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.     

Production of tylosin and nikkomycin by immobilized Streptomyces cells

Summary Mycelia of Streptomyces sp. T 59-235 and Streptomyces tendae Tü 901 (producing the antibiotics tylosin and nikkomycin, resp.) were immobilized in different carriers. With both organisms best antibiotic production was observed in calcium alginate gel.Influence of aeration, cell density and flow rate on antibiotic production was investigated in batch fermentation and in a continuous system (air-bubbled reactor).In batch fermentation, immobilization prolongued the production phase from 72 to 120 h (Streptomyces T 59-235) and from 72 to 96 h (S. tendae). The relative productivity of immobilized cells was 40 to 50% compared to that of free mycelia in both cases.In continuous tylosin fermentation highest production rate was observed in a medium nearly saturated with oxygen.Nikkomycin production by immobilized S. tendae could be maintained for longer than 350 h in a continuous system. The production rate depended on cell density and flow rate of the medium. The maximum specific productivity was 100% compared to that of free mycelium in batch culture.     

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.     

Galactose oxidase production by immobilized cells ofDactylium dendroides

Summary Dactylium dendroides cells were immobilized with calcium alginate, calcium pectate and k-carrageenan. Alginate immobilized cells produced relatively small amounts of (D-galactose: O₂ oxidoreductase, EC 1.1.3.9, GOase). Pectate immobilized cells gave the best yield of GOase, which was comparable with that obtained with free cells, and productivity could be extended up to 28 days (7 cycles). Controlled dosage of phosphate to the medium markedly improved GOase production with higher yields per cycle than with free cells.     

Glucoamylase and α-amylase production by immobilized Aspergillus niger

Summary Aspergillus niger mycelia or spores were immobilized in calcium alginate gel beads and employed for production of glucoamylase and -amylase by repeated batch process. The immobilized mycelium produced lower enzyme activities than immobilized spores germinated in a growth medium and subsequently cultured in an enzyme production medium. In repeated batch experiments, free cells could be used for only 4 4-day batches, whereas with immobilized spores at least 11 4-day batches with a gradual increase in enzyme activities in each successive batch were possible. The activity ratio of glucoamylase and -amylase produced was altered by immobilization.     

A new method for immobilization of acetylcholinesterase

A new method for immobilization of acetylcholinesterase (AChE) to alginate gel beads by activating the carbonyl groups of alginate using carbodiimide coupling agent has been successfully developed. Maximum reaction rate (V _max) and Michaelis–Menten constant (K _m) were determined for the free and binary immobilized enzyme. The effects of pH, temperature, storage stability, reuse number and thermal stability on the free and immobilized AChE were also investigated. For the free and binary immobilized enzyme on the Ca–alginate gel beads, optimum pH values were found to be 7 and 8, respectively. Optimum temperatures for the free and immobilized enzyme were observed to be 30 and 35 °C, respectively. Upon 60 days of storage the preserved activity of free and immobilized enzyme were found as 4 and 68%, respectively. In addition, reuse number, and thermal stability of the free AChE were increased by as a result of binary immobilization.     

Transformation of chlorinated benzoates and other benzene derivatives by Aspergillus niger and Aspergillus japonicus

Summary Aspergillus niger and Aspergillus japonicus mycelia immobilized in calcium alginate could transform aromatic hydrocarbons at rates comparable to those of free mycelia. Conditions for the dehalogenation of chlorobenzoates by immobilized A. niger mycelia were investigated.     

Penicillin G production by immobilized fungal vesicles

Summary The activity of penicillin G production was compared in a well defined medium by native vesicles as well as by calcium alginate gel immobilized vesicles isolated from the protoplasts of Penicillium chrysogenum PQ-96. The activity yield of the immobilized vesicles was 44% in comparison with native vesicles. After 60 h of storage at 4° C the native vesicles showed a rapid decrease in penicillin G production. The storage stability of these vesicles was improved after entrapment inside the calcium alginate gel. After 240 h of storage 1 mg of immobilized vesicular protein catalyzed the production of about 140 nmoles of penicillin G in 1 h.     

Cellulase secretion by immobilized protoplasts of Trichoderma reesei

Protoplasts from Trichoderma reesei were immobilized in alginate and induced to produce cellulase (endoglucanase and β-glucosidase) enzymes. The specific activities of the synthesized enzymes were higher in immobilized protoplasts than in both free and immobilized mycelia. Immobilized protoplasts show an enhanced rate of exocellular β-glucosidase production compared to intact mycelia due to the lack of cell wall. The ratio of the exocellular/intracellular β-glucosidase was 5.9 for immobilized protoplasts and 0.32 for free mycelia.     

Production of alkaline protease with immobilized cells of bacillus subtilis PE-11 in various matrices by entrapment technique

The purpose of this investigation was to study the effect ofBacillus subtilis PE-11 cells immobilized in various matrices, such as calcium alginate, k-Carrageenan, ployacrylamide, agar-agar, and gelatin, for the production of alkaline protease. Calcium alginate was found to be an effective and suitable matrix for higher alkaline protease productivity compared to the other matrices studied. All the matrices were selected for repeated batch fermentation. The average specific volumetric productivity with calcium alginate was 15.11 U/mL/hour, which was 79.03% higher production over the conventional free-cell fermentation. Similarly, the specific volumetric productivity by repeated batch fermentation was 13.68 U/mL/hour with k-Carrageenan, 12.44 U/mL/hour with agar-agar, 11.71 U/mL/hour with polyacrylamide, and 10.32 U/mL/hour with gelatin. In the repeated batch fermentations of the shake flasks, an optimum level of enzyme was maintained for 9 days using calcium alginate immobilized cells. From the results, it is concluded that the immobilized cells ofB subtilis PE-11 in calcium alginate are more efficient for the production of alkaline protease with repeated batch fermentation. The alginate immobilized cells ofB subtilis PE-11 can be proposed as an effective biocatalyst for repeated usage for maximum production of alkaline protease. Published: October 21, 2005     

Production of lysine by using immobilized livingCorynebacterium sp cells

Summary Lysine production by immobilizedCorynebacterium sp cells in alginate gel beads was investigated in flasks. ImmobilizedCorynebacterium sp cells exhibited a slightly greater lysine production than free cells and accumulated 60 g/l of L-lysine at maximum, when cultured for 120h in a medium containing 200g/l glucose as carbon source. Several factors, such as inoculum size, incubation time and alginate gel concentration were examined in order to improve lysine production by immobilized growing cells.     

Comparison of ethanol tolerance of free and immobilizedSaccharomyces uvarum yeasts

O₂ consumption and CO₂ production of free and immobilizedSaccharomyces uvarum in the presence of ethanol were compared. The protective effect of immobilization on the yeast ethanol tolerance at 5–20% of ethanol was more evident in CO₂ production than in O₂ consumption. CO₂ production by the yeast immobilized in calcium alginate and calcium pectate gel beads was approximately 2.5-times higher than by the free yeast at 5 and 10% of ethanol. 4-Fold increase of CO₂ production was observed at 15% ethanol. Immobilization in calcium-containing carriers (alginate, pectate) resulted in enhanced activities of yeasts compared to the κ-carrageenan carrier.     

产糖化酶黑曲霉固定化方法比较的研究

采用海藻酸钙凝胶电埋法、以沸石、多孔聚酯等材料为固定化载体的吸附法固定黑曲霉（Aspergillus niger AS3.4309)菌丝细胞,以游离菌丝体作为对照,进行发酵产糖化酶的比较,结果表明：以聚酯泡沫作为固定化载体吸附固定化菌丝细胞产糖化酶活力最高。在产糖化酶的发酵过程中,与游离菌丝体细胞相比,固定化黑曲霉持续产酶时间有一定程度的延长。     

Production of nikkomycin by immobilized Streptomyces cells — Physiological properties

Summary The antibiotic nikkomycin can be produced by calcium alginate immobilized mycelium of Streptomyces tendae Tü 901 in batch and continuous culture.Scanning electron micrographs show the porous structure of the matrix and the distribution of the cells in the gel.Some physiological properties of free and immobilized mycelia were compared. Immobilization does not change the relative amounts of nikkomycin compounds in the culture broth. DNA and protein content were the same in free and immobilized cells. The specific activity of fructosediphosphate aldolase dropped during fermentation and was lower for entrapped than for free cells. The specific activity of mannitol dehydrogenase increased up to the end of the fermentation and was the same for free and immobilized mycelium.In continuous culture the relative amount of mannitol consumed decreased with increasing flow rate. When the medium was supplemented with amino acids mannitol consumption increased significantly.Dedicated to Prof. Dr. L. Acker on the occasion of his 70th birthday     

Production of ℓ-malic acid by Paecilomyces varioti

Summary Preliminary data for production of -malic acid from calcium acetate byPaecilomyces varioti is presented. Shake flask cultures with free cells and with cells immobilised in calcium alginate beads gave comparable results, acid concentrations of approximately 8 g/l being produced after 5 days from a medium containing 4% w/w of calcium acetate. A packed bed reactor, operated as an extended batch with product recycle, produced maximum acid concentrations of 32.6 g/l, equivalent to 73% of the maximum theoretical yield, after 3 days. Evidence obtained indicated that spores were more active than mycelia in the production of malic acid.     

Cultivation characteristics of immobilized Aspergillus oryzae for kojic acid production

Aspergillus oryzae in situ grown from spores entrapped in calcium alginate gel beads was used for the production of kojic acid. The immobilized cells in flask cultures produced kojic acid in a linear proportion while maintaining the stable metabolic activity for a prolonged production period. Kojic acid was accumulated up to a high concentration of 83 g/L, at which the kojic acid began to crystallize, and, thus, the culture had to be replaced with fresh media for the next batch culture. The overall productivities of two consecutive cultivations were higher than that of free mycelial fermentation. However, the production rate of kojic acid by the immobilized cells was suddenly decreased with the appearance of central cavernae inside the immobilized gel beads after 12 days of the third batch cultivation.     

Ethanol production at 45°C using preparations of Kluyveromyces marxianus IMB3 immobilized in calcium alginate and kissiris

The thermotolerant yeast, K. marxianus IMB3, was grown in free and immobilized states in batch-fed culture at 45°C and ethanol production was examined over a 61-day period. The organism was grown in the free state, in the free state with mineral kissiris, immobilized in calcium alginate and immobilized in calcium alginate together with kissiris. Initially, reactors were fed every two days with 10% (w/v) glucose-containing media and no significant difference in ethanol production was observed. In subsequent refeeding experiments, reactors were re-fed every two days with 15% (w/v) sucrose-containing media. Although overall ethanol concentrations decreased, production in the immobilized systems was higher. In the final stages fermentations were re-fed every 3 days and although overall ethanol production decreased further, production remained highest in the systems containing calcium alginate and kissiris.     

Low molecular weight dextran: Immobilization of cells of Leuconostoc mesenteroides KIBGE HA1 on calcium alginate beads

Dextran is a long chain polymer of d-glucose produced by different bacterial strains including Leuconostoc, Streptococcus and Acetobacter. The bacterial cells from Leuconostoc mesenteroides KIBGE HA1 were immobilized on calcium alginate for dextran production. It was observed that dextran production increases as the temperature increases and after reaching maxima (30 °C) production started to decline. It was also observed that at 50 °C free cells stopped producing dextran, while immobilized cells continued to produce dextran even after 60 °C and still not exhausted. It was found that when 10 g% substrate (sucrose) was used, maximum dextran production was observed. Immobilized cells produced dextran upto 12 days while free cells stopped producing dextran only after 03 days. Molecular mass distribution of dextran produced by immobilized cells is low as compared to free cells.     

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.