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Applied and Environmental Microbiology, March 2001, p. 1102-1106, Vol. 67, No. 3
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.3.1102-1106.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Emulsifying Activities of Purified Alasan Proteins
from Acinetobacter radioresistens KA53
Amir
Toren,
Shiri
Navon-Venezia,
Eliora Z.
Ron, and
Eugene
Rosenberg*
Department of Molecular Microbiology and
Biotechnology, The George S. Wise Faculty of Life Sciences, Tel
Aviv University, Ramat Aviv 69978, Israel
Received 24 July 2000/Accepted 15 November 2000
 |
ABSTRACT |
The bioemulsifier of Acinetobacter radioresistens KA53,
referred to as alasan, is a high-molecular-weight complex of
polysaccharide and protein. The emulsifying activity of the purified
polysaccharide (apo-alasan) is very low. Three of the alasan proteins
were purified by preparative sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and had apparent molecular masses of 16, 31, and 45 kDa. Emulsification assays using the isolated alasan proteins
demonstrated that the active components of the alasan complex are the
proteins. The 45-kDa protein had the highest specific emulsifying
activity, 11% higher than the intact alasan complex. The 16- and
31-kDa proteins gave relatively low emulsifying activities, but they were significantly higher than that of apo-alasan. The addition of the
purified 16- and 31-kDa proteins to the 45-kDa protein resulted in a
1.8-fold increase in the specific emulsifying activity and increased
stability of the oil-in-water emulsion. Fast-performance liquid
chromatography analysis indicated that the 45-kDa protein forms a dimer
in nondenaturing conditions and interacts with the 16- and 31-kDa
proteins to form a high-molecular-mass complex. The 45-kDa protein and
the three-protein complex had substrate specificities for
emulsification and a range of pH activities similar to that of alasan.
The fact that the purified proteins are active emulsifiers should
simplify structure-function studies and advance our understanding of
their biological roles.
| |
INTRODUCTION |
Microorganisms synthesize a wide
variety of high- and low-molecular-mass bioemulsifiers
(16). The low-molecular-mass bioemulsifiers are generally
glycolipids, such as trehalose lipids, sophorolipids, and rhamnolipids,
or lipopeptides, such as surfactin, gramicidin S, and polymyxin. The
high-molecular-mass bioemulsifiers are amphipathic polysaccharides,
proteins, lipopolysaccharides, lipoproteins, or complex mixtures of
these biopolymers. Bioemulsifiers have several important advantages
over chemical surfactants, which should allow them to become prominent
in industrial and environmental applications. The potential commercial
applications of bioemulsifiers include bioremediation of oil-polluted
soil and water (25), enhanced oil recovery
(1), replacement of chlorinated solvents used in
cleaning-up oil-contaminated pipes, vessels and machinery (15), use in the detergent industry (17),
formulations of herbicides and pesticides (14), and the
formation of stable oil-in-water emulsions for the food
(23) and cosmetic industries (6).
The majority of Acinetobacter strains, including both
hospital and environmental isolates, produce high-molecular-mass
bioemulsifiers (22). The best studied are the bioemulsans
of Acinetobacter calcoaceticus RAG-1 and A. calcoaceticus BD4 (17). RAG-1 emulsan is a complex of
an anionic heteropolysaccharide and protein (19). Its
surface activity is largely due to the presence of fatty acids, comprising 15% of the emulsan dry weight, which are attached to the
polysaccharide backbone via O-ester and N-acyl linkages
(3). The protein component of RAG-1 emulsan stimulates the
emulsifying activity (27). A. calcoaceticus
BD4, initially isolated by Taylor and Juni (24), produces
a large polysaccharide capsule. Under certain growth conditions, the
capsule is released together with the bound protein, producing a highly
active emulsifier complex (4). The purified polysaccharide
and protein components have no emulsifying activity by themselves.
However, mixing the polysaccharide and protein led to the
reconstitution of the emulsifying activity (5). Other
Acinetobacter surfactants that have been reported include
biodispersan from A. calcoaceticus A2 (20), an
emulsifier effective on heating oil (8), and whole cells
of A. calcoaceticus 2CA2 (12).
The present study deals with the purification and characterization of
protein components of alasan, the bioemulsifier complex of A. radioresistens KA53. Alasan is composed of a polysaccharide (apo-alasan) containing covalently bound alanine and proteins (9). The proteins of alasan appeared to play an essential
role in both the structure and surface activity of the complex, because apo-alasan had no emulsifying activity and did not show the large temperature-induced hydrodynamic shape changes that were characteristic of alasan (10). Furthermore, treatment of alasan with
specific proteases inactivated the emulsifying activity. Purification
of the alasan proteins, described in this report, provide the basis for
demonstrating that the proteins by themselves and synergistically are
active emulsifiers. Using the potent tools of protein structure analysis, it should now be possible to examine the structure-function properties of bioemulsans with regard to both their ability to stabilize oil-in-water emulsions and their role in the interaction of
bacteria with interfaces (11).
| |
MATERIALS AND METHODS |
Bacterial strain and production of alasan and apo-alasan.
A. radioresistens KA53 (NCIMB 40692), isolated previously on
acetate medium (9), was maintained on brain heart infusion agar (Difco Laboratories, Detroit, Mich.). After incubation for 3 days,
the plates were stored at 4°C. For emulsifier production, inocula
were prepared in ethanol medium (EM) containing (per liter of deionized
water) 5 ml of ethanol, 1.8 g of urea, 13.7 g of Na2HPO4, 7.26 g of
KH2PO4, 0.4 g of MgSO4
· 7H2O, and 1 ml of trace elements. Alasan production was
carried out at 30°C in a 2.5-liter fermentor (Multigen; New Brunswick
Scientific Co., Inc.) containing 1.4 liters of EM. Bacterial growth was
initiated by introducing a 0.1% inoculum. After 80 h of batch-fed
fermentation, as described previously (9), the culture
reached a turbidity of 30 at 600 nm. The cell-free culture broth was
then obtained by centrifugation and filtration through a
0.45-µm-pore-size membrane filter. After the addition of ammonium
sulfate to 65% saturation and standing overnight at 4°C, the turbid
suspension was centrifuged at 10,000 × g for 20 min.
The pellets were dissolved in water, dialyzed extensively against
deionized water, and lyophilized, yielding 3.5 g of alasan.
Apo-alasan (deproteinized alasan) was obtained by the hot-phenol method
(26). After two successive hot phenol treatments, the
apo-alasan contained less than 1% protein. Stock solutions of alasan
and apo-alasan were prepared fresh by hydration at 0°C in 20 mM Tris
HCl (pH 8.5).
Determination of emulsifying activity.
A micromodification
of the standard emulsification assay (21) was used to
measure emulsifying activity. Samples to be tested were introduced into
10-ml glass tubes containing TM buffer (20 mM Tris-HCl buffer, pH 7.0;
10 mM MgSO4) to a final volume of 1.5 ml, and then 0.02 ml
of a 1:1 (vol/vol) mixture of hexadecane and 2-methylnaphthalene was
added. The tubes were then vortexed at room temperature for 30 min. The
turbidity was determined after standing for 30 s, using a Gilford
spectrophotometer (model 240). One unit of emulsifying activity was
defined as the amount of biopolymer that yielded an
A600 of 0.1 in the assay. The average and
standard error of triplicate samples are reported.
Protein electrophoresis.
Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed for
(i) estimating the molecular weight and determining the composition of
various alasan protein fractions and (ii) preparation of milligram
quantities of purified alasan proteins. The former was carried out by
the method of Laemmli (7). Samples were dissolved in 2%
SDS, 4% 
-mercaptoethanol, 8% glycerol, 50 mM Tris-HCl (pH 6.8),
and 0.02% bromphenyl blue and then heated at 100°C for 10 min.
Prestained broad-range SDS-PAGE standards (Bio-Rad Co., Hercules,
Calif.) were used as molecular mass markers. The running buffer was
0.1% SDS, 192 mM glycine, and 25 mM Tris-HCl (pH 8.3). Gels were
stained with Coomassie brilliant blue.
Preparative SDS-PAGE was performed with the model 491 Prep Cell
(Bio-Rad), using continuous elution electrophoresis. The same sample
treatment and buffers were used as described above. After up to 100 mg
of the treated alasan was loaded, the proteins were run at 12 W of
constant power for ca. 11 h on a 12.5% gel, until the ion front
reached the lower part of the gel. At this point the peristaltic pump
was activated, and 5-ml fractions were collected. After we determined
the protein content of the fractions spectrophotometrically (A280) and by the Bradford reaction (Bio-Rad),
we precipitated the alasan proteins with cold trichloroacetic acid
(TCA) and then centrifuged them. The pellets were washed twice with
cold acetone, air dried, and dissolved in water. The protein solutions
were adjusted to pH 8.0 with saturated Tris. For further purification, pooled fractions (5 to 10 mg of protein) were rerun as described above
on the Bio-Rad Mini Prep Cell column.
Size exclusion chromatography of alasan proteins.
Purified
protein fractions (ca. 0.5 mg) obtained by preparative SDS-PAGE were
applied to a Hi Load 16/60 Superdex 200 Prepgrade fast-performance
liquid chromatography (FPLC) column (Pharmacia Biotech, Inc.) and
developed with 50 mM Tris (pH 11.0) containing 0.17 M NaCl. Flow rates
were adjusted to 1 ml/min; 2.5-ml fractions were then collected and
analyzed for protein and emulsifying activity as described above. The
column was standardized with molecular mass markers of from 12 to 200 kDa (Sigma Chemical Co.).
| |
RESULTS |
Isolation of alasan proteins.
SDS-PAGE analysis of alasan
indicated the existence of three major proteins with apparent molecular
masses of 16, 31, and 45 kDa (Fig. 1,
lane a). In addition to the three major proteins, a small amount of
Coomassie blue-staining remained near the origin (not shown in Fig. 1).
Purification of these proteins from 100 mg of alasan, containing 20 mg
of total protein, yielded ca. 1.5, 1.75, and 1.9 mg of the 16-, 31-, and 45-kDa proteins, respectively, after two sequential SDS-PAGE
preparative column runs. After the first preparative run, 59% of the
input protein was recovered in the 16-, 31-, and 45-kDa peaks. Another
38% of the input protein were recovered as mixtures of the 31- and
45-kDa proteins and of the 16- and 31-kDa proteins. The relatively low
recovery of the final purified proteins was due to the fact that only
the peak fractions were pooled from the first column (to enhance
purity) and that TCA precipitation may not have been quantitative. The purity of the three alasan proteins are shown in Fig. 1, lanes b to d.
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FIG. 1.
SDS-PAGE of alasan and purified alasan proteins. Numbers
to the left indicate molecular mass markers in kilodaltons. Samples
were treated at 100°C for 10 min in Laemmli buffer (7),
resolved by SDS-PAGE, and stained with Coomassie blue. Lane a is
alasan; lanes b, c, and d are the purified 45-, 31-, and 16-kDa
proteins.
|
|
Emulsifying activity of the purified alasan proteins.
Table
1 summarizes the emulsifying activities
of the alasan proteins compared to alasan, apo-alasan, and bovine serum
albumin (control). The 45-kDa protein was the most active, yielding a higher specific emulsifying activity (792 U/mg) than alasan (712 U/mg).
The 16- and 31-kDa proteins gave relatively low emulsifying activities,
but they were significantly higher than those obtained with apo-alasan
and bovine serum albumin. The emulsion induced by the 45-kDa protein
was considerably less stable than that produced by alasan. The
emulsifying activity of alasan and each of the alasan proteins was
proportional to concentrations up to 10 µg per assay (data not
shown).
The synergistic effect of the alasan proteins on emulsifying activity
and stability is shown in Table
2. The
mixture of the
16- and 31-kDa proteins gave the emulsifying activity
predicted
from the data in Table
1. However, all of the mixtures that
contained
the 45-kDa protein showed a synergistic effect, with values
higher
than those predicted from the sum of the individual proteins.
This was observed both for emulsifying activity and emulsion stability.
The highest activity was obtained with equal molar concentrations
of
the three alasan proteins (1,300 U/mg and 96% stability). Addition
of
the alasan polysaccharide, apo-alasan, to the three alasan
proteins had
no effect.
Emulsifying properties of the 45-kDa protein.
The effect of pH
on the emulsifying activities of alasan and the 45-kDa protein are
summarized in Fig. 2. Both emulsifiers were active from pH 3 to 10. Alasan showed a peak at pH 9.0, whereas the 45-kDa protein had maximum activity from pH 8.0 to 9.5.
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FIG. 2.
Effect of pH on the emulsifying activity of the 45-kDa
protein ( ) compared to alasan
( ).
The standard microemulsion assay was used except that the pH was
varied. Values ± the standard error (SE) relative to pH 7.0 are
presented.
|
|
One of the unique features of alasan is its heat stability. As seen in
Fig.
3, the alasan emulsifying activity
increased 30%
after heating at 100°C. The emulsifying activity of
the 45-kDa
protein was less stable to heat, losing ca. 40% of its
activity
at 100°C. A mixture of the three alasan proteins retained
90%
of its activity after treatment at 100°C.
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FIG. 3.
Effect of heat treatment on the emulsifying activity of
alasan ( ), the 45-kDa protein (), and a mixture of the three
alasan proteins
(). Each
of the samples was heated at the indicated temperature for 10 min prior
to the standard microemulsion assay. Average values ± the SE are
presented.
|
|
The substrate specificity for emulsification of the 45-kDa protein
compared to alasan is summarized in Table
3. Similar to
alasan, the 45-kDa protein
was more effective in emulsifying hexylbenzene,
hexadecane, and crude
oil than in emulsifying low-molecular-weight
aliphatic and aromatic
hydrocarbons. It should be noted that emulsion
turbidity using
different substrates may not be a direct measure
of emulsification.
However, this property is useful for comparative
studies.
Physical interactions between the alasan proteins.
The
individual SDS-PAGE-purified 45-, 31-, and 16-kDa alasan proteins
eluted from an FPLC column as single peaks, with apparent molecular
masses of ca. 100, 31, and 16 kDa, respectively (data not presented).
Thus, in the absence of SDS, the 45-kDa protein appears to be a dimer,
while the 31- and 16-kDa proteins elute as monomers.
When the three purified alasan proteins were mixed and then run on the
FPLC column, four protein peaks were observed (Fig.
4). Peak A, eluting in the void volume,
had most of the emulsifying
activity and the highest specific activity.
SDS-PAGE analysis
of peak A indicated the presence of the 16-, 31-, and
45-kDa proteins.
Peak B eluted with an apparent molecular mass of 100 kDa and is
probably a residual 45-kDa dimer. It showed ca. 50% of the
emulsifying
activity of peak A. Peaks C and D eluted at the positions
of the
31- and 16-kDa proteins and showed little emulsifying activity.
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FIG. 4.
FPLC analysis of a mixture of the three alasan proteins.
The 45-, 31-, and 16-kDa proteins (0.1 mg of each) were mixed in the
elution buffer and then run on the column as described in Materials and
Methods. An aliquot (0.5 ml) of each of the peak tubes was assayed for
emulsifying activity by the microemulsion assay. The elution volumes
(Ve) of the molecular weight markers are indicated by the vertical
arrows.
|
|
| |
DISCUSSION |
To our knowledge this study represents the first detailed
investigation of the role of proteins of a polymeric bioemulsifier complex. Most Acinetobacter bioemulsans are complexes of
polysaccharides and proteins (17). In the case of alasan,
the active component of the emulsifier complex is protein. The specific
emulsifying activity of the 45-kDa protein was 11% higher than the
intact alasan complex. However, 45-kDa-protein-induced oil-in-water
emulsions were less stable than alasan-induced emulsions, suggesting
that the polysaccharide (apo-alasan) and/or the other alasan proteins played a role in emulsion stability. Addition of the purified 16- and
31-kDa proteins to the 45-kDa protein resulted in a large increase in
specific emulsifying activity and stability of the emulsion, whereas
addition of apo-alasan to the 45-kDa protein or the mixture of the
three proteins had no effect on its emulsifying activity. The 45-kDa
protein and the three-protein complex had substrate specificities and a
range of pH activities similar to that of alasan. Protein-protein
interactions may play an important role in producing the surface active
complex. The role of the polysaccharide is not clear. It may play a
role in the release of the proteins into the medium and in protecting
the protein complex against proteolytic activities. In this regard it
is interesting that the purified 45-kDa protein was readily hydrolyzed
by trypsin, whereas the protein in the alasan complex was resistant.
What are the special structural properties of the 45-kDa protein that
allow it to be such an effective emulsifier? To begin with, it is an
extremely stable molecule. It retained its activity after successive
treatments with hot SDS, TCA precipitation, and 10 min at 100°C. At
present, the only information we have on the structure of the 45-kDa
protein is that it forms a dimer in nondenaturing conditions and
interacts with the 16- and 31-kDa proteins to form a complex with a
molecular mass greater than 400 kDa. The stoichiometry and optimum
conditions for producing the complex have not been determined. Each of
the three alasan proteins contains a unique N-terminal amino acid
sequence (data not presented). The N-terminal amino acid sequence of
the 45-kDa showed high similarity to the OmpA protein of
Acinetobacter spp. (13) and other gram-negative bacteria (2).
The finding that the active emulsifiers of the alasan complex are
proteins will simplify the structural analysis of alasan. It should be
possible to perform a series of defined mutations in the genes coding
for the alasan proteins. The effect of these mutations on the activity
of alasan will be important in structure-function studies. Such studies
will contribute to our understanding of the activity and functions of
alasan and can be extended to the study of additional
high-molecular-weight microbial emulsifiers.
| |
ACKNOWLEDGMENTS |
This investigation was supported by the Ministry of Science,
Israel, the Pasha Gol Chair for Applied Microbiology, and the Manja and
Moris Chair for Biophysics and Biotechnology.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Microbiology and Biotechnology, The George S. Wise Faculty of
Life Sciences, Tel Aviv University, Ramat Aviv 69978, Israel. Phone:
972-3-640-9838. Fax: 972-3-642-9377. E-mail:
eueqene{at}ccsg.tau.ac.il.
| |
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Applied and Environmental Microbiology, March 2001, p. 1102-1106, Vol. 67, No. 3
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.3.1102-1106.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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[Abstract]
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Abstract
Introduction
