Hydrolysis of native poly(hydroxybutyrate) granules (PHB)

International Journal of Biological Macromolecules25(1999)129–134Hydrolysis of native poly(hydroxybutyrate)granules(PHB),crystalline PHB,and artificial amorphous PHB granules by intracellular andextracellular depolymerasesJoseph M.Merrick*,Robert Steger,Darlene DombroskiDepartment of Microbiology,School of Medicine and Biomedical Sciences,State Uni6ersity of New York at Buffalo,Buffalo,NY14214,USAReceived9September1998;accepted2October1998AbstractNative poly(hydroxybutyrate)(PHB)granules,purified PHB and artificial amorphous PHB granules were examined as putative substrates for hydrolysis by the intracellular depolymerase system of Rhodospirillum rubrum and the extracellular depolymerase of Pseudomonas lemoignei.The R.rubrum depolymerizing system requires pretreatment of granules with a heat stable‘activator’fraction;the activator can be replaced by mild trypsin treatment.Artificial granules were prepared with a cationic detergent, cetyltrimethylammonium bromide(CTAB)and an anionic detergent,(sodium cholate).Cholate and CTAB PHB granules were hydrolyzed by both enzyme systems;however,some differences were noted.Cholate granules were hydrolyzed in the absence of the R.rubrum activator fraction.Activator was required for the hydrolysis of CTAB granules but could be replaced by heparin in the extracellular depolymerase system but not in the intracellular depolymerase system.A Triton X-114extract of native PHB granules inhibited the hydrolysis of trypsin-activated granules by the intracellular depolymerase.The inhibition was reversed by the activator fraction.Detergent extracts of granules activated with the R.rubrum activator were unable to inhibit the hydrolysis of trypsin-activated granules.These data suggest that the activator acts to modify an inhibitor present on native granules.©1999 Elsevier Science B.V.All rights reserved.Keywords:Depolymerase;Granules;Poly(hydroxybutyrate)1.IntroductionThe overall mechanism of the enzymatic depolymer-ization of endogenous PHB has not as yet been defined. The only system examined in some detail is an artificial one in which soluble enzymes from Rhodospirillum ru-brum have been used to degrade native PHB granules isolated from Bacillus megaterium KM[1].R.rubrum extracts prepared from cells that had depleted their own polymer stores readily attacked B.megaterium KM native PHB granules but not purified polymer.Native PHB granules were extremely labile and lost their ca-pacity to be depolymerized by the degradative system unless suitable precautions were taken.For example,they were rendered unsuitable as a substrate by a variety of chemical and physical treatments such as their close packing by centrifugation,freezing and thawing,exposure to heat,weak acids,certain deter-gents and extensive digestion by trypsin[1].All at-tempts to reactivate denatured granules were unsuccessful.Recently,new insights into this perplexing phenomenon have been provided[2].High resolution 13C NMR spectroscopy has demonstrated that PHB in native granules is in a mobile amorphous state and not in a crystalline state,as had been suggested by earlier studies[3].It was proposed that‘inactivation’of native granules was associated with crystallization of the poly-mer[2],a state in which the polymer was not suscepti-ble to degradation by the intracellular PHB depolymerizing system.Horowitz and Sanders[4]have*Corresponding author.0141-8130/99/$-see front matter©1999Elsevier Science B.V.All rights reserved. PII:S0141-8130(99)00026-4J.M.Merrick et al./International Journal of Biological Macromolecules25(1999)129–134 130developed procedures by which pure crystalline PHB can be reconstituted into artificial amorphous granules that are in many ways indistinguishable from native PHB granules.In the present study we examined the ability of these artificial granules to serve as a substrate for the R.rubrum depolymerizing system.The R.rubrum PHB depolymerizing system consisted of a heat-stable protein activator fraction and a heat-labile depolymerase fraction[1].Since the activator could be replaced by mild tryptic treatment of native PHB granules,it might be anticipated that the acti-vator fraction acted as a protease;however,proteolytic activity has so far not been detected.To reconcile the differences in activation between trypsin and activator,we postulated that there was an inhibitor protein on the surface of the granule that prevented the depolymerase from accessing its substrate[5].The inhibitor was presumably destroyed by trypsin proteol-ysis or modified by the activator in some as yet un-defined manner.Further studies on the role of the activator and the putative inhibitor are presented in this study.2.Experimental2.1.Bacterial strains and culture conditionsB.megaterium KM was grown as described by Mer-rick and Doudoroff[1].R.rubrum was grown in Difco Trypticase Soy Broth containing sodium acetate(10 mM).The cultures(1000ml of medium in Fernbach cultureflasks)were incubated on a New Brunswick gyrotary shaker at30°C for48h.Escherichia coli DH5a/pAeT41obtained from JoAnne Stubbe(Massa-chusetts Institute of Technology)was grown as described by Sim et al.[6]in LB medium supple-mented with glucose(20g/l)and ampicillin(50mg/l). Plasmid pAeT412contains the PHB biosynthetic operon from Alcaligenes eutrophus.Pseudomonas lemoignei was grown as described by Stinson and Mer-rick[7].2.2.Isolation of PHBNative PHB granules from B.megaterium or from E. coli were isolated as described by Merrick and Doudo-roff[1].Purified PHB was either prepared from B. megaterium PHB granules as described by Delafield et al.[8]or by repeated extraction of B.megaterium granules with1%sodium dodecyl sulfate and removal of the detergent by washing the polymer repeatedly with acetone.Artificial amorphous granules were pre-pared from B.megaterium purified polymer as de-scribed by Horowitz and Sanders[4].2.3.Preparation of intracellular and extracellular depolymerase systemsThe preparation of activator and depolymerase frac-tions from R.rubrum extracts was carried out as de-scribed[1].The ammonium sulfate-precipitated depolymerase was purified by passage through a Phar-macia HiTrap Q column and concentrated with an Amicon Centriprep-10concentrator.The extracellular depolymerase was isolated from the culture medium of Pseudomonas lemoignei essentially as described by Sim et al.[6]with minor modifications.The proteins in the culture medium were precipitated with ammonium sul-fate(75%saturation),centrifuged at25000×g for20 min,resuspended in10mM Tris–HCl,pH7.0,contain-ing1mM CaCl2and5%(v/v)glycerol and dialyzed against the same buffer.Partial purification was achieved by passage through a Hitrap Q column.2.4.Isolation of an inhibitor fraction from PHB granulesThe solubilization of the inhibitor fraction from PHB granules was performed by the detergent extraction procedure described by Wieczorek et al.[9]with minor modifications.PHB granules were suspended in20mM Tris–HCl buffer,pH8.0,containing phenylmethylsul-fonylfluoride(l.0mM),and an equal volume of Triton X-114(3.0%,w/v)was added.The suspension was shaken for60min at4°C and the extracted components were separated from the granules by centrifugation 9000×g for20min on a cushion of glycerol.The supernatantfluid was removed and centrifuged at 33000×g for10min.The clear supernatantfluid was incubated at37°C for5min and the Triton X-114 micelles collected by centrifugation(3000×g,10min). The upper phase was further treated with Bio-Rad SM-2beads to remove traces of the detergent and finally passed through a heparin–agarose column to remove any contaminating lysozyme that may still be present.The unbound fraction which contained the inhibitor was concentrated with an Amicon Centriprep-10concentrator.2.5.Extraction of acti6ator-treated PHB granules with Triton X-114Native PHB granules(20mg)were incubated with or without R.rubrum activator(200m g)for30min at 30°C in afinal volume of3.0ml of20mM Tris–HCl buffer,pH8.0.Following incubation,the granules were centrifuged on a layer of glycerol at9000×g for20 min.The supernatantfluids from activated and non activated granules were removed and the granules were resuspended in20.0ml of the same buffer and recen-trifuged.The washing of the granules was repeated oneJ.M.Merrick et al./International Journal of Biological Macromolecules25(1999)129–134131 Table1Hydrolysis of native PHB granules and purified PHB aNative PHB granules(%hydrolysis after30min)Purified PHB(%hydrolysis after30min) Additions during preincubationDepolymerase I Depolymerase I Depolymerase EDepolymerase E56.8 4.9None 6.051.89.96.334.125.6Activator6.954.5Trypsin43.991.2––Heparin 5.7–a The conditions of incubation and assay were described in Section2.Activator,depolymeraseI,depolymerase E and trypsin were added at22.5, 6.4,0.4and15m g of protein per5ml,respectively;heparin was added at0.8mg per5ml.more time.Finally,activated and native granules were extracted with Triton-X-114as described above,and the extracts were examined for their ability to inhibit hydrolysis(Expt3in Table3).Activated and non-acti-vated granules were also tested as substrates for the intracellular depolymerase.Activator activated granules were hydrolyzed without further addition of activator while the non-activated granules required pretreatment with activator(data not shown).2.6.Assay proceduresReaction mixtures for the depolymerization of native PHB granules or purified PHB contained1.0mg of native PHB granules or purified polymer,125m mol of Tris–HCl,pH8.0,and5m mol of MgCl2in a total volume of 5.0ml.The degree of hydrolysis of the polymer at30°C was determined by the percent de-crease in absorbance of the PHB suspension in a Klett colorimeter(redfilter,no.66).The granules were prein-cubated with activator or with trypsin for15min, followed by the addition of the intracellular depoly-merase(depolymerase I)or the extracellular depoly-merase(depolymerase E).The assay for hydrolysis of the artificial granules was similar except that MgCl2was omitted unless indicated otherwise.Inhibition of PHB hydrolysis by the granule extract containing the inhibitor fraction was performed by preincubation of native PHB granules(1.0mg)with trypsin(15m g)for15min.The assay mixture contained 125m mol of Tris–HCl,pH8.0,and5m mol of MgCl2in a total volume of 1.0ml.At the end of15min, ovomucoid(25m g)was added,followed by the in-hibitor fraction and the incubation continued for an additional10min.In experiments with activator,the inhibitor was incubated for10min with the PHB suspension followed by the addition of activator for an additional5min.The mixture was then diluted to5.0 ml and depolymerase added.3.Results3.1.Hydrolysis of nati6e PHB granules and purified PHBThe hydrolysis of native PHB granules and purified PHB by the intracellular depolymerase of R.rubrum and the extracellular depolymerase of P.lemoignei (depolymerase I and depolymerase E,respectively)is shown in Table 1.Depolymerase E hydrolyzed both native granules and purified PHB in the absence of trypsin or activator pretreatment.In fact,activator-in-hibited hydrolysis of either native or purified PHB, while trypsin pretreatment increased the rate of hydrol-ysis of native PHB granules suggesting that bound protein components interfere with PHB hydrolysis by depolymerase E.As previously reported[1],hydrolysis of native PHB granules by depolymerase I required pre-treatment with activator or trypsin.We also prepared native PHB granules from recombinant E.coli and found that these granules as well,required pretreatment with either activator or trypsin prior to hydrolysis by the R.rubrum depolymerase(data not shown).3.2.Hydrolysis of artificial PHB granulesArtificial amorphous granules,prepared with either cetyltrimethylammonium bromide(CTAB)or sodium cholate,were examined as putative substrates for the intracellular and extracellular depolymerases.The re-sults are shown in Table2.The hydrolysis of cholate PHB granules did not require activator pretreatment with either the depolymerase I or depolymerase E system. In fact,some inhibition was noted in the presence of activator and may be due to nonspecific binding of protein components which mask the substrate sites for the depolymerase.These data suggest that PHB in an appropriate environment(e.g.as negatively charged granules)can interact directly with either of the posi-J.M.Merrick et al./International Journal of Biological Macromolecules25(1999)129–134132Table2Hydrolysis of CTAB and cholate PHB granules aCTAB PHB granules(%hydrolysis after20min)Cholate PHB granules(%hydrolysis after20min) AdditionsDepolymerase I Depolymerase E Depolymerase I Depolymerase E39.08.96.151.1None29.648.429.345.9Activator27.59.9Activator+MgCl2Heparin 4.671.1Heparin+MgCl2 4.458.0a The conditions of incubation and assay were the same as those described in Table1except that MgCl2was added at5m mol as indicated.tively charged depolymerases.It was somewhat surpris-ing tofind that the positively charged CTAB granules required pretreatment with the activator fraction since we had assumed that the activator was interacting with a protein inhibitor present on native granules but absent from the artificial granules.Based on our study with cholate granules we examined whether activator may be acting to depress or neutralize the positive charge on CTAB granules.We found that the addition of heparin, a highly negatively charged molecule,could,in fact, replace the activator fraction in the case of the depolymerase E system but not in the depolymerase I system.Similarly,heparin could not replace the activator in the depolymerization of native granules by depolymerase I(Table1).Thus,in the depolymerase E system,activator likely neutralizes the positive charge on the granules and permits attack by depolymerase E.The explanation,however,for the depolymerase I system appears to be more complex and will require further study.3.3.Inhibition of hydrolysis by granule extract andre6ersal of inhibition by acti6atorIn an earlier study[5],we had demonstrated that mild alkaline-extracted,native PHB granules were directly susceptible to hydrolysis by depolymerase I without pre-treatment by activator or trypsin.In addition,the alkaline extract contained a component that prevented direct hydrolysis and that the addition of activator or trypsin reversed the inhibition.The extracted granules were highly unstable,usually losing their capacity to act as a substrate within24–48h,and no further studies on this system were carried out.We recently decided to reinvestigate the role of the activator and the putative inhibitor.For this purpose we adapted the procedure described by Wieczorek et al.[9]for the extraction of proteins from PHB granules of Alcaligenes eutrophus.To assay for the putative inhibitor,we utilized trypsin-acti-vated granules.After activation,further trypsin action was prevented by the addition of ovomucoid.As seen in Table3,Expt1,Triton X-114extraction solubilized a component(presumably a protein)that was capable of inhibiting hydrolysis and the inhibition could be reversed by the addition of the activator.To determine if the inhibition was specific for the solubilized granule protein extract,we added various other proteins to the hydroly-sis system.Table3,Expt2,demonstrates that of the proteins tested none were able to inhibit the hydrolysis of PHB,suggesting that the inhibition was specific. 3.4.Does acti6ator treatment of nati6e PHB granules modify the inhibitor?The activation of native granules by activator has been poorly understood since it had not been possible to demonstrate any proteolytic activity with this fraction. Presumably it modifies the putative inhibitor,but no information concerning its mechanism is available.To determine if the inhibitor on the granules is modified by activator treatment,we pretreated granules with the activator fraction and then extracted the activated gran-ules with Triton X-114.Native granules that were treated in a similar way but without the activator were also extracted.The results are shown in Expt3in Table 3.Extracts prepared from native granules but not from granules pretreated with activator were able to inhibit hydrolysis,suggesting that the inhibitor component had indeed been altered by pretreatment of the granules with the activator.However,on examination of the solubi-lized protein components by SDS–PAGE,no differ-ences were observed in the protein components from either the activated or non-activated granules.Perhaps the inhibitor(presumably a protein)was at concentra-tions below the detection sensitivity of our elec-trophoretic system.Furthermore,there was no evidence that a protein component from the activator fraction had been absorbed on granules.We also could not detect any loss in activity of the activator fraction following its incubation with the granules and subsequent recovery.4.DiscussionA comparative study of the hydrolysis of native PHB granules and purified polymer by the intracellular andJ.M.Merrick et al./International Journal of Biological Macromolecules25(1999)129–134133 Table3Inhibition of PHB hydrolysis by granule extract a%Inhibition after20 Additions after trypsin activationExptmin b140.7 Granule extract(0.85m g)71.6Granule extract(1.7m g)Granule extract(3.4m g)81.40.0Granule extract(1.7m g)+activator(10.0m g)Granule extract(2.2m g)64.72Tropomyosin(57m g),bovine IgG(40m g),protein A(40m g),fetuin(40m g)and albumin(40m g)0.075.6Granule extract from native granules(4m g)c30.0Granule extract from activated granules(4m g)ca The conditions of incubation and assay are described in Section2.Intracellular depolymerase was added at6.4m g in Expt1,at4.0m g in Expt 2and at3.2m g in Expt3.b The percent hydrolysis of PHB after20min in the presence of ovomucoid alone was38.1in Expt1,34.4in Expt2and29.3in Expt3.c The preparation of activated granules and native granules used in this experiment is described in Section2.extracellular depolymerases showed some interesting differences between the two enzyme systems.The intra-cellular depolymerase cannot hydrolyze purified PHB, while the extracellular depolymerase hydrolyzes either PHB granules or purified polymer.The activator which activates native granules as a substrate for the intracel-lular depolymerase inhibits the hydrolysis of either PHB granules or purified polymer by the extracellular enzyme system.These results strongly suggest that bound protein components may inhibit hydrolysis of purified PHB.This conclusion is also supported by the observation that hydrolysis of native granules by the extracellular depolymerase is markedly enhanced by prior tryptic treatment.The role of the activator in the activation of native granules remains elusive.We had proposed earlier that activator may modify an inhibitor (which is destroyed by trypsin)on native granules. Evidence for this proposed model was obtained by the solubilization of an inhibitor from native granules by detergent extraction.The inhibitor effectively blocked hydrolysis of trypsin-activated granules and the inhibi-tion could be reversed by the addition of the activator. Furthermore,Triton X-114extracts prepared from acti-vated granules were unable to inhibit hydrolysis,sug-gesting that some type of modification of the inhibitor component on native granules had occurred.In our earlier studies on the biochemistry of PHB,we were perplexed by the lability of native granules to act as a substrate for the intracellular depolymerase system. Native granules were irreversibly inactivated by a vari-ety of treatments and required special techniques to isolate granules that were suitable as a substrate for the intracellular depolymerase system.Active substrate preparations were achieved by centrifugation on a cush-ion of glycerol.Although several hypotheses were pro-posed to explain this intriguing but experimentally troubling phenomenon,it was not until1989[2],34 years after we initially published these observations that it was shown that native PHB granules are amorphous, and that inactivation resulted in crystallization of the polymer.Further,the important paper by Horowitz and Sanders[4]demonstrated that it was possible to produce artificial amorphous granules from the crys-talline polymer in which a surfactant has been substi-tuted for the in vivo membrane coating.In this communication we examined whether artificially pre-pared amorphous PHB could serve as a substrate for the intracellular and extracellular depolymerizing sys-tems.Cholate granules were hydrolyzed by both the intracellular and extracellular depolymerases without the requirement of activator.These results were of considerable interest and supported our original hy-pothesis that activator modified an inhibitor associated with native granules,but which was presumably absent on artificial granules.However,similar studies with the CTAB granules did not lead to such an obvious conclu-sion.Both the intracellular and extracellular depoly-merases required activator pretreatment for hydrolysis. Since these granules were positively charged we added heparin,a negatively charged polymer,to determine if the role of the activator might simply be an electrostatic one,that is to neutralize the positive charge.This appeared to be the case with regard to hydrolysis by the extracellular depolymerase.However,in the case of the intracellular depolymerase,heparin cannot replace the activator either with artificial granules or native gran-ules as the substrate.If activator does plays a role in neutralizing a positive charge,it must do so in such a way that permits the intracellular depolymerase to ac-cess its substrate,a condition which presumably does not occur with heparin.Our work has left many questions unanswered.We presume the inhibitor on the granule is a protein be-cause activation of granules can be achieved by mild tryptic treatment.The solubilization of the inhibitor will now permit its purification and identification,aJ.M.Merrick et al./International Journal of Biological Macromolecules25(1999)129–134 134necessary prerequisite to define the mechanism of acti-vation by the activator.The role of the activator re-mains one of the most intriguing aspects of the intracellular breakdown of PHB.How does it interact with the inhibitor so that the inhibitory function is lost? Does it play a role in the regulation of PHB break-down?Is its action enzymatic or is it a physical one such as was required in the CTAB artificial granule system?It is clear that an understanding of the mecha-nism of action of the activator will be essential for an understanding of endogenous PHB metabolism.We can look forward to answers to these questions in the very near future References[1]Merrick JM,Doudoroff M.J Bacteriol1964;88:60.[2]Barnard GN,Sanders JKM.J Biol Chem1989;264:3286.[3]Ellar D,Lundgren DG,Okamura K,Marchessault RH.J MolBiol1968;35:489.[4]Horowitz DM,Sanders JKM.J Am Chem Soc1994;116:2695.[5]Griebel RJ,Merrick JM.J Bacteriol1971;108:782.[6]Sim SJ,Snell KD,Hogan SA,Stubbe J,Rha C,Sinskey AJ.NatBiotechnol1997;15:63.[7]Stinson MW,Merrick JM.J Bacteriol1974;119:152.[8]Delafield FP,Doudoroff M,Palleroni NJ,Lusty CJ,ContopoulosR.J Bacteriol1965;90:1455.[9]Wieczorek R,Pries A,Steinbuchel A,Mayer F.J Bacteriol1995;177:2425..。