A Novel Polyester Hydrolase From the Marine Bacterium Pseudomonas aestusnigri – Structural and Functional Insights

Bollinger, Alexander; Thies, Stephan; Knieps-Grünhagen, Esther; Gertzen, Christoph G. W.; Kobus, S.; Höppner, Astrid; Ferrer, Manuel; Gohlke, Holger; Smits, Sander H. J.; Jaeger, Karl‐Erich

doi:10.3389/fmicb.2020.00114

Cited by 229 publications

(176 citation statements)

References 70 publications

Supporting

Mentioning

171

Contrasting

Unclassified

Order By: Relevance

“…S1). Eight of these CEs were first described by Martínez-Martínez et al (17), one CE was identified by Hajighasemi et al (18), one CE was identified recently by Bollinger et al (26), and 15 CEs were newly identified in this study. Of the CEs used in this study, 16 CEs were recovered from genome libraries after naive screening; additionally, 9 CEs were identified through genome sequence searches.…”

Section: Resultssupporting

confidence: 55%

Organic-Solvent-Tolerant Carboxylic Ester Hydrolases for Organic Synthesis

Bollinger

Molitor

Thies

et al. 2020

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Resultssupporting

confidence: 55%

Organic-Solvent-Tolerant Carboxylic Ester Hydrolases for Organic Synthesis

Bollinger

Molitor

Thies

et al. 2020

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

show abstract

“…Νon-biodegradable, alipharomatic polyesters such as PET and PEF can be hydrolyzed by specific enzymes [ 126 , 127 ]. PEF with different Mws was successfully hydrolyzed by Cutinase 1 from Thermobifida cellulosilytica , opening the possibility for the functionalization and recycling of monomers, as FDCA and its oligomers were released during incubation [ 128 ].…”

Section: Biodegradationmentioning

confidence: 99%

Tuning the Properties of Furandicarboxylic Acid-Based Polyesters with Copolymerization: A Review

et al. 2020

View full text Add to dashboard Cite

Polyesters based on 2,5-furandicarboxylic acid (FDCA) are a new class of biobased polymers with enormous interest, both from a scientific and industrial perspective. The commercialization of these polymers is imminent as the pressure for a sustainable economy grows, and extensive worldwide research currently takes place on developing cost-competitive, renewable plastics. The most prevalent method for imparting these polymers with new properties is copolymerization, as many studies have been published over the last few years. This present review aims to summarize the trends in the synthesis of FDCA-based copolymers and to investigate the effectiveness of this approach in transforming them to a more versatile class of materials that could potentially be appropriate for a number of high-end and conventional applications.

show abstract

“…This article is protected by copyright. All rights reserved Purification of PtEst1 was performed by immobilized metal ion affinity chromatography (IMAC) followed by size exclusion chromatography (SEC) as described earlier [28], with minor modifications. The lysis buffer for IMAC was used without addition of imidazole (20 mM Na2HPO4 pH 7.4, 500 mM NaCl) and the washing buffer was used with 10 mM imidazole (20 mM Na2HPO4 pH 7.4, 500 mM NaCl, 10 mM imidazole).…”

Section: Accepted Articlementioning

confidence: 99%

Crystal structures of a novel family IV esterase in free and substrate‐bound form

et al. 2021

Self Cite

View full text Add to dashboard Cite

Bacterial lipolytic enzymes of family IV are homologs of the mammalian hormone‐sensitive lipases (HSL) and have been successfully used for various biotechnological applications. The broad substrate specificity and ability for enantio‐, regio‐, and stereoselective hydrolysis are remarkable features of enzymes from this class. Many crystal structures are available for esterases and lipases, but structures of enzyme–substrate or enzyme–inhibitor complexes are less frequent although important to understand the molecular basis of enzyme–substrate interaction and to rationalize biochemical enzyme characteristics. Here, we report on the structures of a novel family IV esterase isolated from a metagenomic screen, which shows a broad substrate specificity. We solved the crystal structures in the apo form and with a bound substrate analogue at 1.35 and 1.81 Å resolution, respectively. This enzyme named PtEst1 hydrolyzed more than 60 out 96 structurally different ester substrates thus being substrate promiscuous. Its broad substrate specificity is in accord with a large active site cavity, which is covered by an α‐helical cap domain. The substrate analogue methyl 4‐methylumbelliferyl hexylphosphonate was rapidly hydrolyzed by the enzyme leading to a complete inactivation caused by covalent binding of phosphinic acid to the catalytic serine. Interestingly, the alcohol leaving group 4‐methylumbelliferone was found remaining in the active site cavity, and additionally, a complete inhibitor molecule was found at the cap domain next to the entrance of the substrate tunnel. This unique situation allowed gaining valuable insights into the role of the cap domain for enzyme–substrate interaction of esterases belonging to family IV.DatabaseStructural data of PtEst1 are available in the worldwide protein data bank (https://www.rcsb.org) under the accession codes: 6Z68 (apo‐PtEst1) and 6Z69 (PtEst1‐inhibitor complex).

show abstract

A Novel Polyester Hydrolase From the Marine Bacterium Pseudomonas aestusnigri – Structural and Functional Insights

Cited by 229 publications

References 70 publications

Organic-Solvent-Tolerant Carboxylic Ester Hydrolases for Organic Synthesis

Organic-Solvent-Tolerant Carboxylic Ester Hydrolases for Organic Synthesis

Tuning the Properties of Furandicarboxylic Acid-Based Polyesters with Copolymerization: A Review

Crystal structures of a novel family IV esterase in free and substrate‐bound form

Contact Info

Product

Resources

About