The lipase of Aspergillus niger was examined for partial glycerides synthesis. A

significant degree of synthesis (more than 50%) was achieved using lamate as

one of the substrates. This can be further improved by optimizing the reaction

conditions. A synthesis degree of almost 80% was obtained in the presence of

3.0 g of molecular sieve pellets for water absorption in the reaction system

Che Omar, Ibrahim

H. L., Chin

Universiti Putra Malaysia Institutional Repository

Pertanika J. Sci. & Techno!. 3(2): 383-388 (1995)ISSN:0128-' i)© Penerbit Universiti Pertanian Malay,iaCOMMUNICATION IPartial Glycerides Synthesis by Lipase ofAspergillus nigerReceived 28 July 1993ABSTRAKLipase daripada Aspergillus niger tetah dikaji untuk sintesis gliserida separa.Darjah sintesis yang bererti melebihi 50% telah dicapai menggunakan lauratesebagai salah satu daripada substrat. DaIjah ini dapat diperbaiki lagi denganmengoptimumkan keadaan tindakbalas. Darjah sintesis yang menghampiri80% telah diperoleh dengan kehadiran 3.0 g penapis molekul dalam sistemtindakbalas untuk pe~erapan air.ABSTRACTThe lipase of Aspergillus niger was examined for partial glycerides synthesis. Asignificant degree of synthesis (more than 50%) was achieved using lamate asone of the substrates. This can be further improved by optimizing the reactionconditions. A synthesis degree of almost 80% was obtained in the presence of3.0 g of molecular sieve pellets for water absorption in the reaction system.INTRODUCTIONIn Malaysia, partial glycerides are in great demand for use in food andpharmaceutical industries and also in polymer industries. These glyceridesare presently produced using chemical methods under high temperature(170-220°C) and pressure (3-5 x 10-3 mmHg) in the presence of a chemicalcatalyst such as sodium methoxide. Nevertheless, the products normallycontain high impurities with low recovery yield. A milder alternativerequires the use of lipases, but such a process is still not available inMalaysia. Lipases have been used in partial glycerides and esters synthesis(Ibrahim et al. 1987; Ibrahim and Tan 1991), monoglyceride production(Ibrahim et al. 1989), fatty acid production (Ibrahim and Nagai 1989) andthe production of other value-added products (Ibrahim et al. 1988a). Withthe aim of introducing the application of lipase in the palm oil industry,we have isolated Aspergillus niger SBS K020 as a potential producer ofexogenous lipase. The production of the enzyme by fermentation proc-esses by both the free and immobilized cells has already been reported(Ibrahim et al. 1991; Ibrahim and Noor Izani 1991; Noor Izani andIbrahim 1992). In this paper, some of the characteristics and importantparameters governing the synthesis of partial glycerides by A. niger lipaseare described.MATERIALS AND METHODSThe lipase used throughout the experiment was obtained from A. nigergrown in a palm oil medium as described previously (Ibrahim and NoorH. L. Chin & Ibrahim Che OmarIzani 1991). The crude powder preparation contained 4400 U / g of activitydetermined by the method of Ibrahim and Noor Izani (1991) usingpolyvinyl alcohol-refined, bleached, deodorized (RBD) palm oil as thesubstrate. The enzyme was then used for the partial glycerides synthesisusing a basal reaction mixture of glycerol (4 ml, 75%, v/v) , fatty acid (0.3 g)and lipase solution (1.0 ml, 4 U) based on the method of Tsujisaka et al.(1977). The reaction mixture was placed in a 50-ml conical flask with asilicone cap and incubated for 5 h at 50°C. Mter the reaction, the degreeof synthesis was calculated from the fatty acid consumed in the syntheticreaction by the titration method (Ibrahim et al. 1987).RESULTS AND DISCUSSIONFig. 1 shows the substrate specificity profiles of A. niger lipase for glyceridesynthesis. The enzyme exhibited good specificity with laurate (CI2) givinga maximum degree of synthesis of 53% after about 20 h. A lower degreeof synthesis was observed as the molecular weight of the acid used wasincreased. Similarly, the degree of synthesis dropped with increasingunsaturation in the fatty acid. Thus the degree of glyceride synthesis by thelipase can be arranged in the order of C12 (laurate) > C14 (myristate) >C18:1 (oleate) > C18:2 (linoleate).•o 10 20 30 40 50Reaction time (h)Fig. 1. Substrate specificity Profiles of lipase fromAspergillUS niger for glyceride synthesis (....)laurate; (D) myristate; .) oleate; (0) linoleateSome governing parameters on the formation of glycerides fromlaurate are shown in Fig. 2. The optimum temperature was between 35 to40°C. Although the optimum temperature of hydrolysis was 50°C (deter-384 Pertanika J. Sci. & Techno!. Vo!. 3 No.2, 1995Partial Glycerides Synthesis by Lipase of Aspergillus nigermined using RBD palm oil), the lower temperature for synthesis indicatesthat the physical properties of the reactants might play significant roles. Inhydrolysis, a high temperature is needed to liquefy the solid lipid substrate.A maximum degree of synthesis (57%) was obtained with a glycerolconcentration of 80% in the reaction mixture. Lower synthesis degree wasobtained when less than or higher than 80% concentration of the glycerolwas used. Yamane et at. (1986) demonstrated that glycerol plays twoimportant roles in the reaction system, i.e., as a matrix for lipase and beinghydrophilic in nature, acts as an effective agent for water control. Theseroles, as suggested by Yamane et at. (1986), were also observed in thesynthesis reaction by A. niger lipase (Fig. 2). At a high glycerol concentra-tion, the water concentration is reduced, consequently increasing theenzyme synthetic activity. On the other hand, a low glycerol concentrationis associated with a high water concentration. Under conditions of highwater concentration, the equilibrium of the lipase-catalysed reaction wasshifted towards hydrolysis on the formed glycerides. At a glycerol concen-tration of 80%, an equilibrium between the synthesis and hydrolysisreaction was achieved. In the case of fatty acid, 0.4 g was found to beoptimum. Higher acid concentration may be inhibitory to the enzyme(Loh and Ibrahim 1991). In the same figure, an enzyme concentration of8 Ulml was optimal for maximum synthesis."g- 20o10o3~0-"-;3';--5-4;!;:;0--.!;;45-;;';;50-7.55c-'Reaction temperaTure • C 0(GlycerOl} used in the reaction (-.4) •0.1 0:2 O~3 0:4 0.5LotrCte- content in reoction mixture (01 i:::J.Lipase concentration (U/mll ...Fig. 2. Governing parameters on the degree ofglyceride synthesisUsing the optimized conditions described in Fig. 2, the synthesis ofpartial glycerides in the presence of protective compounds or stabilizersfor the enzyme, such as polyvinyl alcohol, albumin, casein and calciumPertanika J. Sci. & Techno!. Vo!. 3 No.2, 1995 385H. L. Chin & Ibrahim Che Omarchloride, was investigated. The results obtained indicated that the catalyticactivity of the A. niger lipase was not improved in the presence of thesecompounds (data not shown). Tsujisaka et al. (1977) reported that thesecompounds acted as stabilizers to the lipases from Rhizopus delemar, Aspergillusniger, Geotrichum candidum and Penicillium cyclopium which enhanced thesynthesis of glycerides.Based on the parameters described above, it is clearly shown that thewater concentration is the most crucial factor which determines theequilibrium of a lipase-eatalysed reaction. Ibrahim et al. (1988b) havedemonstrated that molecular sieve pellets can effectively control the waterconcentration in the reaction mixture for the synthesis of geranyl laurate.The use of molecular sieve pellets as water absorbents (Fluka, UnionCarbide, Type 4.A) for the glyceride synthesis was thus examined. Com-pared to the system without the addition of molecular sieve pellets, thedegree of synthesis increased rapidly with the addition of more pellets.Water which was produced as a by-product of the reaction was simultane-ously absorbed by the pellets, resulting in a shift in the equilibrium towardsthe synthetic reaction. A maximun of almost 80% degree of synthesis wasobtained after 30 h when 3.0 g of molecular sieve pellets were added (Fig. 3).80~ 60'"'u;'".<::C;; 40~o'"~'"~ 20•.' ---k.:'O--u-•o"--__....L-__-=-"::__-::'-,--__-:'-::----'o 10 20 30 40Reaction time (h)Fig. 3. Effect of molecular sieve pellets as wateradsorbents in the reaction system; molecularsieve pellets (g) added at (0 ) 0.5, (L\) 1.0,C.& ) 2.0 and (0 ) 3. O. Arrow indicates thetime of additionProduct formations were qualitatively reconfirmed on thin layer chro-matography (TLC) using the preparation method and solvent systemdescribed previously (Ibrahim et at. 1987). From such experiments, synthe-386 Pertanika J. Sci. & Techno!. Vo!. 3 No.2, 1995Partial Glycerides Synthesis by Lipase of Aspergillus nigersis of lauric acid glycerides appeared as spots on the TLC plates (data notshown). The intensity of laurate spot decreased with a correspondingincrease in the spot intensity for trilaurin and dilaurin. Nevertheless,monolaurin was not detected, indicating that the rapid synthetic reactiontook place without the formation of a monoglyceride intermediate.CONCLUSIONThis short communication demonstrates that the lipase from a newlyisolated strain of A. niger can be used in the synthesis of glycerides of fattyacids. The degree of glycerides formation can be improved by optimizingseveral reaction parameters such as the temperature and the concentra-tion of glycerol, acid, and enzyme and also by reducing the water concen-tration in the reaction mixture.Although Tsujisaka et al. (1977) have reported the synthesis of glycer-ides by a strain of A. niger, comparative studies between the lipaseperformance of their strain and the new strain that we have isolated is nottotally possible. In both cases, reaction conditions with respect to thesubstrate and enzyme concentration, reaction temperature and stabilizerrequirements are different. Nevertheless, under their respective optimumconditions, the performance of the two lipases is almost similar.H.L. CHIN and IBRAHIM CHE OMARSchool of Biological SceinesUniversiti Sains Malaysia11800 Minden, Penang, MalaysiaREFERENCESIBRAHIM, C.O. and S. NAGAI. 1989. Hydrolysis of fats and oil in an immobilised lipase·continuous stirred tank reactor (CSTR). Sains Malaysiana 18: 115-127.IBRAHIM, e.O., . NISHIO and S. AGAI. 1987. Fat hydrolysis and esterification by a lipase fromHumicola lanuginosa. Agricultural Biological Chemistry 51: 2153-2159.IBRAHIM, e.O., N. NISHIO and S. NAGAI. I988a. Acidolysis reaction by a thermostable lipasefrom Humicola lanuginosa. Agricultural Biological Chemistry 52: 2923-2925.IBRAHIM, e.O., N. NISHIO and S. NAGAI. 1988b. The role of water on the equilibrium ofesterification by immobilized lipase packed-bed column reactor. Biotechnology Letters10: 799-804.IBRAHIM, C.O. and NJ. NoOR !zAN!. 1991. Production of an exogenous lipase by Aspergillusniger grown on a palm oil medium. Joumal ofBioscience 1: 15-28.IBRAHIM, C.O., NJ. NOOR IZANI and 1. DARAH. 1991. Isolation and identification ofexogenouslipase producing fungi using palm oil medium. Joumal ofBioscience 1: 59-69.IBRAHIM, e.O., H. SAEKI, N. NISHIO and S. NAGAI. 1989. Synthesis ofacetone glycerol acyl estersby immobilized lipase of Mucor miehei. Biotechnology Letters 11: 161-166.Pertanika J. Sci. & Techno!. Vo!. 3 No.2, 1995 387H. L. Chin & Ibrahim Che OmarIBRAHIM, e.O. and H.L. TAN. 1991. Esterification by lipase of Candida 0'lindraceawith fattyacid distillates (FAD) as acyl donors. Joumal ofMalaysian Applied Biology 20: 11-23.LOH, Y.S. and e.o. IBRAHIM. 1991. Hydrolysis of palm oil by calcium-alginate entrappedlipase of Candida 0'lindracea. Journal ofBioscience 2: 47-58.NOOR IZANI, J. and e.o. IBRAHIM. 1992. Lipase production using immobilized Aspergillusniger. Joumal ofBioscience 3: 14-24.TSUJISAKA, Y., S. OKUMURA. and M. IWAl. 1977. Glyceride synthesis by four kinds of microbiallipases. Biochimica Biophysica Acta 489: 415-422.YA1\1ANE, T., M.M. HoQ, S.lTOH and S. SHIMIDZU. 1986. Glycerolysis oHat by lipase. Journal ofJapan Oil and Chemical Society 35: 45-51.388 Pertanika J. Sci. & Techno!. Vo!. 3 No.2, 1995

Partial Glycerides Synthesis by Lipase of

Aspergillus niger

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Partial Glycerides Synthesis by Lipase of Aspergillus niger

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