DOI: 10.1002/cctc.201000382 Autodisplay of Nitrilase from Alcaligenes faecalis in E. coli Yields a Whole Cell Biocatalyst for the Synthesis of Enantiomerically Pure (R)-Mandelic Acid Christian Detzel, Ruth Maas, and Joachim Jose* [a] Introduction Enzymes as biocatalysts show some outstanding advantages for the synthesis of chemicals, pharmaceutical, and agrochemi- cal intermediates as well as active pharmaceutical and agro- chemical compounds. Unlike conventional organic chemistry, enzymes can be used under mild conditions concerning tem- perature, pressure, and pH and usually they convert a sub- strate with high regio- and enantioselectivity without protect- ing and deprotecting steps as necessary in conventional or- ganic chemistry. [1] In many cases, the use of enzymes in chemi- cal synthesis requires less substrate, and reduces waste. The use of enzymes displayed on microbial whole cell bio- catalysts extends the advantages of enzymatic applications in biotechnology. The self-replicating microbial cell serves as an enzyme factory in which the produced enzymatic activity is linked and immobilized on the surface of an intact cellular system, avoiding laborious and expensive downstream proc- essing, such as separation and purification of the enzyme prior to its application. The intact cell also provides a stabilizing and protecting environment for the enzyme and the use of whole cell biocatalysts in a synthesis reaction allows the elimination of the enzyme by a simple centrifugation step. In addition, sur- face display allows an enzyme or protein free access to sub- strates or binding partners, without the need to cross a mem- brane. [2] Autodisplay represents an elegant tool for the surface dis- play of recombinant proteins in E. coli. [3] It is based on AIDA-I, the adhesin involved in diffuse adherence in enteropathogenic E. coli (EPEC), which belongs to the autotransporter family of proteins. [4] The autotransporter proteins are synthesized as pre- cursor proteins containing all structural requirements for the transport to the cell surface. [4] They contain an N-terminal signal peptide typical for the Sec pathway, enabling export across the inner membrane. After truncation of the signal pep- tide in the periplasm, the C-terminal domain folds into the outer membrane as a porine-like structure, a so called b-barrel. The passenger domain can be efficiently translocated to the extracellular milieu by this structure. [5] Recombinant passengers can be transported to the surface by simple insertion of the corresponding coding sequence into a distinct position of the precursor gene. One of the most striking features of the Autodisplay system is the flexibility of the anchoring b-barrel within the fluidic outer membrane, enabling passenger driven self association of subunits to an active enzyme. This has been verified for the di- meric enzyme sorbitol dehydrogenase and for the dimeric bovine adrenodoxin. [5, 6] As a model for a multihomomeric enzyme the nitrilase from Alcaligenes faecalis subsp. faecalis ATCC 8750 was used for expression on the surface of E. coli by Autodisplay. Nitrilases (EC 3.5.5.1) are enzymes that convert ni- triles to the corresponding carboxylic acid and ammonia in a single step. [7] The use of nitrilase enzymes has attracted sub- stantial industrial interest. The carboxylic acids produced by this reaction are often used intermediates in a great variety of chemical production processes. They are often enantiomeri- cally pure and can be produced under mild conditions. Nitrilas- In the present study, a whole cell biocatalyst for the synthesis of (R)-mandelic acid from mandelonitrile was constructed. For this purpose, nitrilase from Alcaligenes faecalis subsp. faecalis ATCC 8750 was displayed on the surface of Escherichia coli by using Autodisplay. Localization of the nitrilase in the cell enve- lope of E. coli was monitored by SDS-PAGE and surface expo- sure was verified by its accessibility to externally added pro- tease. The whole cell biocatalyst converted up to 2.6 mm of (R)-mandelic acid under optimum conditions at pH 7.5 and 45 8C within 24 h (1 mL culture, OD 578 = 10). By using chiral HPLC, the ee value of the product was determined to be > 99 %. The surface displayed nitrilase showed an apparent K m value of 3.6 mm and an apparent V max value of 1 nmol min À1 mL À1 when a bacterial suspension of OD 578 3 was used. Substrate inhibition by benzaldehyde was similar to that of the free enzyme. The whole-cell biocatalyst retained 55 % of its ini- tial (R)-mandelic acid production after 5 cycles of repeated use, and could be stored at À70 8C for 180 d without a substantial loss of activity. In addition the whole cell biocatalyst converted 9.3 mm phenylacetonitrile within 16 h. [a] C. Detzel, Dr. R. Maas, + Prof. Dr. J. Jose Institute of Pharmaceutical and Medicinal Chemistry Heinrich-Heine-University Düsseldorf Universitätsstraße 1, 40225 Düsseldorf (Germany) Fax: (+ 49)211-8113847 E-mail : joachim.jose@uni-duesseldorf.de [ + ] Present address: Autodisplay Biotech GmbH Merowinger Platz 1a, 40225 Düsseldorf (Germany) ChemCatChem 2011, 3, 719 – 725  2011 Wiley-VCH Verlag GmbH& Co. KGaA, Weinheim 719