Screening for novel lipolytic enzymes from uncultured soil microorganisms.
The construction and screening of metagenomic libraries constitute a valuable resource for obtaining novel biocatalysts. In this work, we present the construction of a metagenomic library in Escherichia coli using fosmid and microbial DNA directly isolated from forest topsoil and screened for lipolytic enzymes. The library consisted of 33,700 clones with an average DNA insert size of 35 kb. Eight unique lipolytic active clones were obtained from the metagenomic library on the basis of tributyrin hydrolysis. Subsequently, secondary libraries in a high-copy-number plasmid were generated to select lipolytic subclones and to characterize the individual genes responsible for the lipolytic activity. DNA sequence analysis of six genes revealed that the enzymes encoded by the metagenomic genes for lipolytic activity were novel with 34-48% similarity to known enzymes. They had conserved sequences similar to those in the hormone-sensitive lipase family. Based on their deduced amino acid similarity, the six genes encoding lipolytic enzymes were further divided into three subgroups, the identities among which ranged from 33% to 45%. The six predicted gene products were successfully expressed in E. coli and secreted into the culture broth. Most of the secreted enzymes showed a catalytic activity for hydrolysis of p-nitrophenyl butyrate (C(4)) but not p-nitrophenyl palmitate (C(16)).
Lee
Seon-Woo
SW
Biological Function Research Team, Korea Research Institute of Chemical Technology, 100 Jang-Dong, Yuseong-Gu, 305-343, Daejeon, South Korea. seonlee@krict.re.kr.
Won
Keehoon
K
Lim
He Kyoung
HK
Kim
Jin-Cheol
JC
Choi
Gyung Ja
GJ
Cho
Kwang Yun
KY
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D002087:Butyrates / Q000378:metabolism; D003001:Cloning, Molecular; D017124:Conserved Sequence / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010168:Palmitates / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology; D013379:Substrate Specificity; D014280:Triglycerides / Q000378:metabolism*
D002087:Butyrates; D004269:DNA, Bacterial; D010168:Palmitates; D011994:Recombinant Proteins; D014280:Triglycerides; C050015:4-nitrophenyl palmitate; C033592:4-nitrophenyl butyrate; D008049:Lipase; C005830:tributyrin