Baath JA

References (5)

Title : Reaction Pathways for the Enzymatic Degradation of Poly(Ethylene Terephthalate): What Characterizes an Efficient PET-Hydrolase? - Schubert_2022_Chembiochem__e202200516
Author(s) : Schubert S , Schaller K , Baath JA , Hunt C , Borch K , Jensen K , Brask J , Westh P
Ref : Chembiochem , :e202200516 , 2022
Abstract : Bioprocessing of polyester waste has emerged as a promising tool in the quest for a cyclic plastic economy. One key step is the enzymatic breakdown of the polymer, and this entails a complicated pathway with substrates, intermediates, and products of variable size and solubility. We have elucidated this pathway for poly(ethylene terephthalate) (PET) and four enzymes. Specifically, we combined different kinetic measurements and a novel stochastic model and found that the ability to hydrolyze internal bonds in the polymer (endo-lytic activity) was a key parameter for overall enzyme performance. Endo-lytic activity promoted the release of soluble PET fragments with two or three aromatic rings, which, in turn, were broken down with remarkable efficiency (k(cat) /K(M) values of about 10(5) M(-1) s(-1) ) in the aqueous bulk. This meant that approximatly 70 % of the final, monoaromatic products were formed via soluble di- or tri-aromatic intermediates.
ESTHER : Schubert_2022_Chembiochem__e202200516
PubMedSearch : Schubert_2022_Chembiochem__e202200516
PubMedID: 36399069
Gene_locus related to this paper: idesa-peth , humin-cut , 9bact-g9by57 , thefu-q6a0i4

Title : Structure-function analysis of two closely related cutinases from Thermobifida cellulosilytica - Baath_2021_Biotechnol.Bioeng__
Author(s) : Baath JA , Novy V , Carneiro LV , Guebitz GM , Olsson L , Westh P , Ribitsch D
Ref : Biotechnol Bioeng , : , 2021
Abstract : Cutinases can play a significant role in a biotechnology-based circular economy. However, relatively little is known about the structure-function relationship of these enzymes, knowledge that is vital to advance optimized, engineered enzyme candidates. Here, two almost identical cutinases from Thermobifida cellulosilytica DSM44535 (Thc_Cut1 and Thc_Cut2) with only 18 amino acids difference were used for a rigorous biochemical characterization of their ability to hydrolyze PET, PET-model substrates, and cutin-model substrates. Kinetic parameters were compared with detailed in-silico docking studies of enzyme-ligand interactions. The two enzymes interacted with, and hydrolyzed PET differently, with Thc_Cut1 generating smaller PET-degradation products. Thc_Cut1 also showed higher catalytic efficiency on long-chain aliphatic substrates, an effect likely caused by small changes in the binding architecture. Thc_Cut2, in contrast, showed improved binding and catalytic efficiency when approaching the glass transition temperature of PET, an effect likely caused by longer amino acid residues in one area at the enzyme's surface. Finally, the position of the single residue Q93 close to the active site, rotated out in Thc_Cut2, influenced the ligand position of a trimeric PET-model substrate. In conclusion, we illustrate that even minor sequence differences in cutinases can affect their substrate binding, substrate specificity, and catalytic efficiency drastically. This article is protected by copyright. All rights reserved.
ESTHER : Baath_2021_Biotechnol.Bioeng__
PubMedSearch : Baath_2021_Biotechnol.Bioeng__
PubMedID: 34755331
Gene_locus related to this paper: thefu-q6a0i4 , thefu-q6a0i3

Title : Adsorption of enzymes with hydrolytic activity on polyethylene terephthalate - Badino_2021_Enzyme.Microb.Technol_152_109937
Author(s) : Badino SF , Baath JA , Borch K , Jensen K , Westh P
Ref : Enzyme Microb Technol , 152 :109937 , 2021
Abstract : Polyethylene terephthalate (PET) degrading enzymes have recently obtained an increasing interest as a means to decompose plastic waste. Here, we have studied the binding of three PET hydrolases on a suspended PET powder under conditions of both enzyme- and substrate excess. A Langmuir isotherm described the binding process reasonably and revealed a prominent affinity for the PET substrate, with dissociation constants consistently below 150 nM. The saturated substrate coverage approximately corresponded to a monolayer on the PET surface for all three enzymes. No distinct contributions from specific ligand binding in the active site could be identified, which points towards adsorption predominantly driven by non-specific interactions in contrast to enzymes naturally evolved for the breakdown of insoluble polymers. However, we observed a correlation between the progression of enzymatic hydrolysis and increased binding capacity, probably due to surface modifications of the PET polymer over time. Our results provide functional insight, suggesting that rational design should target the specific ligand interaction in the active site rather than the already high, general adsorption capacity of these enzymes.
ESTHER : Badino_2021_Enzyme.Microb.Technol_152_109937
PubMedSearch : Badino_2021_Enzyme.Microb.Technol_152_109937
PubMedID: 34749019
Gene_locus related to this paper: idesa-peth , humin-cut , thefu-q6a0i4

Title : A suspension-based assay and comparative detection methods for characterization of polyethylene terephthalate hydrolases - Baath_2020_Anal.Biochem__113873
Author(s) : Baath JA , Borch K , Westh P
Ref : Analytical Biochemistry , :113873 , 2020
Abstract : Enzymatic breakdown of plastic has emerged as a promising green technology, and its implementation will require assays that are accurate, reliable and convenient. Here, we assess two principles to monitor the hydrolysis of the common polyester, polyethylene terephthalate (PET). Hydrolysis of PET gives rise to heterogeneous products of different sizes and solubility, and as a result, specific experimental methods detect different activity levels. To avoid errors and to get a thorough picture of enzyme reactions, it is beneficial to combine several detection techniques. The two methods described herein are quantitative and complementary, and detect respectively the amount of soluble aromatic products and the formation of the constitutive aromatic monomers. A combined quantification approach identifies pitfalls in the characterization of these enzymes and provides mechanistic insight, but for screening and/or comparative studies of PET hydrolases we recommend a plate reader-based assay with suspended PET powder. This assay is rapid and simple, but still provides a good measure of the initial rates, which may be used in comparative biochemical analyses of these enzymes.
ESTHER : Baath_2020_Anal.Biochem__113873
PubMedSearch : Baath_2020_Anal.Biochem__113873
PubMedID: 32771375

Title : Comparative biochemistry of four polyester (PET) hydrolases - Baath_2020_Chembiochem_22_1627
Author(s) : Baath JA , Borch K , Jensen K , Brask J , Westh P
Ref : Chembiochem , 22 :1627 , 2020
Abstract : The potential of bioprocessing in a circular plastic economy has strongly stimulated research in enzymatic degradation of different synthetic polymers. Particular interest has been devoted to the commonly used polyester, poly(ethylene terephthalate) (PET), and a number of PET hydrolases have been described. However, a kinetic framework for comparisons of PET hydrolases (or other plastic degrading enzymes) acting on the insoluble substrate, has not been established. Here, we propose such a framework and test it against kinetic measurements on four PET hydrolases. The analysis provided values of kcat and KM, as well as an apparent specificity constant in the conventional units of M-1s-1. These parameters, together with experimental values for the number of enzyme attack sites on the PET surface, enabled comparative analyses. A variant of the PET hydrolase from Ideonella sakaiensis was the most efficient enzyme at ambient conditions, which relied on a high kcat rather than a low KM. Moreover, both soluble and insoluble PET fragments were consistently hydrolyzed much faster than intact PET. This suggests that interactions between polymer strands slow down PET degradation, while the chemical steps of catalysis and the low accessibility associated with solid substrate were less important for the overall rate. Finally, the investigated enzymes showed a remarkable substrate affinity, and reached half the saturation rate on PET, when the concentration of attack sites in the suspensi.
ESTHER : Baath_2020_Chembiochem_22_1627
PubMedSearch : Baath_2020_Chembiochem_22_1627
PubMedID: 33351214
Gene_locus related to this paper: idesa-peth