Immobilization of BSA, enzymes and cells of Bacillus stearothermophilus onto cellulose, polygalacturonic acid and starch based graft copolymers containing maleic anhydride

Poly(maleic anhydride styrene) graft copolymers of cellulose, pectin polygalacturonic acid salt, calcium polygalacturonate, and starch were prepared and used to immobilize proteins. The cellulose grafts coupled quite appreciable quantities of acid phosphatase, glucose oxidase, and trypsin. However, the general retention of activity was somewhat disappointing. Further investigation with acid phosphatase showed that the amount of enzyme immobilized increased as the amount of anhydride in the graft copolymer increased but no such relationship existed for the enzymic activity. The cellulose graft copolymers were hydrolyzed and it appeared that the carboxyl group aided adsorption of the enzyme. Attempts to couple acid phosphatase using CMC through the free carboxyl groups, created by hydrolysis, gave only a small increase in the extent of protein coupling. However, the unhydrolyzed system gave a useful degree of immobilization of cells of Bacillus stearothermophilus, as did a poly(maleic anhydride/styrene)-cocellulose system. Attempts to improve the activity by using grafts based on other polysaccharide supports met with mixed success. Pectin products were soluble. Polygalacturonic acid products were partially soluble and extremely high levels of enzymic activity were obtained. This was probably due in part to the hydrophilic nature of the system, which also encouraged absorption of the enzyme. Attempts were made to reduce the solubility by using the calcium pectinate salt. Immobilization of acid phosphatase and trypsin resulted in inceased protein coupling but relatively poor activities were attained. A starch based system gave similar results. Calcium polygalacturonate was used to prepare an insoluble graft copolymeric system containing acrylonitrile-comaleic anhydride. The resulting gels gave excellent coupling with acid phosphatase which had a very good retention of activity.     

The immobilizaton of enzymes, bovine serum albumin, and phenylpropylamine to poly(acrylic acid)-polyethylene-based copolymers

A poly(acrylic acid)-polyethylene graft copolymer was prepared and used initially to couple to acid phosphatase, using soluble carbodiimides. Yields which were quite good were obtained with CMC but not with EDAC. The copolymers was used to couple trypsin using EEDQ. Several organic solvents were investigated for the preparation of the "activated" poly(acrylic acid) intermediate. Using the activated system, high concentrations of trypsin were bound but the relative activities were not very high. The yield was good with bovine serum albumin (BSA). When the method was used for invertase, acid phosphatase, and alkaline phosphatase, the yields were poor and the copolymer was shown to absorb protein by an ion-exchange mechanism. However, the activated system gave a good yield of coupling to phenylpropylamine. A polyethylene-coacrylic-acid polymer containing 13% of acrylic acid (by weight) was then converted to the acid chloride by refluxing with thionyl chloride. The chlorinated copolymer which contained 0.7% chlorine and a thionyl-chloride-treated polyethylene control which contained no chlorine were investigated in immobilization studies. Such coupling involved bovine serum albumin (BSA), alkaline phosphatase, trypsin, beta-galactosidase, and invertase. Bovine serum albumin coupled well to the support, but none of the enzymes gave high levels of enzymes activity. Phenylpropylamine coupled well and all of the acid chloride groups were involved. Tyrosine reacted with 63% of the available acid chloride groups.     

Coupling of enzymes to modified styrene-maleic anhydride copolymers]

A copolymer of styrene and maleic anhydride is modified with low molecular weight polyoles in a very convenient manner, so that it can be applied as a carried for the fixation of biologically active compounds without further pretreatment. The modified products show storage stability over a long time without loss of coupling capacity. Examples for the fixation of trypsin, alkaline phosphatase and a bacterial glutamate dehydrogenase are given, and some properties of the resulting enzyme products are described.     

Stabilization of trypsin by modification with comb-shaped copolymers of poly(ethylene glycol) derivative and maleic anhydride

Summary Trypsin from bovine pancreas was coupled with copolymers of poly(ethylene glycol) derivative and maleic anhydride with the molecular weights of 13 kDa and 100 kDa (activated PM₁₃ and PM₁₀₀). The modified trypsins were more stable towards autolysis and heat- or urea-treatment than nonmodified trypsin. Stabilization of trypsin caused by the chemical modification with activated PMs is discussed in relation to the protein conformation.     

Covalent immobilization of cellulose layers onto maleic anhydride copolymer thin films

Thin films of cellulose are advantageous for analytical studies in aqueous environments to investigate various factors determining the performance of cellulose-based products. However, the weak fixation of cellulose layers on common carrier materials often limits this approach. To address this problem, we suggest a novel maleic anhydride copolymer precoating technique which allows for the covalent attachment of cellulose thin films through esterification. Maleic anhydride copolymers were deposited and covalently bound onto planar, aminosilane-modified glass or silicon oxide surfaces. Cellulose was subsequently immobilized on top of the copolymer precoatings by spin coating from N-methylmorpholine-N-oxide/dimethyl sulfoxide solutions. The resulting cellulose films were thoroughly characterized with respect to layer thickness, morphology, chemical constitution, and electrical charging. The stability of the layers against shear stress was demonstrated in aqueous solutions and the covalent attachment of the cellulose to the copolymer films was proven by means of dissolution experiments followed by ellipsometry and high-resolution X-ray photoelectron spectroscopy.     

Maleic anhydride copolymers--a versatile platform for molecular biosurface engineering

A platform of thin polymer coatings was introduced for the functional modulation of immobilized bioactive molecules at solid/liquid interfaces. The approach is based on covalently attached alternating maleic acid anhydride copolymers with a variety of comonomers and extended through conversion of the anhydride moieties by hydrolysis, reaction with functional amines, and other conversions of the anhydride moieties. We demonstrate that these options permit control of the physicochemical constraints for bioactive molecules immobilized at interfaces to influence important performance characteristics of biofunctionalized materials for medical devices and molecular diagnostics. Examples concern the impact of the substrate-anchorage of fibronectin on the formation of cell-matrix adhesions, the orientation of endothelial cells according to lateral anti-adhesive micropatterns using grafted poly(ethylene oxide), and the spacer-dependent activity of immobilized synthetic thrombin inhibitors.     

Mechanical properties of poly(lactic acid)/starch composites compatibilized by maleic anhydride

Blending poly(lactic acid) (PLA) with wheat starch compatibized by maleic anhydride (MA) was performed with a lab-scale co-extruder. An initiator, 2,5-bis(tert-butylperoxy)-2,5 dimethylhexane (L101), was used to improve compatability among PLA, starch and MA. Interfacial adhesion between PLA and starch was significantly improved. Mechanical properties increased markedly compared to the virgin composites of PLA/starch. The PLA/starch composites at a constant ratio of 55/45 compatibilized by 1% MA and initiated by 10% L101 (MA basis) resulted in the highest tensile strength and elongation.     

Chemical modification of lipase with a comb-shaped synthetic copolymer of polyoxyethylene allyl methyl diether and maleic anhydride

Summary Lipase fromPseudomonas fluorescens was coupled with a copolymer of polyoxyethylene allyl methyl diether and maleic anhydride, activated PM. The PM-lipase became soluble and active in organic solvents, and also heat stable. It catalyzed the ester synthesis in benzene and ester hydrolysis in an aqueous system with high enzymic activity.     

Synthesis of a poly(vinylpyrrolidone-co-dimethyl maleic anhydride) co-polymer and its application for renal drug targeting

We have synthesized a polymeric drug carrier, polyvinylpyrrolidone-co-dimethyl maleic anhydride [poly(VP-co-DMMAn)], for use in renal drug delivery. About 80% of the 10-kDa poly(VP-co-DMMAn) selectively accumulated in the kidneys 24 h after intravenous administration to mice. Although this accumulated poly(VP-co-DMMAn) was gradually excreted in the urine, about 40% remained in the kidneys 96 h after treatment. Poly(VP-co-DMMAn) was taken up by the renal proximal tubular epithelial cells and no cytotoxicity was noted. Higher doses did not produce toxicity in the kidneys or other tissues. In contrast, polyvinylpyrrolidone of the same molecular weight did not show any tissue-specific distribution. Poly(VP-co-DMMAn)-modified superoxide dismutase accumulated in the kidneys after intravenous administration and accelerated recovery from acute renal failure in a mouse model. In contrast, polyvinylpyrrolidone-modified superoxide dismutase and native superoxide dismutase were not as effective. Thus, poly(VP-co-DMMAn) is a useful candidate as a targeting carrier for renal drug delivery systems.     

Antifouling potential of Subtilisin A immobilized onto maleic anhydride copolymer thin films

The proteinaceous nature of the adhesives used by most fouling organisms to attach to surfaces suggests that coatings incorporating proteolytic enzymes may provide a technology for the control of biofouling. In the present article, the antifouling (AF) and fouling release potential of model coatings incorporating the surface-immobilized protease, Subtilisin A, have been investigated. The enzyme was covalently attached to maleic anhydride copolymer thin films; the characteristics of the bioactive coatings obtained were adjusted through variation of the type of copolymer and the concentration of the enzyme solution used for immobilization. The bioactive coatings were tested for their effect on the settlement and adhesion strength of two major fouling species: the green alga Ulva linza and the diatom Navicula perminuta. The results show that the immobilized enzyme effectively reduced the settlement and adhesion strength of zoospores of Ulva and the adhesion strength of Navicula cells. The AF efficacy of the bioactive coatings increased with increasing enzyme surface concentration and activity, and was found to be superior to the equivalent amount of enzyme in solution. The results provide a rigorous analysis of one approach to the use of immobilized proteases to reduce the adhesion of marine fouling organisms and are of interest to those investigating enzyme-containing coating technologies for practical biofouling control.     

The synthesis of poly(MA-alt-NIPA) copolymer, spectroscopic characterization, and the investigation of solubility profile-viscosity behavior

Alternating copolymer of maleic anhydride (MA) with N-isopropylacrylamide (NIPA) has been synthesized. Spectral characterization of this copolymer has been achieved by FTIR, Raman, ¹H, ¹³C NMR spectroscopic methods. Besides hydrodynamic behavior of the copolymer has been also investigated by viscometric method for comparison with that obtained from the solubility profile through algorithmic calculations. As a consequence of the algorithmic calculations DMSO has been determined as the best solvent for the copolymer, poly(MA-alt-NIPA), which is also supported by the viscometric results.     

Preclinical toxicity study of a male injectable antifertility agent (styrene maleic anhydride) in rhesus monkeys, Macaca mulatta

Polymer styrene maleic anhydride dissolved in dimethyl sulphoxide was injected intravasally to rhesus monkeys at the dose levels of 100 mg, 250 mg, and 500 mg in each vas deferens in low, high, and highest dose groups respectively, while control group received 0.5 ml DMSO in each vas deferens. The systemic toxicity study of the implant was carried out for 90 d. There had been no alteration in any of the toxicity parameters, ie, body weight, haematological, biochemical or histopathological, in the test animals as compared to control animals. Hence, it is concluded that the polymer SMA injected at above doses appears to be safe in our experiment.     

Improvement of tautomycin production in <Emphasis Type="Italic">Streptomyces spiroverticillatus</Emphasis> by feeding glucose and maleic anhydride

Optimization of the feeding process for tautomycin production by Streptomyces spiroverticillatus was performed using glucose and/or maleic anhydride. The feeding of glucose was based on the reducing sugar content (lower than 8 g/L) at a cultivation time of 40 h. After addition of 2% (w/v) glucose, the biomass increased from 21 to 28 g/L, and that of tautomycin from 572.06 to 837.6 mg/L. Moreover, 1723.1 mg/L of tautomycin (increased by 201.21%) was obtained by feeding 0.2% (w/v) maleic anhydride solution at a pH between 4 and 7 in the broth. For the experiments in the 15 L fermentor, tautomycin content reached its highest level (1714.7 mg/L), which was 199.7% higher than that of control by feeding both glucose and maleic anhydride.     

Complexation of DNA with poly(methacryl oxyethyl trimethylammonium chloride) and Its poly(oxyethylene) grafted analogue

Intermolecular complexes of genomic polydisperse DNA with synthetic polycations have been studied. Two cationic polymers have been used, a homopolymer poly(methacryl oxyethyl trimethylammonium chloride) (PMOTAC) and its analogue grafted with poly(oxyethylene). The amount of poly(oxyethylene) grafts in the copolymer was 15 mol % and Mw of the graft was 200 g/mol. Salmon DNA (sodium salt) was used. The average molecular weight (Mw) of DNA was 10.4 x 10(6) g/mol. Conductivity, pH, and dynamic light scattering studies were used to characterize the complexes. The size and shape of the polyelectrolyte complex particles have been studied as a function of the cation-to-anion ratio in aqueous solutions of varying ionic strengths. The polyelectrolyte complexes have extremely narrow size distributions taking into account the polydispersity of the polyelectrolytes studied. The poly(oxyethylene) grafts on PMOTAC promote the formation of small colloidally stabile complex particles. Addition of salt shifts the macroscopic phase separation toward lower polycation content; that is, complexes partly phase separate with the mixing ratios far from 1:1. Further addition of salt to the turbid, partly phase separated solution results in the dissociation of complexes and the polycation and DNA dissolve as individual chains.     

Immobilization of laccase onto spacer-arm attached non-porous poly(GMA/EGDMA) beads: application for textile dye degradation

Non-porous poly(glycidyl methacrylate/ethyleneglycol dimetacrylate) (poly(GMA/EGDMA)) beads were prepared by suspension polymerization. The enzyme (i.e. laccase) was covalently immobilized onto plain and spacer-arm attached poly(GMA/EGDMA) beads. The amount of immobilized enzyme on the plain and spacer-arm attached beads was determined as 5.6 and 4.9 mg/g, respectively. The maximum activity (V(max)) and Michaelis constant (K(m)) of laccase immobilized on the spacer-arm attached beads, were found to be 77.6 U/min and 0.47 mM, respectively. Finally, the immobilized laccase was operated in a batch system, and textile dye Reactive Red 120 was successfully decolorized in the enzyme reactor.     

Factors affecting the production of prednisolone by immobilization of Bacillus pumilus E601 cells in poly(vinyl alcohol) cryogels produced by radiation polymerization

To increase the yield percent of prednisolone from hydrocortisone (cortisol), Bacillus pumilus E601 (a radioresistant microorganism) was incorporated into poly(vinyl alcohol) (PVA) cryogel grafted with hydroxyethyl-methacrylate (HEMA) as a crosslinking agent. The polymer was prepared by a radiation polymerization technique at 20 kGy from Co-60 source. The optimum temperature for the biotransformation of hydrocortisone by free cells, poly(PVA)/HEMA, and poly(PVA)/HEMA /N-isopropylacrylamide (N-IPAAm) was 30 °C. The highest yield % of prednisolone was obtained by immobilization of the cells on poly(PVA/HEMA), the addition of N-IPAAm to poly(PVA/HEMA) protected the immobilized cells from temperatures above 35 °C during the fermentation process. The optimal pH (buffered pH) of the biotransformation of hydrocortisone by immobilized and free cells was 7.0, but the maximum yield of prednisolone (60%) was obtained by immobilized cells in comparison with free cells (42%) also at pH 7.0. The prednisolone yield reached 60–65% with 1,2-propanediol cosolvent containing media and 60–62% in the case of ethanediol cosolvent containing media at 1% (v/v) of both cosolvents. 10 mg/50 ml Tween 80 the medium increased the prednisolone yield by only 1.1-fold compared with the control. The maximum bioconversion efficiency was obtained at a substrate concentration of 20 mg/50 ml medium. Stability studies showed that the immobilized cells can be used for seven times without any significant decrease in activity.     

Citric acid and maleic anhydride as compatibilizers in starch/poly(butylene adipate-co-terephthalate) blends by one-step reactive extrusion

Starch/poly(butylene adipate-co-terephthalate) films were obtained by one-step reactive extrusion using maleic anhydride (MA) and citric acid (CA) as compatibilizers. The mechanical, structural, optical and barrier properties of the films were analyzed when glycerol (GLY), CA and MA were added to the starch/PBAT (55:45, w/w) according to mixture design. FTIR analysis showed that CA and MA were able to promote esterification/transesterification reactions and that CA induced them more efficiently. When a greater proportion of compatibilizer (1.5 wt%) was used, the resulting films were more opaque and had a greater tensile strength. A greater proportion of GLY (10.0%, w/w) improved the elongation at the break of the films. The barrier properties to water vapor of the films were improved by high levels of CA (1.5 wt%) and intermediate levels of GLY (9.25 wt%). The inclusion of compatibilizers resulted in blends with improved properties, representing a potential replacement for non-biodegradable films.     

Solvent free vapor phase photografting of maleic anhydride onto poly(ethylene terephthalate) and surface coupling of fluorinated probes, PEG, and an RGD-peptide

Poly(ethylene terephthalate) (PET) was photografted in a solvent free vapor of maleic anhydride and benzophenone. After hydrolysis of the initially grafted succinic anhydride groups, the carboxylic PET surfaces were modified by coupling reactions in organic and aqueous solutions. 2,2,2-Trifluoroethylamine and diamino PEGs of molecular weight 3400 and 2000 were reacted with acid chloride groups obtained by treating the PET-COOH surface with PCl(5). Furthermore, fluoro substituted thiols and a cystein terminated RGD containing peptide were bound to PET-COOH surfaces via a disulfide link by a three step coupling sequence. Coupling yields and surface concentrations of the fluoro substituted ligands were calculated from ESCA data. The RGD-peptide surfaces were evaluated by cultivation with rat smooth muscle cells.     

New sorbent for bilirubin removal from human plasma: Cibacron Blue F3GA-immobilized poly(EGDMA–HEMA) microbeads

Cibacron Blue F3GA-immobilized poly(EGDMA–HEMA) microbeads were investigated as a specific sorbent for bilirubin removal from human plasma. The poly(EGDMA–HEMA) microbeads were prepared by a modified suspension copolymerization technique. Cibacron Blue F3GA was covalently coupled to the poly(EGDMA–HEMA) microbeads via the nucleophilic reaction between the chloride of its triazine ring and the hydroxyl groups of the HEMA molecule, under alkaline conditions. Bilirubin adsorption was investigated from hyperbilirubinemic human plasma on the poly(EGDMA–HEMA) microbeads containing different amounts of immobilized Cibacron Blue F3GA, (between 5.0–16.5 μmol/g). The non-specific bilirubin adsorption on the unmodified poly(EGDMA–HEMA) microbeads were 0.32 mg/g from human plasma. Higher bilirubin adsorption values, up to 14.8 mg/g, were obtained with the Cibacron Blue F3GA-immobilized microbeads. Bilirubin molecules interacted with these sorbents directly. Contribution of albumin adsorption on the bilirubin adsorption was pronounced. Bilirubin adsorption increased with increasing temperature.     

Particle-by-particle quantification of protein adsorption onto poly(ethylene glycol) grafted surfaces

The use and advantage of flow cytometry as a particle-by-particle, low sampling volume, high-throughput screening technique for quantitatively examining the non-specific adsorption of proteins onto surfaces is presented. The adsorption of three proteins: bovine serum albumin (BSA), immunoglobulin gamma (IgG) and protein G, incubated at room temperature for 2 h onto organosilica particles modified with poly(ethylene glycol) (PEG) of increasing MW (2000, 3400, 6000, 10,000 and 20,000 g mol(-1)) and grafted amounts (0.14-1.4 mg m(-2)) was investigated as a model system. Each protein exhibited Langmuir-like, high affinity monolayer limited adsorption on unmodified particles with the proteins reaching surface saturation at 1.8, 4.0 and 2.5 mg m(-2) for BSA, IgG and protein G, respectively. Protein adsorption on PEG-modified surfaces was found to decrease with increasing amounts of grafted polymer. PEG grafting amounts >0.6 mg m(-2) effectively prevented the adsorption of the larger two proteins (BSA and IgG) while a PEG grafting amount >1.3 mg m(-2) was required to prevent the adsorption of the smaller protein G.