Screening and its potential application of lipolytic activity from a marine environment: characterization of a novel esterase from Yarrowia lipolytica CL180.
To develop an enantioselective lipase/esterase hydrolyzing racemic ofloxacin ester to levofloxacin, samples were collected from a variety of marine environments such as cold sea, hydrothermal vent area, sediment, tidal flat area, arctic sea, marine organisms, and so on. Microorganisms were isolated by plating on an enrichment medium with simultaneous detection of lipolytic activities and screened for the hydrolysis of ofloxacin ester. Three candidates among isolates were selected, and one of them, identified as Yarrowia lipolytica CL180, hydrolyzed preferentially S-enantiomer of racemic ofloxacin ester. The lipase/esterase gene (yli180) was cloned by screening a genomic library. The sequence analysis revealed an open reading frame consisting of 1,431 bp that encoded a protein of 476 amino acids with a molecular mass of 53 kDa. The yli180 gene was expressed in Escherichia coli and purified to homogeneity. The optimum activity of the recombinant protein (rYli180) occurred at pH 7.5 and 35 degrees C, respectively. rYli180 preferentially hydrolyzed p-nitrophenyl esters of fatty acids with short chain lengths of < or =10 carbon atoms. This study represents a novel esterase of type B1 carboxylesterase/lipase family from a marine isolate, showing a potential usage as a biocatalyst because of enantioselectivity toward racemic ofloxacin ester.
Kim
Jun-Tae
JT
Marine Biotechnology Center, Korea Ocean Research & Development Institute, Ansan, South Korea.
Kang
Sung Gyun
SG
Woo
Jung-Hee
JH
Lee
Jung-Hyun
JH
Jeong
Byeong Chul
BC
Kim
Sang-Jin
SJ
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli; D004950:Esterases / Q000302:isolation & purification* / Q000378:metabolism*; D015698:Genomic Library; D006863:Hydrogen-Ion Concentration; D064704:Levofloxacin; D008066:Lipolysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015242:Ofloxacin / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature; D014871:Water Microbiology; D025062:Yarrowia / Q000201:enzymology* / Q000302:isolation & purification
D004271:DNA, Fungal; D011994:Recombinant Proteins; D064704:Levofloxacin; D015242:Ofloxacin; D004950:Esterases