Cloning and biochemical characterization of a novel lipolytic gene from activated sludge metagenome, and its gene product.
In this study, a putative esterase, designated EstMY, was isolated from an activated sludge metagenomic library. The lipolytic gene was subcloned and expressed in Escherichia coli BL21 using the pET expression system. The gene estMY contained a 1,083 bp open reading frame (ORF) encoding a polypeptide of 360 amino acids with a molecular mass of 38 kDa. Sequence analysis indicated that it showed 71% and 52% amino acid identity to esterase/lipase from marine metagenome (ACL67845) and Burkholderia ubonensis Bu (ZP_02382719), respectively; and several conserved regions were identified, including the putative active site, GDSAG, a catalytic triad (Ser203, Asp301, and His327) and a HGGG conserved motif (starting from His133). The EstMY was determined to hydrolyse p-nitrophenyl (NP) esters of fatty acids with short chain lengths (≤C8). This EstMY exhibited the highest activity at 35°C and pH 8.5 respectively, by hydrolysis of p-NP caprylate. It also exhibited the same level of activity over wide temperature and pH spectra and in the presence of metal ions or detergents. The high level of stability of esterase EstMY with unique substrate specificities makes it highly valuable for downstream biotechnological applications.
Mianyang Normal University, PR China. hanwenjun_79@hotmail.com
Li
L
JunGang
Zhang
Z
KeGui
Han
H
WenJun
D000595:Amino Acid Sequence; D002210:Caprylates / Q000378:metabolism; D020134:Catalytic Domain; D003001:Cloning, Molecular; D003902:Detergents / Q000737:chemistry; D004950:Esterases / Q000737:chemistry* / Q000145:classification / Q000235:genetics; D015698:Genomic Library; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008066:Lipolysis / Q000235:genetics*; D054892:Metagenome; D008670:Metals / Q000737:chemistry; D008969:Molecular Sequence Data; D010802:Phylogeny; D016415:Sequence Alignment; D012722:Sewage / Q000382:microbiology*; D013379:Substrate Specificity; D013696:Temperature
D002210:Caprylates; D003902:Detergents; D008670:Metals; D012722:Sewage; D004950:Esterases