(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) > cellular organisms: NE > Bacteria: NE > environmental samples: NE > uncultured bacterium FLS12: NE
Molecular evidence
Database
No mutation 1 structure: 4RGY: Structural and functional analysis of a low-temperature-active alkaline esterase from South China Sea marine sediment microbial metagenomic library No kinetic
LegendThis sequence has been compared to family alignement (MSA) red => minority aminoacid blue => majority aminoacid color intensity => conservation rate title => sequence position(MSA position)aminoacid rate Catalytic site Catalytic site in the MSA MALFQCDFFSDVLGLSTSMTVILPQETTGQIGMAGGSERREHPTLFLLHG LSDDHTIWLRRTSIERYVAEMGLAVVMPAVHRSFYTDMAHGLQYWTFISE ELPALARSFFPLATAREDTFVAGLSMGGYGALKLGMRHPERFAAAASLSG ALDITFDPAEHIAMEDDVWVAEQRNIFGDLAALPGSDHDLFALAERMAQS DGPVPKLYQCCGTEDFLYEDNVRFRDHVRGLGLDFMYEESPGEHEWGYWD AQIQRVLAWLPLRPPGTAPA
References
Title: Structural and functional analysis of a low-temperature-active alkaline esterase from South China Sea marine sediment microbial metagenomic library Hu Y, Liu Y, Li J, Feng Y, Lu N, Zhu B, Xue S Ref: J Ind Microbiol Biotechnol, 42:1449, 2015 : PubMed
A low-temperature-active alkaline esterase, Est12, from a marine sediment metagenomic fosmid library was identified. Est12 prefers short- and middle-chain p-nitrophenol esters as substrate with optimum temperature and pH value of 50 degrees C and 9.0, respectively, and nearly 50 % of maximum activity retained at 5 degrees C. The hydrolysis activity of Est12 was stable at 40 degrees C. Ca(2+) especially activated the activity of Est12 to about 151 % of the control. DEPC and PMSF inhibited the activity of Est12 to 34 and 25 %, respectively. In addition, Est12 was more tolerable to methanol compared to other organic solvents tested. The crystal structure of Est12 at 1.39 A resolution showed that the cap domain which is composed of an alpha-helix and a flexible region resulted in a relatively wide spectrum of substrate, with p-nitrophenol caproate as the preferred one. Furthermore, the flexible cap domain and the high percentage of Gly, Ser, and Met may play important roles in the adaptation of Est12 to low temperature.
Metagenomic cloning is a powerful tool for the discovery of novel genes and biocatalysts from environmental microorganisms. Based on activity screening of a marine sediment microbial metagenomic library, a total of 19 fosmid clones showing lipolytic activity were identified. After subcloning, 15 different lipolytic genes were obtained; their encoded proteins showed 32-68% amino acid identity with proteins in the database. Multiple sequence alignment and phylogenetic tree analysis demonstrated that most of these predicted proteins are new members of known families of bacterial lipolytic enzymes. However, two proteins, FLS18C and FLS18D, could not be assigned to any known family, thus probably representing a novel family of the bacterial lipolytic enzyme. The activity assay results indicated that most of these lipolytic enzymes showed optimum temperature for hydrolysis at 40-50 degrees C with p-nitrophenol butyrate as a substrate. The lipolytic gene fls18D was overexpressed, and the resulting protein FLS18D was characterized as an alkaline esterase. Furthermore, the whole sequence of fosmid pFL18 containing FLS18C and FLS18D was shotgun sequenced, and a total of 26 ORFs on it were analyzed and annotated.
uncultured bacterium FLS12 Esterase
