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.

Autodisplay of glucose‐6‐phosphate dehydrogenase for redox cofactor regeneration at the cell surface

“…Since G6PDH is active as a dimer (Rowland et al, ), activity at the cell surface underlines a particular feature of Autodisplay, namely that autotransporter fusion proteins are able to multimerize after transport. This is coherent with previous results showing the active display of homo‐ or heteromultimers such as nitrilase (Detzel et al, ), prenyltransferase (Kranen et al, ), Hsp90 (Bopp et al, ), or CK2 (Gratz et al, ). Treatment of whole cells displaying G6PDH with proteinase K led to a complete loss of the enzymatic activity indicating that the NADP + ‐reducing protein is indeed surface located and the increase in absorption is not due to NADP + diffusion and accompanying intracellular reduction or leakage of intracellular G6PDH to the extracellular space (Fig.…”

“…The exact mechanism is still subject to research and has been discussed in detail elsewhere (Gawarzewski et al, ; Ieva et al, ; Nicolay et al, ). Replacing the natural passenger by enzymes of choice enables their display on the cell surface, which was successfully realized in numerous cases (Detzel et al, ; Kranen et al, ; Tozakidis et al, ). While the approach mimics immobilized enzymes with all their advantages like increased stability, easy separation, and direct substrate contact, it also poses new challenges such as loss of access to the intracellular cofactor pools, a major advantage of whole cell biocatalysts (Schüürmann et al, ).…”

A simplified process design for P450 driven hydroxylation based on surface displayed enzymes

“…The translocation mechanism was enlightened recently (Gawarzewski et al, ; Pavlova et al, ). The Autodisplay system was already successfully used for the utilization of, for example, hyaluronidases (Kaessler et al, ), lipases (Kranen et al, ), as well as nitrilases (Detzel et al, , ). Additionally the display of a functional bacterial and human P450 enzyme with the Autodisplay system had also been performed before (Schumacher et al, ; Schumacher and Jose, ).…”

Modification of auxinic phenoxyalkanoic acid herbicides by the acyl acid amido synthetase GH3.15 from Arabidopsis

“…Although both IAA and 2,4-D target the auxin receptor, 2,4-D is metabolized more slowly than IAA, which enhances herbicidal effects through elevated expression of auxin responsive genes leading to plant death (6,15,16). For agricultural biotechnology applications, herbicide tolerance traits have relied on the identification of enzymes that either chemically inactivate the herbicide or prevent inhibition of a target by the herbicide (17)(18)(19)(20)(21)(22)(23)(24). For example, isolation of a microbial aryloxyalkanoate dioxygenase that cleaves 2,4-D provides tolerance to this auxinic herbicide and is the basis for 2,4-D-resistant crops currently entering the market (23).…”