Indian Journal of Biotechnology Vol. 4, April 2005, pp 241-245 Enzymatic hydrolysis of castor oil: An approach for rate enhancement and enzyme economy Samir R Kulkarni 1 and Aniruddha B Pandit 2* 1 College of Pharmacy, University of Cincinnati Medical Center, Cincinnati, OH 45040, USA 2 Chemical Engineering Division, Institute of Chemical Technology, University of Mumbai, Matunga, Mumbai 400 019, India Received 7 October 2003; revised 12 April 2004; accepted 25 April 2004 Lipase has been used to catalyze the hydrolysis of castor oil. The effect of solvent, temperature, pH and the enzyme concentration on the rate of reaction has been investigated. The rate of reaction could be considerably improved by modifying the reaction protocol with the help of addition of a solvent. The amount of enzyme used for the reaction was found to increase the rate of reaction in a logarithmic relation. By optimizing the enzyme addition protocol, the total amount of enzyme required for the reaction could significantly be reduced. Keywords: castor oil, lipase, enzyme economy, hydrolysis, solvent effect IPC Code: Int. Cl. 7 A01N63/04; C12N9/16, 9/20, 15/09 Introduction The biocatalysts find applications for industrial purposes because they possess high specific catalytic activity over the conventional chemical catalysts especially under milder conditions. Lipases (triacylglycerol ester hydrolase, E.C. 3.1.1.3.) are ubiquitous enzymes whose biological function is to catalyze the hydrolysis of triacylglycerols. The microbial lipases have been used in hydrolysis of fats 1 , esterification and transesterification 2,3 and resolution of chiral compounds 4 . Fatty acids and glycerol are the products of fat hydrolysis with variety of end uses as chemical intermediates 5 . Commercially accepted process for fat hydrolysis operates with superheated steam at 250°C and 50 atm 6 . This high- energy process during hydrolysis also induces polymerization and colour development and requires subsequent purification 7 . An enzymatic process obviates these drawbacks associated with the thermal process. Castor oil is the most ideal candidate for the enzymatic hydrolysis process 8 . In the present investigation, a commercial lipase was used to hydrolyse castor oil with an aim of optimizing the process conditions to get maximum yield of castor fatty acids in minimum time using as little enzyme as feasible. Materials and Methods Materials Lipase, gifted by Novo Nordisk Ltd. (Bagsvaerd, Denmark), was obtained from a genetically engineered species of Aspergillus oryzae. Tributryin and castor oil were purchased in Mumbai, from Loba Chemie and IPCA Chemicals and Cosmetics Ltd., respectively. Proline was purchased from Himedia Laboratories Ltd., Mumbai. All other chemicals used were of reagent grade. Determination of Lipase Activity Lipase activity was determined by performing controlled hydrolysis of tributryin substrate 9 (tributyrin was mixed with water using sonication to obtain a fine emulsion to which buffered enzyme was added. The resultant mixture was incubated for 10 min and the product was analyzed for the quantity of fatty acids formed). Enzyme activity was then expressed as the amount of enzyme required to liberate one μmole of fatty acid per min under the given conditions. Reaction System without Solvent Castor oil (1 g) was sonicated with 8 ml of water for 10 min at 60 W intensity for 5 sec on/off pulse on an ultrasonic processor. To this solution, 1 ml of enzyme solution (U i.e. 2 mg of enzyme) in phosphate buffer (pH-7.0) was added. The reaction mixture was incubated for different time intervals (1 to 24 hrs) ___________ *Author for correspondence: Tel & Fax: 91-22-24145614 E-mail: abp@udct.org