OVERPRODUCTION OF D-AMINOACYLASE FROM ALCALIGENES XYLOSOXYDANS SUBSP XYLOSOXYDANS A-6 IN ESCHERICHIA-COLI AND ITS PURIFICATION

We constructed the high-expression plasmid for D-aminoacylase from Alc aligenes xylosoxydans subsp. xylosoxydans A-6. The appropriate Shine-D algarno sequence (AAGGAG) was introduced to the eight bases upstream o f start codon (ATG) of D-aminoacylase structural gene by site-directed mutagenesis, and then the 1.75-kb DNA fragment including the open rea ding frame was inserted into the downstream of the tac promoter of pla smid vector pKK223-3. The resultant plasmid, which was named pKNSD2, s howed a high D-aminoacylase activity in Escherichia coli JM109 cells t ransformed with it. The enzyme was purified to homogeneity in only two steps with a final yield of 24% (sp act, 2023 U/mg). (C) 1996 Academi c Press, Inc.