Autodisplay for the co-expression of lipase and foldase on the surface of E. coli: washing with designer bugs

Eva Kranen,Joachim Jose,Thomas Weber,Christian Detzel

doi:10.1186/1475-2859-13-19

Eva Kranen, Joachim Jose + Show 2 more

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https://doi.org/10.1186/1475-2859-13-19

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Abstract

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.

Highlights

Lipases 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
polymerase chain reaction (PCR)-primers were designed according to the deposited sequence of the B. cepacia lipase [GenBank: FJ638612] and added an XhoI (5′end) and a KpnI restriction site (3′end) to the PCR fragment in order to enable an in frame fusion with the plasmid DNA encoding the autodisplay domains
After insertion into plasmid pCD003 [25] cleaved with XhoI and KpnI as well, plasmid pATLipBc was obtained encoding a fusion protein comprising the signal peptide of cholera toxin β-subunit (CtxB) at the N terminus followed by the lipase as a passenger, the linker region and the β-barrel from the AIDA-I autotransporter needed for outer membrane translocation and full surface accessibility (Figure 1B)

Summary

Introduction

Lipases 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. To express lipases from Burkholderia and Pseudomonas species in an active form, lipases which have advantageous features regarding thermal stability, alkaline pH tolerance and high substrate selectivity, and making them promising industrial biocatalysts [8,9,10], bears an additional problem. These enzymes are dependent on the presence of a personal chaperon, the so-called lipase-specific foldase (Lif ), responsible for correct folding of the lipase [1,11]. Significant amounts of active lipase were only achieved by applying an additional in-vitro refolding protocol [12]

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