Gene cloning, sequencing, and expression of an esterase from Acinetobacter lwoffii I6C-1.
The esterase-encoding gene, estA, was cloned from Acinetobacter lwoffii I6C-1 genomic DNA into Escherichia coli BL21(DE3) with plasmid vector pET-22b (pEM1). pEM1 has a 4.4-kb EcoRI insert that contained the complete estA gene. A 2.4-kb AvaI- SphI DNA fragment was subcloned (pEM3) and sequenced. estA gene encodes a protein of 366 amino acids (40,687 Da) with a pI of 9.17. The EstA signal peptide was 31 amino acids long, and the mature esterase sequence is 335 amino acids long (37.5 kDa). The conserved catalytic serine residue of EstA is in position 210. The EstA sequence was similar to that of the carboxylesterase from Acinetobacter calcoaceticus (75% identity, 85% similarity), Archaeoglobus fulgidus (37% identity, 59% similarity), and Mycobacterium tuberculosis (35% identity, 51% similarity). These enzymes contained the conserved motif G-X(1)-S-X(2)-G carrying the active-site serine of hydrolytic enzyme. The EstA activity in A. lwoffii I6C-1 remains constant throughout the stationary phase, and the activity in E. coil BL21 (DE3) with pEM1 was similar to A. lwoffii I6C-1.
Kim
Hye Eun
HE
Department of Microbiology, Han Nam University, 133 Ojung-dong, Daeduk-ku, Daejeon, 306-791 Korea.
Lee
In Soo
IS
Kim
Joo Hwan
JH
Hahn
Kyu Woong
KW
Park
Ul Jae
UJ
Han
Hyon Soo
HS
Park
Kyeong Ryang
KR
D000150:Acinetobacter / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004950:Esterases / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D007218:Industrial Microbiology; D013379:Substrate Specificity
D001426:Bacterial Proteins; D004950:Esterases