Lipase diversity in glacier soil based on analysis of metagenomic DNA fragments and cell culture.
Lipase diversity in glacier soil was assessed by culture independent metagenomic DNA fragment screening and confirmed by cell culture experiments. A set of degenerate PCR primers specific for lipases of the hormone-sensitive lipase family was designed based on conserved motifs and used to directly PCR amplify metagenomic DNA from glacier soil. These products were used to construct a lipase fragment clone library. Among the 300 clones sequenced for the analysis, 201 clones encoding partial lipases shared 51-82%identity to known lipases in GenBank. Based on a phylogenetic analysis, five divergent clusters were established, one of which may represent a previously unidentified lipase subfamily. In the culture study, 11 lipase-producing bacteria were selectively isolated and characterized by 16S rDNA sequences. Using the above mentioned degenerate primers, seven lipase gene fragments were cloned, but not all of them could be accounted for by the clones in the library. Two full-length lipase genes obtained by TAIL-PCR were expressed in Pichia pastoris and characterized. Both were authentic lipases with optimum temperatures of <or=40 degrees. Our study indicates the abundant lipase diversity in glacier soil as well as the feasibility of sequence-based screening in discovering new lipase genes from complex environmental samples.
Yuhong
Zhang
Z
Key Laboratory of Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P. R. China.
Shi
Pengjun
P
Liu
Wanli
W
Meng
Kun
K
Bai
Yingguo
Y
Wang
Guozeng
G
Zhan
Zhichun
Z
Yao
Bin
B
D000595:Amino Acid Sequence; D002681:China; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D017931:DNA Primers; D014644:Genetic Variation; D046448:Ice Cover; D008049:Lipase / Q000235:genetics*; D008969:Molecular Sequence Data; D010802:Phylogeny; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics; D015183:Restriction Mapping; D012240:Rhodococcus / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012987:Soil / Q000032:analysis*
D017931:DNA Primers; D012987:Soil; D004247:DNA; D008049:Lipase