Isolation of a low-temperature adapted lipolytic enzyme from uncultivated micro-organism.
OBJECTIVE
The aim of the study was to isolate a novel lipolytic enzyme from the activated sludge of uncultured micro-organisms.


RESULTS
The metagenomic DNA was directly extracted from the activated sludge, and a metagenomic library was constructed by using the pUC vector. The library was screened for lipolytic enzyme activity on 1% tributyrin agar plate. A clone among c. 100 000 recombinant libraries showed the lipolytic activity. The putative lipolytic gene encoding lipo1 from the metagenomic library was subcloned and expressed in Escherichia coli BL21 using the pET expression system. The expressed recombinant enzyme was purified by Ni-nitrilotriacetic acid affinity chromatography and characterized using general substrates of lipolytic property. The gene consisted of 972 bp encoding a polypeptide of 324 amino acids with a molecular mass of 35.6 kDa. Typical residues essential for lipolytic activity such as penta-peptide (GXSXG) and catalytic triad sequences (Ser166, Asp221 and His258) were detected. The deduced amino acid sequence of lipo1 showed low identity with amino acid sequences of esterase/lipase (32%, ZP_01528487) from Pseudomonas mendocina ymp and esterase (31%, AAY45707) from uncultured bacterium. This lipolytic enzyme exhibited the highest activity at pH 7.5 and 10 degrees C. At thermal stability analysis, lipo1 was more unstable at 40 degrees C than 10 degrees C.


CONCLUSIONS
An activity based strategy has been an effective method for fishing out a low-temperature adapted lipolytic enzyme from the metagenomic library. This lipo1 enzyme can be considered to belong to the hormone-sensitive lipase family due to the enzyme's oxyanion hole by the sequence HGGG.


CONCLUSIONS
Lipo1 is a novel psychrophilic esterase obtained directly from the metagenomic library. Owing its support of significant activity at low temperature, this enzyme is expected to be useful for potential application as a biocatalyst in organic chemistry.
Roh
C
C
School of Chemical and Biological Engineering, Institute of Molecular Biology and Genetics, Seoul National University, Seoul, South Korea. chroh21@snu.ac.kr
Villatte
F
F
D000222:Adaptation, Physiological; D000595:Amino Acid Sequence; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000302:isolation & purification*; D003001:Cloning, Molecular; D003080:Cold Temperature; D003902:Detergents / Q000494:pharmacology; D004783:Environmental Microbiology; D016680:Genome, Bacterial; D015698:Genomic Library; D006863:Hydrogen-Ion Concentration; D008066:Lipolysis; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012722:Sewage
D003902:Detergents; D012722:Sewage; D002265:Carboxylic Ester Hydrolases