Impact of Synergy Partner Cel7B on Cel7A Binding Rates: Insights from Single-Molecule Data.

Enzymatic degradation of cellulosic biomass is a well-established route for the sustainable production of biofuels, chemicals, and materials. A strategy employed by nature and industry to achieve an efficient degradation of cellulose is that cellobiohydrolases (or exocellulases), such as Cel7A, work synergistically with endoglucanases, such as Cel7B, to achieve the complete degradation of cellulose. However, a complete mechanistic understanding of this exo-endo synergy is still lacking. Here, we used single-molecule fluorescence microscopy to quantify the binding kinetics of Cel7A on cellulose when it is acting alone on the cellulose fibrils and in the presence of its synergy partner, the endoglucanase Cel7B. To this end, we used a fluorescently tagged Cel7A and studied its binding in the presence of the unlabeled Cel7B. This provided the single-molecule data necessary for the estimation of the rate constants of association kON and dissociation kOFF of Cel7A for the substrate. We show that the presence of Cel7B does not impact the dissociation rate constant, kOFF. But, the association rate of Cel7A decreases by a factor of 2 when Cel7B is present at a molar proportion of 10:1. This ratio has previously been shown to lead to synergy. This decrease in association rate is observed in a wide range of total enzyme concentrations, from sub nM to µM concentrations. This decrease in kON is consistent with the formation of cellulase clusters recently observed by others using atomic force microscopy.

Relationships of crystallinity and reaction rates for enzymatic degradation of poly (ethylene terephthalate), PET.

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 determining enzymatic kinetic parameters for soluble PET fragments. The results indicate that an efficient 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 are released. 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.

ITC analysis of polydisperse systems: Unravelling the impact of sample heterogeneity.

Binding interactions often involve heterogeneous samples displaying a distribution of binding sites that vary in affinity and binding enthalpy. Examples include biological samples like proteins and chemically produced samples like modified cyclodextrins. Experimental studies often ignore sample heterogeneity and treat the system as an interaction of two homogeneous species, i.e. a chemically well-defined ligand binding to one type of site. The present study explores, by simulations and experiments, the impact of heterogeneity in isothermal titration calorimetry (ITC) setups where one of the binding components is heterogeneous. It is found that the standard single-site model, based on the assumption of two homogeneous binding components, provides excellent fits to simulated ITC data when the binding free energy is normally distributed and all sites have similar binding enthalpies. In such cases, heterogeneity can easily go undetected but leads to underestimated binding constants. Heterogeneity in the binding enthalpy is a bigger problem and may result in enthalpograms of increased complexity that are likely to be misinterpreted as two-site binding or other complex binding models. Finally, it is shown that heterogeneity can account for previously observed experimental anomalies. All simulations are accessible in Google Colab for readers to experiment with the simulation parameters.

Improved Hydrolysis of Granular Starches by a Psychrophilic α-Amylase Starch Binding Domain-Fusion.

Degradation of starch granules by a psychrophilic α-amylase, AHA, from the Antarctic bacterium Pseudoalteromonas haloplanktis TAB23 was facilitated by C-terminal fusion to a starch-binding domain (SBD) from either Aspergillus niger glucoamylase (SBDGA) or Arabidopsis thaliana glucan, water dikinase 3 (SBDGWD3) via a decapeptide linker. Depending on the waxy, normal or high-amylose starch type and the botanical source, the AHA-SBD fusion enzymes showed up to 3 times higher activity than AHA wild-type. The SBD-fusion thus increased the density of enzyme attack-sites and binding-sites on the starch granules by up to 5- and 7-fold, respectively, as measured using an interfacial catalysis approach that combined conventional Michaelis-Menten kinetics, with the substrate in excess, and inverse kinetics, having enzyme in excess, with enzyme-starch granule adsorption isotherms. Higher substrate affinity of the SBDGA compared to SBDGWD3 was accompanied by the superior activity of AHA-SBDGA in agreement with the Sabatier principle of adsorption limited heterogenous catalysis.

Interfacial Catalysis during Amylolytic Degradation of Starch Granules: Current Understanding and Kinetic Approaches.

Enzymatic hydrolysis of starch granules forms the fundamental basis of how nature degrades starch in plant cells, how starch is utilized as an energy resource in foods, and develops efficient, low-cost saccharification of starch, such as bioethanol and sweeteners. However, most investigations on starch hydrolysis have focused on its rates of degradation, either in its gelatinized or soluble state. These systems are inherently more well-defined, and kinetic parameters can be readily derived for different hydrolytic enzymes and starch molecular structures. Conversely, hydrolysis is notably slower for solid substrates, such as starch granules, and the kinetics are more complex. The main problems include that the surface of the substrate is multifaceted, its chemical and physical properties are ill-defined, and it also continuously changes as the hydrolysis proceeds. Hence, methods need to be developed for analyzing such heterogeneous catalytic systems. Most data on starch granule degradation are obtained on a long-term enzyme-action basis from which initial rates cannot be derived. In this review, we discuss these various aspects and future possibilities for developing experimental procedures to describe and understand interfacial enzyme hydrolysis of native starch granules more accurately.

Purification and biochemical characterization of SM14est, a PET-hydrolyzing enzyme from the marine sponge-derived Streptomyces sp. SM14.

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°C, 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.

Rate Response of Poly(Ethylene Terephthalate)-Hydrolases to Substrate Crystallinity: Basis for Understanding the Lag Phase.

The rate response of poly(ethylene terephthalate) (PET)-hydrolases to increased substrate crystallinity (XC ) of PET manifests as a rate-lowering effect that varies significantly for different enzymes. Herein, we report the influence of XC 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 XC . The recently discovered PET-hydrolase PHL7 worked efficiently on "amorphous" PET disks (XC ≈10 %), but this enzyme was extremely sensitive to increased XC , whereas the enzymes LCCICCG , LCC, and DuraPETase had higher tolerance to increases in XC and had activity on PET disks having XC of 24.4 %. Microscopy revealed that the XC -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.

Structural and functional characterization of a multi-domain GH92 α-1,2-mannosidase from Neobacillus novalis.

Many secreted eukaryotic proteins are N-glycosylated with oligosaccharides composed of a high-mannose N-glycan core and, in the specific case of yeast cell-wall proteins, an extended α-1,6-mannan backbone carrying a number of α-1,2- and α-1,3-mannose substituents of varying lengths. α-Mannosidases from CAZy family GH92 release terminal mannose residues from these N-glycans, providing access for the α-endomannanases, which then degrade the α-mannan backbone. Most characterized GH92 α-mannosidases consist of a single catalytic domain, while a few have extra domains including putative carbohydrate-binding modules (CBMs). To date, neither the function nor the structure of a multi-domain GH92 α-mannosidase CBM has been characterized. Here, the biochemical investigation and crystal structure of the full-length five-domain GH92 α-1,2-mannosidase from Neobacillus novalis (NnGH92) with mannoimidazole bound in the active site and an additional mannoimidazole bound to the N-terminal CBM32 are reported. The structure of the catalytic domain is very similar to that reported for the GH92 α-mannosidase Bt3990 from Bacteroides thetaiotaomicron, with the substrate-binding site being highly conserved. The function of the CBM32s and other NnGH92 domains was investigated by their sequential deletion and suggested that whilst their binding to the catalytic domain was crucial for the overall structural integrity of the enzyme, they appear to have little impact on the binding affinity to the yeast α-mannan substrate. These new findings provide a better understanding of how to select and optimize other multi-domain bacterial GH92 α-mannosidases for the degradation of yeast α-mannan or mannose-rich glycans.

The interplay between lytic polysaccharide monooxygenases and glycoside hydrolases.

In nature, enzymatic degradation of recalcitrant polysaccharides such as chitin and cellulose takes place by a synergistic interaction between glycoside hydrolases (GHs) and lytic polysaccharide monooxygenases (LPMOs). The two different families of carbohydrate-active enzymes use two different mechanisms when breaking glycosidic bonds between sugar moieties. GHs employ a hydrolytic activity and LPMOs are oxidative. Consequently, the topologies of the active sites differ dramatically. GHs have tunnels or clefts lined with a sheet of aromatic amino acid residues accommodating single polymer chains being threaded into the active site. LPMOs are adapted to bind to the flat crystalline surfaces of chitin and cellulose. It is believed that the LPMO oxidative mechanism provides new chain ends that the GHs can attach to and degrade, often in a processive manner. Indeed, there are many reports of synergies as well as rate enhancements when LPMOs are applied in concert with GHs. Still, these enhancements vary in magnitude with respect to the nature of the GH and the LPMO. Moreover, impediment of GH catalysis is also observed. In the present review, we discuss central works where the interplay between LPMOs and GHs has been studied and comment on future challenges to be addressed to fully use the potential of this interplay to improve enzymatic polysaccharide degradation.

Protein-plastic interactions: The driving forces behind the high affinity of a carbohydrate-binding module for polyethylene terephthalate.

Polyethylene terephthalate (PET) waste is a common pollutant in the environment, mainly due to resistance of the plastic to bio-degradation. Nevertheless, hydrolytic enzymes have been identified with activity on this substrate, which are continually being engineered to increase activity. Some insoluble biological polymers are degraded by enzymes with a multi-domain architecture, comprising of a catalytic domain, and a substrate-binding domain, such as a carbohydrate-binding module (CBM). Enzymes that degrade PET have been shown to have a higher activity when fused with these CBMs, indicating a promising route for engineering better enzymes for plastic bioprocessing. However, no detailed study of the affinity and binding mechanism of these domains on PET has yet been made. Here, we perform an in depth analysis of a binding domain from CBM family 2 on PET, showing that the affinity of the protein for the plastic is highly dependent on temperature and crystallinity of the plastic. We also investigate the mechanism of the interaction, and show how affinity may be engineered in both directions. CBM affinity for other synthetic polymers is also demonstrated for the first time. Our results demonstrate that the substrate affinity of fusion enzymes with binding modules can be tuned to the desired level.

Tuning alginate ß-lactoglobulin complex coacervation by modulating pH and temperature.

The use of biomolecules in food matrices and encapsulation systems is, as in other areas, moving towards greener solutions and a center piece here is the complex coacervation between natural anionic polysaccharides and proteins. Both alginate and ß-lactoglobulin (ß-Lg) are used in different sectors and have been shown to coacervate at pH < 5.2. Albeit with increased interest, complex coacervation has almost exclusively been studied from a macromolecular perspective, and described as an interaction based on charge-charge attraction. Here, we show that through changes in pH and temperature, alginate ß-Lg complex coacervation can be tuned to purpose. By detailed biophysical and chemical characterization of coacervation and coacervate particles, insights into the molecular interaction and effect of external factors are obtained. We find that carboxylate resonance stabilization causes a release of protons at pH < pKa,alginate and an uptake of protons at pH > pKa,alginate upon coacervation. Proton release and uptake were quantified at pH 2.65 and 4.00 by isothermal titration calorimetry to be 4 and 2 protons per ß-Lg molecule, respectively. By increasing the temperature to 65 °C, we discovered a secondary ß-Lg concentration dependent coacervation step, where the formed particles change into large assemblies driven by entropy. These findings bring new insights to complex coacervation and its applicability in microencapsulation and drug delivery.

A new continuous assay for quantitative assessment of enzymatic degradation of poly(ethylene terephthalate) (PET).

Enzymatic degradation of poly(ethylene terephthalate) (PET) has emerged as a promising route for ecofriendly biodegradation of plastic waste. Several discontinuous activity assays have been developed for assessing PET hydrolyzing enzymes, usually involving manual sampling at different time points during the course of the enzymatic reaction. In this work, we present a novel, compartmentalized UV absorbance assay for continuous detection of soluble hydrolysis products released during enzymatic degradation of PET. The methodology is based on removal of the walls separating two diagonally adjacent wells in UV-transparent microplates, to ensure passage of soluble enzymatic hydrolysis products between the two adjacent wells: One well holds an insoluble PET disk of defined dimensions and the other is used for continuous reading of the enzymatic product formation (at 240 nm). The assay was validated by quantifying the rate of mixing of the soluble PET degradation product BHET (bis(2-hydroxyethyl) terephthalate) between the two adjacent wells. The assay validation also involved a simple adjustment for water evaporation during prolonged assays. With this new assay, we determined the kinetic parameters for two PET hydrolases, DuraPETase and LCCICCG, and verified the underlying assumption of steady-state reaction rates. This new continuous assay enables fast exploration and robust kinetic characterization of PET degrading enzymes.

Molecular insights into alginate ß-lactoglobulin A multivalencies-The foundation for their amorphous aggregates and coacervation.

For improved control of biomaterial property design, a better understanding of complex coacervation involving anionic polysaccharides and proteins is needed. Here, we address the initial steps in condensate formation of ß-lactoglobulin A (ß-LgA) with nine defined alginate oligosaccharides (AOSs) and describe their multivalent interactions in structural detail. Binding of AOSs containing four, five, or six uronic acid residues (UARs), either all mannuronate (M), all guluronate (G), or alternating M and G embodying the block structural components of alginates, was characterized by isothermal titration calorimetry, nuclear magnetic resonance spectroscopy (NMR), and molecular docking. ß-LgA was highly multivalent exhibiting binding stoichiometries decreasing from five to two AOSs with increasing degree of polymerization (DP) and similar affinities in the mid micromolar range. The different AOS binding sites on ß-LgA were identified by NMR chemical shift perturbation analyses and showed diverse compositions of charged, polar and hydrophobic residues. Distinct sites for the shorter AOSs merged to accommodate longer AOSs. The AOSs bound dynamically to ß-LgA, as concluded from saturation transfer difference and 1 H-ligand-targeted NMR analyses. Molecular docking using Glide within the Schrödinger suite 2016-1 revealed the orientation of AOSs to only vary slightly at the preferred ß-LgA binding site resulting in similar XP glide scores. The multivalency coupled with highly dynamic AOS binding with lack of confined conformations in the ß-LgA complexes may help explain the first steps toward disordered ß-LgA alginate coacervate structures.

Reaction Pathways for the Enzymatic Degradation of Poly(Ethylene Terephthalate): What Characterizes an Efficient PET-Hydrolase?

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 (kcat /KM values of about 105  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.

The Sabatier principle as a tool for discovery and engineering of industrial enzymes.

The recent breakthrough in all-atom, protein structure prediction opens new avenues for a range of computational approaches in enzyme design. These new approaches could become instrumental for the development of technical biocatalysts, and hence our transition toward more sustainable industries. Here, we discuss one approach, which is well-known within inorganic catalysis, but essentially unexploited in biotechnology. Specifically, we review examples of linear free-energy relationships (LFERs) for enzyme reactions and discuss how LFERs and the associated Sabatier Principle may be implemented in algorithms that estimate kinetic parameters and enzyme performance based on model structures.

Unaided efficient transglutaminase cross-linking of whey proteins strongly impacts the formation and structure of protein alginate particles.

There is a dogma within whey protein modification, which dictates the necessity of pretreatment to enzymatic cross-linking of ß-lactoglobulin (ß-Lg). Here microbial transglutaminase (MTG) cross-linked whey proteins and ß-Lg effectively in 50 mM NaHCO3, pH 8.5, without pretreatment. Cross-linked ß-Lg spanned 18 to >240 kDa, where 6 of 9 glutamines reacted with 8 of 15 lysines. The initial isopeptide bond formation caused loss of ß-Lg native structure with t1/2 = 3 h, while the polymerization occurred with t1/2 = 10 h. Further, cross-linking effects on protein carbohydrate interaction have been overlooked, leaving a gap in understanding of these complex food matrices. Complexation with alginate showed that ß-Lg cross-linking decreased onset of particle formation, hydrodynamic diameter, stoichiometry (ß-Lg/alginate) and dissociation constant. The complexation was favored at higher temperatures (40 °C), suggesting that hydrophobic interactions were important. Thus, ß-Lg was cross-linked without pretreatment and the resulting polymers gave rise to altered complexation with alginate.

Sabatier Principle for Rationalizing Enzymatic Hydrolysis of a Synthetic Polyester.

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.

Analysis of fungal high-mannose structures using CAZymes.

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 nonnative glycan chains that may not fulfill 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 exoglycosidases. Here, we demonstrate an enzymatic O-deglycosylation toolbox of a GH92 α-1,2-mannosidase from Neobacillus novalis, a GH2 ß-galactofuranosidase from Amesia atrobrunnea and the jack bean α-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.

Structure-function analysis of two closely related cutinases from Thermobifida cellulosilytica.

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 poly(ethylene terephthalate) (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.

Adsorption of enzymes with hydrolytic activity on polyethylene terephthalate.

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.