Autodisplay of nitrilase from Klebsiella pneumoniae and whole-cell degradation of oxynil herbicides and related compounds
- 388 Downloads
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.
KeywordsAutodisplay Nitrilase Bromoxynil Biocatalyst Surface display Biodeterioration
The present study arose from a larger project within the scope of a patent value fund. In this context, we acknowledge the financial support provided by the Zyrus Beteiligungsgesellschaft mbH & Co. Patente I KG (Germany). In addition, the authors thank Hans J. Gross, Würzburg, and Mark Teese, Münster for the helpful discussions and Peter Proksch, Düsseldorf for providing the marine sponge extracts.
- Benz I, Schmidt MA (1989) Cloning and expression of an adhesin (AIDA-I) involved in diffuse adherence of enteropathogenic Escherichia coli. Infect Immun 57:1506–1511Google Scholar
- Holtze MS, Sørensen SR, Sørensen J, Aamand J (2008) Microbial degradation of the benzonitrile herbicides dichlobenil, bromoxynil and ioxynil in soil and subsurface environments—insights into degradation pathways, persistent metabolites and involved degrader organisms. Environ Pollut 154:155–168CrossRefGoogle Scholar
- Maurer J, Jose J, Meyer TF (1997) Autodisplay: one-component system for efficient surface display and release of soluble recombinant proteins from Escherichia coli. J Bacteriol 179:794–804Google Scholar
- Sambrook J, Fritsch EF, Maniatis T (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Press, New YorkGoogle Scholar
- Stalker DM, Malyj LD, McBride KE (1988) Purification and properties of a nitrilase specific for the herbicide bromoxynil and corresponding nucleotide sequence analysis of the bxn gene. J Biol Chem 263:6310–6314Google Scholar