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Biodiesel derived from vegetable oils has drawn considerable attention with increasing environmental consciousness. We attempted continuous methanolysis of vegetable oil by an enzymatic process. Immobilized Candida antarctica lipase was found to be the most effective for the methanolysis among lipases tested. The enzyme was inactivated by shaking in a mixture containing more than 1.5 molar equivalents of methanol against the oil. To fully convert the oil to its corresponding methyl esters, at least 3 molar equivalents of methanol are needed. Thus, the reaction was conducted by adding methanol stepwise to avoid lipase inactivation. The first step of the reaction was conducted at 30°C for 10 h in a mixture of oil/methanol (1:1, mol/mol) and 4% immobilized lipase with shaking at 130 oscillations/min. After more than 95% methanol was consumed in ester formation, a second molar equivalent of methanol was added and the reaction continued for 14 h. The third molar equivalent of methanol was finally added and the reaction continued for 24 h (total reaction time, 48 h). This three-step process converted 98.4% of the oil to its corresponding methyl esters. To investigate the stability of the lipase, the three-step methanolysis process was repeated by transferring the immobilized lipase to a fresh substrate mixture. As a result, more than 95% of the ester conversion was maintained even after 50 cycles of the reaction (100 d).Paper no. J9045 in JAOCS 76, 789-793 (July 1999). KEY WORDS:Biodiesel, Candida antarctica lipase, immobilized enzyme, methanolysis, vegetable oil.Biodiesel (fatty acid methyl esters) is produced by alcoholysis of vegetable oils and animal fats. The main advantages of biodiesel are its biodegradability, renewability, and improved exhaust emissions. Because of these environmental advantages, biodiesel has drawn considerable attention. Currently, biodiesel is produced from vegetable oils in Europe and North America (1,2), and from waste edible oil in Japan. At present, chemical methods are used for biodiesel production, but these methods have drawbacks, such as, difficulties in the recovery of glycerol, the need for removal of catalyst, and the energyintensive nature of the processes. Furthermore, oils containing free fatty acids and/or water are incompletely transesterified using chemical methods. A biochemical approach can overcome these problems, but has not been adopted industrially because of the high cost of the enzyme catalyst. The establishment of a continuous production process using an immobilized enzyme is strongly needed to decrease the production cost of biodiesel using this approach. Several studies report alcoholyses of vegetable oils and animal fats with primary and secondary alcohols and straightand branched-chain alcohols using lipases as catalysts (3-6). However, these reports do not contain the continuous methanolysis of oils and fats in an organic solvent-free environment. A continuous reaction system without organic solvent is necessary for the industrial production of biodiesel. Our ...

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Candida antarctica lipase is inactivated in a mixture of vegetable oil and more than 1:2 molar equivalent of methanol against the total fatty acids. We have revealed that the inactivation was eliminated by three successive additions of 1:3 molar equivalent of methanol and have developed a three-step methanolysis by which over 95% of the oil triacylglycerols (TAG) were converted to their corresponding methyl esters (ME). In this study, the lipase was not inactivated even though 2:3 molar equivalent of methanol was present in a mixture of acylglycerols (AG) and 33% ME (AG/ME33). This finding led to a two-step methanolysis of the oil TAG: The first-step was conducted at 30°C for 12 h with shaking in a mixture of the oil, 1:3 molar equivalent of methanol, and 4% immobilized lipase; the second-step reaction was done for 24 h after adding 2:3 molar equivalent of methanol (36 h in total). The two-step methanolysis achieved more than 95% of conversion. When two-step reaction was repeated by transferring the immobilized lipase to a fresh substrate mixture, the enzyme could be used 70 cycles (105 d) without any decrease in the conversion. From the viewpoint of the industrial production of biodiesel fuel production, the two-step reaction was conducted using a reactor with impeller. However, the enzyme carrier was easily destroyed, and the lipase could be used only several times. Thus, we attempted flow reaction using a column packed with immobilized Candida lipase. Because the lipase packed in the column was drastically inactivated by feeding a mixture of AG/ME33 and 2:3 molar equivalent of methanol, three-step flow reaction was performed using three columns packed with 3.0 g immobilized lipase. A mixture of vegetable oil and 1:3 molar equivalent of methanol was fed into the first column at a constant flow rate of 6.0 mL/h. The eluate and 1:3 molar equivalent of methanol were mixed and then fed into the second column at the same flow rate. The final step reaction was done by feeding a mixture of eluate from the second column and 1:3 molar equivalent of methanol at the same flow rate. The ME content in the final-step eluate reached 93%, and the lipase could be used for 100 d without any decrease in the conversion.

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The conversion of waste edible oil to biodiesel fuel in a fixed‐bed bioreactor was investigated. Three‐step methanolysis of waste oil was conducted using three columns packed with 3 g of immobilized Candida antarctica lipase. A mixture of waste oil and 1/3 molar equivalent of methanol against total fatty acids in the oil was used as substrate for the first‐step reaction, and mixtures of the first‐ and second‐step eluates and 1/3 molar equivalent of methanol were used for the second‐ and third‐step reactions, respectively. Ninety percent of waste oil was converted to the corresponding methyl esters (ME) by feeding substrate mixtures into the first, second, and third reactors at flow rates of 6, 6 and 4 mL/h, respectively. We also attempted one‐step methanolysis of waste oil. When a mixture of waste oil and 90% ME‐containing eluate (1∶3, wt/wt) and an equimolar amount of methanol against total fatty acids in the waste oil was fed into a reactor packed with 3 g of immobilized C. antarctica lipase at a flow rate of 4 mL/h, the ME content in the eluate reached 90%. The immobilized biocatalyst could be used for 100 d in the two reaction systems without significant decrease in its activity. Waste oil contained 1980 ppm water and 2.5% free fatty acids, but these contaminants had little influence on enzymatic production of biodiesel fuel.

A thermostable lipase from Pseudomonas cepacia has been purified to homogeneity as judged by SDS-PAGE and isoelectric focusing. The purification included treatment of the culture supernatant with acrinol, hydrophobic interaction chromatography, and gel filtration. The enzyme was a monomeric protein with M(r) of 36,500 and pI of 5.1. The optimal pH at 50 degrees C and optimal temperature at pH 6.5 were 5.5-6.5 and 55-60 degrees C, respectively, when olive oil was used as the substrate. Simple triglycerides of short and middle chain fatty acids (C < or = 12) were the preferred substrates over those of long chain fatty acids. The enzyme cleaved all the ester bonds of triolein, with some preference for the 1,3-ester bonds. The enzyme retained all its activity even after incubation at 75 degrees C (pH 6.5) for 30 min. Further, the activity was not impaired during 21 h storage at pH 6.5 in 40% water-miscible solvents including methanol, ethanol, acetone, acetonitrile, dimethylformamide, dimethylsulfoxide, and dioxane. The addition of dimethylsulfoxide or acetone to the assay mixture in the range of 0-35% stimulated the enzyme, whereas benzene or n-hexane had an inhibitory effect. These properties together with the N-terminal amino acid sequence confirmed that the enzyme differs from the known Pseudomonas sp. lipases.

Three lipases, isolated previously in our laboratory, each with different fatty acid and positional specificities, and a known lipase from Candida cylindracea were screened for concentrating docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids in glycerides. Geotrichum candidum lipase was found to be suitable for their concentration in glycerides. Tuna oil was treated at 30~ with this lipase for 16 h, and 33.5% hydrolysis resulted in the production of glycerides containing 48.7% of DHA and EPA. The hydrolysis was not increased despite adding further lipase, so the glycerides were extracted, and the reaction was repeated. The second hydrolysis produced glycerides containing 57.5% of DHA and EPA in a 54.5% yield, with recovery of 81.5% of initial DHA and EPA. Of the total glycerides, 85.5% were triglycerides. These results showed that G. candidum lipase was effective in producing glycerides that contained a high concentration of polyunsaturated fatty acids in good yield.