Poly(styrene-divinylbenzene) beads surface functionalized with di-block polymer grafting and multi-modal ligand attachment: performance of reversibly immobilized lipase in ester synthesis

Fibrous poly(styrene-b-glycidylmethacrylate) brushes were grafted on poly(styrene–divinylbenzene) (P(S–DVB)) beads using surface-initiated atom transfer radical polymerization. Tetraethyldiethylenetriamine (TEDETA) ligand was incorporated on P(GMA) block. The ligand attached beads were used for reversible immobilization of lipase. The influences of pH, ionic strength, and initial lipase concentration on the immobilization capacities of the beads have been investigated. Lipase adsorption capacity of the beads was about 78.1 mg/g beads at pH 6.0. The K _m value for immobilized lipase was about 2.1-fold higher than that of free enzyme. The thermal, and storage stability of the immobilized lipase also was increased compared to the native lipase. It was observed that the same support enzyme could be repeatedly used for immobilization of lipase after regeneration without significant loss in adsorption capacity or enzyme activity. A lipase from Mucor miehei immobilized on styrene–divinylbenzene copolymer was used to catalyze the direct esterification of butyl alcohol and butyric acid.     

Application of preformed cellulose beads as a support in cell immobilization

Summary A new cell immobilization technique, using preformed cellulose beads, has been developed. Corynebacterium sp. and Saccharomycss cerevisiae cells were grown on the beads and were used for tryptophan and ethanol production.     

Technical scale production of encapsulated Saccharomyces cerevisiae and Metarhizium brunneum attractive to wireworms

Wireworms (Coleoptera: Elateridae) have recently become an increasing problem as agricultural insect pests due to the phasing out of effective control options. Entomopathogenic fungi such as Metarhizium brunneum have proven to be a promising microbial antagonist for wireworm control. Here, we tested whether the efficacy of M. brunneum can be increased through a combination with CO₂, emitted by Saccharomyces cerevisiae, as an attractant (=attract-and-kill). We aimed at a technical scale production of a formulated biological control agent offering a practical and economically feasible application for wireworm control. Therefore, a novel technical formulation process for encapsulated S. cerevisiae (Attract beads) and M. brunneum (Kill beads) was investigated. For the bead production by jet cutting, the parameters nozzle diameter, pump speed, cutting device speed and collecting distance were evaluated. In order to dry the beads in a short time while maintaining a high cell viability, different drying temperatures during fluidised-bed drying were tested and the best results were obtained with an inlet air temperature profile between 50°C and 40°C. CO₂ production of the beads in the soil was highest for co-applied Attract and Kill beads. The potential of beads to modify wireworm behaviour (Agriotes sputator) was tested in a rhizotron experiment. The Attract-and-Kill treatment (co-applied beads) significantly attracted wireworms, whereas Attract beads and Kill beads alone showed a weak, but non-significant attraction. Wireworm mortality could not be enhanced due to a low rate of mycosis from M. brunneum infection.     

Recyclable chaperone-conjugated magnetic beads for in vitro refolding of <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> lipase

Polymer-coated magnetic beads have become widely used in biological applications because of their facile recovery and easily modifiable surface. Herein, we report the application of magnetic beads to in vitro refolding of B. cepacia lipase. Magnetic particles (Fe₃O₄) prepared by co-precipitation of Fe²⁺ and Fe³⁺ ions under basic conditions were subsequently coated with carboxylic acid-containing polystyrene by emulsion polymerization. The polymer-coated magnetic beads were then conjugated with molecular chaperone proteins to assist with refolding. The chaperone-conjugated magnetic beads efficiently refolded B. cepacia lipase and were easily reused. The beads showed comparable refolding activity to the soluble chaperone, and retained more than 95% of their refolding activity after five cycles of refolding B. cepacia lipase.     

Ex situ bioremediation of phenol contaminated soil using polymer beads

Polymer beads have been used to absorb high concentrations of phenol from soil decreasing the initial concentration of 2.3 g kg⁻¹ soil to 100 mg kg⁻¹ soil and achieving a phenol loading within the polymer beads of 27.5 mg phenol g⁻¹ beads. The phenol-loaded polymer beads were removed from the soil and placed in a bioreactor, which was then inoculated with a phenol-degrading microbial consortium. All of the phenol contained within the polymer beads was shown to desorb from the polymer matrix and was degraded by the microbial consortium. The beads were used again (twice) in a similar manner with no loss in performance.     

Host recognition by the egg–larval parasitoid Chelonus inanitus: effects of physical and chemical cues

Parasitoid wasps are widely used as biocontrol agents. For successful deployment, it is important to know how they search for and recognize their hosts. They often use chemical cues in host searching and recognition. Little attention has been paid to the use of physical cues, particularly when combined with chemical cues, though a combination of cues may improve searching efficiency. Chelonus inanitus L. (Hymenoptera: Braconidae) is a solitary, egg–larval parasitoid wasp that parasitizes various lepidopteran species, including serious crop pests. In this study, effects of physical and/or chemical cues on host recognition were investigated in C. inanitus by observing its antennal searching, ovipositor probing, and the duration of arrestment. In a physical cue test, egg-mass models with glass beads differing in size were used, and a single-egg model with a specific size of bead. Egg-mass models with beads of 0.4–1.0 mm in diameter arrested C. inanitus, in accordance with egg size and arrangement of the host. In a chemical cue test, extract from egg masses of the host Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae), equivalent to half of a host egg mass and extract diluted 100-fold were used, and the undiluted extract stimulated C. inanitus. Untreated egg-mass models (0.2-mm-diameter glass beads) and diluted egg extract arrested C. inanitus less effectively; however, when glass beads were treated with the diluted extract, arrestment increased to the same degree as the undiluted extract. These results suggest that C. inanitus uses both physical and chemical cues to recognize eggs of its host, S. litura, and that these stimuli act synergistically.     

Production of glucoamylase using Aspergillus phoenicus immobilized in calcium alginate beads

Summary As a means of better exploiting the growth-dissociated nature of glucoamylase synthesis, a production process in which the growth phase was separated from the enzyme synthesis phase has been developed. Immobilized mycelia arising from a 6-day-old culture of conidia immobilized in calcium alginate beads could be subsequently used repeatedly to produce glucoamylase in a second step using a Dextran T-10 medium. Glucoamylase production was sustained over five sequential batches in a 19-day period and immobilized mycelia remained confined to the subsurface of the beads. Offprint requests to: C. Kuek     

Determination of the intrinsic fermentation kinetics of Saccharomyces cerevisiae cells immobilized in Ca-alginate beads and observations on their growth

Continuous fermentation experiments in a well-stirred fermentor with Saccharomyces cerevisiae cells immobilized in Ca-alginate beads of small diameter (approx. 1 mm) have been performed in order to discover their intrinsic fermentation kinetics, and compare them to the fermentation kinetics for free cells, by fitting both sets of results to the same model. The results show similar kinetic parameters for free and immobilized cells. The changes in cell concentration inside the beads and microscopical observations of transverse sections throughout the experiments, allowed discernment of two different scenarios of cell growth inside the beads: low cell density and fully developed growth. Correspondence to: F. Gòdia     

Design of innovated lipid-based floating beads loaded with an antispasmodic drug: in-vitro and in-vivo evaluation

Context: Drotaverine hydrochloride (DRT) is used to treat gastrointestinal spasms accompanied with diarrhoea. Hence, the drug suffers from brief residence in the highly moving intestine during diarrhoea which leads to poor bioavailability and frequent dosing.

Objective: This study aimed to extend DRT residence in the stomach.

Methods: Calcium alginate floating beads were prepared using sodium alginate, isopropylmyristate (oil), and Gelucire® 43/01 (lipid) adopting emulsion gelation technique. The beads were evaluated for their floating ability, DRT entrapment efficiency and in-vitro release. Gelucire® 43/01 /oil-based beads of the selected formula were coated using ethylcellulose and different plasticizers as polyethylene glycol 400 and triethyl citrate to retard the drug release. The coated beads were re-characterized. Finally, the best formulae were investigated for their in-vivo floating ability in dogs besides their delivery to the systemic circulation compared to drug powder in human volunteers.

Results: Incorporation of Gelucire® 43/01 to oil-based beads enhanced the in-vitro performance of the beads. Coated beads prepared using drug:sodium alginate ratio of 1:3 (w/w), 20% (w/v) isopropylmyristate, 20% (w/v) Gelucire® 43/01 showed promising in-vitro performance. The beads floated for 12?h in the dogs’ stomach and produced three-fold increase of the total amount of DRT absorbed within 24?h compared to that of DRT powder.

Conclusions: Gelucire® 43/01 /isopropylmyristate-based calcium alginate floating beads coated with ethylcellulose using either PEG 400 or TEC as plasticizers proved to be a successful dosage form in extending DRT release.     

Isolation and characterisation of dog uropathogenic Escherichia coli strains and their fimbriae

A number of Escherichia coli strains have been isolated from dogs with urinary tract infections. These strains have been characterised with respect to their O, K, H, and fimbrial antigens, colicin production, antibiotic resistance, plasmid content and their ability to haemagglutinate erythrocytes from various species. Crossed immunoelectrophoresis of fimbrial extracts, as well as the reaction of partly purified fimbriae of a number of these strains with monoclonal antibodies revealed homology or a strong crossereaction with an F12 fimbrial subunit protein of human uropathogenic E. coli strains. Unlike human F12 fimbriae producing strains, the dog isolates did agglutinate dog erythrocytes in the presence of D-mannose but not human erythrocytes, indicating that the adhesin carried by these strains is different from the adhesin on fimbriae of human uropathogenic E. coli. Similar indications were obtained from experiments with latex beads coated with the receptor for P-fimbriae. These beads were agglutinated by Escherichia coli strains from human urinary tract infections, but not by the dog isolates described here. Preliminary adhesion experiments of human and dog Escherichia coli to human bladder epithelial and canine kidney epithelial cells also showed differences in adhesion depending on the origin of the strain tested.     

Effect of Macrolide Antibiotics on Macrophage Functions

Macrolide antibiotics have a variety of actions other than antimicrobial activities. Recently, it has been suggested that macrolide antibiotics act as immunomodulators. In this study, we evaluated the effects of macrolide antibiotics on macrophage functions. For the macrophage, we used the mouse macrophage cell line J774.1. The following effects of macrolide antibiotics on macrophage functions were evaluated: the effect of macrolide antibiotics on macrophage growth; the phagocytosis of beads; cytocidal activity against Candida albicans; and chemotaxis to lipopolysaccharide (LPS). Macrolide antibiotics except for azithromycin significantly stimulated the growth of the macrophage. In addition, pretreatment with macrolide antibiotics except for roxithromycin significantly stimulated the macrophage phagocytosis of beads, macrophage chemotaxis to LPS, and macrophage cytocidal activity against Candida albicans. These results suggest that macrolide antibiotics stimulate macrophage functions.     

Improvement of efficiency of genetic transformation for <Emphasis Type="Italic">Dunaliella salina</Emphasis> by glass beads method

Dunaliella salina has been exploited as a new type of bioreactor due to its unique advantages. However, this bioreactor application was restricted for absence of a high-efficiency and stable transformation method at present. In the present study, the cells of D. salina were transformed by glass beads. The results of histochemical staining revealed that the GUS gene was successfully expressed in the positive transformants, and PCR and PCR-Southern blot analysis further demonstrated that the bar gene was integrated into the D. salina genome. Moreover, the three transformation methods, including glass beads, bombardment particle and electroporation, were compared for screening a high-efficiency transformation method for gene engineering of D. salina. The results showed that transformation efficiency of the glass beads was the highest, approximately 10² transformants/μg DNA. It is concluded that the established glass beads method has been demonstrated to be an optimal transformation way for D. salina.     

Stomach-Specific Controlled Release Gellan Beads of Acid-Soluble Drug Prepared by Ionotropic Gelation Method

The purpose of the present work was the development and evaluation of stomach-specific controlled release mucoadhesive drug delivery system prepared by ionotropic gelation of gellan beads, containing acid-soluble drug amoxicillin trihydrate, using 3² factorial design with concentration of gellan gum and quantity of drug as variables. The study showed that beads prepared in alkaline cross-linking medium have higher entrapment efficiency than the acidic cross-linking medium. The entrapment efficiency was in the range of 32% to 46% w/w in acidic medium, which increased up to 60% to 90% w/w in alkaline medium. Batches with lowest, medium, and highest drug entrapment were subjected to chitosan coating to form a polyelectrolyte complex film. As polymer concentration increases, entrapment efficiency and particle size increases. Scanning electron microscopy revealed spherical but rough surface due to leaching of drug in acidic cross-linking solution, dense spherical structure in alkaline cross-linking solution, and rough surface of chitosan-coated beads with minor wrinkles. The in vitro drug release up to 7 h in a controlled manner following the Peppas model (r = 0.9998). In vitro and in vivo mucoadhesivity study showed that beads have good mucoadhesivity and more than 85% beads remained adhered to stomach mucosa of albino rat even after 7 h. In vitro growth inhibition study showed complete eradication of Helicobacter pylori. These results indicate that stomach-specific controlled release mucoadhesive system of amoxicillin gellan beads may be useful in H. pylori treatment.     

Sustained ammonia production by immobilized filaments of the nitrogen-fixing cyanobacterium Anabaena 27893

Summary Whole filaments of the N₂-fixing cyanobacterium Anabaena ATCC 27893 have been immobilized by entrapment in calcium alginate gel beads. In a continuous flow fluidized bed reactor sustained photosynthesis, N₂-fixation, and ammonia production have been achieved over a 130 hour period, the longest tested.     

Encapsulation of Ketoprofen and Ketoprofen Lysinate by Prilling for Controlled Drug Release

In this paper, ketoprofen and ketoprofen lysinate were used as model drugs in order to investigate release profiles of poorly soluble and very soluble drug from sodium alginate beads manufactured by prilling. The effect of polymer concentration, viscosity, and drug/polymer ratio on bead micromeritics and drug release rate was studied. Ketoprofen and ketoprofen lysinate loaded alginate beads were obtained in a very narrow dimensional range when the Cross model was used to set prilling operative conditions. Size distribution of alginate beads in the hydrated state was strongly dependent on viscosity of drug/polymer solutions and frequency of the vibration. The release kinetics of the drugs showed that drug release rate was related with alginate concentration and solubility of the drug. Alginate solutions with concentration higher than 0.50% (w/w) were suitable to prepare ketoprofen gastro-resistant formulation, while for ketoprofen lysinate alginate, concentration should be increased to 1.50% (w/w) in order to retain the drug in gastric environment. Differential scanning calorimetry thermograms and Fourier transform infrared analyses of drug-loaded alginate beads indicated complex chemical interactions between carboxyl groups of the drug and polymer matrix in drug-loaded beads that contribute to the differences in release profile between ketoprofen and ketoprofen lysinate. Total release of the drugs in intestinal medium was dependent on the solubility of the drug and was achieved between 4 and 6 h.     

A Rapid Method for the Pre‐Enrichment and Detection of Salmonella Typhimurium by Immunomagnetic Separation and Subsequent Fluorescence Microscopical Techniques

Detection of food‐borne pathogens is of great importance in order to minimize the risk of infection for customers. These analyses should be as fast as possible. Any detection method requires enrichment and quantitative analysis of the enriched microbes. Conventional enrichment methods, which take several days, need to be replaced by faster techniques such as immunomagnetic separation (IMS). This technique is based on the use of paramagnetic microspheres coated with antibodies as ligands that have specific affinity to the microbes that have to be detected. In the studies reported here, a rapid method for the detection of Salmonella enterica serovar Typhimurium (Salmonella Typhimurium), combining IMS and Direct Epifluorescence Filter Technique (DEFT), was developed. It was focused on releasing the target cells from the magnetic beads after IMS, because this is a premise for combining IMS, as an alternative pre‐enrichment, with DEFT. Otherwise, the high number of beads form a layer on the filter membrane that makes the following microscopic analysis for the detection of the contaminants impossible. The CELLection^TM Dynabeads^® used in this study, are coated with recombinant streptavidin (rSA) via a DNA linker. The rSA binds biotinylated antibodies that are able to capture target cells. The DNA linker provides the cleavable site, so that the beads can be removed from the captured cells after isolation. In this study a releasing procedure was developed. This procedure allows for an average 74 % ± 4 % of the bead‐bound Salmonella Typhimurium cells to be released from the beads after IMS, so that the detection of the separated cells by DEFT will be possible.     

Production of extracellular native and foreign proteins by immobilized growing cells of Myxococcus xanthus

Summary Several strains of the protein-secreting, Gram negative bacterium Myxococcus xanthus were immobilized in carrageenan beads and the production of extracellular proteins was followed.The extracellular proteolytic activity was enhanced and concentrated in the beads. In contrast, the amount of total protein secreted by the cells was not modified by immobilization, but it was also retained and concentrated in the beads, the more, the harder the gel. The amount of slime produced by the cells did not seem to influence protein retention.Foreign proteins expressed from genes cloned in Myxococcus xanthus chromosome can be secreted into the medium by immobilized recombinant strains. A polygalacturonate lyase, expressed from the pelC gene from Erwinia chrysanthemi was only detectable outside of the beads. The pH 2.5 acid phosphatase expressed from the appA gene from Escherichia coli was secreted by immobilized cells at the same rate than did the free cells. It was predominantly found in the medium outside of the beads which represented a first purification and facilitated the continuous production of this protein by immobilized recombinant cells packed in a reactor.     

Alginate beads provide a beneficial physical barrier against native microorganisms in wastewater treated with immobilized bacteria and microalgae

When the freshwater microalga Chlorella sorokiniana and the plant growth-promoting bacterium Azospirillum brasilense were deployed as free suspensions in unsterile, municipal wastewater for tertiary wastewater treatment, their population was significantly lower compared with their populations in sterile wastewater. At the same time, the numbers of natural microfauna and wastewater bacteria increased. Immobilization of C. sorokiniana and A. brasilense in small (2–4 mm in diameter), polymer Ca-alginate beads significantly enhanced their populations when these beads were suspended in normal wastewater. All microbial populations within and on the surface of the beads were evaluated by quantitative fluorescence in situ hybridization combined with scanning electron microscopy and direct measurements. Submerging immobilizing beads in wastewater created the following sequence of events: (a) a biofilm composed of wastewater bacteria and A. brasilense was created on the surface of the beads, (b) the bead inhibited penetration of outside organisms into the beads, (c) the bead inhibited liberation of the immobilized microorganisms into the wastewater, and (d) permitted an uninterrupted reduction of ammonium and phosphorus from the wastewater. This study demonstrated that wastewater microbial populations are responsible for decreasing populations of biological agents used for wastewater treatment and immobilization in alginate beads provided a protective environment for these agents to carry out uninterrupted tertiary wastewater treatment.     

Plant regeneration from alginate-encapsulated shoot tips of Phylianthus amarus schum and thonn, a medicinally important plant species

Summary A method was developed for plant regeneration from alginate-encapsulated shoot tips of Phyllanthus amarus. Shoot tips excised from in vitro proliferated shoots were encapsulated in calcium alginate beads. The best gel complexation was achieved using 3% sodium alginate and 75 mM CaCl₂·2H₂O. Maximum percentage response for conversion of encapsulated shoot tips into plantlets was 90% after 5 wk of culture on Murashige and Skoog (MS) medium without plant growth regulator. The regrowth ability of encapsulated shoot tips was affected by the concentration of sodium alginate, storage duration, and the presence or absence of MS nutrients in calcium alginate beads. Plantlets with well-developed shoot and roots were transferred to pots containing an autoclaved mixture of soilrite and peat moss (1∶1). The conversion of encapsulated shoot tips into plantlets also occurred when calcium alginate beads were directly sown in autoclaved soilrite moistened with 1/4-MS salts. Encapsulation of vegetative propagules in calcium alginate beads can be used as an alternative to synthetic seeds derived from somatic embryos.     

Cytokinin biosynthesis in roots of corn

Removal of the quiescent center (QC) from the root apex of maize (Zea mays L., cv. Kelvedon 33) initiates a set of events which culiminate in the regeneration of an intact apex with a newly formed QC. Concomitant with the formation of a new QC is a marked reduction in extractable cytokinins in the tissue of the proximal meristem. Replacing the excised QC with a Dowex (acidic cation-exchange resin) bead affects both root growth and QC regeneration. Root growth is inhibited by plain Dowex beads and Dowex beads treated with zeatin; this inhibition is reversed if the beads have been treated with CaCl₂ (±zeatin). Dowex beads treated with zeatin delay the formation of a new QC; this effect is the same whether or not the beads also contain CaCl₂. The results of this investigation support the notions that cytokinin biosynthesis in roots is a result of activities of both the QC and the proximal meristem, and that cytokinins, at least if supplied exogenously, can play a role in root morphogenesis by delaying the regeneration of the QC.Abbreviations used throughout the text PM proximal meristem - QC quiescent center - RC root cap