Puri¢cation and characterization of cyanophycin and cyanophycinsynthetase from the thermophilic Synechococcus sp. MA19

Tran Hai, Fred Bernd Oppermann-Sanio, Alexander Steinbu«chel *Institut fu«r Mikrobiologie der Westfa«lischen Wilhelms-Universita«t, CorrensstraMe 3, 48149 Mu«nster, Germany

Received 23 August 1999; accepted 27 September 1999

Abstract

The biosynthesis and accumulation of cyanophycin in the thermophilic cyanobacterium Synechococcus sp. MA19 werestudied. By growing the cells in a 80-l closed tubular photobioreactor under controlled conditions, the cells accumulatedcyanophycin amounting up to 3.5% of the dry cell matter. The cyanophycin was purified and chemical analysis showed that itwas composed of arginine and aspartic acid occurring at a molar ratio of 1:0.9. Sodium dodecyl sulfate-polyacrylamide gelelectrophoresis revealed a broad distribution of the apparent molecular masses ranging from 20 to 130 kDa with a maximum at50 kDa. During a three-step purification procedure involving ion exchange chromatography and gel filtration, the cyanophycinsynthetase from strain MA19 was purified 144-fold to electrophoretic homogeneity. It consisted of only one single type ofsubunit exhibiting an apparent molecular mass of 130 kDa. The enzyme catalyzed the polymerization of arginine and aspartateat elevated temperatures and was even active at 80³C. ß 1999 Federation of European Microbiological Societies. Publishedby Elsevier Science B.V. All rights reserved.

Keywords: Cyanophycin; Cyanophycin synthetase; Thermophilic cyanobacterium; Tubular glass photobioreactor; Synechococcus sp. MA19

1. Introduction

Cyanophycin is a protein-like polymer, consistingof equimolar amounts of arginine and aspartic acidarranged as a polyaspartate backbone, with an argi-nine moiety linked to the L-carboxyl group of eachaspartate by its K-amino group [1]. The polymer isunique to cyanobacteria and has been reported tooccur in many species of cyanobacteria since its dis-covery more than 100 years ago [2], but not in strainsof the unicellular genus Synechococcus (Anacystis)

[3,4]. Cyanophycin serves as a temporary reserve ma-terial of newly assimilated nitrogen. It accumulatesusually during the transition from the exponential tothe stationary growth phase and disappears whenbalanced growth resumes [5,6]. The cyanophycincontent can be enhanced by (i) reduction of lightand temperature and (ii) by the addition of transla-tional or transcriptional inhibitors as well as of argi-nine and/or aspartic acid [3]. The regulation of cya-nophycin accumulation in Synechocystis sp.PCC6803 and Nostoc ellipsosporum mutant NE1

might be controlled by the gene product of sll0736[7] and L-Arg [8], respectively. In Anabaena cylindri-ca, cyanophycin synthetase and cyanophycinase,which catalyzes the polymerization and degradation

0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 5 4 4 - 3

* Corresponding author. Tel. : +49 (251) 8339821;Fax: +49 (251) 8338388; E-mail: steinbu@uni-muenster.de

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of the polymer, respectively, are constitutively ex-pressed. The mechanism regulating the net contentof cyanophycin is discussed to be a kinetic [9] ore¡ector-mediated [5] control of both enzyme activ-ities. Cyanophycin synthetases were enriched from A.cylindrica by [10] and puri¢ed from Anabaena varia-bilis by [11]. The latter enzyme consists of two iden-tical subunits of 100 kDa. The polymerization reac-tion yields polydisperse polymers with molecularmasses ranging from 25 to 100 kDa and dependson the presence of both monomers, ATP, Mg2�,K�, a sulfhydryl reagent and cyanophycin as primer[1,10]. Since the polymer is insoluble at the pH andionic strength occurring inside the cells [9], the nas-cent cyanophycin strands assemble in vivo into mem-brane-less granules amounting up to 16% of the celldry mass [3,12]. Cyanophycin is of biotechnologicalinterest since a derivative with a reduced argininecontent can be obtained by partial chemical hydrol-ysis, which can be applied as a substitute for poly-acrylic acid to various technical and chemical proc-esses [13]. In this paper, we describe the puri¢cationand characterization of cyanophycin as well as ofcyanophycin synthetase from the thermophilic Syne-chococcus sp. MA19, which was recently isolatedfrom the surface of a volcanic rock [14]. This is the¢rst demonstration of cyanophycin biosynthesis andaccumulation in a member of the genus Synechococ-cus (Anacystis) and in a thermophilic microorganism.

2. Materials and methods

2.1. Bacterial strains and growth conditions

Synechococcus sp. MA19 [14] was a gift from theculture collection of the Molecular BioenergeticsLaboratory of the National Institute of Bioscienceand Human Technology (Tsukuba, Ibaraki, Japan).Synechocystis sp. PCC6803 and Aphanocapsa sp.PCC6308 were obtained from the Pasteur CultureCollection (PCC, France). The cells were cultivatedphotoautotrophically and irradiated with Natura de-Lux Interna lamps at a quantum £ow of 300 WE s31

m32. The cultivation was carried out in a 80-l closedtubular glass photobioreactor, which was describedrecently [15], at 30³C (for Aphanocapsa sp. PCC6308and Synechocystis sp. PCC6803) or at 50³C (Syne-

chococcus sp. MA19) in BG11 medium [16]. A gasmixture consisting of air and CO2 (98:2, v/v) wasconstantly provided at a £ow rate of 0.4 l min31.For stimulating cyanophycin accumulation, L-argi-nine (200 mg l31) and chloramphenicol (7 mg l31)were added to the cultures when the cells hadreached the stationary growth phase. The cells werethan incubated for an additional 33 h under reducedlight intensity of 50 WE s31 m32. In the case of Syn-echococcus sp. MA19, the temperature was reducedto 40³C during this period. Cells were harvested at20 000 rpm by using a continuous £ow throughCEPA Z41 centrifuge (Carl Padberg, Lahr, Ger-many). Growth was monitored by measuring the tur-bidity at 665 nm. The quantum £ow of irradiationwas measured with a Li-CoR Quantum No. 13512-meter (Walz Mess-und Regeltechnik, E¡eltrich, Ger-many).

2.2. Quantitative determination of cyanophycin,protein and cell dry mass

To quantify the cyanophycin content of the cellsduring cultivation, 50-ml samples of cell suspensionwith known cell dry matter were taken and the cellswere harvested by centrifugation. After washing,cells were disintegrated by sonication for 1 minml31 cell suspension by using a Sonoplus GM200soni¢er (Bandelin electronic, Berlin, Germany). Thepellet obtained was then extracted twice with 0.1 NHCl. The cyanophycin content was determined col-orimetrically with Sakagushi reagent as described by[17]. The contents of carbohydrates and nucleic acidsof the puri¢ed cyanophycin were determined withthe Anthron reagent [18] and spectrophotometricallyat 260 nm by using an extinction coe¤cient of10 mM31 cm31, respectively. Protein was determinedas described by [19]. Cell dry mass was determinedfrom washed and dried cell pellets as described by[8].

2.3. Puri¢cation of cyanophycin

Cyanophycin was isolated from the cells by takingadvantage of its molecular size, density and solubil-ity properties following the procedure described by[10], which was slightly modi¢ed by an additional0.5-min centrifugation at 200Ug and 4³C to improve

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the separation of the cell debris and unbroken cellsfrom cyanophycin.

2.4. High performance liquid chromatography(HPLC) analysis

Puri¢ed cyanophycin was treated for 15 h at 95³Cin the presence of 6 N HCl. After lyophilization, thedry material was dissolved in starting eluent (37 mMsodium acetate, pH 7.0, 26% methanol). The solu-tion was clari¢ed by 5 min centrifugation at10 000Ug. To 460 Wl of this solution, 200 Wl 0.5 Msodium borate (pH 9.5) and 100 Wl OPA reagent(100 mg ortho-phthaldialdehyde, 9 ml methanol,1 ml 0.5 M sodium borate, pH 9.5, 100 Wl 2-mercap-toethanol) were added. After exactly 200 s, 60 Wl of0.75 N HCl was added. To 100 Wl of this mixture,400 Wl starting eluent was added. Twenty Wl of thissolution was applied onto a reversed phase column(0.46 by 12.5 cm, RP18 Techsphere ODS-2, KontronInstruments, Neufahrn, Germany) equilibrated withstarting eluent. OPA amino acids were eluted with amethanol gradient (26^100%) at 40³C and with a£ow rate of 1.0 ml min31. OPA amino acids weremonitored £uorometrically at 330/450 nm (excita-tion/emission) by using a model 1046A £uorescencedetector (Hewlett Packard, Germany). Calibrationwas done with chromatographically pure aminoacids (Kollektion AS-10 from Serva Feinbiochemica,Heidelberg, Germany).

2.5. Cyanophycin synthetase assay

Enzyme activity was measured by following theincorporation of [U-14C]-L-arginine into cyanophycinas described by [10]. To determine the temperaturedependency, each 125 Wl of the complete assay mix-ture containing 5 Wg puri¢ed enzyme from Synecho-coccus sp. MA19 or 50 Wg protein from a highlyenriched preparation from Synechocystis sp.PCC6803 was overlaid with silicone oil to preventevaporation and incubated for 1 h at temperaturesbetween 20 and 80³C.

2.6. Puri¢cation of cyanophycin synthetase

All steps were carried out at 4³C. A 20 mM Tris/5mM 2-mercaptoethanol/1 mM EDTA bu¡er, pH 8.2,

was used throughout the puri¢cation procedure. Teng cells (wet weight) was suspended in 20 ml bu¡er(+1 mM MgCl2) and disrupted by sonication as de-scribed above. A 10-ml portion of the supernatantcontaining the soluble cell fraction (250 mg protein),which was obtained after centrifugation for 1 h at100 000Ug and 4³C, was applied onto a gel ¢ltrationcolumn (2.6 by 60 cm, 330-ml bed volume (BV)) ofSuperdex 200 prep grade (Pharmacia-Biotech, Upp-sala, Sweden) equilibrated with bu¡er (+150 mMNaCl). Protein was eluted with a constant £ow rateof 2.5 ml min31. Fractions of 3.0 ml containing ahigh enzyme activity were combined and appliedonto an anion exchange column Mono Q HR5/5(1-ml BV, Pharmacia-Biotech), equilibrated withbu¡er. After the column was washed with 2 BV ofbu¡er, the protein was eluted with a NaCl gradient(0^1000 mM, with a concentration change of 17 mMCl3 ml31) at a constant £ow rate of 0.5 ml min31.The fraction (0.75 ml) containing the highest enzymeactivity was ¢nally applied onto a second gel ¢ltra-tion column (1.0 by 30 cm, 24-ml BV) of Superdex200 HR (Pharmacia-Biotech), equilibrated with buf-fer (+0.1% Triton X-100). Protein was eluted with aconstant £ow rate of 0.25 ml min31. The fraction(0.5 ml) with the highest enzyme activity representedthe ¢nal product of the puri¢cation.

2.7. Electrophoresis

Sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE) was performed in 11.5% gelsas described by [20]. Staining of proteins and cyano-phycin was done with Serva Blue R and silver nitrate[21].

2.8. N-terminal sequence analysis

Determination of N-terminal amino acid se-quences was done with puri¢ed SDS-denatured andelectroblotted proteins as described before [22]. Atthe position where no signal was obtained, cysteinemight be the actual amino acid present, since theprotein was not carboxy-methylated before being se-quenced. The amino acid sequence was comparedwith those of the SwissProt database by using theprograms BLASTP and Best¢t from the HeidelbergUnix Sequence Analysis Resources (release 4.0).

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3. Results

3.1. Induced accumulation of cyanophycin duringgrowth in a closed glass tubular photobioreactor

As can be seen from Fig. 1, in batch culture, Syn-echococcus sp. MA19 accumulated only a negligibleamount of cyanophycin during the exponential andstationary growth phase. After changing the cultureconditions by reducing the light intensity to 50 WEs31 m32 and the temperature to 40³C and by addingarginine and also the translational inhibitor chloram-phenicol, which are all known to stimulate synthesisof cyanophycin in various cyanobacteria [3], cellsimmediately started accumulation of cyanophycinup to a ¢nal content of approximately 3.5% of thecell dry mass.

3.2. Puri¢cation and characterization of cyanophycin

Puri¢cation of the accumulated material wasachieved by following the standard procedure as de-scribed for the isolation of cyanophycin from variousother cyanobacteria [10].

SDS-PAGE of cyanophycin puri¢ed from MA19revealed a broad band indicating a polydispersepolymer with apparent molecular masses from20 kDa to more than 130 kDa, which is similar tothe molecular mass distributions reported for cyano-phycins from other sources [1] like, e.g., Aphanocap-sa sp. PCC6308 (Fig. 2A). The cyanophycin materialfrom Synechococcus sp. MA19 did not react withsilver stain. This is a common phenomenon for cya-nophycin puri¢ed by us from 10 other sources, in-cluding that of Aphanocapsa sp. PCC6308 andSynechocystis sp. PCC6803, and is due to its speci¢camino acid content. In order to determine the aminoacid composition of the isolated cyanophycin, itwas completely hydrolyzed and HPLC analysiswas performed. As shown in Fig. 3, the two aminoacids aspartic acid and arginine were detected atalmost equimolar amounts (1:0.90) as constituentsof the polymer. Other amino acids comprised lessthan 0.7% and the isolated material contained eachless than 0.01% carbohydrates, 0.1% nucleic acidsand 0.5% of other substances like N-acetyl-glucos-amine.

These results provided clear evidence that the ma-

Fig. 1. Growth and accumulation of cyanophycin. Synechococcus sp. MA19 was grown in BG11 medium under full light (300 WE s31

m32) at 50³C. At the time point indicated (Ts, `lamp's), culture conditions were changed as described in Section 2. Cell dry mass (a),turbidity (E), protein (O) and cyanophycin (b) were determined as described in Section 2. Abbreviations: Cm, chloramphenicol ; Arg,L-arginine.

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terial puri¢ed from Synechococcus sp. MA19 is cya-nophycin.

3.3. Puri¢cation and characterization of cyanophycinsynthetase

Cyanophycin synthetase was puri¢ed 144-foldfrom the soluble cell fraction in a three-step proce-dure (Table 1). The increase of activity after gel ¢l-tration (Table 1), which was also observed duringdialysis and during gel ¢ltration of cell extractsfrom Aphanocapsa sp. PCC6308 and Synechocystissp. PCC6803 (data not shown), is probably due tothe removal of a low molecular mass negative e¡ec-tor. The presence of Triton X-100 during the ¢nalstep facilitated the separation of cyanophycin synthe-tase from dominant phycoerythrin-like impurities, ¢-nally resulting in a color-less protein preparation(Fig. 2B). SDS-PAGE of the ¢nal enzyme prepara-tion resulted in one single protein band, indicatingthe presence of only one type of subunit with an

apparent molecular mass of 130 000 þ 10 000 Da(Fig. 2B).

The temperature dependency of the enzymatic pol-ymerization catalyzed by the enzyme puri¢ed fromstrain MA19, which is shown in Fig. 4, revealed abroad peak with a maximum at 30³C and a shoulderat around 50³C, which is the optimum temperatureof growth for this strain. Even at a temperature of80³C, the reaction proceeded at 23% of the maxi-

Fig. 3. HPLC chromatogram of OPA derivatives of hydrolyzedcyanophycin from Synechococcus sp. MA19. Treatment of cyano-phycin and conditions of the chromatography are described inSection 2. Aspartic acid and arginine were identi¢ed and quanti-¢ed by calibration with chromatographically pure amino acids.

Fig. 2. SDS-PAGEs of puri¢ed cyanophycin and cyanophycinsynthetase. Samples were separated in 11.5% SDS-polyacrylamidegels and were stained with Serva Blue R. (A) Puri¢ed cyanophy-cin from Aphanocapsa sp. PCC6308 (lane 1, 50 Wg), Synechocystissp. PCC6803 (lane 2, 50 Wg) and Synechococcus sp. MA19 (lane3, 200 Wg). (B) Puri¢cation of cyanophycin synthetase from Syne-chococcus sp. MA19. Soluble protein fraction (lane 1, 100 Wgprotein), after gel ¢ltration on Superdex S200 prep grade (lane 2,10 Wg), after chromatography on Mono Q HR5/5 (lane 3, 7.5 Wg),after chromatography on Superdex S200 HR (lane 4, 5 Wg). Theposition of the protein band corresponding to cyanophycin syn-thetase is marked by an arrow. The sizes of molecular massstandard proteins (Std) are provided.

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mum rate. In contrast, cyanophycin synthetase prep-arations from the mesophilic Synechocystis sp.PCC6803 showed a steep decrease of the polymer-ization rates, when the incubation temperature wasraised from 20 to 40³C, and at 70³C, the rate wasalmost zero.

During the second puri¢cation step on Mono QHR5/5, a second protein exhibiting weak cyanophy-cin synthetase activity was eluted in a distinct peakat higher ionic strength from the column. SDS-PAGE of this preparation resulted in one major pro-tein band with an apparent molecular mass of51 000 þ 2000 Da.

3.4. N-terminal sequence analysis

No N-terminal amino acid sequence could be ob-tained from the puri¢ed 130 000-Da cyanophycinsynthetase protein. Determinations of N-terminialso failed with cyanophycin synthetase prepara-tions, which we puri¢ed before from other sources(i.e. from Aphanocapsa sp. PCC6308, Synechocystissp. PCC6803 and A. cylindrica, data not shown).This may indicate the presence of a common struc-tural feature of the corresponding cyanophycin syn-thetase N-termini, which prevents Edman degrada-tion.

Fig. 4. Temperature optima of cyanophycin synthetase-catalyzed polymerization rates. The polymerization of cyanophycin was determinedwith the puri¢ed enzyme from Synechococcus sp. MA19 (b) and with a highly enriched enzyme preparation from Synechocystis sp.PCC6803 (R) at di¡erent incubation temperatures as described in Section 2.

Table 1Puri¢cation of cyanophycin synthetase from Synechococcus sp. MA19

Step Volume(ml)

Protein(mg ml31)

Total protein(mg)

Total activity(mU)

Speci¢c activity(U g31)

Puri¢cation(fold)

Recovery(%)

Soluble protein fraction 10.0 25.0 250.0 2.42 0.0097 1 100Superdex 200 prep grade 25.0 0.40 10.0 10.0 1.0 103 413Mono Q HR5/5 0.5 0.30 0.15 0.17 1.1 113 7.0Superdex 200 HR 0.5 0.18 0.09 0.13 1.4 144 5.4

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The ¢rst 15 amino acid residues of the N-terminalsequence of the 51 000-Da protein (TTPEEVL(T orK)LII?KGI) exhibited 60% identity to the N-termi-nus of the glutamine synthetase from the cyanobac-terium Phormidium lapideum [23]. Less similarity wasdetected to the slr2001-encoded hypothetical proteinfrom Synechocystis sp. PCC6803 [24], which is mostlikely a cyanophycinase [11].

4. Discussion

This is the ¢rst report on the synthesis and accu-mulation of cyanophycin in a member of the genusSynechococcus (Anacystis). In a comparative study,Lawry and Simon reported the absence of cyanophy-cin in di¡erent species of this unicellular group ofcyanobacteria even under conditions known to stim-ulate cyanophycin synthesis [3]. This was furthercon¢rmed by [11], who reported the absence of ahybridization signal with genomic DNA of Synecho-coccus sp. PCC7942 when they employed a geneprobe from A. variabilis. Future careful examinationson the accumulation of cyanophycin in other Syne-chococci will show whether the occurrence of cyano-phycin in strain MA19 is exceptional for the genusSynechococcus.

The molecular mass of the cyanophycin synthetasesubunit from Synechococcus sp. MA19, which wasestimated to be 130 000, is higher than those reportedfor the enzymes from A. variabilis and Synechocystissp. PCC6803, which were approximately 100 000 Da[11,24]. However, the enzyme from all three speciesconsisted of only one type of subunit.

The 51 000-Da protein exhibiting weak cyanophy-cin synthetase activity showed no N-terminal se-quence similarity to known cyanophycin synthetases[11,24]. Similarities were, however, obtained to a glu-tamine synthetase [23] and to a cyanophycinase [24].Under the conditions of the cyanophycin synthetaseenzyme assay used in this study (i.e. presence of ex-cess aspartic acid, labelled arginine and unlabelledcyanophycin as primer), the latter enzyme may cata-lyze monomer exchange reactions, which would re-sult in labelled cyanophycin thus simulating synthe-tase activity. Further enzymatic investigations haveto be done with the 51 000-Da protein to ¢nd out ifthis is the physiological function.

Only very limited knowledge is available on thebiochemical characteristics of cyanophycin synthe-tases and no data existed before about the temper-ature optima of cyanophycin synthetases. Althoughthe results of the temperature dependency of the cy-anophycin polymerization rate as shown in Fig. 4re£ects the e¡ects of temperature on both (i) onthe enzyme stability as well as (ii) on the reactionrate due to the necessary long incubation time, whichis required for the assay, the results showed that theenzyme from the thermophilic bacterium is able toproceed polymerization at a signi¢cant rate at tem-peratures as high as 80³C. On the other hand, thesigni¢cant decrease of the polymerization rate ob-tained with the enzyme preparation from the meso-philic Synechocystis sp. PCC6803 can be explainedby the instability of this enzyme at elevated temper-atures (data not shown). The latter seems to be atypical feature of cyanophycin synthetases. Both en-zymes, which were enriched or puri¢ed and charac-terized before from A. cylindrica [10] and A. varia-bilis [11], exhibited a high degree of instability undervarious conditions of storage and puri¢cation.Therefore, the enzyme from the thermophilic Syne-chococcus sp. MA19 may be a good candidate todevelop a biotechnological process for the in vivoand in vitro synthesis of cyanophycin at higher tem-peratures.

Acknowledgements

The authors thank Drs Miyake and Asada (Mo-lecular Laboratory of the National Institute of Bio-science and Human-Technology, Tsukuba, Ibaraki,Japan) for providing Synechococcus sp. MA19. De-termination of the N-terminal amino acid sequenceby Dr Bernhard Schmidt (Zentrum Biochemie undMolekulare Zellbiologie der Georg-August-Universi-ta«t Go«ttingen, Germany) is gratefully acknowledged.

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