Bacillus pumilus Cyanide Dihydratase Mutants with Higher Catalytic Activity.

Mary A Crum,Michael J Benedik,B Trevor Sewell

doi:10.3389/fmicb.2016.01264

Mary A Crum, Michael J Benedik + Show 1 more

Open Access

https://doi.org/10.3389/fmicb.2016.01264

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Abstract

Cyanide degrading nitrilases are noted for their potential to detoxify industrial wastewater contaminated with cyanide. However, such application would benefit from an improvement to characteristics such as their catalytic activity and stability. Following error-prone PCR for random mutagenesis, several cyanide dihydratase mutants from Bacillus pumilus were isolated based on improved catalysis. Four point mutations, K93R, D172N, A202T, and E327K were characterized and their effects on kinetics, thermostability and pH tolerance were studied. K93R and D172N increased the enzyme’s thermostability whereas E327K mutation had a less pronounced effect on stability. The D172N mutation also increased the affinity of the enzyme for its substrate at pH 7.7 but lowered its kcat. However, the A202T mutation, located in the dimerization or the A surface, destabilized the protein and abolished its activity. No significant effect on activity at alkaline pH was observed for any of the purified mutants. These mutations help confirm the model of CynD and are discussed in the context of the protein–protein interfaces leading to the protein quaternary structure.

Highlights

Cyanide has been used for decades by the mining industry (La Brooy et al, 1994) owing to its high affinity for metals; cyanide binds and extracts metal ions from the ore by carrying them into solution
Using random mutagenesis followed by in vivo activity screening, we identified three B. pumilus Cyanide dihydratases (CynDs) mutants that converted cyanide at pH 7.7 at a higher rate than E. coli expressing the wild-type enzyme
Thermostability and pH activity profiles were used to characterize the effect of these mutations on enzyme activity and stability

Summary

Introduction

Cyanide has been used for decades by the mining industry (La Brooy et al, 1994) owing to its high affinity for metals; cyanide binds and extracts metal ions from the ore by carrying them into solution. Traditional decontamination methods involve oxidation of cyanide via hydrogen peroxide, chlorine, or sulfur dioxide (Knorre et al, 1984; Devuyst et al, 1989). Even though these are successful in lowering the concentration of cyanide, they have several disadvantages, notably the cost of reagents, the special handling needed, and the production of adverse byproducts. These limitations lead to significant interest in the potential for the biodegradation of cyanide using microbial or enzymatic methods. One significant success in this respect has been the biological treatment of cyanide solution in the mining environment, using rotating biological vessels (Baxter and Cummings, 2006) incorporating a microbial consortium for cyanide transformation

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