Commercial dry lipase fromCandida rugosa (formerlyC. cylindracea) was used to catalyze hydrolysis of tallow, coconut oil and olive oil at 26–40 C. A methodology was developed to yield results reproducible within ±10% and to achieve essentially complete hydrolysis. From the hydrolysis data, an empirical relationship was developed that shows that the percentage of free fatty acid formed is almost a linear function of the logarithm of reaction time and the logarithm of enzyme concentration. A 95–98% hydrolysis of the 3 substrates was achieved experimentally in 72 hr, requiring 15 units lipase per milliequivalent (U/meq) of coconut oil or tallow and 6 U/meq of olive oil. The kinetics of lipolysis were determined for all 3 substrates and were found to approximate first order. Lipolysis rate was higher for olive oil than for tallow and coconut oil; no significant differences were observed between the latter 2 substrates. No statistically significant change in overall reaction rate was found when the hydrolysis was run at 26 C, 36 C or 46 C. Although the literature cites calcium or sodium ions and albumin as beneficial adjuvants to enzymatic lipolysis, these additives appeared to have no significant beneficial effect on the reaction. On the other hand, hydrocarbon solvents and nonionic surfactants showed an adverse effect.

The hydrolysis of tallow, coconut oil and olive oil, by lipase from Candida rugosa, was studied. The reaction approximates a firstorder kinetics model. Its rate is unaffected by temperature in the range of 26-46 C. Olive oil is more rapidly hydrolyzed compared to tallow and coconut oil. tlydrolysis is adversely affected by hydrocarbon solvents and a nonionic surfactanL Since amounts of fatty acids produced are almost directly proportional to the logarithms of reaction time and enzyme concentration, this relationship provides a simple means of determining these parameters for a desired extent of hydrolysis. All three substrates can be hydrolyzed, almost quantitatively, within 72 hr. Lipase from Aspergillus niger performs similarly. The lipase from Rbizopus arrbizus gives a slow hydrolysis rate because of its specificity for the acyl groups attached to the c~hydroxyl groups of glycerol. Esterification of glycerol with fatty acid was studied with the lipase from C rugosa and A. niger. All expected five glycerides are formed at an early stage of the reaction. Removal of water and use of excess fatty acid reverse the reaction towards esterification. However, esterification beyond a 70% triglyceride content is slow.

A simplified procedure for the measurement of activity was developed for lipases (EC 3.1.1.3) fromChromobacterium viscosum, Candida rugosa, Aspergillus niger andRhizopus arrhizus. It differs from existing procedures in that olive oil, refined with a bleaching clay, was used as the substrate, periodic sonication was applied to promote efficient emulsification, the hydrolysis time was extended to 1 hr, and no additive such as protective colloids and surfactants were used. The activity was determined arbitrarily for that amount of lipase required to bring about 24% hydrolysis/hr at room temperature Unlike currently used assay procedures, the present method gives results which are highly reproducible with a maximum standard deviation of 2.5% and, more commonly, less than 1.00%.

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