Pepsin immobilized on inorganic supports for the continuous coagulation of skim milk.

The milk-clotting enzyme pepsin was immobilized onto beads of alumina, titania, glass, stainless steel, iron oxide, and Teflon for treating skim milk in a fluidized-bed reactor. Two covalent attachment procedures using silanized supports and glutaraldehyde and two adsorption procedures were evaluated. The three best catalysts were titania and glass, using the covalent attachment procedure, and alumina, using the adsorption procedure at pH 1.2. The pepsin adsorbed on alumina catalyst has commercial potential compared to the previously used glass catalyst. Attempts to increase the stability of pepsin adsorbed on alumina by cross-linking with glutaraldehyde were unsuccessful owing to the low pH necessary for optimum pepsin adsorption; Desorption of pepsin from alumina during reactor operation was determined. Regeneration of spent catalysts was only partially successful.     

Standardization of Human Diploid Fibroblast Cultivation: Centrifugation Procedure

The effect of varying centrifugal forces on the growth rate, longevity, and adsorption on glass of human embryonic diploid lung fibroblasts was studied. Cells centrifuged at 120, 500, or 1,500 × g at each passage had similar growth rates but their longevity decreased slightly with increasing force. These forces had no influence on the proportion of cells attaching to the glass. When the material in the first supernatant was recentrifuged at 2,000 × g for 30 min and added to the cells precipitated in the first centrifugation, the longevity of these cells was increased by several cell divisions. Cells which were not centrifuged but added directly from the cell suspension in trypsin to the new culture grew at a slightly slower rate than the centrifuged cells and became senescent at an earlier time. However, the noncentrifuged cells adsorbed to glass better than those centrifuged.     

A microautoradiographic study of the activity of attached and free-living bacteria

Microautoradiography, combined with epifluorescent microscopy, was used to evaluate the uptake of tritiated amino acids by a marine Pseudomonas sp. A comparison was made between the activity of bacteria free-living in the medium and bacteria which were attached to glass, polyethylene or polystyrene substrata. The proportion of active bacteria was lower for free-living cells (53–82%) and those attached to polystyrene (53–76%) than for those attached to glass (77–99%) or polyethylene (73–96%). For bacteria attached to glass, assimilated labelled substrate was retained within the cell over 3 h, whereas with polyethylene, labelled material was released from the cells and adsorbed on the surrounding substratum. Hence the physiological activity of attached bacteria depended on the chemical composition of the substratum.     

Adsorption of HSA, IgG and laminin-1 on model titania surfaces--effects of glow discharge treatment on competitively adsorbed film composition

This study investigated the effect of glow discharge treatment of titania surfaces on plasma protein adsorption, by means of ellipsometry and mechanically assisted SDS elution. The adsorption and film elution of three plasma proteins, viz. human serum albumin (HSA), human immunoglobulin G (IgG) and laminin-1, as well as competitive adsorption from a mixture of the three proteins, showed that the adsorbed amount of the individual proteins after 1 h increased in the order HSA 相似文献   

Water-insoluble papain conjugates of titanium (IV)-activated supports

Glass fibre paper, treated with titanium (IV) chloride and subsequently water-washed, has a significant quantity (27.7 μg/cm²) of surface-bound titanium (IV) which may be removed by exposure to acidic hydrogen peroxide solution. This observation supports the view that materials that have been treated with titanium (IV) chloride by the method of Emery et al.¹possess a surface layer of hydrous titanium (IV) oxide which is able to bind protein in a manner analogous to that of free hydrous titanium (IV) oxide. Titanium (IV)-activated derivatives of polypropylene netting, zirconia fibre sheet and glass fibre paper have each been found able to bind papain. The papain conjugate of each titanium (IV)-activated support exhibited a high ratio of specific proteolytic activity to specific esterolytic activity. These ratios ranged from 29 to 64% of the ratio for the soluble papain employed; the highest ratio obtained was for the papain conjugate of the titanium (IV)-activated glass fibre support. This conjugate was also very satisfactory with regard to its protein content, specific activities, catalytic stability, pH range of activity, mechanical stability and ease of separation from substrate solution. The papain conjugate of titanium (IV)-activated polypropylene was more active and more stable than a papain conjugate of non-activated polypropylene. However, both preparations lost their activity during prolonged storage in substrate solutions, even though the activity of the papain conjugate of titanium (IV)-activated polypropylene was initially maintained for about 20 days. In each case the loss of activity is attributed to protein desorption during storage. The papain conjugate of titanium (IV)-activated zirconia was catalytically stable to storage in substrate solutions but suffered from poor wet strength.     

Effect of Different Supports on the Reaction Rate of Rhizomucor Miehei Lipase in Organic Media

Five different aluminas, a silica and a zirconia support were used to adsorb lipase (E.C. 3.1.1.3) from Rhizomucor miehei. The activity of the immobilised lipase was measured by esterification of dodecanol and decanoic acid in hexane. The immobilised lipase and the organic phase were pre-equilibrated separately to known water activities before mixing them to commence the reactions. The aluminas, which varied in pore sizes and surface areas, adsorbed similar amounts of enzyme. However, the esterification activities varied about 10-fold, increasing with increasing surface area. The silica and zirconia supports adsorbed about half as much lipase as the aluminas. The esterification reaction rates per unit quantity of enzyme adsorbed were compared with those for aluminas with similar surface areas; this specific rate was about 2 times higher for the zirconia, but the difference with silica was only small. There was no clear correlation between the esterification rates at fixed water activity and the amount of water adsorbed by the support used.     

Effect of receptor-ligand affinity on the strength of endothelial cell adhesion.

The objective of this study was to determine the effect of receptor-ligand affinity on the strength of endothelial cell adhesion. Linear and cyclic forms of the fibronectin (Fn) cell-binding domain peptide Arg-Gly-Asp (RGD) were covalently immobilized to glass, and Fn was adsorbed onto glass slides. Bovine aortic endothelial cells attached to the surfaces for 15 min. The critical wall shear stress at which 50% of the cells detached increased nonlinearly with ligand density and was greater with immobilized cyclic RGD than with immobilized linear RGD or adsorbed Fn. To directly compare results for the different ligand densities, the receptor-ligand dissociation constant and force per bond were estimated from data for the critical shear stress and contact area. Total internal reflection fluorescence microscopy was used to measure the contact area as a function of separation distance. Contact area increased with increasing ligand density. Contact areas were similar for the immobilized peptides but were greater on surfaces with adsorbed Fn. The dissociation constant was determined by nonlinear regression of the net force on the cells to models that assumed that bonds were either uniformly stressed or that only bonds on the periphery of the contact region were stressed (peeling model). Both models provided equally good fits for cells attached to immobilized peptides whereas the peeling model produced a better fit of data for cells attached to adsorbed Fn. Cyclic RGD and linear RGD both bind to the integrin alpha v beta 3, but immobilized cyclic RGD exhibited a greater affinity than did linear RGD. Receptor affinities of Fn adsorbed to glycophase glass and Fn adsorbed to glass were similar. The number of bonds was calculated assuming binding equilibrium. The peeling model produced good linear fits between bond force and number of bonds. Results of this study indicate that 1) bovine aortic endothelial cells are more adherent on immobilized cyclic RGD peptide than linear RGD or adsorbed Fn, 2) increased adhesion is due to a greater affinity between cyclic RGD and its receptor, and 3) the affinity of RGD peptides and adsorbed Fn for their receptors is increased after immobilization.     

Adsorption of HSA,IgG and laminin-1 on model titania surfaces – effects of glow discharge treatment on competitively adsorbed film composition

This study investigated the effect of glow discharge treatment of titania surfaces on plasma protein adsorption, by means of ellipsometry and mechanically assisted SDS elution. The adsorption and film elution of three plasma proteins, viz. human serum albumin (HSA), human immunoglobulin G (IgG) and laminin-1, as well as competitive adsorption from a mixture of the three proteins, showed that the adsorbed amount of the individual proteins after 1 h increased in the order HSA <IgG <laminin-1 ≤ protein mixture. Film elutability showed that 30 min of SDS interaction resulted in almost complete removal of adsorbed films. No difference in the total adsorbed amounts of individual proteins, or from the mixture, was observed between untreated and glow discharge treated titania surfaces. However, the composition of the adsorbed films from the mixture differed between the untreated and glow discharge treated substrata. On glow discharge-treated titania the fraction of HSA increased, the fraction of laminin-1 decreased and the fraction of IgG was unchanged compared to the adsorption on the untreated titania, which was attributed to protein–protein interactions and competitive/associative adsorption behaviour.     

In-situ upgrading of biomass pyrolysis vapors: catalyst screening on a fixed bed reactor

In-situ catalytic upgrading of biomass fast pyrolysis vapors was performed in a fixed bed bench-scale reactor at 500 °C, for catalyst screening purposes. The catalytic materials tested include a commercial equilibrium FCC catalyst (E-cat), various commercial ZSM-5 formulations, magnesium oxide and alumina materials with varying specific surface areas, nickel monoxide, zirconia/titania, tetragonal zirconia, titania and silica alumina. The bio-oil was characterized measuring its water content, the carbon-hydrogen-oxygen (by difference) content and the chemical composition of its organic fraction. Each catalytic material displayed different catalytic effects. High surface area alumina catalysts displayed the highest selectivity towards hydrocarbons, yielding however low organic liquid products. Zirconia/titania exhibited good selectivity towards desired compounds, yielding higher organic liquid product than the alumina catalysts. The ZSM-5 formulation with the highest surface area displayed the most balanced performance having a moderate selectivity towards hydrocarbons, reducing undesirable compounds and producing organic liquid products at acceptable yields.     

Chromophore of Bacteriorhodopsin is Closer to the Cytoplasmic Surface of Purple Membrane: Fluorescence Energy Transfer on Oriented Membrane Sheets

Transmembrane location of the retinal chromophore, either native or reduced in situ to a fluorescent derivative, of the purple membrane of Halobacterium halobium was investigated with fluorescence energy transfer techniques. Single sheets of purple membrane, either native or reduced with borohydride, were adsorbed on polylysine-coated glass; the orientation, whether the exposed surfaces were cytoplasmic or extracellular, was controlled by adjusting the pH of the membrane suspension before the adsorption. On the exposed surface of the reduced membrane, a layer of cytochrome c, hemoglobin, or ferritin was deposited. The rate of excitation energy transfer from the fluorescent chromophore in the membrane to the colored protein was greater when the protein was on the cytoplasmic surface of the membrane than when it was on the extracellular surface. Analysis in which uniform distribution of the protein on the surface was assumed showed that the reduced chromophore is situated at a depth of <1.5 nm from the cytoplasmic surface. The location of the native retinal chromophore was examined by depositing a small amount of tris(2,2′-bipyridyl)ruthenium(II) complex on the native membrane adsorbed on the glass. Energy transfer from the luminescent complex to the retinal chromosphore was more efficient on the cytoplasmic surface than on the extracellular surface, suggesting that the native chromophore is also on the cytoplasmic side. From these and previous results we conclude that the chromophore, whether native or reduced, of bacteriorhodopsin is located at a depth of 1.0 ± 0.3 nm from the cytoplasmic surface of purple membrane.     

Cell growth on immobilized cell growth factor. 8. Protein-free cell culture on insulin-immobilized microcarriers

In order to develop a new protein-free cell culture system, microcarriers immobilized with insulin were synthesized. For the synthesis, glass and polyacrylamide beads were treated for the introduction of amino groups on the surface, and insulin was immobilized on the surface by using several method. Anchorage-dependent cells. mouse fibroblast cells STO and fibroic sarcoma cells HSDM(1)C(1), and the anchorage-independent cells, mouse hybridoma cells SJK132-20 and RDP 45/20 were cultivated on the microcarriers immobilized with insulin. The insulin-immobilized microcarriers did not have any effect on the proliferation of the anchorage independent cells but promoted the growth of anchorage-dependent cells remarkably. The activity of immobilized insulin was larger than that of free or adsorbed insulin. The repeated use of the insulin-immobilized microcarrier was possible, and the promotion activity in the the repeated use was greater than that in the use. (c) 1992 John Wiley & Sons, Inc.     

Specific elimination of cytotoxic cells. II. Reduction of cytotoxic activity by adsorption on monolayers results from removal of cytotoxic cells and not from inhibition of their activity.

Cytotoxic lymphocytes produced by in vitro sensitization to H-2 alloantigens may be adsorbed efficiently and specifically on monolayers of spleen lymphocytes attached to poly-l-lysine-coated polystyrene. Greater than 16-fold median reduction in activity resulted from adsorption to the target-type monolayer while adsorption to the attacker-type monolayer resulted in a less than two-fold median reduction. Since this reduction could have resulted from competitive inhibition by detached monolayer cells rather than from sticking of the cytotoxic cells to the monolayer, experiments were performed to distinguish between these possibilities. The inhibitory properties of a nonadherent population were tested directly by addition to a different attacker-target combination in which its killing properties were irrelevant but in which the H-2 genotype of detached monolayer cells was appropriate to cause competitive inhibition. No significant inhibition was observed; and this indicated that reduction of activity in an adsorbed population results from removal of the cytotoxic cells and not from inhibition by detached monolayer cells competing with the labeled targets for attackers.     

Adsorption of coliphages T1 and T7 to host and non-host microbes and to clay minerals

The clay minerals, kaolinite (K) and montmorillonite (M), suspended in either distilled water (DW) or a minimal medium (M-9), were better adsorbents for coliphages T1 or T7 than were bacteria (including early log, late log, or stationary phase cultures of the hosts), actinomycetes, and yeasts. Except for the host bacteria, the microbial cells (regardless of their type, phase of growth, viability, weight or number of cells, and volume of the suspension medium) adsorbed few or no coliphages. Although early log phase cultures (3 h) ofEscherichia coli B, suspended in DW, adsorbed an appreciable amount of T1 (94%), washing the cells with DW reduced the amount of T1 adsorbed (48%); 3-h cultures ofE. coli B/1,5, suspended in DW, adsorbed 15% of a T7 inoculum, and washing the cells with DW reduced the amount of T7 adsorbed to 1%. There was appreciable adsorption (35 to 97%) of both coliphages (with the exception of T1 on K) to 1 mg K or M suspended in either DW or M-9. These results suggest that clays are more important than microbes as adsorbents of viruses in environments of low ionic strength and that microbes do not inactivate coliphages T1 and T7.     

The effect of substrate and adsorbed proteins on adhesion, growth and shape of CaCo-2 cells

Extracellular matrix (ECM) proteins play a critical role in many cellular functions, from spreading, migration and proliferation to apoptosis. This role can be altered when proteins of the native ECM are adsorbed to different substrates which cause structural modifications that can influence their biological function. The effects on CaCo-2 cells of laminin-1, fibronectin, collagen-1 and ECM gel adsorbed to glass and to tissue culture polystyrene (PS) were compared in terms of adhesion, proliferation, shapes and spreading of cells in culture. Significant differences between glass and PS surfaces were observed for proliferation and cell shape. Protein surfaces prepared on PS substrates had, in most cases, more pronounced effects on cells than uncoated PS, especially if coated by collagen-1. Adsorbed ECM gel was the most adhesive for cells, but its effect on cell proliferation was not notably different from the controls (glass or PS). These findings indicate that the choice of the substrate can have a significant effect on experimental results and should be taken into consideration when comparing results obtained on different surfaces.     

Reaction Rates in Organic Media Show Similar Dependence on Water Activity with Lipase Catalyst Immobilized on Different Supports

Lipase (E.C. 3.1.1.3) from Rhizomucor miehei was adsorbed on silica, zirconia and five alumina support materials. The immobilised preparations were used to catalyse esterincation reactions of decanoic acid and dodecanol in hexane. The immobilised lipase and the organic phase were separately preequilibrated to the desired water activities. The various support materials adsorbed widely different amounts of water at a given water activity. The reaction rates with all the support materials show similar dependence on water activity when the rates were normalised with the optimal rate for that support material. Hence water activity predicts the optimal conditions much better than water content.     

The use of graft copolymers to inhibit the adhesion of bacteria to solid surfaces

Twelve graft copolymers have been evaluated for their ability to prevent the adhesion of bacteria to substrata. The copolymers had polyethylene glycol (PEG) side-chains (‘teeth’) and a backbone that was either uncharged, acidic, basic or amphoteric. The copolymers were adsorbed onto glass, stainless steel and hydroxyapatite substrata, and 2-hpetri-dish adhesion experiments performed with bacteria isolated from marine (Pseudomonas sp. NCMB 2021), paper mill (S. marcescens NCIB 12211) and oral (S. mutans NCTC 10449) environments. The copolymers containing the most charged groups in the backbone had the most significant effect on bacterial adhesion levels, with anti-adhesive effects up to 99% achieved. An amphoteric copolymer (Compound 12) on glass, and acidic copolymer (Compound 11) on stainless steel and hydroxyapatite gave the most impressive anti-adhesive effects. These copolymers had non-specific bacterial anti-adhesive properties.It is proposed that the graft copolymers adsorbed onto hydrophilic surfaces via their charged backbone in such a way that the PEG side-chains were pointing out into the aqueous phase, and it was this orientation that was responsible for the observed anti-adhesive effect.     

Lipase immobilization on modified zirconia nanoparticles: Studies on the effects of modifiers

Zirconia nanoparticles modified with carboxylic acid with a long alkyl chain can significantly enhance the activity of immobilized lipases used for asymmetric synthesis in organic media. In this study, various carboxylic acids with different alkyl chain lengths were grafted to zirconia. Lipase from Pseudomonas cepacia (PCL) was immobilized on the modified zirconia nanoparticles and used for the resolution of (R,S)-1-phenylethanol through acylation in isooctane. The results showed that among the straight-chained carboxylic acids that formed a closely packed layer on the support, stearic acid with a long alkyl chain gave the best activity/enantioselectivity. The initial activity obtained was about 10 times higher than that from lipase loaded onto unmodified zirconia. This improvement could be attributed to the interaction between the long hydrophobic chain of the modifier and the lipase, leading to an interfacial activation effect. The stability of the immobilized PCL was investigated, and the lipase was found to be stable for at least ten consecutive runs with a substrate concentration of 5 g/L. At a high substrate concentration of ca. 100 g/L, the activity of the lipase gradually decreased, which suggests that deactivation was induced by substrate toxicity.     

Epoxidation of propylene utilizing Nocardia corallina immobilized by gel entrapment or adsorption

Nocardia corallina B-276 cells are capable of catalysing the direct epoxidation of propylene to propylene oxide, through a monooxygenase enzyme system. The present work was undertaken to see how immobilization of the whole cells by adsorption or entrapment on solid supports would influence the rate and duration of the epoxidation activity. With immobilization by adsorption, the propylene oxide forming activity was highest on hydrophobic supports, such as polypropylene or polyethylene. Under certain conditions the activity was three times that of the free control cells. However, the duration of epoxidation activity was considerably less for the adsorbed cells. The cell loading by adsorption, determined with¹⁴C-labelled cells was 1.0–2.8 mg dry cell weight g^?1of support. The cells, whether immobilized or not (controls), were tested using a continuous gas flow packed-bed or bubble-type reactor. Immobilization of the cells by entrapment in calcium alginate beads gave about the same propylene oxide forming activity and stability as with control cells, provided the reactor was operated at low temperature (30°C) and low oxygen content (20%) of the feed stream. The results suggest that entrapment in a hydrophobic matrix might be a more favourable system; although additional investigation of the rate limiting steps as well as the cause of the activity loss with time is needed.     

Nanoparticle-aided external beam radiotherapy leveraging the Čerenkov effect

This study investigates the feasibility of exploiting the Čerenkov radiation (CR) present during external beam radiotherapy (EBRT) for significant therapeutic gain, using titanium dioxide (titania) nanoparticles (NPs) delivered via newly designed radiotherapy biomaterials. Using Monte Carlo radiation transport simulations, we calculated the total CR yield inside a tumor volume during EBRT compared to that of the radionuclides. We also considered a novel approach for intratumoral titania delivery using radiotherapy biomaterials (e.g. fiducials) loaded with NPs. The intratumoral distribution/diffusion of titania released from the fiducials was calculated. To confirm the CR induced enhancement in EBRT experimentally, we used 6 MV radiation to irradiate human lung cancer cells with or without titania NPs and performed clonogenic assays. For a radiotherapy biomaterial loaded with 20 μg/g of 2-nm titania NPs, at least 1 μg/g could be delivered throughout a tumor sub-volume of 2-cm diameter after 14 days. This concentration level could inflict substantial damage to cancer cells during EBRT. The Monte Carlo results showed the CR yield by 6 MV radiation was higher than by the radionuclides of interest and hence greater damage might be obtained during EBRT. In vitro study showed significant enhancement with 6 MV radiation and titania NPs. These preliminary findings demonstrate a potential new approach that can be used to take advantage of the CR present during megavoltage EBRT to boost damage to cancer cells. The results provide significant impetus for further experimental studies towards the development of nanoparticle-aided EBRT powered by the Čerenkov effect.     

Transformation of 2- and 4-cyanopyridines by free and immobilized cells of nitrile-hydrolyzing bacteria

The transformation dynamics of 2- and 4-cyanopyridines by cells suspended and adsorbed on inorganic carriers has been studied in the Rhodococcus ruber gt1 possessing nitrile hydratase activity and the Pseudomonas fluorescens C2 containing nitrilase. It was shown that both nitrile hydratase and nitrilase activities of immobilized cells against 2-cyanopyridine were 1.5–4 times lower compared to 4-cyanopyridine and 1.6–2 times lower than the activities of free cells against 2-cyanpopyridine. The possibility of obtaining isonicotinic acid during the combined conversion of 4-cyanopyridine by a mixed suspension of R. ruber gt1 cells with a high level of nitrile hydratase activity and R. erythropolis 11-2 cells with a pronounced activity of amidase has been shown. Immobilization of Rhodococcus cells on raw coal and Pseudomonas cells on kaolin was shown to yield a heterogeneous biocatalyst for the efficient transformation of cyanopyridines into respective amides and carboxylic acids.