Characterizing Isozymes of Chlorite Dismutase for Water Treatment

Abstract

This work investigated the potential for biocatalytic degradation of micropollutants, focusing on chlorine oxyanions as model contaminants, by mining biology to identify promising biocatalysts. Existing isozymes of chlorite dismutase (Cld) were characterized with respect to parameters relevant to this high volume, low-value product application: kinetic parameters, resistance to catalytic inactivation, and stability. Maximum reaction velocities (V_max) were typically on the order of 10⁴ μmol min^-1 (μmol heme)^-1. Substrate affinity (K_m) values were on the order of 100 μM, except for the Cld from Candidatus Nitrospira defluvii (NdCld), which showed a significantly lower affinity for chlorite. NdCld also had the highest susceptibility to catalytic inactivation. In contrast, the Cld from Ideonella dechloratans was least susceptible to catalytic inactivation, with a maximum turnover number of approximately 150,000, more than sevenfold higher than other tested isozymes. Under non-reactive conditions, Cld was quite stable, retaining over 50% of activity after 30 days, and most samples retained activity even after 90-100 days. Overall, Cld from I. dechloratans was the most promising candidate for environmental applications, having high affinity and activity, a relatively low propensity for catalytic inactivation, and excellent stability.

Keywords: biocatalyst; catalytic inactivation; chlorite; drinking water; perchlorate.

FIGURE 1

Maximum theoretical turnover number. Comparison of maximum theoretical turnover calculated via activity decay (A) and heme bleaching (B). The average of three biological replicates is shown for each isozyme, with error bars showing standard deviation. Horizontal half circles denote significant difference (p < 0.05) in mean as compared with rIdCld. Vertical half circles denote significant difference (p < 0.05) as compared to rNdCld. rNwCld was not measured by the heme bleaching method because of poor expression.

FIGURE 2

Shelf life of Cld. Stability of stored chlorite dismutase, referred to as shelf life, is presented for (A) soluble protein fraction containing rIdCld, (B) soluble protein fraction from A. oryzae PS, and (C) purified, heterologously expressed Cld from A. oryzae PS. Storage temperatures were 4 (open squares) and 22°C (solid circles). The average decay in activity from three biological replicates over time was normalized to day zero activity at the specified storage temperature. Error bars represent standard deviation.