Immobilization of pectawamorine G10x by gel entrapment

Polyacrylamide gel immobilization of pectawamorine G10x was investigated. Its pectinesterase and polygalacturonase activity and stability in storage were measured. The degree of pectawamorine binding during gel immobilization was 80--90%, 55% of initial activity being retained. Thermal stability of the immobilized and native preparations was equal. Pectinesterase activity of the gel immobilized enzyme increased during storage.     

Immobilization of alcohol dehydrogenase by gel entrapment of cells of Saccharomyces cerevisiae

A stable immobilized preparation of alcohol dehydrogenase (ADH) (EC 1.1.1.1) was obtained by entrapment of ADH-containing Saccharomyces cerevisiae cells in polyacrylamide, polymerized by gamma-rays (100 kR). The permeability barrier for the substrate through the cell membrane was found to be eliminated on entrapment. The stability characteristics, pH-activity profile and other properties of the entrapped ADH are presented. A four-fold enhancement in K_m for NAD⁺ was observed on entrapment, whereas K_m for ethanol was not altered.     

Immobilization of fumarase by entrapment of rat liver mitochondria in polyacrylamide gel using gamma rays

A stable immobilized preparation of fumarase (EC 4.2.1.2) was obtained by entrapment of rat liver mitochondria in acrylamide polymerized by using gamma irradiation (100 kR). The enhanced stability and the efficiency of the entrapped enzyme have shown potential for repeated use for the production of L-malic acid from fumaric acid. The possible formation of succinic acid in the system could be controlled by incorporating malonate along with detergents such as sodium deoxycholate or sodium dodecylsulfate in the reactor system.     

Immobilization of cell-associated enzymes by entrapment in polymethacrylamide beads

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.     

Immobilization of lymphocytes at surfaces by lectins

Phytohemagglutinin (PHA) and Concanavalin A (ConA) cause normal chicken lymphocytes to adhere to glass and plastic surfaces. Pokeweed mitogen (PWM) does not cause adherence. The effect of ConA was studied in detail. The reaction begins within 15 min at 25 °C and proceeds to completion by 2 h. It is independent of pH and resembles in this respect the spontaneous adherence which can occur in protein-depleted suspensions of chicken lymphocytes. It is distinguished from spontaneous adherence by conducting the reaction in 1% or more serum protein; high concentrations exert a slight restraint, which can be overcome by increasing the concentration of ConA. The reaction is slightly greater at high cell concentrations, is inhibited by 3 mM sodium cyanide, and is effectively blocked by 3 mM iodoacetamide and the α-methyl- -glycosides of glucose and mannose. The reaction is not affected by 2-deoxy- -glucose or N-acetyl glucosamine. Adherent lymphocytes detach when the lectin solution is replaced with lectin-free saline; they readhere when reexposed to ConA or to alloantibody directed against lymphocyte surface antigen. At low concentrations of ConA the large lymphocytes of the bursa of Fabricius adhere more rapidly than the small lymphocytes of the blood and thymus. Mouse lymph node lymphocytes adhere in the same manner as small chicken lymphocytes.     

Immobilization of yeast cells by entrapment and adhesion using siliceous materials

Yeast cells (Saccharomyces cerevisiae) have been immobilized by entrapment in silica hydrogel, without significantly changing their biological activity; a simple model describes the rate of oxygen uptake by a film of immobilized cells. The cells have also been immobilized by direct adhesion to a glass surface; this is achieved by a well-controlled drying procedure, sufficient to bring the cells into close contact with the support, but without cell dehydration. The immobilized cells consume glucose at a rate which is about half of the rate obtained in suspension and they are resistant to strong mechanical strains.     

Immobilization of Candida rugosa lipase by cross-linking with glutaraldehyde followed by entrapment in alginate beads

Candida rugosa lipase was immobilized by first cross-linking with glutaraldehyde and then entrapping in calcium alginate beads. The presence of 2-propanol during cross-linking markedly improved the enzyme activity and activity recovery. Maximal enzyme activity (2.1?mmol?h^?1?g^?1 immobilized conjugate, wet weight) and activity recovery (117%) were observed at 30% (v/v) 2-propanol for hydrolysis of olive oil, which were 1.7 and 2.0 times higher than those of the immobilized enzyme prepared in the absence of 2-propanol. The half-life of the immobilized lipase prepared by entrapment after cross-linking in 30% 2-propanol was 1.6 times higher than that prepared by entrapment of the native lipase without cross-linking and 2-propanol pretreatment. The enantioselectivity of the former was 11 times higher than that of the latter for hydrolysis of racemic ketoprofen ethyl ester.     

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.     

Activation of murine B lymphocytes by suramin

Suramin stimulated DNA synthesis in spleen cell cultures of all inbred strains of mice tested, including, for example, CBA, DBA/2, C57BL/6, and the lipopolysaccharide (LPS)-nonresponsive strain C3H/HeJ. The cells responding to the drugs were removed by passage through nylon wool columns, but they were not eliminated by in vivo treatment of the mice with anti-Thy 1.2 antibody. Spleen cells of homozygous nude mice (C57BL/6 or BALB/c background) were as reactive as those of their heterozygous littermates. Collectively the data show that suramin is a B-cell mitogen in the mouse.     

Immobilization of catalase by entrapment of permeabilized yeast cells in hen egg white using glutaraldehyde

An immobilized preparation of whole cell-based catalase was obtained by cross-linking the yeast cells permeabilized with toluene in hen egg white using glutaraldehyde. Optimal preparations were obtained when cross-linking was carried out for 2 h at 4 degrees C. Immobilized cells could be reused for the removal of H2O2 from milk.     

Optimization by experimental design of polyacrylamide gel composition as support for enzyme immobilization by entrapment

We have developed a methodology based on experimental design, to optimize a polyacrylamide gel as the support for enzyme immobilization, taking advantage of all the properties which this type of gel has. Monomer and crosslinking agent proportions are responsible for both the porous structure and pore size of the gel. A correct selection of those variables and suitable synthesis conditions leads to an increase in the activity retained by the gel. The path of steepest ascent method was used to obtain the relative maximum activity. The maximum retained activity was chosen with a central composite design in terms of the gel composition. The retained activity in the network, loss activity in the wash water, and loss activity due to steric impediment or blockage was modeled in terms of the variables responsible for the gel structure. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 497-506, 1997.     

Immobilization of E. coli beta-galactosidase and its derivatives by polyacrylamide gel

We have recently prepared some crosslinked derivatives of Escherichia coli beta-galactosidase by treating the enzyme with bisimidoesters. In this article, we report the results obtained when the native and these crosslinked derivatives are entrapped in polyacrylamide gel lattice. It was found that use of combination of three protective agents, viz., bovine serum albumin, cysteine, and lactose, during immobilization gave an increased yield of 190% in the case of DMA crosslinked preparation. In the case of native enzyme, the K(m), pH optimum, and temperature optimum were found to remain unchanged on immobilization. The DMA crosslinked preparation entrapped in polyacrylamide in the presence of BSA, lactose, and cysteine was found to be a significantly better catalyst and hydrolyzed 47% milk lactose as compared to 31% hydrolysis by entrapped native enzyme in 6 h.     

Immobilization of papain on cotton fabric by sol–gel method

A method has been developed to immobilize papain on cotton fabric by means of sol–gel technique. The activity of free papain and papain in silica sol under sonication was studied. Scanning electron microscopy, energy dispersive spectrometer and the Bradford method were used to characterize papain immobilization. The efficiency of the immobilization was investigated by examining the relative enzymatic activity of free and immobilized papain, respectively. The results show that the optimum pH value in the medium for immobilized papain is shifted to alkaline side. In addition, the adaptability of papain to environmental acidity is significantly increased. The thermostability of immobilized papain shows no significant change compared to the free enzyme. The papain immobilized on fabric by sol–gel technique retains more than 30% of the original activity after six reuses continuously.     

Assessment of radiation-induced DNA damage caused by the incorporation of 99mTc-radiopharmaceuticals in murine lymphocytes using single cell gel electrophoresis

The DNA damage induced by the 99mTc-radiopharmaceuticals incorporation to the cell was determined by the single-cell gel electrophoresis in murine lymphocytes in vitro. The 99mTc-hexamethyl-propylene amine oxime (99mTc-HMPAO) and 99mTc-2, 5-dihydroxybenzoic acid (99mTc-gentisic acid) induced nearly 100% of cells with breaks and/or alkali labile sites, which is explained by the action of the Auger electrons produced by the decay of the 99mTc. These results agree with the doses of 1.6 and 1.0 Gy estimated by subcellular dosimetry for 99mTc-HMPAO that is incorporated in the cytoplasm, and the 99mTc-gentisic acid, which remains bonded to the cell membrane, respectively. The results imply that Auger electrons are able to cause important DNA damage, when the radionuclide is incorporated in the range of a few microns from the nuclei.     

Novel autotrophic nitrogen removal system using gel entrapment technology

A pilot plant involving a nitritation-anammox process was operated for treating digester supernatant. In the preceding nitritation process, ammonium-oxidizing bacteria were immobilized in gel carriers, and the growth of nitrite-oxidizing bacteria was suppressed by heat-shock treatment. For the following anammox process, in order to maintain the anammox biomass in the reactor, a novel process using anammox bacteria entrapped in gel carriers was also developed. The nitritation performance was stable, and the average nitrogen loading and nitritation rates were 3.0 and 1.7 kg N m⁻³ d⁻¹, respectively. In the nitritation process, nitrate production was completely suppressed. For the anammox process, the startup time was about two months. Stable nitrogen removal was achieved, and an average nitrogen conversion rate of 5.0 kg N m⁻³ d⁻¹ was obtained. Since the anammox bacteria were entrapped in gel carriers, stable nitrogen removal performance was attained even at an influent suspended solids concentration of 1500 mg L⁻¹.     

Poly-N-vinylcaprolactam gel: A novel matrix for entrapment of microorganisms

Summary A new gel-type support poly-N-vinylcaprolactam for microbial cell immobilization is presented. The method allows one to obtain beads of biocatalyst in a single step. The properties of beads obtained using different types of gel stabilizers were compared; the best stabilizer was found to be tannin. The method developed was used for entrapment of viable bacterial cells and fungal spores. The biocatalysts obtained were used for transformations of both hydrophilic (sorbitol, indolyl-3-acetic acid) and lipophilic (cortexolone, hydrocortisone) substrates.Abbreviations PVC poly-N-vinylcaprolactam - ImC immobilized cells - IAA indolyl-3-acetic acid - TLC thin layer chromatography     

Polyclonal activation of murine B lymphocytes by immune complexes

Murine splenic B lymphocytes are stimulated by homologous immune complexes to proliferate and secrete polyclonal antibody. The use of antibody from whole serum or monoclonal antibodies to form complexes resulted in the stimulation of mouse B lymphocytes. The ratio of antibody to antigen appears to be critical for the generation of the polyclonal antibody response. Because antigen and antibody are added independently at culture initiation, the exact nature of the complex is unknown, but optimal polyclonal antibody formation occurs in slight antigen excess. Immune complex-induced polyclonal antibody production requires the presence of both macrophages and T cells, whereas B cell proliferation requires only macrophages. The role of the macrophage appears to be to cleave a low m.w. (17,000) fragment from the complex, which is responsible for lymphocyte activation.     

Expression of phospholipase C isozymes by murine B lymphocytes

Cross-linking of membrane (m) Ig, the B cell receptor for Ag, activates protein tyrosine phosphorylation and hydrolysis of phosphotidylinositol 4,5-bisphosphate. The latter signal transduction pathway is an important mediator of antigen receptor engagement. The initial event in this pathway is the activation of phospholipase C (PLC). The identity of the isozyme of PLC used in B cells and the mechanism by which it becomes activated are currently unknown. The cDNA encoding five different isozymes have been cloned. As a first step in identifying the isozyme of PLC that is coupled to mIgM, murine cDNA fragments for the five cloned PLC isozymes were generated by the polymerase chain reaction (PCR), cloned, and used to screen a panel of B cell lines representing different stages of development for PLC mRNA expression. All the B cell lines tested expressed high levels of PLC alpha and PLC gamma 2 mRNA, whereas PLC beta and PLC delta mRNA expression were undetectable by both Northern blot and PCR analysis. PLC gamma 1 had a more complicated pattern of mRNA expression. PLC gamma 1 mRNA expression was lower than that observed for PLC alpha or PLC gamma 2 mRNA and varied widely among different cell lines. The pattern of PLC gamma 1 mRNA expression did not correlate with the developmental stage of the cell lines. The pattern of PLC gamma 1 protein expression in the panel of B cell lines correlated with the pattern of PLC gamma 1 mRNA expression. PLC gamma 1 expression was very low in several B cell lines, despite the fact that these cell lines show mIgM-stimulatable PLC activity. The variable and in some cases very low expression of PLC gamma 1 suggests that it may not be the form of PLC that is activated by mIgM. In contrast, PLC alpha and PLC gamma 2 were abundantly expressed in all B cell lines tested. This observation is consistent with the possibility that PLC alpha or PLC gamma 2 is activated by mIgM.     

Immobilization of thermoalkalophilic recombinant esterase enzyme by entrapment in silicate coated Ca-alginate beads and its hydrolytic properties

Thermoalkalophilic esterase enzyme from Bal?ova (Agamemnon) geothermal site were aimed to be immobilized effectively via a simple and cost-effective protocol in silicate coated Calcium alginate (Ca-alginate) beads by entrapment. The optimal immobilization conditions of enzyme in Ca-alginate beads were investigated and obtained with 2% alginate using 0.5mg/ml enzyme and 0.7 M CaCl(2) solution. In order to prevent enzyme from leaking out of the gel beads, Ca-alginate beads were then coated with silicate. Enzyme loading efficiency and immobilization yield for silicate coated beads was determined as 98.1% and 71.27%, respectively and compared with non-coated ones which were 68.5% and 45.80%, respectively. Surface morphologies, structure and elemental analysis of both silicate coated and non-coated alginate beads were also compared using Fourier Transform Infrared Spectroscopy (FT-IR) and Scanning Electron Microscope (SEM) equipped with Energy-dispersive X-ray spectroscopy (EDX). Moreover, silicate coated alginate beads enhanced reusability of esterase in continuous processes compared to non-coated beads. The hydrolytic properties of free and immobilized enzyme in terms of storage and thermal stability as well as the effects of the temperature and pH were determined. It was observed that operational, thermal and storage stabilities of the esterase were increased with immobilization.     

Possible mechanism of entrapment of neuraminidase-treated lymphocytes in the liver

Neuraminidase-treated rat lymphocytes adhere strongly to rat hepatocytes in vitro. Binding between cells is due to stereo-specific interactions between a mammalian hepatic membrane lectin and galactosyl residues which are exposed on the lymphocyte surface after removal of sialic acid residues. The hepatic galactose specific lectin may play a role in the trapping of recirculating desialylated lymphocytes in the liver.