Solubilization of phenanthrene by recombinant protein bioemulsans

Biochemical Engineering Journal 16 (2003) 169–174

Solubilization of phenanthrene by recombinant protein bioemulsans Amir Toren, Yuval Gefen, Eliora Z. Ron, Eugene Rosenberg∗ Department of Molecular Microbiology and Biotechnology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978, Israel Received 28 June 2002; accepted after revision 11 October 2002

Abstract AlnA is a 35.77 kDa protein responsible for the hydrocarbon emulsifying and solubilizing activity of the Acinetobacter radioresistens KA53 bioemulsifier alasan. Deletion and substitution derivatives of AlnA were produced by site-directed polymerase chain reaction (PCR) mutagenesis and then used to study their ability to solubilize phenanthrene. AlnA contains four hydrophobic regions. Deletions of three or more amino acids from the hydrophobic N-terminus of AlnA caused a large decrease in solubilizing activity, whereas deletions from the C-terminus of 4, 7, 18 and 35 amino acids resulted in the loss of only 9, 22, 35 and 46% of the activity, respectively. Deletions of any of the three internal hydrophobic regions of AlnA caused a greater than 50% loss in solubilizing activity. The solubilizing activity of chimeric proteins, containing sequences from both AlnA and the homologous (but not surface active) Escherichia coli outer membrane protein A (OmpA), indicated that the most important sequences needed for solubilizing phenanthrene are on the N-terminal half of AlnA, especially the two hydrophobic loops (amino acids 37–45 and 164–171) on the ␤-barrel structure. Gel electrophoresis experiments demonstrated that AlnA and derivatives which retained high phenanthrene-solubilizing activity formed 210 kDa complexes in the presence of phenanthrene. The relationship between AlnA structure and its surface activity is discussed. © 2003 Elsevier Science B.V. All rights reserved. Keywords: AlnA; Bioemulsifier; OmpA; Phenanthrene; Alasan

1. Introduction Microorganisms synthesize a wide variety of high and low molecular-mass-bioemulsifiers [1]. The high-molecular-mass bioemulsifiers, referred to as bioemulsans [2], are amphipathic polysaccharides, proteins, lipopolysaccharides, lipoproteins or complex mixtures of these biopolymers that stabilize oil-in-water emulsions. Although most research on bioemulsans has focused on potential industrial and environmental applications [3–9], there is also a growing interest in the natural role of bioemulsans for the producing microorganism [10]. To better understand how bioemulsans function in the growth and survival of microorganisms it is essential to elucidate their detailed chemical structures as well as the genes required for their biosynthesis. Alasan, the bioemulsifier of Acinetobacter radioresistens KA53, is a high molecular-mass complex of an alanine-containing polysaccharide and three proteins [11]. The protein fraction is essential for emulsifying activity and maintaining the structure of the complex [12]. Separation and purification of the three alasan proteins demonstrated that one of the proteins, with an apparent molecular mass of ∗ Corresponding author. Tel.: +972-3-640-9838; fax: +972-3-642-9377. E-mail address: [email protected] (E. Rosenberg).

45 kDa, was the surface active component of the complex [13]. The 45 kDa protein had a higher specific emulsifying activity than the alasan complex. The gene coding for the 45 kDa protein was cloned, sequenced and expressed in Escherichia coli [14]. The recombinant protein AlnA (35.77 kDa without the leader sequence) had an amino acid sequence homologous to the E. coli outer membrane protein A (OmpA) and contained the emulsifying activity of the active 45 kDa glycoprotein. In addition to their emulsifying activities, alasan, the 45 kDa protein and recombinant AlnA were highly effective in solubilizing polyaromatic hydrocarbons (PAHs) [15,16]. Furthermore, alasan stimulated the growth of Sphingomonas paucimobilis on phenanthrene and fluoranthene [15]. Solublization of hydrophobic substances by low-molecular-mass surfactants has been studied extensively. The mechanisms proposed for enhanced solubility include solubilization in the hydrophobic core of multi-molecular surfactant structures formed at above-aggregation concentrations, such as micelles [17,18] and liposomes [19], decreased surface tension of the solvent [20], and interaction with hydrophobic tails of surfactant monomers [21]. Except for alasan, there has only been one report of a high-molecular-mass bioemulsifier that solubilizes PAHs. Burd and Ward [22] demonstrated that a protein/

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A. Toren et al. / Biochemical Engineering Journal 16 (2003) 169–174

lipopolysaccharide bioemulsifier produced by Pseudomonas marginalis PD-14B enhanced the solubilization of PAH crystals. The nature of the solubilization mechanism was not addressed by these authors. In order to begin to understand the relationship between the structure of AlnA and its surface activity, we constructed deletions and substitutions in AlnA by site-directed polymerase chain reaction (PCR) mutagenesis [23]. In this study, several of these mutant proteins were tested for their ability to enhance the solubility of phenanthrene. The strategy used in this study was based on: (i) AlnA has a homologous amino acid sequence and transmembrane hydrophobicity profile to the outer membrane protein A (OmpA) of E. coli; (ii) the ␤-barrel structure of the E. coli OmpA transmembrane domain has been determined to a resolution of 1.65 Å [24,25]; (iii) in spite of its structural similarity to AlnA, E. coli OmpA has no emulsifying activity [14]. Accordingly, we hypothesized that regions of AlnA that showed large increases in hydrophobicity compared to E. coli OmpA were likely to be involved in binding to hydrocarbons and required for solubilization of hydrocarbons. Four hydrophobic regions are present in AlnA and absent from OmpA [23]: (1) the N-terminal tail (amino acids 2–9, VTITPLML), (2) and (3) two loops on the ␤-barrel region (amino acids 37–45 and 47–53, LFVGAALGV and LTPWLGF, respectively), and (4) the C-terminal end of the ␤-barrel (amino acids 164–171, LAGLNVVL). Thus, we constructed modifications of AlnA in these hydrophobic regions and tested their solubilizing activities.

2. Materials and methods 2.1. Recombinant AlnA and derivatives Recombinant AlnA was prepared from E. coli ER2566 that had been transformed with pAT1 as previously described [14]. pAT1 is a high expression vector containing a T7 promoter and the full length 1.044 kb (minus the signal peptide) open reading frame of alnA. The preparation of all of the deletion mutants used in this study was described previously [23], using the overlap extension PCR method [26]. PCR reactions that contain OmpA fragments had genomic OmpA as their template. All mutations were confirmed by sequencing the whole ligated PCR fragment. 2.2. Solubilization of phenanthrene To determine the kinetics of solubilization, 100 ␮g phenanthrene (Aldrich, Milwaukee, WS, USA) was crystallized in the bottom of a 1 ml quartz cuvette. The cuvette was placed in a six-compartment holder of an Ultraspec 2000 spectrophotometer (Pharmacia, Uppsala, Sweden), and 1 ml of assay buffer (20 mm Tris, pH 8.0) containing 40 ␮g AlnA, or an AlnA derivative, was added to the cuvette. Solubilization was performed without shaking. A252

measurements were taken at room temperature every 5 min for 2 h with the parallel non-synchronized mode of the kinetics software package provided by the manufacturer. The phenanthrene and assay buffer (without any emulsifier) was used as the control. Absorbancy reading was converted to concentration of phenanthrene from a standard calibration curve. The data presented are the average values of three determinations after equilibrium had been reached (2 h). The standard deviation for all measurements was less than 5% of the average value. 2.3. Gel electrophoresis of protein–phenanthrene complexes Polyacrylamide gel (8%) electrophoresis was performed to estimate the size of the soluble complexes formed between phenanthrene and AlnA derivatives. Approximately 100 ␮g of phenanthrene in chloroform was evaporated to dryness in glass tubes. To the dry material was added 20 ␮g AlnA, or an AlnA derivative, in 0.1 ml 20 mM Tris–HCl buffer, pH 8.0. After incubating the mixture for 2 h at room temperature, 30 ␮l samples were loaded onto the gel. The running buffer was 192 mM glycine and 25 mM Tris–HCl (pH 8.3). Sweet potato ␤-amylase (200 kDa), purchased from Bio-Rad Co. Hercules, CA, was used as a molecular mass marker. Gels were stained with Coomassie brilliant blue. 3. Results An increase in the aqueous solubility of phenanthrene was obtained in the presence of AlnA and all AlnA derivatives tested. A typical kinetic experiment is shown in Fig. 1. The aqueous solubility of phenanthrene was low, 0.13 ␮g/ml, similar to values reported in the literature [27]. As reported previously [16], 40 ␮g/ml AlnA increased the solubility of phenanthrene to 3.7 ␮g/ml, or 28.5-fold. Deletion of 2 amino acids from the N-terminus of AlnA yielded a derivative that had similar solubilizing activity as AlnA. The derivative with deletion of amino acids 310–319 solubilized only 1.7 ␮g/ml phenanthrene, and the rate of solubilization was slower. The E. coli OmpA protein solubilized only 0.6 ␮g/ml phenanthrene. Table 1 summarizes the solubilization activity of the 15 AlnA deletion mutants studied. Deletion of one, two or three amino acids from the hydrophobic N-terminus of AlnA did not significantly change the activity. However, removal of the fourth and fifth amino acids caused a 24–26 and 59–61%, respectively, decrease in activity. Deletions from the C-terminus of 4, 7, 18, 35 and 67 amino acids resulted in the loss of 21, 26, 37, 53 and 62% of the solubilizing activity, respectively, based on molarity. Deletion of 143 amino acids from the C-terminus deletion resulted in a protein with only 12–19% of the solubilizing activity of the parent protein AlnA. The three deletions in the interior of AlnA caused a 51–64% decrease in activity, the largest decrease occurring with the deletion in a hydrophobic region (161–172).

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Fig. 1. Kinetics of phenanthrene solubilization in the presence of buffer alone (–), and 40 ␮g/ml each of AlnA (䊉), AlnA minus amino acids 1 and 2 (䊐), AlnA minus amino acids 310–319 (䊊) and E. coli OmpA ().

The solubilizing activity of chimeric proteins, containing sequences from both AlnA and E. coli OmpA are shown in Table 2. The OmpA control had very little activity (16% of AlnA). The mutant protein which contained the N-terminal half (1–176) from OmpA and the C-terminal half (177–327) from AlnA also had little activity (5%). Replacement of one of the hydrophobic regions of AlnA (164–171) with the homologous (non-hydrophobic) sequence of OmpA caused

Table 1 Solubilization of phenanthrene by deletion mutants of AlnA Position in AlnA of deleted amino acidsa

Equilibrium solubilityb (␮g/ml)

(M/M)c

(%)d

(%)e

None (AlnA)

3.7

19

100

100

N-terminal region 1 1 and 2 1–3 1–4 1–5 1–6

3.6 3.2 3.8 2.8 1.5 1.8

18 16 19 14 7.4 8.9

97 86 103 76 41 49

95 84 100 74 39 47

Internal region 161–172 201–212 310–319

1.4 1.8 1.7

6.8 8.8 8.3

38 49 46

36 46 44

C-terminal region 324–327 321–327 310–327 293–327 260–327 184–327

3.0 2.9 2.4 2.0 2.6 0.7

15 14 12 10 7.3 2.2

81 78 65 54 70 19

79 74 63 47 38 12

Relative to AlnA

In all experiments, protein concentration was 40 ␮g/ml. Solubility of phenanthrene in the assay buffer was 0.13 ␮g/ml. c Moles phenanthrene solubilized per mole protein. d Based on wt/wt. e Based on M/M. a

b

a 68% loss in activity compared to AlnA. Replacement of the other hydrophobic loop of the ␤-barrel structure of AlnA (37–45) with the corresponding OmpA sequence caused a 35% loss of activity. The mutant which contained the N-terminal half (1–171) from AlnA and the remainder (172–327) from OmpA retained 41% of the solubilizing activity of intact AlnA. Clearly, the most important sequences needed for solubilizing phenanthrene are on the N-terminal half of AlnA, especially the two hydrophobic loops (164–171 and 37–45) on the ␤-barrel structure. The interaction of AlnA and derivatives with phenanthrene leads to the formation of an oligomeric complex with an apparent molecular mass of ca. 220 kDa (Fig. 2). AlnA without the hydrocarbon is a monomer in aqueous solution (Fig. 2, lane G). Incubation of AlnA with phenanthrene led to the formation of an AlnA–Phe complex of ca. 220 kDa, as determined by native gel electrophoresis (Fig. 2, lane F). Based on net the amount of phenanthrene solubilized by AlnA (3.6 ␮g/ml, Table 1) and the known molecular weights of AlnA and phenanthrene, the 220 kDa complex would consist of six molecules of AlnA and 130 molecules of phenanthrene. Deletion 184–327, which showed a very low solubilizing activity, did not form a complex with Table 2 Solubilization of phenanthrene by E. coli OmpA/AlnA chimeric proteinsa Amino acid sequences from AlnA

OmpA

Equilibrium solubility (␮g/ml)

0 177–327 1–7 and 177–327 1–163 and 172–327 1–36 and 46–327 1–171

1–217 1–176 8–176 141–171 37–45 172–327

0.6 0.2 0.6 1.2 2.4 1.5

Relative to AlnA (%) 16 5 16 32 65 41

a Protein concentration was 40 ␮g/ml. Data are presented as wt/wt since all proteins are of approximately the same size.

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A. Toren et al. / Biochemical Engineering Journal 16 (2003) 169–174 Table 3 Interaction of AlnA derivatives with hydrocarbons

Fig. 2. Native gel electrophoresis of mixtures of phenanthrene and AlnA derivatives. Numbers to the left indicate molecular mass in kilodaltons: lane A, ␤-amylase molecular marker; lane B, AlnA minus amino acids 1–4; lane C, AlnA minus amino acids 260–327; lane D, AlnA minus amino acids 184–327; lane E, AlnA minus amino acids 1–5; lane F, AlnA; lane G, AlnA without phenanthrene. Each of the proteins was mixed with phenanthrene prior to loading on the gel as described in Section 2.

phenanthrene (Fig. 2, lane D), whereas deletion 1–4, which had relatively high solubilizing activity, formed the 220 kDa complex (Fig. 2, lane B). Deletions 1–5 and 260–327, which showed intermediate values of solubilization showed mixtures of the monomeric and hexameric forms of AlnA in the presence of phenanthrene (Fig. 2, lanes C and E). Thus, it appears that the ability of AlnA to interact with phenanthrene and form the 220 kDa complex is associated with the enhanced solubilizing activity of the bioemulsifier. A 220 kDa complex was also formed when AlnA was incubated with a number of different polyaromatic hydrocarbons, including anthracene, fluorine, hexaphenylbenzene, perylene and pyrene. Following native gel electrophoresis, only a single protein band was observed with an apparent molecular size of 220 kDa (data not presented). Thus, AlnA is converted from a monomer to a hexamer in the presence of a variety of PAHs.

4. Discussion Table 3 compares the solubilization data obtained in this study with recently reported results on hexadecane-binding and emulsifying activity of the identical AlnA derivatives [23]. For any bioemulsan to solubilize or emulsify a water-insoluble hydrophobic material it must first bind to the surface of that material, presumably via hydrophobic regions on the bioemulsan. AlnA contains 4 hydrophobic regions on the N-terminal half of the molecule (amino acids 2–9, 37–45, 47–53 and 164–171). From amino acid 172 to the C-terminus, hydrophobic sequences are absent [14]. These sequence analyses are consistent with the fact that all derivatives which contained one or more of these hydrophobic regions bound avidly to hexadecane (Table 3, column 2). The only two derivatives which did not bind hexadecane were the chimeric proteins which lacked all 4 hydrophobic

AlnA derivative

Phenanthrene solubilizationa (% of AlnA)

Adhesion to hexadecaneb (%)

Emulsifying activity (% of AlnA)b

AlnA OmpA

100 16

97 13

100 0

N-terminal deletions 1 97 1 and 2 86 1–3 103 1–4 76 1–5 41 1–6 49

90 89 92 90 92 90

94 94 55 6 7 12

Internal deletions 161–172 201–212 310–319

38 49 46

90 91 91

0 95 36

C-terminal deletions 324–327 321–327 310–327 293–327 260–327 184–327

81 78 65 54 70 19

89 92 77 80 68 65

93 95 75 6 5 8

Chimeric proteins (position of OmpA insertion) 1–176 5 15 8–176 16 13 164–171 32 28 37–45 65 81 172–327 41 47 a b

1 6 33 12 12

Data from Tables 1 and 2, using wt/wt. Data from Toren et al. [23].

regions (OmpA insertion 1–176) and 3 hydrophobic regions (OmpA insertion 8–176). The chimeric protein in which the entire C-terminal half (172–327) was replaced by OmpA still bound to hexadecane. Removal of single hydrophobic regions did not interfere with hexadecane binding, e.g. deletions 1–6 and 161–172 and chimeric protein substitution 37–45. Thus, there are multiple hexadecane-binding sites on AlnA, any one of which is sufficient to ensure binding of the protein to hydrocarbons. Emulsifying activity of AlnA (Table 3, column 3) is generally more sensitive to small changes in structure than hydrocarbon-binding. Deletion of the 4–6 N-terminal amino acids caused a 88–94% loss in emulsifying activity, whereas these derivatives still showed 90% binding to hexadecane. Similarly, deletion of the hydrophobic region at 161–172 caused a complete loss of emulsifying activity, while the derivative retained hexadecane-binding properties. Also, the chimeric protein in which the AlnA hydrophobic sequence at 37–45 was replaced by the corresponding (non-hydrophobic) sequence of OmpA showed 81% hexadecane-binding and only 12% emulsifying activity. The conclusion of the studies of the emulsifying activity of AlnA as a function of structure is that all four hydrophobic regions are required for activity, suggesting that multiple hydrophobic binding interactions are used to

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ensure that the protein assumes the correct conformation on the oil droplet surface to prevent coalescence. Emulsifying activity and hexadecane-binding studies of AlnA and derivatives were performed at high hydrocarbon to protein ratios (ca. 2000:1), whereas the solubilization studies reported here were carried out at phenanthrene to protein ratios of 2.5. A key to understanding the mechanism of solubilization was the finding that AlnA and the active derivatives form 220 kDa complexes in the presence of phenanthrene, as determined by native gel electrophoresis. Based on the size of the complex, the amount of phenanthrene solubilized and the molecular weights of AlnA and phenanthrene, it can be calculated that the complex contains six molecules of AlnA and 130 molecules of phenanthrene. Two points should be emphasized: (1) in the presence of phenanthrene, AlnA was quantitatively converted to the octamer, with no sign of monomer or intermediates, and (2) once formed, the complex appears to be stable in aqueous solution, since it migrated as a sharp, single band during electrophoresis. No phenanthrene was present in the running buffer. Formation of the 220 kDa complex appears to be essential for high phenanthrene-solubilizing activity. AlnA and derivatives which showed high solubilizing activity formed the complex, whereas mutant proteins which showed relatively low solubilizing activity did not form the complex. Furthermore, induction of AlnA complex formation is not limited to phenanthrene since a variety of other PAHs, including anthracene, fluorine, hexaphenylbenzene, perylene and pyrene, also induced formation of the 220 kDa complex. Based on these findings, we suggest that the following working hypothesis to explain the mechanism of solubilization of hydrocarbons by AlnA: (1) initial binding of a few hydrocarbon molecules to hydrophobic regions on the protein, (2) conformational changes in AlnA, leading to aggregation and the formation of a specific hexamer structure which contains one or more hydrophobic “pockets”, and (3) uptake of additional hydrocarbon into the “pockets”. The data presented here represent the first structure/function study of the solubilization of a hydrocarbon by a chemically defined bioemulsan. The ability of a pure protein to increase the water solubility of hydrophobic compounds has numerous potential applications, such as in the preparation of drug delivery systems, cosmetic formulations and bioremediation.

Acknowledgements This investigation was supported by the Pasha Gol Chair for Applied Microbiology and the Manja and Morris Leigh Chair in Biophysics and Biotechnology. References [1] E. Rosenberg, E.Z. Ron, High- and low-molecular-mass microbial surfactants, Appl. Microbiol. Biotechnol. 52 (1999) 154–162.

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[2] E. Rosenberg, E. Ron, Bioemulsans: microbial polymeric emulsifiers, Curr. Opin. Biotechnol. 8 (1997) 313–316. [3] I.M. Banata, Biosurfactants production and possible use in microbial enhanced oil recovery and oil pollution remediation: a review, Biosource Technol. 51 (1995) 1–12. [4] V. Klekner, N. Kosaric, Biosurfactants for cosmetics, In: N. Kosaric (Ed.), Biosurfactants: Production, Properties, Applications, Surfactant Science Series, vol. 48, Dekker, New York, p. 329–372. [5] M.N. Patel, K.P. Gopinathan, Lysozyme-sensitive bioemulsifier for immiscible organophosphorus pesticides, Appl. Environ. Microbiol. 52 (1986) 1224–1226. [6] K. Robinson, M. Gosh, Z. Shi, Mineralization enhancement of non-aqueous phase and soil-bound PCB using biosurfactant, Water Sci. Technol. 34 (1996) 303–309. [7] E. Rosenberg, E.Z. Ron, Surface active polymers from the genus Acinetobacter, In: D.L. Kaplan (Ed.), Biopolymers from Renewable Resources, Springer, Berlin, Heidelberg, New York, 1998, p. 281–289. [8] R. Shepherd, J. Rockey, I.W. Sutherland, S. Roller, Novel bioemulsifiers from microorganisms for use in foods, J. Biotechnol. 40 (1995) 207–217. [9] F. Volkering, A. Breure, W. Rulkens, Microbiological aspects of surfactant use for biological soil remediation, Biodegradation 8 (1997) 401–417. [10] E. Ron, E. Rosenberg, Natural roles of biosurfactants, Environ. Microbiol. 3 (2001) 229–326. [11] S. Navon-Venezia, Z. Zosim, A. Gottlieb, R. Legmann, S. Carmeli, E.Z. Ron, E. Rosenberg, Alasan, a new bioemulsifier from Acinetobacter radioresistens, Appl. Environ. Microbiol. 61 (1995) 3240–3244. [12] S. Navon-Venezia, E. Banin, E.Z. Ron, E. Rosenberg, The bioemulsifier alasan: role of protein in maintaining structure and activity, Appl. Microbiol. Biotechnol. 49 (1998) 382–384. [13] A. Toren, S. Navon-Venezia, E.Z. Ron, E. Rosenberg, Emulsifying activity of purified alasan proteins from Acinetobacter radioresistens KA53, Appl. Environ. Microbiol. 67 (2001) 1102–1106. [14] A. Toren, E. Orr, Y. Paitan, E.Z. Ron, E. Rosenberg, The active component of the bioemulsifier alasan from Acinetobacter radioresistens KA53 is an OmpA-like protein, J. Bacteriol. 184 (2002a) 165–170. [15] T. Barkay, S. Navon-Venezia, E.Z. Ron, E. Rosenberg, Enhancement of solubilization and biodegradation of polyaromatic hydrocarbons by the bioemulsifier alasan, Appl. Environ. Microbiol. 65 (1999) 2697–2702. [16] A. Toren, E.Z. Ron, R. Bekerman, E. Rosenberg, Solubilization of polyaromatic hydrocarbons by recombinant bioemuslifier AlnA, Appl. Microbiol. Biotechnol. 59 (2002) 580–584. [17] D.A. Edwards, R.G. Luthy, Z. Liu, Solubilization of polycyclic aromatic hydrocarbons in micellar non-ionic surfactant solutions, Environ. Sci. Technol. 25 (1991) 127–133. [18] F. Volkering, A.M. Breure, J.G. van Andel, W.H. Rulkens, Influence of non-ionic surfactants on bioavailability and biodegradation of polycyclic aromatic hydrocarbons, Appl. Environ. Microbiol. 61 (1995) 1699–1705. [19] R.M. Miller, R. Bartha, Evidence from liposome encapsulation for transport-limited microbial metabolism of solid alkanes, Appl. Environ. Microbiol. 55 (1989) 269–274. [20] Y.M. Zhang, R.M. Miller, Enhanced octadecane dispersion and biodegradation of a Pseudomonas rhamnolipid surfactant (biosurfactant), Appl. Environ. Microbiol. 58 (1992) 3276–3282. [21] M. Almgren, G. Greiser, J.R. Powel, J.K. Thomas, A correlation between the solubility of aromatic hydrocarbons in water and micellar solutions, with their normal boiling points, J. Chem. Eng. Data 24 (1979) 285–287. [22] G. Burd, O.P. Ward, Bacterial degradation of polycyclic aromatic hydrocarbons on agar plates: the role of biosurfactants, Biotechnol. Tech. 10 (1996) 371–374.

174

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[23] A. Toren, G. Segal, E.Z. Ron, E. Rosenberg, Structure/function studies of the recombinant protein bioemulsifier AlnA, Environ. Microbiol. 4 (2002) 257–261. [24] A. Pautsch, G.E. Schultz, Structure of the outer membrane protein a transmembrane domain, Nature Struct. Biol. 5 (1998) 1013–1017. [25] A. Pautsch, G.E. Schultz, High-resolution structure of the OmpA membrane domain, J. Mol. Biol. 298 (2000) 273–282.

[26] S.N. Ho, H.D. Hunt, R.M. Horton, J.K. Pallen, L.R. Pease, Site-directed mutagenesis by overlap extension using the polymerase chain reaction, Gene 77 (1989) 55–59. [27] H.B. Klevens, Solubilization of polycyclic hydrocarbons, J. Phys. Colloid. Chem. 54 (1954) 283–298.