Intramolecular Cross-linking Research Articles

Intramolecular crosslinks in titin-advanced glycation end products stiffen cardiomyocytes

Intramolecular crosslinks in titin-advanced glycation end products stiffen cardiomyocytes

Effect of magnetic field-assisted fermentation on the in vitro protein digestibility and molecular structure of rapeseed meal.

There has been a significant growth in demand for plant-derived protein, and this has been accompanied by an increasing need for sustainable animal-feed options. The aim of this study was to investigate the effect of magnetic field-assisted solid fermentation (MSSF) on the in vitro protein digestibility (IVPD) and functional and structural characteristics of rapeseed meal (RSM) with a mutant strain of Bacillus subtilis. Our investigation demonstrated that the MSSF nitrogen release rate reached 86.3% after 96 h of fermentation. The soluble protein and peptide content in magnetic field feremented rapeseed meal reached 29.34 and 34.49 mg mL-1 after simulated gastric digestion, and the content of soluble protein and peptide in MF-FRSM reached 61.81 and 69.85 mg mL-1 after simulated gastrointestinal digestion, which significantly increased(p > 0.05) compared with the fermented rapeseed meal (FRSM). Studies of different microstructures - using scanning electron microscopy (SEM) and atomic force microscopy (AFM) - and protein secondary structures have shown that the decline in intermolecular or intramolecular cross-linking leads to the relative dispersion of proteins and improves the rate of nitrogen release. The smaller number of disulfide bonds and conformational alterations suggests that the IVPD of RSM was improved. Magnetic field-assisted solid fermentation can be applied to enhance the nutritional and protein digestibility of FRSM. © 2024 Society of Chemical Industry.

Thermokinetic and Chemorheology of the Geopolymerization of an Alumina-Rich Alkaline-Activated Metakaolin in Isothermal and Dynamic Thermal Scans.

Alkaline sodium hydroxide/sodium silicate-activating high-purity metakaolin geopolymerization is described in terms of metakaolin deconstruction in tetrahedral hydrate silicate [O[Si(OH)3]]- and aluminate [Al(OH)4]- ionic precursors followed by their reassembling in linear and branched sialates monomers that randomly copolymerize into an irregular crosslinked aluminosilicate network. The novelty of the approach resides in the concurrent thermo-calorimetric (differential scanning calorimetry, DSC) and rheological (dynamic mechanical analysis, DMA) characterizations of the liquid slurry during the transformation into a gel and a structural glassy solid. Tests were run either in temperature scan (1 °C/min) or isothermal (20 °C, 30 °C, 40 °C) cure conditions. A Gaussian functions deconvolution method has been applied to the DSC multi-peak thermograms to separate the kinetic contributions of the oligomer's concurrent reactions. DSC thermograms of all tested materials are well-fitted by a combination of three overlapping Gaussian curves that are associated with the initial linear low-molecular-weight (Mw) oligomers (P1) formation, oligomers branching into alumina-rich and silica-rich gels (P2), and inter- and intra-molecular crosslinking (P3). The loss factor has been used to define viscoelastic behavioral zones for each DMA rheo-thermogram operated in the same DSC thermal conditions. Macromolecular evolution and viscoelastic properties have been obtained by pairing the deconvoluted DSC thermograms with the viscoelastic behavioral zones of the DMA rheo-thermograms. Two main chemorheological behaviors have been identified relative to pre- and post-gelation separation of the viscoelastic liquid from the viscoelastic solid. Each comprises three behavioral zones, accounting for the concurrently occurring linear and branching oligomerization, aluminate-rich and silica-rich gel nucleations, crosslinking, and vitrification. A "rubbery plateau" in the loss factor path, observed for all the testing conditions, identifies a large behavioral transition zone dividing the incipient gelling liquid slurry from the material hard setting and vitrification.

Comparison of gels synthesized by controlled radical copolymerization and free radical copolymerization: molecular dynamics simulation.

The structures of gels synthesized by controlled radical copolymerization (CRP) and conventional free radical copolymerization (FRP) were studied by a coarse-grained molecular dynamics simulation. It was confirmed that the CRP gel has a larger number of elastically effective chains and fewer cyclic structures and entanglements than the FRP gel, i.e., the network structure of the CRP gel is more uniform than that of the FRP gel. However, the difference in the shear modulus between the two gels was small due to the opposing changes in the number of elastically effective chains and that of entanglements. The relatively uniform structure of the CRP gel is attributed to the suppression of intramolecular cross-linking by the fast initiation and slow propagation, and the development of cross-linked structures in the post-gel region due to the limited termination. The effects of these CRP characteristics were studied in detail. From the results, it was found that all of these characteristics of CRP cooperatively act to improve the homogeneity of the structure of the CRP gel.

Preparation of poly(oxanorbornene) based single and double-folding polymers via nucleophilic aromatic substitution reaction

A facile approach is introduced to prepare single-chain nanoparticles (SCNPs) utilizing intramolecular crosslinking based on nucleophilic aromatic substitution reaction. The linear polymeric precursor comprising dichlorotriazine (DCT) moiety as a reactive pendant group is synthesized via ring-opening metathesis polymerization (ROMP). The reactive chlorine atoms on DCT motifs are substituted with a dithiol nucleophile in varying degrees to yield individually intramolecularly crosslinked polymer nanoparticles. In addition, double-collapse and modification reactions are exploited through the substitution reaction from the remaining unreacted chlorine atoms. A significant reduction in hydrodynamic volumes of the corresponding SCNPs is observed compared to their linear analogues. The obtained nanoparticles are characterized by size exclusion chromatography (SEC), proton nuclear magnetic resonance spectroscopy (1H NMR), diffusion-ordered spectroscopy (DOSY), dynamic light scattering (DLS), and transmission electron microscopy (TEM) techniques.

Effects of Phenolics on the Physicochemical and Structural Properties of Collagen Hydrogel.

In the current era, the treatment of collagen hydrogels with natural phenolics for the improvement in physicochemical properties has been the subject of considerable attention. The present research aimed to fabricate collagen hydrogels cross-linked with gallic acid (GA) and ellagic acid (EA) at different concentrations depending on the collagen dry weight. The structural, enzymatic, thermal, morphological, and physical properties of the native collagen hydrogels were compared with those of the GA/EA cross-linked hydrogels. XRD and FTIR spectroscopic analyses confirmed the structural stability and reliability of the collagen after treatment with either GA or EA. The cross-linking also significantly contributed to the improvement in the storage modulus, of 435 Pa for 100% GA cross-linked hydrogels. The thermal stability was improved, as the highest residual weight of 43.8% was obtained for the hydrogels cross-linked with 50% GA in comparison with all the other hydrogels. The hydrogels immersed in 30%, 50%, and 100% concentrations of GA also showed improved swelling behavior and porosity, and the highest resistance to type 1 collagenase (76.56%), was obtained for 50% GA cross-linked collagen hydrogels. Moreover, GA 100% and EA 100% obtained the highest denaturation temperatures (Td) of 74.96 °C and 75.78 °C, respectively. In addition, SEM analysis was also carried out to check the surface morphology of the pristine collagen hydrogels and the cross-linked collagen hydrogels. The result showed that the hydrogels cross-linked with GA/EA were denser and more compact. However, the improved physicochemical properties were probably due to the formation of hydrogen bonds between the phenolic hydroxyl groups of GA and EA and the nitrogen atoms of the collagen backbone. The presence of inter- and intramolecular cross-links between collagen and GA or EA components and an increased density of intermolecular bonds suggest potential hydrogen bonding or hydrophobic interactions. Overall, the present study paves the way for further investigations in the field by providing valuable insights into the GA/EA interaction with collagen molecules.

“To Be or Not to Be” of a Polymer Nanogel—Unravelling the Relationship of Product Properties vs. Synthesis Conditions Governing the Radiation Crosslinking of Poly(acrylic acid) Using GPC/SEC—MALLS

In this paper, a state-of-the-art multi-detection gel permeation chromatography/size exclusion chromatography (GPC/SEC) system including multi-angle laser light scattering (MALLS) is applied to monitor radiation-induced synthesis of internally crosslinked nanostructures from poly(acrylic acid) (PAA). The aim is to demonstrate that this modern tool yields a more detailed picture of reaction mechanism and product structure than the techniques used to date. The prevailing intramolecular crosslinking narrows the molecular weight distribution from Mw/Mn = 3.0 to 1.6 for internally crosslinked structures. A clear trend from over 0.7 to 0.5 in the Mark–Houwink exponent and a decrease in Rg/Rh from 1.7 to 1.0 point to the formation of nanogels, more rigid and less permeable than the starting coils. Changes in the coil contraction factor (g′ = [η]irradiated/[η]linear) as a function of the radical density revealed the existence of two modes in intramolecular crosslinking, the initial one (up to 0.075 radicals per monomer unit) where the compactness of products changes strongly with progressing crosslinking and a second one where further compacting is suppressed by the lower flexibility of the partially crosslinked chain segments. This indicates a transition from soft, still internally crosslinkable nanogels to more rigid structures, less prone to further intramolecular loop formation. Our findings provide means for the tailored design of new PAA nanomaterials.

Chitosan Oligosaccharide Based Waterborne Polyurethane with UV Resistance and Oxidation Resistance for Functional Fabric

AbstractChitosan oligosaccharide (COS) is an abundant polysaccharide in nature, which has various functions such as antioxidant and antibacterial. The possibilities of using COS can be extended by applying it to polyurethane materials. Herein, Chitosan oligosaccharide‐based waterborne polyurethane (CWPU) with intramolecular crosslinking network is prepared by using COS as chain extender. The mechanical properties of CWPU are improved, with tensile strength and elongation at break reaching 14.83 MPa and 449.63%, respectively. The DPPH free radical scavenging rate of CWPU increases from 8.43% to 70.98%, indicating that CWPU has favorable antioxidant performance. The tensile strength of the CWPU‐treated fabrics obtained by the padding method improve from 542.77 to 605.33N and there is no significant change in the whiteness of the fabric. Furthermore, the CWPU‐treated fabrics, with the UV protection factor of 30.39 and the UVA transmittance below 3%, can be used as UV‐protective fabrics. This work introduces COS into the molecular structure of polyurethanes to obtain functional PUs, expanding the application of COS in polyurethanes.

High solid content waterborne polyurethane and the coatings with double crosslinking structure and multiple hydrogen bonds

The development of coating and emulsion with excellent scratch resistance, friction resistance, high transparency, and yellowing resistance is of great significance for protecting the substrate, reducing the wear of the material, and improving the curing and. drying speed of the coating. Therefore, a double-crosslinked waterborne polyurethane (DCWPU) was prepared in this paper to make up for the shortcomings of slow drying speed and poor toughness of the coating film in a single cross-linked system. Meanwhile, DCWPU utilizes chemical bonding crosslinking network and high-density hydrogen bonding array to cooperate with each other, which is beneficial to improve the mechanical properties of the double-crosslinking system. Herein, 3 (2-hydroxyethyl) isocyanurate (THEIC) was introduced as the intramolecular crosslinking agent, and isophorone isocyanate trimer (IPDI-3), which has the same keto azocyclic structure as THEIC, was added as the post crosslinking agent to further increase the crosslinking degree of the system. The effects of cross-linked monomer content in waterborne polyurethane on scratch resistance, friction resistance, and corresponding emulsion solid content of the coating were studied in the work. It was found that with the increase of double cross-linked monomer content, the hydrogen bond force could be effectively improved, and the mechanical properties, wear resistance and solid content of the emulsion could be significantly improved. It is worth mentioning that DCWPU-3 showed the best comprehensive properties. The emulsion solid content was 48.9%, the tensile strength of the film was increased to 21.8 MPa, the elongation at break was 627.2%, the hardness was 4 H, and the wear amount was 1.2 mg.

Thermoresponsive Helical Dendronized Poly(phenylacetylene)s: Remarkable Stabilization of Their Helicity via Photo-Dimerization of the Dendritic Pendants.

Dynamic helical polymers can change their helicity according to external stimuli due to the low helix-inversion barriers, while helicity stabilization for polymers is important for applications in chiral recognition or chiral separations. Here, we present a convenient methodology to stabilize dynamic helical conformations of polymers through intramolecular cross-linking. Thermoresponsive dendronized poly(phenylacetylene)s (PPAs) carrying 3-fold dendritic oligoethylene glycol pendants containing cinnamate moieties were synthesized. These polymers exhibit typical features of dynamic helical structures in different solvents, that is, racemic contracted conformations in less polar organic solvents and predominantly one-handed stretched helical conformations in highly polar solvents. This dynamic helicity can be enhanced through selective solvation by increasing the polarity of the organic solvents or simply via their thermally mediated dehydration in water. However, through photocycloaddition of the cinnamate moieties between the neighboring pendants via UV irradiation, these dendronized PPAs adopt stable helical conformations either below or above their phase transition temperatures in water, and their helical conformations can even be retained in less polar organic solvents. Spectroscopic and atomic force microscopy measurements demonstrate that photocycloaddition between the cinnamate moieties occurs on the individual molecular level, and this is found to be helpful in restraining the photodegradation of the PPA backbones. Molecular dynamics simulations reveal that the spatial orientation of the pendants along the rigid polyene backbone is crucial for the photodimerization of cinnamates within one helix pitch.

Domain structure and cross-linking in a giant adhesin from the Mobiluncus mulieris bacterium.

Cell-surface proteins known as adhesins enable bacteria to colonize particular environments, and in Gram-positive bacteria often contain autocatalytically formed covalent intramolecular cross-links. While investigating the prevalence of such cross-links, a remarkable example was discovered in Mobiluncus mulieris, a pathogen associated with bacterial vaginosis. This organism encodes a putative adhesin of 7651 residues. Crystallography and mass spectrometry of two selected domains, and AlphaFold structure prediction of the remainder of the protein, were used to show that this adhesin belongs to the family of thioester, isopeptide and ester-bond-containing proteins (TIE proteins). It has an N-terminal domain homologous to thioester adhesion domains, followed by51 immunoglobulin (Ig)-like domains containing ester- or isopeptide-bond cross-links. The energetic cost to the M. mulieris bacterium in retaining such a large adhesin as a single gene or protein construct suggests a critical role in pathogenicity and/or persistence.

Merging non-covalent and covalent crosslinking: En route to single chain nanoparticles

Single-chain nanoparticles represent an emerging class of nanomaterials designed to mimic protein's folding paradigm. Intrachain covalent crosslinking toward the formation of single-chain nanoparticles encounters complex energy landscapes, leading to the potential occurrence of misfolding issues. While non-covalent crosslinking can circumvent this issue, the resulting single-chain nanoparticles exhibit lower structural stability compared to their covalently crosslinked counterparts. In this study, we present a novel approach for the synthesis of single-chain nanoparticles, achieved through the combination of non-covalent and covalent intramolecular crosslinking. Cyanostilbenes grafted onto the linear polymer form intrachain non-covalent stacks aided by hydrogen bonds, leading to the formation of non-covalently crosslinked single-chain nanoparticles. These nanoparticles undergo conversion to covalently crosslinked nanostructures through subsequent photo-irradiation using [2 + 2] photocycloaddition, a process facilitated by the supramolecular confinement effect. Consequently, the resulting single-chain nanoparticles demonstrate both intrachain folding efficiency and substantial stability, offering significant potential for advancing applications across diverse fields.

Autoreactive B cells against malondialdehyde-induced protein cross-links are present in the joint, lung, and bone marrow of rheumatoid arthritis patients

Autoantibodies to malondialdehyde (MDA) proteins constitute a subset of anti-modified protein autoantibodies in rheumatoid arthritis (RA), which is distinct from citrulline reactivity. Serum anti-MDA IgG levels are commonly elevated in RA and correlate with disease activity, CRP, IL6, and TNF-α. MDA is an oxidation-associated reactive aldehyde that together with acetaldehyde mediates formation of various immunogenic amino acid adducts including linear MDA-lysine, fluorescent malondialdehyde acetaldehyde (MAA)-lysine, and intramolecular cross-linking. We used single-cell cloning, generation of recombinant antibodies (n= 356 from 25 donors), and antigen-screening to investigate the presence of class-switched MDA/MAA+ B cells in RA synovium, bone marrow, and bronchoalveolar lavage. Anti-MDA/MAA+ B cells were found in bone marrow plasma cells of late disease and in the lung of both early disease and risk-individuals and in different B cell subsets (memory, double negative B cells). These were compared with previously identified anti-MDA/MAA from synovial memory and plasma cells. Seven out of eight clones carried somatic hypermutations and all bound MDA/MAA-lysine independently of protein backbone. However, clones with somatic hypermutations targeted MAA cross-linked structures rather than MDA- or MAA-hapten, while the germline-encoded synovial clone instead bound linear MDA-lysine in proteins and peptides. Binding patterns were maintained in germline converted clones. Affinity purification of polyclonal anti-MDA/MAA from patient serum revealed higher proportion of anti-MAA versus anti-MDA compared to healthy controls. In conclusion, IgG anti-MDA/MAA show distinct targeting of different molecular structures. Anti-MAA IgG has been shown to promote bone loss and osteoclastogenesis invivo and may contribute to RA pathogenesis.

All-polymer nanocomposites having superior strength, toughness and ultralow energy dissipation

Toughening polymers has attracted significant interest. Traditionally, polymer toughness is enhanced by constructing polymer networks or introducing sacrificial bonds into the chains between crosslink points. These strategies, though, introduce pronounced energy dissipation and associated heat, both of which are undesirable under long-term cyclic loading, for example at the interface of implants in the human body. By incorporating single-chain nanoparticles (SCNPs) into linear polymer chains to generate all-polymer nanocomposites (APNCs), we have been able to achieve high strength, high toughness with low energy dissipation. Using a combination of simulation and experimental results, we are advancing a “SCNPs effect” where tightly cross-linked SCNPs produce a modulus contrast to achieve strengthening and toughening. Benefitting from the soft interface, the penetrable and deformable SCNPs cause the surrounding polymer chains to move in concert, significantly reducing the interfacial friction to achieve low energy dissipation. The intramolecular cross-linking of the SCNPs and adhesion between the SCNPs and polymer matrix are critical for realizing such high-performance systems. Based on a Gaussian regression model and back propagation (BP) neural network, the mechanical strength can be predicted and is supported by simulations. The APNC concept described can be applied to elastomers and gels, broadening its utilization in high-cycle and low-dissipation applications, like soft robots, flexible sensors and cartilage replacements, and artificial heart valves.

Molecular dynamics of poly(methyl methacrylate) single-chain nanoparticles

Polymer Single-chain nanoparticles (SCNPs) with different cross-linking densities were synthetized from poly (methyl methacrylate-co-(2-acetoacetoxy) ethyl methacrylate) (PMMA-co-PAEMA) random copolymers, and the molecular dynamics of SCNPs was investigated using differential scanning calorimetry (DSC) and broadband dielectric spectroscopy (BDS). The DSC results show that the glass transition temperature (Tg) of SCNPs formed after intra-molecular cross-linking is higher than that of linear precursor, and increases with increasing the cross-linking density. The BDS results reveal that the segment (α) relaxation of SCNPs is significantly inhibited, as evidenced by an increase in the relaxation time and a decrease in the dielectric strength. This is due to the intra-molecular topological constraints which limits the mobility of the polymer segments. Moreover, the distribution width of α relaxation is broader, indicating a more pronounced dynamical heterogeneity. The relaxation time of Johari-Goldstein β (βJG) relaxation increases slightly, neither the dynamics nor the peak width of βJG relaxation is affected by intra-molecular cross-linking.

Wheat bran arabinoxylan-soybean protein isolate emulsion-filled gels as a β-carotene delivery carrier: Effect of polysaccharide content on textural and rheological properties

This study aimed to investigate the effects of different wheat bran arabinoxylan (WBAX) concentrations (1, 2, 3, and 4 wt%) on the structural and physicochemical properties of WBAX-soybean protein isolate (SPI) emulsion-filled gels (EFGs) prepared using laccase and heat treatment. The properties of the various gels as well as their microstructure, rheology, and in vitro digestion behaviors were investigated. Results showed that WBAX-SPI EFGs with a 3 wt% WBAX concentration had a smooth and uniform appearance, high water holding capacity (98.5 ± 0.2 %), and enhanced mechanical properties. Rheological experiments suggested that a stronger and closer gel network was formed at 3 wt% WBAX concentration. Fourier transform infrared spectroscopy showed that laccase and heat treatment not only catalyzed the intramolecular crosslinking of WBAX and SPI, respectively, but also promoted the interaction between WBAX and SPI. Confocal laser scanning microscopy revealed that the WBAX gel network was interspersed within the SPI network. The interactions contributing to the gelation analysis revealed that chemical (disulfide bond) and physical (hydrogen bond and hydrophobic) interactions promoted the formation of denser EFGs. Furthermore, the WBAX-SPI EFGs provided a β-carotene bioaccessibility of 21.8 ± 0.6 %. Therefore, our study suggests that WBAX-SPI EFGs hold promising potential for industrial applications in the delivery of β-carotene.

Transglutaminase in Foods and Biotechnology.

Stabilization and reusability of enzyme transglutaminase (TGM) are important goals for the enzymatic process since immobilizing TGM plays an important role in different technologies and industries. TGM can be used in many applications. In the food industry, it plays a role as a protein-modifying enzyme, while, in biotechnology and pharmaceutical applications, it is used in mediated bioconjugation due to its extraordinary crosslinking ability. TGMs (EC 2.3.2.13) are enzymes that catalyze the formation of a covalent bond between a free amino group of protein-bound or peptide-bound lysine, which acts as an acyl acceptor, and the γ-carboxamide group of protein-bound or peptide-bound glutamine, which acts as an acyl donor. This results in the modification of proteins through either intramolecular or intermolecular crosslinking, which improves the use of the respective proteins significantly.

Single‐chain nanoparticle catalyzed polymerization toward composite nanoparticles

AbstractSingle‐chain nanoparticles (SCNPs) as a confined space are promising to synthesize structured nanoparticles. We report a facile synthesis of SCNP colloidal initiators by loading reducing agents during the formation of the SCNPs and the application in redox free radical polymerization to achieve composite nanoparticles with tunable compositions and microstructures. The SCNP initiators could be large‐scale achieved by electrostatic‐mediated intramolecular crosslinking polymers by metallic complexation with protonated PVP at ambient temperature. The catalytic polymerization occurs inwardly under facile conditions to large scale fabricate composite nanoparticles with tunable microstructures from core‐shell to tadpole‐shapes. The facile approach could be extended to derive functional composite nanoparticles by loading desired materials such as paramagnetic Fe3O4, catalytic Pt, and enzymes.

Indirect determination of partial depolymerization reactions in dialdehyde celluloses (DAC) by gel permeation chromatography of their oxime derivatives

Owing to a supposed quantitative transformation, oximation of dialdehyde cellulose (DAC) with hydroxylamine hydrochloride is commonly employed in chemical DAC analysis, e.g., for the determination of the degree of oxidation (DO) by titration or elemental analysis. In this study, this modification was utilized for the indirect determination of molecular weight distributions (MWD) by gel permeation chromatography (GPC). The presumably quantitative conversion of aldehyde groups in DAC to the corresponding oxime also breaks up the intermolecular and intramolecular hemiacetal crosslinks, which were associated with solubility issues in the DMAc/LiCl solvent system in previous studies. The limits of the procedure and the material's stability during oximation were investigated. For samples with a DO up to approximately 9% a good applicability was observed, before at higher DO values residual crosslinks led to solubility problems. The oximation/GPC protocol was used to examine the development of the MWD in the early stages of DAC formation under different reaction conditions. The time-dependent partial depolymerization of the polymer backbone was observed. Furthermore, the stability of DAC towards different pH conditions ranging from strongly acidic to strongly alkaline was tested. The depolymerization of DAC in alkaline media occurred with concomitant degradation of aldehyde moieties. In turn, DAC proved to be remarkably stable in acidic and neutral solutions up to a pH of 7.

Glutaraldehyde modification of lipases immobilized on octyl agarose beads: Roles of the support enzyme loading and chemical amination of the enzyme on the final enzyme features

Lipase B from Candida antarctica (CALB) and lipase from Thermomyces lanuginosus (TLL) have been immobilized on octyl agarose at low loading and at a loading exceeding the maximum support capacity. Then, the enzymes have been treated with glutaraldehyde and inactivated at pH 7.0 in Tris-HCl, sodium phosphate and HEPES, giving different stabilities. Stabilization (depending on the buffer) of the highly loaded biocatalysts was found, very likely as a consequence of the detected intermolecular crosslinkings. This did not occur for the lowly loaded biocatalysts. Next, the enzymes were chemically aminated and then treated with glutaraldehyde. In the case of TLL, the intramolecular crosslinkings (visible by the apparent reduction of the protein size) increased enzyme stability of the lowly loaded biocatalysts, an effect that was further increased for the highly loaded biocatalysts due to intermolecular crosslinkings. Using CALB, the intramolecular crosslinkings were less intense, and the stabilization was lower, even though the intermolecular crosslinkings were quite intense for the highly loaded biocatalyst. The stabilization detected depended on the inactivation buffer. The interactions between enzyme loading and inactivating buffer on the effects of the chemical modifications suggest that the modification and inactivation studies must be performed under the target biocatalysts and conditions.