Abstract
Enzymes can be used in nonpolar reaction media to modify waterinsoluble substrates. A variety of pretreatments, applied to the enzyme prior to introduction to the nonpolar media, can improve enzyme activity. However, the various pretreatments have not been studied using directly comparable conditions, nor have they been applied simultaneously to test for interactive effects. This work evaluates pretreatment of lipase with various classes of additives. The pretreated lipase is used to catalyze esterification between citronellol and acetic acid in a medium of n-hexane. The effectiveness of a particular pretreatment is presented in terms of relative performance (RP), which is equal to the number of times faster the pretreated lipase catalyzes the reaction relative to untreated lipase. The individual and interactive effects of the pretreatment factors were studied and compared. Buffer salts had a much stronger performance-enhancing effect than nonbuffer salts; pretreatment with 90% (w/w) sodium phosphate yielded lipase with an RP of approx 64. A strong interaction was found between the treatments with sodium phosphate and pH adjustment. These treatments may mitigate the inhibitory effect of acetic acid. Activating effects of phase interfaces and active-site protectants are shown to be complementary to other treatments, demonstrating that they likely act by distinct mechanisms.
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Zaks, A. and Klibanov, A. M. (1988), J. Biol. Chem. 263(7), 3194–3201.
Zaks, A. (1991), in Biocatalysis for Industry, Dordick, J. S., ed., Plenum, New York, pp. 161–180.
Klibanov, A. M. (2001), Nature 409, 241–246.
Persson, M., Mladenoska, I., Wehtje, E., and Aldercreutz, P. (2002), Enzyme Microb. Technol. 31, 833–841.
Griebenow, K. and Klibanov, A. M. (1995), Proc. Natl. Acad. Sci. USA 92, 10,969–10,976.
De Castro, H. F., Napoleao, D. A. S., and Oliveira, P. C. (1998), Appl. Biochem. Biotechnol. 70–72, 667–675.
Claon, P. A. and Akoh, C. C. (1994), Enzyme Microb. Technol. 16, 835–838.
Laboret, F. and Perraud, R. (1999), Appl. Biochem. Biotechnol. 82, 185–198.
Brady, L., Brzozowski, A. M., Derewenda, Z. S., et al. (1990), Nature 343, 767–770.
Mingarro, I., Abad, C., and Braco, L. (1995), Proc. Natl. Acad. Sci. USA 92, 3308–3312.
Louwrier, A., Drtina, G. J., and Klibanov, A. M. (1996), Biotechnol. Bioeng. 50(1), 1–5.
Gonzalez-Navarro, H. and Braco, L. (1998), Biotechnol. Bioeng. 59(1), 122–127.
Guo, Y. and Clark, D. S. (2001), Biochim. Biophys. Acta 1546, 406–411.
Dai, L. and Klibanov, A. M. (1999), Proc. Natl. Acad. Sci. USA 96, 9475–9478.
Ru, M. T., Dordick, J. S., Reimer, J. A., and Clark, D. S. (1997), Biotechnol. Bioeng. 63(2), 233–241.
Rich, J. O. and Dordick, J. S. (1997), J. Am. Chem. Soc. 119, 3245–3252.
Triantafyllou, A. O., Wehtje, E., Aldercruz, P., and Mattiasson, B. (1997), Biotechnol. Bioeng. 54(1), 67–76.
Anthonsen, T. and Sjursnes, B. J. (2001), in Methods in Non-aqueous Enzymology, Gupta, M. N., ed., Birkhauser Verlag, Boston, MA, pp. 14–35.
Valivety, R. H., Halling, P. J., Peilow, A. D., and Macrae, A. R. (1992), Biochim. Biophys. Acta 1122, 143–146.
Zaks, A. and Klibanov, A. M. (1988), J. Biol. Chem. 263(17), 8017–8021.
Gorman, L. A. S. and Dordick, J. S. (1992), Biotechnol. Bioeng. 39, 392–397.
Dabulis, K. and Klibanov, A. M. (1993), Biotechnol. Bioeng. 41(5), 566–571.
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Garcia, R.A., Riley, M.R. Relative effectiveness of pretreatments on performance of Rhizomucor miehei lipase in nonpolar reaction media. Appl Biochem Biotechnol 120, 81–95 (2005). https://doi.org/10.1385/ABAB:120:2:081
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DOI: https://doi.org/10.1385/ABAB:120:2:081