Enzyme-mediated bioremediation is an eco-friendly process for removing hazardous toxic heavy metals from the environment. The potential use of mutant alkaline phosphatase H412R for bioprecipitation of heavy metals such as Co2+, Cd2+, Cr6+, Ni2+, Mn2+ and Zn2+ from single-ion solutions and electroplating efuents was analysed in the present study. Purifed wild-type and H412R mutant alkaline phosphatase enzymes were incubated with an initial concentration of 100 ppm metal solutions for various time periods along with the substrate p-nitrophenol phosphate. Upon catalysis, the enzyme–substrate reaction liberates inorganic phosphate which in turn binds to heavy metals and precipitates them as metal-phosphates. The amount of metal ions precipitated as a result of formation of metal ion-phosphate complexes was determined by estimating the amount of free metal ions present in the solution using atomic absorption spectroscopy. Based on the results obtained, maximum bioprecipitation of metal ions, in general, was observed at 180 min of incubation period. The H412R mutant enzyme exhibited higher efciency and precipitated 96% of Mn2+ from electroplating efuent and 92% of Co2+ from the metal ion solution. The pattern of precipitation of various metal ions was in the order Co2+>Cr6+>Ni2+>Cd2+>Mn2+>Zn2+ for H412R mutant enzyme and Co2+>Cr6+>Zn2+>Ni2+>Cd2+>Mn2+ for wild-type enzyme. The results emphasise the use of novel H412R, a mutated alkaline phosphatase enzyme in its catalytic site, as an efcient way of achieving bioremediation of heavy metals from real-time efuents.

Introduction

Release of hazardous wastes into the environment as a result of tremendous increase in industrialisation and urbanisation is of major public health concern. Contamination of ground water occurs because of discharge of efuents from industries such as tannery and electroplating which are known to contain considerable amount of toxic elements causing toxicity in the ecosystem with consequences to health of humans and animals (Bai et al. 2008; Benazir et al. 2010). Investigations with animal models revealed severe damage to the kidney in mice when exposed to heavy metals at levels beyond the limits set by WHO (Wasana et al. 2017). Among various metals, the trivalent and hexavalent chromium is the metal of concern that has reached a hazardous level of 120 µg/L in the ground water which is alarmingly high (Kazakis et al. 2017). It has been reported that upon continuous exposure chromium could increase the risk of bladder cancer in humans (Wise et al. 2016), while cadmium, even when present in low concentration, accumulates in kidney eventually causing renal failure and cardiovascular diseases (Burke et al. 2016). On the other hand, co-contamination of groundwater with other heavy metals such as iron, cobalt and zinc was found to increase the toxicity of nickel causing severe eczema of skin (Sankhla et al. 2016). Heavy metals are known also to disrupt endocrine system when exposed to prolonged periods of time (Chiu et al. 2016). Because of long persistence of heavy metals and their associated health hazards several methods have come into existence for the treatment of efuents. Diverse techniques such as physical, chemical, phytoremediation (Huang et al. 2017; Sharma et al. 2016; Zheng et al. 2017), bioleaching (Xu et al. 2017) and nanoparticles (Yurekli 2016) were employed during wastewater treatment. Some of these methods involve membrane fltration, ion exchange, adsorption (Zhao et al. 2016; Zhang et al. 2016) and electro-chemical techniques such as electro-winning, electro-dialysis and electro-deionisation (Dermentzis et al. 2011; Xu et al. 2017). Most of these methods are often considered to be expensive, time-consuming and requiring utmost energy during the process. Further, bioremediation processes involving whole organisms face limitations with respect to the availability of metal interactive sites, metal toxicity, biosorption and bioaccumulation. The search for alternate methods which are resourceful, cost-efective, biological and environment-friendly has led to what is known as the feld of the green chemistry with emphasis to use of biocatalysts such as enzymes for bioremediation of pollutants (Tischer and Wedekind 1999; Alcalde et al. 2006). Enzymatic bioremediation has become an attractive alternative for the treatment of pollutants since enzymes provide simpler systems than using whole organisms. The added beneft of employing enzymes is that they remain relatively stable during the reactions and being proteins they are readily biodegradable (Ruggaber and Talley 2006).