Fernandez-Lopez L

References (7)

Title : Transforming an esterase into an enantioselective catecholase through bioconjugation of a versatile metal-chelating inhibitor - Fernandez-Lopez_2023_Chem.Commun.(Camb)__
Author(s) : Fernandez-Lopez L , Cea-Rama I , Alvarez-Malmagro J , Ressmann AK , Gonzalez-Alfonso JL , Coscolin C , Shahgaldian P , Plou FJ , Modregger J , Pita M , Sanz-Aparicio J , Ferrer M
Ref : Chem Commun (Camb) , : , 2023
Abstract : Metal complexes introduced into protein scaffolds can generate versatile biomimetic catalysts endowed with a variety of catalytic properties. Here, we synthesized and covalently bound a bipyridinyl derivative to the active centre of an esterase to generate a biomimetic catalyst that shows catecholase activity and enantioselective catalytic oxidation of (+)-catechin.
ESTHER : Fernandez-Lopez_2023_Chem.Commun.(Camb)__
PubMedSearch : Fernandez-Lopez_2023_Chem.Commun.(Camb)__
PubMedID: 37376994
Gene_locus related to this paper: 9zzzz-a0a2k8jn75

Title : Enhancing the Hydrolytic Activity of a Lipase towards Larger Triglycerides through Lid Domain Engineering - Fernandez-Lopez_2023_Int.J.Mol.Sci_24_13768
Author(s) : Fernandez-Lopez L , Roda S , Robles-Martin A , Munoz-Tafalla R , Almendral D , Ferrer M , Guallar V
Ref : Int J Mol Sci , 24 : , 2023
Abstract : Lipases have valuable potential for industrial use, particularly those mostly active against water-insoluble substrates, such as triglycerides composed of long-carbon chain fatty acids. However, in most cases, engineered variants often need to be constructed to achieve optimal performance for such substrates. Protein engineering techniques have been reported as strategies for improving lipase characteristics by introducing specific mutations in the cap domain of esterases or in the lid domain of lipases or through lid domain swapping. Here, we improved the lipase activity of a lipase (WP_075743487.1, or Lip(MRD)) retrieved from the Marine Metagenomics MarRef Database and assigned to the Actinoalloteichus genus. The improvement was achieved through site-directed mutagenesis and by substituting its lid domain (FRGTEITQIKDWLTDA) with that of Rhizopus delemar lipase (previously R. oryzae; UniProt accession number, I1BGQ3) (FRGTNSFRSAITDIVF). The results demonstrated that the redesigned mutants gain activity against bulkier triglycerides, such as glyceryl tridecanoate and tridodecanoate, olive oil, coconut oil, and palm oil. Residue W89 (Lip(MRD) numbering) appears to be key to the increase in lipase activity, an increase that was also achieved with lid swapping. This study reinforces the importance of the lid domains and their amino acid compositions in determining the substrate specificity of lipases, but the generalization of the lid domain swapping between lipases or the introduction of specific mutations in the lid domain to improve lipase activity may require further investigation.
ESTHER : Fernandez-Lopez_2023_Int.J.Mol.Sci_24_13768
PubMedSearch : Fernandez-Lopez_2023_Int.J.Mol.Sci_24_13768
PubMedID: 37762071
Gene_locus related to this paper: 9pseu-a0a1l7F3D7

Title : Assigning Functions of Unknown Enzymes by High-Throughput Enzyme Characterization - Molina-Espeja_2023_Methods.Mol.Biol_2555_181
Author(s) : Molina-Espeja P , Fernandez-Lopez L , Golyshin PN , Ferrer M
Ref : Methods Mol Biol , 2555 :181 , 2023
Abstract : The discovery of new enzymes is strongly enabled by the implementation of high-throughput screening methods to detect enzymatic activity in single organisms or clone expression libraries, or to benchmark their performances against known prototypes. In this chapter, a number of methods, applicable at high-throughput scale, are described that allow the screening and characterization of enzymes relevant to biotechnology, particularly, ester-hydrolases (esterases, lipases, phospholipases, and polyester hydrolases).
ESTHER : Molina-Espeja_2023_Methods.Mol.Biol_2555_181
PubMedSearch : Molina-Espeja_2023_Methods.Mol.Biol_2555_181
PubMedID: 36306087

Title : Metagenomic Mining for Esterases in the Microbial Community of Los Rueldos Acid Mine Drainage Formation - Vidal_2022_Front.Microbiol_13_868839
Author(s) : Vidal P , Martinez-Martinez M , Fernandez-Lopez L , Roda S , Mendez-Garcia C , Golyshina OV , Guallar V , Pelaez AI , Ferrer M
Ref : Front Microbiol , 13 :868839 , 2022
Abstract : Acid mine drainage (AMD) systems are extremely acidic and are metal-rich formations inhabited by relatively low-complexity communities of acidophiles whose enzymes remain mostly uncharacterized. Indeed, enzymes from only a few AMD sites have been studied. The low number of available cultured representatives and genome sequences of acidophiles inhabiting AMDs makes it difficult to assess the potential of these environments for enzyme bioprospecting. In this study, using naive and in silico metagenomic approaches, we retrieved 16 esterases from the alpha/beta-hydrolase fold superfamily with the closest match from uncultured acidophilic Acidobacteria, Actinobacteria (Acidithrix, Acidimicrobium, and Ferrimicrobium), Acidiphilium, and other Proteobacteria inhabiting the Los Rueldos site, which is a unique AMD formation in northwestern Spain with a pH of -2. Within this set, only two polypeptides showed high homology (99.4%), while for the rest, the pairwise identities ranged between 4 and 44.9%, suggesting that the diversity of active polypeptides was dominated not by a particular type of protein or highly similar clusters of proteins, but by diverse non-redundant sequences. The enzymes exhibited amino acid sequence identities ranging from 39 to 99% relative to homologous proteins in public databases, including those from other AMDs, thus indicating the potential novelty of proteins associated with a specialized acidophilic community. Ten of the 16 hydrolases were successfully expressed in Escherichia coli. The pH for optimal activity ranged from 7.0 to 9.0, with the enzymes retaining 33-68% of their activities at pH 5.5, which was consistent with the relative frequencies of acid residues (from 54 to 67%). The enzymes were the most active at 30-65 degreesC, retaining 20-61% of their activity under the thermal conditions characterizing Los Rueldos (13.8 +/- 0.6 degreesC). The analysis of the substrate specificity revealed the capacity of six hydrolases to efficiently degrade (up to 1,652 +/- 75 U/g at pH 8.0 and 30 degreesC) acrylic- and terephthalic-like [including bis(2-hydroxyethyl)-terephthalate, BHET] esters, and these enzymes could potentially be of use for developing plastic degradation strategies yet to be explored. Our assessment uncovers the novelty and potential biotechnological interest of enzymes present in the microbial populations that inhibit the Los Rueldos AMD system.
ESTHER : Vidal_2022_Front.Microbiol_13_868839
PubMedSearch : Vidal_2022_Front.Microbiol_13_868839
PubMedID: 35663881
Gene_locus related to this paper: 9zzzz-t1a3k4 , 9zzzz-t1ci96 , 9zzzz-t1b379 , 9zzzz-t1be47 , 9zzzz-t1d4I7 , 9actn-KY010298 , 9bact-KY010297

Title : EP-Pred: A Machine Learning Tool for Bioprospecting Promiscuous Ester Hydrolases - Xiang_2022_Biomolecules_12_
Author(s) : Xiang R , Fernandez-Lopez L , Robles-Martin A , Ferrer M , Guallar V
Ref : Biomolecules , 12 : , 2022
Abstract : When bioprospecting for novel industrial enzymes, substrate promiscuity is a desirable property that increases the reusability of the enzyme. Among industrial enzymes, ester hydrolases have great relevance for which the demand has not ceased to increase. However, the search for new substrate promiscuous ester hydrolases is not trivial since the mechanism behind this property is greatly influenced by the active site's structural and physicochemical characteristics. These characteristics must be computed from the 3D structure, which is rarely available and expensive to measure, hence the need for a method that can predict promiscuity from sequence alone. Here we report such a method called EP-pred, an ensemble binary classifier, that combines three machine learning algorithms: SVM, KNN, and a Linear model. EP-pred has been evaluated against the Lipase Engineering Database together with a hidden Markov approach leading to a final set of ten sequences predicted to encode promiscuous esterases. Experimental results confirmed the validity of our method since all ten proteins were found to exhibit a broad substrate ambiguity.
ESTHER : Xiang_2022_Biomolecules_12_
PubMedSearch : Xiang_2022_Biomolecules_12_
PubMedID: 36291739

Title : Computationally Driven Rational Design of Substrate Promiscuity on Serine Ester Hydrolases - Roda_2021_ACS.Catal_11_3590
Author(s) : Roda S , Fernandez-Lopez L , Canadas R , Santiago G , Ferrer M , Guallar V
Ref : ACS Catal , 11 :3590 , 2021
Abstract : Enzymes with a broad substrate specificity are of great interest both at the basic and applied level. Understanding the main parameters that make an enzyme substrate ambiguous could be thus important not only for their selection from the ever-increasing amount of sequencing data but also for engineering a more substrate promiscuous variant. This issue, which remains unresolved, was herein investigated by targeting a serine ester hydrolase (EH102), which exhibits a narrow substrate spectrum, being only capable of hydrolyzing 16 out of 96 esters tested. By using a modeling approach, we demonstrated that one can rationalize active site parameters defining substrate promiscuity, and that based on them the substrate specificity can be significantly altered. This was accomplished by designing two variants, EH102DM2 and EH102TM2, that hydrolyze 51 and 63 esters, respectively, while maintaining similar or higher turnover rates compared to the original enzyme. We hypothesized that the parameters identified here (the volume, size, exposure, enclosure, hydrophobicity, and hydrophilicity of the active site cavity and its tightness) can serve in the future to expand the substrate spectra of esterases and thus expand their use in biotechnology and synthetic chemistry.
ESTHER : Roda_2021_ACS.Catal_11_3590
PubMedSearch : Roda_2021_ACS.Catal_11_3590

Title : Genetically engineered proteins with two active sites for enhanced biocatalysis and synergistic chemo- and biocatalysis - Alonso_2020_Nat.Catal_3_319
Author(s) : Alonso S , Santiago G , Cea-Rama I , Fernandez-Lopez L , Coscolin C , Modregger J , Ressmann A , Martinez-Martinez M , Marrero H , Bargiela R , Pita M , Gonzalez-Alfonso JL , Briand M , Rojo D , Barbas C , Plou FJ , Golyshin PN , Shahgaldian P , Sanz-Aparicio J , Guallar V , Ferrer M
Ref : Nature Catalysis , 3 :319 , 2019
Abstract : Enzyme engineering has allowed not only the de novo creation of active sites catalysing known biological reactions with rates close to diffusion limits, but also the generation of abiological sites performing new-to-nature reactions. However, the catalytic advantages of engineering multiple active sites into a single protein scaffold are yet to be established. Here, we report on pro-teins with two active sites of biological and/or abiological origin, for improved natural and non-natural catalysis. The approach increased the catalytic properties, such as enzyme efficiency, substrate scope, stereoselectivity and optimal temperature window, of an esterase containing two biological sites. Then, one of the active sites was metamorphosed into a metal-complex chemocatalytic site for oxidation and Friedel-Crafts alkylation reactions, facilitating synergistic chemo- and biocatalysis in a single protein. The transformations of 1-naphthyl acetate into 1,4-naphthoquinone (conversion approx. 100%) and vinyl crotonate and benzene into 3-phenylbutyric acid (>=83%; e.e. >99.9%) were achieved in one pot with this artificial multifunc-tional metalloenzyme.
ESTHER : Alonso_2020_Nat.Catal_3_319
PubMedSearch : Alonso_2020_Nat.Catal_3_319
Gene_locus related to this paper: 9bact-LAE6