亚栖热菌透性化细胞的耦合固定化研究

将海藻酸盐凝胶包埋法与交联法和聚电解质静电自组装覆膜法相耦合,对含有海藻糖合酶活性的亚栖热菌的透性化细胞进行了固定化研究。结果表明,利用重氮树脂和聚苯乙烯磺酸钠对海藻酸凝胶微球交替覆膜,可以显著提高凝胶微球在磷酸盐缓冲液中的稳定性,以碳二亚胺对固定化细胞进行交联处理则可以提高固定化细胞中海藻糖合酶的热稳定性。透性化细胞经包埋－交联－覆膜耦合固定化后,酶活回收率为32％,最适酶反应pH值由6.5左右升至7.0左右,最适反应温度未变,仍为60℃。所得固定化细胞间歇反应时,催化麦芽糖转化为海藻糖的转化率可达60％,重复使用4次（每次50℃、反应24h）,酶活损失小于20％,转化率可保持在50％以上。     

Production of dextransucrase by Leuconostoc mesenteroides immobilized in calcium-alginate beads: I. Batch and fed-batch fermentations

In batch fermentation Leuconostoc mesenteroides immobilized in calcium alginate beads produced a total dextransucrase activity equal to about 93% of that by free, suspended bacterial cells under comparable conditions in a bubble column reactor. Continuous sucrose feeding (5 g/L h) to the immobilized-cell culture in the airlift bioreactor increased production of enzymatic activity by about 107% compared with ordinary batch operation of this reactor. About 14% of the enzymatic activity produced by the immobilized cells appears as soluble activity in the cell-free broth compared with about 40% in case of free cells. In an airlift bioreactor, both the soluble and the intact (sorbed and entrapped) enzymatic activity produced by the immobilized bacterial cells was about 34% greater under automatic pH control, compared to that produced in a bubble column reactor with only manual pH control. During formation of dextran by intact enzyme within cells and beads, declines are observed in apparent enzymatic activity.     

Optimization of lactic acid production by immobilized <Emphasis Type="Italic">Lactococcus lactis</Emphasis> IO-1

Production of lactic acid from glucose by immobilized cells of Lactococcus lactis IO-1 was investigated using cells that had been immobilized by either entrapment in beads of alginate or encapsulation in microcapsules of alginate membrane. The fermentation process was optimized in shake flasks using the Taguchi method and then further assessed in a production bioreactor. The bioreactor consisted of a packed bed of immobilized cells and its operation involved recycling of the broth through the bed. Both batch and continuous modes of operation of the reactor were investigated. Microencapsulation proved to be the better method of immobilization. For microencapsulated cells at immobilized cell concentration of 5.3 g l⁻¹, the optimal production medium had the following initial concentrations of nutrients (g l⁻¹): glucose 45, yeast extract 10, beef extract 10, peptone 7.5 and calcium chloride 10 at an initial pH of 6.85. Under these conditions, at 37 °C, the volumetric productivity of lactic acid in shake flasks was 1.8 g l⁻¹ h⁻¹. Use of a packed bed of encapsulated cells with recycle of the broth through the bed, increased the volumetric productivity to 4.5 g l⁻¹ h⁻¹. The packed bed could be used in repeated batch runs to produce lactic acid.     

Immobilization of laccase from Streptomyces psammoticus and its application in phenol removal using packed bed reactor

Laccase was produced from Streptomyces psammoticus under solid-state fermentation. The enzyme was partially purified by ammonium sulphate precipitation and was immobilized in alginate beads by entrapment method. Calcium alginate beads retained 42.5% laccase activity, while copper alginate beads proved a better support for laccase immobilization by retaining 61% of the activity. Phenol and colour removal from a phenol model solution was carried out using immobilized laccase. Batch experiments were performed using packed bed bioreactor, containing immobilized beads. Reusability of the immobilized matrix was studied for up to 8 successive runs, each run with duration of 6 h. The system removed 72% of the colour and 69.9% of total phenolics from the phenol model solution after the initial run. The immobilized system maintained 50% of its efficiency after eight successive runs. The degradation of phenolic compounds by immobilized laccase was evaluated and confirmed by Thin layer chromatography and nuclear magnetic resonance spectroscopy.     

Immobilized salt-tolerant yeasts: application of a new polyethylene-oxide support in a continuous stirred-tank reactor for flavour production

Immobilization of salt-tolerant yeasts considerably decreases the total time required for the flavour development in soy-sauce processes. For immobilization of cells, alginate gel is mostly used as support material. However, alginate is not very suitable for use in soy-sauce processes because alginate is sensitive to abrasion and chemically unstable towards the high salt content of the soy-sauce medium. In contrast, a newly developed polyethylene-oxide gel seems to be more suitable, but this gel has not been used so far for flavour production in a bioreactor with a high salt content. Therefore, this gel was applied with immobilized salt-tolerant yeasts in a continuous stirred-tank reactor, containing more than 12.5% (w/v) salt. In this reactor, the polyethylene-oxide gel particles did not show any abrasion for several days, while alginate gel beads were already destroyed within 1 day. In addition, the polyethylene-oxide gel particles with immobilized salt-tolerant yeasts Candida versatilis and Zygosaccharomyces rouxii showed a good flavour production. From this work, it was concluded that the application of polyethylene-oxide gel in long-term soy-sauce processes is attractive in the case the sticking together of polyethylene-oxide gel particles can be controlled.     

Enhancement and stabilization of the production of glucoamylase by immobilized cells of Aureobasidium pullulans in a fluidized-bed reactor

Summary Glucoamylase production by Aureobasidium pollulans A-124 was compared in free-living cells, cells immobilized in calcium alginate gel beads aerated on a rotary shaker (agitation rate 150 rpm), and immobilized cells aerated in an air bubble column reactor. Fermentation conditions in the bioreactor were established for bead concentration, substrate (starch) concentration, calcium chloride addition to the fermentation medium, and rate of aeration. Production of glucoamylase was optimized at approximately 1.5 units of enzyme activity/ml medium in the bioreactor under the following conditions: aeration rate, 2.0 vol air per working volume of the bioreactor (280 ml) per minute; gel bead concentration, 30% of the working volume; substrate (starch) concentration, at 0.3% (w/v); addition of calcium chloride to the medium at a final concentration of 0.01 M. Productivity levels were stabilized through the equivalent of ten batches of medium with the original inoculum of immobilized beads. Offprint requests to: M. Petruccioli     

Design of a biomedical reactor for plasma low-density lipoprotein removal

The purpose of this study was to design a biomedical reactor that reduces plasma cholesterol when incorporated in an in vivo extracorporeal system. Phospholipase A(2), immobilized onto Agarose beads and housed inside the bioreactor, modifies plasma low density lipoprotein (LDL) into a form that is rapidly removed from circulation. In a packed bed reactor, the enzymatic conversion of LDL to the modified form (with plasma taken from hypercholesterolemic New Zealand white rabbits) was relatively low, 25% +/- 6 for a single pass of plasma through the reactor. An extended bed reactor, a hybrid of fluidized and packed bed reactors, was then developed to increase the conversion. This reactor displays a single pass conversion of 60% +/- 5 under optimal flow conditions. An evaluation of the flow rate through the reactor indicates that the system is limited by external mass transfer when employed under in vivo conditions. In addition, this system requires blood separation before the enzyme modification, which complicates the circuit control. Therefore, a new system was designed for in vivo use with rabbits. The resulting design, called the plasma separator reactor (PSR), combines plasma separation and enzymatic conversion in a single chamber. The PSR has three advantages over other studied systems: improved external mass transfer conditions, easy controlability, and simple set-up procedures. Single pass conversion reached 52% +/- 12 in suboptimal flow under simulated in vivo conditions. This reactor was also tested in vivo with hypercholesterolemic New Zealand white rabbits. A continuous conversion of up to 80% +/- 6 of rabbit plasma phospholipids was observed during 90 min of blood circulation (5 mL/min). The decrease in total plasma cholesterol reached a level of 60% of the initial value and was observed to be a function of the bioreactor enzyme activity. (c) 1993 John Wiley & Sons, Inc.     

Gas phase acetaldehyde production in a continuous bioreactor

The gas phase continuous production of acetaldehyde was studied with particular emphasis on the development of biocatalyst (alcohol oxidase on solid phase support materials) for a fixed bed reactor. Based on the experimental results in a batch bioreactor, the biocatalysts were prepared by immobilization of alcohol oxidase on Amberlite IRA-400, packed into a column, and the continuous acetaldehyde production in the gas phase by alcohol oxidase was performed. The effects of the reaction temperature, flow rates of gaseous stream, and ethanol vapor concentration on the performance of the continuous bioreactor were investigated. (c) 1993 John Wiley & Sons, Inc.     

Hydantoinase activity of immobilized non-growing Pseudomonas putida cells

Summary Hydantoinase (dihydropyrimidinase E.C. 3.5.2.2) activity of Pseudomonas putida DSM 84 was evaluated using cells immobilized in alginate beads and in a microporous hollow fibre bioreactor. Conversion of dihydrouracil into N-carbamyl--alanine was most efficient with alginate-immobilized cells. A 40 to 45% conversion was obtained in shake flasks and in continuous mode with packed bed columns. The highest volumetric productivity was obtained with a packed bed column operated at a dilution rate of 0.5 h^-1 (99 g of product. 100 l^-1 per hour). After 96 h the alginate beads began to swell and break apart; no free cells were detected however. Despite some initial loss of cells from the microporous hollow fibre bioreactor, a steady state was later established and maintained for 400 h at dilution rates of 0.1 and 0.25 h^-1.     

Hybridoma perfusion systems: a comparison study

The efficiency of lgM production by hybridoma cells (1) cultured in suspension; (2) entrapped in alginate beads; or (3) packed in hollowfiber cartridge bioreactors, were compared in long-term perfusion cultures. The results showed that steady-state cell concentration and antibody production, per liter of perfused medium per day, were similar when cells were either entrapped in alginate beads of maintained in suspension. These values were also similar whether cells were maintained at high density in a hollowfiber cartridge bioreactro, or at low density in suspension. This work points out that cell behavior and antibody yield are comparable overall in the various perfusion systems currently used. However, a significant reduction of antibody production appeared whenever a part of the viable cells was lost in the filtrate. The reduction was due both to a decrease of viable cell yield and a decline of lgM productivity on a percell basis. This result is well in agreement with the previously presented model of "grow or die" cell cycle system of hybridoma, which proposes that the ratio of arrested to proliferating cells in perfusion cultures, should be increased in proportion to cell retention in the bioreactor, with a concomitant increase of lgM productivity.     

Extractive Synthesis of Ethyl‐Oleate Using Alginate Gel Co‐Entrapped Yeast Cells and Lipase Enzyme

To synthesize ethyl‐oleate ester, a complex Ca‐alginate gel co‐entrapped system was prepared. The gel beads contained two kinds of biocatalysts (living yeast cells and a lipase enzyme) and various amounts of glucose (100–400 g/L). These alginate beads dispersed directly in pure oleic acid. To follow the bioconversion of the cell growth, the glucose uptake of yeast cells, the concentration of ethanol inside the gel beads and the ethyl‐oleate concentration in oleic acid phase was monitored. The glucose was quantitatively taken up by yeast cells during 24–72 h, depending on the concentration of glucose. After this 24–72‐hour period, the glucose uptake was stopped. In accordance with changes in glucose concentration, the concentration of ethanol and ethyl‐oleate increased rapidly during the first day of fermentation and thereafter slowed down. It is supposed that the inhibitory effect of produced ethanol would be resolved by co‐immobilization of lipase in the same gel particles. Using lipase, one is able to transform ethanol to ethyl‐oleate, which is soluble in oleic acid. According to the data obtained a minimum of 4 U/mL lipase is required to increase ethyl‐oleate production significantly. Summing up it can be concluded that by means of this system a maximum yield of ethanol and ethyl‐oleate was achieved when gel beads containing 100 g/L glucose and 4 U/mL lipase enzyme were used.     

Novel method for cell immobilization and its application for production of oligosaccharides from sucrose

AIMS: The purpose of the present investigation was to develop a novel method for cell immobilization. METHODS AND RESULTS: Aureobasidium pullulans cells were mixed with an alginate solution, and the mixture was extruded to form small gel beads as hydrated-immobilized cells. The beads were then placed at -15 degrees C for 6-24 h to induce freeze-dehydration. The freeze-dehydration resulted in shrinkage of beads as a result of water removal reducing bead volume by 82% and bead weight by 85%. The dehydrated beads were successfully used for the production of fructo-oligosaccharides in a model reactor system. CONCLUSIONS: Dehydrated beads may provide some commercial advantages over conventional immobilized cells. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that bioreactor performance can be improved up to two times by the use of the dehydrated beads.     

Operation of packed-bed immobilized cell reactor featuring active β-galactosidase inclusion body-containing recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> cells

In this study, we developed a packed-bed immobilized cell reactor containing active β-gal (β-galactosidase) inclusion body (IB)-containing Escherichia coli (E. coli) cells in alginate beads. This packed-bed reactor was operated using a substrate feed solution 0.72 ∼ 38.4 mM ONPG (o-nitrophenyl-β-D-galactoside) prepared in Z buffer supplemented with chloroform and 0.1% SDS (sodium dodecyl sulfate). The production rate of ONP (o-nitrophenol) in the reactor containing cells that were incubated with α-MG (α-methyl D-glucospyranoside) or D-fucose after induction was superior to those prepared with cells that were not incubated with α-MG or D-fucose. The ONP production rate was increased proportionally with ONPG concentration in the substrate feed up to a concentration of 38.4 mM. However, as the ONPG concentration was increased in the substrate feed solution, galactose inhibition inside the alginate beads was increased. This most likely occurred due to problems with diffusion. In addition, partial breakage of alginate beads was observed during the later periods of operation. In this study, we demonstrated that active β-gal IB-containing E. coli cells were sustained in the immobilized cell reactor during operation. Particularly, these findings demonstrate the feasibility of using active IBs in an enzymatic reaction without the need for any purification step. In addition, we showed that these IB-containing cells could be directly used in an immobilized reactor.     

Performance of an external loop air-lift bioreactor for the production of monoclonal antibodies by immobilized hybridoma cells

Summary The performance of an external loop air-lift bioreactor was investigated by assessing the inter-relationships between various hydrodynamic properties and mass transfer. The feasibility of using this bioreactor for the production of monoclonal antibodies by mouse hybridoma cells immobilized in calcium alginate gel beads and alginate/poly-l-lysine microcapsules was also examined. When the superficial gas velocity, V _g , in the 300 ml reactor was varied from 2 to 36 cm/min, the average liquid velocity increased from 3 to 14 cm/sec, the gas hold-up rose from 0.2 to 3.0%, and the oxygen mass transfer coefficient, k _L a, increased from 2.5 to 18.1 h^-1. A minimum liquid velocity of 4 cm/s was required to maintain alginate gel beads (1000 m diameter, occupying 3% of reactor volume) in suspension. Batch culture of hybridoma cells immobilized in alginate beads followed logarithmic growth, reaching a concentration of 4×10⁷ cells/ml beads after 11 days. Significant antibody production did not occur until day 9 into the culture, reaching a value of 100 g/ml of medium at day 11. On the other hand, bioreactor studies with encapsulated hybridoma cells gave monoclonal antibody concentrations of up to 800 g/ml capsules (the antibody being retained within the semipermeable capsule) and maximum cell densities of 2×10⁸ cells/ml capsule at day 11. The volumetric productivities of the alginate gel immobilized cell system and the encapsulated cell system were 9 and 3 g antibody per ml of reactor volume per day, respectively. The main advantage of the bioreactor system is its simple design, since no mechanical input is required to vary the hydrodynamic properties.     

Performance of rotating packed disk reactor with immobilized glucose oxidase

Summary Reactor performance was studied to investigate whether a rotating packed disk reactor (RPDR) can be used for the enzymatic oxidation of biochemicals. The disks were packed with calcium alginate beads with immobilized glucose oxidase and catalase, which catalyze the reaction of glucose and oxygen. The production rate of gluconic acid increased with the speed of rotation and the bulk flow rate. An optimum submergence for maximum productivity existed.     

Dried alginate-entrapped enzymes (DALGEEs) and their application to the production of fructooligosaccharides

A modification of the classical calcium alginate enzyme entrapment technique is described aiming to overcome some of the limitations of the former gel-based biocatalysts. Dried alginate entrapped enzymes (DALGEEs) were obtained dehydrating calcium alginate gel beads containing entrapped enzymes. A fructosyltransferase from Aspergillus aculeatus, present in Pectinex Ultra SP-L, was entrapped using this technique. The resulting DALGEEs were successfully tested both operating batchwise and in a continuous fixed-bed reactor for fructooligosaccharides (FOS) synthesis from sucrose. Interestingly, DALGEEs did not re-swell upon incubation in concentrated (600 g/L) sucrose solutions, probably due to the lowered water activity (a_w) of such media. Confocal laser scanning microscopy of DALGEEs revealed that the enzyme molecules accumulated preferably in the shell of the particles. DALGEEs showed an approximately 30-fold higher volumetric activity (300 U/mL) compared with the calcium alginate gel beads. Moreover, a significant enhancement (40-fold) of the space-time-yield of fixed-bed bioreactors was observed when using DALGEEs as biocatalyst compared with gel beads (4030 g/day L of FOS vs. 103 g/day L). The operational stability of fixed-bed reactors packed with DALGEEs was extraordinary, providing a nearly constant FOS composition of the outlet during at least 700 h. It was also noticeable their resistance against microbial attack, even after long periods of storage at room temperature. The DALGEEs immobilisation strategy may also be useful for other biotransformations, in particular when they take place in low a_w media.     

A feasible enzymatic process for D-tagatose production by an immobilized thermostable L-arabinose isomerase in a packed-bed bioreactor

To develop a feasible enzymatic process for d-tagatose production, a thermostable l-arabinose isomerase, Gali152, was immobilized in alginate, and the galactose isomerization reaction conditions were optimized. The pH and temperature for the maximal galactose isomerization reaction were pH 8.0 and 65 degrees C in the immobilized enzyme system and pH 7.5 and 60 degrees C in the free enzyme system. The presence of manganese ion enhanced galactose isomerization to tagatose in both the free and immobilized enzyme systems. The immobilized enzyme was more stable than the free enzyme at the same pH and temperature. Under stable conditions of pH 8.0 and 60 degrees C, the immobilized enzyme produced 58 g/L of tagatose from 100 g/L galactose in 90 h by batch reaction, whereas the free enzyme produced 37 g/L tagatose due to its lower stability. A packed-bed bioreactor with immobilized Gali152 in alginate beads produced 50 g/L tagatose from 100 g/L galactose in 168 h, with a productivity of 13.3 (g of tagatose)/(L-reactor.h) in continuous mode. The bioreactor produced 230 g/L tagatose from 500 g/L galactose in continuous recycling mode, with a productivity of 9.6 g/(L.h) and a conversion yield of 46%.     

On-line monitoring of bioreductions via membrane introduction mass spectrometry

Real-time and on-line continuous monitoring of reactants, intermediates, and final products for dicarbonyl compound bioreduction in a continuous plug flow reactor packed with baker's yeast (Saccharomyces cerevisiae) whole cells immobilized on calcium alginate beads was performed by membrane introduction mass spectrometry (MIMS) via selective ion monitoring.     

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.     

Formulation and drying of alginate beads for controlled release and stabilization of invertase

Several alternatives to the conventional alginate beads formulation were studied for encapsulation of invertase. Pectin was added to the alginate/enzyme solution while trehalose and β-cyclodextrin were added to the calcium gelation media. The effect of composition changes, freezing, drying methods (freeze, vacuum, or air drying), and thermal treatment were evaluated on invertase stability and its release kinetics from beads. The enzyme release mechanism from wet beads depended on pH. The addition of trehalose, pectin, and β-cyclodextrin modified the bead structure, leading in some cases to a release mechanism that included the relaxation of the polymer chains, besides Fickian diffusion. Enzyme release from vacuum-dried beads was much faster than from freeze-dried beads, probably due to their higher pore size. The inclusion of β-cyclodextrin and especially of pectin prevented enzyme activity losses during bead generation, and trehalose addition was fundamental for achieving adequate invertase protection during freezing, drying, and thermal treatment. Present results showed that several alternatives such as drying method, composition, as well as pH of the relese medium can be managed to control enzyme release.