Inverse high internal phase emulsion polymerization (i-HIPE) of GMMA, HEMA and GDMA for the preparation of superporous hydrogels as a tissue engineering scaffold.

A series of novel superporous hydrogels for regenerative medicine were prepared by oil-in-water (o/w) or inverse high internal phase emulsion (i-HIPE) copolymerization of glycerol monomethacrylate (GMMA), 2-hydroxy ethyl methacrylate (HEMA) and glycerol dimethacrylate (GDMA) as a cross-linker using a non toxic solvent and a redox initiator system at the physiological temperature (37 °C). The monomer GMMA was synthesized from glycidyl methacrylate (GMA) by an alternative facile method using Amberlyst-15. The described i-HIPEs showed a significantly wider stability window. The polyHIPE hydrogels were characterized by FTIR, BET method for surface area, mercury porosimetry, SEM, DSC, TGA, XRD, compressive strain and strain recovery. The swelling ratio of the hydrogels and their degradation in 0.007 M NaOH and lipase B (Candida antarctica) solutions were determined gravimetrically and the rate of degradation was explained in terms of the molecular structure of the hydrogels. The morphological studies showed that the pore diameter varied between 20 and 30 µm and the pore throats (interconnecting windows) diameter was in the range of 4-8 µm. The described polyHIPE hydrogels were found to have an open cell morphology and interconnected pore architecture, which are important characteristics for scaffold applications. The initial cytotoxicity study performed according to ISO-10993-5 indicated cytocompatibility (97% cell viability) and the subsequent cell seeding and proliferation study exhibited 55-88% cell viability (increased monotonously from GHG-1 to GHG-5), which could be attributed to modulation of the physical and chemical properties of the hydrogels. The described super porous hydrogels are considered as potential candidates for scaffold materials in tissue engineering applications.

Arthrobacter sp. lipase immobilization for preparation of enantiopure masked beta-amino alcohols.

Recent reports on immobilization of lipase from Arthrobacter sp. (ABL, MTCC 5125; IIIM isolate) on insoluble polymers have shown altered properties including stability and enantioselectivity. Present work demonstrates a facile method for the preparation of enantiopure beta-amino alcohols by modulation of ABL enzyme properties via immobilization on insoluble as well as soluble supports using entrapment/covalent binding techniques. Efficacies of immobilized ABL on insoluble supports prepared from tetraethylorthosilicate/aminopropyltriethoxy silane and soluble supports derived from copolymerization of N-vinyl pyrrolidone-allylglycidyl ether (ANP type)/N-vinyl pyrrolidone-glycidyl methacrylate (GNP type) for kinetic resolution of masked beta-amino alcohols have been studied vis-à-vis free ABL enzyme/wet cell biomass. The immobilized lipase on different insoluble/soluble supports has shown 21-110 mg/g protein binding and 30-700 U/g activity for hydrolyzing tributyrin substrate. The findings have shown a significant enhancement in enantioselectivity (ee 99%) vis-à-vis wet cell biomass providing ee 70-90% for resolution of beta-amino alcohols.

Immobilization of Candida rugosa lipase on poly(allyl glycidyl ether-co-ethylene glycol dimethacrylate) macroporous polymer particles.

Macroporous polymer particles containing surface epoxy groups were synthesized for immobilization of Candida rugosa lipase (CRL). The effect of incorporation of two different sets of monomers [allyl glycidyl ether (AGE) and glycidyl methacrylate (GMA)] and the effect of crosslinking density on immobilization of lipase were studied. AGE-co-EGDM polymers gave higher binding and expression of lipase than GMA-co-EGDM polymers. Optimization of immobilization parameters was done with respect to immobilization time and enzyme loading. Amongst AGE-co-EGDM polymer series, AGE-150 polymer found to give maximum lipase activity yield and therefore evaluated for temperature, pH and storage stability. Under optimum conditions, AGE-150 polymer gave 78.40% of activity yield. Immobilized lipase on AGE-150 showed a broader pH, higher temperature and excellent storage stability.

Solution doping of microstructured polymer optical fibres.

Solution doping of microstructured polymer optical fibres [mPOF] is demonstrated, a technique which allows dopants to be introduced after polymerisation through the microstructure. Controlled diffusion is used to disperse the dopant uniformly across the fibre core, and the final concentration can be systematically varied by appropriate choice of conditions. We use this technique to produce a fibre doped with Rhodamine 6G and characterize its loss and fluorescence behavior.

Macroporous copolymer matrix. IV. Expanded bed adsorption application.

Macroporous crosslinked hydroxyethyl methacrylate-ethylene dimethacrylate copolymeric beads (HEG beads) were synthesized by suspension polymerization in the presence of a pore generating agent. These beads were coupled to alpha-cyclodextrin through a urethane spacer. These modified copolymer beads (affinity-HEG beads) so prepared were evaluated for their suitability in expanded bed chromatography. The optimum thickness of the distributor plate for stable expanded bed for use in expanded bed adsorption (EBA) was established. The affinity-HEG beads are comparable in density to Streamline diethyl amino ethane (DEAE) and exhibit better mechanical stability at higher superficial velocity under fluidization. The affinity-BEG beads were used as affinity chromatography matrices for the purification of cyclodextrin glycosyltransferase. Feeding of 5-fold diluted fermented broth to the column containing affinity-HEG beads of settled bed height 7.5 cm (I.D. 26 mm and length 42 cm) at double bed expansion resulted in a sharp breakthrough curve of alpha-cyclodextrin glycosyltransferase (CGTase). The adsorbed enzyme was eluted from the bed in 50 mM Tris-HCl buffer containing 10 mM CaCl2 at 25 degrees C in packed bed configuration.

Macroporous copolymer matrix: 1. Effectiveness of different diisocyanate spacer arms to bind cyclodextrins.

Cross-linked macroporous beaded polymer matrices, with pendant hydroxyl groups, were synthesized by the copolymerization of 2-hydroxyethyl methacrylate and ethylene glycol dimethacrylate using suspension polymerization methodology. Novel affinity chromatography matrices were synthesized using various diisocyanates as bifunctional reagents to couple the macroporous polymeric supports, of controlled particle size distribution, with alpha and beta-cyclodextrins. The optimal conditions to couple the hydroxyl groups of cyclodextrin (ligand) and the polymeric supports through urethane linkages were established iteratively using various diisocyanates. Efficacy of ligand binding on the matrix and nonspecific interactions of the synthesized affinity matrices were evaluated to establish the best support and spacer arm. 2,4-Tolylene diisocyanate was established as the best spacer arm on the basis of high ligand binding and low nonspecific interactions. The characteristics of the synthesized affinity matrices toward the adsorption of alpha and beta-cyclodextrin glycosyltransferase (CGTase) were investigated. The binding of beta-CGTase was the highest on affinity matrices with the polymeric methylene diisocyanate spacer. The optimal conditions to regenerate the matrices were also established.

Development of tailor-made glycidyl methacrylate-divinyl benzene copolymer for immobilization of D-amino acid oxidase from Aspergillus species strain 020 and its application in the bioconversion of cephalosporin C.

A tailor-made glycidyl methacrylate-divinyl benzene (GMA-DVB) copolymer PC-3 was evolved by studying the effect of synthesis variables on binding and expression of D-amino acid oxidase (DAAO) from Aspergillus species strain 020. Almost quantitative binding (100%) and a high yield of immobilization per unit of enzyme loaded was achieved. Optimum pH, optimum temperature and K(m)95% was achieved by using 3% (w/v) solution of ceph C, 48 U of DAAO per g of ceph C, keeping dissolved oxygen level above 50%, maintaining the pH between 7.6 and 7.8 and temperature at 24 degrees C. The immobilized DAAO was used for 60 cycles in a stirred tank reactor.

Role of physical properties of glycidyl methacrylate- pentaerythritol triacrylate copolymers on immobilization of milk clotting protease from aspergillus niger MC4

The effect of cross-link density and amount of porogen on physical properties of the glycidyl methacrylate-pentaerythritol triacrylate (GMA-PETA) copolymer and intern on the immobilization of milk clotting protease from Aspergillus niger MC4 was studied. Almost quantitative expression of the milk clotting protease was achieved with copolymer of 251% cross-link density and generated with 65.4 mL of cyclohexanol as porogen. The pH of 4.0 and 18 h period were the best conditions for binding the milk clotting protease onto the polymer. The immobilized milk clotting protease prepared had an activity of 6000 units/g. The optimum pH and temperature ranges for both soluble and immobilized milk clotting protease preparations were pH 5.6-5.8 and 55-60 degreesC, respectively. Immobilized milk clotting protease was more stable than the soluble enzyme; the half-lives at pH 4.0 and 37 degreesC were 160 and 60 days, respectively. At pH 5.8 and 37 degreesC, the parameters favorable for milk clotting, the immobilized enzyme preparation did not exhibit any aspartic protease activity.

Beaded reactive polymers. 3. Effect of triacrylates as crosslinkers on the physical properties of glycidyl methacrylate copolymers and immobilization of penicillin G acylase.

Various glycidyl methacrylate (GMA) copolymers were synthesized by suspension polymerization, using pentaerythritol triacrylate (PETA), trimethylolpropane triacrylate (TMPTA), and trimethylolpropane trimethacrylate (TRIM) as crosslinking comonomers. These copolymers were evaluated for the immobilization of penicillin G acylase. Broad pore-size distribution that was observed was in the range 5-300 nm. Both surface area and pore volume increased with increase in the mole fraction of crosslinking comonomer (increasing crosslink density). The pore volume of the copolymers was more than doubled by including lauryl alcohol as porogen. Binding of penicillin G acylase (PGA) was quantitative on highly crosslinked copolymers. The expression of bound PGA was better on the relatively more hydrophilic GMA-TMPTA and GMA-PETA copolymer supports compared to the GMA-TRIM copolymers. Among the different copolymers studied, GMA-TMPTA copolymer 7411 exhibited highest activity of immobilized penicillin G acylase (167.4 IU/g) with 35.1% expression.

Polyethyleneimine in immobilization of biocatalysts.

A variety of polymers with amino pendant groups have been accepted as suitable enzyme carriers. A number of synthesis strategies and techniques are adopted to anchor enzymes. The polymers are activated through derivatization either prior to or during adsorption to facilitate the covalent binding of enzyme. Polyethyleneimine (PEI), with the highest concentration of amino groups, has found acceptance as a carrier in a number of industrial immobilized biosystems. The two forms of PEI known are the linear crystalline type and the more important amorphous branched structure with a distribution of primary, secondary, and tertiary amino groups in the ratio 1:2:1. The primary and secondary amino groups are conveniently modified to generate facile enzyme carriers. A number of patents have been filed on the use of PEI to bind a rich variety of enzymes and whole cells. This review is primarily a compilation of these reports and purports to highlight the potential of PEI.

Adsorption and expression of penicillin G acylase immobilized onto methacrylate polymers generated with varying pore generating solvent volume.

Adsorption and expression of penicillin G acylase was studied on macroporous methacrylate polymer beads of differing pore volume, generated with kerosene. The absorption and expression of the penicillin G acylase was dependent on pore volume. Maximum expression of 57% of adsorbed enzyme was obtained on beads synthesized with 40 mL of kerosene, indicating minimum pore-diffusion limitations.

Immobilization of penicillin G acylase on methacrylate polymers.

Macroporous weak cation-exchange methacrylate polymers were synthesized for the immobilization of penicillin G (Pen G) acylase. The role of certain factors such as pore-generating solvent, cross-linking agent, cross-linking density, and comonomer, in enzyme adsorption and expression was studied. Kerosene was a superior pore-generating solvent to paraffin oil. Ethylene glycol dimethacrylate and acrylic acid served as the best cross-linking agent and comonomer, respectively, in the systems studied. 80.3% of the activity of the enzyme adsorbed onto polymer beads prepared with 0.05 mol of acrylic acid (polymer PM-39) was expressed. Properties of the Pen G acylase, immobilized on PM-39 by adsorption and cross-linking with glutaraldehyde (IME-PM-39) were studied. The optimum pH, optimum temperature and Km of Pen G acylase shifted from 8.0 to 7.5-7.8, 50 degrees C to 55 degrees C and 0.038 mol dm-3 to 2.4-3.0 mol dm-3, respectively, as a result of immobilization on PM-39. IME-PM-39 was used repeatedly for 15 cycles in the production of 6-amino penicillanic acid (6-APA).