolyzed P-S class of organophosphates. The genes encoding the wild-type OPH, or the enhanced variant enzyme S308L-OPH, were integrated into the ribosomal operon of the Saccharomyces cerevisiae genome to create a stable yeast biocatalyst. Recombinant yeast lysates expressed functional protein that hydrolyzed, and detoxified the pesticides malathion and demeton-S methyl. Recombinant yeast cells displayed the ability to function as biocatalysts and hydrolyze organophosphate pesticides and the warfare agent VX. Activity was associated with substrate detoxification since the cleaved substrate lost the ability to inhibit its target enzyme, acetylcholinesterase. Yeast biocatalytic activity was dependent on substrate concentration and assay temperature. The recombinant yeast offers a new bioremediation alternative for the removal and detoxification of organophosphates, especially for the poorly hydrolyzed and problematic P-S class of compounds such as malathion, demeton-S and VX. Keywords: Organophosphate hydrolysis; Detoxification; Enzyme can hydrolyze, and thereby reduce the toxicity of organophosphate mediated pesticides and nerve agents by cleaving the various phosphorus-ester bonds (P-O, P-CN, P-F, P-S). The enzyme mediated cleavage of these Introduction bonds, however, occurs with different efficiencies [5]. For example, of 2280 s-1, but Organophosphates are toxic man-made compounds that are OPH catalyzes the P-O bond of paraoxon with a kcat used as chemical warfare agents (nerve agents), and more commonly catalyses the P-S bonds of demeton-S, malathion, phosalone and acephate with k of 0.63-13.16 s-1, which is approximately 1000-fold as pesticides. Worldwide, five billion pounds of pesticides are used cats every year of which, organophosphate pesticides (mostly insecticides) slower. Therefore, while it is very efficient at bioremediating some constitute 20-38% of the total pesticides used; the most commonly organophosphates, the hydrolytic efficiency of OPH towards the P-S used organophosphate insecticide in the U.S. is malathion [1]. As a bond class of organophosphates such as VX is very poor. result, low levels of organophosphate contamination are commonly The goal of our research is to develop environmentally-friendly found in natural habitats, in the food chain, and in our water supplies. enzymes and biocatalysts for the bioremediation of organophosphates. The number of pesticide poisonings caused by inadvertent exposure The P-S class of organophosphates such as malathion, demeton-S, and has been estimated at between 1 and 3 million every year [2-4]. VX was the focus of this study because: (i) these organophosphates Organophosphate compounds exert their toxicity by inhibiting the pose a serious decontamination challenge since they are hydrolyzed enzyme acetylcholinesterase (AChE) which can cause a variety of very inefficiently by OPH; (ii) malathion is the most commonly used ailments, including respiratory failure and death. organophosphate insecticide, and (iii) VX is extremely toxic, and the Organophosphate exposure may also be caused by the accidental or most stable of the warfare nerve agents. Towards this goal, a variant deliberate release of chemical warfare nerve agents such as VX (O-ethyl OPH enzyme (termed S308L-OPH) was generated which displayed a S-[2-diisopropylaminoethyl] methylphosphonothiolate), sarin, soman 25-fold increase in hydrolytic efficiency against P-S organophosphates or tabun. The threat is compounded by the fact that some countries [6]. In this report, the Generally Regarded As Safe (GRAS) yeast have massive chemical weapon stockpiles. World stockpiles are Saccharomyces cerevisiae was utilized for the generation of a stable reported to exceed 200 kilotons, with U.S. reserves alone amounting to 30 kilotons. The Chemical Weapons Convention of 1997 required the *Corresponding author: David A Schofield, Guild Associates Inc., 1313B Ashley destruction of all chemical weapons by the year 2007; in the U.S., this River Road, Charleston, SC 29407, USA, Tel: +(843) 573 0095; Fax: +(843) 573 deadline has passed and is now extended until 2023. This extension has 0707; E-mail: [email protected] been brought about by delays in the development of disposal facilities, Received February 08, 2013; Accepted February 21, 2013; Published February mostly incinerators, and the environmentally regulatory, safety, and 23, 2013 security concerns associated with these facilities. The Pentagon has Citation: Makkar RS, DiNovo AA, Westwater C, Schofield DA (2013) Enzyme- estimated that the cost of destroying U.S. stockpiles at $32 billion. This Mediated Bioremediation of Organophosphates Using Stable Yeast Biocatalysts. J delay raises the risk of an accident or theft by terrorists. Consequently, Bioremed Biodeg 4: 182. doi:10.4172/2155-6199.1000182 the development of effective and environmentally-friendly methods to Copyright: © 2013 Makkar RS, et al. This is an open-a ccess article distributed dispose of these man-made toxic compounds would be of value. under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the The bacterial Organo Phosphorus Hydrolase (OPH) enzyme original author and source are credited. J Bioremed Biodeg ISSN: 2155-6199 JBRBD, an open access journal Volume 4 • Issue 3 • 1000182 Citation: Makkar RS, DiNovo AA, Westwater C, Schofield DA (2013) Enzyme-Mediated Bioremediation of Organophosphates Using Stable Yeast Biocatalysts. J Bioremed Biodeg 4: 182. doi:10.4172/2155-6199.1000182 Page 2 of 7 recombinant yeast biocatalyst expressing OPH or the S308L-OPH. The promoter function in vivo [9]. Therefore, the GAPDH promoter (653 biocatalyst was able to hydrolyze and thereby detoxify organophosphate bp) was generated by PCR using a high fidelity DNA polymerase and S. pesticides (malathion, demeton-S) and warfare agents (VX). cerevisiae genomic DNA as template. The 5’ and 3’ PCR primers were designed to incorporate PstI and HindIII sites, respectively (Table 3). Materials and Methods The resulting PCR product was cloned into the corresponding sites Microorganisms and propagation of pOPH-SK- and pS308L-OPH-SK-, thereby placing the GAPDH promoter upstream of the target genes. The Escherichia coli and S. cerevisiae strains used and generated in this study are described in table 1. E. coli was propagated in Luria The gene encoding neomycin resistance (NEOr) was used as the Bertani (LB) broth or LB agar at 37°C supplemented with 100 µg/mL marker for the selection of recombinant clones. A 916 bp fragment ampicillin when needed. S. cerevisiae was grown and maintained on 1% containing the NEOr gene and an abbreviated (120 bp) 5’ SV40 flanking yeast extract, 2% peptone, and 2% dextrose (YPD, Difco) broth or YPD promoter sequence was PCR-amplified using pEGFP-N1 (Clontech, agar at 30°C, where appropriate, YPD was supplemented with 200 µg/ Mountain View CA, USA) as template. The 5’ and 3’ PCR primers mL G418. contained SpeI and PstI restriction endonuclease sites for cloning Construction of the yeast Oph and S308l-Oph integration into the pGAPDH-OPH-SK- and pGAPDH-S308L-OPH-SK- vectors cassettes (Table 2). This placed the NEOr gene 5’ of the GAPDH promoter. An integration/expression cassette containing preferential yeast The integration/expression cassette was targeted to the non- expression signals and the necessary sequences to direct targeted transcribed spacer in the rDNA between the 5S and 18S subunits genome integration into the yeast rDNA unit was constructed. (located on chromosome XII) by homologous recombination. Two The wild-type gene encoding OPH, but lacking the signal peptide rDNA fragments comprising of 592 bp and 663 bp were PCR amplified sequence, was kindly provided by Mulbry and Karns [7]. The variant using S. cerevisiae genomic DNA as template, and primers R4F/R5R and S308L-OPH was generated from the wild-type gene and harbors the R6F/R7R, respectively (Table 3). The R4F/R5R and R6F/R7R primers following amino acid substitutions; A80V, I106V, F132D, K185R, were designed to contain SacI/SpeI and XhoI/KpnI sites respectively, D208G, H257W, I274N, S308L, R319S [6]. The wild-type and variant for cloning into the 5’ or 3’ ends of the integration cassette, thereby S308L-OPH, originally derived from the bacterium Flavobacterium providing flanking