Metabolic activity of yeast immobilized as supported monolayer

Summary Immobilization of Saccharomyces cerevisiae as a monocellular layer adhering to a glass support has been achieved by adsorbing Al or Fe(III) ions on the cells or by coating the support with Al(OH)₃ or Fe₂O₃ particles. The initial rate of glucose to ethanol conversion was measured in pure glucose solutions for free cells in suspension, either untreated or submitted to the chemical treatments used for immobilization, and for immobilized cells, using a small reactor (50–70 ml) in closed circuit. The conversion rate of immobilized cells is in the range of 3.5 to 5×10^-14 mol/cell·h while that of untreated suspended cells is in the range of 5 to 7×10^-14 mol/cell·h.     

Immobilization of yeast by adhesion to a support without use of a chemical agent

Yeast Saccharomyces cerevisiae has been immobilized by adhesion to plate supports of glass or polycarbonate after a suitable pretreatment, and sedimentation on the support, followed by washing the non-adhering cells. The pretreatment consists of starving the cells in pure water. Its efficiency is related to both a release of substances, decreasing the electrostatic repulsion between the particles involved, and a modification of the cell wall itself. A single layer of immobilized cells is obtained and a large fraction of the cells keep the capacity to reduce methylene blue and to multiply. The influence of various experimental factors (pretreatment, harvesting time, time of contact with the support) is discussed.     

Characterization of Nitrosomonas europaea immobilized in calcium alginate

Nitrosomonas europaea cells have been immobilized in calcium alginate and the resulting preparation was used as a biocatalyst for the oxidation of NH⁺₄ to NO^?₂. Characterization of this immobilized biocatalyst was done according to the guidelines recommended by the Working Party on Immobilized Biocatalysts of the European Federation of Biotechnology. The most important indications obtained from the results are: (a) at low concentrations of substrate, either ammonium ions or oxygen, diffusion limitation will play a role; (b) inhibition by nitrite ions accumulating in the support is not rapidly controlling the efficiency of the immobilized cells; (c) accumulation of hydrogen ions is a rate-limiting factor, especially in unbuffered solutions; (d) the activity of immobilized N. europaea can increase as a result of growth in the support under conditions which would cause washout of free cells. This last result shows the potential of immobilized N. europaea for nitrification of wastewater. The development of a system applying a cheaper and more stable support is, however, a prerequisite for this application.     

Cobra venom phospholipase A2 immobilized to porocus glass beads.

Phospholipase A₂ (EC 3.1.1.4) from cobra venom (Naja naja naja) has been covalently immobilized to aryl amine porous glass beads by diazo coupling. The attachment of the enzyme to the glass beads is apparently through tyrosine. The activity of the immobilized enzyme toward phospholipid substrate has been monitored using the Triton X-100/phospholipid mixed micelle assay system. The activity of the immobilized phospholipase A₂ toward phosphatidylcholine is about 160 μmol min^?1 ml^?1 of glass beads, and the specific activity is about 13 μmol min^?1 mg^?1 of protein in this assay system. The pH rate profile and apparent pK_a in 10 mm Ca²⁺ of the immobilized enzyme parallels that of the soluble enzyme. The substrate specificity of the immobilized enzyme toward individual phospholipid species in mixed micelles is phosphatidylcholine ? phosphatidylethanolamine. In binary lipid mixtures in mixed micelles containing phosphatidylcholine and phosphatidylethanolamine together, a reversal in specificity is observed, and phosphatidylethanolamine is the preferred substrate. This unusual specificity reversal in binary mixtures is also observed for the soluble enzyme. The activity of the immobilized enzyme toward phospholipid inserted in mixed micelles is the same as toward a synthetic phospholipid which forms monomers, while a 20-fold decrease in activity toward monomeric substrate is observed for the soluble enzyme. The immobilized enzyme is stable at temperatures of 90 °C as is the soluble enzyme. However, p-bromphenacyl bromide, a reagent which inactivates the soluble enzyme, does not inactivate the immobilized enzyme. The immobilized enzyme can be stored frozen for several months and is reusable. The mechanism of action of immobilized phospholipase A₂ from cobra venom and the potential usefullness of the bound enzyme as a probe for phospholipids in surfaces of membranes is considered.     

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.     

Continuous production of human erythropoietin by immobilized recombinant L-929 cells

Recombinant L-929 cells transfected with the human erythropoietin (EPO) gene were immobilized in a macroporous cellulosic support and its derivatives in which charged groups or cell attachment factors were introduced. The immobilized cells were cultured in serum-containing and serum-free media. Comparable production of EPO was observed even in the serum-free medium when a support modified by polyethyleneimine was used for immobilization. The cells immobilized on the supports were cultured in fluidized-bed and inner-loop type air-lift bioreactors for continuous production of EPO. A high cell density of more than 2 × 10⁷ cells/cm³-support and high EPO productivity were achieved and maintained for 50 d through the use of the inner-loop type air-lift bioreactor. The productivity was 13.4-fold higher than that of conventional static cultures in petri-dishes.     

Immobilization of firefly luciferase on glass rods: Properties of the immobilized enzyme

Firefly luciferase has been covalently linked with glutaraldehyde to alkylamine glass beads which had been cemented to glass rods. The immobilized enzyme has a lower pH optima than the soluble enzyme and emits light with a major peak at 615 nm, while the soluble enzyme emits light with a peak at 562 nm. The immobilized enzyme is stable and can be used for multiple assays. The peak light intensity is linear with respect to ATP concentration in the range of 1 × 10⁻⁵ to 1 × 10⁻⁸ . The luciferase rods have been used in a coupled assay to measure the rate of ATP production catalyzed by creatine phosphokinase. This immobilized luciferase should be very useful for assaying low levels of ATP in any type of sample.     

Degradation of Carbazole by Microbial Cells Immobilized in Magnetic Gellan Gum Gel Beads

Polycyclic aromatic heterocycles, such as carbazole, are environmental contaminants suspected of posing human health risks. In this study, we investigated the degradation of carbazole by immobilized Sphingomonas sp. strain XLDN2-5 cells. Four kinds of polymers were evaluated as immobilization supports for Sphingomonas sp. strain XLDN2-5. After comparison with agar, alginate, and κ-carrageenan, gellan gum was selected as the optimal immobilization support. Furthermore, Fe₃O₄ nanoparticles were prepared by a coprecipitation method, and the average particle size was about 20 nm with 49.65-electromagnetic-unit (emu) g⁻¹ saturation magnetization. When the mixture of gellan gel and the Fe₃O₄ nanoparticles served as an immobilization support, the magnetically immobilized cells were prepared by an ionotropic method. The biodegradation experiments were carried out by employing free cells, nonmagnetically immobilized cells, and magnetically immobilized cells in aqueous phase. The results showed that the magnetically immobilized cells presented higher carbazole biodegradation activity than nonmagnetically immobilized cells and free cells. The highest biodegradation activity was obtained when the concentration of Fe₃O₄ nanoparticles was 9 mg ml⁻¹ and the saturation magnetization of magnetically immobilized cells was 11.08 emu g⁻¹. Additionally, the recycling experiments demonstrated that the degradation activity of magnetically immobilized cells increased gradually during the eight recycles. These results support developing efficient biocatalysts using magnetically immobilized cells and provide a promising technique for improving biocatalysts used in the biodegradation of not only carbazole, but also other hazardous organic compounds.     

Maintenance and operational stability of immobilized Arthrobacter simplex for the ▵1-dehydrogenation of steroids

The stability of the ?¹-dehydrogenation system of Arthrobacter simplex immobilized in calcium alginate has been studied. A high stability was related to the ability of the cells to utilize a carbon source such as d-glucose or steroid. Inhibition of de novo protein synthesis reduced the ?¹-dehydrogenase [3-oxosteroid: acceptor) ?¹-oxidoreductase, EC 1.3.99.4] stability of the immobilized cells. The operational stability of immobilized cell preparations in the presence of the steroid degradation inhibitor, α,α-dipyridyl, could not be improved significantly by supplementing steroid substrate suspensions with either d-glucose or yeast extract.     

Immobilization of Bacillus subtilis α-amylase on zirconia-coated alkylamine glass with glutaraldehyde

Bacillus subtilis α-amylase (EC 3.2.1.1) has been immobilized on zirconia-coated alkylamine glass by using the process of glutaraldehyde coupling. The immobilized enzyme preparation exhibited 52% of the initial enzyme activity and a conjugation yield of 28 mg/g support. The K_m value of the immobilized α-amylase was decreased by immobilization while V_max was unaltered. E_a of the enzyme was decreased upon conjugation. The soluble enzyme was optimally active at pH 5.6 while the immobilized enzyme exhibited optimal activity in the pH range 5.4–6.2. The alkylamine-immobilized enzyme has also been characterized through its isoelectric point. The industrial importance of this work is discussed.     

Studies on Horseradish Peroxidase Immobilized onto Arylamine and Alkylamine Glass

Peroxidase from horseradish has been immobilized onto zirconia coated arylamine and alkylamine glass through the process of diazotization and glutaraldehyde coupling, respectively. Arylamine glass bound enzyme retained 77% of the initial activity with a conjugation yield of 18 mg g^-1 support, while alkylamine glass bound enzyme retained 38% of the initial activity with a conjugation yield of 16 mg g^-1 support. The immobilized enzyme showed an increase in optimum pH, temperature for maximum activity, energy of activation (Ea), and thermal stability but decrease in time for linearity and Km for H₂O₂. Vmax value of arylamlne conjugated enzyme decreased but Vmax of alkylamine conjugated enzyme was unaltered compared to free enzyme. Both arylamine and alkylamine bound enzyme showed higher stability in cold compared to that of free enzyme. The application of glass bound peroxidase in discrete analysis of serum urate is demonstrated.     

Conversion of Glycerol to Dihydroxyacetone by Immobilized Whole Cells of Acetobacter xylinum

Enzymatic production of dihydroxyacetone (DHA) was studied by immobilization of the whole cells of acetic acid bacteria capable of oxidizing glycerol to DHA. Acetobacter xylinum A-9 cells immobilized in a polyacrylamide gel were selected as the most favorable enzyme preparation. The enzymatic properties of immobilized cells converting glycerol to DHA were investigated and compared with those of intact cells. The optimum pH for the immobilized cells was broad (4.0 to 5.5), whereas the intact cells had a narrow pH optimum at 5.5. The thermal stability of the immobilized cells was somewhat higher than that of the intact cells. Apparent K_m values for glycerol with both intact and immobilized cells were about equal, 6.3 × 10⁻² to 6.5 × 10⁻² M. The complete conversion of glycerol to DHA was achieved within 40 h under optimum conditions, and pure crystalline DHA was readily isolated from the reaction mixture with over 80% yield.     

Studies on sugar isomerases of Lactobacillus sp.: Whole cell immobilization of d-glucose isomerase

A new soil isolate of Lactobacillus sp. grown in Yamanaka medium under submerged conditions showed the presence of d-glucose, d-xylose and d-ribose isomerases in washed cell suspension and cell free extracts. d-Xylose isomerase (d-xylose ketol-isomerase, EC 5.3.1.5) and d-ribose isomerase (d-ribose ketol-isomerase, EC 5.3.1.20) activities reached a maximum in 48 h of growth and then declined. d-Glucose isomerase (d-glucose 6-phosphate isomerase, d-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9) activity was maximum after 72 h and remained constant for ～120 h of growth. d-Glucose isomerase activity increased with the increase in number of generations of culture and reached a maximum in 5–6 generations, whereas d-xylose and d-ribose isomerase activities decreased. The washed and starved whole cells could be heat treated and immobilized on the rough surface of glass rods or glass slides using acetone treatment. The heat treated immobilized cells showed only the presence of d-glucose isomerase activity and showed no d-xylose and d-ribose isomerase activities. d-Glucose isomerase activity of heat treated immobilized cells was inhibited less by sorbitol, mannitol, sodium arsenate, cysteine and calcium ions than the free d-glucose isomerase activity in fresh untreated washed whole cells and cell free extracts. EDTA inhibition had the same effect for both forms. Ca²⁺inhibition could be reversed by adding Mg²⁺ions.     

Enhanced Reactivity of Rhizopus oryzae Lipase Displayed on Yeast Cell Surfaces in Organic Solvents: Potential as a Whole-Cell Biocatalyst in Organic Solvents

Immobilization of enzymes on some solid supports has been used to stabilize enzymes in organic solvents. In this study, we evaluated applications of genetically immobilized Rhizopus oryzae lipase displayed on the cell surface of Saccharomyces cerevisiae in organic solvents and measured the catalytic activity of the displayed enzyme as a fusion protein with α-agglutinin. Compared to the activity of a commercial preparation of this lipase, the activity of the new preparation was 4.4 × 10⁴-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate and 3.8 × 10⁴-fold higher in an esterification reaction with palmitic acid and n-pentanol (0.2% H₂O). Increased enzyme activity may occur because the lipase displayed on the yeast cell surface is stabilized by the cell wall. We used a combination of error-prone PCR and cell surface display to increase lipase activity. Of 7,000 colonies in a library of mutated lipases, 13 formed a clear halo on plates containing 0.2% methyl palmitate. In organic solvents, the catalytic activity of 5/13 mutants was three- to sixfold higher than that of the original construct. Thus, yeast cells displaying the lipase can be used in organic solvents, and the lipase activity may be increased by a combination of protein engineering and display techniques. Thus, this immobilized lipase, which is more easily prepared and has higher activity than commercially available free and immobilized lipases, may be a practical alternative for the production of esters derived from fatty acids.     

Preparation,characterization and laboratory-scale application of an immobilized glucoamylase

Glucoamylase (1,4-α-d-glucan glucohydrolase, EC 3.2.1.3) has been covalently immobilized on a polyacrylamide-type support containing carboxylic groups activated by water-soluble carbodiimide. The activity was 5.5– 6.0 units g^?1solid. The optimum pH for catalytic activity was pH 3.8. The apparent optimum temperature was found at 60°C. With soluble starch as substrate the K_m value was 14 mg ml^?1. The pH for maximum stability was pH 4.0–4.5. In the presence of 8 m urea the immobilized glucoamylase retained most of its catalytic activity but it was more susceptible to guanidinium hydrochloride than the soluble enzyme. The practical applicability of immobilized glucoamylase was tested in batch process and continuous operation.     

Bioanode for a microbial fuel cell based on Gluconobacter oxydans immobilized into a polymer matrix

Acetic acid bacteria Gluconobacter oxydans subsp. industrius RKM V-1280 were immobilized into a synthetic matrix based on polyvinyl alcohol modified with N-vinylpyrrolidone and used as biocatalysts for the development of bioanodes for microbial fuel cells. The immobilization method did not significantly affect bacterial substrate specificity. Bioanodes based on immobilized bacteria functioned stably for 7 days. The maximum voltage (fuel cell signal) was reached when 100–130 μM of an electron transport mediator, 2,6-dichlorophenolindophenol, was added into the anode compartment. The fuel cell signals reached a maximum at a glucose concentration higher than 6 mM. The power output of the laboratory model of a fuel cell based on the developed bioanode reached 7 mW/m² with the use of fermentation industry wastes as fuel.     

Development of a Streptavidin-Conjugated Single-Chain Antibody That Binds Bacillus cereus Spores

Control of microorganisms such as Bacillus cereus spores is critical to ensure the safety and a long shelf life of foods. A bifunctional single chain antibody has been developed for detection and binding of B. cereus T spores. The genes that encode B. cereus T spore single-chain antibody and streptavidin were connected for use in immunoassays and immobilization of the recombinant antibodies. A truncated streptavidin, which is smaller than but has biotin binding ability similar to that of streptavidin, was used as the affinity domain because of its high and specific affinity with biotin. The fusion protein gene was expressed in Escherichia coli BL21 (DE3) with the T7 RNA polymerase-T7 promoter expression system. Immunoblotting revealed an antigen specificity similar to that of its parent native monoclonal antibody. The single-chain antibody-streptavidin fusion protein can be used in an immunoassay of B. cereus spores by applying a biotinylated enzyme detection system. The recombinant antibodies were immobilized on biotinylated magnetic beads by taking advantage of the strong biotin-streptavidin affinity. Various liquids were artificially contaminated with 5 × 10⁴ B. cereus spores per ml. Greater than 90% of the B. cereus spores in phosphate buffer or 37% of the spores in whole milk were tightly bound and removed from the liquid phase by the immunomagnetic beads.     

Immobilization of apricot pectinesterase (Prunus armeniaca L.) on porous glass beads and its characterization

Pectinesterase isolated from Malatya apricot pulp was covalently immobilized onto glutaraldehyde-containing amino group functionalized porous glass beads surface by chemical immobilization at pH 8.0. The amount of covalently bound apricot PE was found 1.721 mg/g glass support. The properties of immobilized enzyme were investigated and compared to those of free enzyme. The effect of various parameters such as pH, temperature, activation energy, heat and storage stability on immobilized enzyme were investigated. Optimum pH and temperature were determined to be 8.0 and 50 °C, respectively. The immobilized PE exhibited better thermostability than the free one. Kinetic parameters of the immobilized enzyme (K_m and V_max values) were also evaluated. The K_m was 0.71 mM and the V_max was 0.64 μmol min^?1 mg^?1. No drastic change was observed in the K_m and V_max values. The patterns of heat stability indicated that the immobilization process tends to stabilize the enzyme. Thermal and storage stability experiments were also carried out. It was observed that the immobilized enzyme had longer storage stability and retained 50% of its initial activity during 30 days.     

Optical spectroscopy of inorganic-organic host-guest nanocrystals organized as oriented monolayers

Oriented and densely packed zeolite L monolayers were prepared on a glass support. The one-dimensional channels of zeolite L, being all oriented perpendicular to the glass and parallel to each other, were sequentially filled by ion exchange with two strongly fluorescent dye molecules. First N-methylacridine (MeAcr⁺) was inserted followed by 3,3′-diethylthiacarbocyanine (DTC⁺). The shorter MeAcr⁺ is oriented perpendicular to the channel axis while the longer DTC⁺ is parallel, due to the constraints imposed by the geometry of the zeolite L channels, as deduced from fluorescence anisotropy of single MeAcr⁺-zeolite L and DTC⁺-zeolite L crystals. The dye molecules can enter the channels only from the top side of the monolayer, since the entrances on the bottom are blocked by the glass support. The resulting ordering has been observed by fluorescence microscopy of single DTC⁺, MeAcr⁺-zeolite L crystals. Conditions were found to suppress the pronounced Rayleigh scattering of zeolite monolayers. Thus high quality absorption spectra of DTC⁺, MeAcr⁺-zeolite L monolayers on glass could be measured at different angles between the incident light and the layer. The results deliver a direct proof that microscopic ordering of the dyes in the channels of zeolite L as well as macroscopic organization of the dye-zeolite L monolayer on the glass support was achieved. Thus a high level of organization was obtained by controlled assembly of the zeolite L crystals into oriented structures followed by subsequent insertion of strongly luminescent dyes.     

Development of Bed Reactor Using Brick Dust Immobilized CIVI-Cellulase from Seeds of Cowpea (Vigna sinensis L)

Cellulase extracted from seeds of Cowpea (Vigna sinensis L var VITA-4) was partially purified and immobilized on brick dust as solid support via glutaraldehyde. The percentage retention of the enzyme activity on brick dust was nearly 85%. After immobilization specific activity of the enzyme increased from 0.275 to 0.557 U mg^?1 protein with about 2 fold enrichment. The optimum pH and temperature of soluble enzyme were determined as pH 4.6 and WC, respectively whereas immobilized enzyme showed at pH 5.0 and 37°C, respectively. The V_max values for soluble and immobilized enzyme were determined as 6.67 and 1.25 mg min^?1, respectively whereas K_m values were 4.35 and 4.76 mg ml^?1, respectively. The immobilized enzyme displayed higher thermal stability than soluble enzyme and retained about 50% of its initial activity after 12 reuses. Immobilized enzyme was packed in an indigenously designed double walled glass bed reactor for continuous production of reducing sugars.