Expression and structural investigation of acid tolerant arsenite oxidase with biosensor potential

Abstract

Groundwater contamination by the two dominant toxic arsenic species, arsenite (As(III)) and arsenate (As(V)) has caused a global epidemic of arsenic poisoning effecting over 100 million people. Currently there is no commercially available As(III) biosensor to specifically detect this most toxic inorganic compound. Arsenite oxidase (Aio) catalyzes the oxidation of As(III) to As(V) by two electron transfer. As a redox enzyme, Aio is potentially suitable for construction of enzyme based electrochemical biosensor to detect As(III) specifically. A moderate acidophilic arsenite oxidizer, Thiomonas delicata DSM 16361 was used as source of Aio in this study. To obtain Aio sufficient for biosensor construction, it was first necessary to optimize the expression and purification of recombinant Aio in E. coli strain. Full length aioBA gene was isolated and deposited to GenBank under accession number KX792110. Expression of the recombinant Aio was successfully performed in ZYM-5052 autoinduction medium at 20 �C for 48 hours in E. coli strain C43(DE3). The Aio was purified to homogeneity with purity > 90% and characterized. The purified Aio was found to be heterodimeric with subunits of 91 and 21 kDa (17 kDa without signal peptide) in size, respectively. Specific activity of purified Aio was 4 U/mg, with substrate Km of 14 �M. The temperature-activity profile of purified Aio was found optimum at 55 �C and Aio retained nearly 45% of its initial activity after pre-incubation at 60 �C for 1 hour. The enzyme was stable in acidic pH ranging from pH 2.5 to 6. Activity of Aio was retained in the presence of 10mMmetal ions (K+, Li+, Co2+, Ca2+, Ni2+, Mn2+, and Zn2+) and anions (NO2??, SO2?? 4 , and Cl??). In order to elucidate the acidophilic adaptation of Aio, homology model of Aio was constructed using X-ray crystallized structure of Aio from Alcaligenes faecalis (PDB ID: 1G8K) owing to the high sequence identity of 64%. The homology model was compared to the two structures from alkaliphilic sources, A. faecalis and Rhizobium sp. NT-26. Notable difference between the structures and model was observed on the surface of the enzyme as well as the existence of a unique loop region in T. delicata Aio. Several structural features that might be implicated in the acidophilic adaptation of T. delicata Aio were: (a) a higher proline content in Aio, (b) positively charged surface protein, (c) a decreased number of salt bridges and hydrogen bonds, and (d) introduction of polar and charged residues distal to catalytic site. When Aio was incorporated in biosensor construction, the DCPIP/Aio electrochemical assay was capable of detecting 10 to 500 ppb As(III) by using carbon screen printed electrode. This revealed the potential of Aio as a biosensing material for determining safe level of As(III) in water systems. This is the first characterization study of acid tolerant Aio from T. delicata DSM 16361 with biosensor potential.