Immobilization of whole Escherichia coli containing penicillin amidase using cross-linking agents and fillers

Abstract: Immobilization of E. coil, Aspergillus niger and Aspergillus japonicus was accomplished by entrapment in polymethacrylamide beads via a two-phase polymerization using soybean oil as the suspension medium. The immobilized E. coli cells containing penicillin G acylase possessed a higher activity than that reported in the literature. The mmobilized mycelia of Aspergillus sp. containing β-fructofuranosidase were effective for catalyzing the formation of fructooligosaccharides.

Abstract: Escherichia coli ATCC 11105 containing the periplasmic penicillin G acylase was entrapped within a copolymer of methacrylamide andN,N’- methylenebisacrylamide. A solution of monomer that was made up from methacrylamide andN,N’-methylenebisacrylamide dissolved in buffer was mixed with lyophilized cells and ammonium persulfate. This suspension was then pumped drop by drop into in soybean oil supplemented with 0.06% (v/v) 3-(dimethylamino)-propionitril. During submerging in the oil phase, the droplets were hardened and induced to polymerize within the droplets. Particles with a volume ranging from 0.013–0.017 mL per bead containing a biomass concentration up to 38.0 g/L were prepared. The optimal condition for the deacylation of penicillin G to 6-aminopencillanic acid (6-APA) catalyzed by the immobilized whole-cell penicillin G acylase was found to be 45‡C and pH 8.0. Product inhibition of this enzyme by 6-APA could be eliminated by controlling pH value at 8 during the course of penicillin G hydrolysis using a pH-stat. Conversion determined by the pH-stat method were 0.3% higher than that by p-dimethylaminobenzaldehyde method. Cell concentration in the matrix was found to be an important factor influencing the maximum velocity and the specific activity retained in the matrix. A kinetic model, in which the mass transfer resistances as a result of external film mass transfer and pore diffusion were assumed to be negligible, could properly describe the hydrolysis of penicillin G by the cells entrapped within the polymethacylamide beads.

Abstract: To compare the efficiency of various whole cell immobilization techniques for the production of gluconic acid by Aspergillus niger were investigated using potassium ferrocyanide-treated cane molasses as the substrate. The techniques followed were: (1) Calcium alginate entrapment, (2) cross-linking with glutaraldehyde after cell permeabilization with (a) acetone, (b) toluene and (c) isopropanol and (3) development of granular catalyst.

Abstract: Gluten is advantageous for use as a new base material for cell and enzyme immobilization because it is biodegradable, cheaper than other natural polymers and readily available. In this work, a procedure for immobilization of cell-associated enzymes by entrapping within gluten matrices was developed. According to this method, cells of E. coli containing penicillin G acylase were mixed with gluten solution during the formation of gel, and the resultant gel was hardened by the addition of oxidized starch or glutaraldehyde. Scanning electron micrographs of the cell-immobilized preparations indicated that they were porous and the pore size decreased with increasing the dosage of cross-linking agent. Oxidized starch was superior to glutaraldehyde as the crosslinker, since the gel matrices hardened by the former were more accessible to the substrate and less harmful to the enzyme. The immobilized preparations in the form of either a single sheet or small pieces containing a biomass concentration up to 10%, w/w w...

Abstract: A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.

Abstract: Publisher Summary This chapter discusses immobilized living cells, their advantages, and their applications in industry. Over the past two decades, there have been rapid developments in the use of enzymes as catalysts for industrial, analytical, and medical purposes and a new field of research called enzyme technology. Current and industrial applications of continuous single-enzyme reactions are carried out using immobilized microbial cells. Immobilized living cells should be preferred to immobilized enzymes for degradative and synthetic reactions in industry; another major advantage of immobilized cells is that the operational stability of the immobilized living cells may often be greatly enhanced by the regeneration of the enzyme activities of the immobilized cells. The use of immobilized cells enables greater control throughout the reaction. The applications of immobilized living cells are not limited to the production of chemical fermentation products but have been extended to the production of viral particles or synchronous cells, the chromatographic separation of special cells, the culturing of animal tissue, and, more recently, the immobilization and use of plant cells in the production of alkaloids.

Abstract: 6-Aminopenicillanic acid from penicillin V is produced by Pleurotus ostreatus immobilized by entrapment in a chitosan matrix. In these carriers the cell concentration increases after network formation by irreversible shrinking of the biocatalyst. Specific activity of the biocatalyst for the hydrolysis reaction is 1,31 μmol.min-1. (g wet weight of catalyst)-1 corresponding to a relative activity of 38%. Catalytic half-life of immobilized Pl. ostreatus is 25 days compared to 2.5 days for free suspended cells.