Rice Straw as a Support for Immobilization of Microbial Lipase Heizir F. de Castro,* ,† Rosemar de Lima, † and Ine ˆ s C. Roberto ‡ Department of Chemical Engineering and Department of Biotechnology, Faculty of Chemical Engineering of Lorena, P.O. Box 116, 12600-970 Lorena, SP, Brazil Candida rugosa lipase was covalently immobilized on rice straw activated with glutaraldehyde using poly(ethylene glycol) (PEG) as the stabilizing agent. The effects of PEG molecular weight and enzyme loading were studied according to a full 2 2 factorial design. Higher immobilization yields (>70%) were attained when the lipase loading was 95 units/mg of dry support, independent of PEG molecular weight. All derivatives showed high hydrolytic and synthetic activities. This work provides preliminary results on the use of agricultural residues as a support matrix for immobilizing lipase and on the application of the resulting derivatives to butyl butyrate synthesis as a study model. 1. Introduction Rice straw can be fitted in a group of agroindustrial residues with suitable feedstock characteristics for the production of several chemical compounds by the bio- technological route. In this regard, attention has been focused on the utilization of the hemicellulosic fraction to produce both ethanol and xylitol by means of suitable organisms (Abbi et al., 1996; Roberto et al., 1995). Rice straw also has application in the cellulose and paper industry (Oy et al., 1996). As a support matrix for microbial cells, it has proven more efficient than its counterparts, namely, sugar cane bagasse, sawdust, and rice husk (Das et al., 1993). More recently, this agricul- tural waste has been employed as an adsorbent for metal and color removal (Johns et al., 1998; Ahmedna et al., 2000). There is no information, however, on the use of rice straw as a support matrix for isolated enzymes, especially those such as lipases (EC 3.1.1.3) with poten- tial application in a number of industrial processes. Important uses of lipases include their addition to detergents, the manufacture of food ingredients, pitch control in the pulp industry, and biocatalysis of stereo- selective transformations (Ghandi, 1997; Jeager and Reetz, 1998). With immobilized lipases, improved stability, reuse, continuous operation, the possibility of better control of reactions, and hence more favorable economical factors can be expected (Yahyla et al., 1998). Lipase has been immobilized by several methods, namely, adsorption, cross-linking, adsorption followed by cross-linking, cova- lent attachment, and physical entrapment using many commercial carriers, e.g., controlled pore silica, natural and synthetic polymers, hollow fibber, activated charcoal, aluminum oxide and diatomaceous earth (Celite) (Balca ˜o et al., 1996). In view of the current high cost of some available commercial support matrixes, the possibility of using cheap and/or alternative supports for lipase im- mobilization such as rice husk (Tantrakulsiri et al., 1997) and CaCO 3 powder (Rosu et al., 1998) has also been considered. The search for an inexpensive support has motivated our group to select matrixes, which can be recognized as solid surfaces by lipases, at the molecular level. The present study investigates the feasibility of using rice straw for immobilizing Candida rugosa lipase using poly- (ethylene glycol) to protect the enzyme from inactivation during the immobilization step. This methodology is recommended when a bifunctional reagent, such as glutaraldehyde, is used to activate solid matrixes (Rocha et al., 1998). The enzymatic activity was determined by both hydrolysis of olive oil and esterification of 1-butanol with butyric acid. 2. Materials and Methods Materials. Commercial C. rugosa lipase (type VII) and bovine serum albumin (BSA) were purchased from Sigma Chemical Co. (St. Louis, MO). The lipase was a crude preparation with a nominal specific activity of 950 units/ mg of solid. Olive oil (low acidity) was purchased at a local market. The substrates for esterification reactions were dehydrated with 0.32 cm molecular sieves (alumi- num sodium silicate, type 13, X-BHD Chemicals, Toronto, Canada), previously activated in an oven at 350 °C for 6 h. The solvents were standard laboratory grade. Alcohol, organic acid, and other reagents were purchased either from Aldrich Chemical Co. (Milwaukee, WI) or Sigma. Support. Rice straw “in natura” with a 10% (w/w) moisture content, supplied by local farmers, was ground and sieved to obtain particle sizes between 80 and 100 mesh. This material was then washed with distilled water and dried at 100 °C before being used as the support matrix. Immobilization Procedure. The immobilization pro- cedure was a three-step sequential process. The first step was the support activation with glutaraldehyde, following the methodology previously described (Soares et al., 1999). The activated support (1.5 g, dry weight) was soaked in hexane (10 mL) for 2 h. The excess solvent was discharged, and an amount of enzyme necessary to give lipase loadings in the range of 95-285 units/mg of dry support was dissolved in 10 mL of distilled water, mixed * To whom correspondence should be addressed. Fax: (+55) 12- 5533224. E-mail: heizir@dequi.faenquil.br. † Department of Chemical Engineering. ‡ Department of Biotechnology. 1061 Biotechnol. Prog. 2001, 17, 1061-1064 10.1021/bp010099t CCC: $20.00 © 2001 American Chemical Society and American Institute of Chemical Engineers Published on Web 11/16/2001