Yeast Yarrowia lipolytica CL180로 부터 광학이성질적 Ofloxacin ester분해에 관련한 Esterase 특성분석

Abstract

Levofloxacin, (S)-isomer of the (R),(S)-ofloxacin, is a new fluoroquinolone antibiotic. To screen strains producing an esterase that hydrolyzes the racemic ofloxacin ester to levofloxacin, 151 isolates showing lipolytic activity on tributyrin agar plate were tested for the hydrolysis of ofloxacin ester. 3 strains among them were selected, and one of them, identified as the yeast Yarrowia lipolytica CL180, was found to enantioselectively hydrolyze the (S)-enantiomer of the (R),(S)-ofloxacin ester. The enantioselective esterase-encoding gene was cloned by screening genomic library of Y. lipolytica CL180 and the sequence was determined. Similarity analysis revealed that the esterase gene consisted of 1431-bp (476 aa) with an estimated molecular mass of 52 kDa. The consensus sequence G–X1–S–X2–G in most serine-esterase was also conserved. The amino acids comparison showed a high similarity with the yeast enzymes of type B1 carboxylesterase/lipase family. The esterase was overexpressed in E. coli. and purified to homogeneity. The purified esterase was further characterized. The novel esterase in this study suggested that it might be effectively used as a novel biocatalyst for production of levofloxacin in the pharmaceutical industry.