Identification and characterization of a novel nitrilase from Pseudomonas fluorescens Pf-5

Jung-Soo Kim, Manish Kumar Tiwari, Hee-Jung Moon, Marimuthu Jeya, Thangadurai Ramu, Deok-Kun Oh, In-Won Kim, Jung-Kul Lee

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Nitrile groups are catabolized to the corresponding acid and ammonia through one-step reaction involving a nitrilase. Here, we report the use of bioinformatic and biochemical tools to identify and characterize the nitrilase (NitPf5) from Pseudomonas fluorescens Pf-5. The nitPf5 gene was identified via sequence analysis of the whole genome of P. fluorescens Pf-5 and subsequently cloned and overexpressed in Escherichia coli. DNA sequence analysis revealed an open-reading frame of 921 bp, capable of encoding a polypeptide of 307 amino acids residues with a calculated isoelectric point of pH 5.4. The enzyme had an optimal pH and temperature of 7.0A degrees C and 45A degrees C, respectively, with a specific activity of 1.7 and 1.9 mu mol min(-1) mg protein(-1) for succinonitrile and fumaronitrile, respectively. The molecular weight of the nitrilase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography was 33,000 and 138,000 Da, respectively, suggesting that the enzyme is homotetrameric. Among various nitriles, dinitriles were the preferred substrate of NitPf5 with a K (m) = 17.9 mM and k (cat)/K (m) = 0.5 mM(-1) s(-1) for succinonitrile. Homology modeling and docking studies of dinitrile and mononitrile substrate into the active site of NitPf5 shed light on the substrate specificity of NitPf5. Although nitrilases have been characterized from several other sources, P. fluorescens Pf-5 nitrilase NitPf5 is distinguished from other nitrilases by its high specific activity toward dinitriles, which make P. fluorescens NitPf5 useful for industrial applications, including enzymatic synthesis of various cyanocarboxylic acids.
Original languageEnglish
JournalApplied Microbiology and Biotechnology
Volume83
Issue number2
Pages (from-to)273-283
Number of pages11
ISSN0175-7598
DOIs
Publication statusPublished - 2009
Externally publishedYes

Keywords

  • Amino Acid Sequence
  • Aminohydrolases
  • Bacterial Proteins
  • Cloning, Molecular
  • Escherichia coli
  • Kinetics
  • Molecular Sequence Data
  • Molecular Weight
  • Pseudomonas fluorescens
  • Sequence Alignment
  • Substrate Specificity
  • EC 3.5.4.- Aminohydrolases
  • EC 3.5.5.1 nitrilase
  • Amines
  • Amino acids
  • Chromatographic analysis
  • Cloning
  • Cyanides
  • Electrophoresis
  • Enzymes
  • Gel permeation chromatography
  • Gelation
  • Gels
  • Industrial applications
  • Nucleic acids
  • Organic acids
  • Polymers
  • Sodium
  • Sodium sulfate
  • Substrates
  • amino acid
  • dinitrile
  • DNA
  • dodecyl sulfate sodium
  • fumaronitrile
  • mononitrile
  • nitrilase
  • nitrile
  • polyacrylamide gel
  • protein
  • succinonitrile
  • unclassified drug
  • ammonia
  • bacterium
  • carboxylic acid
  • clone
  • enzyme
  • genome
  • homology
  • molecular analysis
  • pH
  • substrate
  • article
  • bacterial gene
  • bioinformatics
  • DNA determination
  • DNA sequence
  • enzyme activity
  • enzyme specificity
  • gel filtration chromatography
  • gene
  • gene overexpression
  • isoelectric point
  • molecular weight
  • nitPf5 gene
  • nonhuman
  • nucleotide sequence
  • open reading frame
  • polyacrylamide gel electrophoresis
  • protein analysis
  • sequence analysis
  • synthesis
  • Pseudomonas fluorescens Pf-5
  • Active sites
  • Aliphatic nitrilase
  • Biochemical tools
  • Dinitrile
  • DNA sequence analysis
  • Enzymatic synthesis
  • Gel-filtration chromatographies
  • Homology modeling
  • Iso-electric points
  • Nitrile groups
  • One-step reactions
  • Sequence analysis
  • Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • Specific activities
  • Substrate specificity
  • Succinonitrile
  • BIOTECHNOLOGY
  • RHODOCOCCUS-RHODOCHROUS K22
  • ALIPHATIC NITRILASE
  • CRYSTAL-STRUCTURE
  • HYDROLYZING ENZYMES
  • GENE CLONING
  • PURIFICATION
  • EXPRESSION
  • PROTEIN
  • ACIDS
  • DINITRILES
  • Facultatively Anaerobic Gram-Negative Rods Eubacteria Bacteria Microorganisms (Bacteria, Eubacteria, Microorganisms) - Enterobacteriaceae [06702] Escherichia coli species expression system strain-DH-5-alpha
  • Gram-Negative Aerobic Rods and Cocci Eubacteria Bacteria Microorganisms (Bacteria, Eubacteria, Microorganisms) - Pseudomonadaceae [06508] Pseudomonas fluorescens species strain-Pf-5
  • Pseudomonas fluorescens nitPf5 gene [Pseudomonadaceae] expression
  • cyanocarboxylic acid synthesis
  • monomitrile
  • nitrilase NitPf5 82391-37-5 active site, homotetramer
  • open-reading frame
  • polypeptide 307 amino acid residues
  • succinonitrile 110-61-2
  • 03502, Genetics - General
  • 10062, Biochemistry studies - Nucleic acids, purines and pyrimidines
  • 10802, Enzymes - General and comparative studies: coenzymes
  • 31000, Physiology and biochemistry of bacteria
  • 31500, Genetics of bacteria and viruses
  • Enzymology
  • Molecular Genetics
  • docking study
  • specific activity
  • substrate specificity
  • Biochemistry and Molecular Biophysics
  • YP260015 GenBank, EMBL, DDJB nucleotide sequence
  • cloning laboratory techniques, genetic techniques
  • DNA sequence analysis laboratory techniques, genetic techniques
  • gel filtration chromatography laboratory techniques, chromatographic techniques
  • homology modeling mathematical and computer techniques
  • sodium dodecyl sulfate-polyacrylamide gel electrophoresis electrophoretic techniques, laboratory techniques

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