Gene cloning and characterization of a novel esterase from activated sludge metagenome.
A metagenomic library was prepared using pCC2FOS vector containing about 3.0 Gbp of community DNA from the microbial assemblage of activated sludge. Screening of a part of the un-amplified library resulted in the finding of 1 unique lipolytic clone capable of hydrolyzing tributyrin, in which an esterase gene was identified. This esterase/lipase gene consists of 834 bp and encodes a polypeptide (designated EstAS) of 277 amino acid residuals with a molecular mass of 31 kDa. Sequence analysis indicated that it showed 33% and 31% amino acid identity to esterase/lipase from Gemmata obscuriglobus UQM 2246 (ZP_02733109) and Yarrowia lipolytica CLIB122 (XP_504639), respectively; and several conserved regions were identified, including the putative active site, HSMGG, a catalytic triad (Ser92, His125 and Asp216) and a LHYFRG conserved motif. The EstAS was overexpressed, purified and shown to hydrolyse p-nitrophenyl (NP) esters of fatty acids with short chain lengths (< or = C8). This EstAS had optimal temperature and pH at 35 degrees C and 9.0, respectively, by hydrolysis of p-NP hexanoate. It also exhibited the same level of stability over wide temperature and pH ranges and in the presence of metal ions or detergents. The high level of stability of esterase EstAS with its unique substrate specificities make itself highly useful for biotechnological applications.
Zhang
Tao
T
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China. zhangt218@hotmail.com
Han
Wen-Jun
WJ
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000378:metabolism; D004950:Esterases / Q000235:genetics* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D054892:Metagenome; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012722:Sewage / Q000382:microbiology*; D013379:Substrate Specificity; D013696:Temperature; D014280:Triglycerides / Q000378:metabolism; D025062:Yarrowia / Q000145:classification / Q000201:enzymology
D004269:DNA, Bacterial; D012722:Sewage; D014280:Triglycerides; D004950:Esterases; C005830:tributyrin