Biotechnological Process for Production of {beta}-Dipeptides from Cyanophycin on a Technical Scale and Its Optimization

A triphasic process was developed for the production of βdipeptides from cyanophycin (CGP) on a large scale. Phase Icomprises an optimized acid extraction method for technicalisolation of CGP from biomass. It yielded highly purified CGPconsisting of aspartate, arginine, and a little lysine. PhaseII comprises the fermentative production of an extracellularCGPase (CphE_al) from Pseudomonas alcaligenes strain DIP1 ona 500-liter scale in mineral salts medium, with citrate as thesole carbon source and CGP as an inductor. During optimization,it was shown that 2 g liter^–1 citrate, pH 6.5, and 37°Care ideal parameters for CphE_al production. Maximum enzyme yieldswere obtained after induction in the presence of 50 mg liter^–1CGP or CGP dipeptides for 5 or 3 h, respectively. Aspartateat a concentration of 4 g liter^–1 induced CphE_al productionwith only about 30% efficiency in comparison to that with CGP.CphE_al was purified utilizing its affinity for the substrateand its specific binding to CGP. CphE_al turned out to be a serineprotease with maximum activity at 50°C and at pH 7 to 8.5.Phase III comprises degradation of CGP to β-aspartate-arginineand β-aspartate-lysine dipeptides with a purity of over99% (by thin-layer chromatography and high-performance liquidchromatography), employing a crude CphE_al preparation. Optimumdegradation parameters were 100 g liter^–1 CGP, 10 g liter^–1crude CphE_al powder, and 4 h of incubation at 50°C. Theoverall efficiency of phase III was 91%, while 78% (wt/wt) ofthe used CphE_al powder with sustained activity toward CGP wasrecovered. The optimized process was performed with industrialmaterials and equipment and is applicable to any desired scale.

INTRODUCTION

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INTRODUCTION

Cyanophycin granule polypeptide (CGP), or multi-L-arginyl-poly(L-asparticacid), was discovered in cyanobacteria about 130 years ago (6).The branched polymer consists of a poly(aspartic acid) backbonewith arginine moieties linked to the β-carboxyl group ofeach aspartic acid by its ${alpha}$ -amino group (34, 42) and accumulatesduring the transition from the exponential to the stationarygrowth phase (23, 40) and under limiting conditions, includinglow temperature, low light intensity, and phosphorus or sulfurlimitation (44). CGP functions as a temporary nitrogen, energy,and possibly also carbon reserve (10, 21). Because CGP containsfive nitrogen atoms in every building block, it is an idealintracellular nitrogen reserve (43). Most genera of cyanobacteria(5, 22, 23, 43, 48) and some heterotrophic bacteria (12, 16,51) harbor a cyanophycin synthetase gene (cphA) and synthesizeCGP. The polymer is insoluble at neutral pH as well as at physiologicalionic strength (4). In cyanobacteria, CGP has a molecular massof 25 to 100 kDa (41), while in recombinant strains it exhibitsa molecular mass of 25 to 30 kDa and a lower polydispersityand contains lysine, which partially replaces arginine (2, 50).

CGP degradation (intra- or extracellularly) leads mainly tothe release of dipeptides. Intracellular degradation of CGPis catalyzed by cyanophycinases (CphB); the first cyanophycinasewas described for Anabaena cylindrica by Gupta and Carr (14).The enzyme is a monomeric, 29.4-kDa, serine-type CGP-specificexopeptidase with an ${alpha}$ -cleavage mechanism (35). In the last fewyears, aerobic and anaerobic bacteria capable of degrading CGPby extracellular cyanophycinases (CphE) have been identified(28, 29, 31, 36). Similar to CphB, the extracellular CGPasesCphE_Pa and CphE_Bm, from Pseudomonas anguilliseptica B1 and Bacillusmegaterium BAC19, respectively, were identified as serine-typeCGP-specific enzymes and produced mainly CGP dipeptides as degradationproducts. Labeling studies of CphE_Pa showed that the enzymehydrolyzes CGP at the carboxyl terminus and successively releasesβ-Asp-Arg dipeptides from the degraded polymer chain end(30). Moreover, extracellular CGPases were also found in strictlyand facultatively anaerobic bacteria, such as Sedimentibacterhongkongensis KI and Pseudomonas alcaligenes DIP1, respectively(31, 36).

Until recently, no practical applications for CGP itself orfor the dipeptides derived from it were known. In contrast,economically important applications have been established forpoly(aspartic acid) as a substitute for nonbiodegradable polyacrylates(38) or as an additive in the paper, paint, and oil industries(15). Biomedical applications have also been described for poly(asparticacid) (20, 49). Only recently were biomedical applications forCGP dipeptides and possibly CGP itself proposed; these applicationsdepend on the astonishingly widespread occurrence of CGP-degradingbacteria in the digestive tracts of various vertebrates (A.Sallam and A. Steinbüchel, submitted for publication),thus indicating that CGP is probably degraded in these habitats.On the other hand, di- and tripeptides are more efficientlyutilized than intact proteins or free amino acids, have a greaternutritional value, and are better absorbed (24). Moreover, administrationof amino acids as dipeptides or in mixtures is clinically approved,and products containing these preparations have been commercialized(7, 9, 19, 39). Thus, CGP and/or its dipeptides are consideredpotential natural food additives and/or therapeutics (Sallamand Steinbüchel, submitted for publication).

The production and efficient isolation of CGP in gram amountswere established only during recent years. Several recombinantstrains of Escherichia coli, Ralstonia eutropha, Pseudomonasputida, and Acinetobacter baylyi strain ADP1 were used; thelatter showed a maximum CGP content of about 46% (wt/wt) (32).To provide enough CGP dipeptides for in vivo experiments andto approach industrial-scale production, we developed an economicallarge-scale process which provides pure CGP dipeptides fromCGP-containing biomass. Furthermore, the last two phases ofthe initial triphasic process were largely optimized for futureapplications, CphE_al was technically purified from a powderobtained from lyophilized crude supernatant, and the biochemicalcharacteristics thereof were revealed.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Media.
The following simplified medium (SM) was used for fermentativeproduction of CphE_al by P. alcaligenes DIP1: 1.0 g NH₄Cl, 5.0g KH₂PO₄, 1 g MgSO₄·7H₂O (sterilized and added separately),and 10 ml of trace element stock solution (8) per liter of tapwater. The pH of the medium was adjusted to 7.0, and the mediumwas subsequently sterilized by autoclaving at 121°C for20 min. For optimization experiments and for easier preparation,a trace element solution containing the following compoundswas used (13): KI (0.02 g liter^–1), CoCl₂·6H₂O(0.8 g liter^–1), MnSO₄·H₂O (0.64 g liter^–1),FeSO₄·7H₂O (0.4 g liter^–1), ZnSO₄·7H₂O (0.24g liter^–1), CuSO₄ (0.038 g liter^–1), H₃BO₃ (0.060g liter^–1), Na₂MoO₄·2H₂O (0.06 g liter^–1),and CaCl₂·2H₂O (0.46 g liter^–1). The compositionof SM proved to be suitable for monitoring turbidity changeswhich occur due to degradation of the inductor (CGP), therebyrepresenting a definite sign for the release of CphE_al.

Additionally, Luria-Bertani (LB) medium (37) was used for maintenanceof viable cultures. CGP-overlaid agar plates were prepared usinga sterile mixture of CGP and 1.2% (wt/vol) Bacto agar, whichwas poured as thin layers onto SM agar plates (36).

Cultivations on a 500-liter scale.
Cultivations on a 500-liter scale were done in a Biostat D650stainless steel bioreactor (B. Braun Biotech International,Melsungen, Germany) with a total volume of 650 liters (64-cminner diameter and 198-cm height) and a d/D value (stirrer diameter/vesseldiameter) of 0.375. This bioreactor was equipped with threestirrers, each containing six paddles, and a Funda-Foam mechanicalfoam destroyer (B. Braun Biotech International, Melsungen, Germany).Sterilizable ports were used to measure dissolved oxygen (pO₂)(model 25; Mettler-Toledo, Steinbach, Switzerland), pH (modelPa/25; Mettler-Toledo), foam (model L300/Rd. 28; B. Braun BiotechInternational, Melsungen, Germany), temperature (pt 100 electrode;M. K. Juchheim, Fulda, Germany), and optical density at 850nm (OD₈₅₀) (model CT6; Sentex/Monitek Technology, CA). Operationswere controlled and recorded by a digital control unit in combinationwith the MFCS/win software package (B. Braun Biotech International,Melsungen, Germany).

Cell separation and concentration and desalting of supernatant proteins.
Cells were harvested by centrifugation with a type Z41 or typeZ61 CEPA continuous centrifuge (Carl Padberg ZentrifugenbauGmbH, Lahr, Germany). Proteins were concentrated and desaltedusing a cross-flow Sartocon polyethersulfone cassette with acutoff point (COP) of 30 kDa and a Sartocon 2 Plus stainlesssteel holder (Sartorius AG, Göttingen, Germany). To identifya suitable filtration system for CGP dipeptides, several 20-mlaliquots were filtered with different membranes having the followingCOPs: 30, 10, 5, 1, and 0.5 kDa.

Sources of CGP.
To obtain a large amount of CGP for preliminary tests and forprocess design, 4,776 g cell dry biomass of CGP-containing cellsof R. eutropha H16-PHB-4- ${Delta}$ eda (pBBR1MCS-2::cphA₆₃₀₈/edaH16) wereused; these cells were previously produced during one 500-literfermentation and had a CGP content of 32% (wt/wt) (46). Additionally,several other lyophilized biomass batches from lab-scale fermentationsof R. eutropha H16-PHB-4- ${Delta}$ ldh/ ${Omega}$ Km-cphA₆₃₀₈ were mixed (a totalof 2,490 g) and treated separately. After CGP extraction accordingto a modified acid extraction method (11; see below), 621 and82 g of dry CGP were obtained from both charges, respectively.

Fermentative production of CGP on a 500-liter scale.
For optimization experiments for the designed technical process,CGP was produced using the recombinant strain E. coli DH1(pMa/c5-914::cphA_PCC6803)(11) in a 500-liter fermentation according to the method ofElbahloul et al. (10). For this process, 7% (vol/vol) protamylasse(Avebe, Veendam, The Netherlands), a residual compound of industrialstarch production, was used. Microscopic examination of samplestaken each hour revealed progressive intracellular accumulationof CGP (Fig. 1B), with a maximum after 13 h. When the OD₆₀₀was 18.3, after 15 h, fermentation was terminated. Analysisrevealed a maximum CGP content of the cells of 13% (wt/wt ofcell dry mass) after 13 h of fermentation, which dropped toabout 10% (wt/wt) during the next 2 hours. This extraction yielded135 g of CGP powder from the resulting 4,626 g wet mass (1,372g cell dry mass).

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FIG. 1. (A) Degradation halos caused by the extracellular CGPase from P. alcaligenes strain DIP1 on a CGP-overlaid agar plate. CphE, the crude enzyme powder before the degradation phase (phase III); CphE R, the recovered powder after the degradation phase. (B) Phase-contrast micrograph of cells of E. coli DH1(pMA/c5.914::cphA_PCC6803) after 15 h of fermentation in a Biostat D650 reactor in 7% (vol/vol) protamylasse medium. CGP grana appear as light-reflecting accumulations in the cells. Bar, 10 µm. (C) CGP degradation in a 100 g liter^–1 water suspension of the polymer by the action of 10 g liter^–1 crude CphE_al at 50°C. Tube 1, control suspension (no CphE_al); tube 2, after CGP degradation.

Technical-scale isolation of CGP from biomass.
To isolate and purify CGP on larger scales, the acid extractionmethod (11) was optimized as follows. The CGP-containing drymass was suspended in tap water to give a final concentrationof 100 g liter^–1. The pH was reduced to 1 with concentratedHCl (32%), and the suspension was stirred overnight and thencentrifuged at 17,000 rpm, using a CEPA Z61 continuous centrifuge(Carl Padberg Zentrifugenbau GmbH, Lahr, Germany). The pelletwas resuspended in 20 liters of HCl (0.1 N), stirred for 1 h,and centrifuged again, and the supernatant was added to thefirst charge, while the pellet was discarded. The CGP-containingsupernatant was neutralized (pH 7.3) with NaOH (50%), and CGPwas precipitated. The milky suspension was left to settle overnightat 4°C before the supernatant was discarded. CGP was dissolvedand neutralized three more times to remove all impurities whichwere insoluble in diluted HCl. The resulting CGP was resolvedin 30 liters of 0.1 N HCl and was then passed through a 0.2-µmtype 00 Sartobran-P filter unit (Sartorius AG, Göttingen,Germany). The solution was neutralized again (pH 7.3) with NaOHand left overnight to settle, whereas the supernatant was discarded.To remove any water-soluble impurities and to desalt CGP, thepellet was washed three successive times with five bed volumesof distilled water. Finally, the CGP pellet was centrifugedat 20,000 rpm (CEPA Z41 continuous centrifuge), frozen at –30°C,and lyophilized in a BETA 1-16 freeze-dryer (Christ Gefriertrocknungsanlagen,Osterode, Germany). CGP was sterilized by diethyl ether treatmentor by solvation in 0.1 N HCl and sterile filtration as describedbefore (36).

Substrate utilization.
Substrate utilization was investigated in 100-ml Klett flaskswith baffles; each flask contained 10 ml of SM and 1 g liter^–1of one of the following substrates (sterile filtered [pore size,0.2 µm]; Millipore GmbH, Eschborn, Germany): lactate,citrate, succinate, acetate, propionate, gluconate, glucose,fructose, sucrose, and glycerol. Experiments were performedin duplicate over an incubation period of 24 h at 30°C andwere inoculated from a preculture grown under the same testconditions. The following potential inductors were tested: CGP,β-dipeptides from CGP, synthetic dipeptides ( ${alpha}$ -arginine-aspartate, ${alpha}$ -lysine-aspartate, and ${alpha}$ -ornithine-aspartate) (Sigma-Aldrich,St. Louis, MO), ${alpha}$ -polyaspartate (Bayer AG, Leverkusen, Germany),poly- ${varepsilon}$ -lysine (Chisso, Tokyo, Japan), the amino acids L-aspartate,L-arginine, L-lysine, L-citrulline, and L-ornithine, and theaspartate analogues N-acetyl-aspartate, ureidosuccinic acid,and N-carbamoyl-aspartate.

Analytical techniques.
Bacterial growth and CGP degradation were monitored by measuringchanges in turbidity in Klett flasks, using a Klett photometer(Manostat Corporation, NY) or an Eppendorf 1101 M spectrophotometer(578-nm wavelength; Eppendorf, Hamburg, Germany). Otherwise,the OD₆₀₀ of 1-ml fermentation samples was estimated using aLibra S5 photometer (Biochrom Ltd., Cambridge, United Kingdom).Thin-layer chromatography was done on silica gel 60 plates (Merck,Darmstadt, Germany), and the starting eluent for high-performanceliquid chromatography (HPLC) (81% [vol/vol] 50 mM sodium acetate,19% [vol/vol] methanol) was also used here as a run buffer;for staining, 20% (wt/vol) ninhydrin solution in acetone wasused. DNA concentrations were determined photometrically at260 nm, and the concentrations of carbohydrates were measuredby the anthrone reagent method (45).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)was performed on 11.5% (wt/vol) gels according to the methodof Laemmli (18). In-gel renaturation of proteins was appliedaccording to the method of Lacks and Springhorn (17). Proteinsand CGP were visualized by Coomassie blue staining (47) or silverstaining (only for proteins) (27). Concentrations of total proteinand CGP were determined using Bradford reagent (10). Proteinsin cultivation samples were concentrated, desalted, and separatedfrom CGP dipeptides by ultrafiltration, using 10-kDa-membraneVivaspin tubes (Vivascience AG, Hannover, Germany) or an Amiconchamber (Amicon, Beverly, MA) with 10-kDa membranes (MilliporeCorporation, Bedford, MA) for volumes of up to 500 ml. Freeamino acids and dipeptides were detected by HPLC (Kontron Instruments,Neufahrn, Germany) after precolumn derivatization with orthophthaldialdehyde(OPA) as described before (1, 36). CGP samples were subjectedin advance to acid hydrolysis (6 N HCl, 95°C, overnight).

Determination of enzyme activity and concentration.
The activity of cyanophycinase (CphE_al) was inspected by theformation of degradation halos on CGP-overlaid agar plates.For this assay, 5-ml culture samples were centrifuged at 2,800x g for 30 min (Megafuge 1.0 R; Heraeus Sepatech GmbH, Osterode,Germany), and 4 ml of the supernatant was concentrated 100-fold.For quantitative determination of the enzyme in culture samples,a photometric method was developed as follows. Two microlitersof the concentrated culture supernatant was added to 1 ml ofa CGP suspension (100 mg liter^–1) and incubated for 30min at 30°C in a tube rotator (3 rpm). Finally, the OD₆₀₀of samples indicated the decrease in CGP, which in turn determinedthe concentration of CphE_al through a respective calibrationcurve.

Optimal concentrations and conditions for CGP degradation.
To determine the optimum ratio between CGP and crude CphE_alconcentrations in relation to the incubation time, a seriesof dilutions (10 to 100 g liter^–1) of pure CGP suspensions(in water, pH 7.3) were prepared in plastic tubes with a totalvolume of 1 ml each. Different amounts of crude CphE_al powder(4.6% [wt/wt] CphE_al content) were added to each tube, and thetubes were incubated at 30°C in a tube rotator (3 rpm) toreveal the required incubation periods for complete CGP degradation.Experiments to determine the optimum pH for CGP degradationwere conducted using plastic tubes containing 1 ml CGP suspension(100 g liter^–1) with pH values between 5.0 and 9.0 inaddition to 10 g liter^–1 crude CphE_al, and the tubes wereincubated at 30°C for 30 min. Reaction mixtures for determinationof the optimum degradation temperature contained similar concentrationsof CGP (pH 7.0) and CphE_al and were incubated for 30 min at15, 20, 25, 30, 35, 37, 40, 50, 60, or 70°C. After bothexperiments, CGP degradation activities in all tubes were calculatedas percentages and compared.

Purification of CphE_al with organic solvents and ammonium sulfate precipitation.
Each 1 ml of cold ethanol, acetone, or methanol at a concentrationof 10 to 100% (vol/vol) was added to a 50-µl aliquot ofconcentrated crude CphE_al solution (14 g liter^–1) in aplastic tube and incubated for 60 min at –20°C. After5 min of centrifugation at 16,000 x g, the pellets were driedand resuspended in 50 µl sodium phosphate buffer (50 mM,pH 7.0). Ammonium sulfate fractionation was done by stepwiseincreasing the solubility saturations from 10 to 100% in 10ml of crude CphE_al solution (3.5 g liter^–1). Tubes wereincubated at room temperature for 30 min and centrifuged for10 min at 16,000 x g. Pellets were suspended in sodium phosphatebuffer (pH 7.0) and then desalted by ultrafiltration. All pelletswere assayed for CGP degradation on CGP-overlaid agar platesas well as for protein content by SDS-PAGE.

Substrate affinity purification of CphE_al.
A purification method for CphE_al was developed which dependedon the strong affinity of the enzyme in the crude extract forits insoluble substrate (CGP). To determine the time requiredfor complete binding of the enzyme to CGP at pH 7.0, 0.5 mlof concentrated crude CphE_al solution (14 g liter^–1) wasadded to 0.5 ml of a CGP suspension (100 g liter^–1). Sampleswere withdrawn every 10 min, and 2-µl aliquots of thesupernatant were tested for activity. The degree of diminishmentof degradation halos on CGP-overlaid agar plates indicated theextent of binding of CphE_al to CGP. The actual purificationprocess was performed in a similar 1-ml reaction mixture andproceeded as follows: after complete binding of CphE_al to CGP,the mixture was centrifuged for 0.5 min (16,000 x g), the supernatantwas discarded, and the pellet was washed five times with 10ml of sodium phosphate buffer. Afterwards, the pellet was suspendedin 1 ml phosphate buffer and incubated overnight at 30°Cwith rotation (3 rpm) until complete degradation of CGP hadoccurred. The mixture was centrifuged (5 min, 16,000 x g), CGPdipeptides were then removed by ultrafiltration, and the concentratedprotein fraction of the supernatant was analyzed for purityby SDS-PAGE.

Characterization of CphE_al.
To determine the temperature stability of purified CphE_al, 10-µlaliquots were incubated for 20 min at different temperatures(10 to 80°C); 3 µl thereof was then tested for degradationactivity on CGP-overlaid agar plates at 30°C. For the optimumdegradation temperature, 3-µl aliquots of the purifiedCphE_al solution and 1-ml CGP suspensions (100 g liter^–1in sodium phosphate buffer, pH 7.0) were mixed and incubatedfor 20 min at different temperatures (10 to 60°C), whereasthe optimum pH was determined in similar mixtures with differentpH values (5 to 9), which were incubated for 30 min at 30°C.Finally, CGP degradation was determined photometrically forboth experiments, as described above.

The substrate specificity of purified CphE_al was tested as describedbefore (29), employing the following polypeptide substrates:CGP, bovine casein (Hammersten-grade; Merck, Darmstadt, Germany),bovine serum albumin (BSA) (Roth, Karlsruhe, Germany), and poly( ${alpha}$ ,β-D/L-asparticacid) (Bayer AG, Leverkusen, Germany). To reveal the effectsof enzyme inhibitors, 50-µl aliquots of the purified enzymesolution were added to 450 µl of sodium phosphate bufferand incubated for 2 h at 30°C in the presence of one ofthe following group-specific inhibitors: leupeptin (thiol proteases),EDTA (metalloproteases), Pefabloc (serine proteases), phenylmethylsulfonylfluoride (PMSF) (serine proteases), or N-bromosuccinimide (tryptophanresidues). Five-microliter samples of each reaction mixturewere assayed for activity on CGP-overlaid agar plates. Afterwards,5-µl aliquots of a CGP suspension (50 g liter^–1)were added to the reaction tubes, incubated for a further 15min, centrifuged, and screened for degradation products viaHPLC.

RESULTS

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RESULTS

A triphasic technical process was designed and optimized forthe production of β-dipeptides from highly concentratedCGP suspensions. Phase I provided CGP and comprised large-scaleisolation and purification of CGP from biomass. Phase II providedcyanophycinase and comprised fermentative production of crudeCphE_al powder from P. alcaligenes strain DIP1. In phase III,the products of phases I and II were combined, and degradationof CGP in highly concentrated CGP suspensions to its dipeptideswas achieved.

Phase I (large-scale isolation and purification of CGP).
The optimized extraction method was applied to CGP-containingdry biomass of R. eutropha (7,266 g) and E. coli (1,372 g).Total amounts of 704 g and 135 g of purified CGP, respectively,were obtained. HPLC analysis of the individual amino acid constituentsof the isolated CGPs revealed that the polymer was highly pureand consisted of aspartate, arginine, and lysine. The lattercomprised approximately 6.8 mol% of the polymer constituents.SDS-PAGE analysis of the isolated CGP showed a molecular massof about 25 to 30 kDa. These characteristics are in strong agreementwith those of CGP previously produced by both strains (10, 11,46).

Phase II (large-scale production of crude CphE powder).
To obtain enough crude CGPase for CGP degradation (phase III),P. alcaligenes DIP1 (36) was cultivated on a 500-liter scale.A preculture was cultivated in 2-liter baffled flasks containing1 liter of SM with 1 g liter^–1 sodium citrate; the flaskswere incubated with shaking for 12 h at 30°C. The bioreactorwas filled with 420 liters of the same medium, sterilized, andinoculated with 4% (vol/vol) preculture. The initial pH wasadjusted to 6.9, and an increase to 7.5 during growth was tolerated.The pO₂ was adjusted to exceed 40% saturation in the mediumand was automatically controlled by stirring; the aeration ratewas kept constant at 0.7 volume per volume per minute. Duringfermentation, cells of strain DIP1 started to grow after 1 hourand reached a maximum OD₆₀₀ value of 0.4 after 9 h. Excretionof CphE_al was induced when 0.25 g liter^–1 sterile CGPsuspension was added after 11 h of incubation. CGP increasedthe OD₆₀₀ immediately from 0.55 to 0.98 and was completely degradedwithin 2 h. About 1 hour later, cells started to grow slightlyon the released CGP dipeptides. The fermentation was terminatedafter a total cultivation period of 14 h (Fig. 2).

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FIG. 2. Batch fermentation for CphE_al production from P. alcaligenes DIP1 in a Biostat D650 stirred tank reactor containing 420 liters SM with 1 g liter^–1 sodium citrate. The reactor was inoculated with 4% (vol/vol) of a preculture that was cultivated in 2-liter baffled flasks containing 1 liter of the same medium and was incubated for 12 h at 30°C. The fermentation parameters and cultivation conditions in the Biostat D650 reactor were as follows: pH of 6.9 to 7.5, temperature of 30°C, and aeration rate of 0.2 volume per volume per minute. pO₂ was set to a minimum of 40% and was adjusted automatically by increasing the stirring rate, which otherwise was kept at 100 rpm. The arrow indicates the time of induction by CGP, which caused a sudden increase in medium turbidity followed by a return to the initial value after the release of CphE_al and CGP degradation. ${blacklozenge}$ , OD₆₀₀; ${perp}$ , pH; ${blacktriangleup}$ , stirrer speed (rpm); –, pO₂ (% of saturation); •, airflow (liters min^–1).

For harvesting, concentration, and desalting of proteins inthe cell-free supernatant, including the excreted cyanophycinase,a continuous process was installed (Fig. 3). Cells were continuouslyseparated in a CEPA Z41 continuous centrifuge. The supernatantwas collected in a 100-liter tank and was simultaneously concentratedby a cross-flow unit with a 30-kDa filter cassette. The temperatureof the solution was kept at 20°C or below by adding iceto the tank. The final 5 liters of the concentrated supernatantwas desalted with 25 liters of H₂O, frozen at –30°C,and lyophilized to obtain 17.5 g of a crude protein powder.

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FIG. 3. Continuous system for harvesting, concentration, and desalting of proteins in the supernatants of fermentation broth of P. alcaligenes strain DIP1 (phase II). For harvesting, a CEPA Z41 continuous centrifuge was used to separate the cells from the medium. The supernatant was collected in a central 100-liter tank. For concentration, a cross-flow unit with a 30-kDa cassette was connected to the central tank; the concentrated retentate was pumped back into the tank, while the permeant material was directly discarded. The flow rate of the cross flow was adjusted to maintain only 50 liters in the tank. The final concentrated enzyme solution of 5 liters was desalted with 5 bed volumes of H₂O, frozen at –30°C, and lyophilized.

Phase III (degradation of purified CGP to its dipeptides).
Large-scale degradation of CGP to its dipeptides was startedfrom 250 g purified CGP in tap water (final concentration, 50g liter^–1) and 10 g of the crude CphE_al powder (finalconcentration, 2 g liter^–1). The mixture was stirred slowlyat 30°C to ensure complete degradation within 12 h, as estimatedfrom the results of small-scale experiments (Fig. 4). Afterwards,the dipeptide solution was sterilized by filtration, separatedfrom the crude protein components by use of the same cross-flowsystem as in phase II, frozen at –30°C, and finallylyophilized. Loss in the resulting dry weight with differenttested filtration systems increased with a decreasing COP ofthe filter. This indicated that the applied cross-flow systemclearly provided the most suitable COP to purify CGP dipeptidesand also to recover the crude CphE portion of the degradationmixture. The recovery rate for the crude enzyme was 78% (wt/wt),and the regained protein powder showed retained cyanophycinaseactivity on CGP-overlaid agar plates (Fig. 1A).

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FIG. 4. Incubation periods required for complete degradation of CGP at different concentrations (10 to 50 g liter^–1) by crude CphE_al powder applied at different concentrations (1 to 10 g liter^–1). The reaction tubes were incubated at 30°C in a tube rotator rotating at 3 rpm. Suspensions with a CGP concentration of 50 g liter^–1 could be degraded within 10 h in the presence of 2 g liter^–1 crude CphE_al powder. Each value represents a mean time period measured for two parallel experimental repetitions, with an estimated standard deviation maximum of 10%.

Quality control.
For final quality control of the resulting dipeptide powder,samples were examined, directly and also after acid-catalyzedhydrolysis, by thin-layer chromatography and HPLC. Both methodsrevealed the amino acids aspartate, arginine, and lysine asthe only constituents in the analyzed samples. Because DNA andtotal carbohydrate contents did not exceed 0.0002 x 10^–5%± 4 x 10^–5% and 0.05 x 10^–3% ± 8 x10^–3%, respectively, the purity of the dipeptides in theobtained powder was estimated to be over 99%. The process yielded227.5 g purified dipeptides from 250 g CGP, representing anoverall efficacy of the process of 91%.

Process optimization.
The conditions applied during phase II and phase III were furtherinvestigated in order to enhance the parameters and effectivenessof the process.

Optimum conditions for growth and CphE_al production by P. alcaligenes DIP1.
Optimum growth for strain DIP1 occurred in the presence of 6g liter^–1 citrate and at pH 6.5 and 37°C. However,no CphE_al production was observed at citrate concentrationsbelow 0.5 or above 4 g liter^–1. Because the maximum CphE_alproduction was monitored at 2 g liter^–1 citrate, the followingconditions were considered optimal: SM with 2 g liter^–1citrate, pH 6.5, 37°C, induction after 13 h, and harvestingafter 3 h of further incubation.

Optimum induction of CphE_al with CGP and alternative substances.
All potential inductors (see Materials and Methods) were testedfirst at a concentration of 0.25 g liter^–1. Only CGP,its dipeptides, and aspartate induced the formation of significantamounts of CphE_al (Fig. 5A). At CGP concentrations between 0.001and 3.0 g liter^–1, the inductive effect increased withconcentrations of up to 0.05 g liter^–1, which led to maximumCphE_al production after 5 h of induction (Fig. 5B). CGP dipeptidesused in the same concentration range as CGP showed similar results;however, maximum CphE_al production was obtained after only 3h (Fig. 5C). Maximum induction by aspartate was obtained at4 g liter^–1 after 5 h of induction (Fig. 5D), but witha lower productivity than that with the optimum CGP concentration.

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FIG. 5. CGP degradation by concentrated supernatant samples from cultures of P. alcaligenes DIP1 after induction. CGP degradation was determined photometrically and was used as an indicator of the CphE_al concentration. (A) Samples from cultures induced with different potential inductors, indicated on the x axis, at a concentration of 0.25 g liter^–1. (B) Samples from cultures induced with different concentrations of CGP. (C) Samples from cultures induced with different concentrations of CGP dipeptides. (D) Samples from cultures induced with different concentrations of aspartate. CGP, cyanophycin; Arg-Asp, ${alpha}$ -arginine-aspartate; Lys-Asp, ${alpha}$ -lysine-aspartate; Orn-Asp, ${alpha}$ -ornithine-aspartate; CGP-Dip, CGP dipeptides; poly-Asp, poly(aspartic acid); poly-Lys, poly(lysine); Asp, L-aspartate; Lys, L-lysine; Cit, L-citrulline; Orn, L-ornithine; Arg, L-arginine; control, noninduced culture.

Optimum conditions for CGP hydrolysis to dipeptides by crude CphE_al.
To decrease the reaction volumes and the duration of the thirdprocess phase (CGP degradation), the time required for CGP hydrolysisstarting with significantly higher concentrations (50 to 100g liter^–1) of the polymer was investigated at 30°C.These studies showed an increase in degradation time with decreasingconcentrations of crude CphE_al and with increasing concentrationsof CGP. In the presence of 10 g liter^–1 crude CphE_al,complete degradation of CGP applied at concentrations of 50g liter^–1 and 100 g liter^–1 was observed after 4and 16 h of incubation, respectively. Maximum CGP degradationoccurred at pH 6.5, but at pH values between 5.5 and 7.5, thedegradation rates were not much lower. CGP degradation increasedwith increasing temperature, with a maximum at 50°C. Applyingthese parameters (50°C, pH 6.5) (Fig. 1C) reduced the degradationtime for 50 and 100 g liter^–1 CGP to only 1 and 4 h, respectively.

Purification of CphE_al from crude supernatant powder, utilizing specific substrate binding.
Standard methods for technical protein purification using ammoniumsulfate or solvents gave only low recovery rates and low purificationfactors and were therefore not satisfactory. In contrast, usingthe insoluble CGP as a matrix to specifically bind CphE_al gavevery promising results. Initial experiments demonstrated thatan incubation time of 5 min was enough to bind CphE_al quantitativelyto the CGP matrix. SDS-PAGE of protein samples from all purificationsteps showed a gradual purification of the enzyme (Fig. 6A)and that the first two washing steps are necessary to removeother proteins. The purified enzyme exhibited an apparent molecularmass of 45 kDa by SDS-PAGE and regained activity by in-gel renaturation.The concentration of the purified CphE_al was 43.2 µg ml^–1,while the total protein content in the initial crude solutionwas 944 µg ml^–1. SDS-PAGE analysis of highly concentratedsamples and intensive silver staining revealed the presenceof only a few other protein bands, occurring at much lower concentrations(Fig. 6B).

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FIG. 6. SDS-PAGE of samples obtained during purification of CphE_al from P. alcaligenes strain DIP1 via specific binding of CGP. (A) SDS-polyacrylamide gel stained by the silver nitrate method. M, molecular mass standard proteins; C, control of crude CphE_al; S₁, supernatant sample obtained immediately after mixing CGP and crude CphE_al; S₂, supernatant sample obtained after 6 min of binding time; S₂', same as S₁ but after 10-fold concentration; W₁ and W₂, supernatant samples obtained after the two washing steps. (B) SDS-polyacrylamide gel with triple the volume of purified CphE_al as that used for panel A and extended staining with silver nitrate. Only a few other low-concentration protein bands can be observed in addition to that of CphE_al at 45 kDa.

Determination of CphE_al content in fermentation supernatants.
If an application of CphE_al is desired, then the concentrationof this CGPase in the produced supernatants must be known. Forthis purpose, an easy and rapid assay was developed in which3-µl solutions containing different concentrations (2.7to 43.2 µg ml^–1) of the purified enzyme were addedto 1 ml of CGP suspension with a fixed concentration (100 mgliter^–1, corresponding to an OD₆₀₀ of 0.215). Suspensionswere incubated for 60 min at 30°C, and the extent of CGPdegradation was determined by measuring the OD₆₀₀. The followingformula could be applied if test parameters were kept constantand if measurements were made in an OD₆₀₀ range of 0.05 to 0.215:E = (0.2404 – D)/0.0017, where E is the CphE_al contentin crude extract (µg ml^–1) and D is the OD₆₀₀ after60 min.

Required CGP amounts for technical purification of CphE_al.
To integrate the enzyme purification procedure into the mainproduction process, the amount of CGP which is necessary tobind all CphE_al (in supernatants with known CphE_al content)has to be determined. In a total volume of 0.5 ml, differentconcentrations of purified CphE_al solutions (1.4 to 19 µgml^–1) in 50 µl were added to different CGP concentrations(0.1 to 6 g liter^–1). The reaction mixtures were thenincubated for 10 min at 30°C and slowly rotated at 3 rpm.After 2 min of centrifugation at 16,000 x g, the supernatantswere screened for activity on CGP-overlaid agar plates to detectthe minimum CGP concentration which did not induce degradationhalos (i.e., complete binding). The determined data were integratedin the following formula: C = (E – 2.4392)/0.9432, whereC is the required CGP concentration (g liter^–1) and Eis the CphE_al content in crude extract (µg ml^–1).

Biochemical characterization of purified CphE_al.
The purified enzyme showed maximum CGP degradation activityat 50°C and complete inactivation at 68°C. The optimumpH range for CGP degradation was 7 to 8.5, with an optimum at8.5. Among the tested substrates, the purified CphE_al showedthe highest degradation activity with CGP. However, BSA wasalso partially degraded (65% in comparison to CGP). Bovine casein(17%) and poly(aspartic acid) (1.5%) were minimally affectedby the enzymatic activity of the purified CphE_al.

Inhibition experiments employing different group-specific inhibitors(Table 1) revealed that extracellular CphE_al is strongly inhibitedby the serine protease inhibitors Pefabloc and PMSF. Also, N-bromosuccinimide(tryptophan oxidant) led to a total inhibition of the enzyme.In contrast, CphE_al was not negatively affected by the thiolprotease inhibitor leupeptin or by the metalloprotease inhibitorEDTA. HPLC analysis of CGP degradation products (Fig. 7) confirmedthese results, except for samples that were treated with EDTA,which showed inhibition of CphE_al (75%).

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TABLE 1. Effects of several group-specific inhibitors on purified CphE_al from P. alcaligenes strain DIP1

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FIG. 7. Effects of different protease inhibitors on CphE_al are indicated by the decrease in CGP degradation products in comparison to the control (without inhibitor), as determined by HPLC.

DISCUSSION

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DISCUSSION

Economic factors are generally of great importance for technicalprocesses. Strains of P. alcaligenes, including strain DIP1,are known to grow on simple media and on minimal amounts ofa wide range of substrates (36). Citrate is a cheap substrate,that is available in technical quantities; it was previouslyused for the fermentative production of extracellular lipasesfrom P. alcaligenes. This, in addition to the high enzyme productivityof P. alcaligenes strains, was the reason for their applicationin fermentative production processes (3, 13, 26). Besides thesecharacteristics, the high stability and activity of the CGPasefrom strain DIP1 (36) rendered this strain ideal for the designedtechnical process.

The CGP acid extraction method of Frey et al. (11) was optimizedfor kilogram amounts of biomass. Optimization comprised thelogistics applied and an integrated filtration step which improvedCGP purity. In contrast, increasing the number of other purificationsteps (with diluted acid and water) may unnecessarily increasethe loss of CGP. Alternatively, the application of an effectiveinstrument, such as in cross-flow filtration, would increasethe overall productivity of this extraction method. However,the relatively high prices and limited life of such ultrafiltrationcassettes leave their application to be a matter of cost.

The identical purity grade of the resulting CGP dipeptides beforeand after filtration is clearly due to the initial lack of impuritiesin the resulting dipeptide solution. This represents an advantagefor applying a defined enzymatic process in comparison to directcultivation strategies with bacterial cells. On the other hand,the quantitative loss of dipeptides during filtration was expectedand, unfortunately, inevitable. Several general factors, includingfilter material, COPs, saturation of the membrane surface, and/orthe characteristics of the filtered substance itself, are knownto cause such losses. This also explains the loss of 9% of CGPand 22% of crude CphE_al during the degradation phase (phaseIII).

Maximum production of CphE_al by strain DIP1 in the presenceof 2 g liter^–1 citrate and a lack of enzyme formationin the presence of 6 g liter^–1 citrate (which was optimalfor growth) indicated that the enzyme is induced only duringsubstrate limitation. Furthermore, the fact that the dipeptidescan render the same inductive effect (Fig. 5C), but in a muchshorter time (3 h) than that with CGP (5 h) (Fig. 5B), indicatedthat CGP dipeptides are the actual inductors for CphE_al. Onthe other hand, the ability of aspartate to induce CphE_al, butwith a lower efficiency (Fig. 5D), makes the choice of the inductorcase and cost dependent.

The developed procedure for purification of CphE_al by specificbinding to CGP proved to be highly effective and has the advantageof separating CphE_al from other proteins in crude solutionsby use of one substance (CGP). The purification method endswith the degradation of the CGP matrix to its dipeptides. Atthe same time, these are the valuable end products of the processand can be directed further to the main production stream (noloss). The purification method is easy to scale up and to integrateinto the process if desired.

The efficiency of the second phase of the process and the possiblepurification of the enzyme could be enhanced by the applicationof two formulas that were developed in this study. The firstformula depends on the described photometric test and enablesrapid determination of CphE_al content in crude supernatants.The determined enzyme concentration can then be integrated intothe second formula to estimate the required amount of CGP tobind the complete content of CphE_al in the supernatant for purification.The scheme provides a reliable instrument for future batchesof crude CphE_al, which might differ in their protein composition.

The third phase of the process was found to be much more effectiveat 50°C than at 30°C. This optimization rendered muchhigher concentrations of CGP (up to 100 g liter^–1) tobe easily degraded in only about 25% of the time required at30°C. Also, the volume of degradation mixtures and the riskof their contamination are minimized at the elevated temperature.Apart from temperature, degradation time showed a colinear increasewith decreasing concentrations of crude CphE_al and with increasingCGP concentrations. Thus, the respective formula will be helpfulto apply optimum degradation parameters. The following formulawas calculated for applications at 50°C and is suitablefor CGP concentrations of up to 100 g liter^–1: P = 17.55– 29.891E, where P is the degradation time at 50°Cand E is the desired concentration of pure CphE_al (g liter^–1).

Substrate specificity experiments employing purified CphE_alrevealed that the enzyme degraded not only CGP but also BSA,but to a lesser extent. This indicated that the CGPase fromstrain DIP1 might be less specific than the previously characterizedCphE_Pa and CphE_Bm enzymes, from P. anguilliseptica B1 and B.megaterium BAC19, respectively. Another cause for this unspecificitymay be the presence of a few other proteins in the purifiedenzyme solution. Although these proteins were present at verylow concentrations, even a small amount of an unspecific proteasemight have partially degraded BSA.

The inhibition of CphE_al by serine protease inhibitors indicatesthat this CGPase most probably also belongs to the serine-typeproteases, as do the previously characterized CphB, CphE_Pa,and CphE_Bm enzymes. Moreover, total inhibition of CphE_al byN-bromosuccinimide shows that a tryptophan residue might beinvolved in its catalytic mechanism, in accord with the casefor CphE_Pa and CphE_Bm. CphE_al samples which were treated withleupeptin or EDTA showed no activity inhibition on CGP-overlaidagar plates; the positive results of HPLC analysis (Fig. 7)were due to precipitate formation during OPA derivatization(28, 29).

Although several characteristics of the purified CphE_al wererelatively similar to those of CphE_Pa and CphE_Bm (Table 2),some relevant differences were observed. Among these, the optimumtemperature for CphE_al (50°C), which is the highest temperatureoptimum for all known CGPases, provides a great advantage forlarge-scale application. The shift in optimum pH between thepurified enzyme (7 to 8.5, with an optimum at 8.5) and the enzymein crude preparations (5.5 to 7.5, with an optimum of 6.5) ismost probably due to the presence of many other proteins inthe crude preparations, representing a complex milieu. Interactionswithin such milieus may in turn affect the structure and/orproperties of the CGPase.

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TABLE 2. Comparison of biochemical characteristics of CphE_al from P. alcaligenes strain DIP1 and previously characterized extracellular cyanophycinases

Besides the ecological importance of enzymatic processes ingeneral, and in contrast to direct cultivation strategies withbacterial cells, defined enzymatic processes such as the onedescribed in this article have several advantages, includingsmall losses of end product (consumption during growth is excluded),smaller reaction volumes, fewer contaminants, and time savings.On the other hand, the commercial value of CGP dipeptides isdirectly related to the production costs of CGP itself. Overthe last few years, increasing CGP contents have been achievedusing new and more suitable economical substrates (25); thesedevelopments seem to move fast toward commercial CGP production.Such a development was also needed for other known bacterialpoly(amino acids), such as poly(glutamic acid) and poly( ${varepsilon}$ -lysine),to become commercialized (30, 33). Until that point, the biomedicalvalue of CGP dipeptides, currently under investigation (Sallamand Steinbüchel, submitted for publication), may in factprovide a balanced relationship to the current production costsof CGP.

FOOTNOTES

* Corresponding author. Mailing address: Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, D-48149 Münster, Germany. Phone: 49-251-8339821. Fax: 49-251-8338388. E-mail: steinbu{at}uni-muenster.de

Published ahead of print on 31 October 2008.

REFERENCES

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