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 envelope of E. coli was monitored by SDS‐PAGE and surface exposure was verified by its accessibility to externally added protease. The whole cell biocatalyst converted up to 2.6 mM of (R)‐mandelic acid under optimum conditions at pH 7.5 and 45 °C within 24 h (1 mL culture, OD578=10). By using chiral HPLC, the ee value of the product was determined to be >99 %. The surface displayed nitrilase showed an apparent Km value of 3.6 mM and an apparent Vmax value of 1 nmol min−1 mL−1 when a bacterial suspension of OD578 3 was used. Substrate inhibition by benzaldehyde was similar to that of the free enzyme. The whole‐cell biocatalyst retained 55 % of its initial (R)‐mandelic acid production after 5 cycles of repeated use, and could be stored at −70 °C for 180 d without a substantial loss of activity. In addition the whole cell biocatalyst converted 9.3 mM phenylacetonitrile within 16 h.

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.

BackgroundLipases including the lipase from Burkholderia cepacia are in a main focus in biotechnology research since many years because of their manifold possibilities for application in industrial processes. The application of Burkholderia cepacia lipase for these processes appears complicated because of the need for support by a chaperone, the lipase specific foldase. Purification and reconstitution protocols therefore interfere with an economic implementation of such enzymes in industry. Autodisplay is a convenient method to express a variety of passenger proteins on the surface of E. coli. This method makes subsequent purification steps to obtain the protein of interest unnecessary. If enzymes are used as passengers, the corresponding cells can simply be applied as whole cell biocatalysts. Furthermore, enzymes surface displayed in this manner often acquire stabilization by anchoring within the outer membrane of E. coli.ResultsThe lipase and its chaperone foldase from B. cepacia were co-expressed on the surface of E. coli via autodisplay. The whole cell biocatalyst obtained thereby exhibited an enzymatic activity of 2.73 mU mL-1 towards the substrate p-nitrophenyl palmitate when applied in an OD578 =1. Outer membrane fractions prepared from the same culture volume showed a lipase activity of 4.01 mU mL-1. The lipase-whole cell biocatalyst as well as outer membrane preparations thereof were used in a standardized laundry test, usually adopted to determine the power of washing agents. In this test, the lipase whole cell biocatalyst and the membrane preparation derived thereof exhibited the same lipolytic activity as the purified lipase from B. cepacia and a lipase preparation which is already applied in commercial washing agents.ConclusionsCo-expression of both the lipase and its chaperone foldase on the surface of E. coli yields a lipid degrading whole cell biocatalyst. Therefore the chaperone supported folding process, absolutely required for the lipolytic activity appears not to be hindered by surface display. Furthermore, the cells and the membrane preparations appeared to be stable enough to endure a European standard laundry test and show efficient fat removal properties herein.

An increasing number of chemicals and materials, like base chemicals and polymers, as well as high value products, such as consumer chemicals and specialty chemicals, are produced using biotechnology in one or more of the process steps. In 2010, the sale volume of biotechnology products was around 92 billion Euro worldwide. Sales are estimated to increase to around 228 billion Euro in 2015 and to around 515 billion Euro in 2020. On a sector level, the largest market potential lies in the production of biopolymers and active pharmaceutical ingredients. As a rule, commercial development is mainly driven by multinational enterprises, whereas small and medium enterprises contribute primarily to the technological development. Especially the latter group faces several challenges during their development. These mainly concern business models and growth strategies as well as financing strategies and resources. Investors have not yet fully identified the area of industrial biotechnology as an attractive investment field but they could become a major capital source as they start to understand more the potential of industrial biotechnology.

Die Biopolymer-Enkapsulierung von Biokatalysatoren bietet eine Steigerung der bereits hohen Substratspezifität bzw. Selektivität durch das gezielte Einstellen der diffusiven Trenneigenschaften des Kapselmaterials und somit eine neue Alternative bei der Reaktionsführung biokatalytischer Systeme. Vorteilhaft ist dabei, dass die Modifizierung nicht am Biokatalysator selbst, sondern an dessen Umgebung durchgeführt und zudem dessen leichtere Abtrennung und Wiederverwendung ermöglicht wird. Die Er-weiterung dieses Verfahrens auf die Anwendung in schwerlöslichen bzw. zweiphasigen Systemen bietet ein sehr groûes Potenzial zur Prozessintensivierung. Mikrokapseln (Vollkugel-und Schalenkapseln) wurden für verschiedene Biopolymere in Bezug auf Partikelgröûenverteilung, Sphärizität, Membrandicke sowie Innenvolumen untersucht und charakterisiert. Um eine reaktionstechnische Beschreibung der enkapsulierten Modell-Biokatalysatoren (En-zyme und Zellen) zu ermöglichen, wurden diese hinsichtlich ihrer Aktivität, Stabilität und Substratspezifität mit dem ¹freien Biokatalysatorª verglichen und anhand von Modell-Reaktionen batchweise und kontinuierlich untersucht, wobei der Stofftransport von Substraten und Produkten berücksichtigt wurde. Anhand der experimentellen Daten (mit Modellrechnungen validiert) konnte auch die Permeabilität bzw. Permselektivität der Kapselmaterialien charakterisiert werden.