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.     

Kinetic and thermodynamic properties of purified alkaline protease from Bacillus pumilus Y7 and non‐covalent immobilization to poly(vinylimidazole)/clay hydrogel

The characterization of the hydrogel was performed using Fourier‐transform infrared spectroscopy, X‐ray diffraction, and scanning electron microscopy. Purified Bacillus pumilus Y7‐derived alkaline protease was immobilized in Poly (vinylimidazole)/clay (PVI/SEP) hydrogel with 95% yield of immobilization. Immobilization decreased the pH optimum from 9 to 6 for free and immobilized enzyme, respectively. Temperature optimum 3°C decreased for immobilized enzyme. The K_m, V_m, and k_cat of immobilized enzyme were 4.4, 1.7, and 7.5‐fold increased over its free counterpart. Immobilized protease retained about 65% residual activity for 16^th reuse. The immobilized protease endured its 35% residual activity in the material after six cycle's batch applications. The results of thermodynamic analysis for casein hydrolysis showed that the ΔG^≠ (activation free energy) and ΔG^≠_E‐T (activation free energy of transition state formation) obtained for the immobilized enzyme decreased in comparison to those obtained for the free enzyme. On the other hand, the value of ΔG^≠_ES (free energy of substrate binding) was observed to have increased. These results indicate an increase in the spontaneity of the biochemical reaction post immobilization. Enthalpy value of immobilized enzyme that was 2.2‐fold increased over the free enzyme indicated lower energy for the formation of the transition state, and increased ΔS^≠ value implied that the immobilized form of the enzyme was more ordered than its free form.     

Enzymatic preparation of fatty acid esters of sugar alcohols by condensation in acetone using a packed-bed reactor with immobilized Candida antarctica lipase

Mono- and dilauroyl arabitols, ribitols, xylitols and sorbitols were synthesized batchwise or continuously at 50°C or 60°C by condensation catalyzed by an immobilized Candida antarctica lipase in acetone. Continuous production was realized using a system where a column packed with sugar alcohol and a packed-bed reactor with the immobilized lipase were connected in series. The concentrations of the mono- and dilauroyl esters of each sugar alcohol became almost constant at mean residence times of 15 min or longer in the packed-bed reactor. The monolauroyl, monomyristoyl and monopalmytoyl arabitols, ribitols, xylitols and sorbitols were continuously produced using the reactor system at 60°C, and the productivity was in the range of 1.3–2.0 kg L^?1-reactor·day except for the fatty acid esters of sorbitol, the productivity of which was 0.6–0.8 kg L^?1-reactor·day.     

Immobilization of the serine protease from Thermomonospora fusca YX on porous glass

As much as 84% of the thermostable serine protease from Thermomonospora fusca strain YX was covalently attached to silanized glass using glutaraldehyde. The immobilized protease exhibited a higher temperature optimum (86 degrees C) and pH optimum (9.4) for activity compared to soluble YX-protease (80 degrees C and pH 9.0, respectively). Immobilization improved enzyme thermo-stability above 90 degrees C and reduced inactivation during prolonged storage (9% loss of activity after 90 days at 12 degrees C). A continuous-flow column reactor packed with immobilized protease readily hydrolyzed casein over broad ranges of temperature and pH.     

Continuous production of high degree casein hydrolysates by immobilized proteases in column reactor

The enzymatic complete hydrolysis of casein was investigated by using immobilized endopeptidase and exopepti dase packed in the jacketed column reactors. The mass transfer efficiency of proteins was improved by using sliced shrimp chitin hull as enzyme support, which formed a network structure inside the column reactor that prevented the formation of protein precipitate and increased the line flow rate of protein solution. The specificity of the protease was of crucial importance for both the hydrolysis degree and the free amino acid content of the hydrolysates. Of the enzymes tested, the immobilized A. oryzae protease was the most effective enzyme in breaking down the casein molecules and releasing the free amino acid from casein hydrolysates. The immobilized pancreatic and kidney exopeptidase could lead to a 20% increase of free amino acids. The free amino acid content of casein hydrolysates was 34.81% after processing and could reach to 64% if the column length was doubled, but 100% hydrolysis was impossible as the reverse reaction was also taking place. The casein hydrolysates was characterized by its high degree of hydrolysis and high content of free amino acids. It can be applied in infant formula, element diet, and as a protein ingredient for food industry.     

Denaturable immobilized enzymatic reaction in a packed-bed reactor with and without substrate inhibitions

Summary An immobilized enzymatic reaction in a packed-bed reactor is investigated in this paper. The thermal denaturation of immobilized enzyme caused by excessive reacting temperature rise is considered. An unsteady state dispersion model is employed to examine the dynamic behaviors of the substrate concentration, temperature and enzyme activity along the reactor. Also included in the present paper is the effect of substrate inhibition which occurs rather frequently in many enzymatic reactions. Comparison of results of the immobilized enzymatic reactions with and without substrate inhibitions are made to show the extent the substrate inhibition affects the enzymatic reaction. Furthermore, the effects of heat reaction and the Peclet number which characterize the reaction and flow behaviors, respectively, on the system considered are analyzed in detail.     

Palatinose production by free and Ca-alginate gel immobilized cells of Erwinia sp.

Palatinose is a non-cariogenic disaccharide obtained from the enzymatic conversion of sucrose, used in food industries as a sugar substitute. Free and Ca-alginate immobilized cells of Erwinia sp. D12 were used to produce palatinose from sucrose. Palatinose production was studied in a repeated-batch process using different immobilized biocatalysts: whole cells, disrupted cells and glucosyltransferase. Successive batches were treated with the immobilized biocatalyst, but a decrease in palatinose production was observed. A continuous process using a packed-bed reactor was investigated, and found to produce 55–66% of palatinose during 17 days using immobilized cells treated with glutaraldehyde and a substrate flow speed of 0.56 ml min⁻¹. However, immobilized cells in a packed-bed reactor failed to maintain the palatinose production for a prolonged period. The free cells showed a high conversion rate using batch fermentation, obtaining a palatinose yield of 77%. The cells remained viable for 16 cycles with high palatinose yields (65–77%). Free Erwinia sp. D12 cells supported high production levels in repeated-batch operations, and the results showed the potential for repeated reuse.     

A packed-bed reactor utilizing porous resin enables high density culture of hepatocytes

Summary To enable high density culture of hepatocytes for use as a hybrid artificial liver support system or a bioreactor system, a packed-bed reactor using collagen-coated reticulated polyvinyl formal (PVF) resin was applied to a primary culture of hepatocytes. Cubic PVF resins (2×2×2 mm, mean pore size: 100, 250 or 500 m) were used as supporting substrates to immobilize hepatocytes. Two hundred and fifty cubes were packed in a cylindrical column, and 2.6–11.3×10⁷ hepatocytes were seeded in the column by irrigating with 3 ml of the medium containing hepatocytes. Perfusion culture experiments using this packed-bed reactor, as well as monolayer cultures using conventional collagen-coated petri dishes as control experiments, were performed. Sufficient amounts of hepatocytes were found to be immobilized in the reticulated structure of the PVF resins. The highest density of immobilized hepatocytes attained with PVF resin was 1.2×10⁷ cells/cm³ PVF, which showed levels of ammonium removal and urea-N secretion comparable to those in the monolayer culture. It is concluded that the packed-bed reactor system utilizing PVF resin is a promising process for developing a bioreactor or a bioartificial organ using hepatocytes. Correspondence to: N. Ohshima     

Production of l-rhamnulose,a rare sugar,from l-rhamnose using commercial immobilized glucose isomerase

Abstract

A commercial immobilized d-glucose isomerase from Streptomyces murines (Sweetzyme) was used to produce l-rhamnulose from l-rhamnose in a packed-bed reactor. The optimal conditions for l-rhamnulose production from l-rhamnose were determined as pH 8.0, 60?°C, 300?g L^?1 l-rhamnose as a substrate, and 0.6?h^?1 dilution rate. The half-life of the immobilized enzyme at 60?°C was 809?h. Under the optimal conditions, the immobilized enzyme produced an average of 135?g L^?1 l-rhamnulose from 300?g L^?1 l-rhamnose after 16 days at pH 8.0, 60?°C, and 0.6?h^?1 dilution rate, with a productivity of 81?g/L/h and a conversion yield of 45% in a packed-bed reactor.     

Twin-core packed-bed reactors for organic-phase enzymatic esterification with water activity control

A method for the removal of water and the control of water activity, a _w, during enzymatic esterification is the use of salt hydrate pairs. When this technique is used on a laboratory scale, the recovery and reuse of the salt are not critical. Potential problems, such as the reactivity of some salts, can also be overcome simply by substituting another salt. However, if this technique is to be used on a larger scale, economic constraints would require salt recovery and restric the range of salts that could be used. In this article a twin-core packed-bed reactor — used for the esterification of an equimolar mixture of decanoic acid and dodecanol catalysed by lipase from Candida rugosa — which facilitates salt recovery and permits a _w control without direct contact between immobilized enzyme and salt, has been described. a _w control was maintained by using suitable salt hydrate mixtures in the inner core of the reactor. The substrate mixture was esterified by pumping it through the outer core of the reactor, which contained enzyme immobilized on a macroporous polypropylene support. Complete conversion, albeit at different rates, was obtained with a _w buffering at 0.48 and 0.8 by using hydrates of Na₄P₂O₇ and Na₂HPO₄.     

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.     

Purification and Some Properties of Crystalline Acid Protease from Acrocylindrium sp.

A crystalline acid protease produced by a strain of Acrocylindrium in a submerged culture was prepared by treatment with acetone (60%), salting out with ammonium sulfate (saturated) and, after chromatography on Duolite GS-101 column, dialysis against distilled water. This preparation was homogeneous on sedimentation analysis, starch-gel electrophoresis and gel filtration with Sephadex G-75. The optimum pH was 2.0 for milk casein digestion and the pH stability was for 2.0~5.0 at 30°C for one day. The crystalline enzyme was completely stable below 50°C, but lost the activity at 70°G in ten minutes. The acid protease was almost equal to pepsin on specific activity when milk casein solution (pH 2.0) was used as substrate.     

Biodegradation and detoxification of dyes and industrial effluents by Ganoderma weberianum B-18 immobilized in a lab-scale packed-bed bioreactor

White-rot fungi are considered to be promising biotechnological tools to complement or replace the current technologies for the treatment of effluents from textile production plants. The aim of this work was to investigate the decolorization capacity of Ganoderma weberianum B-18 in solid state fermentation with sugarcane bagasse as a substrate and ligninolytic inducer as well as to decolorize and detoxify industrial effluents by this strain in a laboratory scale packed-bed bio-reactor. The results demonstrated that G. weberianum B-18 indeed showed to possess decolorization capacity in solid state fermentation with sugarcane bagasse supplemented with synthetic dyes. Moreover, fungal biomass of G. weberianum B-18 immobilized in sugarcane bagasse in a packed-bed bioreactor was shown to efficiently decolorize and detoxify different dyes and authentic industrial effluents in semi-continuous conditions. In this decolorization process, laccase enzymes secreted by the fungus played the main role. Hence, a packed-bed reactor with G. weberianum B-18 immobilized in sugarcane bagasse seems to be a suitable system for the further development of an efficient bioprocess for large-scale treatment of dye-containing wastewaters.     

Some properties of a papain–agarose reactor provided with a pH monitor

A small reactor of immobilized papain was used to gain some knowledge about the effect of immobilization upon the reactivity of the enzyme towards one substrate and various types of inhibitors. A buffer solution containing benzoyl–arginine ethyl ester as substrate was run through a small column of papain immobilized by attachment to agarose beads. The pH of the effluent was measured continuously and provided the data used to calculate the substrate conversion during passage through the reactor. The operation of the system was checked by determining the substrate conversion as a function of flow rate. It proved to operate as theory demanded. The rate and extent of inhibition were measured after addition of various inhibitors to the buffer–substrate solution. The following quantities of immobilized papain were found to be equal within ±20% to those of the free enzyme in solution: the overall activity, the K_m of benzoyl–arginine ethyl ester, the K_i of the competitive inhibitor benzoylamino-acetonitrile, the rate of inactivation by chloroacetic acid and by chloroacetamide, the rate of activation by cysteine of the mixed disulfide of papain and cysteine, and the rate of spontaneous reactivation of the KCNO–papain adduct. The inactivation by KCNO proved to be strongly pH dependent. This may explain why the rate of the latter reaction is only 66% of the rate with free enzyme. It is concluded that the rates and equilibrium constants measured in the present reactor system are within ±20% of the values of the dissolved enzyme, provided that the reactions are not strongly pH dependent. Calculation showed there was no diffusion limitation.     

Evaluation of alginate-immobilized Lactobacillus casei for lactate production

Summary Lactate production by immobilized Lactobacillus casei has been studied. The cells were immobilized in alginate and the effect of variations in different parameters on product formation and productivity was investigated. The performance of the reaction was evaluated in stirred batch as well as in packed-bed conditions. pH control was a problem in the packed-bed reactor. In stirred batch experiments, nearly total glucose utilization was observed with a lactate yield of 90–99% and a total productivity of 1.6 g·l^–1·h^–1. Under standard conditions only a low percentage (3–4%) of the total lactate formed was the abetd-isomer. When immobilized cells were reused, increased formation of abetd-lactate took place, especially when the cell conditions were sub-optimal. After revitalization by exposure to growth nutrients the balance was restored.On leave from Microbiology Institute, Zhejiang Academy of Agricultural Sciences, Hangzhou-310021, China Offprint requests to: Bo Mattiasson     

Hydrolysis of lactose in whey permeate by immobilized β-galactosidase from Penicillium notatum

A new low-cost β-galactosidase (lactase) preparation for whey permeate saccharification was developed and characterized. A biocatalyst with a lactase activity of 10 U/mg, a low transgalactosylase activity and a protein content of 0.22 mg protein/mg was obtained from a fermenter culture of the fungus Penicillium notatum. Factors influencing the enzymatic hydrolysis of lactose, such as reaction time, pH, temperature and enzyme and substrate concentration were standardized to maximize sugar yield from whey permeate. Thus, a 98.1% conversion of 5% lactose in whey permeate to sweet (glucose-galactose) syrup was reached in 48 h using 650 β-galactosidase units/g hydrolyzed substrate. After the immobilization of the acid β-galactosidase from Penicillium notatum on silanized porous glass modified by glutaraldehyde binding, more than 90% of the activity was retained. The marked shifts in the pH value (from 4.0 to 5.0) and optimum temperatures (from 50°C to 60°C) of the solid-phase enzyme were observed and discussed. The immobilized preparation showed high catalytic activity and stability at wider pH and temperature ranges than those of the free enzyme, and under the best operating conditions (lactose, 5%; β-galactosidase, 610–650 U/g lactose; pH 5.0; temperature 55°C), a high efficiency of lactose saccharification (84–88%) in whey permeate was achieved when lactolysis was performed both in a batch process and in a recycling packed-bed bioreactor. It seems that the promising results obtained during the assays performed on a laboratory scale make this immobilizate a new and very viable preparation of β-galactosidase for application in the processing of whey and whey permeates.     

Biodiesel synthesis in a solvent-free system by recombinant Rhizopus oryzae: comparative study between a stirred tank and a packed-bed batch reactor

A simultaneous synthesis of biodiesel, as fatty acid methyl esters, and monoacylglycerols catalysed by the recombinant Rhizopus oryzae lipase immobilized by adsorption on Relizyme OD/403M is presented. The use of this 1(3)-positional specific lipase prevents the formation of glycerol as a by-product, thus avoiding its drawbacks. The synthesis was carried out in a solvent-free system and it has been studied in two different reactor systems: stirred tank and packed-bed reactor. Stirred tank reactor presented a high-initial reaction rate and achieved a 33.6% yield, which corresponds to a value of 50.4% of the maximum yield that can be achieved with a 1(3)-positional specific lipase. In packed-bed reactor there was a smaller initial reaction rate, but it was achieved a 49.1% yield, which corresponds to a 73.6% of the maximum yield. When a second batch is performed, the yield decreased only 4% when packed-bed reactor is employed whereas a drastic decrease is observed in a stirred tank operation. Therefore, packed-bed reactor showed a best performance and minor damage to the biocatalyst.     

β-Galactosidase from Talaromyces thermophilus immobilized on to Eupergit C for production of galacto-oligosaccharides during lactose hydrolysis in batch and packed-bed reactor

The β-galactosidase from Talaromyces thermophilus CBS 236.58 immobilized onto Eupergit C produced galacto-oligosaccharides (GalOS) in batchwise and continuous packed-bed mode of operation. A maximum yield of GalOS of 12, 39 and 80 g l⁻¹ was obtained for initial lactose concentrations of 50, 100 and 200 g l⁻¹, respectively, for batch conversion experiments. The immobilized enzyme could be re-used for several cycles for lactose hydrolysis and transformation. The maximum GalOS concentration of approximately 50 g l⁻¹ was obtained with the dilution rate of 0.375 h⁻¹ in a packed-bed reactor, when using an initial lactose concentration of 200 g l⁻¹. Continuous conversion of lactose in the packed-bed reactor resulted in the formation of relatively more trisaccharides than when employing the immobilized enzyme in discontinuous mode of operation.     

Comparative studies on immobilized laccase behaviour in packed-bed and batch reactors

Extracellular laccase produced by the wood-rotting fungus Cerrena unicolor was immobilized covalently via glutaraldehyde to cellulose-based carrier Granocel. Laccase was partially purified by membrane concentration and diafiltration followed by precipitation with acetone. Five-fold increase in the measured activity of immobilized enzyme was obtained when six times purer laccase was used for immobilization. For the best preparation, with very high activity of 2053 U per 1 mL of the carrier, thermal- and pH-stability, and activity profiles were determined. Experiments carried out in a batch reactor showed that k_cat/K_m for immobilized enzyme (0.65) is three times lower than the value obtained for the native laccase (2.19) whereas k_cat/K_m estimated from continuous reactor (1.50) is notably closer to that for the native enzyme. Continuous process probably reflects more precisely kinetics of the reaction accompanied by simultaneous product precipitation on the carrier’s surface. Operational stability of immobilized laccase was tested in continuous mode operation with ABTS, guaiacol and trichlorophenol as substrates and showed that packed-bed reactor is unprofitable system for laccase immobilized on Granocel carrier due to the high bed compaction. However, excellent stability of the preparation was noted under 20 successive runs in the well mixed tank reactor and better ability towards trichlorophenol biotransformation was observed in the case of immobilized laccase.     

Studies on the Proteolytic Action of Dairy Lactic Acid Bacteria

Intracellular protease (IPLB) of Streptococcus cremoris was extracted from the cells, which were cultivated in liquid media, by momentarily disrupting between two disks by high pressure.

The hydrolyzing modes of α_s-, crude k-, β-, and whole casein by IPLB of Str. cremoris or rennet were observed through the released amounts of tyrosine, sialic acid, NPN, and calcium insensitive substance. Relative specific turbidity of casein solution and dissymmetry coefficient of casein were measured. Particle weight and UV absorption spectrum of each high molecular hydrolyzate of whole casein were also determined.

Among four kinds of casein fractions, α_s- or crude k-casein was most easily hydrolyzed by IPLB of Str. cremoris or rennet. Relative specific turbidity of crude k-casein solution was remarkably, but those of α_s-, β-, and whole casein slightly increased by the action of IPLB of Str. cremoris or of rennet. Changes of dissymmetry coefficients were negligibly induced by these two enzymes. Absorption spectrum of IPLB-Str. cremoris-casein showed some conformational change.

It was recognized that intracellular protease (IPLB) of L. bulgaricus, L. helveticus or Str. lactis, all together, more easily hydrolyzed α_s-casein than crude k-, β-, and whole casein. By the actions of three IPLBs, relative specific turbidity of crude k-casein solution remarkably but those of α_s-, β-, and whole casein slightly increased, and dissymmetry coefficients of these casein fractions changed negligibly.

Particle weight of whole casein hydrolyzed by each IPLB for five days was larger than that of control casein. UV absorption of each whole casein hydrolyzed by a IPLB increased at the wave length range of 280~250 mμ.