Pfaff L

References (9)

Title : High-Throughput Screening for Thermostable Polyester Hydrolases - Branson_2023_Methods.Mol.Biol_2555_153
Author(s) : Branson Y , Badenhorst CPS , Pfaff L , Buchmann C , Wei R , Bornscheuer UT
Ref : Methods Mol Biol , 2555 :153 , 2023
Abstract : Due to the promise of more sustainable recycling of plastics through biocatalytic degradation, the search for and engineering of polyester hydrolases have become a thriving field of research. Furthermore, among other methods, halo formation assays have become popular for the detection of polyester-hydrolase activity. However, established halo-formation assays are limited in their ability to screen for thermostable enzymes, which are particularly important for efficient plastic degradation. The incubation of screening plates at temperatures above 50 degreesC leads to cell lysis and death. Therefore, equivalent master plates are commonly required to maintain and identify the active strains found on the screening plates. This replica plating procedure necessitates 20- to 60-fold more plates than our method, assuming the screened library is transferred to 384-well microtiter plates or 96-well microtiter plates, respectively, to organize the colonies in a retraceable manner, thus significantly lowering throughput. Here, we describe a halo formation assay that is designed to screen thermostable polyesterases independent of master plates and colony replication, thereby markedly reducing the workload and increasing the throughput.
ESTHER : Branson_2023_Methods.Mol.Biol_2555_153
PubMedSearch : Branson_2023_Methods.Mol.Biol_2555_153
PubMedID: 36306085

Title : Structural Insights into (Tere)phthalate-Ester Hydrolysis by a Carboxylesterase and Its Role in Promoting PET Depolymerization - von Haugwitz_2022_ACS.Catal_12_15259
Author(s) : von Haugwitz G , Han X , Pfaff L , Li Q , Wei H , Gao J , Methling K , Ao Y , Brack Y , Jan Mican J , Feiler CG , Weiss MS , Bednar D , Palm GJ , Lalk M , Lammers M , Damborsky J , Weber G , Liu W , Bornscheuer UT , Wei R
Ref : ACS Catal , 12 :15259 , 2022
Abstract : TfCa, a promiscuous carboxylesterase from Thermobifida fusca, was found to hydrolyze polyethylene terephthalate (PET) degradation intermediates such as bis(2-hydroxyethyl) terephthalate (BHET) and mono-(2-hydroxyethyl)-terephthalate (MHET). In this study, we elucidated the structures of TfCa in its apo form, as well as in complex with a PET monomer analogue and with BHET. The structurefunction relationship of TfCa was investigated by comparing its hydrolytic activity on various ortho- and para-phthalate esters of different lengths. Structure-guided rational engineering of amino acid residues in the substrate-binding pocket resulted in the TfCa variant I69W/V376A (WA), which showed 2.6-fold and 3.3-fold higher hydrolytic activity on MHET and BHET, respectively, than the wild-type enzyme. TfCa or its WA variant was mixed with a mesophilic PET depolymerizing enzyme variant [Ideonella sakaiensis PETase (IsPETase) PM] to degrade PET substrates of various crystallinity. The dual enzyme system with the wild-type TfCa or its WA variant produced up to 11-fold and 14-fold more terephthalate (TPA) than the single IsPETase PM, respectively. In comparison to the recently published chimeric fusion protein of IsPETase and MHETase, our system requires 10% IsPETase and one-fourth of the reaction time to yield the same amount of TPA under similar PET degradation conditions. Our simple dual enzyme system reveals further advantages in terms of cost-effectiveness and catalytic efficiency since it does not require time-consuming and expensive cross-linking and immobilization approaches.
ESTHER : von Haugwitz_2022_ACS.Catal_12_15259
PubMedSearch : von Haugwitz_2022_ACS.Catal_12_15259
PubMedID: 36570084
Gene_locus related to this paper: thefu-1831

Title : Engineering and evaluation of thermostable IsPETase variants for PET degradation - Brott_2022_Eng.Life.Sci_22_192
Author(s) : Brott S , Pfaff L , Schuricht J , Schwarz JN , Bottcher D , Badenhorst CPS , Wei R , Bornscheuer UT
Ref : Eng Life Sciences , 22 :192 , 2022
Abstract : Polyethylene terephthalate (PET) is a mass-produced petroleum-based synthetic polymer. Enzymatic PET degradation using, for example, Ideonella sakaiensis PETase (IsPETase) can be a more environmentally friendly and energy-saving alternative to the chemical recycling of PET. However, IsPETase is a mesophilic enzyme with an optimal reaction temperature lower than the glass transition temperature (T (g)) of PET, where the amorphous polymers can be readily accessed for enzymatic breakdown. In this study, we used error-prone PCR to generate a mutant library based on a thermostable triple mutant (TM) of IsPETase. The library was screened against the commercially available polyester-polyurethane Impranil DLN W 50 for more thermostable IsPETase variants, yielding four variants with higher melting points. The most promising IsPETaseTM(K95N/F201I) variant had a 5.0 degreesC higher melting point than IsPETaseTM. Although this variant showed a slightly lower activity on PET at lower incubation temperatures, its increased thermostability makes it a more active PET hydrolase at higher reaction temperatures up to 60 degreesC. Several other variants were compared and combined with selected previously published IsPETase mutants in terms of thermostability and hydrolytic activity against PET nanoparticles and amorphous PET films. Our findings indicate that thermostability is one of the most important characteristics of an effective PET hydrolase.
ESTHER : Brott_2022_Eng.Life.Sci_22_192
PubMedSearch : Brott_2022_Eng.Life.Sci_22_192
PubMedID: 35382549
Gene_locus related to this paper: idesa-peth

Title : Multiple Substrate Binding Mode-Guided Engineering of a Thermophilic PET Hydrolase - Pfaff_2022_ACS.Catalysis_12_9790
Author(s) : Pfaff L , Gao J , Li Z , Jackering A , Weber G , Mican J , Chen Y , Dong W , Han X , Feiler CG , Ao YF , Badenhorst CPS , Bednar D , Palm GJ , Lammers M , Damborsky J , Strodel B , Liu W , Bornscheuer UT , Wei R
Ref : ACS Catal , 12 :9790 , 2022
Abstract : Thermophilic polyester hydrolases (PES-H) have recently enabled biocatalytic recycling of the mass-produced synthetic polyester polyethylene terephthalate (PET), which has found widespread use in the packaging and textile industries. The growing demand for efficient PET hydrolases prompted us to solve high-resolution crystal structures of two metagenome-derived enzymes (PES-H1 and PES-H2) and notably also in complex with various PET substrate analogues. Structural analyses and computational modeling using molecular dynamics simulations provided an understanding of how product inhibition and multiple substrate binding modes influence key mechanistic steps of enzymatic PET hydrolysis. Key residues involved in substratebinding and those identified previously as mutational hotspots in homologous enzymes were subjected to mutagenesis. At 72 C, the L92F/Q94Y variant of PES-H1 exhibited 2.3-fold and 3.4-fold improved hydrolytic activity against amorphous PET films and pretreated real-world PET waste, respectively. The R204C/S250C variant of PES-H1 had a 6.4 C higher melting temperature than the wild-type enzyme but retained similar hydrolytic activity. Under optimal reaction conditions, the L92F/Q94Y variant of PES-H1 hydrolyzed low-crystallinity PET materials 2.2-fold more efficiently than LCC ICCG, which was previously the most active PET hydrolase reported in the literature. This property makes the L92F/ Q94Y variant of PES-H1 a good candidate for future applications in industrial plastic r"cycling processes.
ESTHER : Pfaff_2022_ACS.Catalysis_12_9790
PubMedSearch : Pfaff_2022_ACS.Catalysis_12_9790
PubMedID: 35966606
Gene_locus related to this paper: 9firm-PHL7

Title : Molecular and Biochemical Differences of the Tandem and Cold-Adapted PET Hydrolases Ple628 and Ple629, Isolated From a Marine Microbial Consortium - Meyer-Cifuentes_2022_Front.Bioeng.Biotechnol_10_930140
Author(s) : Meyer-Cifuentes IE , Wu P , Zhao Y , Liu W , Neumann-Schaal M , Pfaff L , Barys J , Li Z , Gao J , Han X , Bornscheuer UT , Wei R , Ozturk B
Ref : Front Bioeng Biotechnol , 10 :930140 , 2022
Abstract : Polybutylene adipate terephthalate (PBAT) is a biodegradable alternative to polyethylene and can be broadly used in various applications. These polymers can be degraded by hydrolases of terrestrial and aquatic origin. In a previous study, we identified tandem PETase-like hydrolases (Ples) from the marine microbial consortium I1 that were highly expressed when a PBAT blend was supplied as the only carbon source. In this study, the tandem Ples, Ple628 and Ple629, were recombinantly expressed and characterized. Both enzymes are mesophilic and active on a wide range of oligomers. The activities of the Ples differed greatly when model substrates, PBAT-modified polymers or PET nanoparticles were supplied. Ple629 was always more active than Ple628. Crystal structures of Ple628 and Ple629 revealed a structural similarity to other PETases and can be classified as member of the PETases IIa subclass, alpha/beta hydrolase superfamily. Our results show that the predicted functions of Ple628 and Ple629 agree with the bioinformatic predictions, and these enzymes play a significant role in the plastic degradation by the consortium.
ESTHER : Meyer-Cifuentes_2022_Front.Bioeng.Biotechnol_10_930140
PubMedSearch : Meyer-Cifuentes_2022_Front.Bioeng.Biotechnol_10_930140
PubMedID: 35935485
Gene_locus related to this paper: 9zzzz-Ple628 , 9zzzz-Ple629

Title : Mechanism-Based Design of Efficient PET Hydrolases - Wei_2022_ACS.Catal_12_3382
Author(s) : Wei R , von Haugwitz G , Pfaff L , Mican J , Badenhorst CPS , Liu W , Weber G , Austin HP , Bednar D , Damborsky J , Bornscheuer UT
Ref : ACS Catal , 12 :3382 , 2022
Abstract : Polyethylene terephthalate (PET) is the most widespread synthetic polyester, having been utilized in textile fibers and packaging materials for beverages and food, contributing considerably to the global solid waste stream and environmental plastic pollution. While enzymatic PET recycling and upcycling have recently emerged as viable disposal methods for a circular plastic economy, only a handful of benchmark enzymes have been thoroughly described and subjected to protein engineering for improved properties over the last 16 years. By analyzing the specific material properties of PET and the reaction mechanisms in the context of interfacial biocatalysis, this Perspective identifies several limitations in current enzymatic PET degradation approaches. Unbalanced enzyme-substrate interactions, limited thermostability, and low catalytic efficiency at elevated reaction temperatures, and inhibition caused by oligomeric degradation intermediates still hamper industrial applications that require high catalytic efficiency. To overcome these limitations, successful protein engineering studies using innovative experimental and computational approaches have been published extensively in recent years in this thriving research field and are summarized and discussed in detail here. The acquired knowledge and experience will be applied in the near future to address plastic waste contributed by other mass-produced polymer types (e.g., polyamides and polyurethanes) that should also be properly disposed by biotechnological approaches.
ESTHER : Wei_2022_ACS.Catal_12_3382
PubMedSearch : Wei_2022_ACS.Catal_12_3382
PubMedID: 35368328

Title : Rapid depolymerization of poly(ethylene terephthalate) thin films by a dual-enzyme system and its impact on material properties - Tarazona_2022_Chem.Catal_2_3573
Author(s) : Tarazona NA , Wei R , Brott S , Pfaff L , Bornscheuer UT , Lendlein A , Machatschek R
Ref : Chem Catal , 2 :3573 , 2022
Abstract : Enzymatic hydrolysis holds great promise for plastic waste recycling and upcycling. The interfacial catalysis mode, and the variability of polymer specimen properties under different degradation conditions, add to the complexity and difficulty of understanding polymer cleavage and engineering better biocatalysts. We present a systemic approach to studying the enzyme-catalyzed surface erosion of poly(ethylene terephthalate) (PET) while monitoring/controlling operating conditions in real time with simultaneous detection of mass loss and changes in viscoelastic behavior. PET nanofilms placed on water showed a porous morphology and a thickness-dependent glass transition temperature (T(g)) between 40 degreesC and 44 degreesC, which is >20 degreesC lower than the T(g) of bulk amorphous PET. Hydrolysis by a dual-enzyme system containing thermostabilized variants of Ideonella sakaiensis PETase and MHETase resulted in a maximum depolymerization of 70% in 1 h at 50 degreesC. We demonstrate that increased accessible surface area, amorphization, and T(g) reduction speed up PET degradation while simultaneously lowering the threshold for degradation-induced crystallization.
ESTHER : Tarazona_2022_Chem.Catal_2_3573
PubMedSearch : Tarazona_2022_Chem.Catal_2_3573
PubMedID: 37350932
Gene_locus related to this paper: idesa-mheth , idesa-peth

Title : Biosensor and chemo-enzymatic one-pot cascade applications to detect and transform PET-derived terephthalic acid in living cells - Bayer_2022_iScience_25_104326
Author(s) : Bayer T , Pfaff L , Branson Y , Becker A , Wu S , Bornscheuer UT , Wei R
Ref : iScience , 25 :104326 , 2022
Abstract : Plastic waste imposes a serious problem to the environment and society. Hence, strategies for a circular plastic economy are demanded. One strategy is the engineering of polyester hydrolases toward higher activity for the biotechnological recycling of polyethylene terephthalate (PET). To provide tools for the rapid characterization of PET hydrolases and the detection of degradation products like terephthalic acid (TPA), we coupled a carboxylic acid reductase (CAR) and the luciferase LuxAB. CAR converted TPA into the corresponding aldehydes in Escherichia coli, which yielded bioluminescence that not only semiquantitatively reflected amounts of TPA in hydrolysis samples but is suitable as a high-throughput screening assay to assess PET hydrolase activity. Furthermore, the CAR-catalyzed synthesis of terephthalaldehyde was combined with a reductive amination cascade in a one-pot setup yielding the corresponding diamine, suggesting a new strategy for the transformation of TPA as a product obtained from PET biodegradation.
ESTHER : Bayer_2022_iScience_25_104326
PubMedSearch : Bayer_2022_iScience_25_104326
PubMedID: 35602945

Title : Enzymatic degradation of polyethylene terephthalate nanoplastics analyzed in real time by isothermal titration calorimetry - Vogel_2021_Sci.Total.Environ_773_145111
Author(s) : Vogel K , Wei R , Pfaff L , Breite D , Al-Fathi H , Ortmann C , Estrela-Lopis I , Venus T , Schulze A , Harms H , Bornscheuer UT , Maskow T
Ref : Sci Total Environ , 773 :145111 , 2021
Abstract : Plastics are globally used for a variety of benefits. As a consequence of poor recycling or reuse, improperly disposed plastic waste accumulates in terrestrial and aquatic ecosystems to a considerable extent. Large plastic waste items become fragmented to small particles through mechanical and (photo)chemical processes. Particles with sizes ranging from millimeter (microplastics, <5 mm) to nanometer (nanoplastics, NP, <100 nm) are apparently persistent and have adverse effects on ecosystems and human health. Current research therefore focuses on whether and to what extent microorganisms or enzymes can degrade these NP. In this study, we addressed the question of what information isothermal titration calorimetry, which tracks the heat of reaction of the chain scission of a polyester, can provide about the kinetics and completeness of the degradation process. The majority of the heat represents the cleavage energy of the ester bonds in polymer backbones providing real-time kinetic information. Calorimetry operates even in complex matrices. Using the example of the cutinase-catalyzed degradation of polyethylene terephthalate (PET) nanoparticles, we found that calorimetry (isothermal titration calorimetry-ITC) in combination with thermokinetic models is excellently suited for an in-depth analysis of the degradation processes of NP. For instance, we can separately quantify i) the enthalpy of surface adsorption delta(Ads)H = 129 +/- 2 kJ mol(-1), ii) the enthalpy of the cleavage of the ester bonds delta(EB)H = -58 +/- 1.9 kJ mol(-1) and the apparent equilibrium constant of the enzyme substrate complex K = 0.046 +/- 0.015 g L(-1). It could be determined that the heat production of PET NP degradation depends to 95% on the reaction heat and only to 5% on the adsorption heat. The fact that the percentage of cleaved ester bonds ( = 12.9 +/- 2.4%) is quantifiable with the new method is of particular practical importance. The new method promises a quantification of enzymatic and microbial adsorption to NP and their degradation in mimicked real-world aquatic conditions.
ESTHER : Vogel_2021_Sci.Total.Environ_773_145111
PubMedSearch : Vogel_2021_Sci.Total.Environ_773_145111
PubMedID: 33940717