Hyvonen M

References (2)

Title : From a metagenomic source to a high-resolution structure of a novel alkaline esterase - Pereira_2017_Appl.Microbiol.Biotechnol_101_4935
Author(s) : Pereira MR , Maester TC , Mercaldi GF , de Macedo Lemos EG , Hyvonen M , Balan A
Ref : Applied Microbiology & Biotechnology , 101 :4935 , 2017
Abstract : Esterases catalyze the cleavage and formation of ester bonds and are members of the diverse family of alpha/beta hydrolase fold. They are useful in industries from different sectors, such as food, detergent, fine chemicals, and biofuel production. In a previous work, 30 positive clones for lipolytic activity were identified from a metagenomic library of a microbial consortium specialized in diesel oil degradation. In this study, a putative gene encoding an esterase/lipase, denominated est8, has been cloned and the corresponding protein expressed recombinantly, purified to homogeneity and characterized functional and structurally. We show that the protein codified by est8 gene, denominated Est8, is an alkaline esterase with high catalytic efficiency against p-nitrophenyl acetate and stable in the presence of up to 10% dimethyl sulfoxide. The three-dimensional structure of Est8 was determined at 1.85-A resolution, allowing the characterization of the substrate-binding pocket and features that rationalize the preference of Est8 for short-chain substrates. In an attempt to increase the size of ligand-binding pocket and enzyme activity against distinct substrates of long chain, we mutated two residues (Met213 and Phe217) that block the substrate channel. A small increase in the reaction velocity for p-nitrophenyl butyrate and p-nitrophenyl valerate hydrolysis was observed. Activity against p-nitrophenyl acetate was reduced. The functional and structural characterization of Est8 is explored in comparison with orthologues.
ESTHER : Pereira_2017_Appl.Microbiol.Biotechnol_101_4935
PubMedSearch : Pereira_2017_Appl.Microbiol.Biotechnol_101_4935
PubMedID: 28331945
Gene_locus related to this paper: 9zzzz-Est8

Title : Ultrahigh-throughput discovery of promiscuous enzymes by picodroplet functional metagenomics - Colin_2015_Nat.Commun_6_10008
Author(s) : Colin PY , Kintses B , Gielen F , Miton CM , Fischer G , Mohamed MF , Hyvonen M , Morgavi DP , Janssen DB , Hollfelder F
Ref : Nat Commun , 6 :10008 , 2015
Abstract : Unculturable bacterial communities provide a rich source of biocatalysts, but their experimental discovery by functional metagenomics is difficult, because the odds are stacked against the experimentor. Here we demonstrate functional screening of a million-membered metagenomic library in microfluidic picolitre droplet compartments. Using bait substrates, new hydrolases for sulfate monoesters and phosphotriesters were identified, mostly based on promiscuous activities presumed not to be under selection pressure. Spanning three protein superfamilies, these break new ground in sequence space: promiscuity now connects enzymes with only distantly related sequences. Most hits could not have been predicted by sequence analysis, because the desired activities have never been ascribed to similar sequences, showing how this approach complements bioinformatic harvesting of metagenomic sequencing data. Functional screening of a library of unprecedented size with excellent assay sensitivity has been instrumental in identifying rare genes constituting catalytically versatile hubs in sequence space as potential starting points for the acquisition of new functions.
ESTHER : Colin_2015_Nat.Commun_6_10008
PubMedSearch : Colin_2015_Nat.Commun_6_10008
PubMedID: 26639611
Gene_locus related to this paper: 9bact-KP212148