KINETICS OF HYDROLYSIS OF TRIBUTYRIN BY LIPASE

Transcription

1 Journal of Engineering Science and Technology Vol. 1, No. 1 (26) 5-58 School of Engineering, Taylor s College KINETICS OF HYDROLYSIS OF TRIBUTYRIN BY LIPASE SULAIMAN AL-ZUHAIR School of Chemical Engineering, Faculy of Engineering, he Universiy of Noingham Malaysia Campus, 435 Semenyih, Malaysia alzuhair.sulaiman@noingham.edu.my Absrac Kineics of he enzymaic hydrolysis of ribuyrin using lipase has been invesigaed. The iniial rae of reacion was deermined experimenally a differen subsrae concenraion by measuring he rae of buyric acid produced. Michaels-Menen kineic model has been proposed o predic he iniial rae of hydrolysis of ribuyrin in micro-emulsion sysem. The kineic parameers were esimaed by fiing he daa o he model using hree mehods, namely, he Lineweaver-Burk, Edie-Hofsee and Hanes mehods. The Michaels-Menen model wih he consan prediced by Edie- Hofsee and Hanes mehods prediced he iniial rae of reacion a various subsrae concenraions beer han he model wih he consan prediced Lineweaver-Burk mehod, especially a high subsrae concenraions. Keywords: Lipase hydrolysis, Tribuyrin, Michaelis-Menen kineics. 1. Inroducion The applicaions, imporance and significance of lipase in oleochemical indusry have been horoughly demonsraed in lieraure [1-4]. The mos imporan among hese applicaions is he use of lipase for he producion of fay acids from oils. I is recenly aemped as an energy-saving mehod, especially for producing high valueadded producs or hea sensiive fay acids [1-4]. 5

2 Kineics of Hydrolysis of Trbuyrin by Lipase 51 Nomenclaures a Toal specific inerfacial area [m -1 ] [E] Free enzyme concenraion [klu m -3 ] [E] Toal acive enzyme concenraion [klu m -3 ] k ca Caalyic rae consan [min -1 ] kd Desorpion rae consan [min -1 ] kp Adsorpion rae consan [m 2.min -1 ] Ke Equilibrium consan of ES [mol m -3 ] Ki Produc inhibiion consan [mol m -3 ] LU Lipase Uni [P] Produc concenraion [mol m -3 ] [S] Subsrae concenraion [mol m -3 ] Greek Symbols υ Reacion rae [mol m -3 min -1 ] Lipase caalysed reacions ake place a he inerface beween he aqueous phase conaining he enzyme and he oil phase, where he enzyme has o penerae he inerface as a firs sep in he reacion [1,2]. Al-Zuhair e al. [1] derived a mahemaical model o predic he hydrolysis rae of oils by lipase, from a proposed kineic mechanism of he reacion, assuming low enzyme concenraion (Eq.1). kca υ = kd K e 2 k p a [ E] [ S] [ S] Where he subsrae [S], represens he concenraion of eser bonds in he oil. Comparison of Eq(1) and he Michaelis-Menen kineic equaion (Eq.2) shows ha he K m in Eq(1) is no a consan and is a funcion of he oal specific inerfacial area, a. [ E] [ S] + [ S] kca υ = (2) K m In a mechanically sirred bi-phasic sysems, he oal free inerfacial area changes wih changes in operaing condiions, such as subsrae concenraion, agiaion speed and emperaure [1]. Tha is he reason behind he discrepancy found in previous models ha assumed Michaelis-Menen kineics in he predicion of he hydrolysis rae of oils by lipase in mechanically agiaed sysems [3]. However, oil-waer micro-emulsions, aided by emulsificaion reagen, are sable sysems wih much larger inerfacial area. This would resul in a consan oal free inerfacial area, independen on operaing condiions. Therefore, based in Eq (1), he Michaelis-Menen model could successfully be adoped o such sysems as found in he work of Knezevic e al [4] on he hydrolysis of palm oil in lecihin/isoocane reversed micelles. (1)

3 52 S. Al-Zuhair On he oher hand, Malcaa e al [5,6] proposed o describe he rae expression of he hydrolysis of oils by lipase by Ping-Pong-Bi-Bi mechanism. This leads o a rae expression associaed wih a Michaelis-Menen mechanism in he presence of produc inhibiion, (Eq. 3): [ E] [ S] [ P] + [ S] k ca υ = K m 1 + K i However, considering he iniial rae of reacion, he produc concenraion [P] is assumed o be negligible wih comparison o he iniial subsrae concenraion [S] and he K m value, hence, Eq (3) is simplified again o Eq (2). I is herefore clear ha he Michaels-Menen kineic model (Eq. 2) is appropriae o predic he iniial rae of hydrolysis of ribuyrin in micro-emulsion sysem. In his paper, experimenal daa are fied o he Michaelis-Menen model using hree mehods, namely, he Lineweaver-Burk, Edie-Hofsee and Hanes mehods. The model wih he consan prediced are validaed agains he experimenal resuls. (3) 2. Chemicals -1 Liquid lipase (EC ) from Mucor miehei (claimed aciviy 1 klu ml ) was obained from Novo Nordisk, Denmark. Analyical grade Tribuyrin (98%) was obained from Acros Organics, USA. NaOH was obained from Mallinckrod, Sweden. Gum Arabic, NaCl, KH 2 PO 4, and glycerol were obained from Fisher Scienific, UK. 3. Experimenal Procedure The mehod is based on he hydrolysis of ribuyrin by lipase, and iraing he buyric acids produced wih.5 NaOH in disilled waer [7]. The alkali consumpion was regisered as a funcion of ime a ph 7. using an auo iraor (Merohm 794 Basic Tirino, Swaziland). 3.1 Preparaion of emulsifying reagen 8.95 g NaCl and.2 g KH 2 PO 4 were dissolved in 2 ml demineralised waer wih 27 ml glycerol. Under vigorous sirring using homogeniser (X12, CAT Ingenieuburo, Germany) 3. g gum Arabic was added o he mixure. The homogeniser was capable of sirring up o 3, rpm. The mixure was hen ransferred o a 5 ml measuring flask and demineralised waer was added o make up he volume o 5 ml.

4 Kineics of Hydrolysis of Trbuyrin by Lipase Preparaion of subsrae emulsion Under homogeniser sirring of up o 3, rpm, 5 ml of emulsifying reagen was mixed wih 25 ml of ribuyrin-waer mixure having differen concenraions of ribuyrin. The concenraions used were prepared o give final eser bond concenraions, [S], of he following values: 6.8, 13.7, 2.5, 9.11, 27.3, 68.4, 12.6, and 171. mol m Deerminaion of he reacion rae Small samples of he subsrae emulsion having a volume of 2 ml were wihdrawn ino a small beaker, which was placed in he iraion se-up. The ph of he soluion was hen adjused o 7. ±.1 wih.5 N NaOH. 2 ml of enzyme soluion, having a concenraion of 1. LU ml -1 of enzyme, was added o he subsrae mixure o iniiae he reacion and he ph sa iraion was sared. The alkali addiion was carried ou for 5 minues. This reacion was slow enough o assume ha wihin he firs five minues he changes in he iniial concenraion of subsrae was negligible (i.e, small amouns of subsrae have been reaced). The rae of he alkali addiion was used o deermine he iniial reacion rae, using Eq (4). ml mol -3 L Slope Normaliy NaOH 1 mol min L ml Rae of reacion,υ ( ) = (4) 3 3 m min Volume of he sample ( m ) The abovemenioned procedure was repeaed en imes for each prescribed subsrae concenraion. The resuls are presened in Fig. 1, where he error bars give an indicaion of he accuracy and reproducibiliy of he resuls. 4. Validaions of Kineic Model Equaions The Michaelis-Menen in is original form (Eq.2), is no well suied o esimae he kineic parameers k ca and K m. By rearranging Eq (2) he following opions for daa ploing and graphical parameer evaluaion can be derived: 1 1 K m 1 = + (5) υ k ca [ E] k [ E] [ S] [ E] ca [ S] υ υ = k K (6) [ S] υ ca m K 1 = (7) k ca m + [ ] [ ] [ S] E k E ca

5 54 S. Al-Zuhair Iniial Rae of Reacion, υ (mol m -3 s -1 ) Tribuyrin Concenraion, [S] (mol m -3 ) Fig.1. The change of iniial rae of reacion a differen iniial subsrae concenraion, [S] Each equaion suggess an appropriae linear plo. In evaluaion of he kineic parameers of he model using such plos, however, several poins should be noed. Figure 5 shows he plo of Eq (5), as 1/ υ vs 1/[S] (known as a Lineweaver-Burk plo). Noe ha, he mos accurae rae values are hose a high subsrae concenraion. This is because a high subsrae concenraions, i is more accurae o assume he change in subsrae concenraion o be negligible during he course of he experimen. Fig. 2, however, shows he mos accurae rae values clusered near he origin, while hose less accurae rae values are far from he origin and dominanly deermine he slope K m / k ca[e]. Therefore, he value of K m deermined by his mehod is subjec o large errors. Subsiuing he value of enzyme concenraion used of 9. klu m -3, he Michaelis-Menen kineic model equaion wih he esimaed consans from Lineweaver-Burk plo is given by Eq (8): [ E] [ S] υ = ± S Figure 3 and 4 shows he plos of Eqs (6) and (7) (known as Edie-Hofsee and Hanes plos, respecively). The plos end o spread ou he daa poins for higher values of [S], so ha he slope, and hence he value of K m can be deermined accuraely. The Michaelis-Menen kineic model equaions wih he esimaed consans from Edie- Hofsee and Hanes plos are given by Eqs (9) and (1), respecively, (8)

6 Kineics of Hydrolysis of Trbuyrin by Lipase S [ E] [ S] -5 5 υ = ± [ E] [ S] υ = ± S 3 I is clearly seen ha he consans prediced by he Edie-Hofsee and Hanes mehods are very close, however, hose found by he Lineweaver-Burk mehod differs considerably. Figure 5 show he comparison beween he experimenal resuls and he Michaelis-Menen kineic equaion wih he esimaed consans Eqs (8-1), o measure how closely he experimenal resuls are presened. The figure shows ha he Michaels-Menen model curves, wih he consans deermined by he hree mehods, prediced fairly well he iniial rae of reacion a low subsrae concenraions. However, a high subsrae concenraion, he Lineweaver-Burk model curve deviaes from he experimenal daa and did no predic he subsrae sauraion. (9) (1) 1 8 y = 5742x /υ (m 3 s mol -1 ) /[S] (m 3 mol -1 ) Fig.2. The Lineweaver and Burk mehod, 1/υ (s m 3 mol -1 ) versus 1/[S] (m 3 mol -1 ) On he oher hand, he Edie-Hofsee and Hanes model curves followed beer he rend of he experimenal daa a high subsrae concenraions. The deviaion beween he experimenal daa and he model equaions can also be noiced from he absolue error shown in Eqs (8-1). The absolue error of he Lineweaver-Burk equaion is 1.4x1-4 mol m -3 s -1, hree imes higher han he absolue error of he Edie-Hofsee and Hanes equaions, 4.6x1-5 mol m -3 s -1. I can be concluded herefore, ha he Lineweaver-

7 56 S. Al-Zuhair Burk mehod is limied o low subsrae concenraions, in he regions prior o he subsrae sauraion..1.8 υ (mol m -3 s -1 ) y = x υ/[s] (s -1 ) Fig. 3. The Edie-Hofsee mehod, υ (mol m -3 s -1 ) versus υ/[s] (s -1 ) [S]/υ (s) y = x [S] (mol m -3 ) Fig. 4. The Hanes mehod, [S]/υ (s) versus [S] (mol m -3 )

8 Kineics of Hydrolysis of Trbuyrin by Lipase 57 Iniial rae of racion, υ (mol m -3 s -1 ) Experimenal resuls Edie-Hofsee and Hanes Lineweaver-Burk Subsrae concenraion, [S] (mol m -3 ) Fig. 5. Comparison beween he experimenal resuls and he Michaelis-Menen kineic equaion wih he esimaed consans Eqs (5-7) 5. Conclusions An experimen was conduced o deermine he iniial rae of enzymaic hydrolysis of ribuyrin in micro-emulsion sysem using lipase a differen subsrae concenraion. Michaels-Menen kineic model has been considered o predic he iniial rae of reacion. Lineweaver-Burk, Edie-Hofsee and Hanes mehods were used o esimae he kineic parameers of he Michaels-Menen model by fiing he experimenal daa. I was shown ha Edie-Hofsee and Hanes models prediced he iniial rae of reacion a various subsrae concenraions beer han he Lineweaver-Burk model, especially in he subsrae sauraion region. References 1. Al-Zuhair, S., Hasan, M. & Ramachandran, K.B. (23). Kineic Hydrolysis of palm Oil Using Lipase. Process Biochemisry. 38, Tsai, S.W. & Chang, C.S. (1993). Kineics of Lipase-Caalysed Hydrolysis of Lipids in Biphasic Organic-Aqueous Sysems. Journal of Chemical Technology and Bioechnology. 57, Mukaaka, S., Tesuo, K. & Joji, T. (1985). Kineics of Enzymaic Hydrolysis of Lipids in Biphasic Organic-Aqueous Sysems. J. Fermen. Technol. 63 (5),

9 58 S. Al-Zuhair 4. Knezevic, Z.D., Siler-Marinkovic, S.S. & Mojovic, L.V. (1998). Kineics of Lipase-Caalysed Hydrolysis of Palm Oil in Lecihin/isoocane Reversed Micelles. Appl. Microbiol. Bioechnol 49, Malcaa, F.X., Hill, Jr, C.G. & Amundson, C.H. (1992). Hydrolysis of Bueroil by Immobilised Lipase using a Hollow-Fibre Reacor. Par II. Uniresponse Kineic Sudies. Bioech. and Bioeng. 39, Malcaa, F.X., Hill, Jr, C.G. & Amundson, C.H. (1992). Hydrolysis of Bueroil by Immobilised Lipase using a Hollow-Fibre Reacor. Par III. Muliresponse Kineic Sudies. Bioech. and Bioeng. 39, NOVO Indusrials. Analyical mehods handou (1995). Enzyme Process Division, NOVO indusrials, Denmark.