Hydrolytic and synthetic activities of esterases produced by Bacillus sp. A60 isolated from an oil contaminated soil

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1 Vol. 12(47), pp , 20 Novemer, 2013 DOI: /AJB ISSN Ademi Journls Afrin Journl of Biotehnology Full Length Reserh Pper Hydrolyti nd syntheti tivities of esterses produed y Billus sp. A60 isolted from n oil ontminted soil Flvi del Vlle Loto 1, Cinti Mrin Romero 1,2, Alfonso Emnuel Crrizo 1, Mrio Domingo Bigori 1,2 nd Lii Mrí Per 1 * 1 PROIMI-CONICET, Av. Belgrno y Psje Cseros, T4001 MVB, Tuumán, Argentin. 2 Fultd de Bioquími, Quími y Frmi, Btll de Ayuho 471, T4000INI, Tuumán, Argentin. Aepted 9 Otoer, 2013 A novel esterse produer strin nmed Billus sp. A60 ws isolted from soil smple ontminted with hydrorons. It ws found to elong to Billus sutilis speies through morphologil, iohemil nd 16S rrna gene sequene nlyses. This strin whih n tolerte 15% (w/v) NCl nd growth t 55 C, produed n interesting esterse tivity in Luri-Bertni medium. Two different moleulr weight esterse tivities were deteted in zymogrphi ssys. Culture superntnt nd whole ells showed speifi hydrolyti tivities of 2.67 ± 0.11 U/mg of protein nd 7.07 ± 0.09 U/mg of dry weight, respetively. Conerning ethyl ette prodution, onversions of ± 0 nd ± 0.78% were otined with ulture superntnt entrpped in polyrylmine gel nd whole ells, respetively. In ddition, the effet of different onentrtion of LB medium omponents on oth growth nd extrellulr esterse hydrolyti tivity ws lso disussed. Key words: Billus, esterses, hydrolyti tivity, syntheti tivity, ethyl ette. INTRODUCTION The rod metoli diversity exhiited y the genus Billus oupled to the low pthogeniity of severl speies, mke it ommerilly importnt nd n environmentl friendly soure of enzymes, vitmins nd other produts. In this onnetion, the sreening of sporeforming teri for the prodution of useful metolites ontinues to e n importnt spet of iotehnology. Among the enzymes, esterses (E.C ) represent diverse group of hydrolses tlyzing the levge nd the formtion of ester onds. They hve een extensively exploded in the synthesis of flvor esters, in the resolution of remi mixtures, nd in the degrdtion of nturl mteril s well s industril pollutnts (Pnd nd Gowrishnkr, 2005). However, the enzymti prodution of ethyl ette, n importnt environmentl friendly solvent, ws reltively little investigted (Alvrez-Mrie nd Brtti, 2000; Bélfi-Bkó et l., 2003). Thus, the serh of new miroil esterses suitle for this pplition is therefore, very ttrtive. As n id inhiition during the esterifition of eti id nd ethnol y Novozyme 435 lipse ws lso reported y Bélfi-Bkó et l. (2003), the enzyme immoiliztion ould improve its utiliztion t extreme ph, nd lso in different solvents, tempertures nd high sustrte onentrtions (Hnefeld et l., 2009). Alterntively, the use of ell-ound enzymes (tht is, nturlly immoilized enzymes) is n interesting *Corresponding uthor. E-mil: lym@rnet.om.r.

2 6626 Afr. J. Biotehnol. strtegy euse it elimintes omplex proedures of purifitions, so this kind of iotlysts is ost effetive sine the iomss n e diretly utilized in the retion (Stergiou et l., 2013). In ddition, the ell struture my t s nturl mtrix proteting the enzymes from the possile negtive tion of externl gents. On the other hnd, the seletion of n pproprited fermenttion tehnique s well s the optimiztion of ulture onditions for the prodution of n industrilly importnt trget produt must e done to ensure good yields nd qulity (Pnd nd Gowrishnkr, 2005). In our lortory, the purifition nd hrteriztion of esterses nd lipses from oth spore-forming teri nd filmentous fungi were evluted. In ddition, the enzymti synthesis of isomy ette nd ethyl sterete were lso explored (Bigorí et l., 1996; Torres et l., 2009; Romero et l., 2012). In this work, hydrolyti nd syntheti tivities of esterses produed y Billus sp A60 isolted from n oil ontminted soil were reported. Some spets of the enzyme prodution were lso nlyzed. MATERIALS AND METHODS Miroorgnisms nd fermenttion onditions Bteri were isolted from different soures suh s soils ontminted with hydrorons nd soils from river ost in the North West of Argentine. Soil suspensions were heted t 80 C for 15 min, plted on Luri-Bertni (LB) gr nd inuted t 37 C. Liquid ultures were rried out in flsks of 125 ml ontining 10 ml of LB medium on n oritl shker t 37 C. The referene strin Billus sutilis 1A571 (Lindgren nd Ruterg, 1974), derivtive of B. sutilis wild-type strin 168 (trp C2), ws kindly provided y the Billus Geneti Stok Center, Columus, Ohio. Sreening of esterse-produing spore-forming miroorgnisms Seprted olonies of isolted teri were grown 48 h on LB gr t 37 C. The enzyme tivity ws studied y pouring the pltes with α-nphtil ette nd Fst Blue dissolved in 50 mm phosphte uffer ph 7 supplemented with 0.75% gr. A rown hlo round the olonies ws onsidered s positive result for extrellulr enzyme tivity. The rtio etween the dimeter of eh olony nd the orresponding hydrolysis hlo (Rtio C/H) ws evluted s n inditor of the strins effiieny to produe n esterse tivity; the vlues rnged from 0 to 1, nd the highest level of enzymti tivity ws the nerest to 0. Identifition of the spore-forming strin A60 Phenotypi hrteriztion Grm stining, motility ssys, tlse test nd strh hydrolysis were performed using stndrds protools. Growth t different ph vlues, tempertures, NCl onentrtions, nd motility were rried out s desried y Grrity et l. (2005). The API 50 CHB system (iomérieux) ws used ording to the mnufturer s instrutions. Zymogrms were performed in ntive- polyrylmide gel eletrophoresis (PAGE) using 10% polyrylmide gel (Dvis, 1964). Moleulr hrteriztion Totl DNA ws extrted from ells hrvested in the mid-exponentil growth phse s desried y Miller (1972). Polymerse hin retion (PCR) mplifition ws performed in 25 µl retion mix ontining 2.5 µl 10X STR retion uffer (Promeg), 20 ng totl DNA, 0.5 µm of eh primer nd 1 U of Tq DNA polymerse (Promeg). Primers 27 F (5 - AGAGTTTGATCMTGGCTCAG-3 ) nd 1492 R (5 - GGTTACCTTGTTACGACTT-3 ) were used to generte prtil sequenes of 16S rdna. Smples were mplified s follows: 5 min t 94 C; 35 yles of 1 min t 94 C, 2 min t 50 C nd 2 min t 72 C, nd t the end, 7 min t 72 C for finl extension. PCR produts were nlyzed y eletrophoresis in 2% (wt/vol) grose gels. DNA sequening ws rried out y Mrogen Servies. Sequenes were ompred nd ligned with those from the GenBnk dtse with BLAST softwre. The method of Jukes nd Cntor (1969) ws used to lulte evolutionry distnes. Phylogeneti dendrogrm ws onstruted with the neighor-joining method, nd tree topology ws evluted y performing ootstrp nlysis of 1000 dt sets using MEGA 4 softwre ( Prtil nuleotide sequene of the 16S rdna gene of Billus sp. A60 ws deposited in GenBnk dtse under ession numer EF Sustrte speifiity of esterse tivity on LB gr pltes The seleted strin Billus sp. A60 s well s the referene strin B. sutilis 1A571 were grown 48 h on LB gr t 37 C. The sustrte speifiity of the hydrolyti tivity ws lso studied y pouring the pltes with α-nphtil derivtives (α-ette, α-propionte, α-prote, α-lurte, α-miristte, α-plmitte nd α-sterte) s desried for sreening ssys. Determintion of hydrolyti tivity Hydrolyti tivity ws mesured spetrophotometrilly t 405 nm y using p-nitrophenyl ette (pnpa) s sustrte. All ssys were performed t 37 C, nd ontrols were inluded in whih no enzyme or no sustrte ws dded. One unit of enzyme tivity (U) ws defined s the mount of enzyme tht relesed 1 μmol of p- nitro phenol per min. For extrellulr hydrolyti tivity, speifi tivity ws expressed s U per mg of protein. Protein onentrtion ws determined with Brdford (1976) regent. For ell-ound hydrolyti tivity determintion, ell pellets were wshed twie with 50 mm phosphte uffer ph 7, entrifuged nd resuspended in the retion mixture. The retion ws shken t 150 rpm, nd the sorne of the superntnt ws then mesured. Speifi tivity ws expressed s U per mg dry weight. Determintion of syntheti tivity Ester synthesis ws performed with either entrpped ulture superntnt in polyrylmide gels or dried ells. In the first se, 4.25 ml of ulture superntnt were dded to solution ontining 0.75 ml of rylmide-is rylmide (30:0.8), 20 μl of mmonium persulfte solution (0.1 g/ml) nd 6 μl of TEMED solution. Polymeriztion ws performed on 1 mm width glss se. Piees of gel (1 x 1 m) were then ut nd used for enzymti synthesis. Ester synthesis ws lso evluted in the presene of miroil iomss; in this experiment, the ells were first wshed with n- hexne. Then, 1 mg of ells s well s gel slie were resuspended

3 Loto et l Tle 1. Medium omposition tested for esterse tivity. Medium NCl (g/l) Yest extrt (g/l) Tryptone (g/l) (LB) LB, Luri Bertni medium. 2 Figure 1 3 in 10 ml of n-hexne ontining 20 mm of eti id nd n exess of ethnol. Retions were inuted t 37 C on n oritl shker 4 t 150 rpm. After retion time of 24 h, the residul id ontent ws determined y titrtion with 0.1 N sodium hydroxide solution. The molr onversion ws determined from the vlues otined for 5 the lnk nd the test smples. Qulittive nlysis of ethyl ette prodution ws rried out y thin lyer hromtogrphy (TLC) 6 using sili gel 60 s support nd hloroform s developing solvent. Spots were visulized in iodine vpor. All ssys were rried out t lest in duplite. 7 Effet of LB omponents on Billus sp. A60 growth nd 8 hydrolyti esterse prodution 9 Different onentrtions of LB omponents were evluted. The proportions of the omponents were vried one t time s detiled in Tle 1. All experiments were rried out in triplite nd 10 verge vlues of speifi hydrolyti esterse tivity s well s iomss were lulted. 11 Sttistil nlysis Sttistil nlyses ws performed using the Minit (version 14; Minit In) softwre for windows. Sttistil signifine vlues of the mens were evluted using one-wy nlysis of vrine. Susequent omprisons were performed using Tukey s post-ho test. Results were presented s the men ± SD. Differenes were epted s signifint when P<0.05. RESULTS AND DISCUSSION Isoltion nd seletion of the indigenous sporeforming strin A60 A totl of 44 spore-forming teri showing esterse tivity in LB gr were isolted from nturl environments. Thirty-four strins (78%) showed oth, extrellulr nd iomss-ound esterse tivity (Figure 1,), nd 10 strins (22%) only displyed iomss-ound esterse tivity (Figure 1). Interestingly, onsidering the rtio etween the dimeter of eh olony nd the orresponding hydrolysis hlo, 17 strins were etter produers thn the referene strin B. sutilis 1A571 (Tle 2). Figure 1. Hydrolyti esterse tivity on LB gr plte. Esterse tivity ws deteted using 1.3 mm of α-nphthyl ette (C2) nd 1 mm Fst Blue RR dissolved in 50 mm phosphte uffer ph 7., Billus sutilis 1A571;, Billus sp. A60;, Billus sp. M2. Sle: 2 m. Esterses from this speies hydrolyze different sustrtes s ntiioti esters, nd were utilized in hirl drug resolution, mong other pplitions (Bornsheuer, 2002). In our study, the most promising strin nmed A60 showed the lowest olony dimeter to hlo dimeter rtio (Rtio C/H = 0.33), nd ws then seleted for further studies. Phenotypi nd moleulr hrteriztion of Billus sp. A60 The spore-forming strin A60 stined Grm-positively nd formed entrl ovl spores. Growth ws oserved t ph 5.8, not t ph 10, nd the strin ws le to grow in LB gr supplemented with 7, 10 nd 15% NCl. Mximum growth temperture ws 55 C. Aording to API 50CH system, rohydrtes fermented were: Glyerol, riose, D-gluose, D-frutose, N-etyl-gluosmine,

4 6628 Afr. J. Biotehnol. Tle 2. Rtio etween the dimeter of eh isolted olony nd the orresponding hydrolysis hlo produed y n esterse tivity (Rtio C/H). Strin Rtio C/H Strin Rtio C/H A6 0.7 A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A M A M A M A M A M A M A A A mygdline, rutine, esuline, sliine, elloiose, mltose, shrose, trehlose, strh, glyogene nd gentioiose. Phenotypi nlyses nd morphologil hrteristis strongly suggested A60 strin s B. sutilis. Additionlly, the prtil sequening of 16S rdna gene onfirmed the iohemil nd morphologil hrteriztion. Bsi Alignment Serh Tool (BLAST) serh nd lignment nlyses showed similrity of 99% to B. sutilis susp. sutilis str The phylogeneti tree is shown in Figure 2. It is importnt to mention the iotehnologil importne of B. sutilis s n orgnism whih lks of toxiity (onsidered generlly regrded s sfe (GRAS) y FDA, USA). Moreover, it is reognized s soure of different metolites potentilly useful in food nd detergent industry, phrmeutil nd helth tehnologies nd griulture produts (Hrwood nd Wipt, 1996; Shllmey et l., 2004). Hydrolyti esterse tivity from Billus sp. A60 The sustrte speifiity of the hydrolyti tivities produed in LB gr y Billus sp. A60 s well s y the referene strin B. sutilis 1A571 ws ompred. Both esterse tivities were le to hydrolyze ette, propionte nd prote α-nphthyl derivtives. However, only the esterse from Billus sp A60 ws ple to hydrolyze the lurte derivtive. In ddition, none of them were le to hydrolyze hromogeni sustrtes with longer ron hins. These results strongly suggested tht the enzyme tivity produed y Billus sp. A60 orresponds to n esterse tivity (Bornsheuer, 2002). Speifiity of esterse from Billus sp. A60 s well s the esterse speifiity reported from Billus sp. 4 (Ateslier nd Metin, 2006) were different to tht from the referene strin nd other known esterses, whih ommonly showed no tivity on sustrtes with hins longer thn 10 rons. However, it nnot e ruled out tht other proteins or rohydrtes originting from the ulture roth ould ontriute to more roust tlyti system. Zymogrms of Billus sp. A60 superntnt showed two nds of tivity with pprent moleulr weights of ± 0.31 nd ± 0.07 kd, respetively (Figure 3). Higerd nd Spizizen (1973) lso reported two etyl esterses from B. sutilis 168 extrts. By employing gel filtrtion hromtogrphy, the estimted moleulr weights for these esterses, nmed A nd B, re 160,000 nd 51,000, respetively. Finlly, no signifint differene ws deteted etween the hydrolyti tivities of the oth whole ell iotlysts tested. However, the superntnt tivity otined from Billus sp. A60 lmost douled tht otined from referene B. sutilis 1A571 (Tle 3).

5 Loto et l Billus sutilis A60 (EF ) B. sutilis KU201-7 (EU ) 67 B. sutilis GH38 (AB ) B. sutilis susp. sutilis CICC (GQ ) B. sutilis susp. sutilis str. 168 (AL009126) Billus ereus ATCC53522 (AF ) 0.01 Figure 2. Neighor-joining phylogeneti tree of prtil 16S rdna sequenes. The method of Jukes nd Cntor ws used to lulte evolutionry distnes, nd tree topology ws onstruted using MEGA 4. Bootstrp vlues (n = 1000 replites) were indited t the nodes. Sle r represents oserved numer of hnges per nuleotide position. In prenthesis, ession numers of the referene strins 16S rdna sequenes. Tle 3. Comprison of hydrolyti nd syntheti tivities from superntnt nd whole ells etween Billus sp. A60 nd the referene strin B. sutilis 1A571. Retion Biotlyst Billus sp. A60 B. sutilis 1A571 Hydrolysis Superntnt 2.67 ± 0.11 () 1.62 ± 0.08 () (U/mg) Whole ell 7.07 ± 0.09 () 6.94 ± 0.75 () Synthesis Superntnt 88 ± 0 () 74.5 ± 0.35 () (% onversion) Whole ell ± 0.78 () ± 0 () Vlues ross lines followed y the sme letters do not differ signifintly (p<0.05). Syntheti retions tlyzed y ulture superntnts nd whole ells One of the proess for produing ethyl ette is y esterifition of ethnol with eti id. As shown in Tle 3, enzymti esterifitions of ethnol with eti id in n-hexne using severl iotlysts preprtions were investigted. The immoilized superntnt of Billus sp A60 hd etter performne (88.0% of onversion) thn tht of B. sutilis 1A571 (74.50% of onversion) (p<0.0001). In ontrst, whole ells of B. sutilis 1A571 showed higher level of onversion (66.47%) thn those of Billus sp. A60 (55.58%). In ddition, the TLC profiles of syntheti retions showed new spot tht ould orrespond to the ethyl ette prodution (dt not shown). It is interesting to note tht lthough high tolerne towrds orgni solvents hs een desried for B. sutilis 168 (Ktok et l., 2011), the proposed iotrnsformtion tlyzed y either immoilized superntnt or whole ells from B. sutilis 1A571 onstitutes n importnt ontriution to the knowledge of this referene strin. Conerning the enzymti synthesis of ethyl ette, the onversions otined in this work were similr to or higher thn those reported in some hemil proesses suh s the ethnol dehydrogention to ethyl ette y using opper nd opper hromite (Sntesri et l., 2012) or Cu-Zn-Zr-Al-O (Inui et l., 2004) s tlysts. Effet of medium omponents on Billus sp. A60 growth nd hydrolyti esterse prodution Esterse prodution requires omplex nitrogen nd ron soures nd its pproprite type nd onentrtion differ from orgnism to orgnism (Kdemi et l., 1999). To effiiently utilize Billus sp. A60 s iotlyst, high iomss density nd high esterse tivity would e neessry. In our ssys, the onentrtions of the omponents of LB medium were vried in order to determine ny effet on either growth or hydrolyti esterse tivity produed y Billus sp. A60. As shown in Figure 4, the nutrient medi omposition strongly ffeted oth growth nd enzyme tivity under study. As expeted, the LB medium represented the right proportions for growth. Conerning the extrellulr esterse prodution, it ws inresed s the onentrtion of either tryptone or yest extrt inresed. Others uthors hve otined similr results, where tryptone nd yest extrt t s enhners for esterse nd lipse prodution, without growth

6 Speifi tivity (U/mg) Growth (OD 610nm) 6630 Afr. J. Biotehnol. 1 MW kd potentil utility of esterses from Billus sp. A60 with gret pplition in food nd hemil industries. ACKNOWLEDGEMENT This work ws supported y grnts PICT (FONCyT), CIUNT 26/D409 (UNT) nd PIP 297 (CONICET). REFERENCES Figure 3. Ntive-PAGE using 10 % polyrylmide gel reveled for esterse tivity. Lne 1, Billus sp. A60 superntnt; MW, moleulr weight mker. Growth Speifi Ativity Medium Figure 4. Effet of medium omponents on Billus sp. A60 growth nd hydrolyti esterse prodution. orreltion (Kdemi et l., 1999; Mimur nd Ngt, 1998). Under our ssy onditions, the effet of yest extrt ws the most notorious; its sene ment slower growth nd lower esterse hydrolyti tivity. Adding yest extrt involved the reovery of oth prmeters, espeilly the esterse hydrolyti tivity. On the other hnd, ording to its hlotolernt ondition, the presene of NCl influened positively on the prodution of the esterse tivity. This is n interesting issue, sine high onentrtion of NCl ould hve n effet of therml protetion, s desried for Breviterium sp. (Mimur nd Ngt, 1998). For these resons it is possile to improve the esterse tivity of Billus sp.a60 y seleting pproprite onentrtion of the ulture medium. These results omined with the previous ones prove the Alvrez-Mrie E, Brtti J (2000). Short hin flvour ester synthesis y new esterse from Billus liheniformis. J. Mol. Ctl. B Enzym. 10: Ateslier ZBB, Metin K (2006). Prodution nd prtil hrteriztion of novel thermostle esterse from thermophili Billus sp. Enzyme Miro. Teh. 38: Bigorí M, Cstro GR, Siñeriz F (1996). Purifition nd hrteriztion of n extrellulr esterse from Billus sutilis MIR- 16. Biotehnol. Appl. Biohem. 24: Bélfi-Bkó K, Kiri Bdr A, Nemestóthy N, Erenstein U, Guiz L (2003). Kinetis of ethyl ette formtion y lipse in orgni solvent nd solvent-free system. Chem. Pp. 57: Bornsheuer UT (2002). Miroil roxyl esterses: lssifition, properties nd pplition in iotlysis. FEMS Miroiol. Rev. 26: Brdford MM (1976). A rpid nd sensitive method for the quntittion of mirogrm quntities of protein utilizing the priniple of protein-dye inding. Anl. Biohem. 72: Dvis BJ (1964). Dis eletrophoresis. II. Method nd pplition to humn serum proteins. Ann. N. Y. Ad. Si. 121: Grrity GM, Bell JA, Lilurn TG (2005). Txonomi Outline of the Prokryotes: Bergey s Mnul of Systemti Bteriology. Doi No. Pulished online y Springer-verlg, New York. Hnefeld U, Grdossi L, Mgner E (2009). Understnding enzyme immoiliztion. Chem. So. Rev. 38: Hrwood CR, Wipt A (1996). Sequening nd funtionl nlysis of the genome of Billus sutilis strin 168. FEBS Lett. 389: Higerd TB, Spizizen J (1973). Isoltion of two etyl esterses from extrts of Billus sutilis. J. Bteriol. 114: Inui K, Kuryshi T, Sto S, Ihikw N (2004). Effetive formtion of ethyl ette from ethnol over Cu-Zn-Zr-Al-O tlyst. J. Mol. Ctl. A Chem. 216: Jukes TH, Cntor CR (1969). Evolution of protein moleules. In Munro HN, editor, Mmmlin Protein Metolism, pp , Ademi Press, New York. Kdemi A, Fkhreddine L, Ait-Adelkder N, Brtti JC (1999). Effet of ulture onditions on growth nd esterse prodution y the moderte thermophile Billus irulns MAS2. J. Industril Miroiol. Biotehnol. 23: Ktok N, Tjim T, Kto J, Rhdeh W, Vngni AS (2011). Development of utnol-tolernt Billus sutilis strin GRSW2-B1 s potentil ioprodution host. AMB Express 1:10. Lindgren V, Ruterg L (1974). Glyerol metolism in Billus sutilis: gene-enzyme reltionships. J. Bteriol. 119: Miller JH (1972). Experiments in moleulr genetis. Cold Spring Hror Lortory, Cold Spring Hror, NY. Mimur H, Ngt S (1998). Effet of hyper-slt stress on the het resistne of hlotolernt Breviterium sp. JCM6894. J. Ferment. Bioeng. 85: Pnd T, Gowrishnkr BS (2005). Prodution nd pplitions of esterses. Appl. Miroiol. Biotehnol. 67: Romero CM, Per LM, Loto F, Vllejos C, Cstro G, Bigori MD (2012). Purifition of n orgni solvent-tolernt lipse from Aspergillus niger MYA 135 nd its pplition in ester synthesis. Biotl. Agri. Biotehnol. 1: Sntesri E, Crotenuto G, Tesser R, Di Serio M (2012). Ethnol dehydrogention to ethyl ette y using opper nd opper hromite tlysts. Chem. Eng. J. 179:

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