TY - JOUR
T1 - Overproduction of D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 in Escherichia coli and its purification
AU - Wakayama, Mamoru
AU - Hayashi, Shin ichi
AU - Yatsuda, Yukinori
AU - Katsuno, Yutaka
AU - Sakai, Kenji
AU - Moriguchi, Mitsuaki
PY - 1996/6
Y1 - 1996/6
N2 - 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).
AB - 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).
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U2 - 10.1006/prep.1996.0059
DO - 10.1006/prep.1996.0059
M3 - Article
C2 - 8776758
AN - SCOPUS:0030175183
SN - 1046-5928
VL - 7
SP - 395
EP - 399
JO - Protein Expression and Purification
JF - Protein Expression and Purification
IS - 4
ER -