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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 ...

Afferent and efferent connections of the fastigial oculomotor region (FOR) were studied in macaque monkeys by using axonal transport of wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP). When injected HRP is confined to the FOR, retrogradely labeled cells appear in lobules VIc and VII of the ipsilateral vermis and in group b of the contralateral medial accessory olive (MAO). In reference to the maps of topographical organization, the extent of the effective site in the fastigial nucleus (FN) could be assessed from the distributions of labeled Purkinje cells (P cells) in the vermis and labeled olivary neurons in the MAO. In contrast to the unilateral nature of the P-cell and climbing-fiber projections, those from the other brainstem regions to the FOR were bilateral. Following the injection of HRP into the FOR, the largest number of retrogradely labeled cells appeared in the pontine nuclei. Although the number of labeled cells was greater on the contralateral side in both the peduncular and dorsomedial pontine nuclei (DMPN), the number of each side was virtually identical in the dorsolateral pontine nucleus (DLPN). In the nucleus reticularis tegmenti pontis (NRTP), labeled cells were located only in its medial and dorsolateral portions bilaterally. In the vestibular complex, labeled cells appeared in the superior (SVN), medial (MVN), and inferior vestibular nuclei (IVN) bilaterally. The lateral vestibular nucleus (LVN), including y group and the ventrolateral vestibular nucleus, were free of labeled cells. Labeled cells appeared also in the perihypoglossal nucleus (PHN) bilaterally. In the pontine raphe (PR) and paramedian pontine reticular formation (PPRF), labeled cells appeared bilaterally in the caudal third of the area between the oculomotor and abducens nuclei. Labeled cells appeared also in the mesencephalic and medullary reticular formation. Tracing of anterogradely labeled axons demonstrated that most fibers from the FOR decussated within the cerebellum and entered the brainstem via the contralateral uncinate fasciculus. Some crossed fibers ascended with the contralateral brachium conjunctivum and terminated in the midbrain tegmentum. A small contingent of fibers advanced further to the thalamus. In the mesodiencephalic junction, labeled terminals were found contralaterally in the rostral interstitial nucleus of medial longitudinal fasciculus (riMLF) and a medial portion of FOrel's H Field. They appeared also in the central mesencephalic reticular formation (cMRF), the periaqueductal gray (PAG), the posterior commissure nucleus, and the superior colliculus. The oculomotor and trochlear nuclei, the red nucleus, and the interstitial nucleus of Cajal were free of labeled terminals.(ABSTRACT TRUNCATED AT 400 WORDS)

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.