Kim Hye Eun, Lee In Soo, Kim Joo Hwan, Hahn Kyu Woong, Park Ul Jae, Han Hyon Soo, Park Kyeong Ryang
Department of Microbiology, Han Nam University, 133 Ojung-dong, Daeduk-ku, Daejeon, 306-791 Korea.
Curr Microbiol. 2003 Apr;46(4):291-5. doi: 10.1007/s00284-002-3886-3.
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.
酯酶编码基因estA从鲁氏不动杆菌I6C-1基因组DNA中克隆出来，与质粒载体pET-22b（pEM1）一起导入大肠杆菌BL21(DE3)。pEM1有一个4.4 kb的EcoRI插入片段，其中包含完整的estA基因。一个2.4 kb的AvaI - SphI DNA片段被亚克隆（pEM3）并测序。estA基因编码一个由366个氨基酸组成的蛋白质（40,687 Da），其等电点为9.17。EstA信号肽长31个氨基酸，成熟酯酶序列长335个氨基酸（37.5 kDa）。EstA保守的催化丝氨酸残基位于第210位。EstA序列与乙酸钙不动杆菌的羧酸酯酶序列相似（同一性75%，相似性85%），与嗜热栖热放线菌的羧酸酯酶序列相似（同一性37%，相似性59%），以及与结核分枝杆菌的羧酸酯酶序列相似（同一性35%，相似性51%）。这些酶含有保守基序G-X(1)-S-X(2)-G携带水解酶的活性位点丝氨酸。鲁氏不动杆菌I6C-1中的EstA活性在整个稳定期保持恒定，带有pEM1的大肠杆菌BL21(DE3)中的活性与鲁氏不动杆菌I6C-1相似。
