Redirecting cellular metabolism by immobilization of cultured plant cells: A model study with Coffea arabica

Suspension-cultured cells of Coffea arabica have been immobilized by entrapment in calcium alginate gels to mimic natural aggregation. The production of methylxanthine alkaloid was increased up to 13-fold by the immobilization. This increased production has been ascribed to organization of the entrapped cells through physicochemical interactions between the polymer (alginate) and the plant cell wall. It has been shown that the metabolic changes induced by the immobilization are reversible.     

Immobilization of native and dextran-free dextransucrases from Leuconostoc mesenteroides NRRL B-512F for the synthesis of glucooligosaccharides

Dextransucrase from Leuconostoc mesenteroides NRRL B-512F was immobilized using two different methods: covalent attachment to activated silica and entrapment in calcium alginate. For immobilization on silica, native enzyme and dextran-free enzyme were compared. However, the entrapment in calcium alginate beads gave the best results in terms of immobilization yield and stability. This biocatalyst was employed in the acceptor reaction with maltose showing similar glucooligosaccharide production than the native enzyme but increased operational stability.     

Immobilization of murine lymphocytes by gel entrapment

Summary Murine non-transformed lymphocytes were immobilized by alginate and agarose entrapment. After lipopolysaccharide activation, immunoglobulin production was followed as a criterion of viability of the cells. In alginate beads, diffusion limitations result in cell death. In agarose, the production level of specific antibodies is 40% lower than with suspended cells while immobilization does not alter polyclonal antibody production.     

Immobilization of whole cells ofStreptomyces kanamyceticus containing glucose isomerase by a combination of heat treatment in the presence of minerals and entrapment in calcium alginate gels

A method of immobilization of whole cells ofStreptomyces kanamyceticus containing glucose isomerase was devised, based on techniques of heat fixation in the presence of minerals and, entrapment in calcium alginate gels. The optimum activity of the enzyme was obtained when the cells were heat-fixed at 60°C for 10 min in the presence of 50 mmol/L MgSO₄·7H₂O and 5 mmol/L CoCl₂·6H₂O and then cast into calcium alginate beads using 2% sodium alginate.     

Induction of stress metabolites in immobilized Glycyrrhiza echinata cultured cells

Transfer into a fresh medium or immobilization by entrapment in calcium alginate gels of cultured Glycyrrhiza echinata cells caused a rapid and transient accumulation of a retrochalcone, echinatin, in both the cells and the medium. The higher level and longer duration of echinatin production was observed in the immobilized cells than in freely suspended cells. Transfer of the cells into the medium containing either sodium alginate or calcium chloride, and the addition of sodium alginate into the suspension culture, caused the same effect as observed in cell immobilization. A novel metabolite was also detected in the induced cells. Activities of the enzymes involved in echinatin biosynthesis were shown to rapidly increase by immobilization of the cells.Abbreviations IAA indole-3-acetic acid - LMT S-adenosylmethionine: licodione 2-O-methyltransferase - CHS chalcone synthase     

Production of alkaline protease by entrapped Bacillus licheniformis cells in repeated batch process

In this study, Bacillus licheniformis cells were immobilized by entrapment in calcium alginate beads and were used for production of alkaline protease by repeated batch process. In order to increase the stability of the beads, the immobilization procedure was optimized by statistical full factorial method, by which three factors including alginate type, calcium chloride concentration, and agitation speed were studied. Optimization of the enzyme production medium, by the Taguchi method, was also studied. The obtained results showed that optimization of the cell immobilization procedure and medium constituents significantly enhanced the production of alkaline protease. In comparison with the free-cell culture in pre-optimized medium, about 7.3-fold higher productivity was resulted after optimization of the overall procedure. Repeated batch mode of operation, using optimized conditions, resulted in continuous production of the alkaline protease for 13 batches in 19 days.     

Production of cephalosporin C by immobilized cells of Cephalosporium acremonium

Cephalosporium acremonium ATCC 48272 cells were immobilized on various adsorbents and in various entrapment matrices. The influence of the incubation period, the best immobilization technique and the optimum concentrations of the selected matrices were investigated. From the results of the repeated batch fermentation in shake flasks, a good level of antibiotic was maintained for a period of about 19 days using 4% calcium alginate and 1% glass wool as entrapment and adsorbent supports, respectively.     

An optimized process for manufacturing anAzospirillum inoculant for crops

Summary An optimized process for manufacturing a crop moculant was developed with anAzospirillum lipoferum strain. This process involves the entrapment of living cells in alginate beads and dehydration. The influence of several parameters, alginate concentration, additions of adjuvants at different stages, dilution of culture broth, water activity and dehydration method on bacterial survival is presented. The highest survival was obtained by addition of skim milk and controlled air-dehydration of the alginate beads. Finally, a powdered inoculant was obtained, containing more than 10 billion cells/g, easy to store and to handle, which can be used in the field as a microgranule or as a seed coating. Furthermore, the biodegradability insures that there is no environmental pollution.     

The effect of immobilization on the course of biotransformation reactions by plant cells

Summary The influence of different immobilization methods on the biotransformation of verbenol bySolanum aviculare plant cells has been studied. The biotransformation course was compared using free and immobilized cells. Immobilization techniques used included entrapment in alginate, pectate and carrageenane gels, in polyurethane foam, and on the surface of polyphenylenoxide.     

Calcium alginate beads as a slow-release system for delivering angiogenic molecules in vivo and in vitro.

A method previously used in this laboratory for entrapment of tumor cells in alginate beads has been extended to provide a slow release delivery system for growth factors with known in vivo angiogenic activity. Protein growth factors were entrapped in alginate beads in amounts sufficient to cause incorporation of 3H-thymidine by COMMA-D cells in vitro, and in vivo neovascularization when injected subcutaneously into Balb/c mice. Entrapment of 125I-labelled growth factors showed that the amount of molecule entrapped in alginate beads may vary with the charge of the molecule. In vitro cell proliferation studies showed that entrapment in alginate beads may provide a slow-release system or a stabilizing environment for the protein. In some cases biological activity of the growth factor in solution was increased by the presence of control alginate beads. When alginate-entrapped growth factors were injected into Balb/c mice, induction of new blood vessels could be monitored qualitatively by macroscopic photography and assessed quantitatively by measuring the pooling of radiolabelled red blood cells at the experimental site. Subcutaneous injection of purified angiogenic factors not entrapped in alginate beads did not cause neovascularization. Diffusion of 125I-labelled growth factors from alginate beads in the animal showed that release in vivo may depend on the charge of the protein molecule. These results indicate that injection of purified molecules entrapped in alginate beads provides an effective localized and slow-release delivery of biologically active molecules. This delivery system may extend the time of effectiveness of biologically active molecules in vivo compared to direct injection without alginate entrapment. The method of entrapment and injection has potential for identifying active factors in tumor-induced angiogenesis and testing new compounds as modulators of neovascularization.     

Synthesis and characterization of a pyrrole-alginate conjugate and its application in a biosensor construction

N-(3-Aminopropyl)pyrrole was covalently coupled with alginate in an aqueous-phase reaction by means of carbodiimide-mediated activation chemistry to provide a pyrrole-alginate conjugate for subsequent use in biosensor applications. The pyrrole-alginate conjugate was quantified by UV spectroscopy at 230 nm, by an HPSEC-MALLS analytical method, as well as by FTIR and 13C NMR spectroscopies. The new pyrrole-alginate conjugate was used for the immobilization of polyphenol oxidase (PPO) onto an electrode surface by physical entrapment resulting from the gellification process and electrochemical polymerization of the pyrrole groups. The efficiency of this cross-linking approach (chemical and electrochemical) was investigated by comparing the amount of enzyme released from polypyrrole-alginate and regular alginate. In addition, biosensors were prepared by entrapment of the PPO in polypyrrole-alginate and regular alginate matrixes and their performance for the amperometric determination of catechol chosen as a model analyte was examined, yielding a sensitivity of 350 and 80 microA M(-1) cm(-2), respectively, for polypyrrole-alginate and alginate biosensors.     

Production of tannase by the immobilized cells of Bacillus licheniformis KBR6 in Ca-alginate beads

AIMS: The present study was aimed at finding the optimal conditions for immobilization of Bacillus licheniformis KBR6 cells in calcium-alginate (Ca-alginate) beads and determining the operational stability during the production of tannin-acyl-hydrolase (tannase) under semicontinous cultivation. METHODS AND RESULTS: The active cells of B. licheniformis KBR6 were immobilized in Ca-alginate and used for the production of tannase. The influence of alginate concentration (5, 10, 20 and 30 g l(-1)) and initial cell loading on enzyme production were studied. The production of tannase increased significantly with increasing alginate concentration and reached a maximum enzyme yield of 0.56 +/- 0.03 U ml(-1) at 20 g l(-1). This was about 1.70-fold higher than that obtained by free cells. The immobilized cells produced tannase consistently over 13 repeated cycles and reached a maximum level at the third cycle. Scanning electron microscope study indicated that the cells in Ca-alginate beads remain in normal shape. CONCLUSIONS: The Ca-alginate entrapment is a promising immobilization method of B. licheniformis KBR6 for repeated tannase production. Tannase production by immobilized cells is superior to that of free cells because it leads to higher volumetric activities within the same period of fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of tannase production from immobilized bacterial cells. The bacterium under study can produce higher amounts of tannase with respect to other fungal strains within a short cultivation period.     

New processes and actual trends in biotechnology

Abstract: A survey of immobilization techniques with special emphasis on alginate entrapment and micro hollow spheres is presented. The advantages and disadvantages of immobilization are discussed. Industrial applications of immobilization are demonstrated, especially biosensors and production of acrylamide. The fundamentals of bioprocesses with reduced water content and application as well as process examples are presented.     

Doubly entrapped baker's yeast survives during the long-term stereoselective reduction of ethyl 3-oxobutanoate in an organic solvent

To attain long-term bioreaction in organic solvents with living microorganisms, we tried to protect the microorganisms from the toxicity of the solvent by immobilization. In this study, baker's yeast, which is not tolerant to organic solvents such as isooctane, was selected as a model microorganism and the immobilized living yeast cells were examined for activity in the steroselective reduction of ethyl 3-oxobutanoate to ethyl (S)-3-hydroxybutanoate in isooctane; an activity that correlated well with the viability of the yeast cells. It was found that double entrapment, that is, further entrapment of calcium-alginate-gel-entrapped cells with a urethane prepolymer, made it possible for the yeast to remain viable in isooctane, although other conventional immobilization methods, such as single entrapment using polysaccharide or synthetic resin prepolymers, were insufficient for its protection. Furthermore, doubly entrapped living yeast cells could carry out the stereoselective reduction in isooctane repeatedly for a long period (more than 1200 h) with occasional cultivation. Thus, double entrapment enabled a microorganism sensitive to organic solvents to survive over long-term bioreaction in an organic solvent. Received: 29 August 1997 / Received last revision: 24 December 1997 / Accepted: 13 January 1998     

Application of response surface methodology to cell immobilization for the production of palatinose

Response surface methodology (RSM), based on multivariate non-linear model, was applied to study the interactions and optimization of the immobilization parameters for cell entrapment, namely alginate concentration, cell loading and bead diameter using Erwinia rhapontici NCPPB 1578 that produced palatinose. ANOVA analysis and statistical parameters calculations showed that RSM could be used effectively to model and improve a complex system like cell immobilization. Palatinose yield was increased by 40%. The maximum yield of 140 mg/ml was achieved in a batch of 1h at alginate concentration of 5% w/v, cell loading of 5 g l(-1) and 2.25 mm bead diameter. Thus, the E. rhapontici NCPPB 1578 immobilization in alginate bead and subsequent palatinose yield was successfully improved by application of RSM technique.     

Immobilization of cells by electrostatic droplet generation: a model system for potential application in medicine

The process of electrostatic extrusion as a method for cell immobilization was investigated that could be used for potential applications in medicine. An attempt was made to assess the effects of cell addition and polymer concentration on the overall entrapment procedure, ie, on each stage of immobilization: polymer-cell suspension rheological characteristics, electrostatic extrusion process, and the process ofgelation. The findings should contribute to a better understanding of polymer-cell interactions, which could be crucial in possible medical treatments. Alginate-yeast was used as a model system for carrier-cells. The electrostatic extrusion was considered as a complex two-phase flow system and the effects of cell and alginate concentrations on the resulting microbead size and uniformity were assessed. Under investigated conditions, microbeads 50-600 microm in diameter were produced and the increase in both alginate and cell concentrations resulted in larger microbeads with higher standard deviations in size. We attempted to rationalize the findings by rheological characterization of the cell-alginate suspensions. Rheological characterization revealed non-Newtonian, pseudoplastic behavior of cell-alginate suspensions with higher viscosities at higher alginate concentrations. However, the presence of cells even at high concentrations (5x10(8) and 1x10(9) cells/mL) did not significantly affect the rheological properties of Na-alginate solution. Lastly, we investigated the kinetics of alginate gelation with respect to the quantity of Ca2+ ions and cell presence. The gelation kinetics were examined under conditions of limited supply with Ca2+ ions, which can be essential for immobilization of highly sensitive mammalian cells that require minimal exposure to CaCl2 solution. The molar ratio of G units to Ca2+ ions of 3.8:1 provided complete crosslinking, while the increase in alginate concentration resulted in prolonged gelation times but higher strength of the resulting gel. The cell presence decreased the rate of network formation as well as the strength of the obtained Ca-alginate hydrogel.     

Entrapment of a Trigonopsis variabilis D‐amino acid oxidase variant F54Y for oxidative deamination of cephalosporin C

Trigonopsis variabilis D ‐amino acid oxidase (TvDAAO) is an enzyme used in the industrial bioconversion of cephalosporin C (CPC) into 7‐aminocephalosporanic acid, a crucial biosynthetic nucleus for a wide spectrum of semi‐synthetic cephem antibiotics. Using homology modeling and site‐directed mutagenesis, we have previously shown that the TvDAAO variant F54Y possesses improved catalytic activity and thermostability. To further explore its industrial application, the conditions for immobilization of the enzyme were examined in the present investigation. The results showed that entrapment in a calcium alginate (Ca‐alginate) matrix using 2% alginate, 500 mM CaCl₂, and 15 min stabilization appeared to be optimal for the immobilization of F54Y. The entrapped enzyme allowed complete CPC conversion. The entrapped enzyme also showed good operational stability and retained at least 90% of its original activity after 20 reaction cycles. To conclude, the entrapment of F54Y in Ca‐alginate appeared to be a simple and efficient biocatalysis system with potential application in the antibiotics industry.     

Biotransformation of (−)-limonene using Solanum aviculare and Dioscorea deltoidea immobilized plant cells

The biotransformation course of S-(–)-limonene by Solanum aviculare and Dioscorea deltoidea plant cells was studied using 5 different immobilization methods (entrapment into alginate, carrageenan and pectate gels, binding to polyphenyleneoxide and permeation into polyurethane foam). All techniques, with exception of permeation into pre-formed matrix, affected the product ratio. The main products were cis- and trans-carveol and carvone.     

Alginate as immobilization matrix and stabilizing agent in a two-phase liquid system: application in lipase-catalysed reactions.

Alginate was evaluated as an immobilization matrix for enzyme-catalyzed reactions in organic solvents. In contrast to most hydrogels, calcium alginate was found to be stable in a range of organic solvents and to retain the enzyme inside the gel matrix. In hydrophobic solvents, the alginate gel (greater than 95% water) thus provided a stable, two-phase liquid system. The lipase from Candida cylindracea, after immobilization in alginate beads, catalysed esterification and transesterification in n-hexane under both batch and continuous-flow conditions. The operational stability of the lipase was markedly enhanced by alginate entrapment. In the esterification of butanoic acid with n-butanol, better results were obtained in the typical hydrophilic calcium alginate beads than in less hydrophilic matrices. The effects of substrate concentration, matrix area, and polarity of the substrate alcohols and of the organic solvent on the esterification activity were examined. The transesterification of octyl 2-bromopropanoate with ethanol was less efficient than that of ethyl 2-bromopropanoate with octanol. By using the hydrophilic alginate gel as an immobilization matrix in combination with a mobile hydrophobic phase, a two-phase liquid system was achieved with definite advantages for a continuous, enzyme-catalysed process.     

Studies on immobilization of the thermophilic bacterium Thermus aquaticus YT-1 by entrapment in various matrices

Summary Immobilization of the thermophilic bacterium Thermus aquaticus YT-1 has been studied using various entrapment techniques. Alginate, -carrageenan, agar, agarose and polyacrylamide were tested as supports during operation at 65°C at which the cells are known to produce protease when grown free in solution. Alginate showed toxic effects and no viability was observed after entrapment in Ca alginate or even after exposure of free living cells to sodium alginate. Polyacrylamide was observed to be the best support. Protease activity was closely related to the appearance of free cells in the medium.