Investigations on the Solubility Behavior of Cyanophycin. Solubility of

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Investigations on the Solubility Behavior of Cyanophycin. Solubility of Cyanophycin in Solutions of Simple Inorganic Salts Gregor Fu¨ser and Alexander Steinbu¨chel* Institut fu¨r Molekulare Mikrobiologie und Biotechnologie, Westfa¨lische Wilhelms-Universita¨t Mu¨nster, Corrensstrasse 3, 48149 Mu¨nster, Germany Received October 5, 2004; Revised Manuscript Received January 3, 2005

On the basis of a previous report on the occurrence of water-soluble cyanophycin (CGP, cyanophycin granule polypeptide) in a recombinant strain of Escherichia coli expressing the cyanophycin synthetase (CphA) of Desulfitobacterium hafniense published by others, the conditions of its production were investigated in this study. Although the incubation temperature, aeration level, and NaCl concentration during cultivation had effects on the in vivo production of water-soluble CGP, it could be isolated as a major variant irrespective of the cultivation conditions. The occurrence of the soluble variant was also not dependent on the E. coli host or on the origin of cphA. Furthermore, it was shown that water-insoluble CGP can be in vitro solubilized to extents of up to about 80% (w/w) in solutions of different inorganic salts such as LiCl, NaCl, KCl, RbCl, KBr, MgCl2, or CaCl2. Evidence was obtained that the salt ions bind tightly to CGP. If the ions were not removed from the salt solution by dialysis or dilution, the CGP remained stable in solution. This method to solubilize water-insoluble CGP could also be applied to high concentrations of the polymer. CGP that remained insoluble after the first treatment could only marginally be solubilized in following treatments. The polydisperse CGP molecules were solubilized to the same extent over the whole molecular weight range with no preference of a particular molecular weight. Introduction The poly(amino acid) cyanophycin (CGP, cyanophycin granule polypeptide) is a nonribosomally synthesized biopolymer, which consists of equimolar amounts of arginine and aspartic acid arranged as a polyaspartate backbone, with arginine moieties linked to the β-carboxyl group of each aspartate by its R-amino group.1 In nature, CGP occurs in most cyanobacteria2,3 and also nonphotosynthetic bacteria.4,5 Biosynthesis of CGP is catalyzed by cyanophycin synthetase encoded by cphA.6,7 Recent analysis of genome sequence data showed that ORFs with considerable homology to cphA are present in several noncyanobacterial bacteria.4,5 Cyanophycin synthetases from Anabaena Variabilis strain ATCC 29413,6,7 Synechocystis sp. strains PCC 68036,8 and PCC 6308,9 Acinetobacter calcoaceticus strain ADP110 and the thermophilic Synechococcus sp. strain MA1911 have been purified and consist of identical subunits of 100-130 kDa. In contrast to CGP isolated from cyanobacteria, which consists of polymer strands with an apparent molecular mass range of 25-100 kDa,1,12 in vitro synthesized CGP and the polymer isolated from recombinant cells harboring cphA exhibited lower average molecular masses and a relatively narrower molecular mass range of 25-30 kDa.6-9,13,14 CGP is insoluble at neutral pH and under physiological (low) ionic strength, but it is soluble in diluted acids and bases.15 Furthermore, the polymer can be dissolved in 1% * To whom correspondence should be addressed. Phone: +49-2518339821. Fax: +49-251-8338388. E-mail: [email protected].

(w/v) SDS, 4.0 M urea, and 8.3 M cesium chloride, whereas it remains insoluble in organic solvents (e.g., methanol, ethylene glycol, dimethyl sulfoxide, formamide), 0.5% (w/ v) glycine, 0.01 M EDTA, 2% (v/v) Triton X-100, and 1% (w/v) sodium deoxycholate.16 The expression of the cphA gene from Desulfitobacterium hafniense in Escherichia coli led to the formation of a hitherto unobserved water-soluble CGP-like polymer in the cells.5 Detailed chemical and mass spectroscopic analyses and also hydrolysis of the polymer by cyanophycinase showed that this polymer does not differ from CGP with regard to amino acid composition and chemical structure, except for its solubility properties.5 However, the reasons for these differences were not revealed by the authors. The chemical structure of polyaspartate is contained in CGP. Polyaspartate can be applied in various technical processes as a biodegradable substitute for polyacrylate.17,18 Because of the polyanionic character, both polymers are widely used as a dispersing agent. However, the chemical production of polyaspartate holds disadvantages; e.g., polyaspartates obtained by thermal polymerization processes contain a mixture of R- and β-moieties.17 Poly-R-L-aspartate is the backbone of CGP, but CGP also contains arginine residues at an about equimolar ratio to aspartate. So far it is only possible to convert CGP to a derivative with reduced arginine content.19 If an enzyme is found that selectively cleaves the arginine moieties from the CGP, it is very likely that an enzymatic reaction will be more effective on solubilized CGP. Furthermore, soluble CGP may provide

10.1021/bm049371o CCC: $30.25 © 2005 American Chemical Society Published on Web 03/03/2005

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Table 1. Bacterial Strains and Plasmids Used in This Study strains and plasmids

E. coli BL21 (DE3) E. coli Origami B(DE3) pLysS E. coli top10

E. coli DH1 pET-19b::cphADh pBBR1MCS-2::cphAMA19 pMa/c5-914::cphAPCC6803

relevant characteristics Bacterial Strains F-, dcm, ompT, hsdS (rB-, mB-) gal, λ (DE3) F-, ompT, hsdSB (rB-, mB-), gal, dcm, lacY1, ahpC, gor522:Tn10 (TcR), trxB::kan, (DE3), pLysS (CmR) F , mcrA, ∆(mrr hsdRMS mcrBC), Φ80lacZ∆M15, ∆lacX74, deoR, recA1, araD139, ∆(ara leu)7697, galU, galK, rpsL, (StrR), endA1, nupG F-, endA1, gyrA96, hsdR17 (rK- mK+), recA1, supE, thi-1, λPlasmids pET-19b carrying a 2.6-kb PCR product from D. hafniense DSM10664 genomic DNA harboring cphA, ApR pBBR1MCS-2 carrying a 2.9-kb PCR product from Synechococcus sp. MA19 genomic DNA harboring cphA, KmR pMa/c5-914 carrying a 2.6-kb PCR product from Synechocystis sp. PCC6803 genomic DNA harboring cphA, ApR, CmR

additional applications. Moreover, studies on water-soluble CGP might gain further insight into the solubility properties of CGP. This study was done to investigate and describe the solubility of the cyanophycin-like polymer synthesized by the cyanophycin synthetase of D. hafniense and others in more detail. It reports on (i) the expression of the cphA homologue from D. hafniense in E. coli under various culture conditions and (ii) the solubilization of water-insoluble CGP in solutions of various simple inorganic salts. Experimental Section Bacterial Strains and Cultivation Conditions. Strains and plasmids used in this study are listed in Table 1. E. coli cells were grown in Erlenmeyer flasks without baffles following the procedure of Ziegler et al.5 (with specifications according to a personal communication). Material of several colonies was used to inoculate a 45 mL preculture (LB medium containing glucose, 10 g/L, and ampicillin, 125 µg/ mL, at 37 °C) in a 250 mL Erlenmeyer flask. The culture was grown to an OD600 of about 1.0. Cells were harvested by centrifugation (4000g, 15 min, 20 °C) and used to inoculate two 100 mL precultures in 250 mL Erlenmeyer flasks; the cultures were grown to an OD600 of about 1.0. Cells were then harvested by centrifugation, washed with double-strength LB medium (20 g of tryptone, 10 g of yeast extract, 20 g of NaCl, in 1000 mL of H2O, pH 7.5) and used to inoculate two 300 mL double-strength LB solutions with 250 µg/mL ampicillin in 1 L Erlenmeyer flasks. Cells were incubated at 16 °C with vigorous shaking for 15 h, harvested by centrifugation (4000g, 15 min, 4 °C), washed with 20 mM Tris-HCl (pH 8.0), frozen, and lyophilized. Isolation of CGP. CGP was isolated as indicated in the text following the methods described by Simon and Weathers1 or Frey et al.22 for water-insoluble CGP or Ziegler et al.5 for water-soluble CGP. Reversed-phase high-performance liquid chromatography (HPLC) was used to identify and analyze the isolated material after hydrolysis and derivatization of the amino groups with o-phthaldialdehyde (OPA) reagent as described before.13

reference or source Novagen (Madison, WI) Novagen (Madison, WI) Invitrogen (Carlsbad, CA)

20 5 21 22

Isolation and Transfer of Plasmids. Plasmid DNA was isolated from E. coli cells by the alkaline lysis method.23 Competence was induced using calcium chloride, and E. coli cells were transformed as described by Sambrook et al.24 General Protocol for Solubilization. Water-insoluble CGP was isolated from cells of E. coli DH1 (pMa/c5-914:: cphAPCC6803) and purified by the previously described largescale isolation method.22 CGP (80 mg, if not indicated otherwise) was incubated with 3.5 mL of a 1.71 M salt solution (if not indicated otherwise) for 75 min at 30 °C with shaking (preliminary tests had shown that 75 min of incubation ensures maximal possible solubility, data not shown.). Samples were centrifuged at the highest possible speed for each centrifuge and/or rotor. The resulting supernatant with a temperature of 24 °C was directly transferred into a weighed test tube; the pellet was resuspended in H2Odd and also transferred into a weighed test tube. The tubes were then closed with dialysis membranes, dialyzed for 24 h against a 100-fold volume of H2Odd at 9 °C, frozen, and lyophilized. Quantification of Solubilized CGP. After lyophilization the test tube was weighed again. The solubility was defined as the percentage of the mass of solubilized CGP with regard to the mass of the originally used, water-insoluble CGP. In addition, the solubilized CGP was measured with Bradford reagent.25 The lyophilized CGP was dissolved in 0.1 M HCl and subsequently diluted and quantified. Calibration curves were done with water-insoluble CGP, which was isolated following the method of Frey et al.22 and dissolved in 0.1 M HCl. Preparation of Dialysis Tubing. Regenerated cellulose dialysis tubes (Carl Roth GmbH & Co., Karlsruhe, Germany) were cut into the desired length, boiled in 2% (w/v) sodium hydrocarbonate, washed in H2Odd, boiled in 1 mM EDTA, and washed twice with H2Odd. The tubes were stored at 4 °C after autoclaving. Electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed in an 11.5% (w/v) polyacrylamide gel as described by Laemmli.26 The CGP was stained with Serva Blue R.

Cyanophycin Solubility in Inorganic Salt Solutions

Figure 1. CGP variants occurring in cells of E. coli after cultivation at different temperatures in the main culture. Cells of E. coli BL21 (DE3) (pET-19b::cphADh) were cultivated as described in the Experimental Section. The main cultures containing double-strength LB medium were cultivated at the indicated temperatures under otherwise identical conditions in Erlenmeyer flasks without baffles (for further details see the Experimental Section). (A) Total CGP in cells shown as a percentage of the cell dry mass (CDM). (B) Fractions of CGP isolated by the method of Ziegler et al.5 (water-soluble CGP, [) and the method of Simon and Weathers1 (water-insoluble CGP, 9) shown as a percentage of the total CGP.

Reproducibility. All experiments were done in duplicate. We never observed significant deviations within a set of two experiments. Results and Discussion The reasons for formation of water-soluble CGP in cells of a recombinant E. coli strain expressing the cyanophycin synthetase (CphA) from D. hafniense are unknown.5 Therefore, we investigated in detail the conditions at which soluble CGP with a chemical structure virtually identical to that of insoluble CGP occurred. Some unusual cultivation conditions and medium compositions to grow this strain were noticed in the study of Ziegler et al.5 Heterologous Expression of the D. hafniense cphA Homologue in E. coli under Various Culture Conditions. Recombinant cells of E. coli BL21 (DE3) were cultivated as described in the Experimental Section. The main cultures were incubated at temperatures of 9, 16, 23, 30, or 37 °C. The highest content of total CGP and the highest fraction of water-soluble CGP were measured in cells cultivated at 23 or 30 °C, respectively (Figure 1). Although both CGP variants were found at any tested temperature, water-

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Figure 2. CGP variants occurring in cells of E. coli after cultivation in Erlenmeyer flasks with different baffles in the main culture. Cells of E. coli BL21 (DE3) (pET-19b::cphADh) were cultivated as described in the Experimental Section. The main cultures containing doublestrength LB medium were cultivated at 16 °C in Erlenmeyer flasks without baffles or with baffles of varying sizes under otherwise identical conditions (for further details see the Experimental Section). (A) Total CGP in cells shown as a percentage of the CDM. (B) Fractions of CGP isolated by the method of Ziegler et al.5 (watersoluble CGP, pale column) and the method of Simon and Weathers1 (water-insoluble CGP, dark column) shown as a percentage of the total CGP.

insoluble CGP contributed to a maximum of about only 1.3% (w/w) of the total CGP (Figure 1B). The main cultures were also incubated at different aeration levels, which were achieved by the absence or presence of baffles of varying sizes in the Erlenmeyer flasks.27,28 Baffles reduced the amounts of total CGP accumulated by the cells (Figure 2A), thus indicating that an increased oxygen supply diminished CGP synthesis. Soluble CGP was again the most predominant CGP variant occurring in the cells, whereas insoluble CGP contributed only about 5-6% of the total CGP (Figure 2B). At a lower aeration level less oxygen is available, resulting in a decreased oxidation of reduction equivalents and accumulation of TCA intermediates. This probably provides an abundance of precursors of aspartic acid and arginine, which are derived from oxaloacetate and 2-oxoglutarate, respectively. When the hitherto used double-strength LB medium was replaced by a medium containing 20 g/L tryptone and 10 g/L yeast extract plus NaCl at varying concentrations of 0%, 0.5%, 1%, 2%, or 3% (w/v), cells cultivated in the presence

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Figure 3. CGP variants occurring in cells of E. coli after cultivation at different NaCl concentrations in the main culture. Cells of E. coli BL21 (DE3) (pET-19b::cphADh) were cultivated as described in the Experimental Section. The main cultures containing a double-strength LB-based medium were cultivated at 16 °C and at the indicated concentrations of NaCl (created by the addition of NaCl to the other two components), under otherwise identical conditions in Erlenmeyer flasks without baffles (for further details see the Experimental Section). (A) CGP content of cells shown as a percentage of the CDM. (B) Fractions of CGP isolated by the method of Ziegler et al.5 (watersoluble CGP, [) and the method of Simon and Weathers1 (waterinsoluble CGP, 9) shown as a percentage of the total amount of isolated CGP.

Figure 4. Solubility of CGP in solutions of various alkali-metal chlorides, KBr, and alkaline-earth-metal chlorides. An 80 mg sample of water-insoluble CGP was incubated in 3.5 mL of solutions of the indicated salts for 75 min. After centrifugation, the supernatants were dialyzed for 24 h against H2Odd and then lyophilized. The solubilized CGP was quantified by two independent analyses according to Bradford.25 The solubilities are shown as a percentage of solubilized CGP with regard to the initially used CGP dependent on the concentrations of the salt solutions. (A) Solubility in solutions of various alkali-metal chlorides and KBr: LiCl ([), NaCl (9), KCl (2), RbCl (×), CsCl (b), KBr (4). (B) Solubility in solutions of alkalineearth-metal chlorides: MgCl2 (*) or CaCl2 (+).

of 2% (w/v) NaCl contained the highest amount of total CGP, whereas the fraction of the insoluble CGP variant was lowest (Figure 3). The fractions of the insoluble variant varied between 7.2% and 20.2% of the total CGP. Therefore, the presence of NaCl affected the formation of CGP and of its two variants in E. coli. This may be caused by the uptake of amino acids such as the CGP constituents aspartic acid and arginine or the flow of metabolites derived from constituents of the medium toward these amino acids. However, a more detailed rationale of the host response to the varying NaCl concentrations with regard to CGP cannot be provided. Accumulation of the two CGP variants was also studied in different strains of E. coli. Plasmid pET-19b::cphADh expressing cphA from D. hafniense was isolated from E. coli BL21 (DE3) and transformed into E. coli Origami B(DE3) pLysS. In addition, E. coli top10 expressing cphA of Synechococcus sp. MA19 was investigated. All three strains were cultivated as described in the Experimental Section. The CGP contents of cells of E. coli Origami B(DE3) pLysS harboring pET-19b::cphADh, E. coli BL21 (DE3), and E. coli top10 harboring pBBR1MCS-2::cphAMA19 were 13.6%, 11.3%, and 7.2% (w/w) of the cell dry matter with fractions of the soluble variant of 96.0%, 87.4%, or 97.5% (w/w), respec-

tively. This clearly demonstrated that the ability to synthesize soluble CGP is not restricted to CphADh of D. hafniense, a specific E. coli strain, or the pET vector system, although minor differences in CGP contents and in the fractions of two CGP variants indicated host-specific effects on CGP accumulation. Interestingly, soluble CGP was also isolated from cells of Pseudomonas putida GPp104 harboring pBBR1MCS-2::cphAMA19 (Simone Diniz, personal communication). Solubilization of Water-Insoluble CGP in Solutions of Inorganic Salts. An influence of NaCl on the production of soluble CGP was shown above. Preliminary tests showed that insoluble CGP isolated from recombinant E. coli cells can be dissolved in aqueous solutions of NaCl, KCl, CaCl2, MgCl2, Na2CO3, NaH2PO4, and NaCH3COOH (data not shown). The in vitro solubility of CGP in solutions of LiCl, NaCl, KCl, CsCl, and KBr as well as of MgCl2 and CaCl2 was then systematically studied (Figure 4). The pH of the salt-cyanophycin solutions changed only marginally during incubation. The solubility of CGP at concentrations of any of these alkali-metal chlorides at 0.2 M or less was marginal (Figure 4A). With rising salt concentrations, the amounts of solubilized CGP increased, and at 1.7 M salt a maximum of


CGP could be solubilized. With KBr the rise was steeper than with solutions of alkali-metal chlorides in general or with KCl in particular, and maximum solubility was already obtained at 1.2 M (Figure 4A). A maximum of 82.5% ( 4.1% or 79.6% (w/w) of the CGP became soluble in solutions of the alkali-metal chlorides or of KBr, respectively. In contrast, significant solubilization of CGP by alkaline-earthmetal chlorides (Figure 4B) occurred already at the lowest concentration tested, i.e., at 0.09 M. With rising concentrations the fraction of solubilized CGP increased, and the maxima were already obtained at concentrations of 0.4-0.5 M. Since CGP is soluble in 8.3 M cesium chloride,16 the solubility of CGP is unlikely to decrease with a further increase of the salt concentrations. As CGP is polyzwitterionic, cations or anions could cause its solubilization. Since the anion was identical when using LiCl, NaCl, KCl, RbCl, or CsCl, it was concluded that the different alkali-metal cations have an equal or no effect. Since maximum solubility of CGP occurred at a lower concentration of KBr than of KCl, it was concluded that the anion has a significant effect and that the observed effect may be due to the larger size of the bromide anion in comparison to the chloride anion. With alkaline-earth-metal chlorides, the maximum solubilization of CGP was reached already at much lower concentrations than with alkali-metal chlorides, and the maximal solubility of CGP depended significantly on the used cation. Both the cations and the anions must contribute to the solubiliziation of CGP, which increases with increasing valence of the cation and with increasing diameter of the anion. There was a discrepancy between the masses of solubilized CGP as determined by weighing directly the solubilized CGP and by calculating its masses from the differences of the nonsolubilized pellet of the used CGP or by the Bradford test;25 the masses of solubilized CGP exceeded that of the used CGP. Therefore, and because dialysis was extensively performed for 24 h, it was concluded, that salt ions bound firmly to CGP, thus explaining the discrepancy. The amounts of the bound salt ions were calculated (Figure 5A) and are displayed as a salt to CGP ratio (Figure 5B). This ratio barely changed at concentrations from 0.7 to 1.7 M with regard to the change of the solubility of CGP, which increased in the solutions of alkali-metal chlorides above 0.7 M from 43% to 82.5% (Figure 4B). Therefore, attachment of the ions seems to be not necessarily essential for solubilization of CGP. Effect of Temperature on the Solubility of CGP. Solubilization of water-insoluble CGP was also studied at a temperature of 9 °C in 1.7 M solutions of LiCl, NaCl, KCl, and RbCl. All other conditions were identical to those described above (Figure 4) except that the 30 min centrifugation step was done in a precooled rotor and centrifuge. Pellets and supernatants were dialyzed before lyophilization. The solubilities of CGP at 9 °C were significantly lower than those at 24 °C and amounted to only 60-70% (w/w) in comparison to about 80% (w/w) at 24 °C (Figure 4A). This effect may be due to complications in the conformation change of CGP, due to a reduced molecular movement of the CGP and salt ions at lower temperatures.


Figure 5. Determination of the amount of salt bound to CGP and of the ratio of bound salt to solubilized CGP. An 80 mg sample of waterinsoluble CGP was incubated in 3.5 mL of solutions of the indicated salts for 75 min. After centrifugation, the supernatant was dialyzed for 24 h against H2Obidest and lyophilized afterward. The lyophilisate was weighed, and the differences between the masses of the starting material and the resulting material were defined as the mass of salt bound to CGP. (A) Amount of salt bound to CGP. (B) Ratio of bound salt to solubilized CGP. Solubility in LiCl ([), NaCl (9), KCl (2), RbCl (×), CsCl (b), MgCl2 (*), CaCl2 (+), or KBr (4).

Effect of the CGP Concentration on Solubility. Saltmediated solubilization of CGP will be done for technical uses only if this method is applicable to higher CGP concentrations. To study the technical applicability of the method, the solubility of CGP was investigated at concentrations up to 40 g/L. CGP was incubated at the indicated concentrations in 3.5 mL of 0.17, 0.86, or 1.71 M solutions of LiCl, NaCl, or KCl (Figure 6). The standard solubilization method as described above employs CGP at a concentration of 22.9 g/L and achieves a solubility of 82.5% ( 4.1% in the presence of 1.71 M alkali-metal chlorides. Although none of the curves exhibited a straight course, they all showed about the same tendency, indicating a limitation of solubilization by salt at higher CGP concentrations. As already shown above, at alkali-metal chloride solutions below 0.2 M almost no CGP was solubilized. At salt concentrations of 0.86 M not more than 60% (w/w) of the initially insoluble CGP was solubilized. Much higher solubilities of CGP were achieved at 1.71 M salt solutions, however with a decreasing fraction of the solubilized CGP at increasing CGP concentrations. The solubility of CGP was also studied at a concentration of 28.6% (w/v). A 4.0 g sample of insoluble CGP was suspended in 14 mL of 1.71 M NaCl in a 100 mL Erlenmeyer flask, and the suspension was incubated for 75 min at 30 °C. After 30 min of centrifugation at 6000g three layers were obtained: (i) a pellet, representing insoluble CGP, (ii) a light

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Fu¨ser and Steinbu¨chel Table 2. Yield of CGP Dissolved in Water from Solid, Previously Solubilized CGPa CGP previously solubilized by treatment with water-soluble CGP yield (%)

LiCl

NaCl

KCl

RbCl

nd 82.7

88.4 nd

84.6 75.5

nd 77.7

a An 80 mg sample of water-insoluble CGP was incubated in 3.5 mL of a 1.7 M concentration of the indicated salt solutions for 75 min. After centrifugation, the supernatant was dialyzed for 24 h against H2Odd and then lyophilized. Such solubilized CGP was incubated in 3.5 mL of H2Odd for 6 h. After centrifugation, supernatants were lyophilized without dialysis. The masses of these lyophilisates were determined. Values represent the percentage of CGP which could be dissolved in water. The two lines show the results of two independent experiments. Abbreviation: nd ) not determined.

Figure 6. Solubility of CGP at various polymer concentrations as a percentage of solubilized CGP with regard to the used CGP depending on the used salt and its concentration. Water-insoluble CGP was incubated in 3.5 mL of solutions of the indicated salts for 75 min. After centrifugation, the supernatant was dialyzed for 24 h against H2Obidest and then lyophilized. The solubilized CGP was quantified by two independent analyses according to Bradford.25 (A) Solubility in LiCl. (B) Solubility in NaCl. (C) Solubility in KCl. CGP dissolved in 0.17 M salt solution ([), CGP dissolved in 0.86 M salt solution (9), CGP dissolved in 1.71 M salt solution (2).

yellow liquid phase on top of the pellet, and (iii) a liquid phase beneath the pellet exhibiting an intensively yellow color. The color probably resulted from minor impurities of the isolated CGP, which were not visible in solid CGP powder and diluted solutions of CGP. The three phases were separated and dialyzed before lyophilization; only the bottom liquid phase could not be dialyzed due to its viscous consistency. A total of 100.4% of the initial CGP was recovered with about 64.7% (w/w) solubilized CGP occurring in the bottom (8.3%) or top (56.4%) liquid phases and with only 35.8% (w/w) insoluble CGP in the pellet. Considering the very high CGP concentration applied in this experiment, the solubility came very close to the value of about 80% (w/w) obtained at much lower CGP concentrations. This demonstrated that the insoluble form of CGP can be converted into the soluble form also at high polymer

concentrations, indicating applicability of the method on a technical scale. The appearance of the bottom liquid phase containing a minor fraction of the solubilized CGP is curious and could not be explained. Stability of CGP Solubility. To test whether CGP retains its water solubility after solubilization, lyophilized powders of CGP previously solubilized in solutions of LiCl, NaCl, KCl, or RbCl were incubated in H2Odd at 30 °C. After 6 h of incubation, the solutions were centrifuged, the pellets and supernatants were lyophilized without dialysis, and the masses of the lyophilized supernatants representing watersoluble CGP and of the lyophilized pellets representing insoluble CGP were determined (Table 2). Most of the CGP was recovered as soluble polymer during this experiment, thus indicating that the solubility of CGP was basically maintained irrespective of the salt solution in which it was previously solubilized. To assay whether solubilized CGP remains in the supernatant when not dialyzed, insoluble CGP was incubated in solutions of LiCl, NaCl, KCl, and RbCl under standard conditions (see the Experimental Section). The supernatants obtained after centrifugation were not dialyzed but further incubated. Even after 300 days of incubation no visible turbidity occurred. Therefore, significant quantities of CGP did not convert into the insoluble form and precipitate, and solubilized CGP is stable if the conditions (i.e., ionic strength) are not changed. For example, addition of water to such stable aqueous solutions of CGP led to partial precipitation of CGP. Thus, lowering the ion concentration obviously results in a reduced solubility. Behavior of Nonsolubilized CGP. It was investigated whether CGP that remained in its insoluble form after a first treatment with salts can be solubilized by a second or third treatment. For this, 140 mg of CGP was incubated in solutions of NaCl and KCl under standard conditions, and the solubilized CGP was removed by 30 min of centrifugation. The nonsolubilized CGP in the pellet was then incubated in the presence of fresh salt solution for 90 min. This procedure was repeated for a third time, and all supernatant samples and the final pellet were then dialyzed and lyophilized. The percentages of CGP solubilized in the first step were 78.8% (NaCl) and 78.2% (KCl); the second treatment solubilized only 1.6% or 2.1% and the third only 0.09% and 0.08% of the remaining insoluble CGP, respectively. These experiments clearly showed that additional attempts solubi-


lized only little or almost no additional amounts of CGP, indicating that no equilibrium exists between solubilized and nonsolubilized CGP. It was also investigated whether nonsolubilized, lyophilized CGP can be solubilized in a subsequent experiment. For this, previously nonsolubilized, lyophilized CGP was incubated in NaCl solution under standard conditions (see the Experimental Section). After centrifugation for 30 min, the supernatants and pellets were dialyzed and lyophilized. The CGP, which was not solubilized in the first experiment, and which became soluble in the second experiment, contributed to only about 10.5% (w/w) of the total CGP, which was clearly less than the amount of initially solubilized CGP (82.5% ( 4.1%, w/w). Therefore, a second solubilization occurs to a much lesser extent. When the nonsolubilized CGP was “newly isolated” by the acid extraction method described by Frey et al.22 and then incubated in NaCl solution under standard conditions (see the Experimental Section), about 27.6% (w/w) of the CGP became soluble; sporadically, up to a maximum of 52.8% (w/w) of the CGP was obtained. These experiments led to the conclusion that a certain fraction of CGP can generally not be solubilized. This may be due to a too dense package of the CGP molecules, leading to very stable aggregates. During the first solubilization experiment most of the solubilizable CGP will be solubilized, and the nonsolubilizable form will be enriched, whereby the yields of solubilized CGP decrease in subsequent solubilization experiments. In the case of previously lyophilized CGP, it is possible that the aggregates were partially rearranged by the lyophilization process, thus yielding slightly higher fractions of solubilized CGP. It remained unclear why a rearrangement of the CGP molecules, which should appear to a great extent during the acid extraction method, had only little effect. Effect of Molecular Weight on the Solubility of CGP. Since CGP is a polydisperse polymer, it was investigated whether CGP molecules of a particular molecular weight solubilize preferentially. CGP was incubated in the presence of NaCl under standard conditions, both variants were separated by centrifugation, and the resulting pellets and supernatants were dialyzed and then lyophilized. The molecular weights of the initial CGP, of the solubilized CGP (in the supernatants), and of the nonsolubilized CGP (in the pellets) were analyzed by SDS-PAGE. The apparent molecular weights of the CGP molecules ranged from 25000 to 32000 and were identical in all three samples. Therefore, the polydisperse CGP molecules were solubilized to the same extent over the whole range with no apparent preference of a particular molecular weight, and CGP solubilizes independently of its molecular weight. Conclusion The discovery of water-soluble CGP in bacterial cells by Ziegler et al.5 was very intriguing. In this study, we investigated the formation and occurrence of soluble CGP in vivo and in vitro. The in vitro solubilization of CGP is most probably due to a salting-in effect. The phenomenon of salting-in or -out is not yet fully understood, but according


to the conventional view29 opposed electrical charges of different proteins interact with each other to form an electrostatic complex at low ionic strength. When many molecules form such complexes, the protein precipitates. Increasing ionic strength helps break these complexes. Studies on the solubilities of model peptides in salt solutions have shown that there is a salting-in effect due to an electrostatic interaction between the salt ions and the peptide groups which is attributed to the large dipole moment of the peptide group.30 This effect should also salt-in CGP, because CGP is a poly(amino acid) with two peptide groups per repeating unit. The causes of the in vivo solubilization of CGP are less clear. Synthesis of water-soluble CGP was in vivo independent of the expression system and of the origin of cphA and occurred at all tested temperatures. It seemed to be increased under conditions of low aeration, and although addition of salt had a certain positive effect on the in vivo production of soluble CGP, it was not essential. Since soluble CGP was synthesized in all tested variations, it is curious that this was never observed before with the exception of only one study.5 Since the solubilization method could be applied even at high polymer concentrations, and since CGP remains soluble in water over a long period once solubilized, this method can most probably also be applied on a technical scale, making CGP better applicable as a substrate for chemical or enzymatic conversions of CGP. Such enzymes would have to resist high ionic strength. Since many enzymes have been described, which are active at high salt concentrations,31-33 application of solubilized CGP for enzymatic conversion may be feasible. Acknowledgment. We thank Dr. Karl Ziegler (Humboldt-Universita¨t, Berlin) for providing plasmid pET-19b:: cphADh and Kay M. Frey (Westfa¨lische Wilhelms-Universita¨t, Mu¨nster) for providing the cell dry mass of E. coli DH1 (pMa/c5-914::cphAPCC6803). We thank Martin Krehenbrink for helpful discussions and for critically reading the manuscript. This study was supported by a collaborative research grant provided by the Fachagentur Nachwachsende Rohstoffe e.V. (FNR) (FKZ: 00NR125) and Bayer AG (Leverkusen, Germany). References and Notes (1) Simon, R. D.; Weathers, P. Biochim. Biophys. Acta 1976, 420, 165. (2) Allen, M. M. Annu. ReV. Microbiol. 1984, 38, 1. (3) Simon, R. D. Inclusion bodies in the cyanobacteria: cyanophycin, polyphosphate, polyhedral bodies. In The cyanobacteria; Fay, P., van Baalen, C., Eds.; Elsevier: Amsterdam, New York, 1987. (4) Krehenbrink, M.; Oppermann-Sanio, F. B.; Steinbu¨chel, A. Arch. Microbiol. 2002, 177, 371. (5) Ziegler, K.; Deutzmann, R.; Lockau, W. Z. Naturforsch. 2002, 57c, 522. (6) Ziegler, K.; Diener, A.; Herpin, C.; Richter, R.; Deutzmann, R.; Lockau, W. Eur. J. Biochem. 1998, 254, 154. (7) Berg, H.; Ziegler, K.; Piotukh, K.; Baier, K.; Lockau, W.; VolkmerEngert, R. Eur. J. Biochem. 2000, 267, 5561. (8) Oppermann-Sanio, F. B.; Hai, T.; Aboulmagd, E.; Hezayen, F. F.; Jossek, S.; Steinbu¨chel, A. In Biochemical principles and mechanisms of biosynthesis and biodegradation of polymers; Steinbu¨chel A., Ed.; Wiley-VCH: Weinheim, Germany, 1999; p 185. (9) Aboulmagd, E.; Oppermann-Sanio, F. B.; Steinbu¨chel, A. Appl. EnViron. Microbiol. 2001, 67, 2176. (10) Krehenbrink, M.; Steinbu¨chel, A. Microbiology 2004, 150, 2599.

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(11) Hai, T.; Oppermann-Sanio, F. B.; Steinbu¨chel, A. FEMS Microbiol. Lett. 1999, 181, 229. (12) Simon, R. D. Biochim. Biophys. Acta 1976, 422, 407. (13) Aboulmagd, E.; Oppermann-Sanio, F. B.; Steinbu¨chel, A. Arch. Microbiol. 2000, 174, 297. (14) Aboulmagd, E.; Voss, I.; Oppermann-Sanio, F. B.; Steinbu¨chel, A. Biomacromolecules 2001, 2, 1338. (15) Simon, R. D. Proc. Natl. Acad. Sci. U.S.A. 1971, 68, 265. (16) Lang, N. J.; Simon, R. D.; Wolk, C. P. Arch. Microbiol. 1972, 83, 313. (17) Schwamborn, M. Polym. Degrad. Stab. 1998, 59, 39. (18) Joentgen, W.; Mu¨ller, N.; Mitschker, A.; Schmidt, H. In Biopolymers Vol. 7 (Polyamides and complex proteinaceous Materials I); Fahnestock, S. R., Steinbu¨chel A., Eds.; Wiley-VCH: Weinheim, Germany, 2003; p 175. (19) Joentgen, W.; Groth, T.; Steinbu¨chel, A.; Hai, T.; Oppermann, F. B. International Patent Application WO 98/39090, 1998. (20) Hanahan, D. J. Mol. Biol. 1983, 166, 557. (21) Voss, I.; Cardoso Diniz, S.; Aboulmagd, E.; Steinbu¨chel, A. Biomacromolecules 2004, 5, 1588.

Fu¨ser and Steinbu¨chel (22) Frey, K. M.; Oppermann-Sanio, F. B.; Schmidt, H.; Steinbu¨chel, A. Appl. EnViron. Microbiol. 2002, 68, 3377. (23) Birnboim, H. C.; Doly, J. Nucleic Acids Res. 1979, 7, 1513. (24) Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular cloning: a laboratory manual, 2nd ed.; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 1989. (25) Bradford, M. M. Anal. Biochem. 1976, 72, 248. (26) Laemmli, U. K. Nature (London) 1970, 227, 680. (27) Smith, C. G.; Johnson, M. J. J. Bacteriol. 1954, 68, 346. (28) Tunac, J. B. J. Ferment. Eng. 1989, 68, 157. (29) Zubay, G. L. Biochemie, translation of the 4th ed.; McGraw-Hill International (UK) Ltd.: London, 1999. (30) Nandi, P. K.; Robinson, D. R. J. Am. Chem. Soc. 1972, 94, 1299. (31) Suzuki, H.; Kumagai, H. Challenges In Taste Chemistry And Biology. ACS Symp. Ser. 2004, 867, 223. (32) Ozaki, A. Patent Application JP 4370094, 1992. (33) Kembhavi, A. A.; Kulkarni, A.; Pant, A. Appl. Biochem. Biotechnol. 1993, 38, 83.

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Investigations on the Solubility Behavior of Cyanophycin. Solubility of

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