木糖氧化产碱杆菌木糖氧化亚种A-6来源的D-氨基酰化酶在大肠杆菌中的过量表达及其纯化

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

作者信息

Wakayama M, Hayashi S, Yatsuda Y, Katsuno Y, Sakai K, Moriguchi M

机构信息

Department of Applied Chemistry, Faculty of Engineering, Oita University, Japan.

出版信息

Protein Expr Purif. 1996 Jun;7(4):395-9. doi: 10.1006/prep.1996.0059.

DOI:10.1006/prep.1996.0059

PMID:8776758

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).

摘要

我们构建了来自木糖氧化产碱杆菌木糖氧化亚种A-6的D-氨基酸酰化酶的高表达质粒。通过定点诱变，将合适的Shine-Dalgarno序列（AAGGAG）引入到D-氨基酸酰化酶结构基因起始密码子（ATG）上游的八个碱基处，然后将包含开放阅读框的1.75 kb DNA片段插入质粒载体pKK223-3的tac启动子下游。所得质粒命名为pKNSD2，在用其转化的大肠杆菌JM109细胞中显示出高D-氨基酸酰化酶活性。该酶仅通过两步就被纯化至均一，最终产率为24%（比活性，2023 U/mg）。