Autodisplay of enzymes--molecular basis and perspectives.

To display an enzyme on the surface of a living cell is an important step forward towards a broader use of biocatalysts. Enzymes immobilized on surfaces appeared to be more stable compared to free molecules. It is possible by standard techniques to let the bacterial cell (e.g. Escherichia coli) decorate its surface with the enzyme and produce it on high amounts with a minimum of costs and equipment. Moreover, these cells can be recovered and reused in several subsequent process cycles. Among other systems, autodisplay has some extra features that could overcome limitations in the industrial applications of enzymes. One major advantage of autodisplay is the motility of the anchoring domain. Enzyme subunits exposed at the cell surface having affinity to each other will spontaneously form dimers or multimers. Using autodisplay enzymes with prosthetic groups can be displayed, expanding the application of surface display to the industrial important P450 enzymes. Finally, up to 105-106 enzyme molecules can be displayed on a single cell. In the present review, we summarize recent achievements in the autodisplay of enzymes with particular attention to industrial needs and process development. Applications that will provide sustainable solutions towards a bio-based industry are discussed.

Escherichia coli with autodisplayed Z-domain of protein A for signal amplification of SPR biosensor.

Generally, an immunoaffinity SPR biosensor detects a target analyte in a sample through highly selective adsorption by using the antigen-antibody interaction. For improving the sensitivity, various kinds of particles have been added to the already bound analytes on the SPR biosensor (sandwich assay). In this work, signal amplification was demonstrated by the expression of the IgG-binding Z-domain of protein A on the outer membrane of Escherichia coli via "Autodisplay". The amount of Z-domain of protein A expressed on the outer membrane was calculated to be 280,000 molecules per cell. In addition, the IgG-binding ability of the expressed protein was characterized using FACS analysis. The signal amplification of the SPR biosensor was performed in the sandwich assay format using a model of horseradish peroxidase (HRP); the limit of detection was determined to be significantly improved from 1 microg/ml to 1 ng/ml. Finally, myoglobin analysis was demonstrated for the medical diagnosis of cardiac diseases. The detection limit was estimated to be improved from 10 ng/ml to <1 ng/ml. These results show that Z-domain-displaying E. coli can be successfully used for the signal amplification of immunoaffinity biosensors, thereby improving the sensitivity and the limit of detection.

The conjugative plasmid pSG5 from Streptomyces ghanaensis DSM 2932 differs in its transfer functions from other Streptomyces rolling-circle-type plasmids.

The Streptomyces ghanaensis plasmid pSG5 is self-transmissible but does not form the growth-retardation zones (pocks) normally characteristic of the Streptomyces plasmid-transfer process. The complete nucleotide sequence of pSG5 was determined on both strands. pSG5 is 12,208 bp in length and has a GC content of 68 mol%. Characterization of the open reading frames by insertion and deletion analysis revealed that only a single gene, traB, is involved in the transfer of pSG5. The deduced amino acid sequence of TraB is similar to the SpoIIIE protein that is responsible for chromosome translocation during prespore formation of Bacillus subtilis. In contrast to the tra genes of the other Streptomyces plasmids, the pSG5 traB does not represent a kill function. Although pSG5 transfer is not associated with pock formation, pSG5 was shown to possess putative spd genes that are responsible for the pock phenotype of other Streptomyces plasmids. However, promoter-probe experiments revealed that the spd genes of pSG5 are not transcribed, thus explaining the deficiency in pock formation.