Characterization of two metagenome-derived esterases that reactivate chloramphenicol by counteracting chloramphenicol acetyltransferase.
Function-driven metagenomic analysis is a powerful approach to screening for novel biocatalysts. In this study, we investigated lipolytic enzymes selected from an alluvial soil metagenomic library, and identified two novel esterases, EstDL26 and EstDL136. EstDL26 and EstDL136 reactivated chloramphenicol from its acetyl derivates by counteracting the chloramphenicol acetyltransferase (CAT) activity in Escherichia coli. These two enzymes showed only 27% identity in amino acid sequence to each other; however both preferentially hydrolyzed short-chain p-nitrophenyl esters (< or =C5) and showed mesophilic properties. In vitro, EstDL136 catalyzed the deacetylation of 1- and 3- acetyl and 1,3-diacetyl derivates; in contrast, EstDL26 was not capable of the deacetylation at C1, indicating a potential regioselectivity. EstDL26 and EstDL136 were similar to microbial hormone-sensitive lipase (HSL), and since chloramphenicol acetate esterase (CAE) activity was detected from two other soil esterases in the HSL family, this suggests a distribution of CAE among the soil microorganisms. The isolation and characterization of EstDL26 and EstDL136 in this study may be helpful in understanding the diversity of CAE enzymes and their potential role in releasing active chloramphenicol in the producing bacteria.
Department of Medical Bioscience, Dong-A University, Busan 604-714, Korea.
Weixin
W
Tao
Myung Hwan
MH
Lee
Mi-Young
MY
Yoon
Jin-Cheol
JC
Kim
Shweta
S
Malhotra
Jing
J
Wu
Eul Chul
EC
Hwang
Seon-Woo
SW
Lee
D000595:Amino Acid Sequence; D001419:Bacteria / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D055162:Biocatalysis; D002701:Chloramphenicol / Q000737:chemistry / Q000378:metabolism*; D015500:Chloramphenicol O-Acetyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004950:Esterases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054892:Metagenome; D008969:Molecular Sequence Data; D010802:Phylogeny; D016415:Sequence Alignment; D012988:Soil Microbiology; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D002701:Chloramphenicol; D015500:Chloramphenicol O-Acetyltransferase; D004950:Esterases