Westh P

References (16)

Title : Unveiling PET Hydrolase Surface Dynamics through Fluorescence Microscopy - Rennison_2024_Chembiochem__e202300661
Author(s) : Rennison AP , Nousi A , Westh P , Marie R , Moller MS
Ref : Chembiochem , :e202300661 , 2024
Abstract : PET hydrolases are an emerging class of enzymes that are being heavily researched for their use in bioprocessing polyethylene terephthalate (PET). While work has been done in studying the binding of PET oligomers to the active site of these enzymes, the dynamics of PET hydrolases binding to a bulk PET surface is an unexplored area. Here, methods were developed for total internal reflection fluorescence (TIRF) microscopy and fluorescence recovery after photobleaching (FRAP) microscopy to study the adsorption and desorption dynamics of these proteins onto a PET surface. TIRF microscopy was employed to measure both on and off rates of two of the most commonly studied PET hydrolases, PHL7 and LCC, on a PET surface. It was found that these proteins have a much slower off rates on the order of 10-3 s-1, comparable to non-productive binding in enzymes such as cellulose. In combination with FRAP microscopy, a dynamic model is proposed in which adsorption and desorption dominates over lateral diffusion over the surface. The results of this study could have implications for the future engineering of PET hydrolases, either to target them to a PET surface or to modulate interaction with their substrate.
ESTHER : Rennison_2024_Chembiochem__e202300661
PubMedSearch : Rennison_2024_Chembiochem__e202300661
PubMedID: 38224131
Gene_locus related to this paper: 9firm-PHL7 , 9bact-g9by57

Title : Relationships of crystallinity and reaction rates for enzymatic degradation of poly (ethylene terephthalate), PET - Schubert_2024_ChemSusChem__e202301752
Author(s) : Schubert SW , Thomsen TB , Clausen KSR , Malmendal A , Hunt CJ , Borch K , Jensen K , Brask J , Meyer AS , Westh P
Ref : ChemSusChem , :e202301752 , 2024
Abstract : Biocatalytic degradation of plastic waste is anticipated to play an important role in future recycling systems. However, enzymatic degradation of crystalline poly (ethylene terephthalate) (PET) remains consistently poor. Herein, we employed functional assays to elucidate the molecular underpinnings of this limitation. This included utilizing complementary activity assays to monitor the degradation of PET disks with varying crystallinity (XC), as well as kinetic parameters for soluble PET fragments. The results indicate that a proficient PET-hydrolase, LCCICCG, operates through an endolytic mode of action, and that its activity is limited by conformational constraints in the PET polymer. Such constraints become more pronounced at high XC values, and this limits the density of productive sites on the PET surface. Endolytic chain-scissions are the dominant reaction type in the initial stage, and this means that little or no soluble organic product occurs here. However, endolytic cuts gradually and locally promote chain mobility and hence the density of attack sites on the surface. This leads to an upward concave progress curve; a behavior sometimes termed lag-phase kinetics.
ESTHER : Schubert_2024_ChemSusChem__e202301752
PubMedSearch : Schubert_2024_ChemSusChem__e202301752
PubMedID: 38252197
Gene_locus related to this paper: 9bact-g9by57

Title : Purification and biochemical characterization of SM14est, a PET-hydrolyzing enzyme from the marine sponge-derived Streptomyces sp. SM14 - Carr_2023_Front.Microbiol_14_1170880
Author(s) : Carr CM , Keller MB , Paul B , Schubert SW , Clausen KSR , Jensen K , Clarke DJ , Westh P , Dobson ADW
Ref : Front Microbiol , 14 :1170880 , 2023
Abstract : The successful enzymatic degradation of polyester substrates has fueled worldwide investigation into the treatment of plastic waste using bio-based processes. Within this realm, marine-associated microorganisms have emerged as a promising source of polyester-degrading enzymes. In this work, we describe the hydrolysis of the synthetic polymer PET by SM14est, a polyesterase which was previously identified from Streptomyces sp. SM14, an isolate of the marine sponge Haliclona simulans. The PET hydrolase activity of purified SM14est was assessed using a suspension-based assay and subsequent analysis of reaction products by UV-spectrophotometry and RP-HPLC. SM14est displayed a preference for high salt conditions, with activity significantly increasing at sodium chloride concentrations from 100 mM up to 1,000 mM. The initial rate of PET hydrolysis by SM14est was determined to be 0.004 s(-1) at 45 degreesC, which was increased by 5-fold to 0.02 s(-1) upon addition of 500 mM sodium chloride. Sequence alignment and structural comparison with known PET hydrolases, including the marine halophile PET6, and the highly efficient, thermophilic PHL7, revealed conserved features of interest. Based on this work, SM14est emerges as a useful enzyme that is more similar to key players in the area of PET hydrolysis, like PHL7 and IsPETase, than it is to its marine counterparts. Salt-tolerant polyesterases such as SM14est are potentially valuable in the biological degradation of plastic particles that readily contaminate marine ecosystems and industrial wastewaters.
ESTHER : Carr_2023_Front.Microbiol_14_1170880
PubMedSearch : Carr_2023_Front.Microbiol_14_1170880
PubMedID: 37250061

Title : Rate Response of Poly(Ethylene Terephthalate)-Hydrolases to Substrate Crystallinity: Basis for Understanding the Lag Phase - Thomsen_2023_ChemSusChem_16_e202300291
Author(s) : Thomsen TB , Schubert S , Hunt CJ , Borch K , Jensen K , Brask J , Westh P , Meyer AS
Ref : ChemSusChem , 16 :e202300291 , 2023
Abstract : The rate response of poly(ethylene terephthalate) (PET)-hydrolases to increased substrate crystallinity (X(C) ) of PET manifests as a rate-lowering effect that varies significantly for different enzymes. Herein, we report the influence of X(C) on the product release rate of six thermostable PET-hydrolases. All enzyme reactions displayed a distinctive lag phase until measurable product formation occurred. The duration of the lag phase increased with X(C) . The recently discovered PET-hydrolase PHL7 worked efficiently on "amorphous" PET disks (X(C) =10 %), but this enzyme was extremely sensitive to increased X(C) , whereas the enzymes LCC(ICCG) , LCC, and DuraPETase had higher tolerance to increases in X(C) and had activity on PET disks having X(C) of 24.4 %. Microscopy revealed that the X(C) -tolerant hydrolases generated smooth and more uniform substrate surface erosion than PHL7 during reaction. Structural and molecular dynamics analysis of the PET-hydrolyzing enzymes disclosed that surface electrostatics and enzyme flexibility may account for the observed differences.
ESTHER : Thomsen_2023_ChemSusChem_16_e202300291
PubMedSearch : Thomsen_2023_ChemSusChem_16_e202300291
PubMedID: 37073816

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 : Sabatier Principle for Rationalizing Enzymatic Hydrolysis of a Synthetic Polyester - Arnling Baath_2022_JACS.Au_2_1223
Author(s) : Arnling Baath J , Jensen K , Borch K , Westh P , Kari J
Ref : JACS Au , 2 :1223 , 2022
Abstract : Interfacial enzyme reactions are common in Nature and in industrial settings, including the enzymatic deconstruction of poly(ethylene terephthalate) (PET) waste. Kinetic descriptions of PET hydrolases are necessary for both comparative analyses, discussions of structure-function relations and rational optimization of technical processes. We investigated whether the Sabatier principle could be used for this purpose. Specifically, we compared the kinetics of two well-known PET hydrolases, leaf-branch compost cutinase (LCC) and a cutinase from the bacterium Thermobifida fusca (TfC), when adding different concentrations of the surfactant cetyltrimethylammonium bromide (CTAB). We found that CTAB consistently lowered the strength of enzyme-PET interactions, while its effect on enzymatic turnover was strongly biphasic. Thus, at gradually increasing CTAB concentrations, turnover was initially promoted and subsequently suppressed. This correlation with maximal turnover at an intermediate binding strength was in accordance with the Sabatier principle. One consequence of these results was that both enzymes had too strong intrinsic interaction with PET for optimal turnover, especially TfC, which showed a 20-fold improvement of k (cat) at the maximum. LCC on the other hand had an intrinsic substrate affinity closer to the Sabatier optimum, and the turnover rate was 5-fold improved at weakened substrate binding. Our results showed that the Sabatier principle may indeed rationalize enzymatic PET degradation and support process optimization. Finally, we suggest that future discovery efforts should consider enzymes with weakened substrate binding because strong adsorption seems to limit their catalytic performance.
ESTHER : Arnling Baath_2022_JACS.Au_2_1223
PubMedSearch : Arnling Baath_2022_JACS.Au_2_1223
PubMedID: 35647598
Gene_locus related to this paper: 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 : Analysis of fungal high mannose structures using CAZymes - Kolaczkowski_2021_Glycobiology__
Author(s) : Kolaczkowski BM , Jorgensen CI , Spodsberg N , Stringer MA , Supekar NT , Azadi P , Westh P , Krogh K , Jensen K
Ref : Glycobiology , : , 2021
Abstract : Glycoengineering ultimately allows control over glycosylation patterns to generate new glycoprotein variants with desired properties. A common challenge is glycan heterogeneity, which may affect protein function and limit the use of key techniques such as mass spectrometry. Moreover, heterologous protein expression can introduce non-native glycan chains which may not fulfil the requirement for therapeutic proteins. One strategy to address these challenges is partial trimming or complete removal of glycan chains, which can be obtained through selective application of exo-glycosidases. Here, we demonstrate an enzymatic O-deglycosylation toolbox of a GH92 alpha-1,2-mannosidase from Neobacillus novalis, a GH2 beta-galactofuranosidase from Amesia atrobrunnea and the jack bean alpha-mannosidase. The extent of enzymatic O-deglycosylation was mapped against a full glycosyl linkage analysis of the O-glycosylated linker of cellobiohydrolase I from Trichoderma reesei (TrCel7A). Furthermore, the influence of deglycosylation on TrCel7A functionality was evaluated by kinetic characterization of native and O-deglycosylated forms of TrCel7A. This study expands structural knowledge on fungal O-glycosylation and presents a ready-to-use enzymatic approach for controlled O-glycan engineering in glycoproteins expressed in filamentous fungi.
ESTHER : Kolaczkowski_2021_Glycobiology__
PubMedSearch : Kolaczkowski_2021_Glycobiology__
PubMedID: 34939126

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 : Surface display as a functional screening platform for detecting enzymes active on PET - Heyde_2021_Microb.Cell.Fact_20_93
Author(s) : Heyde SAH , Arnling Baath J , Westh P , Norholm MHH , Jensen K
Ref : Microb Cell Fact , 20 :93 , 2021
Abstract : Poly(ethylene terephthalate) (PET) is the world's most abundant polyester plastic, and its ongoing accumulation in nature is causing a global environmental problem. Currently, the main recycling processes utilize thermomechanical or chemical means, resulting in the deterioration of the mechanical properties of PET. Consequently, polluting de novo synthesis remains preferred, creating the need for more efficient and bio-sustainable ways to hydrolyze the polymer. Recently, a PETase enzyme from the bacterium Ideonella sakaiensis was shown to facilitate PET biodegradation, albeit at slow rate. Engineering of more efficient PETases is required for industrial relevance, but progress is currently hampered by the dependency on intracellular expression in Escherichia coli. To create a more efficient screening platform in E. coli, we explore different surface display anchors for fast and easy assaying of PETase activity. We show that PETases can be functionally displayed on the bacterial cell surface, enabling screening of enzyme activity on PET microparticles - both while anchored to the cell and following solubilization of the enzymes.
ESTHER : Heyde_2021_Microb.Cell.Fact_20_93
PubMedSearch : Heyde_2021_Microb.Cell.Fact_20_93
PubMedID: 33933097
Gene_locus related to this paper: idesa-peth

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 : The structural basis of fungal glucuronoyl esterase activity on natural substrates - Ernst_2020_Nat.Commun_11_1026
Author(s) : Ernst HA , Mosbech C , Langkilde AE , Westh P , Meyer AS , Agger JW , Larsen S
Ref : Nat Commun , 11 :1026 , 2020
Abstract : Structural and functional studies were conducted of the glucuronoyl esterase (GE) from Cerrena unicolor (CuGE), an enzyme catalyzing cleavage of lignin-carbohydrate ester bonds. CuGE is an alpha/beta-hydrolase belonging to carbohydrate esterase family 15 (CE15). The enzyme is modular, comprised of a catalytic and a carbohydrate-binding domain. SAXS data show CuGE as an elongated rigid molecule where the two domains are connected by a rigid linker. Detailed structural information of the catalytic domain in its apo- and inactivated form and complexes with aldouronic acids reveal well-defined binding of the 4-O-methyl-a-D-glucuronoyl moiety, not influenced by the nature of the attached xylo-oligosaccharide. Structural and sequence comparisons within CE15 enzymes reveal two distinct structural subgroups. CuGE belongs to the group of fungal CE15-B enzymes with an open and flat substrate-binding site. The interactions between CuGE and its natural substrates are explained and rationalized by the structural results, microscale thermophoresis and isothermal calorimetry.
ESTHER : Ernst_2020_Nat.Commun_11_1026
PubMedSearch : Ernst_2020_Nat.Commun_11_1026
PubMedID: 32094331
Gene_locus related to this paper: cerui-gce

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

Title : Thermal stability of Humicola insolens cutinase in aqueous SDS - Nielsen_2007_J.Phys.Chem.B_111_2941
Author(s) : Nielsen AD , Borch K , Westh P
Ref : J Phys Chem B , 111 :2941 , 2007
Abstract : Cutinase from Humicola insolens (HiC) has previously been shown to bind anomalously low amounts of the anionic surfactant sodium dodecylsulfate (SDS). In the current work, we have applied scanning and titration calorimetry to investigate possible relationships between this weak interaction and the effect of SDS on the equilibrium and kinetic stability of HiC. The results are presented in a "state-diagram," which specifies the stable form of the protein as a function of temperature and SDS concentration. In comparison with other proteins, the equilibrium stability HiC is strongly decreased by SDS. For low SDS concentrations (SDS:HiC molar ratio, MR < 8) this trait is also found for the kinetically controlled thermal aggregation of the protein. At higher MR, however, SDS stabilizes noticeably against irreversible aggregation. We suggest that this relies on electrostatic repulsion of the increasingly negatively charged HiC-SDS complexes. The combined interpretation of calorimetric and binding data allowed the calculation of the changes in enthalpy and heat capacity for the association of HiC and SDS near the saturation point. The latter function was about -410 J mol(-1) K(-1) or similar to the heat capacity change for micelle formation (-470 J mol(-1) K(-1)). This suggests that SDS is hydrated to a similar extent in the micellar and protein associated forms. The results are discussed in terms of the Wyman theory for linked equilibria. Quantitative analysis along these lines suggests that the reversible thermal unfolding of the protein couples to the binding of 2-3 additional SDS molecules. This corresponds to a 15-20% increase in the binding number. Wyman theory also rationalizes relationships between low affinity and high susceptibility observed in this study.
ESTHER : Nielsen_2007_J.Phys.Chem.B_111_2941
PubMedSearch : Nielsen_2007_J.Phys.Chem.B_111_2941
PubMedID: 17319710
Gene_locus related to this paper: humin-cut

Title : Analysis of protein-surfactant interactions--a titration calorimetric and fluorescence spectroscopic investigation of interactions between Humicola insolens cutinase and an anionic surfactant - Nielsen_2005_Biochim.Biophys.Acta_1752_124
Author(s) : Nielsen AD , Arleth L , Westh P
Ref : Biochimica & Biophysica Acta , 1752 :124 , 2005
Abstract : We have studied interactions of cutinase (HiC) from Humicula insolens and sodium dodecyl sulphate (SDS) by parallel calorimetric and fluorescence investigations of systems in which the concentration of both components was changed systematically. Results from the two methods exhibit a number of synchronous characteristics, when plotted against the total SDS concentration, [SDS]tot. The molecular origin of several of these anomalies was assigned, and five intervals of [SDS]tot in which different modes of interactions dominated were identified. Going from low to high [SDS]tot, these modes were: binding of (a few) SDS to native HiC, formation of oligomeric protein aggregates, denaturation of HiC and adsorption of SDS on denatured protein. For [SDS]tot>3-6 mM (depending on the protein concentration), the adsorption saturated, and no further protein-detergent interaction could be detected. Two particularly conspicuous anomalies in the calorimetric data were ascribed to respectively denaturation and saturation. It was found that [SDS]tot at these points depended linearly on the (total) protein concentration, [HiC]. We suggest that this reflects the balance between bound and free SDS [SDS]tot=[SDS]aq+[HiC] Nb where [SDS]aq and Nb are, respectively, the aqueous ("free") concentration of SDS and the average number of SDS bound per protein. Interpretation of the results along these lines showed that at 22 degrees C and pH 7.0, HiC denatures with approximately 14 bound surfactant molecules at [SDS]aq=1.0 mM. Saturation is characterized by Nb approximately 39 and [SDS]aq=2.2 mM. The latter value is equal to CMC in the (protein free) buffer. These results are discussed with respect to the SDS-binding capacity of HiC and the origin and location of the saturation point.
ESTHER : Nielsen_2005_Biochim.Biophys.Acta_1752_124
PubMedSearch : Nielsen_2005_Biochim.Biophys.Acta_1752_124
PubMedID: 16162423
Gene_locus related to this paper: humin-cut

Title : Interactions of Humicola insolens cutinase with an anionic surfactant studied by small-angle neutron scattering and isothermal titration calorimetry - Nielsen_2005_Langmuir_21_4299
Author(s) : Nielsen AD , Arleth L , Westh P
Ref : Langmuir , 21 :4299 , 2005
Abstract : The interaction of cutinase from Humicula insolens (HiC) and sodium dodecyl sulfate (SDS) has been investigated by small-angle neutron scattering (SANS) and isothermal titration calorimetry (ITC). The concerted interpretation of structural and thermodynamic information for identical systems proved valuable in attempts to elucidate the complex modes of protein-detergent interaction. Particularly so at the experimental temperature 22 degrees C, where the formation of SDS micelles is athermal (deltaH = 0), and the effects of protein-detergent interactions stand out clearly in the thermograms. It was found that the effect of SDS on cutinase depended strongly on the sample composition. Thus, addition of SDS corresponding to a molar ratio, n(s) = n(SDS)/n(HiC) of about 10, was associated with the formation of HiC/SDS aggregates, which include more than one protein molecule. The SANS results suggested that on the average such adducts contained two HiC, and the ITC traces showed that they form and break down slowly. At slightly higher SDS concentrations (n(s) = 10-25) these "dimers" dissociated, and the protein denatured. The denaturation showed the characteristic positive enthalpy change, but the SDS denatured state of HiC was unusually compact with a radius of gyration close to that of the native conformation. Further titration with SDS was associated with exothermic binding to the denatured protein until the saturation point at about n(s) = 90. At this point, the free monomer concentration was 2.2 mM and the binding number was approximately 40 SDS/HiC. Interestingly, this degree of SDS binding (approximately 0.5 g of SDS/g of HiC) is less than half the amount bound to typical water-soluble proteins.
ESTHER : Nielsen_2005_Langmuir_21_4299
PubMedSearch : Nielsen_2005_Langmuir_21_4299
PubMedID: 16032839
Gene_locus related to this paper: humin-cut