A new esterase EstD2 isolated from plant rhizosphere soil metagenome.
Soil metagenome constitutes a reservoir for discovering novel enzymes from the unculturable microbial diversity. From three plant rhizosphere metagenomic libraries comprising a total of 142,900 members of recombinant plasmids, we obtained 14 recombinant fosmids that exhibited lipolytic activity. A selected recombinant plasmid, pFLP-2, which showed maximum lipolytic activity, was further analyzed. DNA sequence analysis of the subclone in pUC119, pELP-2, revealed an open reading frame of 1,191 bp encoding a 397-amino-acid protein. Purified EstD2 exhibited maximum enzymatic activity towards p-nitrophenyl butyrate, indicating that it is an esterase. Purified EstD2 showed optimal activity at 35 °C and at pH 8.0. The K(m) and K(cat) values were determined to be 79.4 μM and 120.5/s, respectively. The esterase exhibited an increase in enzymatic activity in the presence of 15% butanol and 15% methanol. Phylogenetic analysis revealed that the lipolytic protein EstD2 may be a member of a novel family of lipolytic enzymes. Several hypothetical protein homologs of EstD2 were found in the database. A hypothetical protein from Phenylobacterium zucineum HLK1, a close homolog of EstD2, displayed lipolytic activity when the corresponding gene was expressed in Escherichia coli. Our results suggest that the other hypothetical protein homologs of EstD2 might also be members of this novel family.
Lee
Myung Hwan
MH
Department of Applied Biology, Dong-A University, Busan, Republic of Korea.
Hong
Kyung Sik
KS
Malhotra
Shweta
S
Park
Ji-Hye
JH
Hwang
Eul Chul
EC
Choi
Hong Kyu
HK
Kim
Young Sup
YS
Tao
Weixin
W
Lee
Seon-Woo
SW
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D058448:Biota; D002087:Butyrates / Q000378:metabolism*; D042063:Caulobacteraceae / Q000235:genetics; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D004950:Esterases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D015870:Gene Expression; D016680:Genome, Bacterial; D015698:Genomic Library; D054892:Metagenome; D008969:Molecular Sequence Data; D010802:Phylogeny; D010957:Plasmids / Q000235:genetics; D056910:Republic of Korea; D058441:Rhizosphere; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology; D013379:Substrate Specificity
D001426:Bacterial Proteins; D002087:Butyrates; D004269:DNA, Bacterial; C033592:4-nitrophenyl butyrate; D004950:Esterases