Enzymatic activity and conformational and morphological studies of four commercial lipases treated with supercritical carbon dioxide

1 Ti le Enzyma ic ac i i y and con o ma ional and mo phological s udies o ou comme cial lipases ea ed wi h supe c i ical ca bon dioxide Au ho names Melgosa, R. ( m[email p o ec ed]) Sanz, M. T. ([email p o ec ed]) Bel án, S. ([email p o ec ed]) Solaesa, A. G. ([email p o ec ed]) Bucio, S.L. ([email p o ec ed]) A ilia ion Depa men o Bio echnology and Food Science (Chemical Enginee ing Sec ion), Uni e si y o Bu gos, 09001 Bu gos, Spain Co esponding au ho Sanz, M. Te esa Depa men o Bio echnology and Food Science (Chemical Enginee ing Sec ion), Uni e si y o Bu gos, Pza. Misael Bañuelos s/n 09001 Bu gos, Spain Tel.: +0034 947 258810. Fax: +0034 947 258831. E-mail add ess: e [email protected] 2 Abs ac This wo k in es iga es he e ec o supe c i ical ca bon dioxide (SC-CO2) ea men on ou comme cial lipases. The in luence o expe imen al condi ions: empe a u e (35 – 70 ºC), p essu e (100 – 250 ba ), exposu e ime (60 – 360 min) and dep essu iza ion cycles (1 – 3) on he esidual ac i i y was s udied. Ac i i y enhancemen was e i ied o ee enzymes (Pala ase 20000 L and Lipozyme CALB L) ea ed unde mild condi ions; while he highes empe a u e and p essu e and he longes exposu e ime assayed led o ac i i y losses. On he o he hand, ac i i y losses we e obse ed in he enzyme ac i i y o immobilized enzymes (Lipozyme RM IM and Lipozyme 435). Addi ional quali a i e s udies we e pe o med: Fluo escence emission spec a showed changes in he con o ma ional s uc u e o bo h wo ee enzymes a e SC-CO2 ea men . Scanning elec on mic og aphs showed mo phological al e a ions in he immobiliza ion suppo s o he ea ed enzymes; while in a ed spec a did no show signi ican chemical modi ica ions. Keywo ds: Candida an a c ica lipase B; Rhizomuco miehei lipase; Immobilized/ ee lipases; Enzyme ac i i y; Supe c i ical ca bon dioxide 1. In oduc ion The u iliza ion o enzymes as bioca alys s in many p ocesses, ei he in hei ee o immobilized o m, has become an inc easingly impo an esea ch ield in ecen yea s. Conside able a en ion has been paid o lipases, due o hei high speci ici y in lipid biomodi ica ion and hei selec i i y owa ds a y acid posi ions on he glyce ol backbone [1]. 3 Since he wo k o Zaks and Klibano [2], o ganic sol en s ha e been employed ex ensi ely in enzyma ic eac ions. Howe e , hey p esen se ious en i onmen al d awbacks and addi ional pu i ica ion s eps a e needed o ob ain he p oduc s o in e es in a sa e o m. To cope wi h hese issues, biochemical ca alysis wi h lipases can be conduc ed in supe c i ical ca bon dioxide (SC-CO2), a non oxic, eadily a ailable, inexpensi e, and easily emo able sol en . In addi ion, he singula p ope ies o supe c i ical luids, such as liquid-like and unable sol a ing powe , and gas-like iscosi y, di usi i y and su ace ension, ha e a posi i e e ec on he eac ion pe o mance [3]. Addi ionally, ac iona ion o he eac ion p oduc s is possible by coupling a se ies o sepa a o s a e he eac o essel. The s udy o enzyme deac i a ion o he imp o emen o enzyme s abili y is impo an in he implemen a ion o bioca alysis in supe c i ical sys ems. Ac i i y changes can be obse ed when enzymes a e ea ed wi h SC-CO2. In he case o immobilized enzymes, enzyme s abili y is ela ed o many ac o s, including he sou ce and na u e o he enzyme, he cha ac e is ics o he suppo , and he immobiliza ion me hod. Essen ial wa e in he enzyme mic oen i onmen can be emo ed as a esul o un a o able pa i ioning be ween he suppo and he sol en , causing enzyme inac i a ion [4–6]. O he pa ame e s du ing SC-CO2 ea men , such as p essu e and empe a u e o he sys em, can also a ec he enzyme ac i i y. In high p essu e-ba ch s i ed eac o s, exposu e ime, dep essu iza ion a e, and he numbe o p essu iza ion/dep essu iza ion cycles a e also impo an pa ame e s ha mus be conside ed [7]. F ee enzymes ea ed wi h SC-CO2 ha e been also s udied. They a e less expensi e han immobilized enzymes and, since p o eins a e no soluble in SC-CO2, ee enzymes can be mo e easily sepa a ed om he eac ion p oduc s han in con en ional bioca alysis. 4 O he eason is ha he enzyma ic ac i i y can be imp o ed when ee enzymes a e exposed o SC-CO2 media. Al hough some esea che s epo ed ha ce ain ee enzymes can be inac i a ed when exposed o SC-CO2, mainly because o a pH dec ease [8], o he ee enzymes ha e been epo ed o inc ease hei ac i i y a e incuba ion in SC-CO2 (up o 760% esidual ac i i y, depending on he na u e o he enzyme) [9]. Changes in he con o ma ional s uc u e due o enzyme esidues and SC-CO2 in e ac ions [10–14], and/o ex ac ion o wa e and impu i ies om he enzyme p epa a ion [15] could explain his enzyme ac i i y enhancemen . This wo k in es iga es he in luence o empe a u e, p essu e, exposu e ime and dep essu iza ion cycles on he ac i i y o ou comme cial lipases ea ed wi h SC-CO2: wo immobilized lipases (Lipozyme RM IM and Lipozyme 435); and wo ee lipases (Pala ase 20000 L and Lipozyme CALB L). The aim o his s udy is o help o unde s and he e ec s o SC-CO2 exposu e on he ac i i y o di e en comme cial enzymes, as well as o help o selec he bes condi ions o ca y ou u he enzyma ic eac ions unde SC-CO2 medium. The analysis includes s udies o he possible chemical, mo phological and con o ma ional modi ica ions caused by SC-CO2 ea men . Fluo escence emission spec oscopy was applied in o de o s udy he con o ma ional changes ha could ha e occu ed in he s uc u e o he ee enzymes. Fou ie ans o m-in a ed spec oscopy (FT-IR) and scanning elec on mic oscopy (SEM) we e also used o e alua e he possible chemical and physical al e a ions in he immobilized enzymes, espec i ely. 5 2. Ma e ials and Me hods 2.1. Enzymes and chemicals Lipozyme RM IM and Pala ase 20000 L we e pu chased om Sigma Ald ich (S . Louis, MO). Lipozyme 435 and Lipozyme CALB L we e kindly p o ided by No ozymes (Bags æ d, Denma k). Lipozyme RM IM is a lipase om Rhizomuco miehei, immobilized on Duoli e A568 [16], a mac o-po ous hyd ophilic g anula weak base anion exchange esin, based on c osslinked phenol- o maldehyde polycondensa e. The mois u e con en o Lipozyme RM IM was 3.8 ± 0.2 g/100 g, de e mined by ex ac ion wi h d y me hanol du ing 24 h and u he i a ion by Ka l-Fische me hod (Mi subishi CA-20 au oma ic i a o ). A simila alue (3.3 ± 0.2 g/100 g) was epo ed by Jenab e al. [17] by using a g a ime ic me hod. Lipozyme 435 is a ecombinan lipase om Candida an a c ica, exp essed on Aspe gillus nige , and adso bed on o Lewa i VP OC 1600 [16], a mac o-po ous hyd ophobic esin p esen ed in sphe ical beads and based on c oss-linked me hac ylic es e s. The mois u e con en o Lipozyme 435 was 0.7 ± 0.2 g/100 g, de e mined by he same Ka l-Fische i a ion me hod. Pala ase 20000 L ( ee R. miehei lipase exp essed on A. o yzae) and Lipozyme CALB L ( ee C. an a c ica lipase exp essed on A. nige ) we e p o ided in aqueous solu ion con aining glyce ol, so bi ol and o he excipien s and p ese a i es. Ca bon dioxide (99.9%) was supplied by Ca bu os Me álicos S.A. (Spain). All o he chemicals used we e o analy ical g ade. 6 2.2. Enzyme ea men unde SC-CO2 A schema ic diag am o he expe imen al appa a us used in he enzyme ea men is depic ed in Figu e 1. Basically, i consis s o a CO2 ese oi , a high p essu e sy inge pump wi h a p essu e con olle (ISCO 260 D) and 3 high p essu e cells imme sed in a he mos a ic wa e ba h. In a ypical expe imen , he enzyme p epa a ion was cha ged in o he high p essu e cell, which was hen placed in he he mos a ic wa e ba h a he es ablished empe a u e. A e wa ds, he sys em was p essu ized and main ained a cons an empe a u e and p essu e o a p e-es ablished exposu e ime. Typically, he du a ion o he p essu iza ion s ep was less han 0.5 min and acco dingly was no included in he p essu e holding ime. Dep essu iza ion s eps we e pe o med a a cons an decomp ession a e o 240 kg m-3 min-1. Expe imen s we e done in a empe a u e and p essu e ange commonly used in enzyma ic eac ions: empe a u e (T) om 35 o 75 ºC and p essu e (p) om 100 o 250 ba . Exposu e ime ( ) was ex ended om 1 o 6 h o all enzymes. Addi ionally, se e al dep essu iza ion cycles (1 – 3) we e ca ied ou in he case o immobilized enzymes (see Table 1). 2.3. Residual enzyme ac i i y The enzyme ac i i y o ee enzymes a e SC-CO2 ea men was de e mined as he ini ial a e in he hyd olysis eac ion o he oli e oil iglyce ides [12]. In a ypical assay, 1 mL o enzyme p epa a ion was added o he subs a e, consis ing on 4 mL o 10 % homogenized oli e oil and 5 mL o 50 mM phospha e bu e pH = 7.0. The eac ion was ca ied ou a 50 ºC o 15 min. A e incuba ion, 15 mL o a mix u e o e hanol:ace one (1:1) was added o s op he eac ion. Libe a ed a y acids we e i a ed wi h KOH 0.1 N 7 in e hanol. As a blank con ol, he eac ion mix u e wi hou he enzyme was i a ed in he same way. The enzyme ac i i y o immobilized enzymes a e SC-CO2 ea men was de e mined as he ini ial a e in he es e i ica ion eac ion o lau ic acid wi h p opanol a a mola a io o 3:1, 60 % w . hexane as eac ion medium and enzyme concen a ion o 5 % w . based on he subs a es. A he beginning o he eac ion, samples con aining he mix u e o lau ic acid and p opanol we e collec ed and he lau ic acid con en was de e mined by i a ion wi h KOH 0.1 N in e hanol by using an au oma ic i a o (Me h om Ti ando 905). A e he addi ion o he enzyme o he subs a es, he mix u e was kep a 50 ºC o 15 min. Then, he lau ic acid consump ion was de e mined by he same expe imen al p ocedu e. In all cases, esidual ac i i y was calcula ed as he ela ionship be ween he enzyme ac i i y a e SC-CO2 exposu e and he ini ial enzyme ac i i y, exp essed as pe cen age: Residual ac i i y =Ac i i y a e SC −CO2 ea men Ac i i y o he un ea ed enzyme ·100 % All enzyme ac i i y de e mina ions we e pe o med a leas in iplica e. 2.4. Enzyme assays The e ia y s uc u e o he ee enzymes was measu ed by luo escence spec oscopy using a Va ian Ca y Eclipse spec o luo ime e (Agilen Technologies) he mos a ed a 25 ºC. The exci a ion wa eleng h was 280 nm, and he emission was ead a 290 – 450 nm. All he spec a we e scanned con inuously wi h i e eplica es. All samples we e dilu ed 10 imes in pu e wa e p io o analysis. In a ed spec oscopy analyses we e made o ollow possible chemical al e a ions a e exposu e o SC-CO2 since p o eins abso b in a ed wa eleng hs due o he pep ide bond 8 ib a ions. Fou ie ans o m-in a ed spec oscopy (FT-IR) was pe o med in he 4000 – 400 cm-1 ange using a The mo-Nicole Nexus 670 FT-IR spec opho ome e . Scanning elec on mic oscopy (SEM) was pe o med o check possible changes in he mo phological p ope ies o he immobilized enzymes. Mic og aphs we e ob ained using a a iable-p essu e scanning elec on mic oscope JEOL JSM-6460LV. 3. Resul s and discussion 3.1. F ee enzymes 3.1.1. Residual ac i i y The expe imen al esul s ob ained in he esidual ac i i y de e mina ion o he wo ee enzymes a e compiled in Table 1. The e ec o p essu e, empe a u e and exposu e ime is p esen ed in Figu es 2 a,b,c. An inc ease in he esidual enzyme ac i i y o he wo ee lipases was obse ed when SC-CO2 ea men s we e pe o med a mild condi ions (exp. 1, 2, 4, and 6). In any case, he inc ease in he enzyme ac i i y a e SC-CO2 ea men is lowe he highe he ope a ing p essu e and empe a u e, as well as he exposu e ime. T ea men s a he highes p essu e s udied in his wo k (exp. 3) esul ed in low ac i i y losses o bo h wo ee enzymes (up o app oxima ely 96 % ini ial ac i i y). Pala ase 20000 L gained ac i i y e en a e being exposed o high exposu e ime (exp 7) while sligh ac i i y loss was obse ed o Lipozyme CALB L. In gene al, esul s ob ained in Lipozyme CALB L esidual ac i i y de e mina ion a e in ag eemen wi h hose epo ed in he li e a u e s udying he same enzyme [12]. Howe e , expe imen al condi ions in he wo k by Liu e al. (6-10 MPa, 35-40 ºC and 20-30-150 min) [12] p omo ed sligh ly lowe ac i i y enhancemen s han he obse ed 9 in his s udy, sugges ing ha expe imen al condi ions, i.e. p essu e and empe a u e o he sys em, as well as exposu e ime, should no be neglec ed. The highes ac i i y imp o emen was ound when ea ing Pala ase 20000 L a mild condi ions (maximum o 134.8 % ini ial ac i i y a exp. 6). No e e ences abou he e ec o he SC-CO2 ea men on Pala ase 20000 L ac i i y we e ound in he a ailable li e a u e. The highes ac i i y losses (86.3 o Pala ase 20000 L and 80.9 % ini ial ac i i y o Lipozyme CALB L) we e ound a he highes empe a u e assayed (exp. 5). The e o e, hey could be mainly a ibu ed o he mal deac i a ion, possibly due o pa ial un olding by b eaking non-co alen in e ac ions [13]. In any case, he esidual ac i i y o bo h ee ea ed lipases emains abo e 80 % a he highes empe a u e s udied in his wo k (expe ience 5, 70 ºC). The mos he mos able lipase was Pala ase 20000 L (86.3 % ini ial ac i i y a 70 ºC). Gieβau and Gamse [15] epo ed ha he mal s abili y o ee enzymes depends on many ac o s such as he p esence o uns able impu i ies o s abilize s. Acco dingly, Baue e al. [18] ela ed he ac i i y loss o es e ase EP10 om Bu kholde ia gladioli a e SC-CO2 ea men a 75 ºC o he p esence o impu i ies in he enzyme p epa a ion. In he li e a u e, i has been epo ed ha SC-CO2 could imp o e enzyme ac i i y and s abili y by solubiliza ion and emo al o many impu i ies like ca bohyd a es, a y acids, and iglyce ides ha can be p esen in ee enzyme p epa a ions while he enzyme is gene ally insoluble in SC-CO2 [15]. To e alua e his e ec , p o ein concen a ion was de e mined by he B ad o d me hod [19], inding ha p o ein concen a ion was no signi ican ly a ec ed (p < 0.05) by he SC-CO2 ea men in any o he expe iences pe o med wi h bo h ee enzyme p epa a ions. These esul s we e expec ed since wa e solubili y in SC-CO2 is low a he p essu e and empe a u e 16 was added (a ound 0.5 – 1 % w . based on enzyme). This beha io was no obse ed on Lipozyme 435. The enzyme suppo o Lipozyme RM IM is hyd ophilic and some wa e can be easily adso bed; howe e , he hyd ophobic cha ac e o he immobiliza ion suppo o Lipozyme 435 migh no allow he enzyme o eco e i s cons i u ion wa e , showing an ini ial pla eau. In bo h enzymes, la ge amoun s o added wa e esul ed in he dec ease o he enzyma ic ac i i y. Since wa e is a eac ion p oduc , i can be assumed ha his excess o ee wa e could slow down he eac ion a e. Con o ma ional changes could also ake place a high wa e concen a ions [33] and, in he case o Lipozyme RM IM, mass ans e limi a ions could ha e occu ed due o wa e adso p ion on he hyd ophilic immobiliza ion suppo . 3.2.3. Con o ma ional and mo phological changes The con o ma ional and mo phological s uc u es o SC-CO2- ea ed and un ea ed immobilized lipases we e also in es iga ed by FT-IR and SEM, espec i ely. In a ed spec a FT-IR analysis o he un ea ed and SC-CO2- ea ed immobilized lipases was pe o med in he ange be ween 4000 and 400 cm-1 (Fig. 6). In he p o ein FT-IR spec a, he majo p o ein abso p ion bands due o he pep ide g oup ib a ions occu be ween 1900 and 1200 cm-1 [34]. Di e en bands can be obse ed in his egion: he amide I band (be ween 1700 and 1600 cm-1) is mainly associa ed wi h ca bonyl s e ching o he pep ide. I consis s o a g oup o o e lapped signals, p o iding in o ma ion abou he seconda y p o ein s uc u e o he enzyme; he amide II band (1580-1510 cm-1) is due o he N-H bending wi h a con ibu ion o he C-N s e ching ib a ions and he amide III egion (1400-1200 cm-1), which has a weake in ensi y [34,35]. The e o e, he spec a 17 da a be ween 1800 and 1500 cm-1 we e aken and plo ed in u he de ail a e baseline co ec ion and no maliza ion. I was ound ha he e was no signi ican di e ence be ween he spec a o he un ea ed immobilized lipases and he spec a o he ones exposed o SC-CO2. These esul s a e in coincidence wi h he wo ks by Oli ei a e al. [6] and Jenab e al. [17] and, based on hem, no signi ican con o ma ional changes in he seconda y s uc u e o he immobilized lipases we e obse ed a e enzyme ea men wi h SC-CO2. Scanning Elec on Mic oscopy (SEM) Scanning Elec on Mic oscopy (SEM) was used o in es iga e any po en ial mo phological changes in bo h immobilized lipases. Mic og aphs om Lipozyme RM IM and Lipozyme 435 a e shown in Figu es 7 and 8, espec i ely. Immobiliza ion suppo o Lipozyme RM IM was also s udied (Fig. 7d). Some au ho s epo ed ha SC-CO2 can in e ac wi h he enzyme suppo , esul ing in de o ma ions such as plas iciza ion and swelling [36]. In some cases, apid dep essu iza ion can lead o he o ma ion o c acks and holes in he suppo su ace and inc eased po osi y [6]. Recen ly, Jenab e al. [17] ha e obse ed s uc u al changes in he SEM mic og aphs o Lipozyme RM IM and Lipozyme TL IM a e SC-CO2 ea men . Ne e heless, i was concluded ha such changes could be a ibu ed o mechanical s esses caused by magne ic s i ing du ing SC-CO2 exposu e and no o p essu iza ion/dep essu iza ion cycles [17]. In his s udy, ex e nal o bi al agi a ion was used in he SC-CO2 ea men o he immobilized enzymes ins ead o in e nal magne ic s i ing; he e o e, he suppo was no subjec ed o in ense mechanical s esses due o ha means o s i ing. Howe e , apid dep essu iza ion (240 kg CO2 m-3 min-1), and se e al dep essu iza ion cycles we e conduc ed. As a consequence, a ough and c acked 18 su ace wi h an appa en inc ease o po osi y can be app ecia ed a e SC-CO2 ea men in 10,000x magni ica ions o Figs. 7 b,c and 8 b,c. 4. Conclusions In he p esen wo k, ou comme cial lipases: ee Pala ase 20000 L and Lipozyme CALB L, and immobilized Lipozyme RM IM and Lipozyme 435, we e subjec ed o SC- CO2 ea men . Based on he esul s ob ained, i can be concluded ha SC-CO2 ea men a he expe imen al condi ions in his s udy can a ec he enzyme ac i i y by means o con o ma ional changes and s uc u al al e a ions. I is also impo an o no ice ha he e ec o he SC-CO2 ea men s ongly depends on he expe imen al condi ions, he na u e and he sou ce o he enzyme and, mainly, whe he he enzyme is p esen ed in a ee o immobilized o m. In he case o immobilized enzymes, he cha ac e is ics o he suppo should be also aken in o accoun . The esul s ob ained in his wo k may help wi h he pu pose o selec ing he mos app op ia e ope a ion condi ions a he ypical eac ions conduc ed in he bio ans o ma ion o lipids unde SC-CO2, wi h he lowes ac i i y loss, o , when possible, he highes ac i i y imp o emen . Acknowledgmen s To he Spanish Go e nmen h ough MINECO and he Eu opean Regional De elopmen Fund (ERDF) o inancial suppo o he p ojec CTQ2012-39131-C02- 01. To No ozymes A/S o kindly supplying he enzymes. RM acknowledges MINECO o a p edoc o al g an ( e e ence BES-2013-063937). SLB acknowledges Mexican Sec e a ia o Public Educa ion and Technological Uni e si y o Mo elia o a 19 ellowship h ough PROMEP p og am. AGS acknowledges Uni e si y o Bu gos o a ellowship. Re e ences [1] K.-E. Jaege , T. Egge , Lipases o Bio echnology, Cu en Opinion in Bio echnology 13 (2002) 390-397. [2] A. Zaks, A.M. Klibano , Enzyme-ca alyzed p ocesses in o ganic sol en s, P oceedings o he Na ional Academy o Sciences o he Uni ed S a es o Ame ica 82 (1985) 3192-3196. [3] K. Nakamu a, Biochemical eac ions in supe c i ical luids, TibTech 8 (1990) 288-292. [4] M.C. Almeida, R. Rui o, C. Maia, L. F ei e, T. Co êa de Sampaio, S. Ba ei os, No ozym 435 ac i i y in comp essed gases. 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E ec o he expe imen al condi ions o he SC-CO2 ea men on he esidual ac i i y o he s udied lipases. a) E ec o p essu e (100 – 250 ba : exp1-exp3); b) E ec o empe a u e (35 – 70 ºC: exp 2, exp4 and exp5); c) E ec o exposu e ime (60 – 360 min: exp6, exp2 and exp7); d) E ec o dep essu iza ion cycles (1 – 3: exp 2, exp8 and exp9). Lines a e d awn o guide he eye. Fig. 3. Fluo escence emission spec a o he s udied ee lipases be o e and a e SC- CO2 ea men unde di e en expe imen al condi ions. Fig. 4. Lipozyme RM IM esidual ac i i y e sus wa e con en a e SC-CO2 ea men . Numbe s indica e es ablished condi ions in each expe imen (see Table 1). Fig. 5. Residual ac i i ies o he s udied immobilized lipases a e SC-CO2 ea men and di e en amoun s o wa e added. Emp y symbols: un ea ed samples; ull squa es: ea ed samples om exp.2. Dashed lines indica e he esidual ac i i y alue o each ea ed lipase (exp. 2) when no wa e was added. Fig. 6. FT-IR spec a o he s udied immobilized lipases be o e and a e SC-CO2 ea men . Le : comple e IR spec a (4000 – 400 cm-1); igh : de ail o he 1800 – 1500 cm-1 egion. T ea ed samples om exp. 2. 25 Fig. 7. SEM mic og aphs o Lipozyme RM IM. Le o igh : 50 x, 600 x and 10,000 x. (a) un ea ed enzyme; (b) ea ed sample om exp. 3; (c) ea ed sample om exp. 9; (d) immobiliza ion suppo alone (Duoli e A568). Fig. 8. SEM mic og aphs o Lipozyme 435. Le o igh : 50 x, 600 x and 10,000 x. (a) un ea ed enzyme; (b) ea ed sample om exp. 3; (c) ea ed sample om exp. 9. 32 Fig. 6. FT-IR spec a o he s udied immobilized lipases be o e and a e SC-CO2 ea men . Le : comple e IR spec a (4000 – 400 cm-1); igh : de ail o he 1800 – 1500 cm-1 egion. T ea ed samples om exp. 2. 33 Fig. 7. SEM mic og aphs o Lipozyme RM IM. Le o igh : 50 x, 600 x and 10,000 x. (a) un ea ed enzyme; (b) ea ed sample om exp. 3; (c) ea ed sample om exp. 9; (d) immobiliza ion suppo alone (Duoli e A568). 34 Fig. 8. SEM mic og aphs o Lipozyme 435. Le o igh : 50 x, 600 x and 10,000 x. (a) un ea ed enzyme; (b) ea ed sample om exp. 3; (c) ea ed sample om exp. 9.