title: Autodisplay of nitrilase from Klebsiella pneumoniae and whole-cell degradation of oxynil herbicides and related compounds.
abstract: Using the Autodisplay system, a recombinant Escherichia coli strain displaying the dimeric nitrilase from Klebsiella pneumoniae subsp. ozaenae (NitKp) on the cell surface was constructed. Localization of the nitrilase in the cell envelope of E. coli was monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis and surface exposure was verified by its accessibility to externally added protease. The whole-cell biocatalyst obtained converted the substrates analyzed in the following order: chloroxynil > bromoxynil > ioxynil > 3-bromo-4-hydroxybenzonitrile (1.67, 0.89, 0.13, and 0.09 mM product formation within 72 h, respectively), indicating the same substrate specificity for the displayed enzyme as for the free enzyme. The whole-cell biocatalyst was also able to convert 3-fluoro-4-hydroxybenzonitrile and 3,5-dimethyl-4-hydroxybenzonitrile to the corresponding carboxylic acids. In contrast, it was not possible to detect any enzyme activity when 4-methoxybenzonitrile was used as substrate. The temperature optimum determined was 45 °C for the surface-displayed enzyme instead of 35 °C for the purified enzyme. In addition, the optimum activity of the displayed nitrilase was shifted to more acidic pH in comparison to the free enzyme.
authors.affiliation: Institut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms-Universität, Hittorfstr. 58-62, 48149 Münster, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Detzel
authors.forename: Ruth
authors.initials: R
authors.lastname: Maas
authors.forename: Agne
authors.initials: A
authors.lastname: Tubeleviciute
authors.forename: Joachim
authors.initials: J
authors.lastname: Jose
mesh_terms: D000619:Aminohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001711:Biotransformation; D061505:Cell Surface Display Techniques / Q000379:methods*; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D006540:Herbicides / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D009570:Nitriles / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D004800:Enzymes, Immobilized; D006540:Herbicides; D009570:Nitriles; D011994:Recombinant Proteins; D000619:Aminohydrolases; C021867:nitrilase