Overproduction of D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 in Escherichia coli and its purification

Mamoru Wakayama, Shin ichi Hayashi, Yukinori Yatsuda, Yutaka Katsuno, Kenji Sakai, Mitsuaki Moriguchi

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

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).

Original languageEnglish
Pages (from-to)395-399
Number of pages5
JournalProtein Expression and Purification
Volume7
Issue number4
DOIs
Publication statusPublished - Jan 1 1996
Externally publishedYes

Fingerprint

Alcaligenes
Plasmids
Escherichia coli
Initiator Codon
Site-Directed Mutagenesis
Open Reading Frames
DNA
Enzymes
Genes
aminoacylase I

All Science Journal Classification (ASJC) codes

  • Biotechnology

Cite this

Overproduction of D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 in Escherichia coli and its purification. / Wakayama, Mamoru; Hayashi, Shin ichi; Yatsuda, Yukinori; Katsuno, Yutaka; Sakai, Kenji; Moriguchi, Mitsuaki.

In: Protein Expression and Purification, Vol. 7, No. 4, 01.01.1996, p. 395-399.

Research output: Contribution to journalArticle

Wakayama, Mamoru ; Hayashi, Shin ichi ; Yatsuda, Yukinori ; Katsuno, Yutaka ; Sakai, Kenji ; Moriguchi, Mitsuaki. / Overproduction of D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 in Escherichia coli and its purification. In: Protein Expression and Purification. 1996 ; Vol. 7, No. 4. pp. 395-399.
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