A stepwise approach to the understanding of extracellular enzyme activity in soil. I. Effect of electrostatic interactions on the conformation of a beta-D-glucosidase adsorbed on different mineral surfaces

The enzymatic activity of sweet almond beta-D-glucosidase adsorbed on various mineral surfaces was studied. Our aim was to elucidate the mechanism responsible for the observed changes in catalytic activity. The results of the investigation are discussed with reference to the hypotheses generally proposed to explain the well-documented shift in optimal pH of the activity of adsorbed enzymes. By separate determinations of enzymatic activity in a mineral suspension and of its supernatant solution, and comparison with a control without mineral added, we obtained accurate measurements of the catalytic activity of the adsorbed enzyme alone. Different pH profiles of activity profiles were found when the enzyme was adsorbed onto montmorillonite, kaolinite and goethite. The activity profiles, were also found to vary with ionic strength, the pH at which enzyme adsorbed onto the mineral surface, and in the case of goethite, on the nature of the anions in the buffer. Our observations cannot be adequately explained by assuming a more acidic microenvironment at the mineral surface. We postulate that on some mineral surfaces a conformational change is induced in the adsorbed protein, which reduces its catalytic activity. We contend that such conformational changes are due primarily to electrostatic forces.     

A screening approach reveals the influence of mineral coating morphology on human mesenchymal stem cell differentiation

“Biomimetic” inorganic coating on biomaterials has been an active area of research with the aim of providing bioactive surfaces that can regulate cell behavior. Previous studies have demonstrated that human mesenchymal stem cell (hMSC) behavior is differentially regulated by the physical and chemical properties of inorganic mineral coatings, indicating that modulation of mineral properties is potentially important in regulating hMSC behavior. However, the lack of an efficient experimental context, in which to study stem cell behavior on inorganic substrates, has made it difficult to systematically study the effects of specific mineral coating parameters on hMSC behavior. In this study, we developed an efficient experimental platform to screen for the effects of mineral coating morphology on hMSC expansion and differentiation. hMSC expansion on mineral coatings was regulated by the micro-scale morphology of these coatings, with greater expansion on small granule-like coatings when compared to plate-like or net-like coatings. In contrast, hMSC osteogenic differentiation was inversely correlated with cell expansion on mineral coatings indicating that mineral coating morphology was a key parameter that regulates hMSC differentiation. The effect of mineral coating morphology on hMSC behavior underlines the utility of this inorganic screening platform to identify optimal coatings for medical devices and bone tissue engineering applications.     

Siloxane-based biocatalytic films and paints for use as reactive coatings

We have developed a new methodology for preparing films and paints suitable for use as biocatalytic coatings. The hydrolytic enzymes pronase and alpha-chymotrypsin were immobilized by either sol-gel entrapment or by covalent attachment into a polydimethylsiloxane (PDMS) matrix and cast into thin films or incorporated into an oil-based paint formulation. All of the coatings retained enzymatic activity and adhered to several different materials. The enzymatic films and paints also exhibited higher thermostability than enzyme free in solution or covalently attached to the outer surface of PDMS. A porous membrane based on a PDMS-immobilized enzyme was also prepared by an immersion precipitation process. Protein adsorption measurements showed that the enzyme-containing films and paints adsorbed less protein than enzyme-free controls, and that protein adsorption decreased with increasing proteolytic activity of the coating. These coatings thus provide the means to apply a stable enzymatic surface to a wide range of materials, and may be generally useful as biocatalytic paints and films.     

Layered inorganic/enzyme nanohybrids with selectivity and structural stability upon interacting with biomolecules

Effective intercalation of protein molecules within the galleries of montmorillonites can be achieved via simple space enlarging and exchange processes while retaining the native conformation of the guest protein and the multilayered structure of the bioinert host plates. The capacity of accommodating protein molecules in the galleries can be markedly larger than that governed by the Langmuir-type adsorption of protein molecules on the external surfaces of particles. The basal spacing in the multilayered structure of clay is abruptly enlarged when the extent of protein intercalation increases to a critical point. Beyond this critical point, the nanohybrids show well-preserved catalytic activity in hydrolyzing small substrates while establishing a barrier to interactions with large biomacromolecules. Furthermore, the structural stability of the inorganic/organic nanohybrids is enhanced such that neither exchange of biomolecules nor exfoliation of layered clay particles occurs when exposed to other proteins. The results indicate that, through the benign accommodation of protein species between the inorganic platelets, this nanoscaled manipulation of protein functions can be highly useful in developing new inorganic/enzyme nanohybrids for protein therapeutics and tissue engineering.     

A conceptual model of organo-mineral interactions in soils: self-assembly of organic molecular fragments into zonal structures on mineral surfaces

In this paper, we propose a structure for organo-mineral associations in soils based on recent insights concerning the molecular structure of soil organic matter (SOM), and on extensive published evidence from empirical studies of organo-mineral interfaces. Our conceptual model assumes that SOM consists of a heterogeneous mixture of compounds that display a range of amphiphilic or surfactant-like properties, and are capable of self-organization in aqueous solution. An extension of this self-organizational behavior in solution, we suggest that SOM sorbs to mineral surfaces in a discrete zonal sequence. In the contact zone, the formation of particularly strong organo-mineral associations appears to be favored by situations where either (i) polar organic functional groups of amphiphiles interact via ligand exchange with singly coordinated mineral hydroxyls, forming stable inner-sphere complexes, or (ii) proteinaceous materials unfold upon adsorption, thus increasing adhesive strength by adding hydrophobic interactions to electrostatic binding. Entropic considerations dictate that exposed hydrophobic portions of amphiphilic molecules adsorbed directly to mineral surfaces be shielded from the polar aqueous phase through association with hydrophobic moieties of other amphiphilic molecules. This process can create a membrane-like bilayer containing a hydrophobic zone, whose components may exchange more easily with the surrounding soil solution than those in the contact zone, but which are still retained with considerable force. Sorbed to the hydrophilic exterior of hemimicellar coatings, or to adsorbed proteins, are organic molecules forming an outer region, or kinetic zone, that is loosely retained by cation bridging, hydrogen bonding, and other interactions. Organic material in the kinetic zone may experience high exchange rates with the surrounding soil solution, leading to short residence times for individual molecular fragments. The thickness of this outer region would depend more on input than on the availability of binding sites, and would largely be controlled by exchange kinetics. Movement of organics into and out of this outer region can thus be viewed as similar to a phase-partitioning process. The zonal concept of organo-mineral interactions presented here offers a new basis for understanding and predicting the retention of organic compounds, including contaminants, in soils and sediments.     

Multilayer adsorption of lysozyme on a hydrophobic substrate.

Macromolecular adsorption is known to occur as a complex process, often in a series of steps. Several models are discussed in the literature which describe the microscopic structure of the adsorbate. In the present study we investigated the adsorption of hen egg white lysozyme on alkylated silicon oxide surfaces. A combination of fluorescence excitation in the evanescent field and fluorescence recovery after photobleaching allowed us to measure the amount of adsorbed fluorescent lysozyme and the equilibrium exchange kinetics with molecules in solution. We found that a model with at least three classes of adsorbed molecules is necessary to describe the experimental results. A first layer is formed by the molecules which adsorb within a short time after the beginning of the incubation. These molecules make up approximately 65% of the final coverage. They are quasi-irreversibly adsorbed and do not measurably exchange with bulk molecules within one day even at temperatures up to 55 degrees C. A second layer, which reaches equilibrium only after several hours of incubation, shows a pronounced exchange with bulk molecules. The on-off kinetics show a distinct temperature dependence from which an activation barrier of delta E approximately 22 kcal/mol is derived. A third layer of molecules that exchange rapidly with the bulk can be seen to comprise approximately 10% of the total coverage. The exchange rate is on the order of fractions of a second. The binding of the latter two classes of adsorbed molecules is exothermic. From the temperature dependence of the coverage, the binding enthalpy of the slowly exchanging layer was estimated to be delta Hads approximately 3.8 kcal/mol. The second and third class of molecules remain enzymatically active as a muramidase, which was tested by the lysis of the cell walls of Micrococcus lysodeiktikus. The molecules in the first layer, on the other hand, showed no enzymatic activity.     

Doped diamond-like carbon coatings for surgical instruments reduce protein and prion-amyloid biofouling and improve subsequent cleaning

Doped diamond-like carbon (DLC) coatings offer potential antifouling surfaces against microbial and protein attachment. In particular, stainless steel surgical instruments are subject to tissue protein and resilient prion protein attachment, making decontamination methods used in sterile service departments ineffective, potentially increasing the risk of iatrogenic Creutzfeldt-Jakob disease during surgical procedures. This study examined the adsorption of proteins and prion-associated amyloid to doped DLC surfaces and the efficacy of commercial cleaning chemistries applied to these spiked surfaces, compared to titanium nitride coating and stainless steel. Surfaces inoculated with ME7-infected brain homogenate were visualised using SYPRO Ruby/Thioflavin T staining and modified epi-fluorescence microscopy before and after cleaning. Reduced protein and prion amyloid contamination was observed on the modified surfaces and subsequent decontamination efficacy improved. This highlights the potential for a new generation of coatings for surgical instruments to reduce the risk of iatrogenic CJD infection.     

Nanoscale reduction in surface friction of polymer surfaces modified with Sc3 hydrophobin from Schizophyllum commune

Hydrophobins are amphipathic self-assembling proteins secreted by filamentous fungi that exhibit remarkable ability to modify synthetic surfaces. Thin coatings of Sc3 hydrophobin isolated from the wood-rotting fungus Schizophyllum commune were prepared via spin coating and adsorption techniques onto polymeric surfaces. Surface morphology and nanotribological characteristics of the films were evaluated using lateral force microscopy (LFM) and nanoindentation techniques. This paper reports the first observation of reduction in nanoscale relative surface friction of Sc3 hydrophobin protein modified polymeric surfaces. Relative friction coefficients were dramatically reduced and hydrophilicity increased for polymer surfaces modified with Sc3 hydrophobin thin films. Morphology of the protein films as well as degree of surface modification was observed to be a function of film formation technique and composition of the substrate.     

Adsorption, desorption, and activity of glucose oxidase on selected clay species.

The adsorption of the enzyme glucose oxidase (EC 1.1.3.4) to clays followed the pattern described for other proteins as being pH dependent. Maximum adsorption occurred at or below the isoelectric point of the enzyme. The amount of enzyme adsorbed to clay was influenced by the type of clay used, and also the saturating cations. Initially adsorbed enzyme showed low specific activities, and as amounts of enzyme adsorbed approached maximum stauration of clay, specific activities increased approaching that determined for free enzyme. The adsorption of glucose oxidase involved a temperature-independent cation-exchange mechanism, and enzyme adsorbed to surfaces of clay could be desorbed in active form by elevation of pH of suspending solution. This was followed by a slower temperature-dependent fixation, probably by hydrogen bonding, which resulted in protein being irreversibly adsorbed to clay surfaces. It is proposed that on adsorption of glucose oxidase to clay surfaces unravelling of the protein structure occurred, which allowed penetration of protein into the interlamellar spaces of montmorillonite. This proposal was based on the observed expansion of montmorillonite to 23 A, and the decreases in amount of a second-protein lysozyme adsorbed with extended incubation times of glucose oxidase - clay complexes at pH 4.5.     

Osteoconductivity and growth factor production by MG63 osteoblastic cells on bioglass‐coated orthopedic implants

We have produced Bioglass coatings for Orthopedic implants by using a novel coating technique, CoBlast. The two resultant surfaces, designated BG and hydroxyapatite (HA)/BG, were compared with their HA counterpart, OsteoZip in terms of osteoblastic cell attachment, adhesion, proliferation, differentiation, and growth factor production. BG and HA/BG were demonstrated by goniometry to be more hydrophilic than OsteoZip. This corresponded to enhanced protein adsorption, cell attachment, and cell adhesion documented by both quantitative and qualitative assessments. BG and HA/BG surfaces had a significant initial release of Si and Ca ions, and this was consistent with elevated cell proliferation and basic fibroblast growth factor levels. However, OsteoZip, being similar to HA/BG, exhibited better osteogenic differentiation than BG did, shown by augmented differentiation marker activity at both protein and mRNA levels. Sandwich ELISA was used to quantify angiopoietin and inducible nitric oxide synthase which are involved in peri‐prosthetic angiogenesis and aseptic loosening of total hip replacement, respectively. Both Bioglass‐derived coatings provide superior initial osteoconductivity to OsteoZip, and HA/Bioglass composite coating outruns in long‐term osteogenic differentiation and prognostic bioprocesses. The novel coatings discovered in this study have significant potential in providing both orthopedic and therapeutic functions. Biotechnol. Bioeng. 2011;108: 454–464. © 2010 Wiley Periodicals, Inc.     

Conformational changes and orientation of Humicola lanuginosa lipase on a solid hydrophobic surface: an in situ interface Fourier transform infrared-attenuated total reflection study

This study was done to better understand how lipases are activated at an interface. We investigated the conformational and solvation changes occurring during the adsorption of Humicola lanuginosa lipase (HLL) onto a hydrophobic surface using Fourier transform infrared-attenuated total reflection spectroscopy. The hydrophobic surfaces were obtained by coating silicon attenuated total reflection crystal with octadecyltrichlorosilane. Analysis of vibrational spectra was used to compare the conformation of HLL adsorbed at the aqueous-solid interface with its conformation in solution. X-ray crystallography has shown that HLL exists in two conformations, the closed and open forms. The conformational changes in HLL caused by adsorption onto the surface were compared with those occurring in three reference proteins, bovine serum albumin, lysozyme, and alpha-chymotrypsin. Adsorbed protein layers were prepared using proteins solutions of 0.005 to 0.5 mg/mL. The adsorptions of bovine serum albumin, lysozyme, and alpha-chymotrypsin to the hydrophobic support were accompanied by large unfoldings of ordered structures. In contrast, HLL underwent no secondary structure changes at first stage of adsorption, but there was a slight folding of beta-structures as the lipase monolayer became complete. Solvation studies using deuterated buffer showed an unusual hydrogen/deuterium exchange of the peptide CONH groups of the adsorbed HLL molecules. This exchange is consistent with the lipase being in the native open conformation at the water/hydrophobic interface.     

Reduction of albumin adsorption onto silicon surfaces by Tween 20

The ability of Tween 20 to reduce the adsorption of albumin on silicon surfaces of different hydrophobicity was investigated by ellipsometry. As expected, protein adsorption was found to depend on the degree of hydrophobicity of the surfaces and on the concentration of the surfactant. A reduction of 90% in albumin adsorption on hydrophobic methylated surfaces by 0.05% Tween 20 was achieved, whereas a reduction of only 15% on hydrophilic surfaces was observed. Experiments of time-dependent protein adsorption in both pure protein and protein-surfactant mixtures were conducted to ascertain the stability of physically adsorbed Tween 20 films on intermediate silicon surfaces. It was found that the adsorbed Tween 20 film was robust and there was no evidence of exchange of the Tween molecules with albumin for up to 240 min exposure. Adsorption minima were confirmed to correlate with minima in contact angle and critical micelle concentration (CMC). (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 618-625, 1997.     

Arginine Inhibits Adsorption of Proteins on Polystyrene Surface

Nonspecific adsorption of protein on solid surfaces causes a reduction of concentration as well as enzyme inactivation during purification and storage. However, there are no versatile inhibitors of the adsorption between proteins and solid surfaces at low concentrations. Therefore, we examined additives for the prevention of protein adsorption on polystyrene particles (PS particles) as a commonly-used material for vessels such as disposable test tubes and microtubes. A protein solution was mixed with PS particles, and then adsorption of protein was monitored by the concentration and activity of protein in the supernatant after centrifugation. Five different proteins bound to PS particles through electrostatic, hydrophobic, and aromatic interactions, causing a decrease in protein concentration and loss of enzyme activity in the supernatant. Among the additives, including arginine hydrochloride (Arg), lysine hydrochloride, guanidine hydrochloride, NaCl, glycine, and glucose, Arg was most effective in preventing the binding of proteins to PS particles as well as activity loss. Moreover, even after the mixing of protein and PS particles, the addition of Arg caused desorption of the bound protein from PS particles. This study demonstrated a new function of Arg, which expands the potential for application of Arg to proteins.     

Adsorption of poly(ethylene glycol)-modified ribonuclease A to a poly(lactide-co-glycolide) surface

Protein adsorption is a source of variability in the release profiles of therapeutic proteins from biodegradable microspheres. We employ optical reflectometry and total internal reflection fluorescence to explore the extent and kinetics of ribonuclease A (RNase A) adsorption to spin-cast films of poly(lactide-co-glycolide) (PLG) and, in particular, to determine how covalent grafting of polyethylene glycol (PEG) to RNase A affects adsorption. Adsorption kinetics on PLG surfaces are surface-limited for RNase A but transport-limited for unconjugated PEG homopolymers and for PEG-modified RNase A, indicating that PEG anchors the conjugates to the surface during the transport-limited regime. PEG modification of RNase A decreases the total number of adsorbed molecules per unit area but increases the areal surface coverage because the grafted PEG chains exclude additional surface area. Total internal reflection fluorescence-based exchange measurements show that there is no exchange between adsorbed and solution-phase protein molecules. This indicates an unusually tenacious adsorption. Streaming current measurements indicate that the zeta potential of the PLG surface becomes increasingly negative as the film is exposed to water for several weeks, as expected. Aging of the PLG surface results in increased adsorption of unmodified RNase A but decreased adsorption of unconjugated PEG homopolymers and of PEG-RNase A conjugates, relative to the extent of adsorption on freshly prepared PLG surfaces. Adsorption results correlate well with an increase in the rate, total extent and preservation of bioactivity of RNase A released from PLG microspheres for the PEG-modified version of RNase A.     

Regulation of phospholipase A2 activity by the lipid-water interface: a monolayer approach

Interfacial regulation of phospholipase A2 activity on lecithin monolayers was investigated by using radioactively labeled enzyme. Labeling of the protein with 125I did not produce a change of the enzyme and protein properties as compared to the 3H fully amidinated phospholipase A2. The induction time observed during pre-steady-state kinetics reflects the rate-limiting step of the penetration of the enzyme in the interface. This penetration is reversible. However, in the surface pressure range where the enzyme is able to hydrolyze the lecithin films, the desorption of the protein from the film is slow as compared to the adsorption. Below a surface pressure of 10 dyn/cm nonspecific adsorption occurs. Using lecithins with fatty acids of different chain lengths, we have shown that the kinetics of the penetration process is governed by the packing density of the substrate molecules independent of the surface pressure. However, the steady-state surface concentration of the enzyme increases with the fatty acyl chain length of the lecithin, indicating that hydrophobic interaction occurs between phospholipase A2 and the lipid molecules at the interface. From the lecithins used pancreatic phospholipase A2 preferentially splits substrate molecules with nine carbon atoms in the acyl chain.     

Purification and properties of an induced beta-D-glucosidase from stachybotrys atra.

We have purified an induced beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) from Stachybotrys atra to apparent homogeneity. The induced enzyme was clearly different from the constitutive beta-D-glucosidase. The molecular weight was 65 500-69 000, the pH optimum was at 6.7 and the isoelectric point at 4.8. Carbohydrate content (related to glucose) was 14.4%. The enzyme showed beta-D-glucosidase, beta-D-xylosidase and beta-D-thioglucosidase activity. These three activities sppear to be due to the same enzyme. The enzyme was strongly inhibited by D-glucono-(1 leads to 5)-lactone and nojirimycin and was very sensitive to sulfhydryl reagents.     

Factors that influence elastomeric coating performance: the effect of coating thickness on basal plate morphology, growth and critical removal stress of the barnacle Balanus amphitrite

Silicone coatings are currently the most effective non-toxic fouling release surfaces. Understanding the mechanisms that contribute to the performance of silicone coatings is necessary to further improve their design. The objective of this study was to examine the effect of coating thickness on basal plate morphology, growth, and critical removal stress of the barnacle Balanus amphitrite. Barnacles were grown on silicone coatings of three thicknesses (0.2, 0.5 and 2 mm). Atypical ("cupped") basal plate morphology was observed on all surfaces, although there was no relationship between coating thickness and i) the proportion of individuals with the atypical morphology, or ii) the growth rate of individuals. Critical removal stress was inversely proportional to coating thickness. Furthermore, individuals with atypical basal plate morphology had a significantly lower critical removal stress than individuals with the typical ("flat") morphology. The data demonstrate that coating thickness is a fundamental factor governing removal of barnacles from silicone coatings.     

Regulation of carboxylester lipase adsorption to surfaces. 1. Chemical specificity

The chemical specificity of the adsorption of porcine pancreatic carboxyl ester lipase to pure lipid surfaces was examined. Adsorption of native and catalytically inactivated enzyme was measured at the argon-buffer interface by using lipid films near the point of collapse. Protein adsorbed readily to films of triolein, 1,3-diolein, methyl oleate, oleonitrile, oleyl alcohol, and 13,16-docosadienoic acid. However, recovery of enzyme activity was variable. These differences and the changes in surface pressure accompanying adsorption indicated the occurrence of enzyme denaturation at the interface. Denaturation was controlled largely by surface free energy but showed some chemical specificity at high surface pressures. Adsorption of protein to the lipids was comparable when measured under either equilibrium or initial rate conditions. Together with surface pressure changes that accompany adsorption, the data indicate a relative lack of specificity for the enzyme-surface interaction. Adsorption to 13,16-docosadienoic acid and 1,3-diolein obeyed the Langmuir adsorption isotherm. Dissociation constants ranged from 10 to 50 nM, depending on enzyme form, ionic strength, and pH. With both lipids, a monolayer of enzyme was adsorbed at saturation. In contrast to these results, adsorption of enzyme activity and protein to films of 1-palmitoyl-2-oleoyl-phosphatidylcholine was less than or equal to 5% of that observed with the other lipids under all conditions. Comparison of rate constants for adsorption to 13,16-docosadienoic and 1,3-diolein as a function of subphase pH indicated a marked dependence on the ionization state of the fatty acid. Overall, the data suggest that the presence of zwitterionic and anionic lipids may regulate the interaction of the enzyme with substrate-containing surfaces in vivo.     

Properties and application of immobilized beta-D-glucosidase coentrapped with Zymomonas mobilis in calcium alginate

The enzyme beta-D-glucosidase has been immobilized on concanavalin A-Sepharose to give a maximum loading of 2050 units/g dry weight of support material. The immobilized beta-D-glucosidase was also entrapped within calcium alginate gel spheres with apparently only 35% retention of activity when assayed with 10mM cellobiose. However, it was discovered that, unlike the immobilized enzyme, the entrapped immobilized enzyme was not subject to substrate inhibition up to 100mM cellobiose, suggesting that a concentration gradient of cellobiose existed between the bulk solution and the interior of the gel sphere. Thus, the activity of the entrapped immobilized enzyme was almost twice as high as that of the immobilized enzyme when assayed with 100mM cellobiose. Concanavalin A-Sepharose-immobilized beta-D-glucosidase and the bacterium Zymomonas mobilis coimmobilized in calcium alginate gel spheres converted cellobiose to ethanol in both batch and continuous-flow fermentation systems.     

The role of mineral surfaces in the origin of life.

Adsorption of nucleoside phosphates on the surfaces of volcanic rocks has been studied. Differences in the absorption of some nucleoside phosphates on the surface of basalt cinder have been found. Differences in the adsorption of similar molecules on different mineral surfaces have also been shown. Different adsorptive capacities may have served as a mechanism for the selection of organic molecules during prebiotic evolution.