Enzyme–Catalysed Synthesis of Palm-Based Wax Esters-A Kinetic Study

Wax esters are long chain esters that are derived from fatty acids and alcohols with chain lengths of 12 carbons or more. Wax esters havea wide range of application in industrial especially in cosmetics. The present work focuses on the synthesis of wax esters by alcoholysisreaction from palm oil and oleyl alcohol in hexane by lipase from Rhizomucor meihei (Lipozyme IM). Effect of various concentrations ofpalm oil and oleyl alcohol were studied to deduce the kinetics and mechanism of the reaction. The alcoholysis reaction followed Michaelis-Menten kinetics. The reaction follows a Ping-pong Bi-Bi mechanism. The maximum rate was estimated to be 6 x 10 -3 mmol/h. mg catalystand the Michaelis-Menten constant for palm oil and oleyl alcohol were 4.145 M and 6.120 M, respectively.


INTRODUCTION
Interest in lipase-catalyzed preparation of wax esters has grown in the last decade, because of the ability to obtain a wide variety of high-quality products under mild reaction condition (Mat Radzi et al. 2005;George et al. 2000). Wax esters consist of a long fatty acid esterified to a long alcohol.
Wax esters are important ingredients in cosmetic formulations (cleansers, conditioners and moisturizers) (Peter & Robert 2001), in pharmaceuticals (as an anti foaming agent in the production penicillin and a timed release in the production pharmaceutical tablet) (Kline 1956), lubricants, plasticizers and polishes (Chen & Wang 1997).
Wax esters can be extracted from animals and plant materials such as beeswax, sperm whale and jojoba oil.
However, it is often either too scarce or expensive for commercial use and the main obstacles to large-scale use them are its availability (Sanchez et al. 1992;Yadav & Lathi 2003). The synthetic wax esters can be synthesized using chemicals and enzymatic catalyzed methods (Gunawan & Suhendra 2008a, 2008b.The use of homogeneous chemical catalysts leads to several problems such as corrosion of equipment, hazards of handling of the corrosive acids, highenergy consumption and degradation of esters, (Knox & Clife 1984), whereas the enzymatic synthesis offers mild reaction conditions and environmentally friendly process.
We have optimized the lipase-catalyzed alcoholysis of palm-based wax ester and oleyl alcohol (Gunawan & Suhendra 2008a, 2008b. In the present study, the kinetics of the alcoholysis of palm oil and oleyl alcohol in hexane, catalyzed by lipozime IM was examined. This study is for understanding the reaction mechanism, as well as for design of alcoholysis reactor for scale-up.  The initial rates were estimated from the slope of plots of the percentage conversion versus reaction period and reported as mg/h.mg catalyst. The maximum initial rate (v max ) and Michaelis-Menten constants (K m ) were determined by linear regression using the Lineweaver-Burk approach.

Immobilized lipase from
Synthesis and Analysis. Different molar ratios of palm oil and oleyl alcohol were added to 10 ml n-hexane, followed by amounts of enzyme. The mixture of palm oil, oleyl alcohol and Lipozyme I M were incubated in a horizontal water bath shaker (150 rpm) at different reaction temperatures and reaction times. The reactions were analyzed by a gas chromatograph (Hitachi model G-3000, Tokyo, Japan), using an Rtx-65TG capillary column (30 m x 0.25 mm). Helium was used as the carrier gas at a flow rate 30 ml/min. The temperature was programmed at 2 min at 150 0 C, 20 0 C/min to 300 0 C and 10 min at 300 0 C. The product composition was quantitated by an internal standard method with methyl linoleate as the internal standard. The concentrations of esters were calculated by equation:

RESULTS AND DISCUSSION
Kinetic is a study concerning with the rates at which reactions occur and with the factors that affect the rates of a reaction, such as pH, temperature, presence of catalyst, and is therefore essential in an experimental investigation. The Oleyl alcohol (mmol) V 0 x 10 -4 mmol/min.mg catalyst PO 1mmol PO 2 mmol PO 3 mmol predicted by a mathematical formula is only valid in an ideal system in which the concentration of both substrates is small. However, the enzymatic catalysis of two substrates can be simplified by maintaining the concentration of one substrate, and a plot of initial rate against the other substrate concentration will follow a Michaelis-Menten type relationship (Lai et al. 1999).
The maximum initial rate of substrates consumption  Table 1. Similar maximum rate values were obtained in this research. Awang et al. (2004), have also reported a similar observation, on the esterification of palm-based 9,10-dihydroxystearic acid and 1-octanol.
Given the nature of enzyme-catalyzed reactions, the case of a single product reaction is very rare. Most cases of enzyme-catalyzed reactions involve more than one substrate and produce two or more products (Lai et al. 1999). The alcoholysis of palm oil with oleyl alcohol can be considered in this research as an enzymatic reaction system, which involves two reactants and two products. There are three possibilities of enzymatic reaction mechanisms that involve two reactants and two products: 1) a random-order mechanism, 2) a ping-pong mechanism, and 3) an ordered bi-bi mechanism. Scheme 1 shows the general reaction schemes for the three possibilities. According to the studies of some researchers about inter-esterification and trans-esterification (Garcia et al. 1999;Zaidi et al. 2002), the active conformation of lipase comes via an acyl-enzyme intermediate. Since the reaction sequence must be initiated with the bond formation between the carboxyl groups, the random mechanism can be considered less likely.
Based on the type of substrates, the most appropriate explanation for the lipase catalysis mechanism is ping-pong mechanism and ordered bi-bi mechanism, which involve acyl-enzyme intermediate. Lai et al. (1999), combine these mechanisms to be pingpong bi-bi (i.e., bi-substrate, bi-product, non-sequential kinetics) mechanism, in which the first reactant would be involved in the reaction mechanism producing the max max  (acyl migration processes) are the lowest rate constants of the whole catalytic process. In other words, acyl migration is the rate-determining step.

CONCLUSION
The kinetics of the reaction was investigated by studying the effect of the concentration of both palm oil and oleyl alcohol on the initial rate of the reaction. The hyperbolic curves obtained indicated that the reaction followed Michaelis-Menten type kinetics. The information obtained regarding the immobilized-lipase mechanism and the kinetic behaviour will be applied to the optimization of the esterification in bioreactors.