Esterases receive special attention because of their wide distribution in biological systems and environments and their importance for physiology and chemical synthesis. The prediction of esterases' substrate promiscuity level from sequence data and the molecular reasons why certain such enzymes are more promiscuous than others remain to be elucidated. This limits the surveillance of the sequence space for esterases potentially leading to new versatile biocatalysts and new insights into their role in cellular function. Here, we performed an extensive analysis of the substrate spectra of 145 phylogenetically and environmentally diverse microbial esterases, when tested with 96 diverse esters. We determined the primary factors shaping their substrate range by analyzing substrate range patterns in combination with structural analysis and protein-ligand simulations. We found a structural parameter that helps rank (classify) the promiscuity level of esterases from sequence data at 94% accuracy. This parameter, the active site effective volume, exemplifies the topology of the catalytic environment by measuring the active site cavity volume corrected by the relative solvent accessible surface area (SASA) of the catalytic triad. Sequences encoding esterases with active site effective volumes (cavity volume/SASA) above a threshold show greater substrate spectra, which can be further extended in combination with phylogenetic data. This measure provides also a valuable tool for interrogating substrates capable of being converted. This measure, found to be transferred to phosphatases of the haloalkanoic acid dehalogenase superfamily and possibly other enzymatic systems, represents a powerful tool for low-cost bioprospecting for esterases with broad substrate ranges, in large scale sequence data sets.

Biodegradation of synthetic polymers, in particular polyethylene terephthalate (PET), is of great importance, since environmental pollution with PET and other plastics has become a severe global problem. Here, we report on the polyester degrading ability of a novel carboxylic ester hydrolase identified in the genome of the marine hydrocarbonoclastic bacterium Pseudomonas aestusnigri VGXO14 T. The enzyme, designated PE-H, belongs to the type IIa family of PET hydrolytic enzymes as indicated by amino acid sequence homology. It was produced in Escherichia coli, purified and its crystal structure was solved at 1.09 Å resolution representing the first structure of a type IIa PET hydrolytic enzyme. The structure shows a typical α/β-hydrolase fold and high structural homology to known polyester hydrolases. PET hydrolysis was detected at 30 • C with amorphous PET film (PETa), but not with PET film from a commercial PET bottle (PETb). A rational mutagenesis study to improve the PET degrading potential of PE-H yielded variant PE-H (Y250S) which showed improved activity, ultimately also allowing the hydrolysis of PETb. The crystal structure of this variant solved at 1.35 Å resolution allowed to rationalize the improvement of enzymatic activity. A PET oligomer binding model was proposed by molecular docking computations. Our results indicate a significant potential of the marine bacterium P. aestusnigri for PET degradation.

Hydrolases acting on polyesters like cutin, polycaprolactone or polyethylene terephthalate (PET) are of interest for several biotechnological applications like waste treatment, biocatalysis and sustainable polymer modifications. Recent studies suggest that a large variety of such enzymes are still to be identified and explored in a variety of microorganisms, including bacteria of the genus Pseudomonas. For activitybased screening, methods have been established using agar plates which contain nanoparticles of polycaprolactone or PET prepared by solvent precipitation and evaporation. In this protocol article, we describe a straightforward agar plate-based method using emulsifiable artificial polyesters as substrates, namely Impranil â DLN and liquid polycaprolactone diol (PLD). Thereby, the currently quite narrow set of screening substrates is expanded. We also suggest optional pre-screening with short-chain and middle-chainlength triglycerides as substrates to identify enzymes with lipolytic activity to be further tested for polyesterase activity. We applied these assays to experimentally demonstrate polyesterase activity in bacteria from the P. pertucinogena lineage originating from contaminated soils and diverse marine habitats.

Biosurfactants are amphiphilic secondary metabolites produced by microorganisms. Marine bacteria have recently emerged as a rich source for these natural products which exhibit surface-active properties, making them useful for diverse applications such as detergents, wetting and foaming agents, solubilisers, emulsifiers and dispersants. Although precise structural data are often lacking, the already available information deduced from biochemical analyses and genome sequences of marine microbes indicates a high structural diversity including a broad spectrum of fatty acid derivatives, lipoamino acids, lipopeptides and glycolipids. This review aims to summarise biosyntheses and structures with an emphasis on low molecular weight biosurfactants produced by marine microorganisms and describes various biotechnological applications with special emphasis on their role in the bioremediation of oil-contaminated environments. Furthermore, novel exploitation strategies are suggested in an attempt to extend the existing biosurfactant portfolio.

Marine habitats represent a prolific source for molecules of biotechnological interest. In particular, marine bacteria have attracted attention and were successfully exploited for industrial applications. Recently, a group of Pseudomonas species isolated from extreme habitats or living in association with algae or sponges were clustered in the newly established Pseudomonas pertucinogena lineage. Remarkably for the predominantly terrestrial genus Pseudomonas, more than half (9) of currently 16 species within this lineage were isolated from marine or saline habitats. Unlike other Pseudomonas species, they seem to have in common a highly specialized metabolism. Furthermore, the marine members apparently possess the capacity to produce biomolecules of biotechnological interest (e.g. dehalogenases, polyester hydrolases, transaminases). Here, we summarize the knowledge regarding the enzymatic endowment of the marine Pseudomonas pertucinogena bacteria and report on a genomic analysis focusing on the presence of genes encoding esterases, dehalogenases, transaminases and secondary metabolites including carbon storage compounds.

Amine transaminases of the class III ω-TAs are key enzymes for modification of chemical building blocks, but finding those capable of converting bulky ketones and (R) amines is still challenging. Here, by an extensive analysis of the substrate spectra of 10 class III ω-TAs, we identified a number of residues playing a role in determining the access and positioning of bulky ketones, bulky amines, and (R)- and (S) amines, as well as of environmentally relevant polyamines, particularly putrescine. The results presented can significantly expand future opportunities for designing (R)-specific class III ω-TAs to convert valuable bulky ketones and amines, as well as for deepening the knowledge into the polyamine catabolic pathways.

Engineering dual-function single polypeptide catalysts with two abiotic or biotic catalytic entities (or combinations of both) supporting cascade reactions is becoming an important area of enzyme engineering and catalysis. Herein we present the development of a PluriZyme, TR 2 E 2 , with efficient native transaminase (k cat : 69.49 � 1.77 min À 1 ) and artificial esterase (k cat : 3908-0.41 min À 1 ) activities integrated into a single scaffold, and evaluate its utility in a cascade reaction. TR 2 E 2 (pH opt : 8.0-9.5; T opt : 60-65 °C) efficiently converts methyl 3-oxo-4-(2,4,5-trifluorophenyl)butanoate into 3-(R)-amino-4-(2,4,5-trifluorophenyl)butanoic acid, a crucial intermediate for the synthesis of antidiabetic drugs. The reaction proceeds through the conversion of the β-keto ester into the β-keto acid at the hydrolytic site and subsequently into the β-amino acid (e.e. > 99 %) at the transaminase site. The catalytic power of the TR 2 E 2 PluriZyme was proven with a set of β-keto esters, demonstrating the potential of such designs to address bioinspired cascade reactions.

Biocatalysis has emerged as an important tool in synthetic organic chemistry enabling the chemical industry to execute reactions with high regio- or enantioselectivity and under usually mild reaction conditions while avoiding toxic waste. Target substrates and products of reactions catalyzed by carboxylic ester hydrolases are often poorly water soluble and require organic solvents, whereas enzymes are evolved by nature to be active in cells, i.e., in aqueous rather than organic solvents. Therefore, biocatalysts that withstand organic solvents are urgently needed. Current strategies to identify such enzymes rely on laborious tests carried out by incubation in different organic solvents and determination of residual activity. Here, we describe a simple assay useful for screening large libraries of carboxylic ester hydrolases for resistance and activity in water-miscible organic solvents. We have screened a set of 26 enzymes, most of them identified in this study, with four different water-miscible organic solvents. The triglyceride tributyrin was used as a substrate, and fatty acids released by enzymatic hydrolysis were detected by a pH shift indicated by the indicator dye nitrazine yellow. With this strategy, we succeeded in identifying a novel highly organic-solvent-tolerant esterase from Pseudomonas aestusnigri. In addition, the newly identified enzymes were tested with sterically demanding substrates, which are common in pharmaceutical intermediates, and two enzymes from Alcanivorax borkumensis were identified which outcompeted the gold standard ester hydrolase CalB from Candida antarctica. IMPORTANCE Major challenges hampering biotechnological applications of esterases include the requirement to accept nonnatural and chemically demanding substrates and the tolerance of the enzymes toward organic solvents which are often required to solubilize such substrates. We describe here a high-throughput screening strategy to identify novel organic-solvent-tolerant carboxylic ester hydrolases (CEs). Among these enzymes, CEs active against water-insoluble bulky substrates were identified. Our results thus contribute to fostering the identification and biotechnological application of CEs.