Radioactive penicillin G production by immobilized fungal vesicles

Summary Radioactive penicillin G production from l-[1-¹⁴C]-valine (1.75 GBq · mmol^-1) by native and by calcium alginate gel immobilized mycelium of Penicillium chrysogenum PQ-96 in a medium for antibiotic production as well as by vesicles isolated from the protoplasts of the same strain in a well-defined reaction mixture was investigated. Specific radioactivity of the penicillin G produced by the native vesicles was 1.45 GBq · mmol^-1 and that of the antibiotic synthesized by the calcium alginate gel immobilized vesicles was 1.48 GBq · mmol^-1. By comparison, the specific radioactivity of penicillin G produced by native mycelium was 0.42 GBq · mmol^-1 and of that synthesized by the immobilized mycelium was 0.96 GBq · mmol^-1. Production of radioactive penicillin G by native and immobilized vesicles in repeated use was also investigated. At the beginning of the production phase, the radioactive penicillin G synthesized by the immobilized vesicles was 25 nmol · mg protein^-1 · h^-1 and decreased after 8 days to a level of 11 nmol · mg protein^-1 · h^-1. The half-life of the immobilized vesicles was 7 days. The native vesicles showed a rapid decrease in radioactive antibiotic production. In comparison, the penicillin G production in a repeated use of immobilized vesicles decreased during 40 days from 140 nmol · mg protein^-1 · h^-1 to 60 nmol · mg protein^-1 · h^-1. The half-life of the immobilized vesicles was 35 days. The native vesicles showed after 4 days a lack of activity of penicillin G production. The stability of immobilized mycelium or vesicles in the process of radioactive penicillin G production is discussed.     

Continuous penicillin production by Penicillium chrysogenum immobilized in calcium alginate beads

Summary A high penicillin-producing Penicillium chrysogenum strain immobilized in calcium alginate beads was used for continuous penicillin fermentation in a bubble column and in a conical bubble fermentor. The fermentation was limited by the growth rate, dilution rates and the stability of the alginate beads. The immobilized cells lost their ability to produce penicillin in the bubble column after 48 h from beginning of the continuous fermentation. In the conical bubble fermentor the immobilized cells remained active for more than 7 days. This bioreactor ensured a good distribution of nutrients and oxygen as well as a higher mechanical stability of the alginate beads.     

Continuous production of isomalto-oligosaccharides from maltose syrup by immobilized cells of permeabilizedAureobasidium pullulans

Continuous production of isomalto-oligosaccharides from maltose syrup by the permeabilized cells ofAureobasidium pullulans immobilized into calcium alginate gel was studied using a column reactor. The immobilized cell column maintained its full activity over 45 days when the reactor was operated at a velocity of 0.1 h^–1 at 50°C using 60%(w/v) maltose syrup as a substrate, and the maximum productivity achieved was around 60 g/1h.     

Stabilization of Aspergillus parasiticus cytosine deaminase by immobilization on calcium alginate beads improved enzyme operational stability

Cytosine deaminase (CD) from Aspergillus parasiticus, which has half-life of 1.10?h at 37°C, was stabilized by immobilization on calcium alginate beads. The immobilized CD had pH and temperature optimum of 5 and 50°C respectively. The immobilized enzyme also stoichiometrically deaminated Cytosine and 5-fluorocytosine (5-FC) with the apparent K_M values of 0.60?mM and 0.65?mM respectively, displaying activation energy of 10.72 KJ/mol. The immobilization of native CD on calcium alginate beads gave the highest yield of apparent enzymatic activity of 51.60% of the original activity and the enzymatic activity was lost exponentially at 37°C over 12?h with a half-life of 5.80?h. Hence, the operational stability of native CD can be improved by immobilization on calcium alginate beads.     

Production of thermostable and neutral glucoamylase using immobilized <Emphasis Type="Italic">Thermomucor indicae-seudaticae</Emphasis>

Thermomucor indicae-seudaticae was immobilized in alginate, κ-carrageenan, agarose, agar, polyacrylamide and loofah (Luffa cylindrica) sponge (as such or coated with alginate/starch/Emerson YpSs agar), and used for the production of glucoamylase in submerged fermentation. The mycelium developed from alginate-immobilized sporangiospores secreted higher glucoamylase titres (22.7 U ml⁻¹) than those immobilized in other gel matrices and the freely growing mycelial pellets (18.5 U ml⁻¹). Loofah network provided a good support for mycelial growth, but the enzyme production was lower than that attained with alginate beads. Glucoamylase production increased with inoculum density and the optimum levels were achieved when 40 calcium alginate beads (∼5 × 10⁶ immobilized spores) were used to inoculate 50 ml production medium. The alginate bead inoculum displayed high storage stability at 4°C and produced comparable enzyme titres up to 120 days. The glucoamylase production by hyphae emerged from the immobilized sporangiospores was almost stable over eight batches of repeated fermentation. Scanning electron micrographs of alginate beads, after batch fermentation, revealed extensive mycelial growth inside and around the beads.     

Semicontinuous cultivation of immobilized Claviceps purpurea

Summary Mycelia of Claviceps purpurea CBS 164.59 were immobilized in 2%, 4%, and 8% calcium alginate. Alkaloid production by free cells declined after 60 days, while immobilized cells retained their activity for 200 days. The cumulative alkaloid production for all fermentation cycles using 8% calcium alginate immobilized mycelia was 25 times higher than that from free cells. The best yields of the ergopeptide ergometrine were reached with 4% gel immobilized mycelia, while higher gel concentrations caused a shift in the alkaloid biosynthesis towards high clavine alkaloid production.Beginning with the third cycle of reincubation the immobilized mycelia showed a marked tendency to fragmentize into vacuolated arthrosporoid-like structures and produced violet-black pigments so that the beads recalled sclerotial structures of parasitically living Claviceps.Dedicated to Prof. Dr. K. Esser to his 60th birthday     

Enhancement of penicillin acylase activity by cultivating immobilized Kluyvera citrophila

Summary Whole cells of Kluyvera citrophila were immobilized in polyacrylamide gel. The penicillin acylase activity of immobilized whole cells was 60%–70% of native cells. When the immobilized cells were continuously cultivated for 40 h in an aerated fermentor containing peptone medium and were treated with alkali in order to remove -lactamase activity, the immobilized cells produced ampicillin up to 4.4 times faster than noncultivated cells.Ampicillin production was investigated in a column system using these cultivated immobilized whole cells. The cultivated immobilized cells showed excellent performance in continuous ampicillin production.     

Improved Properties of Bacillus coagulans β‐Galactosidase through Immobilization

Thermostable β‐galactosidase from Bacillus coagulans RCS3 was purified by successive column chromatography using DEAE‐cellulose and Sephadex G‐50. Immobilization of the purified enzyme was studied with DEAE‐cellulose and calcium alginate. The efficiency of β‐galactosidase retention was 87 % with DEAE‐cellulose (17 mg protein/mL of matrix) and 80 % with calcium alginate (2.2 mg protein/g bead). Comparative studies of immobilization displayed a shift in the optimum temperature from 65 °C to 70 °C provoked by DEAE‐cellulose, although no effect was observed with calcium alginate. The heat inactivation curve revealed an improvement in the stability (t_1/2 of 14.5 h for the immobilized enzyme as compared to 2 h for the free enzyme at 65 °C) in a calcium alginate system. This immobilized enzyme has a wide pH stability range (6.5–11). β‐Galactosidase immobilized by DEAE‐cellulose and calcium alginate allowed a 57 and 70 % lactose hydrolysis, respectively, to be achieved within 48 h after repeated use for twenty times.     

Application of enzyme flow microcalorimetry to the study of microkinetic properties of immobilized biocatalyst

Summary Flow microcalorimeter was used for the study of microkinetic properties of Escherichia coli cells enriched with the penicillin G acylase activity immobilized in calcium pectate gel. The experimental kinetic data were obtained by measurement of the thermometric signal in the microcalorimetric column with immobilized enzyme and described by the introduced mathematical model involving the mass transfer and reaction kinetic phenomena.     

Potential application of immobilized streptokinase extracted from Streptococcus equinus VIT_VB2

Streptokinase purified from Streptococcus equinus VIT_VB2 isolated from bovine milk sample was immobilized in various solid supports namely entrapment in agarose gel, calcium alginate beads and gelatin gel by cross-linking with formaldehyde. Immobilization of streptokinase in calcium alginate beads showed maximum efficiency (81.8?±?1.06%) when compared with entrapment with agarose gel (55.6?±?2.17%) and cross-linked gelatin formaldehyde gel (71.0?±?1.54%). The purified SK activity was expressed maximum in calcium alginate (1%) and gelatin gel (0.25%) with 1292.68?±?1.33 and 1121.9?±?1.2?U?mL^?1, respectively. Similarly, SK entrapped in gelatin gel and calcium alginate showed maximum in vitro blood clot lysis activity with 77.67?±?2.64% and 76.16?±?2.72%, respectively. The immobilized SK in gelatin gel showed complete clot lysis within 15?min; hence, this application of the study could be used in the treatment of superficial thrombophlebitis, phlebitis, and venous thrombosis. These beads were used for three repeated cycles to check the conversion of substrates into their products, and we concluded that SK can be immobilized in the suitable matrices. Therefore, this helps in the drug-delivery strategies in highly efficient way, moreover, economically competent process in the pharmaceutics.     

Protease production by immobilized growing cells of Serratia marcescens and Myxococcus xanthus in calcium alginate gel beads

Summary Serratia marcescens and Myxococcus xanthus cells were immobilized in calcium alginate gel beads. Immobilization under various conditions had no effect on the extracellular proteolytic activity of S. marcescens cells. Protease production seemed rather to depend on the free cells in the medium. However, the stability over time of enzyme production was enhanced, as immobilization increased protease production half-life from 5 to 12 days. On the other hand, Myxococcus xanthus produced proteases inside the gel beads which could diffuse into the medium. The proteolytic activity increased as a function of the initial cell content of the beads and of the bead inoculum. Compared to free cells, immobilized cells of Myxococcus xanthus could produce 8 times more proteolytic activity, with a very low free-cell concentration in the medium.     

L-Lactic acid production from raw cassava starch in a circulating loop bioreactor with cells immobilized in loofa (Luffa cylindrica)

l-Lactic acid was produced from raw cassava starch, by simultaneous enzyme production, starch saccharification and fermentation in a circulating loop bioreactor with Aspergillus awamori and Lactococcus lactis spp. lactis immobilized in loofa sponge. A. awamori was immobilized directly in cylindrical loofa sponge while the L. lactis was immobilized in a loofa sponge alginate gel cube. In the loofa sponge alginate gel cube, the sponge serves as skeletal support for the gel with the cells. The alginate gel formed a hard outer layer covering the soft porous gel inside. By controlling the rate and frequency of broth circulation between the riser and downcomer columns, the riser could be maintained under aerobic condition while the downcomer was under anaerobic condition. Repeated fed-batch l-lactic acid production was performed for more than 400 h and the average lactic acid yield and productivity from raw cassava starch were 0.76 g lactic acid g^–1 starch and 1.6 g lactic acid l^–1 h^–1, respectively.     

Continuous production of L-arginine using immobilized growing Serratia marcescens cells: Effectiveness of supply of oxygen gas

Summary The immobilized growing cell system using Serratia marcescens was applied to continuous L-arginine production. From the determination of oxygen uptake rate, it was shown that the cells entrapped in carrageenan gel were in an oxygen-limited state due to the diffusion barrier to oxygen transport created by the gel layer. This limited state in gel was relieved by supply of oxygen-enriched gas instead of air into the medium. The maximum population of immobilized cells increased to five times that of free cells with the supply of pure oxygen gas. The L-arginine-producing activity of the immobilized growing cells was proportional to the concentration of oxygen gas supplied and was 6 mg/h per millilitre in gel supplied with pure oxyges gas. The continuous L-arginine containing production was constantly maintained by controlling the medium penicillin G at pH 6.5 and more than 10 mg/ml of L-arginine were obtained at 10h of residence time for at least 12 days.     

Production of oxytetracycline by immobilized Streptomyces rimosus cells in calcium alginate gels

Streptomyces rimosus Pfizer 18234–2 cells were immobilized in calcium alginate and used for the production of oxytetracycline. The influence of the incubation period, alginate concentration and storage in CaCl₂ were investigated. From the results of the repeated batch fermentations of the shake flasks, a good level of antibiotic was maintained for a period of about 28 days using 4% calcium alginate. The cell leakage and cell concentration inside the beads were affected by the alginate concentration and storage in CaCl₂ solution.     

Continuous production of gibberellic acid in a fixed-bed reactor by immobilized mycelia of Gibberella fujikuroi in calcium alginate beads

Summary The continuous production of gibberellic acid with immobilized mycelia of Gibberella fujikuroi was maintained over a hundred days in a tubular fixed-bed reactor. Free mycelium at the beginning of the storage phase was harvested from G. fujikuroi shake-flask culture and was immobilized by ionotropic gelation in calcium alginate beads.The continuous recycle production system consisted of a fixed-bed reactor, a container in which the culture medium was heated, stirred and aerated, and valves for sample withdrawal or reactant addition during the first 1320 h (55 days). A two-phase continuous extractor was then added for the last 960 hours (40 days). Free and immobilized mycelium shake-flask cultures with the same strain used in the continuous culture system were also realized to compare growth, maintenance and production parameters. The results show about the same gibberellic acid productivity in both free and immobilized mycelium shakeflask cultures: 0.384 and 0.408 mgGA₃·gBiomass^-1 ·day^-1, respectively, whereas in the continuous system the gibberellic acid production is about twice as large for a similar biomass: 0.768 mgGA₃·gBiomass^-1·day^-1. Several factors affecting the overall productivity of the immobilized systems were found to be: the quality and the quantity of mycelia in the biocatalyst beads and the immobilization conditions.     

Continuous production of l-serine by immobilized growing Corynebacterium glycinophilum cells

Summary Auxotrophic mutant cells of Corynebacterium glycinophilum with high l-serine production activity were immobilized by entrapment with various gel materials, such as synthetic prepolymers and natural polysaccharides. The entrapped cells were used for estimation of l-serine productivity in a medium supplemented with glycine as a precursor. Based on the above criteria, including cell growth in gels and cell leakage from gels, calcium alginate was the most suitable gel material. Continuous l-serine fermentation with calcium alginate-entrapped growing cells was successfully achieved in an air-bubbled reactor for at least 13 days.     

Kinetics of ethanol production by immobilized Kluyveromyces marxianus cells at varying sugar concentrations of Jerusalem artichoke juice

Summary Kinetics of ethanol fermentation at varying sugar concentrations of Jerusalem artichoke tuber extract has been studied using Kluyveromyces marxianus cells immobilized in calcium alginate gel beads. A maximum ethanol concentration of 111 g/l was achieved at an initial sugar concentration of 260 g/l in 20 hours, when the immobilized cell concentration in the calcium alginate beads was 53.3 g dry wt./l bead volume. Ethanol yield remained almost unaffected by initial sugar concentration up to 250 g/l and was found to be about 88% of the theoretical. Maximum rate of ethanol production decreased from 22.5 g ethanol/l/h to 10.5 g ethanol/l/h while the maximum rate of total sugars utilization decreased from 74.9 g sugars/l/h to 28.5 g sugars/l/h as the initial substrate concentration was increased from 100 to 300 g/l. The concentration of free cells in the fermentation broth was low.     

The production of ethanol by immobilized yeast cells

Saccharomyces cerevisiae cells were immobilized in calcium alginate beads for use in the continuous production of ethanol. Yeasts were grown in medium supplemented with ethanol to selectively screen for a culture which showed the greatest tolerance to ethanol inhibition. Yeast beads were produced from a yeast slurry containing 1.5% alginate (w/v) which was added as drops to 0.05M CaCl₂ solution. To determine their optimum fermentation parameters, ethanol production using glucose as a substrate was monitored in batch systems at varying physiological conditions (temperature, pH, ethanol concentration), cell densities, and gel concentration. The data obtained were compared to optimum free cell ethanol fermentation parameters. The immobilized yeast cells examined in a packed-bed reactor system operated under optimized parameters derived from batch-immobilized yeast cell experiments. Ethanol production rates, as well as residual sugar concentration were monitored at different feedstock flow rates.     

Growth performances ofLactobacillus hilgardii immobilized in dextran gel and in continuous fermentation

A variant ofLactobacillus hilgardii was immobilized by its own production of dextran gel, forming grains. The best rate of weight increase of the gel in continuous fermentation was 16.3±3.3%/h, at pH 4.8±0.1 and with a dilution rate of 0.22 to 0.26/h. Observation by scanning electron microscopy located most of the bacteria as microcolonies on the surface. A similar arrangement appeared in calcium alginate beads. The best population density (10¹⁰ cells/g) was obtained in grains at pH 5.8, after 30h. At a similar pH value, 4.8, the growth rate was higher in alginate beads than in dextran gel but the final population density was approximately the same. Acidification rate increased faster with mixed gel at pH 5.2 than with dextran at pH 5.8.     

l-Glutamate production by protoplasts immobilized in carrageenan gel

Protoplastization of Brevibacterium flavum cultured in a medium containing 50 μg l⁻¹ and 5 units penicillin per ml was performed by lysozyme treatment. The protoplasts were immobilized in various polymer matrices, such as agar, polyacrylamide, calcium alginate, and κ-carrageenan and then used for l-glutamate production from glucose and urea in a batch system. The protoplasts immobilized in κ-carrageenan gels showed the highest productivity of l-glutamate being twice that of immobilized whole cells under optimum conditions. The maximum productivity reached 2.3 mg ml⁻¹ initially. The immobilized B. flavum protoplasts could be used 8 times (192 h) for l-glutamate production retaining about 22% of the initial productivity during the last reaction.