Properties and application of poly(methacrylic acid-co-dodecyl methacrylate-cl-N,N-methylene bisacrylamide) hydrogel immobilized Bacillus cereus MTCC 8372 lipase for the synthesis of geranyl acetate

A range of fatty acids esters is now being produced commercially with immobilized microbial lipases (glycerol ester hydrolases; EC) in nonaqueous solvents. In this study, a synthetic hydrogel was prepared by the copolymerization of methacrylic acid and dodecyl methacrylate in the presencce of a crosslinker, N,N-methylene bisacrylamide. A purified alkaline thermotolerant bacterial lipase from Bacillus aereus MTCC 8372 was immobilized on a poly(methacrylic acid-co-dodecyl methacrylate-d-N,N-methylene bisacrylamide) hydrogel by an adsorption method. The hydrogel showed at 95% binding efficiency for the lipase. The bound lipase was evaluated for its hydorlytic potential toward various p-nitrophenyl acyl esters with various C chain lengths. The bound lipase showed optimal hydrolytic activity toward p-nitrophenyl palmitate at a pH of 8.5 and a temperature of 55 degrees C. The hydorlytic activity of the hydrogel-bound lipase was enhanced by Hg2+, Fe3+, and NH4+ ions at a concentration of 1mM. The hydrogel-bound lipase was used to synthesize geranyl acetate from geraniol and acetic acid in n-heptane. The optimization of the reaction conditions, such as catalyst loading, effect of substrate concentration, solvent (n-pentane, n-hexane, n-heptane, n-octane, and n-nonane), reaction time, temperature, molecular sieve (3 angstrom x 1.5 mm) and scale up (at 50-mL level), was studied. The immobilized lipase (25 mg/mL) was used to perform an esterification in n-alkane(s) that resulted in the synthesis of approximately 82.8 mM geranyl acetate at 55 C in n-heptane under continuous shaking (160 rpm) after 15 h when geraniol and acetic acid were used in a ratio of 100 : 100 mM. The additon of molecular sieve (3 angstrom x 1.5 mm) to the reaction system at a concentration of 40 mg/mL in reaction volume(2 mL) resulted in an increase in the conversion of reactants into geranyl acetate (90.0 mM). During the repetitive esterification under optimum conditions, the hydrogel-bound lipase produced ester (37.0 mM) after the eighth cycle of reuse. When the reaction volume was scaled up to 50 mL, the ester synthesized was 58.7 mM under optimized conditions. (c) 2008 Wiley Periodicals, Inc.