Internatinal Research Jurnal f Bitechnlgy (ISSN: 2141-5153) Vl. 3(6) pp. 96-111, June 2012 Available nline http://www.interesjurnals.rg/irjob Cpyright 2012 Internatinal Research Jurnals Full Length Research Paper Effects f ilizatin n the Catalytic Prperties f Extracellular Fructsyltransferase frm Rhdtrula sp. LEB-V10 *Elizama Aguiar-Oliveira 1 and Francisc Maugeri 2 1 Department f Fd Engineering (DEA), Faculty f Fd Engineering (FEA), University f Campinas (UNICAMP). R. Mnteir Lbat 80, Cidade Universitária Zeferin Vaz, Pstal Cde: 13083-862, Campinas - Sã Paul, Brazil 2 Faculty f Fd Engineering (FEA), University f Campinas (UNICAMP), Brazil Accepted 08 June, 2012 The lyphilizatin f fructsyltransferase (FTase) frm Rhdtrula sp. LEB-V10 was carried ut and its significant ptential fr synthesis f fructligsaccharides (FOS) was evaluated and cmpared t the nn-lyphilized enzyme. Twelve cryprtectant additives were selected and analyzed bth individually and tgether; the results generally shwed that lyphilized enzymes have a higher enzymatic activity per gram althugh different levels f relative enzyme activity were bserved, depending n the dilutin f the initial slutin. ilizatin withut additives f an enzymatic slutin diluted in 1:2 (v/v) sdium acetate buffer (200 mm) ph 4.5 presented the greatest increase in enzymatic activity per gram, almst 6.5-fld higher after lyphilizatin and a weight reductin f almst 89%, but this result als indicated a lss f 26% f the initial enzymatic activity. Additives such as CMC at 1.25% (w/v), mannitl, ammnium sulfate, srbitl and xylitl at 2.5% (w/v) increased enzymatic stability after 6 mnths frm 3 t 37%. Effects f the additives alne were better than when mixed. ilizatin als affected the bicatalytic activity f FTase, especially by increasing GF 4 (% cmpsitin) by almst 3-fld. Keywrds: Enzyme ilizatin, Free Enzyme, Freeze Drying, Fructligsaccharides Synthesis, FTase. INTRODUCTION Industrial bitechnlgy (white bitechnlgy) has generated much interest because it is frequently assciated with reduced energy cnsumptin, new fd and bifuels surces, and is frequently able t present ecnmic, plitical and scial slutins (Tang and Zha, 2009). Studies carried ut with extracellular fructsyltransferase (FTase, EC 2.4.1.9) partially purified (ethanl precipitatin) frm Rhdtrula sp. LEB-V10 (Aguiar-Oliveira and Maugeri, 2010; Alvarad-Huallanc and Maugeri, 2010; Hernalsteens and Maugeri, 2008; Maugeri and Hernalsteens, 2007), in free and immbilized frms, have demnstrated great industrial ptential fr the prductin f fructligsaccharides *Crrespnding Authr E-mail: elizamaguiar@yah.cm.br Tel: +55-19-3521-4052, Fax: +55-19-3521-4027 (FOS) - a prebitic sugar (Mairan et al., 2008; Sangeetha et al., 2005) - by prmting cnversins f apprximately 58% f sucrse t FOS under ptimized cnditins with the enzyme immbilized n nibium (Aguiar-Oliveira et al., 2012). In traditinal prcesses using fructsyltransferases purified frm filamentus fungus, cnversins vary frm 50 t 70% with purified enzymes. The use f partially purified fructsyltransferase reduces the csts f FOS prductin, and its immbilizatin techniques (Aguiar-Oliveira and Maugeri, 2010) favr cntinuus industrial use; therefre, the studies presented in the present study regarding the lyphilizatin f fructsyltransferase frm Rhdtrula sp. LEB-V10 are designed t slidify the feasibility f this enzyme in industrial applicatins. Kinetic studies with this same enzyme als immbilized in nibium (Alvarad- Huallanc and Maugeri, 2010) revealed that in either the purified r partially purified frms, sucrse cnversins t FOS were practically the same, and this justifies applicatin f the partially purified enzyme. The present
Aguiar-Oliveira and Maugeri 97 study evaluated the lyphilizatin f fructsyltransferase frm Rhdtrula sp. and made cmparisns with the nn-lyphilized enzyme in terms f activity, synthetic capacity and stability. ilizatin ilizatin (freeze drying) is ne f the mst wellknwn preservatin prcesses, and it cnsists f remving water frm prteins r cells in suspensin by sublimatin (Blanch and Clark, 1997; Cabral et al., 1994; Aehle, 1990; Chaplin and Bucke, 1990). This prcess is highly indicated fr adding cmmercial value t the prtein in questin since it facilitates transprt/distributin, strage, use, etc. (fr bth purpses pharmaceutical and alimentary) despite the fact that it is a high cst technlgy due t energy cnsumptin and preparatin time (Ry and Gupta, 2004; Tang and Pikal, 2004; Partridge et al., 1998; Carpenter et al., 1997; Fágáin, 1997). Cnservatin methds such as refrigerated strage in phsphate buffer (ph = 7.0) and ethanl slutins, r freezing in the presence f a reducing agent (e.g. glutathine) results in lw shelf-life, mrever, these techniques are nt industrially feasible (Kbayashi et al., 1984). Additinally, they can induce prtelysis, chemical degradatin (e.g. deamidatin r xidatin) and/r physical degradatin (e.g. degradatin, aggregatin and precipitatin) (Carpenter et al., 1997; Fágáin, 1997). Varius authrs have bserved different levels f activity and increased stability in lyphilized enzymes in a variety f methdlgies: Ru et al. (2001) described a greater than 20,000 fld increase in the bicatalytic activity f lyphilized enzymes in the presence f salts; Mrgan and Clark (2004) bserved a 10 fld increase in activity f a lyphilized xanthine xidase in the presence f salts; when lyphilizing a glucse dehydrgenase with srbitl and especially with trehalse, Sde and Yasutake (1997) managed t increase thermal stability in relatin t the nn-lyphilized cnditin; Wang and Mei (2007) increased the cnversin f lipase lyphilized with cycldextrins frm 9 t 14%. This activatin and the preservatin f the three-dimensinal structure after lyphilizatin may ccur due t the mst varied mechanisms. Lee and Drdick (2002) and Ru et al. (2001) stated that the use f certain additives increase the slubility f stabilizer slutes arund the prtein, frming a prtective layer. Accrding t Partridge et al. (1998), an efficient enzyme "dehydratin" prcess ccurs when water mlecules are remved, s that the prtein mlecules can maintain their three-dimensinal structure as intact as pssible. Fágáin (1997) stated that the mre an enzyme is able t maintain an inic balance, crrespnding t the ptimum ph level, the better the preservatin f its bicatalytic functins. In rder t reduce the stress suffered by the prtein, varius cry- r lip-prtectant cmpunds can be added (Kbayashi et al., 1984; Ru et al., 2001; Hirakura et al., 2004; Gibsn and Wdward, 1993; Rser, 1990). After freezing, sme buffers have their ph levels altered due t a change in pka r selective crystallizatin and precipitatin f a less sluble salt; the mst inadequate buffers wuld be thse f phsphate and pyrphsphate which present a reductin f 2.3 units in ph after freezing and the Tris buffer shws a ph level increase f the same magnitude (Carpenter et al., 1997; Hirakura et al., 2004; William-Smith et al., 1977). N references were fund regarding the sdium acetate buffer defined fr the fructsyltransferase frm Rhdtrula sp. LEB-V10 (Hernalsteens and Maugeri, 2008), where the nly reference fund analyzed the effects induced by the sdium acetate salt in cncentratins as high as 98% (w/v) (Ru et al., 2001; Brle and Davisn, 2008). The activatin mechanism prmted by high salt cncentratins appears t invlve several factrs including changes in the plarity f the enzyme r the ksmtrpicity f the activating salt (stabilizer capacity). Sme authrs suggest the use f buffers with higher salt cncentratins such as 200 mm, since 98% (w/v) f a salt in a prduct frmulatin with applicatins in the fd industry can interfere with the final prduct's cmpsitin (Ry and Gupta, 2004; Partridge et al., 1998; Ru et al., 2001; Mrgan and Clark, 2004). Pharmaceutical drugs and ther lyphilized prteins ften cntain glucse and/r fructse (hexses), sucrse (dissaccharide) r trehalse (nn-reducing disaccharide) in their frmulatins (Carpenter et al., 1997; Kbayashi et al., 1984; Gibsn and Wdward, 1993), and these cmpunds are capable f reducing the denaturatin impact during lyphilizatin by increasing the medium density r by remving water mlecules. Sucrse is the mst recmmended sugar due t its lw cst cmpared t trehalse (Carpenter et al., 1997), hwever, fr fructsyltransferase frm Rhdtrula sp. LEB-V10 it wuld prbably nt be recmmended t use sucrse, glucse r fructse frmulatins because they are substrates and by-prducts f the FOS synthesis, and at different cncentratins, these three cmpunds can induce kinetic inhibitin (Alvarad-Huallanc and Maugeri, 2010). Althugh Chaplin and Bucke (1990) supprted the use f substrates in frmulatins fr lyphilizatin, Carpenter et al., (1997) argues that even if it is effective, these additives have the prpensity t degrade prteins via Maillard reactins. Trehalse has been widely used due t its high stabilizing effect and ability t preserve the prtein structure (Carpenter et al., 1997; Rser, 1990) at different cncentratins including
98 Int. Res. J. Bitechnl. 100 mm (Carpenter et al., 1997), 300 mm (Sde and Yasutake, 1997) and frm 0.05 t 20% (Rser, 1990). The use f bulking agents such as mannitl r srbitl (hexahydric sugar alchl), and xylitl (pentahydric sugar alchl) are recmmended at varying cncentratins, frm 2 t 10%, 50 mm r in the prprtins f 1.1 r 1:6 (prtein:sugar) (Tang and Pikal, 2004; Carpenter et al., 1997; Kbayashi et al., 1984; Ru et al., 2001; Sde and Yasutake, 1997; Gibsn and Wdward, 1993). It is knwn that glycerl is ften emplyed in pharmaceutical frmulatins due t its viscsity, and icecrystal frmatin at sub subzer temperatures (Chaplin and Bucke, 1990), and as well as ethylene glycl, srbitl and xylitl, have a prtective effect frm direct interactin (whether specific r nt) with enzyme plypeptides, but glycerl has the disadvantage f being a gd bacterial substrate (Fágáin, 1997). Ethylene glycl is used as a prtectin grup fr the carbnyl grups in rganic synthesis, and it is knwn fr its desiccant prperties (Sares et al., 2002). Ply(ethylene glycl) r PEG (amphipathic plymer and lip-prtectant) is ften applied in lyphilized prtein frmulatins at 49% cmbined with KCl (Ru et al., 2001) r sucrse (Msharraf et al., 2007), ranging frm 155 t 323 mm (Mine et al., 2001) r in a 1:2 rati f enzyme:peg (Brle and Davisn, 2008) and is als indicated as a gd mlecular imprinter (Lee and Drdick, 2002). Carbxymethyl cellulse (CMC) is knwn as a viscsity mdifier r thickener, as well as a gd emulsifing agent in varius prducts f the fd industry. Sde and Yasutake (Sde and Yasutake, 1997) investigated the additin f ammnium sulfate at 50 mm, amng ther cmpunds. Accrding t Carpenter et al. (Carpenter et al., 1997), increased enzymatic cncentratin heightens the resistance t degradatin during freezing. It is believed that damage culd be caused by the icewater interface during the freezing prcess, and fr this reasn it is imprtant t evaluate the ptimal cncentratin r dilutin rate fr each system studied. MATERIAL AND METHODS Partial purificatin f the extracellular fructsyltransferase and the cultivatin f Rhdtrula sp. LEB-V10 was carried ut accrding t Aguiar- Oliveira and Maugeri (Aguiar-Oliveira and Maugeri, 2010), as well as the methdlgies fr determinatin f enzymatic activity frm sugars released using the Smgyi-Nelsn methdlgy and Glucsexidase enzymatic kit, and FOS by inexchange chrmatgraphy (HPLC-PAD). Additives Selectin The additives evaluated in this study were: ammnium sulfate, carbxymetyl cellulse (CMC), ethylene glycl, glycerl, insitl, mannitl, plyethylen glycl (PEG 6000), srbitl, trehalse and xylitl added t sdium acetate buffer (ph 4.5) at cncentratins f 100 and 200 mm. These cmpunds were acquired frm trusted and available cmpanies. In the first step, each additive was individually evaluated at the cncentratin f 2.5% (w/v r v/v). Later, sme additives were chsen fr use in the frmulatins: CMC, PEG 6000, mannitl, xylitl, trehalse, and ammnium sulfate, all at 1.25% (w/v), with the dispersin and dilutin slutin being sdium acetate buffer at 200 mm and ph 4.5. Sample preparatin and analysis In the first stage f this study, the individual effect f each additive was studied; all lyphilizatins were perfrmed in duplicate in which each had a starting vlume f 3.0 ml. An enzymatic slutin with transfructsylatin activity f 134.06 ± 13.68 U /ml (ρ = 0.718 ± 0.0050 g/ml) was used withut dilutin and diluted t 1:6 (v/v) in sdium acetate buffer (50 mm), ph 4.5; each additive (listed in Table 1) was assessed as part f bth enzymatic slutins at 2.5% (w/v), where nly CMC was als evaluated at 1.25% (w/v). After selecting the best additives accrding t individual perfrmances, the enzyme slutins used fr the secnd stage f this study (frmulatins f additives) were diluted t 1:2 and 1:6 (v/v) in 200 mm sdium acetate buffer, ph 4.5, and the fllwing experiments were carried ut: three frmulatins cntaining tw additives were chsen fr the dilutin f 1:2 (v/v) and three frmulatins cntaining three additives fr the dilutin f 1:6 (v/v), as presented in Table 3. In this secnd stage, sample preparatin was similar t the first stage, always in duplicate, and the cncentratin f each additive in the frmulatin was 1.25% (w/v). Based n results frm a previus wrk (Aguiar- Oliveira and Maugeri, 2011), fructsyltransferase was pre-activated (perfrmed by 15 min f incubatin at 52 r 60 C fllwed by submersin in an ice bath) befre lyphilizatin. ilizatin f the free fructsyltransferase All samples were previusly frzen at -60 C in an ultrafreezer fr abut 18 h prir t lyphilizatin. These samples were then lyphilized fr 24 h in a Terrni
Aguiar-Oliveira and Maugeri 99 Table 1. Characterizatin f fructsyltransferase frm Rhdtrula sp. LEB-V10, lyphilized in the presence and absence f stabilizing additives. The characterizatin parameters were as fllw: residual enzyme activity [ R ], thermal residual enzyme activity [R.65 C * ], specific enzyme activity [ U ptn = U ptn mg ], FOS yield [Y FOS] and specific FOS prductivity after 24 h [Pr FOS = mg/u.h]. Syntheses were carried ut with 0.02 g f the lyphilized pwder in 5 ml f 50% (w/v) f sucrse in 50 mm sdium acetate buffer, ph 4.5. Initial Enzyme Slutin ilized enzyme withut addictive U /g R 65 C (U 65 C /U ) *U ptn (U /mg) Y FOS (24h) (Eq.04) Pr FOS (24h) (Eq.05) 186.65 ± 19.04 0.37 ± 3.8E -2 5.59 ± 0.16 0.45 17.63 ilized enzyme frm the initial undiluted enzyme slutin R R.65 C *U ptn Y FOS (24h) Pr FOS (24h) (Eq. 01) (Eq. 02) (Eq.03) (Eq.04) (Eq.05) 4.11 ± 0.35 0.58 ± 1.4E -2 5.16 ± 0.15 0.42 2.89 R R.65 C *U ptn Y FOS (24h) Pr FOS (24h) (Eq. 01) (Eq. 02) (Eq.03) (Eq.04) (Eq.05) ilized enzyme with additives Ammnium sulfate - 2.5% (w/v) 3.15 ± 0.34 0.50 ± 5.2E -2 4.55 ± 0.10 0.50 4.61 CMC - 2.5% (w/v) 4.46 ± 0.40 0.38 ± 1.7E -2 6.27 ± 0.08 0.50 4.64 Ethylene glycl - 2.5% (v/v) 2.99 ± 0.14 0.25 ± 2.3E -2 2.96 ± 0.14 0.52 4.41 Glycerl - 2.5% (w/v) 2.54 ± 0.37 0.43 ± 3.5E -2 3.36 ± 0.15 0.49 4.34 Insitl - 2.5% (w/v) 3.47 ± 0.41 0.41 ± 7.9E -3 4.59 ± 0.02 0.41 3.84 Mannitl - 2.5% (w/v) 3.30 ± 0.25 0.35 ± 2.2E -2 4.37 ± 0.06 0.50 5.54 PEG 6000-2.5% (w/v) 4.91 ± 0.19 0.32 ± 8.6E -3 6.42 ± 0.21 0.59 3.33 Srbitl - 2.5% (w/v) 3.04 ± 0.37 0.28 ± 7.8E -2 4.01 ± 0.13 0.30 2.40 Trehalse - 2.5% (w/v) 3.38 ± 0.30 0.35 ± 1.7E -2 4.09 ± 0.07 0.47 3.65 Xylitl - 2.5% (w/v) 3.00 ± 0.29 0.57 ± 3.5E -3 3.95 ± 0.12 0.33 2.74 ilized enzyme frm the initial enzyme slutin diluted at 1:6 (v/v) in sdium acetate buffer, 50 mm and ph 4.5. Y FOS (24h) Pr FOS (24h) ilized enzyme (Eq. 01) (Eq. 02) (Eq.03) (Eq.04) (Eq.05) withut additive 3.32 ± 0.28 0.63 ± 3.6E -2 4.65 ± 0.02 0.44 3.75 ilized enzyme R R.65 C *U ptn Y FOS (24h) Pr FOS (24h) with additives (Eq. 01) (Eq. 02) (Eq.03) (Eq.04) (Eq.05) Ammnium sulfate - 2.5% (w/v) 2.14 ± 0.23 0.26 ± 3.4E -3 3.62 ± 0.11 0.37 4.04 CMC - 2.5% (w/v) 1.85 ± 0.15 0.76 ± 4.2E -2 2.83 ± 0.01 0.44 5.32 CMC - 1.25% w/v 2.99 ± 0.28 0.57 ± 9.8E -3 4.22 ± 0.08 0.45 10.89 Ethylene glycl - 2.5% (v/v) 1.16 ± 0.10 0.58 ± 4.3E -2 1.56 ± 0.03 0.17 2.45 Glycerl - 2.5% (w/v) 2.09 ± 0.25 0.28 ± 1.8E -2 3.94 ± 0.03 0.37 4.89 Insitl - 2.5% (w/v) 2.44 ± 0.31 0.31 ± 6.2E -3 5.35 ± 0.08 0.33 4.79 Mannitl - 2.5% (w/v) 1.95 ± 0.19 0.27 ± 2.3E -2 3.95 ± 0.03 0.38 5.06 PEG 6000-2.5% (w/v) 3.57 ± 0.28 0.71 ± 1.4E -2 4.80 ± 0.08 0.44 3.43 Srbitl - 2.5% (w/v) 2.44 ± 0.31 0.26 ± 8.7E -3 4.99 ± 0.03 0.31 3.58 Trehalse - 2.5% (w/v) 1.90 ± 0.25 0.63 ± 7.2E -2 3.77 ± 0.08 0.47 3.68 Xylitl - 2.5% (w/v) 1.93 ± 0.13 0.61 ± 8.1E -2 3.89 ± 0.05 0.34 5.01 ilized enzyme frm the initial enzyme slutin diluted at 1:6 (v/v) in sdium acetate buffer at ph 4.5 Buffer cncentratin R R.65 C *U ptn Y FOS (24h) Pr FOS (24h) (Eq. 01) (Eq. 02) (Eq.03) (Eq.04) (Eq.05) 100 mm 3.62 ± 0.42 0.52 ± 4.4E -2 4.97 ± 0.08 0.39 3.00 200 mm 3.65 ± 0.45 0.55 ± 7.1E -3 5.04 ± 0.06 0.41 3.12 R R.65 C *U ptn Interprise 1 bench-type lyphilizer, where they were cled t -55 C by a hermetic cmpressr with frced ventilatin (air) and using a vacuum pump with capacity f 5/370 (CFM/W). The lyphilized cakes were gently
100 Int. Res. J. Bitechnl. milled with a glass baguette until turning t dust. ilized enzymes were then stred in standard glass flasks (5 ml), withut vacuum seal, in a freezer (-20 C) until all analyses were perfrmed; analyses were perfrmed within ne week f lyphilizatin (t = 0) fr the initial characterizatin, and 6 mnths after refrigerated strage (t = 6 m) fr the shelf-life analysis. Characterizatin f the lyphilized enzyme Accrding t Carpenter et al. (1997), the final analysis f a lyphilized prteic frmulatin shuld cnsider factrs such as: prtein stability during and after lyphilizatin, final applicatin f the lyphilized material, frmulatin tnicity, cake structure and ther physicchemical prperties. Enzymatic activity f lyphilized free fructsyltransferase was determined frm a slutin cntaining 0.02 g f lyphilized pwder disslved in 10 ml f 50 mm acetate buffer, ph 4.5. Standard FOS syntheses were perfrmed at 50 C using 0.02 g f the pwder in 5 ml f 50% (w/v) f sucrse in sdium acetate buffer (50 mm and ph 4.5); FOS synthesis under ptimized cnditins (Aguiar-Oliveira et al., 2012) was basically carried ut the same way except fr the fllwing cnditins: 48 C, ph 6.0 and 10 U /ml. All lyphilized samples were analyzed and cmpared t a nn-lyphilized standard cnditin withut any additives. The lyphilized enzymes were characterized mainly accrding t residual activity f the lyphilized enzyme (Eq. 01), thermal residual activity f the lyphilized enzyme (Eq. 02), specific activity f the lyphilized enzyme (Eq.03), FOS yield (Eq.04), specific prductivity f FOS (Eq.05) and 6 mnths shelf-life. Residual Enzyme Activity ( R ) Expressed by Equatin 01 accrding t the lyphilized free enzymatic activity ( U free enzymatic activity ( U ). U R = U ) and the nn-lyphilized (01) minutes fllwed by submersin in an ice bath [.65 C U ], and the lyphilized free enzymatic activity f the same sample befre this thermal treatment [ U ]. R.65 C U = U.65 C Specific Enzyme Activity ( U ) (02) Expressed by Equatin 03 accrding t the lyphilized free enzyme activity [ U ] and the prtein value (mg) determined per gram f lyphilized enzyme. Lwry s methdlgy (Lwry et al., 1951) was used t determine the ttal prtein in the lyphilized enzyme frmulatin. * U ptn U = mg FOS yield (Y FOS ) (03) Expressed by Equatin 04 accrding t the ttal FOS cncentratin (g/l) and the initial sucrse cncentratin (500 g/l). 200 µl samples were cllected frm the synthesis prduct during different perids, and analyzed by in-exchange chrmatgraphy (HPLC-PAD) accrding t the methdlgy described in a previus wrk (Aguiar- Oliveira and Maugeri, 2010). Y [ FOS] [ FOS] = = FOS [ sucrse] 500g / L (04) FOS Specific Prductivity (Pr FOS = mg/u.h) Rati expressed by Equatin 05 accrding t the ttal FOS cncentratin (mg/ml) and the cncentratin f free enzyme activity used in the synthesis (U /ml), as a functin f the synthesis elapsed time (h). [ FOS ] Pr FOS = ( U )( h) (05) Thermal Residual Enzyme Activity (R.65 C ) Expressed by Equatin 02 accrding t the lyphilized free enzyme activity after incubatin at 65 C fr 15 Water Activity (Aw) The lyphilized enzymes withut additives had their water activity (Aw) determined in triplicate in an AQUALAB
Aguiar-Oliveira and Maugeri 101 Figure 1. Standard lyphilized material f fructsyltransferase frm Rhdtrula sp. LEB-V10: a) withut dilutin (lyphilized cake with Aw = 0.187 ± 0.001 and with greater resistance t crushing and disslutin), b) diluted t 1:2 (v/v) (lyphilized with Aw = 0.192 ± 0.004, fine pwder aspect and fast disslutin) and c) diluted t 1:6 (v/v) (lyphilized with Aw = 0.276 ± 0.001, lighter clratin, very fine unifrm pwder, easy disslutin). The dilutins were achieved in sdium acetate buffer (50 mm and ph 4.5). Series 3TE device, Decagn Devices Inc. (USA), at 25 C after lyphilizatin (time zer) and after 6 mnths f refrigerated strage. Factr fr Enzyme Activity Reductin after ilizatin ( F ) U The ttal reductin f enzymatic activity after lyphilizatin was determined accrding t Equatin 06 fr the lyphilized free enzyme withut additives, using the enzymatic activities per gram befre (U /g) and after lyphilizatin ( U g ) and the weight f the samples befre (6 g) and after lyphilizatin.. U lyphilize d U slutin FU = * ( g) * (6g) g g RESULTS AND DISCUSSION (06) Accrding t Oetjen and Haseley (2004), freezing f cmplex rganic slutins is ften difficult t predict and the grwth rate f ice crystals is crucial because it is dependent n the temperature f initial freezing and the viscsity f the slutin, which increases significantly with the increasing slutin cncentratin. The water nt prperly frzen can frms highly viscus cclusins between the crystals, and by adding excipients the crystallizatin can again be delayed because f the viscsity f the initial slutin. In the first stage f this study, an enzymatic slutin withut dilutin and an activity f 134.06 ± 13.68 U /ml was used, whse crrected activity based n density (ρ = 0.718 ± 0.050 g/ml) was equivalent t 186.65 ± 19.04 U /g. ilizatin f this slutin (withut additives) resulted in a material with enzymatic activity f 763.64 ± 16.71 U g, a 4-fld increase in enzymatic activity per weight. Weight reductin after lyphilizatin was abut 85%: 1g f the enzymatic slutin resulted in 0.1522 ± 0.026 g f lyphilized pwder. Cncerning the enzymatic activity lss during the prcess, the factr F U (Eq. 06) was 0.62, i.e. abut 38% f enzyme activity was lst during the prcess with the nn-diluted enzymatic slutin. Water activity f this lyphilized enzyme at time zer was Aw = 0.187 ± 0.001. The same cncentrated enzymatic slutin mentined abve was diluted by 1:2 (v/v) with sdium acetate buffer at 50 mm and ph 4.5, whse density was 0.993 ± 0.016 g/ml. Weight reductin after lyphilizatin was abut 89%, indicating that 1g f the diluted slutin prduced 0.1129 ± 0.014 g f lyphilized pwder. The F U factr fr enzymatic activity lss was f 0.73, signifying that abut 26% f the enzymatic activity was lst (this was the smallest F U amng the three cnditins analyzed). Water activity f this lyphilized enzyme at time zer was Aw = 0.192 ± 0.004. Finally, a dilutin f 1:6 (v/v) with the same initial enzymatic slutin led t a density f ρ = 0.996 ± 0.010 g/ml. Weight lss after lyphilizatin was apprximately 96%, meaning that 1g f the diluted slutin resulted in 0.0258 ± 0.009 g f lyphilized pwder. The F U factr fr reductin f enzymatic activity was 0.20, i.e., abut 80% f the initial enzymatic activity was lst during lyphilizatin; this cnditin resulted in the highest activity lss amng the three analyzed cnditins. Water activity f this lyphilized enzyme at time zer was Aw = 0.276 ± 0.001. Effects f single additives were evaluated using the enzymatic slutin withut dilutin and that diluted at 1:6 (v/v), as shwn in Table 1. Afterwards, enzymatic slutins at the 1:2 and 1:6 (v/v) dilutin ratis were used in a secnd step with frmulatins f the best additives
102 Int. Res. J. Bitechnl. previusly selected (Table 3). Visual analysis f the lyphilized cakes (light brwn clr) indicated the differences between the cncentrated slutin and its dilutins. The lyphilizatin f slutins withut dilutin resulted in a harder cake, resistant t crushing, even frming larger and irregular granules (Figure 1a) which were disslved slwly (r resuspended) and requiring lnger stirring times t achieved the ttal disslutin; the lyphilized pwder frm diluted slutins resulted in a sfter cake which was easier t crush, with fine granules and easy disslutin, especially fr the dilutin at 1:6 (v/v) (Figure 1.c). Accrding t several authrs, even after lyphilizatin an enzyme preparatin still cntains water which remains strngly linked and this plays a crucial rle fr the final prduct because water ften acts as a catalyst fr enzymes by increasing the internal flexibility f its mlecules r by activating deterirative reactins. In general, instability is increased when the final lyphilized prduct cntains water abve r belw the ideal limit f the water layer (M) (Cabral et al., 1994; Fágáin, 1997; Msharraf et al., 2007; Cstatin et al., 1998). It shuld als be cnsidered that during r immediately after lyphilizatin (freeze-drying) and/r recnstitutin (rehydratin) it is pssible t frm irreversible aggregates, a phenmenn cmmnly assciated with a prly designed r prepared lyphilized prtein (Carpenter et al., 1997; Hirakura et al., 2004), resulting in lw activity. Individual effect f additives Table 1 shws the results frm the first stage f the study evaluating effects f individual additives. It can be seen that all lyphilized enzyme prducts cntaining additives shwed an increase in enzymatic activity after lyphilizatin ( R. > 1.0) cmpared t the nn-lyphilized standard cnditin. Accrding t Table 1, lyphilizatin f the undiluted enzymatic slutin resulted in a greater than 4 times increase in enzymatic activity per weight; lyphilizatin f the diluted slutin at 1:6 (v/v) resulted in an increase f mre than 3 times. The small difference in R between the lyphilized. cnditins withut additives reflects the fact that very cncentrated slutins induce the frmatin f prtein aggregates that jepardize the final effective enzymatic activity. The undiluted enzymatic slutin cntaining PEG 6000 and CMC bth at 2.5% (w/v), the slutin diluted in sdium acetate buffer at 200 and 100 mm and that diluted cntaining PEG 6000 and insitl at 2.5% (w/v) presented, in descending rder, the highest lyp- hilized enzymatic activity (3.50 < R < 5.0). Literature. reprts that the additin f srbitl, mannitl and sdium acetate, all at 1 ml/l, led t remaining enzyme activities f 75.3, 67.3 and 81.2%, respectively, after lactate dehydrgenase freezing and thawing (Oetjen and Haseley, 2004). The thermal residual activity (R.65 C ) revealed that lyphilizatin f the enzymatic slutin increased thermal stability cmpared t the nn-lyphilized slutin (Table 1); fr the standard lyphilized enzyme, a dilutin f 1:6 (v/v) increased thermal stability by 9 %. Fr lyphilizatin f the undiluted enzymatic slutin, nne f the additives were capable f increasing thermal stability; when cmpared t the lyphilized standard cnditin (R.65 C = 0.58), nly the additin f xylitl resulted in a similar value (R.65 C = 0.57). ilizatin f the diluted enzymatic slutin (1:6 v/v) presented an increased thermal stability with the additin f the PEG 6000 (R.65 C = 0.71) and CMC (R.65 C = 0.76), bth at 2.5% (w/v), when cmpared t the standard lyphilized enzyme. The specific lyphilized activity ( * U ptn ) shuld be analyzed with cautin, since it is a partially purified enzyme slutin and unidentified prteins are als precipitated alng with the fructsyltransferase. Therefre, * U ptn basically reflects the lyphilized enzyme activity, as shwn by Equatin 03. After the lyphilizatin prcedures, the standard lyphilized enzyme frm the undiluted slutin shwed a specific activity similar t the nn-lyphilized slutin; hwever, dilutin and lyphilizatin resulted in a specific activity 10% lwer than in the standard undiluted cnditin. The highest value was btained with the lyphilized enzyme frm the undiluted slutins with the additin f PEG 6000 (Table 1). FOS synthesis perfrmed with bth the lyphilized enzyme withut additives, and with the nn-lyphilized slutin shwed similar cnversins (0.42 < Y FOS < 0.45). Mrever, fr the lyphilized enzymes with additives, mst Y FOS values were near r higher than thse fr the standard undiluted lyphilized enzymes (Table 1). The highest cnversins f sucrse (Y FOS > 0.45) were btained with the lyphilized enzymes (frm the undiluted slutin) supplemented with: PEG 6000, CMC, ethylene glycl, mannitl, glycerl, trehalse, and ammnium sulfate, all at 2.5% (w/v). Regarding the lyphilized enzyme frm a diluted slutin, nly the additin f trehalse led t an increased Y FOS. Hernalsteens and Maugeri (2008) reprted that with free fructsyltransferase under the same synthesis cnditins, and with 3 U /ml, Y FOS values reached 0.45 after 24 h f reactin. Cncerning the FOS specific prductivity (Pr FOS ), after 24 h f synthesis the lyphilized enzyme
Aguiar-Oliveira and Maugeri 103 Table 2. Analysis f the residual activity f the stred lyphilized enzyme ( R 6m ) after 6 mnths f strage at -20 C when cmpared with the enzyme activity at time zer (Table 1) fr the lyphilized free fructsyltransferase frm Rhdtrula sp. LEB-V10. The lyphilized enzymatic activities fr the standards withut additives after 6 mnths were 507.72 U g fr the tw cnditins: diluted and withut dilutin. ilized enzyme frm an initial slutin withut dilutin ilized Enzyme R a 6 m ilized withut additive 0.66 Ammnium Sulfate (2.5% w/v) 1.37 CMC (2.5% w/v) 0.58 Mannitl (2.5% w/v) 1.03 PEG 6000 (2.5% w/v) 0.45 Srbitl (2.5% w/v) 1.10 Trehalse (2.5% w/v) 0.80 Xylitl (2.5% w/v) 1.26 ilized enzyme frm an initial slutin diluted at 1:6 (v/v) in sdium acetate buffer 50 mm at ph 4.5 ilized Enzyme R a 6 m ilized withut additive 0.82 CMC (2.5% w/v) 0.80 CMC (1.25% w/v) 1.07 Insitl (2.5% w/v) 0.92 PEG 6000 (2.5% w/v) 0.67 Srbitl (2.5% w/v) 0.79 ilized frm an initial enzymatic slutin diluted at 1:6 (v/v) in sdium acetate buffer at ph 4.5 Buffer cncentratin R a 6 m 100 mm 0.68 200 mm 0.84 R ( t= 6m) ( t= 0 ) 6m = U U frm a diluted slutin generally shwed higher specific prductivities than the lyphilized enzyme frm an undiluted slutin. Additinally, the additives used with the latter enzyme resulted in higher Pr FOS than the standard undiluted lyphilized enzyme, except fr xylitl (Pr FOS = 2.74 mg U. h ) and srbitl (Pr FOS = 2.40 mg U. h). Cnsidering the enzymes btained frm a 1:6 diluted slutin, the buffer cncentratin factr (100 and 200 mm) and the additin f PEG 6000, ethylene glycl, srbitl and trehalse all led t lwer Pr FOS when cmpared t the lyphilized enzymes withut additives. Shelf-life after 6 mnths Accrding t the results in Table 1, cnditins were selected fr 6 mnth shelf-life evaluatins f lyphilized enzymes that presented at least 70% f the ttal enzymatic activity f the standard lyphilized enzymes,
104 Int. Res. J. Bitechnl. Figure 2a. FOS yield (Y FOS) and FOS specific prductivity (Pr FOS), and b) percent cmpsitin f FOS frm syntheses with lyphilized fructsyltransferase withut dilutin (w.d) (squares and filled lines) and diluted in sdium acetate buffer (50 mm), at a 1:6 (v/v) dilutin factr. Syntheses were perfrmed under the fllwing cnditins: 50% (w/v) sucrse in 50 mm sdium acetate buffer, ph 4.5, 50 C and 10 U ml. withut additives (with and withut dilutin). The results are shwn in Table 2 and evaluated n the basis f residual enzymatic activity, i.e., the maintenance f enzymatic activity cmpared t its initial activity ( R ( t= 6m ) ( t= 0 ) 6 m U U = ), based n Equatin 1. Enzymatic activities f the standard lyphilized enzyme withut additives (with and withut dilutin) after 6 mnths shwed the same values f 507.72 g U (Table 2), which represent enzymatic activity retentins f 66 and 82%, respectively. Amng the ther cnditins evaluated, the tw highest values f lyphilized enzymatic activity were bserved with the fllwing cnditins: ammnium sulfate (802.11 g ) and U xylitl (700.98 U g ) bth at 2.5% and withut dilutin, representing an increase f apprximately 37 and 26%, respectively, in enzymatic activity after 6 mnths. This culd be explained by pssible water reabsrptin during strage, assciated with the type f additive that may smehw have given back sme flexibility t the mlecules, thus increasing activity. It can be bserved in Table 2 that the best results fr maintenance f enzymatic activity after 6 mnths f strage, expressed by the residual strage lyphilized 6m enzymatic activity ( R > 0.9), were btained mstly using the lyphilized enzyme frm an undiluted slutin with the additives f ammnium sulfate, xylitl, mannitl and insitl (at 2.5% w/v) and CMC (at 1.25% w/v). The standard cnditins shwed lss f enzymatic activity after 6 mnths f strage; the lyphilized enzyme frm a diluted slutin shwed a reductin f 18% in the initial enzymatic activity, and the undiluted lyphilized enzyme had a reductin f 34%. FOS syntheses were perfrmed with nly the tw standard lyphilized enzymes (with and withut dilutin) stred fr 6 mnths in rder t separately evaluate the effects f lyphilizatin and strage n transfructsylatin activity. Figure 2a shws the values f FOS yield (Y FOS ) and specific prductivity f FOS (Pr FOS ) fr these tw cnditins. The results were very similar and the enzymatic cncentratins used in bth syntheses were the same: 10 ml. The tw syntheses presented U decreasing Y FOS values after 24 h; fr the lyphilized undiluted enzyme, at 24 h, Y FOS was 0.69 and with the lyphilized diluted enzyme at 1:6 (v/v), Y FOS was 0.66. Specific prductivity f FOS (Pr FOS ) shwed maximum values in 3 hurs fr bth cnditins (arund 7.0 mg U. h ). Figure 2 shws that after 6 mnths f refrigerated strage, the lyphilized enzyme withut additives maintained its transfructsylatin activity. In bth cases large quantities f 1-kestse (GF 2 ) were hydrlyzed reaching 6 t 7% f ttal FOS after 72 h; n the ther hand, 1 F- fructfuransyl-nystse (GF 4 ) shwed higher cmpsitins, reaching values up t almst 70% (~182.49 g/l) after 72 h f synthesis fr bth lyphilized enzymes (Figure 2b). This result has never been previusly bserved with the fructsyltransferase frm Rhdtrula sp. LEB-V10 and may be technlgically interesting since the larger the FOS mlecule, the greater the benefits t the bdy (Stewart et al., 2008). Therefre, lyphilizatin f fructsyltransferase frm Rhdtrula sp. LEB-V10 led t a change in its bicatalytic specificity, in such a way that a significantly change in FOS cmpsitin was bserved, especially with GF 4. Under ptimum cnditins with the nn-lyphilized
Aguiar-Oliveira and Maugeri 105 Table 3. Characterizatin f the lyphilized fructsyltransferase frm Rhdtrula sp. LEB-V10 with additive frmulatins. The characterizatin parameters fr 1g f slutin and lyphilized pwder were: residual lyphilized enzyme activity [ R ],.65 C * thermal residual activity f the lyphilized enzyme [ R ], specific lyphilized enzyme activity [ U ptn ], FOS yield [Y FOS] and FOS specific prductivity [Pr FOS; mg/u h], at 72 h f synthesis. FOS syntheses were carried ut with 0.02g f the lyphilized enzyme in 5 ml f 50% (w/v) sucrse in 50 mm acetate buffer at ph 4.5. Analyses were perfrmed in duplicate. Enzyme slutin btained by dilutin in sdium acetate buffer 50 mm and ph 4.5 (partially purified FTase) * U ptn Y FOS - 72h Pr FOS - 72h Standards nn-lyphilized U /g ( mg f ptn ) (Eq. 04) (Eq. 05) R 65 C ( C U 65 U ) 1:2 (v/v) dilutin 179.47 ± 5.98 0.21 ± 0.01 4.77 ± 0.16 0.41 ± 0.01 3.94 ± 0.14 1:6 (v/v) dilutin 135.02 ± 4.34 0.18 ± 0.01 5.02 ± 0.05 0.59 ± 0.04 15.95 ± 1.03 ilized enzyme frm an initial slutin diluted at 1:2 (v/v) in sdium acetate buffer, 200 mm and ph 4.5. ilized withut additives (standard 1:2) R (Eq. 01) 6.46 ± 0.15 R.65 C U * U ptn Y FOS (72h) (Eq. 04) Pr FOS (72h) (Eq. 05) (Eq. 02) (Eq. 03) 0.36 ± 4.1E - 3 7.54 ± 0.07 0.62 ± 0.02 0.93 ± 0.03 Frmulates (1:2) cmpsitin (% w/v) R (Eq. 01) R.65 C (Eq. 02) * U ptn (Eq. 03) Y FOS (72h) (Eq. 04) Pr FOS (72h) (Eq. 05) PEG 6000 (1.25%) + ammnium Sulfate (1.25 %) + mannitl (1.25 %) 4.26 ± 0.18 0.33 ± 0.01 4.98 ± 0.37 0.63 ± 0.01 1.43 ± 0.03 PEG 6000 (1.25%) + ammnium sulfate (1.25%) + xylitl (1.25%) 3.19 ± 0.21 0.54 ± 0.10 3.73 ± 0.37 0.55 ± 0.01 1.66 ± 0.04 PEG 6000 (1.25%) + ammnium sulfate (1.25%) + trehalse (1.25%) 3.14 ± 0.36 0.41 ± 0.01 3.65 ± 0.29 0.60 ± 0.06 1.85 ± 0.18 ilized enzyme frm an initial slutin diluted at 1:6 (v/v) in sdium acetate buffer 200 mm and ph 4.5. R R.65 C U ptn Y FOS (72h) Pr FOS (72h) ilized withut additives (Eq. 01) (Eq. 02) (Eq. 03) (Eq. 04) (Eq. 05) (standard 1:6) 3.72 ± 0.07 0.61 ± 0.03 3.64 ± 0.04 0.56 ± 0.01 1.94 ± 0.04 Frmulates (1:6) cmpsitin (% w/v) R (Eq. 01) R.65 C (Eq. 02) * U ptn (Eq. 03) Y FOS (72h) (Eq. 04) Pr FOS (72h) (Eq. 05) CMC (1.25%) + mannitl (1.25%) 2.32 ± 0.26 0.67 ± 0.14 2.27 ± 0.29 0.56 ± 0.01 3.14 ± 0.02 CMC (1.25%) + xylitl (1.25%) 1.80 ± 0.29 0.92 ± 0.05 1.75 ± 0.20 0.57 ± 4.0.E -3 3.61 ± 0.28 CMC (1.25%) + trehalse (1.25%) 4.76 ± 0.28 0.55 ± 0.04 4.66 ± 0.11 0.57 ± 0.01 4.09 ± 2.0E -3 and immbilized fructsyltransferase, the relative GF 4 fractin was 4% (11.90 g/l) (Aguiar-Oliveira et al., 2012). Frmulatins Frmulatins with selected additives and new lyphilizatin cnditins were chsen, as shwn in Table 3, taking int accunt the best previus results f residual activities ( R, R (Y FOS )..65 C and R 6 mnths ) and FOS yield Fr this set f experiments the 1:2 dilutin was chsen fr practical reasns, since the undiluted lyphilized cake is hard t dilute, and the 200 mm sdium acetate buffer was selected since it prvided ne f the highest thermal residual activity and imprved shelf-life. Several wrks reprted successful uses f highly cncentrated buffers (Ry and Gupta, 2004; Ru et al., 2001; Mrgan and Clark, 2004). CMC and PEG 6000 resulted in gd residual lyphilized activities and thermal residual activities, therefre they were selected as bulking agents. Ammnium sulfate was selected since it permitted enhanced thermal residual activity and FOS yield (Table
106 Int. Res. J. Bitechnl. Table 4. Analysis f residual activity fr the stred lyphilized enzyme ( R 6m ) after 6 mnths stred at -20 C, cmpared with enzyme activity at time zer (Table 3) fr the lyphilized fructsyltransferase frm Rhdtrula sp. LEB-V10 in frmulatins. The enzyme activity f the standard lyphilized enzyme was 757.14 ± 31.18 U g (dilutin 1:2) and 431.03 ± 44.36 U g (dilutin 1:6) after 6 mnths. The cncentratin f each additive in all frmulatins was 1.25 % (w/v). ilized enzyme frm an initial slutin diluted at 1:2 (v/v) in sdium acetate buffer 200 mm and ph 4.5. ilized Enzymes R a 6 m ilized withut additives 0.65 ± 0.03 PEG + ammnium sulfate + mannitl 0.52 ± 0.02 PEG + ammnium sulfate + xylitl 0.64 ± 0.14 PEG + ammnium sulfate + trehalse 0.77 ± 0.04 ilized frm an initial enzymatic slutin diluted at 1:6 (v/v) in sdium acetate buffer 200 mm and ph 4.5. ilized Enzymes R a 6 m ilized withut additives 0.86 ± 0.08 CMC + mannitl 0.67 ± 0.16 CMC + xylitl 0.90 ± 0.33 CMC + trehalse 0.23 ± 0.02 R (t= 6m) (t= 0) 6m = U U 1). Mannitl, xylitl and trehalse were als selected because f the psitive effects they have n thermal stability, and als because they are reliable cryprtectants agents and effective in cntrlling smtic pressure. Other cnditins analyzed are summarized in Table 3. The dilutin 1:2 (v/v) in the 200 mm buffer resulted in a residual lyphilized enzyme activity ( R ) abut six times higher than the nn-lyphilized enzymatic slutin. This result cnfirms that highly cncentrated prtein slutins may prmte the frmatin f reversible agglmerates, resulting in a lwer apparent enzymatic activity. Nne f the lyphilized frmulated cnditins decreased enzymatic activity in cmparisn t the standard nn-lyphilized slutin (R 65 C < 1.0); n the ther hand, the lyphilized enzyme withut additives shwed lwer residual activity (Table 3). Thermal residual lyphilized activity (R 65 C ) shwed that the 1:6 (v/v) dilutin in 200 mm buffer was mre stable than the 1:2 (v/v) dilutin in the same buffer (Table 3), and was als mre stable than the lyphilized enzyme withut dilutin (Table 1); this reinfrces the need fr knwing the best initial dilutin rate f the enzyme slutin t avid frmatin f aggregates. The frmulatin (CMC + xylitl) at the dilutin f 1:6 (v/v) presented the highest thermal stability amng all tested cnditins (R 65 C = 0.92), prving that these tw additives are, as the literature review affirms, gd stabilizers f prtein slutins. The specific lyphilized enzyme activity ( * U ptn ) increased by almst 1.6 times with the lyphilizatin f an enzymatic slutin diluted at 1:2 (v/v) with a buffer cncentratin 4 times greater (200 mm) than the standard buffer (50 mm). ilizatin f the frmulatins with enzymatic dilutin f 1:6 (v/v) generally displayed the lwest values f * U ptn. FOS synthesis with the lyphilized frmulates revealed an increase in the cnversin f sucrse (Y FOS ) frm 0.41 t 0.62 fr the lyphilized enzymatic slutin diluted at 1:2 (v/v), and a reductin in cnversin frm 0.59 t 0.56 when using the lyphilized enzymatic slutin diluted at 1:6 (v/v). With the dilutin f 1:2 (v/v), the frmulatin [PEG + ammnium sulfate + mannitl] exhibited the highest cnversin (Y FOS = 0.63) amng all cnditins analyzed, with a ttal FOS cncentratin f 314.16 ± 6.89 g/l after 72 h f synthesis. With the dilutin f 1:6 (v/v), the lyphilized frmulates frm dilutins f
Aguiar-Oliveira and Maugeri 107 Figure 3a) FOS yield (Y FOS) and FOS specific prductivities (Pr FOS), and b) FOS percent cmpsitins frm syntheses carried ut with the lyphilized fructsyltransferase frm Rhdtrula sp. LEB-V10 at dilutin f 1:2 v/v (squares and filed lines) and 1:6 v/v (inverted triangles and dtted lines) in 200 mm sdium acetate buffer at ph 4.5. The lyphilized enzymes were stred at - 20 C fr 6 mnths and then FOS syntheses were carried ut under at 50% (w/v) f sucrse, in 50 mm sdium acetate buffer, ph 4.5, 50 C and 10 U ml. 1:6 (v/v) revealed similar cnversins f abut 0.57. Despite the increase in Y FOS, the specific prductivity f FOS (Pr FOS ) was reduced after lyphilizatin by abut 4 times with the enzymatic slutin diluted at 1:2 (v/v) and 8 times at a dilutin f 1:6 (v/v). Frmulatins prepared with a dilutin f 1:2 (v/v) displayed Pr FOS values 2 times greater than with the 1:2 dilutin f the standard lyphilized enzyme; the same was bserved with frmulatins at a dilutin f 1:6. In cmparisn with the FOS specific prductivities with the individual additin f cmpunds (Table 1), the frmulatins resulted in lwer values (Table 3). As reprted by several authrs, these results suggest that lyphilizatin withut any additive and in higher dilutins favr the vulnerability f the enzyme, and it may induce changes in its structure that decrease its cnversin rate, just like the excessive number f additives in a frmulatin may equally affect the enzyme. Fr a better understanding f the effects f lyphilizatin and frmulatins, it wuld be necessary t perfrm specific studies regarding the impact f lyphilizatin n the enzymatic mlecular cnfrmatin, as mentined by Oetjen and Haseley (2004). Accrding t Kaplan and Taralp (1997), sme physicchemical studies shw that smetimes the structure after lyphilizatin can be very similar t that in slutin, r can present sme limited but reversible cnfrmatinal changes; hwever, in several studies it is stated that the majrity f enzymes retain their native structure after lyphilizatin in rganic slvents. Shelf-life after 6 mnths The analyses f the lyphilized frmulatins after 6 mnths f strage at -20 C fllwed the same prcedures used fr the single additives. All lyphilized frmulatins shwn in Table 3 were evaluated accrding t their residual strage activity ( R 6 m ), as presen- ted in Table 4. The standard lyphilized material withut additives, diluted in 200 mm acetate buffer at 1:2 and 1:6 (v/v), shwed a reductin in enzymatic activity f abut 35 and 14%, respectively. Cnsidering the 1:6 dilutin in acetate buffer at 50 mm (Table 2) and 200 mm (Table 2 and 5), it can be bserved that the residual activity f the stred lyphilized enzyme increased by abut 5% after 6 mnths. Amng the frmulatins with the enzyme diluted at 1:2, nly the frmulatin cntaining [PEG + ammnium sulfate + trehalse] shwed a slightly smaller lss f activity ( R 6m = 0.77) cmpared t the lyphilized enzyme withut additives and diluted at 1:2; amng the frmulatins with the enzyme diluted at 1:6 (v/v), that cntaining [CMC + xylitl] maintained the highest
108 Int. Res. J. Bitechnl. Figure 4. FOS syntheses as a functin f reactin time: a) FOS yield (Y FOS) and specific prductivity f FOS (Pr FOS), and b) percent cmpsitins f FOS with nn-lyphilized fructsyltransferase frm Rhdtrula sp. LEB-V10 (squares and filed lines) and lyphilized at a 1:2 dilutin rati, in 200 mm sdium acetate buffer, ph 4.5 (inverted triangles and dtted lines). The syntheses were perfrmed under ptimized cnditins: 48 C, 10 U /ml, 50% (w/v) f sucrse in 50 mm sdium acetate buffer, ph 6.0. 6 m enzymatic activity ( R = 0.90). The standard lyphilized enzyme (withut additives) shwn in Table 4, diluted in sdium acetate buffer (200 mm and ph 4.5) at 1:2 and 1:6 (v/v), were used in FOS syntheses t evaluate their bicatalytic activity after 6 mnths f strage: the enzyme activities were 10 U. ml. Figure 3 shws the results fr Y FOS and Pr FOS alng with the percent cmpsitins f each FOS. Again, the tw cnditins f lyphilized enzymes shwed very similar Y FOS and Pr FOS prfiles ver time; the Y FOS values increased ver time, reaching 0.62 (dilutin 1:2) and 0.63 (dilutin 1:6) at the end f 72 h, and the Pr FOS reached its maximum at 3 h (~14 mg/u.h). The specific prductivity f the lyphilized enzyme diluted in 50 mm buffer (Figure 2.a) and 200 mm buffer (Figure 3a) revealed that with a higher cncentratin f the buffer Pr FOS values dubled in the first hurs f synthesis; sucrse cnversin values (Y FOS ) did nt reach a maximum value as shwn in Figure 2. Syntheses carried ut with the standard lyphilized enzyme diluted at 1:2 and 1:6 (v/v) shwed much lwer GF 4 cmpsitins (less than 8% after 72 h in bth cases), in which nystse (GF 3 ) was the predminant FOS cmpnent, cnsisting f nearly 50% f the glbal cmpsitin (Figure 3b). Effect f a pre-activatin f the enzyme prir t lyphilizatin In a previus study, Aguiar-Oliveira and Maugeri (2011) reprted that when the immbilized fructsyltransferase was incubated at ph 4.5 and 6.0 fr 15 min at 52 C and 60 C, and when the free fructsyltransferase was incubated at ph 4.5 and 52 C fllwed by a 5 min ice bath, different levels f enzymatic activatin were btained. Thus, pre-incubatin f the free enzyme at bth temperatures (52 and 60 C) prir t lyphilizatin was perfrmed in rder t determine the effect f such a treatment n the lyphilized material. An enzymatic slutin f 107.06 ± 4.00 U /g in acetate buffer, 50 mm and ph 4.5, was used fr lyphilizatin withut preincubatin (standard cnditin) and with pre-incubatin at 52 and 60 C. The results shwed that pre-incubatin at 52 C reduced the residual activity f the lyphilized enzyme ( R ) t 0.56 ± 0.09, and interestingly at 60 C this value increased t 1.43 ± 0.18, which is a peculiar result since the 60 C treatment led t the activatin f nly the immbilized frm (Aguiar-Oliveira and Maugeri, 2011). Hwever, with respect t thermal residual activity f the lyphilized enzyme (R.65 C ) and FOS yield (Y FOS ), the results cnfirmed that pre-activatin fllwed by lyphilizatin f the enzyme is nt a gd methdlgy: R.65 C was 0.17 at 52 C and 0.06 at 60 C, and Y FOS
Aguiar-Oliveira and Maugeri 109 values were 0.39 and 0.02, respectively. The preactivatin previusly bserved (Aguiar-Oliveira and Maugeri, 2011), suggests a mmentary alteratin n the cnfrmatinal structure f the mlecule and/r n its energetic levels. Effect f ilizatin n FOS Synthesis In previus studies, (Aguiar-Oliveira et al., 2012) ptimized FOS synthesis with the immbilized fructsyltransferase, whse ptimal cnditins are: 48 C, 20 U i /ml, 24 h and 50% f sucrse in 50 mm sdium acetate buffer and ph 6.0. The perfrmance f free fructsyltransferase was als evaluated under these same cnditins, except fr the enzymatic cncentratin which was 10 U /ml. Therefre, in this wrk FOS was prduced under the cnditins mentined abve with nn-lyphilized enzymes, the latter at a 1:2 dilutin rati in 200 mm sdium acetate buffer at ph 4.5. The results are shwn in Figure 4. In Figure 4.a it can be bserved that the lyphilized enzyme led t Y FOS values f 0.68-0.69 fr reactin times between 24 and 48 h, fllwed by gradual decreases; fr the nn-lyphilized enzyme similar Y FOS values were btained ver a lnger reactin time, between 48 and 72 h. Hwever, the specific prductivities are quite similar in bth cases. Regarding the percent cmpsitin f FOS (Figure 4.b) there is a significant difference, since with the exceptin f GF 2 cntents, the cmpsitins changed at the end f the prcess mre drastically fr GF 4, where the lyphilized enzyme led t abut 67% f the ttal FOS, nearly 3 times mre than the nn-lyphilized enzyme. Studies cnducted with a fructsyltransferase frm Bacillus macerans EG 6 (Taralp and Kaplan, 1998) demnstrated that this enzyme was able t prduce nly the GF 4 frm 500 g/l f sucrse, reaching a maximum f 45.2% f GF 4 after 48 h f synthesis. Brle and Davisn (2008) bserved a 6 t 7-fld increase in the initial rate f cnversin with a salt-lyphilized lipase used in an rganic medium. Mrever, Wang and Wei (2007) als btained the same results with a lyphilized lipase in the presence f certain additives, shwing increases ranging frm 1.2 t 1.7 times the standard cnditin. CONCLUSIONS ilizatin f the free fructsyltransferase partially purified frm Rhdtrula sp. LEB-V10 prved t be an apprpriate methdlgy fr acquiring a stable enzyme with gd transfructsylatin activity and increased enzymatic activity and thermal stability. The density f the initial enzyme slutin prved t be an imprtant factr t take int cnsideratin, since the prpsed bitechnlgical applicatin f this enzyme is in a partially purified frm, thus the ideal dilutin f the starting enzymatic slutin is imprtant in rder t avid hard enzyme agglmerates after lyphilizatin. The mre liquid and unifrm the appearance f the starting enzymatic slutin, the better the characteristics f the lyphilized cake, making it easier t btain a hmgeneus pwder which facilitates recnstitutin. Many authrs suggest that the frmulatins fr lyphilizatin shuld be kept as simple as pssible, and that the additin f single cryprtectant cmpunds allw the maintenance f enzymatic activity fr up t 6 mnths, better than in frmulatins. Changing the cncentratin f the buffer usually emplyed with fructsyltransferase frm Rhdtrula sp. LEB-V10 t a value 4 times higher als prved t be favrable since mre cncentrated buffers are cmmnly utilized in prtein lyphilizatin. ilizatin f the free fructsyltransferase did nt affect its transfructsylatin activity r prductin f fructligsaccharides, where this technique psitively cntributed t the industrial applicatins f the enzyme by facilitating its transprt and strage, withut affecting its functinality. Althugh a reductin in enzyme activity after lyphilizatin was bserved, the final lyphilized pwder still presents advantages including increased enzyme activity per weight and reduced vlume. Additinally, there was a significant change after lyphilizatin in which there was an increase in 1 F -fructfuransylnystse (GF 4 ) cmpsitin with nly the lyphilized enzyme f between 67 and 69% f the ttal FOS cmpared t the typical cmpsitins f 10 t 20% with the nn-lyphilized enzyme. The best results f FOS prductin were bserved with the additives: PEG 6000, ethylene glycl, ammnium sulfate, CMC and mannitl; fr the lyphilized enzyme with additive frmulatins, the best result was btained with a mixture f PEG, ammnuim sulfate and mannitl. ACKNOWLEDGMENT The authrs wuld like t thanks the Fundaçã de Ampar à Pesquisa d Estad de Sã Paul (FAPESP) fr financial supprt. REFERENCES Aehle W (1990). Catalytic Activity f Enzymes. In Enzymes in Industry: Prductin and Applicatin, 2 nd Ed.; Wiley VCH: The Netherlands. 13-34. Aguiar-Oliveira E, Maugeri F (2010). Characterizatin f Immbilized