R E S E A R C H L E T T E R
Amphoteric surfactant N -oleoyl- N -methyltaurine utilized by Pseudomonas alcaligenes with excretion of N -methyltaurine
Karin Denger1, Jutta Mayer1, Klaus Hollemeyer2& Alasdair M. Cook1
1Department of Biology, University of Konstanz, Konstanz, Germany; and2Institute of Biochemical Engineering, University of the Saarland, Saarbr ¨ucken, Germany
Correspondence:Alasdair M. Cook, Department of Biology, University of Konstanz, D-78457 Konstanz, Germany. Tel.:
149 753 188 4247; fax:149 753 188 2966;
e-mail: alasdair.cook@uni-konstanz.de
Editor: Christiane Dahl
Keywords
amidase; amphoteric detergent; degradation of amphoteric surfactant; excretion ofN - methyltaurine;N -oleoyl-N -methyltaurine;
Pseudomonas alcaligenes .
Abstract
The amphoteric surfactantN-oleoyl-N-methyltaurine, which is in use in skin-care products, was utilized by aerobic bacteria as the sole source of carbon or of nitrogen in enrichment cultures. One isolate, which was identified asPseudomonas alcaligenes, grew with the xenobiotic compound as the sole source of carbon and energy. The sulfonate moiety,N-methyltaurine, was excreted quantitatively during growth, while the fatty acid was dissimilated. The initial degradative reaction was shown to be hydrolytic and inducible. This amidase reaction could be demonstrated with crude cell extracts. The excreted N-methyltaurine could be utilized by other bacteria in cocultures. Complete degradation of similar natural compounds in bacterial communities seems likely.
Introduction
There are four major classes of laundry surfactants, nonionic, anionic, cationic, and amphoteric, in descending order of annual production (Knepper & Berna, 2003), and European law requires that they are fully biodegradable.
Most research has been focused on the degradability of these products (Knepperet al., 2003). Little is known about the microbiology and biochemistry of surfactant degradation except that communities of microorganisms are needed (van Ginkel, 1996), and an initial understanding of the microbial communities involved in the degradation of major cationic and anionic surfactants has been established (Kroon & van Ginkel, 2001; Schleheck et al., 2004a).
Amphoteric surfactants have received little attention.
N-Oleoyl-N-methyltaurine (Fig. 1) has had a long commercial life time and is known to be very mild on the skin (Kosswig & Stache, 1993); it now tends to be used in hair shampoos and soap-free skin-care products. It will, then, be widespread in sewage treatment plants. This xenobiotic compound shares structural features with natural taurine-containing lipids and emulsifiers (Fig. 1), and so it
might be a model for the degradation of these compounds, which are available for research in only small amounts.
We now report that N-oleoyl-N-methyltaurine can be utilized byPseudomonas alcaligenesOT as the sole source of carbon and energy for growth. The growth substrate is cleaved by an amidase andN-methyltaurine excreted. The latter could be degraded in coculture.
Materials and methods
Growth medium, cultivation, and growth conditions
Aerobic enrichment cultures to select for microorganisms able to utilizeN-oleoyl-N-methyltaurine as the sole source of carbon and energy, or of nitrogen for growth, were described elsewhere, as was the isolation of pure cultures (Weinitschkeet al., 2005; Mayeret al., 2006). The isolate used, strain OT, was grown aerobically at 301C in a 50 mM potassium phosphate-buffered salts medium, pH 7.2, which contained 0.25 mM magnesium sulfate, 20 mM ammonium chloride, trace elements (Thurnheer et al., 1986), and
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1–2 mM N-oleoyl-N-methyltaurine. Cultures (5 mL in 30-mL tubes) were incubated on a roller.Alcaligenes faecalis MT-1 (DSM 16964) (Weinitschkeet al., 2006) was grown in the same mineral medium with 20 mMN-methyltaurine as the carbon source.
Growth of strain OT withN-oleoyl-N-methyltaurine was followed in 50-mL cultures in 300-mL Erlenmeyer flasks shaken at 301C in a waterbath. Samples were taken at intervals to measure turbidity, to assay protein, and to determine the concentrations of N-methyltaurine, sulfate, and occasionally methylamine. Cells for the preparation of cell-free extracts were grown in 100-mL cultures of
1 mM N-oleoyl-N-methyltaurine- or 20 mM acetate salts medium in 1-L flasks, harvested by centrifugation (30 000g for 10 min at 41C) at the end of the exponential growth phase and disrupted by sonication. Debris and whole cells were removed by centrifugation (30 000gfor 10 min at 41C) and the supernatant fluid was stored frozen.
Analytical methods
Growth was followed as turbidity at 580 nm or quantified as protein in a Lowry-type reaction (Cook & H¨utter, 1981).
N-Methyltaurine and methylamine were determined after
O
N SO
N SO
O N
O
N SO
Dodecanoylsarcosyltaurine
H C O
O N
O N
SO C H NH
O
OH
Cerilipin
Found in the crab alimentary tract
AGluconobacter lipid
OH
1,2-Diacyl-3-α-D- glucuronopyranosyl-sn- glycerol taurineamide
A marine-bacterial lipid
H C O
O N
O
N SO
NH C H
O
AHyphosomonas lipid
O O
C H
O O
C H O
OH OH
O N
SO
O
N-Oleoyl-N- methyltaurine
Commercial surfactant Cell membrane
O
N SO
N SO
O O
β-Oxidation and growth
Oleate N-Methyltaurine
P. alcaligenes Transport
Amidase
Export –
–
–
–
–
–
–
– –
Fig. 1.Structures of some natural-product taurolipids and emulsifiers compared with the commercial surfactantN-oleoyl-N-methyltaurine, and the degradation of the latter byPseudomonas alcaligenesOT. The structures of the natural products are often representative, because several fatty acids may be present as substituents (Taharaet al., 1976; Kishimotoet al., 1993; Batrakovet al., 1996; Abrahamet al., 2004). Evidence is provided in the text for the presence ofN-oleoyl-N-methyltaurine amidase. It is axiomatic that sulfonates require transport into the cell (Grahamet al., 2002), so transport of N-oleoyl-N-methyltaurine and the export ofN-methyltaurine are hypothesized.
derivatization with 2,4-dinitrofluorobenzene and separation using reversed phase HPLC (Weinitschke et al., 2006).
Oleate, as the pentafluorobenzyl ester, was determined after separation using HPLC. The published method (Tsikas et al., 2003, 2004) was altered using (1) a vacuum centrifuge in place of streams of dinitrogen to remove solvents, (2) a smaller HPLC column, and (3) gradient elution (70–100%
acetonitrile in water) rather than isocratic conditions.
The presence of a surfactant in solution was assayed as foam-formation on shaking (Schlehecket al., 2004b). Ma- trix-assisted laser-desorption ionization time-of-flight MS (MALDI-TOF-MS) in the negative-ion mode (Tholeyet al., 2002) was used to prove thatN-oleoyl-N-methyltaurine was consumed during growth. Sulfate was quantified turbidime- trically as a suspension of BaSO4 (S¨orbo, 1987): samples were diluted 1 : 5 to prevent interference by N-oleoyl-N-methyltaurine. Standard methods were used for the Gram reaction, and to assay catalase or cytochrome c-oxidase activity (Gerhardt et al., 1994). Chromosomal DNA was isolated as described elsewhere (Desomeret al., 1991). Amplification of the 16S rRNA gene, sequencing, and sequence analysis were carried out as reported previously (Weinitschkeet al., 2006).
Enzyme assay
The amidase reaction was routinely assayed discontinuously at room temperature in 50 mM Tris/HCl buffer, pH 9.0, including 2 mM N-oleoyl-N-methyltaurine and crude cell extract (about 0.5 mg mL 1) with which the reaction was started. Samples (50mL) were taken at intervals and the reaction was stopped by addition of 1 mL 0.1 M NaHCO3, which was the buffer for the following derivatization reaction. The activity was lower in 50 mM potassium phosphate buffer pH 7.2, 50 mM 2-(N-morpholino) ethane- sulfonic acid buffer pH 5.8, or 50 mM cyclohexylaminopro- panesulfonic acid buffer pH 9.8. When the formation of oleate was examined, the compound in complete reaction mixtures (0.5 mL, brought to pH 5 by addition of 2 M HCl) was extracted into ethylacetate and derivatized as described above.
Chemicals
N-oleoyl-N-methyltaurine was provided by Clariant (Frank- furt, Germany) as the HOSTAPON TPHC [63%
(w/w) active matter]; our analysis of the material showed that it containedN-methyltaurine, which represented some 37% (w/w) of the preparation; correspondingly, no oleate could be detected in the preparation. Oleic acid (cis-9-octadecenoic acid) was from Fluka,N-methlytaurine from Merck, and pentafluorobenzylbromide from Acros.
Other chemicals, at the highest quality available, were provided by Acros, Fluka, Merck, or Sigma-Aldrich.
Results and discussion
Enrichment cultures and the identification of P. alcaligenes OT
Two aerobic enrichment cultures, one nitrogen-limited and one carbon-limited, which involved N-oleoyl-N-methyl- taurine and an inoculum from the local communal sewage works, were initiated as part of a larger experiment, some of which has been reported elsewhere (Styp von Rekowskiet al., 2005; Weinitschkeet al., 2005, 2006). Four isolates were obtained, one from the nitrogen-limited culture and three from the carbon-limited culture, and isolate OT, from the carbon-limited cultures, was chosen for further work. The organism was shown to utilize the surfactant by observing (1) substrate-dependent growth (turbidity and microscopy) and (2) that the foam caused by the presence of the surfactant disappeared during growth.
Strain OT was a strictly aerobic, straight, motile, Gram-negative rod (about 2mm0.5mm), which was oxidase-positive and catalase-positive, and grew not only at 301C but also at 411C. No fluorescent pigment was formed.
This was consistent (see Palleroni, 1984) with the sequence identity (100%) of a 747-bp fragment of the 16S rRNA gene of strain OT with the corresponding sequence from the type strain ofP. alcaligenes(LMG 1224T). The identification was confirmed by growth with acetate, succinate, fumarate, lactate, propionate, andL-malate, whereas glucose, adipate,
D-malate, benzoate, and betaine did not support growth (see Palleroni, 1984). Pseudomonas alcaligenes OT was deposited with the German Culture Collection DSMZ, Braunschweig, Germany, under the accession number DSM 19550.
Growth ofP. alcaligenes OT
Strain OT grew exponentially inN-oleoyl-N-methyltaurine- salts medium with a specific growth rate (m) of 0.2 h 1and a yield of 105 g protein (mol surfactant) 1(Fig. 2a and b). We had no direct determination of the surfactant (M= 403.6), but the foaming was eliminated during growth and the species at m/z= 402.8 = [M-H] 1, detected using MALDI-TOF-MS in the negative-ion mode at the start of the experiment, was absent at the end of growth.
This was interpreted as quantitative disappearance of substrate.
Concomitant with growth, a product was released, which, derivatized, cochromatographed (HPLC) with derivatized authentic N-methyltaurine, and was thus identified as N-methyltaurine, the sulfonate moiety of the surfactant.
N-Methyltaurine was already present at the beginning of growth because the commercial preparation ofN-oleoyl- N-methyltaurine contained it. The additional amount
formed corresponded to the concentration of the surfactant added at the start of the experiment (Fig. 2b). No release of sulfate, the usual product from sulfonate-sulfur in aerobes (Cook & Denger, 2002), or methylamine, a product from N-methyltaurine (Weinitschke et al., 2006), was detected during growth (not shown). These data implied that the sulfonate moiety of the surfactant is not further degraded.
It was inferred from the 1 : 1 stoichiometry ofN-oleoyl- N-methyltaurine toN-methyltaurine that the surfactant was cleaved to N-methyltaurine, which was excreted, and to oleate (Fig. 1), which was further degraded. Assuming complete degradation of the 18 C-atoms of oleate, a yield of 5.8 g protein (mol carbon) 1was calculated, which indicates quantitative utilization (Cook, 1987). The fatty acids oleate, laurate, palmitate, and stearate were tested as sole growth substrates for strain OT. Each supported growth, but the insoluble compounds did not allow satisfactory measure- ment of the molar growth yield.N-Methyltaurine was not a
growth substrate, consistent with the release of this sulfonate after growth withN-oleoyl-N-methyltaurine.
An amidase to cleaveN-oleoyl-N-methyltaurine intoN-methyltaurine and oleate
Hydrolysis of the amide N-oleoyl-N-methyltaurine to N-methyltaurine and oleate by an amidase was hypothe- sized, analogous to the degradation of N-acetyltaurine by Delftia acidovorans NAT (Mayeret al., 2006) and of taur- ocholate by several organisms (R¨oschet al., 2008). Amidase activity could be demonstrated in strain OT: incubation of crude extract of cells grown withN-oleoyl-N-methyltaurine resulted in the formation of N-methyltaurine from the surfactant. Simultaneously, the release of oleate could be detected (c.f. Fig. 1). The reaction mixture contained neither an electron-acceptor nor an amino-group acceptor, and so the reaction was considered to be hydrolytic. The amidase had a specific activity of 0.5 mkat (kg protein) 1in extracts of surfactant-grown cells and 0.1 mkat (kg protein) 1 in extracts of acetate-grown cells (Fig. 3) (1 kat = 1 mol s 1).
The amidase was thus inducible.
This finding is another example of the bacterial degrada- tion of a sulfonated amide with an organosulfonate as an intermediate or an excretion product, as shown for N-acetyltaurine (Mayer et al., 2006), and taurocholate (R¨oschet al., 2008). TheN-acetyltaurine amidase was found to be substrate specific in contrast to many broad substrate- range amidases (Webb, 1992). The substrate range of the N-oleoyl-N-methyltaurine amidase remains to be eluci- dated. Other natural taurine-containing lipids (Fig. 1) might also be degraded by specific amidases as initial reaction liberating taurine or taurine-derivatives that are subject to established taurine-degradative pathways (Cook & Denger, 2006).
0 0.01
0.1 1
A580 nm
Time (h)
5 10 15 20
(a)
0 0.0 0.5 1.0 1.5 2.0 2.5
N-Methyltaurine (mM)
Protein (µg mL–1)
50 100 150 200 250
(b)
Fig. 2.Growth ofPseudomonas alcaligenesOT with 2 mM N-oleoyl- N-methyltaurine as carbon source. (a) Semi-logarithmic plot of growth;
(b) product (N-methyltaurine) concentration as a function of growth (protein).
0 10 20 30 40
0.0 0.3 0.6 0.9 1.2
N-Methyltaurine (mM)
Time (min)
Fig. 3. Formation ofN-methyltaurine fromN-oleoyl-N-methyltaurine in extracts ofN-oleoyl-N-methyltaurine-grown cells () or of acetate-grown cells () ofPseudomonas alcaligenesOT.
Complete degradation ofN-oleoyl-N- methyltaurine in coculture
Outgrown medium of strain OT after growth withN-oleoyl- N-methyltaurine was filter-sterilized and inoculated with A. faecalisMT-1, which is known to degradeN-methyltaur- ine to methylamine (Weinitschke et al., 2006).Alcaligenes faecalis MT-1 grew and methylamine was produced as identified using HPLC after derivatization. This was inter- preted as confirmation of the identity ofN-methyltaurine and a further step in the complete degradation of the amphoteric surfactant. Methylamine was shown to be assimilated by different Paraccocus strains (Weinitschke et al., 2006); thus, complete degradation of the surfactant in a microbial community is anticipated as shown for example the major anionic surfactant linear alkylbenzene- sulfonate (Schleheck et al., 2004a) (see also van Ginkel, 1996). Degradation in cocultures presumably represents the situation in the environment when the system has to cope with complex substrates, be they xenobiotics, as in the current study, or natural compounds.
Acknowledgements
We are grateful to D. Tsikas (Institute of Clinical Pharma- cology, Hannover Medical School, Hannover, Germany) for a preliminary identification of oleate by GC-MS and advice on the determination of oleate. We thank Marijke I. Baldock and Anja Dahler for data generated in a practical course for advanced students, and Theo H.M. Smits for help with 16S rRNA gene sequencing. Bodo Philipp kindly gave us permission to use his L2-facility. The project was funded by the University of Konstanz.
Statement
The partial 16S rRNA gene sequence determined in this paper was identical to bases 1–747 of the entry for the corresponding type strain (Pseudomonas alcaligenes LMG 1224T, GenBank accession no. Z76653), and so no new entry was submitted.
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