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The improvement of lipase secretion and stability by addition of inert compounds into Acinetobacter calcoaceticus cultures

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The improvement of lipase secretion and stability by addition of inert compounds into Acinetobacter calcoaceticus cultures
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  The improvement of lipase secretion and stabilityby addition of inert compounds into  Acinetobacter  calcoaceticus   cultures Daniela A. Martinez and B. Clara Nudel Abstract :  Acinetobacter calcoaceticus  BD413 produces variable amounts of an exocellular lipase that becomes rapidlyinactivated upon secretion. To achieve high yield and protect the enzyme, we assayed the addition of several inert com-pounds to cell-free supernatants, cell fractions, and whole cultures. Glass beads, poly(ethylene glycol) 600, TritonX-100, saccharose, gum arabic, and  β -cyclodextrin were among the compounds tested.  β -Cyclodextrin and gum arabic(and saccharose to a lesser extent) were effective enzyme stabilizers in cell-free supernatants, while gum arabic, glassbeads, and Triton X-100 improved lipase secretion from cells, and, therefore, total lipase yield (30–50%, according tothe additive). In whole cultures,  β -cyclodextrin was the most effective additive, particularly in combination with glassbeads or gum arabic. Indeed, cultures containing  β -cyclodextrin plus gum arabic were able to maintain 95% (±1.5%) of the initial lipase activity for more than 16 h, while control cultures with no additives maintained only 10% (±4%) of the enzyme activity after the same period. In conclusion, the addition of inert compounds in cultures may be consid-ered a useful approach for achieving increased yield and lipase stabilization, amenable for downstream processing. Key words :  Acinetobacter calcoaceticus , lipase, secretion, stabilization, inert additives. M artinez and N udel Résumé  :  Acinetobacter calcoaceticus  BD413 peut produire des quantités variables d’une lipase exocellulaire qui estrapidement inactivée à la suite de sa sécrétion. Afin d’obtenir un rendement élevé et de protéger l’enzyme, nous avonsfait des tentatives d’ajout d’une variété de composés inertes aux surnageants acellulaires, aux fractions cellulaires etaux cultures entières. Parmi les composés testés se retrouvaient des billes de verre, du polyéthylène glycol 600, du Tri-ton X-100, du saccharose, de la gomme arabique et de la  β -cyclodextrine. La cyclodextrine et la gomme arabique (et lesaccharose dans une moindre mesure) furent d’efficaces stabilisateurs enzymatiques, alors que la gomme arabique, lesbilles de verre et le Triton X-100 ont amélioré la sécrétion de lipase par les cellules, et donc le rendement total de li-pase (de 30 à 50 %, selon l’additif). Dans les cultures entières, la  β -cyclodextrine se sont avérées les additifs les plusefficaces, surtout en combinaison avec des billes de verre ou de la gomme arabique. En effet, les cultures contenant dela  β -cyclodextrine et de la gomme arabique furent capables de conserver 95 % (± 1,5 %) de l’activité lipase initialependant plus de 16 heures, alors que les culture témoins sans additifs n’ont conservé que 10 % (± 4 %) de leur activitélipase à la fin de cette même période. En conclusion, l’ajout de composés inertes dans les cultures pourrait être consi-déré comme une approche utile dans le but d’obtenir un rendement supérieur et une stabilisation de la lipase, qui pour-rait se prêter à des traitements subséquents.  Mots clés  :  Acinetobacter calcoaceticus , lipase, sécrétion, stabilisation, additifs inertes[Traduit par la Rédaction]  1061 Introduction Microbial lipases have a considerable industrial potentialas catalysts for hydrolysis, synthesis, and transesterificationof triglycerides. Lipase-catalyzed reactions can be used forhydrolysis of oils and fats (Talon et al. 1993; Dandik andAksoy 1996; Plou et al. 1996), synthesis of fatty acid esters(Bloomer et al. 1992; Lee and Akoh 1998), and the produc-tion of various intermediates for organic synthesis (Wu et al.1996). This latter application of lipases is becoming increas-ingly significant, due to the combination of broad substratespecificity and high regio- and stereoselectivity exhibited bymost microbial lipases.These enzymes have some peculiar traits that affect theiractivity and stability, causing some difficulties in controllingthe conditions for optimal production and isolation. Amongthese features are, as a distinctive characteristic, their inter-facial properties, including their affinity towards solid adsorb-ents, polymers, and surfactants (Wingender et al. 1987; Geluk et al. 1992; Gitlesen et al. 1997; Lin et al. 1995; Rosu et al. Can. J. Microbiol.  48 : 1056–1061 (2002) DOI: 10.1139/W02-108 © 2002 NRC Canada 1056 Received 19 April 2002. Revision received 25 November 2002.Accepted 28 November 2002. Published on the NRC ResearchPress Web site at http://cjm.nrc.ca on 7 January 2003. D.A. Martinez and B.C. Nudel. 1 Cátedra de MicrobiologíaIndustrial y Biotecnología, Facultad de Farmacia yBioquímica, Universidad de Buenos Aires, Junín 956 (1113),Buenos Aires, Argentina. 1 Corresponding author (e-mail: cnudel@ffyb.uba.ar orcnudel@fibertel.com.ar).  1998), their activation or hyperactivation at hydrophobic in-terfaces (Pencreac’h and Baratti 1997; Bastida et al. 1998),and their inactivation due to surface forces (Falk et al. 1991).These peculiarities stimulated the use of several organicand inorganic polymers, such as alginate and Triton X-114,for the purification and immobilization of lipases (Wingenderet al. 1987; Bompensieri et al. 1996; Kunková and Sivel1997; Fernandez-Lafuente et al. 1998).In addition to interfacial behavior, other mechanisms con-tribute to the overall lipase activity, such as size of substrateparticles, induction of a correct folding of the enzyme, acti-vation or removal of repressors and inhibitors, and increasedresistance to degradation. (Cordenons et al. 1996; Reetz andJaeger 1998).The lipase from  Acinetobacter calcoaceticus  BD413 hasbeen isolated, purified, and characterized (Kok et al. 1995).Regulatory aspects of its production regarding the influenceof carbon and nitrogen sources on microbial growth, LipAproduction, and  lipA  expression have been described (Kok etal. 1996; Mahler et al. 2000).The purpose of the present work was to explore condi-tions for improved secretion and stability of this exo-lipase,in order to design simple procedures for efficient culture anddownstream processing. Materials and methods Strains Two strains were used for this study.  Acinetobacter calcoa-ceticus  BD413 is a mini-encapsulated mutant secreting lipaseand esterase(s) (Juni and Janik 1969).  Acinetobacter calcoa-ceticus  AAC320 is a transcriptional  lipA::lacZ   fusion, gener-ated by the insertion of a promoterless lacZ cassette undercontrol of the  lipA  promoter into the chromosome of   A.calcoaceticus  BD413 (Kok et al. 1995). As a consequence of this insertion, AAC320 does not produce lipase (Kok et al.1996). Both strains were obtained from Klaas Hellingwerf,University of Amsterdam. Media and culture conditions The strains were cultivated aerobically at 30°C in a me-dium containing: 37 mM NH 4 Cl, 0.81 mM MgSO 4 , 68  µ MCaCl 2 , 11 mM KH 2 PO 4 , 95 mM Na 2 HPO 4 , 1.8  µ M FeSO 4 ,and 1 mL of a spore solution containing 50 g EDTA, 2.2 gZnSO 4 ·7H 2 O, 5 g FeSO 4 ·7H 2 O, 1.6 g CuSO 4 ·5H 2 O, 5 gMnCl 2 ·4H 2 O, 1.1 g (NH4) 6 Mo 7 O 24 ·4H 2 O, 50 mg H 3 BO 3 ,10 mg KI, and 50 mg CoCl 2 ·6H 2 O (per litre). This base me-dium was adjusted to pH 6.8 and autoclaved at 120°C for20 min. After sterilization, 1% (v/v) Tween 80 (Sigma–Aldrich Corp., St. Louis, Mo., U.S.A.) and 1% (p/v) try-ptone (Oxoid, Basingstoke,U.K.) were added asepticallyfrom 10× sterile stock solutions. Early stationary phasecultures (13 h) were used to inoculate 30 mL of the culturemedium in 250-mL Erlenmeyer flasks at an initial opticaldensity (OD 580 nm ) of approximately 0.1, and these were incu-bated at 30°C on a rotatory shaker at 250 rpm. Samples werecollected in duplicate and analyzed. The inoculum was pre-grown in the same medium during 24 h.Cells and supernatants were prepared from 10-h cultures bycentrifugation at 23 800  ×  g  for 10 min at 4°C and analyzedseparately. The cell pellets were resuspended in the samevolume of 0.1 M Tris (pH 7.2) containing 1mM MgSO 4 .The effect of the additives was assayed on cell suspensions,cell-free supernatants, and whole cultures, as described below.Final concentrations selected for the additives were as fol-lows: 0.5% gum arabic, 5%  β -cyclodextrin, 120 glass beads(3 mm diameter), 1% Triton X-100, 9% saccharose, and 1%poly(ethylene glycol) (PEG) 600. Other concentrations andadditives tested included the following: 0.3–1% gum arabic,0.5–10%  β -cyclodextrin, 30–120 glass beads (3 mm diame-ter), 0.3–3% Triton X-100, 0.3–3% Triton X-114, 1–3% octylsepharose, 0.5–3% PEG 600, and 0.5–3% glass powder (mesh120). Stability of purified lipase  Acinetobacter calcoaceticus  BD413 lipase was purified asdescribed previously (Kok et al. 1995) and lyophilized in2 mM Tris, 10% (v/v) Triton X-100. This preparation wasreconstituted in water (approx. activity 1000 U mL –1 ), and30- µ L fractions (corresponding to 31.6 lipase units) wereused for the tests. The stability of the lipase solution wastested at 4°C and 30°C, and also added to 12-h culturesupernatants, with and without various protease inhibitors,among other conditions studied. The protease inhibitor phe-nylmethylsulfonyl fluoride was used at 4 mM final concen-tration. Analytical methods Biomass production was monitored by measuring OD 580 nm of appropriate dilutions and dry cell weight (expressedin g L –1 ).Extracellular lipase was measured in culture supernatantsand resuspended cells of   A. calcoaceticus  BD413, as previ-ously described (Kok et al. 1995) by using  p -nitrophenylpalmitate (Sigma–Aldrich Corp.) as the substrate. One unit(U) of enzyme activity is defined as the amount of enzymeforming 1 micromole of   p -nitrophenol per minute.Production of   β -galactosidase was measured in the strain  A. calcoaceticus  AAC320  lipA::lacZ  , essentially accordingto Miller (1982), using  o -nitrophenyl  β - D -galactopyranoside(Sigma–Aldrich Corp.) as the substrate.Cells were harvested by centrifugation at 23 800  ×  g  for5 min at 4°C, washed twice, and suspended in a knownvolume of the same buffer as above. After measurementof the OD 580 nm , these samples were frozen until furtheruse. Prior to the  β -galactosidase assay, cells were thawed, di-luted appropriately to reach an OD 580 nm  of less than 3,pelleted via centrifugation in the same conditions as describedabove, and resuspended in the same volume of Z buffer (con-taining 60 mM Na 2 HPO 4 , 40 mM NaH 2 PO 4 , 10 mM KCl,1 mM MgSO 4 , 0.001% sodium dodecyl sulphate, and50 mM  β -mercaptoethanol).Permeabilization of cells was performed in 1-mL samplesby adding 100  µ L ethanol and 20  µ L toluene and incubatingthe samples at 37°C for 20 min. The rest of the procedurewas essentially as described (Miller 1982).  β -Galactosidase-specific yield was calculated in Miller units (M.U.). All testedconditions were analyzed by taking duplicate samples fromone, two, or three independent cultures. Values displayed arethe mean values. © 2002 NRC Canada M artinez and N udel 1057  Lipase and  β -galactosidase measurements were analyzedby ANOVA, LSD, and Fisher’s statistical tests (Box et al.1978) to select significant main effect and interactions, usingstatistical software (© 2001 Statistix 4.1 Analytical Soft-ware). Results The production of exo-lipase by  A. calcoaceticus  is typi-cally an early stationary phase event. As shown in Fig. 1, theenzyme activity reaches a maximum at the moment of tran-sition to the stationary phase, around 8–10 h culture, and de-creases sharply to the levels of the exponential growth phaseas the culture proceeds. Despite this decline in the amount of extracellular lipase produced, transcriptional activity of the lipA  gene remains high, as shown by the level of   β -galactosidase measured in the AAC320  lipA–lacZ   fusionstrain. The same pattern of production and further inactiva-tion has been observed with cultures in rich or minimal me-dia, supplemented with diverse carbon and nitrogen sources(Gonzalez et al. 1996).A significant contribution to the decay in lipase activityhas been attributed to proteases (Gonzalez et al. 1996). In-deed, partially digested lipase fragments have been detectedin culture supernatants using specific antibodies (Kok et al.1996) simultaneously to gelatin hydrolysis in gels.However, the addition of protease inhibitors (phenylmethyl-sulfonyl fluoride, leupeptin, and (or) pepstatin) to culture super-natants only partially protected the enzyme activity, suggestingthat we are dealing with a more complex phenomena. In ef-fect, as shown in Fig. 2, both proteolysis and thermal inactiva-tion played a significant role in the inactivation process, evenwith pure lipase preparations.The complex behavior that evidences the enzyme also im-poses a severe limitation for its biotechnological exploita-tion, calling for improvements in the conditions for recoveryand stabilization. In this work, the addition of several com-pounds (mostly inert additives) was tested with  A.calcoaceticus  whole cultures and isolated fractions.Some of the additives that exhibited a positive responseare listed in Table 1. They included glass beads, PEG 600,Triton X-100, saccharose, gum arabic, and  β -cyclodextrin,among others. To find out the precise level at which theywere acting, the additives were tested first on separated cul-ture fractions, cells, and supernatants.The effect of additives on cell-free culture supernatants isdisplayed in Fig. 3. Typically, supernatants with no additives(control) showed about 30% decrease in lipase activity in2 h incubation and 50% in 4 h. Similar results were obtainedwith the addition of glass beads (20 and 50%, respectively)or Triton X-100 (50% reduction in 2 h), indicating no lipasestabilization effect of these compounds on culture super-natants. A better performance was obtained with the additionof saccharose, because 70% of the initial enzyme activitycould be recovered in supernatants after 6 h incubation.Total lipase activity was recovered only from supernatantscontaining gum arabic or  β -cyclodextrin; with these addi-tives, 100% of the enzymatic activity was maintained after6 h incubation at 30°C. The stabilizing effect of both poly-saccharides was thus established.Several compounds added to washed cell suspensions fromearly stationary phase cultures, particularly gum arabic, glassbeads, and Triton X-100, increased the release of lipase fromthe cells 30–50%. This improvement was not due to in-creased  lipA  transcription, as shown in the strain AAC320,carrying a  lipA–lacZ   transcriptional fusion. Indeed, maximal β -galactosidase activity measured with AAC320 in the pres-ence of these additives was not significantly different fromthe control condition (400 ± 30 M.U., in all cases), indicat-ing that higher lipase yields obtained with these additivesmust be attributed to enhanced secretion and not to increasedlipase transcription.If these incubations were prolonged for 3 h, then lipaseactivity decreased in most cases, except in the suspensionscontaining gum arabic,  β -cyclodextrin, or saccharose. TritonX-100 or glass beads, on the other hand, although improvinglipase secretion at early stages, were not able to prevent en-zyme inactivation in the longer term. PEG 600 neither stim- © 2002 NRC Canada 1058 C an.J.M icrobiol.Vol.48,2002 Fig. 1.  Growth, lipase production, and  lipA  expression by  Acinetobacter calcoaceticus  strains in minimal medium contain-ing Tween as carbon source.   , exo-lipase (U mL –1 );   , growth(OD 580 nm );   , dry cell weight (g L –1 ); ×,  β -galactosidase (MillerUnits). Fig. 2.  The stability of   Acinetobacter calcoacticus  BD413 lipaseat various incubation conditions. Purified lipase was incubated at4°C (  ) and 30°C (  ). The same solution was added to culturesupernatant, with (×) and without (  ) the addition of 4 mMphenylmethylsulfonyl fluoride.  © 2002 NRC Canada M artinez and N udel 1059 ulated secretion nor stabilized the enzyme (Fig. 4). Table 2summarizes the effects of the additives tested, with regard tostimulation of secretion or enzyme stabilization of super-natants.Because of the better performance of   β -cyclodextrin, glassbeads, and gum arabic on the secretion and stabilization of the lipase, these compounds were added, alone or in combi-nations, into whole cultures. A control condition with no ad-ditives was also included. Except for glass beads, whichshowed a similar behavior to the control (10 ± 4% lipase ac-tivity recovered in 16-h cultures), all other additives im-proved significantly ( P  < 0.006) the yield of lipase recovery(average: 75 ± 16%). Moreover,  β -cyclodextrin in combina-tion with gum arabic or glass beads, improved lipase secre-tion and stabilization (Fig. 5). Particularly in culture brothscontaining a combination of gum arabic and cyclodextrin,more than 95% (±1.5) of the lipase activity was recoveredafter prolonged incubations. In addition, only a minor frac-tion of lipase remained adsorbed to the cyclodextrin uponseparation of the additive by centrifugation (less than 5%, inall cases). Discussion For the commercial exploitation of lipases, it is essentialto achieve high yields and maintain enzyme stability. Tomake this process feasible for the  A. calcoaceticus  lipase,various additives, selected on the basis of previous reportson lipase interfacial activation, were assayed.We tested a number of detergents and polymeric matrices,including Triton X-114, octyl-sepharose, glass powder, andseveral PEGs, in various concentrations. Only the ones re-ported have shown an effect on enzyme secretion or stability.The stimulation on lipase secretion by Triton X-100 agreeswith previous observations on  Pseudomonas pseudoalcali-genes  lipase (Lin et al. 1995). Glass beads also stimulatedlipase secretion (as reported in this work), but it was evidentthat neither the detergent nor glass beads could avoid furtherenzyme inactivation and (or) degradation.The unique role of gum arabic in stimulating lipase secre-tion and protecting the enzyme in solutions has far exceededits recognized function as a protective colloid for emulsions.Nevertheless, there is a disadvantage in the use of this poly-mer, because it cannot be easily separated from supernatants,therefore complicating the separation process of the enzyme.On the other hand, glass beads or cyclodextrins can be sepa-rated from cultures by centrifugation without introducing ad-ditional operations.Studies on the interactions between proteins and solventcomponents have identified sucrose as thermodynamicallycontributing to the stabilization of many proteins in watersolutions. (Lee and Timasheff 1981; Exterkate 2000). Thiseffect was also observed in the present study.Although cyclodextrins are known as stabilizers of smallmolecules and are, as such, used in the pharmaceutical andfood industries, there have been no previous reports on its Additive Lipase Activity (%)Control (no additives) 100±9Glass beads (120) 134±12Triton X-100 (1%) 155±22Gum arabic (0.5%) 131±12Saccharose (9%) 84±9 β -Cyclodextrin (5%) 103±10PEG 600 (1%) 93±16 Note:  Lipase activity was measured after 1 h incubation of the cells in0.1 M Tris–1 mM Mg 2+ buffer (pH 7.2) with the corresponding additiveand expressed as the percentage recovered relative to the controlcondition. PEG 600, poly(ethylene glycol) 600. Table 1.  Additives to cell suspensions and their effect on exo-lipase production. Fig. 3.  Lipase activity in cell-free supernatants with various addi-tives.   , control (no additives);   , glass beads;  ∆ , Triton X-100;  , cyclodextrin;   , gum arabic;   , saccharose. Fig. 4.  Lipase activity in cell suspensions was measured 1 and 3 hafter the addition of each compound. Results are expressed as thepercentage of lipase recovered relative to the initial amount. Col-umns: 1, control (no additives); 2, glass beads; 3, Triton X-100 ;4, gum arabic; 5, saccharose; 6,  β -cyclodextrin; 7, poly(ethyleneglycol) (PEG) 600.Additive Secretion StabilityGum arabic positive positive β -Cyclodextrin none positiveGlass beads positive noneTriton X-100 positive negativeSaccharose none positivePEG 600 none none Note:  PEG 600, poly(ethylene glycol) 600. Table 2.  Effect of additives on lipase secretion and stabilization.  use for lipase stabilization. A possible explanation for theobserved stabilization is the sequestration of protease(s) incultures. Another postulated role of cyclodextrins as induc-ers of outer membrane permeability is, in our case, lesslikely (Hozbor et al. 1994), since no significant improve-ment on total extracellular lipase was measured in treatedcells.The removal of free fatty acids (i.e., oleic acid) by forma-tion of cyclodextrin–fatty acid complexes, is, in our case,also unlikely. In effect, free fatty acids are potent  lipA repressors (Mahler et al. 2000), and their removal wouldhave increased the  lipA  transcriptional activity in our  lipA– lacZ   fusion strain, an effect that has not been observed.(Kolossvary and Kolossvary 1996; Frohlich et al. 1996). It has been shown that the binding of lipase to hydropho-bic supports induces enzyme activation or hyperactivation(Bastida et al. 1998). In our case, some of these inert sup-ports were useful for stabilizing the lipase during culture,with the potential of combining, in a single step, stimulationof secretion, separation, stabilization, and activation of theenzyme. Acknowledgements The authors wish to thank Dr. Jorge Florin-Christensenfor his helpful comments. This study was financially sup-ported by a grant from the University of Buenos Aires(UBACYT 1998–2000) and a fellowship to D. Martinez. References Bastida, A., Sabuquillo, P., Armisen, P., Fernandez-Lafuente, R.,Huguet, J., and Guisan, J.M. 1998. A single step purification,immobilization, and hyperactivation of lipases via interfacial ad-sorption on strongly hydrophobic supports. Biotechnol. Bioeng. 58 : 486–493.Bloomer, S., Adlercreutz, P., and Mattiasson, B. 1992. Facile syn-thesis of fatty acids esters in high yields. Enzyme Microb.Technol.  14 : 546–552.Bompensieri, S., Gonzalez, R., Kok, R., Miranda, M.V., Nutgeren-Roodzant, I., Hellingwerf, K., Cascone, O., and Nudel, B.C.1996. Purification of a lipase from  Acinetobacter calcoaceticus AAC323-1 by hydrophobicity-exploiting methods. Biotechnol.Appl. Biochem.  23 : 77–81.Box, G.E.P., Hunter, J.S., and Hunter, W.G. 1978. Statistics for ex-perimenters: an introduction to design, data analysis, and modelbuilding. John Wiley & Sons Inc., N.Y.Cordenons, A., Gonzalez, R., Kok, R., Hellingwerf, K., and Nudel,B.C. 1996. Effect of nitrogen sources on the regulation of extracellular lipase production in  Acinetobacter calcoaceticus strains. Biotechnol. Lett.  18 : 633–638.Dandik, L., and Aksoy, H.A. 1996. Applications of   Nigella sativa seed lipase in oleochemical reactions. Enzyme Microb. Technol. 19 : 277–281.Exterkate, F.A. 2000. Structural changes and interactions involvedin the Ca 2+ -triggered stabilization of the cell-bound cell enve-lope proteinase in  Lactococcus lactis  subsp.  cremoris  SK11.Appl. Environ. Microbiol.  66 : 2021–2028.Falk, M.P.F., Sanders, E.A., and Deckwer, W.D. 1991. Studies onthe production of lipase from recombinant  Staphylococcuscarnosus . Appl. Microbiol. Biotechnol.  35 : 10–13.Fernandez-Lafuente, R., Armisen, P., Sabuquillo, P., Fernandez-Lorente, G., and Guisan, J.M. 1998. Immobilization of lipasesby selective adsorption on hydrophobic supports. Chem. Phys.Lipids,  93 : 185–197.Frohlich, B.T., D’Alarcao, M., Feldberg, R.S., Nicholson, M.I.,Siber, G.R., and Swartz, R.W. 1996. Formation and cell-medium © 2002 NRC Canada 1060 C an.J.M icrobiol.Vol.48,2002 Fig. 5.  Effect of various additives on lipase activity in  Acinetobacter calcoaceticus  cultures.   , control (no additives);   ,glass beads;   ,  β -cyclodextrin; ×, gum arabic;  ∆ , glass beads +  β -cyclodextrin;   , gum arabic +  β -cyclodextrin.
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