Autodisplay for the co-expression of lipase and foldase on the surface of E. coli: washing with designer bugs
© Kranen et al.; licensee BioMed Central Ltd. 2014
Received: 8 January 2014
Accepted: 16 January 2014
Published: 29 January 2014
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. 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.
The 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.
Co-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.
Lipolytic enzymes are attractive biotechnological tools . Among them lipases (triacylglycerol acylhydrolases EC 188.8.131.52), which catalyze the hydrolysis of triglycerides in aqueous media, liberating free fatty acids and glycerol, or the reverse reaction in organic solvents as well, have gained particular interest, since they simultaneously show high enantio- and/or regio-selectivity as well as a high catalytic activity and thermostability in organic solvents [2, 3]. Contrary to esterases, which preferentially break ester bonds of short chain fatty acids, lipases are able to catalyze the hydrolysis of water-insoluble long-chain acylglycerols . Interestingly, activation of lipases often depends on the presence of a lipid-water interface, which can be explained by their three-dimensional structure. In an enzymatically inactive state, a surface loop, the so-called lid, covers the active site of the lipase. Upon contacting the lipid-water interface the lid switches open, and the active site becomes accessible for the substrate .
So far, lipases have been established in numerous industries, such as the food industry, paper manufacturing, pharmaceutical processing , and detergents industry, reflecting their great importance . Despite this enormous industrial interest, not more than around 20 lipases have been established for industrial applications yet . The sometimes troublesome and time-consuming purification procedures to obtain pure enzyme preparations for particular applications seem to be one possible obstacle in broadening the use of lipases in industrial processes . Moreover, 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 therefore making them promising industrial biocatalysts [8–10], 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]. As a consequence, former heterologous expression of the Burkholderia cepacia lipase in E. coli resulted in a very low yield of active soluble lipase, whereas the majority of the enzyme was expressed as insoluble inclusion bodies. Significant amounts of active lipase were only achieved by applying an additional in-vitro refolding protocol .
An innovative way to gain access to the synthetic potential of lipases is their display on the surface of a living cell, in particular an E. coli cell . Since the enzyme is directly accessible for its substrate, costly purifications as mentioned above are not necessary.
Construction of the plasmid for autodisplay of lipase
By analyzing the amino acid sequence of B. cepacia ATCC 21808 lipase using the SignalP computer program , a classical signal peptide was identified at its N terminus. Since this lipase inherent signal peptide is proposed to interfere with the signal peptide used in autodisplay and thus constrain a proper transport across the inner membrane, the lipase signal peptide encoding 120 bp sequence was deleted by PCR. 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. For PCR plasmid pHES8 was used, which resembles pHES12 described by Quyen et al.  and encodes the complete B. cepacia lipase operon (i.e. lipase and its corresponding foldase) for intracellular expression in E. coli. After insertion into plasmid pCD003  cleaved with XhoI and KpnI as well, plasmid pAT-LipBc was obtained encoding a fusion protein comprising the signal peptide of 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).
Surface display of lipase
Construction of the plasmid for autodisplay of foldase
According to Quyen et al.  the gene for foldase contains a possible N-terminal 70 aa membrane anchor. This structure is not required for the chaperone function of foldase, but may interfere with correct surface expression via autodisplay. Therefore foldase also was amplified from plasmid pHES8, which encodes the whole lipase operon , deleting the first 210 bp encoding this particular anchor structure. PCR primers, designed using the deposited sequence of the whole B. cepacia lipase [GenBank:FJ638612] added an XhoI site at the 5′-end and a KpnI site at the 3′-end of the foldase gene, analogously as described for the construction of plasmid pAT-LipBc. The derived fragment was ligated into autodisplay vector pBL001, digested with XhoI and KpnI before. Vector pBL001 is a pCOLA DuetTM derivative, encoding the domains needed for autodisplay. Vector pBL001 furthermore provides a kanamycin resistance. Insertion of the foldase gene into pBL001 resulted in plasmid pAT-FoldBc encoding an in frame fusion of the autodisplay domains with foldase as a passenger (Figure 1C).
Surface display of foldase
E. coli BL21 (DE3) pAT-FoldBc cells were grown to midlog phase and autotransporter fusion protein expression (FoldBc-FP) was induced by adding 1 mM IPTG to the fermentation broth and incubating the culture for another hour. After preparation of the outer membrane fraction, obtained protein samples were subjected to SDS-PAGE. As can be seen in Figure 2B, induction of protein expression resulted in the appearance of a protein band with an apparent molecular mass of around 80 kDa (Figure 2B, lane 2), which is in good accordance with the calculated molecular mass of 78.5 kDa for FoldBc-FP. The SDS-analysis revealed the location of the autotransporter fusion protein in the outer membrane protein fraction. The investigation of surface exposure via FACS was not possible for foldase, since there was no specific antibody against foldase available. Therefore, to elucidate if the passenger domain of FoldBc-FP is truly surface exposed and not directed to the periplasm, the accessibility of the fusion protein for proteases was tested. Since proteases are too large to pass the outer membrane, only surface exposed proteins will be degraded. In order to perform this degradation test whole cells of E. coli BL21(DE3) pAT-FoldBc were incubated with different concentrations of proteinase K. This treatment resulted in degradation of FoldBc-FP (Figure 2B, lanes 3 and 4). To demonstrate the integrity of the outer membrane during protease treatment, outer membrane protein A (OmpA) can be used as a reporter. The C-terminal part of OmpA directs into the periplasmic space while the N-terminal part builds a compact β-barrel structure inside the outer membrane . A digestion of OmpA therefore can only occur from the periplasmic side, indicating that the outer membrane lost its integrity to enable the access for proteases into the periplasm. Thus, the fact, that the performed protease accessibility test led to a strong decrease of FoldBc-FP intensity (Figure 2B, lanes 3 and 4), without affecting OmpA intensity, provides strong evidence for the surface exposure of FoldBc-FP.
Coexpression of both LipBc-FP and FoldBc-FP
Lipase Activity of whole cells co-expressing LipBc-FP and FoldBc-FP
In addition, we investigated whether mixing the cells displaying only the lipase with cells displaying only the foldase could lead to whole cell lipase activity. This approach was somehow similar to that of Wilhelm et al. , who mixed cells displaying foldase with a denatured lipase and ended up with lipase activity. In our investigation, for the combination of both types of cells, E. coli BL21(DE3) pAT-LipBc and E. coli BL21(DE3) pAT-FoldBc were cultivated separately and protein expression was induced as described above. Each type of cells was washed and suspended to an OD578 of 10 as described before. Subsequently E. coli BL21(DE3) pAT-LipBc and E. coli BL21(DE3) pAT-FoldBc were mixed in a ratio of 1:1. Half of the sample was incubated for one hour, the other half was incubated for 24 hours at 20°C with vigorous shaking (200 rpm) to avoid sedimentation. After the incubation enzymatic activity was determined as described for the cells co-expressing lipase and foldase. However, mixing the cells displaying the foldase with cells displaying the lipase did not yield any activity at all, neither after 1 h nor after 24 h. This is to indicate that the surface displayed lipase needs to be co-expressed with its chaperone foldase on the surface of a single cell to gain its enzymatic activity.
Lipase activity of outer membrane preparations from E. Coli BL21(DE3) pAT-LiFoBc
Application of cells and membrane preparations in a standardized laundry test
Since ecologically friendly housekeeping processes become more and more important for a broad public and within a steadily growing biotechnological industry the need for cost efficient and easy accessible lipase preparations increases. By means of Autodisplay a new method to make the challenging lipase from B. cepacia easily available was developed: Within this study we were for the first time able to use Autodisplay for the co-expression of two different proteins, which need to interact with each other, a lipase and its implicitly required chaperone, foldase. By co-expression of both these proteins on the surface of one single E. coli cell we obtained a functional lipase-whole cell biocatalyst. Simply combining two cell types, each displaying one of the proteins, either lipase or foldase was not sufficient to create a functional whole cell biocatalyst. This indicates that the interaction between lipase and foldase can only take place if they are expressed on the surface of a single cell. Therefore, it can be assumed that a certain vicinity of lipase and foldase is needed for the process of folding supported by the chaperone. The novelty of the present investigation is, that the lipase and its specific foldase were expressed separately and both proteins interacted spontaneously and self driven, finally yielding an enzymatically active lipase at the cell surface of E. coli. In this respect the study goes beyond the aims of Wilhelm et al., , which displayed a foldase on the surface of E. coli and added the corresponding lipase as a purified protein subsequently and it goes an important step further than the work of Yang et al.  who obtained the surface display of an active lipase after co-expression with foldase in a single fusion protein. Our report is the first time description of the separate expression and surface display of two enzymes that finally interacted with each other in order to obtain an enzymatic activity. It paves the way for the surface display of other multiprotein or multienzyme complexes by a similar strategy, which was to the best of our knowledge up to now not taken into consideration. Our data show, that this interaction and the anchorage within the E. coli outer membrane deliver a biocatalyst stable enough to endure even a stressing and mechanically demanding procedure like the standardized laundry tests which had been conducted here. The whole cell biocatalyst and the membrane preparations yielded an activity in the same order of magnitude to the purified enzyme and a standard lipase formulation already used in detergents (Lipex®). Taken the activity 0f 4.01 mU/ml at an OD578 = 1 as an example, the whole cell lipase/foldase biocatalyst described here would reduce the costs in a 30 qm fermenter to 35% of those required for the purified enzyme to get the same amount of product, taken into consideration fermentation, purification and stabilization of the catalysts, as well as the necessary raw materials (G. Festel, unpublished). But it would be also possible to gain an even higher enzymatic activity by E. coli BL21(DE3) pAT-LiFoBc which exceeds the activity of purified and reconstituted B. cepacia lipase and the detergent lipase by further optimization of the culturing conditions and culture medium for instance. Moreover directed evolution approaches or site-directed mutagenesis could be applied in order to gain higher lipase activities finally.
Autodisplay offers once more a convenient alternative to obtain a functional biocatalyst without precedent laborious purifying steps and in the special case of B. cepacia lipase and its chaperone foldase without a strongly required reconstitution protocol. The successful removal of fat or grease spots respectively during standard washing procedures was possible by simply applying surface engineered cells and E. coli outer membrane preparations containing active surface displayed lipase. Working with a cell-free preparation which achieves the same activities like the whole cell biocatalyst is therefore also feasible. These results give an outlook of possible applications for enzymes utilized by Autodisplay beyond laboratory scale testing.
Bacterial strains, plasmids and culture conditions
Escherichia coli strains UT5600(DE3) [Fˉ, ara-14, leu B6, sec A6, lac Y1, pro C14, tsx-67, Δ(omp T-fepC)266, ent A403, trp E38, rfb D1, rps L109(Strr), xyl-5, mtl-1, thi-1, λ(DE3)] and E. coli BL21(DE3) [B, Fˉ, dcm, omp T, lon, hsdS(rBˉ mBˉ), gal, λ(DE3)] were used for the expression of autotransporter fusion proteins. E. coli TOP10 (F- mcr A Δ(mrrhsd RMS-mcr BC) ϕ80lac ZDM15 Δlac X74 deo R rec A1 ara D139 Δ(ara-leu) 7697 gal U gal K rps L (StrR) end A1 nup G) and the vector pCR®4-TOPO® were used for subcloning of polymerase chain reaction (PCR) products, using the TOPO-TA cloning kit (Invitrogen, Carlsbad, CA, USA). Site directed mutagenesis of the restriction sites for XhoI and KpnI inside the genes of interest was performed using the QuikChange Site Directed Mutagenesis Kit (Stratagene, Santa Clara, CA, USA) and appropriate mutagenesis primers. Construction of plasmid pCD003 which encodes the AIDA-I autotransporter has been described elsewhere . Plasmid pBL001 is a pCOLA-DuetTM-1–derivative. The second MCS had been removed and the autotransporter cassette was inserted using NcoI and BlpI restriction sites. Plasmid pHES8, encoding the lipase and foldase from Burkholderia cepacia, is a derivative of pHES12, which has been described by Quyen et al. . Bacteria were routinely grown at 37°C in Lysogeny broth (LB) containing carbenicillin (100 mg L-1) or kanamycin (30 mg L-1) or both antibiotics, respectively. For co-expression of both, lipase and foldase, a culture from strain E. coli BL21(DE3) pAT-LipBc, already containing the plasmid encoding for lipase-autotransporter fusion protein, was prepared to obtain electrocompetent cells according to a modified protocol from Sambrook et al. . Plasmid pAT-FoldBc was then transformed into an aliquot of these cells by electroporation resulting in strain BL21(DE3)pAT-LiFoBc which contains both plasmids.
Recombinant DNA techniques
For construction of plasmid pAT-LipBc, which contains the gene encoding LipBc-FP, the lipase gene was amplified by PCR. Plasmid pHES8 served as a template for primers EK009 (CGCTCGAGGCGAGCGCGCCCGCCGAC) and EK010 (GGTACCCACGCCCGCGAGCTTCAGCCG). To facilitate cloning of the lipase-PCR fragment into the autotransporter cassette, a XhoI restriction site was added to the 5′-end and a KpnI restriction site was added to the 3′-end via PCR. For construction of plasmid pAT-FoldBc, containing the gene which encodes for FoldBc-FP, the foldase gene was amplified by PCR, again using pHES8 as a template for primers CD004 (CTCGAGCCGTCGTCGCTGGCCGGCTCC) and CD005 (GGTACCCTGCGCGCTGCCCGCGCCGCG). 5′-XhoI and 3′-KpnI-restriciton sites were attached to the PCR fragment analogously. Both PCR products were each inserted into vector pCR®4-TOPO® and first brought to site directed mutagenesis according to the protocols delivered by Stratagene to remove unwanted restriction sites within the genes of interest. Mutated plasmids were then restricted with XhoI and KpnI. The restriction fragment containing the lipase gene was ligated into pET-derivative pCD003  restricted with the same enzymes. The restriction fragment containing the foldase gene was ligated into pCOLA-DuetTM-1–derivative pBL001 restricted with the same enzymes before. Both ligation steps yielded an in frame fusion of lipase or foldase respectively, with the autotransporter domains under the control of a T7/lac promoter. Plasmid DNA preparation, restriction digestion, ligation, DNA electrophoresis and transformation were performed according to standard protocols . Gel extraction of digested fragments was performed using a gel extraction kit from Qiagen (Hilden, Germany).
Outer membrane protein preparation
E. coli cells were grown overnight and 1 ml of the culture was used to inoculate LB medium (40 ml). Cells were cultured at 37°C with vigorous shaking (200 rpm) for about 2 hours until an OD578 of 0.5 was reached. The culture was separated into two aliquots and protein expression was induced by adding IPTG at a final concentration of 1 mm to one of the aliquots. Cultures then were incubated at 30°C and shaking (200 rpm) for one hour. Induction was stopped by incubating the cells on ice for 15 min. After harvesting and washing of the cells with Tris-HCl (0.2 m, pH 8), differential cell fractionation was performed according to the method of Hantke  as modified by Schultheiss et al.  In detail, cell lysis was obtained by adding lysozyme (0.04 mg/mL end concentration) in the presence of 10 mM saccharose and 1 μM EDTA in a final volume of 1.5 mL of Tris-HCl (0.2 M, pH 8) and incubation for 10 min at room temperature. Subsequently aprotinin (10 μg/mL), phenylmethylsulfonyl fluoride (PMSF) (0.5 mM), as well as 5 mL of extraction buffer (50 mM Tris-HCl pH8.0, 10 mM MgCl2, 2% Triton × 100) and DNAseI (10 μg/mL) were added. After incubation on ice for 30 min the samples were centrifuged (2,460 g, 5 min, 4.0°C) to remove intact bacteria and large cell debris. The supernatants representing the clarified bacterial lysate were retained and centrifuged at higher speed (38,700 × g, 30 min, 4.0°C) in order to obtain the membrane protein fraction. The resulting supernatant, containing soluble cytoplasmic and periplasmic proteins, was completely aspirated. The pellet was suspended in 10 ml phosphate-buffered saline (PBS) plus 1% Sarcosyl (N- lauryl sarcosinate, sodium salt) and centrifuged again (38,700 × g, 60 min, 4°C). The supernatant after this step contained the sarcosyl-soluble cytoplasmic membrane proteins and was completely aspirated. The sediment representing the outer membrane protein fraction was washed twice with 10 ml of water and dissolved in 30 μl water for SDS-PAGE or an adequate volume of 25 mM potassium phosphate buffer pH 7,4 for activity determination. For whole cell protease treatment, E. coli cells were harvested, washed and resuspended in 1 ml Tris-HCl (0.2 m, pH 8). Proteinase K was added to final concentrations between 0.2 mg mL-1 and 0.5 mg mL-1 and cells were incubated for 1 hour at 37°C. Digestion was stopped by washing the cells twice with Tris-HCl (0.2 M, pH 8) containing 10% fetal calf serum (FCS) and outer membrane proteins were prepared as described above.
For outer membrane proteins that were applied for activity assays, cells were not treated with Proteinase K.
Outer membrane isolates were diluted (1:1.5) with sample buffer (100 mm Tris/HCl (pH 6.8) containing 4% SDS, 0.2% bromophenol blue, 200 mm dithiothreitol and 20% glycerol), boiled for 10 minutes and analyzed on 10% polyacrylamid gels. Proteins were stained with Coomassie brilliant blue (R250). To correlate molecular masses of protein bands of interest, a molecular weight standard was used (PageRuler unstained, Fermentas, Burlington, Canada).
Flow cytometer analysis
E. coli BL21(DE3) pAT-LipBc cells were grown and expression of lipase fusion protein was induced as described above by adding IPTG to a final concentration of 1 mM and incubating the cells for another hour at 30°C under shaking (200 rpm). Cells were harvested by centrifugation (2400 g, 2 min, 4°C, Mikro200R, Hettich, Tuttlingen, Germany) and washed twice with filter sterilized (0.2 μm pore size, ethylethersulfone membrane) phosphate buffered saline (PBS, pH 7.4) before suspending to a final OD578 of 0.25/mL for further experiments. 100 μl of these cells were again centrifuged and resuspended in 500 μL PBS (pH 7.4) containing 3% bovine serum albumin (BSA, filter sterilized) and incubated for 10 min at room temperature. After centrifuging the cells for 60 sec with 17,000 g (Mikro200R, Hettich, Tuttlingen, Germany), the obtained cell pellet was suspended with 100 μL of rabbit anti lipase antibody (diluted 1:50 in PBS (pH 7.4) + 3% BSA, filter sterilized) and incubated for another 30 min at room temperature. Subsequently cells were washed twice with 500 μL of PBS (pH 7.4) + 3% BSA. Cell pellets were resuspended in 100 μL of secondary antibody solution (goat-anti-rabbit, DylightTM 633, Thermo Scientific, diluted 1:25 in PBS (pH 7.4) +3% BSA) and incubated for 30 min in the dark at room temperature. After washing twice in 500 μL of PBS (pH 7.4) the cell pellet was finally suspended in 1.5 mL of PBS (pH 7.4, filter sterilized to avoid external particles). The samples were analyzed using a flow cytometer (Cyflow Space, Partec, Münster, Germany) at an excitation wavelength of 647 nm.
Lipase activity assay
Photometrical Assays to determine lipolytic activity of the lipase-whole cell biocatalyst were performed according to a modified protocol by Winkler and Stuckmann  with p-nitrophenylpalmitate (p-NPP) as substrate. For this purpose cells were routinely cultivated in LB medium until an optical density at 578 nm (OD578) of 1.0 was reached. Induction of protein expression was started by adding IPTG at a final concentration of 1 mM and incubating the cells another hour at 30°C and 200 rpm. Cells were then harvested by centrifugation and washed twice in potassium phosphate buffer, 25 mM, pH 7.4, and stored in the same buffer at 4°C in an OD578 = 10 until used for assays. In case of mixing different types of cells, they were used in a 1:1 ratio at OD578 =10 and incubated at 20°C on a rocking platform to avoid sedimentation For activity assays a stock solution of the substrate p-NPP was prepared in ethanol to a final concentration of 7.9 mM) and finally diluted in potassium phosphate buffer, 25 mM, pH 7.4 under constant stirring to a working concentration of 0.29 mM. This working solution was prepared freshly, kept at 25°C for one hour before its application and was not used when a visible turbidity or a yellow coloring occurred. Activity measurement was started by adding 180 μl of this working solution to 20 μl of cells with an OD578 = 10. This yielded a final substrate concentration of 0.26 mM and a final OD578 = 1 of the cells in the assay. The lipolytic production of yellow colored nitrophenylate at 25°C was measured at 405 nm in a 96 well plate using a microplate reader (Mithras LB940, Berthold, Bad Wildbach, Germany). The linear increase in absorption was used to calculate the enzymatic activity according to the law of Lambert and Beer (εnitrophenol = 17,000 L mol-1 cm-1, d = 0,52 cm path length correction for the layer thickness of 200 μL assay volume in one well). One unit was defined as the amount of enzyme which caused the release of 1 μmol of p-NPP per minute . For measuring the lipolytic activity of outer membrane protein preparation we proceeded similarly, with the exception, that the 20 μl which were added to the 180 μl assay buffer with the substrate were derived from an amount of cells corresponding to an OD578 = 2. For these activity measurements, absorption values at 405 nm obtained with outer membrane preparations in potassium phosphate buffer without the addition of p-NPP were used for blank correction.
Laundry tests with lipase-whole cell biocatalyst/ E. coli BL21(DE3) pAT-LipBc
The capability of lipase was tested on five different, standardized, lipase sensitive staining. The staining contained either Biskin® (Peter Kölln KGaA, Elmshorn, Germany), Butaris® (DFF Dairy Fine Food GmbH, Ratzeburg, Germany) or butter oil or a mixture of soot and mineral oil (C01, Center for Test Materials, Vlaardingen, The Netherlands) and a mixture of cutaneous sebum and pigment (20D, wfk Testgewebe GmbH, Krefeld, Germany) respectively. Tested lipases were a) a standard lipase preparation which is already used for washing purposes, b) soluble lipase from B. cepacia, c) the herein described lipase-whole cell biocatalyst and d) a membrane preparation thereof. To allow comparability, all lipases were applied in the same amounts, related to enzymatic activity. The washing process was carried out in a Linitest Plus (Atlas, Rock Hill, SC, USA), which represents the minituarized form of a standard machine washing process. The washing solution was prepared with 3.53 g of an enzyme free liquid detergent similar to a european premium detergent in water (16 °dH) buffered with 50 mM sodium phosphate pH 7.0. The washing process took place in a total volume of 170 mL at 40°C and 45 rpm for 60 minutes. To simulate the mechanism of a standard washing process, 10 steel balls were added and filled up with test cloth to a total amount of 14.3 g textile weight. Subsequently the test cloth was rinsed three times with deionized water and dried at room temperature in the dark. Color measurement of the staining was then carried out with a Minolta colorimeter (Konica-Minolta, München-Neuperlach, Germany), calibrated against producer’s standards, applying CIE L*a*b*, D65/10°/SCI settings. Each staining was measured three times and the average L* value was determined.
We thank T. Voelker for the preparation of cells and membranes used in the laundry tests. In addition, we acknowledge support by Deutsche Forschungsgemeinschaft and Open Access Publication Fund of University of Muenster.
- Arpigny JL, Jaeger KE: Bacterial lipolytic enzymes: classification and properties. Biochem J. 1999, 343: 177-183. 10.1042/0264-6021:3430177.View ArticleGoogle Scholar
- Reetz MT: Lipases as practical biocatalysts. Curr Opin Chem Biol. 2002, 6: 145-150. 10.1016/S1367-5931(02)00297-1.View ArticleGoogle Scholar
- Tadashi E: Mechanism of enantioselectivity of lipases and other synthetically useful hydrolases. Curr Org Chem. 2004, 8: 1009-1025. 10.2174/1385272043370230.View ArticleGoogle Scholar
- Jaeger KE, Reetz MT: Microbial lipases form versatile tools for biotechnology. Trends Biotechnol. 1998, 16: 396-403. 10.1016/S0167-7799(98)01195-0.View ArticleGoogle Scholar
- Sharma R, Chisti Y, Banerjee UC: Production, purification, characterization, and applications of lipases. Biotechnol Adv. 2001, 19: 627-662. 10.1016/S0734-9750(01)00086-6.View ArticleGoogle Scholar
- Shu ZY, Jiang H, Lin RF, Jiang YM, Lin L, Huang JZ: Technical methods to improve yield, activity and stability in the development of microbial lipases. J Mol Catal B-Enzym. 2010, 62: 1-8. 10.1016/j.molcatb.2009.09.003.View ArticleGoogle Scholar
- Gupta R, Gupta N, Rathi P: Bacterial lipases: an overview of production, purification and biochemical properties. Appl Microbiol Biotechnol. 2004, 64: 763-781. 10.1007/s00253-004-1568-8.View ArticleGoogle Scholar
- Gotor-Fernández V, Brieva R, Gotor V: Lipases: useful biocatalysts for the preparation of pharmaceuticals. J Mol Catal B-Enzym. 2006, 40: 111-120. 10.1016/j.molcatb.2006.02.010.View ArticleGoogle Scholar
- Noureddini H, Gao X, Philkana RS: Immobilized pseudomonas cepacia lipase for biodiesel fuel production from soybean oil. Bioresour Technol. 2005, 96: 769-777. 10.1016/j.biortech.2004.05.029.View ArticleGoogle Scholar
- Park DS, Oh HW, Heo SY, Jeong WJ, Shin DH, Bae KS, Park HY: Characterization of an extracellular lipase in Burkholderia sp. HY-10 isolated from a longicorn beetle. J Microbiol. 2007, 45: 409-417.Google Scholar
- Quyen TD, Vu CH, Le GT: Enhancing functional production of a chaperone-dependent lipase in Escherichia coli using the dual expression cassette plasmid. Microb Cell Fact. 2012, 11: 29- 10.1186/1475-2859-11-29.View ArticleGoogle Scholar
- Quyen DT, Schmidt-Dannert C, Schmid RD: High-level formation of active Pseudomonas cepacia lipase after heterologous expression of the encoding gene and its modified chaperone in Escherichia coli and rapid in vitro refolding. Appl Environ Microb. 1999, 65: 787-794.Google Scholar
- Rutherford N, Mourez M: Surface display of proteins by Gram-negative bacterial autotransporters. Microb Cell Fact. 2006, 5: 22- 10.1186/1475-2859-5-22.View ArticleGoogle Scholar
- Baek JH, Han MJ, Lee SH, Lee SY: Enhanced display of lipase on the escherichia coli cell surface, based on transcriptome analysis. Appl Environ Microbiol. 2010, 76: 971-973. 10.1128/AEM.02463-09.View ArticleGoogle Scholar
- Jung HC, Ko S, Ju SJ, Kim EJ, Kim MK, Pan JG: Bacterial cell surface display of lipase and its randomly mutated library facilitates high-throughput screening of mutants showing higher specific activities. J Mol Catal B-Enzym. 2003, 26: 177-184. 10.1016/j.molcatb.2003.05.007.View ArticleGoogle Scholar
- Lee SH, Choi JI, Han MJ, Choi JH, Lee SY: Display of lipase on the cell surface of Escherichia coli using OprF as an anchor and its application to enantioselective resolution in organic solvent. Biotechnol Bioeng. 2005, 90: 223-230. 10.1002/bit.20399.View ArticleGoogle Scholar
- Lee SH, Choi JI, Park SJ, Lee SY, Park BC: Display of bacterial lipase on the Escherichia coli cell surface by using FadL as an anchoring motif and use of the enzyme in enantioselective biocatalysis. Appl Environ Microbiol. 2004, 70: 5074-5080. 10.1128/AEM.70.9.5074-5080.2004.View ArticleGoogle Scholar
- Wilhelm S, Rosenau F, Becker S, Buest S, Hausmann S, Kolmar H, Jaeger KE: Functional cell-surface display of a lipase-specific chaperone. Chembiochem. 2007, 8: 55-60. 10.1002/cbic.200600203.View ArticleGoogle Scholar
- Yang TH, Kwon MA, Song JK, Pan JG, Rhee JS: Functional display of pseudomonas and burkholderia lipases using a translocator domain of EstA autotransporter on the cell surface of escherichia coli. J Biotechnol. 2010, 146: 126-129. 10.1016/j.jbiotec.2010.01.022.View ArticleGoogle Scholar
- Kaessler A, Olgen S, Jose J: Autodisplay of catalytically active human hyaluronidase hPH-20 and testing of enzyme inhibitors. Eur J Pharm Sci. 2011, 42: 138-147. 10.1016/j.ejps.2010.11.004.View ArticleGoogle Scholar
- Jose J, Jahnig F, Meyer TF: Common structural features of IgA1 protease-like outer membrane protein autotransporters. Mol Microbiol. 1995, 18: 378-380. 10.1111/j.1365-2958.1995.mmi_18020378.x.View ArticleGoogle Scholar
- Maurer J, Jose J, Meyer TF: Autodisplay: one-component system for efficient surface display and release of soluble recombinant proteins from Escherichia coli. J Bacteriol. 1997, 179: 794-804.Google Scholar
- Jose J, Bernhardt R, Hannemann F: Cellular surface display of dimeric Adx and whole cell P450-mediated steroid synthesis on E. coli. J Biotechnol. 2002, 95: 257-268. 10.1016/S0168-1656(02)00030-5.View ArticleGoogle Scholar
- Jose J, von Schwichow S: Autodisplay of active sorbitol dehydrogenase (SDH) yields a whole cell biocatalyst for the synthesis of rare sugars. Chembiochem. 2004, 5: 491-499. 10.1002/cbic.200300774.View ArticleGoogle Scholar
- Detzel C, Maas R, Jose J: Autodisplay of nitrilase from alcaligenes faecalis in E. Coli yields a whole cell biocatalyst for the synthesis of enantiomerically pure (R)-mandelic acid. ChemCatChem. 2011, 3: 719--725. 10.1002/cctc.201000382. 10.1002/cctc.201000382.View ArticleGoogle Scholar
- Kranen E, Steffan N, Maas R, Li S-M, Jose J: Development of a whole cell biocatalyst for the efficient prenylation of indole derivatives by autodisplay of the aromatic prenyltransferase FgaPT2. ChemCatChem. 2011, 3: 1200-1207. 10.1002/cctc.201000429. 10.1002/cctc.201000429.View ArticleGoogle Scholar
- Schumacher SD, Hannemann F, Teese MG, Bernhardt R, Jose J: Autodisplay of functional CYP106A2 in escherichia coli. J Biotechnol. 2012, 161: 104-112. 10.1016/j.jbiotec.2012.02.018.View ArticleGoogle Scholar
- Schumacher SD, Jose J: Expression of active human P450 3A4 on the cell surface of escherichia coli by autodisplay. J Biotechnol. 2012, 161: 113-120. 10.1016/j.jbiotec.2012.01.031.View ArticleGoogle Scholar
- Jose J, Maas RM, Teese MG: Autodisplay of enzymes-molecular basis and perspectives. J Biotechnol. 2012, 161: 92-103. 10.1016/j.jbiotec.2012.04.001.View ArticleGoogle Scholar
- Jose J, Meyer TF: The autodisplay story, from discovery to biotechnical and biomedical applications. Microbiol Mol Biol Rev. 2007, 71: 600-619. 10.1128/MMBR.00011-07.View ArticleGoogle Scholar
- Gawarzewski I, Smits SH, Schmitt L, Jose J: Structural comparison of the transport units of type V secretion systems. Biol Chem. 2013, 394: 1385-1398.View ArticleGoogle Scholar
- Economou A: Following the leader: bacterial protein export through the Sec pathway. Trends Microbiol. 1999, 7: 315-320. 10.1016/S0966-842X(99)01555-3.View ArticleGoogle Scholar
- Dyrløv Bendtsen J, Nielsen H, von Heijne G, Brunak S: Improved prediction of signal peptides: SignalP 3.0. J Mol Biol. 2004, 340: 783-795. 10.1016/j.jmb.2004.05.028.View ArticleGoogle Scholar
- Hantke K: Regulation of ferric iron transport in Escherichia coli K12: isolation of a constitutive mutant. Mol Gen Genet. 1981, 182 (2): 288-292. 10.1007/BF00269672.View ArticleGoogle Scholar
- Schultheiss E, Paar C, Schwab H, Jose J: Functional esterase surface display by the autotransporter pathway in Escherichia coli. J Mol Catal B-Enzym. 2002, 18: 89-97. 10.1016/S1381-1177(02)00063-2.View ArticleGoogle Scholar
- Jose J, Handel S: Monitoring the surface display of recombinant protiens by cysteine labeling ad flow cytometry. ChemBioChem. 2003, 4: 396-405. 10.1002/cbic.200200530.View ArticleGoogle Scholar
- Koebnik R, Kramer L: Membrane assembly of circularly permuted variants of the E. coli outer membrane protein OmpA. J Mol Biol. 1995, 250: 617-626. 10.1006/jmbi.1995.0403.View ArticleGoogle Scholar
- Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning. A Laboratory Manual. 1989, Cold Spring Harbour, NY: Cold Spring Harbor PressGoogle Scholar
- Winkler UK, Stuckmann M: Glycogen, hyaluronate, and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens. J Bacteriol. 1979, 138: 663-670.Google Scholar
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