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Lysine acetylation can generate highly charged enzymes with increased resistance toward irreversible inactivation

Bryan F. Shaw¹, Gregory F. Schneider¹, Baar Bilgiçer¹, George K. Kaufman¹, John M. Neveu², William S. Lane², Julian P. Whitelegge³, and George M. Whitesides¹

¹ Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA
² Mass Spectrometry and Proteomics Resource Laboratory, FAS Center for Systems Biology, Harvard University, Cambridge, Massachusetts 02138, USA
³ Department of Psychiatry and Biobehavioral Sciences, University of California at Los Angeles, Los Angeles, California, USA

(RECEIVED February 27, 2008; FINAL REVISION April 24, 2008; ACCEPTED April 24, 2008)

This paper reports that the acetylation of lysine -NH₃ ⁺ groups of -amylase—one of the most important hydrolytic enzymes used in industry—produces highly negatively charged variants that are enzymatically active, thermostable, and more resistant than the wild-type enzyme to irreversible inactivation on exposure to denaturing conditions (e.g., 1 h at 90°C in solutions containing 100-mM sodium dodecyl sulfate). Acetylation also protected the enzyme against irreversible inactivation by the neutral surfactant TRITON X-100 (polyethylene glycol p-(1,1,3,3-tetramethylbutyl)phenyl ether), but not by the cationic surfactant, dodecyltrimethylammonium bromide (DTAB). The increased resistance of acetylated -amylase toward inactivation is attributed to the increased net negative charge of -amylase that resulted from the acetylation of lysine ammonium groups (lysine -NH₃ ⁺ -NHCOCH₃). Increases in the net negative charge of proteins can decrease the rate of unfolding by anionic surfactants, and can also decrease the rate of protein aggregation. The acetylation of lysine represents a simple, inexpensive method for stabilizing bacterial -amylase against irreversible inactivation in the presence of the anionic and neutral surfactants that are commonly used in industrial applications.

Keywords: amylase; charge ladders; industrial biotechnology; protein aggregation; protein engineering; sodium dodecyl sulfate; thermostability

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