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 Alcaligenes xylosoxydans subsp. xylosoxydans A-6. The appropriate Shine-Dalgarno sequence (AAGGAG) was introduced to the eight bases upstream of start codon (ATG) of D-aminoacylase structural gene by site-directed mutagenesis, and then the 1.75-kb DNA fragment including the open reading frame was inserted into the downstream of the tac promoter of plasmid vector pKK223-3. The resultant plasmid, which was named pKNSD2, showed a high D-aminoacylase activity in Escherichia coli JM109 cells transformed with it. The enzyme was purified to homogeneity in only two steps with a final yield of 24% (sp act, 2023 U/mg).
Wakayama
M
M
Department of Applied Chemistry, Faculty of Engineering, Oita University, Japan.
Hayashi
S
S
Yatsuda
Y
Y
Katsuno
Y
Y
Sakai
K
K
Moriguchi
M
M
D000421:Alcaligenes / Q000201:enzymology* / Q000235:genetics; D000581:Amidohydrolases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D007475:Ion Exchange Resins; D007930:Leucine / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012117:Resins, Synthetic; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D014462:Ultrafiltration
D007475:Ion Exchange Resins; D011994:Recombinant Proteins; D012117:Resins, Synthetic; D012967:Sodium Dodecyl Sulfate; D000581:Amidohydrolases; C019672:aminoacylase I; D007930:Leucine