Screening and Improving the Recombinant Nitrilases and Application in Biotransformation of Iminodiacetonitrile to Iminodiacetic Acid

In this study, several nitrilase genes from phylogenetically distinct organisms were expressed and purified in E. coli in order to study their ability to mediate the biotransformation of nitriles. We identified three nitrilases: Acidovorax facilis nitrilase (AcN); Alcaligenes fecalis nitrilase (AkN)...

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Bibliographic Details
Published in:PloS one 2013-06, Vol.8 (6), p.e67197
Main Authors: Liu, Zhi-Qiang, Baker, Peter James, Cheng, Feng, Xue, Ya-Ping, Zheng, Yu-Guo, Shen, Yin-Chu
Format: Article
Language:English
Subjects:
Acetonitriles - metabolism
Acids
Alcaligenes
Alcaligenes faecalis - enzymology
Aminohydrolases - chemistry
Aminohydrolases - genetics
Aminohydrolases - isolation & purification
Aminohydrolases - metabolism
Bacterial Proteins - chemistry
Bacterial Proteins - genetics
Bacterial Proteins - isolation & purification
Bacterial Proteins - metabolism
Bioengineering
Biology
Biotransformation
Catalytic converters
Chemical reactions
Chemistry
Chromatography, High Pressure Liquid
Circular Dichroism
Cloning
Comamonadaceae - enzymology
Cyanide
Deoxyribonucleic acid
DNA
E coli
Environmental impact
Enzymes
Escherichia coli
Genes
Genetic engineering
Homology
Hydrolysis
Imino Acids - metabolism
Iminodiacetic acid
Kinetics
Medical screening
Molecular Docking Simulation
Mutagenesis
Mutants
Mutation
Mutation hot spots
Nitrilase
Nitriles
Phylogeny
Protein Structure, Secondary
Proteins
Recombinant Proteins - chemistry
Recombinant Proteins - isolation & purification
Recombinant Proteins - metabolism
Rhodococcus
Rhodococcus - enzymology
Rhodococcus rhodochrous
Sequence Alignment
Sequence Homology, Amino Acid
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Summary:In this study, several nitrilase genes from phylogenetically distinct organisms were expressed and purified in E. coli in order to study their ability to mediate the biotransformation of nitriles. We identified three nitrilases: Acidovorax facilis nitrilase (AcN); Alcaligenes fecalis nitrilase (AkN); and Rhodococcus rhodochrous nitrilase (RkN), which catalyzed iminodiacetonitrile (IDAN) to iminodiacetic acid (IDA). AcN demonstrated 8.8-fold higher activity for IDAN degradation as compared to AkN and RkN. Based on homology modeling and previously described 'hot spot' mutations, several AcN mutants were screened for improved activity. One mutant M3 (F168V/L201N/S192F) was identified, which demonstrates a 41% enhancement in the conversion as well as a 2.4-fold higher catalytic efficiency towards IDAN as compared to wild-type AcN.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0067197