Continuous production of NADP by immobilized Achromobacter aceris cells.

Several microorganisms having higher nicotinamide adenine dinucleotide kinase (NAD kinase, EC 2.7.1.23) activity were immobilized into polyacrylamide gel lattices. The enzyme activity field by immobilization was highest in Achromobacter aceris AKU 0120. By the incubation of the immobilized A. aceris cells at pH 4.0, the NAD kinase activity increased and the adenosine triphosphate (ATP)-degradation activity disappeared completely. Enzymatic properties of the immobilized A. aceris cells were investigated and compared with those of intact cells. The optimal pH and the optimal temperature of immobilized cells were the same as those of intact cells. Immobilized cell NAD kinase was more stable than that of intact cells. The operational half-life of immobilized cells was 20 days when the substrate solution was passed through a column packed with immobilized cells at a flow rate which gives a space velocity (SV) of 0.1 hr-1 at 37 degrees C. On the other hand, the half-life of the intact cells was only 6 hr.     

Physiological tests for yeast brewery cells immobilized on modified chamotte carrier

In this study yeast cell physiological activity was assessed on the basis of the in situ activity of two important enzymes, succinate dehydrogenase and pyruvate decarboxylase. FUN1 dye bioconversion and cellular ATP content were also taken as important indicators of yeast cell activity. The study was conducted on six brewing yeast strains, which were either free cells or immobilized on a chamotte carrier. The experimental data obtained indicate clearly that, in most cases, the immobilized cells showed lower enzyme activity than free cells from analogous cultures. Pyruvate decarboxylase activity in immobilized cells was higher than in planktonic cell populations only in the case of the Saccharomyces pastorianus 680 strain. However, in a comparative assessment of the fermentation process, conducted with the use of free and immobilized cells, much more favorable dynamics and carbon dioxide productivity were observed in immobilized cells, especially in the case of brewing lager yeast strains. This may explain the higher total cell density per volume unit of the fermented medium and the improved resistance of immobilized cells to environmental changes.     

具有谷氨酸脱羧酶的大肠杆菌固定化细胞

本文研究了用海藻酸钙包埋法制备含谷氨酸脱羧酶固定化细胞的方法以及研究了制备的条件和影响其制备的因素。该法具有包埋细胞活力回收高,方法简便等优点。比较研究了固定化细胞和自然细胞谷氨酸脱羧酶的一些生物化学性质。其中固定化细胞最适pH和pH稳定性增加,最适温度及热稳定性下降;表观米氏常数增大;二价金属离子Zn~(++)、Cu~(++)、Mg~(++)、Fe~(++),Sr~(++)程度不同的抑制酶活性,Ca~(++)激活固定化细胞酶活性,EDTA无抑制作用。对固定化细胞和自然细胞酶活力活化的研究中发现这两种细胞经蒸馏水保温处理后酶活性都上升,且自然细胞酶活的上升较固定化细胞大;而用底物溶液处理后,自然细胞无变化,固定化细胞酶活下降。     

Immobilization of Kluyveromyces marxianus cells containing inulinase activity in open pore gelatin matrix: 1. Preparation and enzymatic properties

Kluyveromyces marxianus cells with inulinase (2,1-β-d-fructan fructanohydrolase, EC 3.2.1.7) activity have been immobilized in open pore gelatin pellets with retention of > 90% of the original activity. The open pore gelatin pellets with entrapped yeast cells were obtained by selective leaching out of calcium alginate from the composite matrix, followed by crosslinking with glutaraldehyde. Enzymatic properties of the gelatin-entrapped cells were studied and compared with those of the free cells. The immobilization procedure did not alter the optimum pH of the enzymatic preparation; the optimum for both free and immobilized cells was pH 6.0. The optimum temperature of inulin hydrolysis was 10°C higher for immobilized cells. Activation energies for the reaction with the free and immobilized cells were calculated to be 6.35 and 2.26 kcal mol^?1, respectively. K_m values were 8 mM inulin for the free cells and 9.52 mM for the immobilized cells. The thermal stability of the enzyme was improved by immobilization. Free and immobilized cells showed fairly stable activities between pH 4 and 7, but free cell inulinase was more labile at pH values below 4 and above 7 compared to the immobilized form. There was no loss of enzyme activity of the immobilized cells on storage at 4°C for 30 days. Over the same period at room temperature only 6% of the original activity was lost.     

利用天然载体固定化活性干酵母细胞的研究

利用天然载体——蛋清固定化市售活性干酵母细胞,再经戊二醛进行共价交联而制备的具有高转化酶活力的新型粒状固定化生物催化剂,菌体包埋量大,活力回收高,机械性能好;特别是固定化生物催化剂经冷冻后,形成了均匀的多孔状颗粒,而酶活力基本不变,机械性能增强之特性．活性干酵母固定化后,其动力学特性表现为：K’m明显增大,热稳定性大大提高．于最适条件下,连续批次搅拌反应达两个月,凝胶颗粒无细胞渗漏,表现出相当稳定的酶活力．     

Production of acrylamide using immobilized cells of<Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> M33

The cellsof Rhodococcus rhodochrous M33, which produce a nitrile hydratase enzyme, were immobilized in acrylamide-based polymer gels. The optimum pH and temperature for the activity of nitrile hydratase in both the free and immobilized cells were 7.4 and 45°C, respectively, yet the optinum temperature for acrylamide production by the immobilized cells was 20°C. The nitrile hydratase of the immobilized cells was more stable with acrylamide than that of the free cells. Under optimal conditions, the final acrylamide concentration reached about 400 g/L with a conversion yield of almost 100% after 8 h of reaction when using 150 g/L of immobilized cells corresponding to a 1.91 g-dry cell weight/L. The enzyme activity of the immobilized cells rapidly decreased with repeated use. However, the quality of the acrylamide produced by the immobilized cells was much better than that produced by the free cells in terms of color, salt content, turbidity, and foam formation. The quality of the aqueous acrylamide solution obtained was found to be of commercial use without further purification.     

Comparison of free and immobilized Pichia pastoris cells for conversion of ethanol to acetaldehyde

Free and immobilized cells of Pichia pastoris were used to convert ethanol to acetaldehyde in small-scale batch reactors. Immobilized cells were less active than free cells (V(max) free = 7.81 g/L h, V(max) immobilized = 3.17 g/L h) due to a number of factors including end product inhibition and diffusional limitations. Immobilized cells were more resistant to heat denaturation both in the presence and absence of ethanol. Immobilized cells retained more of their activity during repeated batch cycles than did free cells.     

Kinetic study of 3,4-dihydroxyphenyl-L-alanine synthesis in Citrobacter freundii cells immobilized in carrageenan

A kinetic study was carried out of the enzymatic synthesis of 3,4-dihydroxyphenyl-L-alanine (DOPA) by the Citrobacter freundii 62 cells, possessing tyrosine-phenol-lyase (TPL) activity, immobilized in carrageenan, and optimum conditions of the reaction were found. The dependence of the TPL activity and its stability on the conditions of the DOPA synthesis was investigated. The TPL activity was higher and more stable in the immobilized cells as compared to free ones.     

The Effects of Immobilization on Photosynthesis,Growth, Nitrogen Fixation and Ammonium Ion Excretion of Mutant and Wild Type Cells of Anabaena variabilis

Ammonium ion photoproduced by nitrogen-fixing cyanobacteria is formed from water, air nitrogen and solar energy under normal temperature and atmospheric pressure. It is reported here that the mutant and its parent wiid type cells of Anabaena variabilis from Shanmugan lab were immobilized into the polyurethane foams. The growth curves measured by cell counting, O.D750 measurement and ch]orophyll a determination showed that the growth of the immobilized wild type cells was more rapid than the immobilized mutant cells, and for both two types of cells the free-living cells grew better than the immobilized ones. The nitrogenase activity (reduction of C2H2) was 44% greater in immobilized mutant cells and 232% greater in immobilized wild type cells than both free-living cells, respectively. Free-living cells of wild type A. variabilis almost did not excrete any ammonium ion and the immobilized cells of the wild type had the ammonium ion excretion activity similar to that of the free-living mutant cells. Moreover, immobilization stimulated the activity of ammonium ion excretion 55.6% greater than the free-living cells, in the mutant strain. The fluorescence enhancement induced by DCMU (3-(3,4-dichlor-benzene)-1, 1-dimethl urea) indicated the photosynthetic capability of wild type ceils in both free-living and immobilized states were higher than that of the mutant cells, and the free-living cells of both two types of cells had higher capability than the immobilized cells. The fluorescence emission spectra at 77K showed that there were four peak emission: F646(F645 or F650) and F662(F664 or F665) from phycobilin, F693(F698,F700 or F702) from PS Ⅱ and F732(F728 or F733 from PS Ⅰ In both two types of immobilized cells the photosynthetic light energy distribution tended to be in PS Ⅰ and it seems to be benefitlal for the nitrogenase activity and ammonium ion excretion. SEM observation indicated that the shape and size of the immobilized cells remained unchanged incompared with the free-living cells, However, the surface of the immobilized cells had accumulated some mucilage, and particularly, the film of mucilage coated both surface of the mutant cells and the foam matrix.     

Production of dextransucrase and dextran by Leuconostoc mesenteroides immobilized in calcium-alginate beads: II. Semicontinuous fed-batch fermentations

Cells of Leuconostoc mesenteroides immobilized in calcium alginate beads were used to produce dextransucrase (DS) in three sequential cycles of semicontinuous fed-batch fermentations. Each cycle consisted of a fed-batch DS production period of 24 h followed by a batch dextran production period for another 24 h. Free, suspended cells were used in only one cycle of fed-batch DS production followed by a dextran production period. It was impractically tedious to separate and reuse free cells. Increasing sucrose feed rate from 5 to 10 g/L h led to increases of the total enzymatic activity by about 88% with immobilized cells and by about 100% with free cells. In DS fed-batch semicontinuous fermentation, total enzymatic activity produced by immobilized cells was 1.35 and 1.56 times greater than that produced by free cells with respective sucrose feeding rates of 10 and 5 g/L h. These increases in enzyme productivity with immobilized cells, however, required total overall operating times three times longer (three cycles) than with free cells (one cycle). Growing the microorganism at optimum conditions for DS production also increased the dextran yield and shortened the time of conversion of sucrose to dextran, regardless of whether the cells were free or immobilized. Moreover, during three cycles of semicontinuous operation (144 h) immobilized cells produced more than three times as much dextran as free cells during one cycle (24 h).     

Trichloroethylene degradation by cells of a methane-utilizing bacterium,Methylocystis sp. M,immobilized in calcium alginate

When Methylocystis sp. M cells were immobilized in calcium alginate, the resulting cell beads showed optimum trichloroethylene (TCE) degradation activity at pH 7.0 and 35°C. In comparison with free cells, the immobilized cells were more stable at low pH, and to some extent, at higher temperatures. Studies on the kinetics and the influence of cell density suggest that oxygen permeation was a rate-limiting step. Investigation of the storage stability and the optimum concentration of dissolved oxygen revealed that the TCE degradability was greater under anaerobic than aerobic conditions. Although a toxic effect caused by TCE was observed, methane seemed to restore activity, suggesting that the development of a two-step reactor system might be advantageous. The finding that the immobilized cells showed TCE degradation activity in actual groundwater suggests that TCE bioremediation could be achieved through the use of bioreactors with such cells.     

Continuous production of L-tryptophan from indole and L-serine by immobilized Escherichia coli cells

Escherichia coli B 10, which has high activity of tryptophan synthetase, was grown in a 50-L batch culture in order to determine in which growth phase the cells have the highest specific tryptophan productivity. Accordingly, whole cells of the stationary phase were used for immobilization in polyacrylamide beads. After immobilization, these immobilized cells had 56% activity of tryptophan synthetase compared with that of free cells. First, the properties of immobilized cells were investigated. Next, discontinuous productions of L-tryptophan were carried out by using immobilized cells. In discontinuous production of L-tryptophan by the batch, the activity remaining of immobilized cells was 76-79% after 30 times batchwise use. In continuous production of L-tryptophan with a continuous stirred tank reactor (CSTR), the activity remaining of the immobilized cells was 80% after continuous use for 50 days. The maximum productivity of L-tryptophan in this CSTR system was 0.12 g tryptophan L(-1) h(-1).     

Effect of PEG-mediated pore forming on Ca-alginate immobilization of nitrilase-producing bacteria Pseudomonas putida XY4

Effect of PEG-mediated pore forming on Ca-alginate immobilization of nitrilase-producing bacteria Pseudomonas putida XY4 was studied. Through using PEG as porogen, the environmental tolerance as well as the biocatalytic reaction efficiency of immobilized cells was greatly improved, i.e., Ca-alginate-PEG immobilized cells got better temperature and substrate concentration tolerance than Ca-alginate immobilized cells and showed similar efficiency with free cells, suggesting that the intrinsic mass transfer resistance of immobilization obviously decreased. It was also observed that the pore diameter and porosity of immobilization beads were related with the molecular weight of PEG. PEG400 was found to be a relatively suitable porogen for Ca-alginate-PEG immobilized cells catalyzed hydrolysis of glycinonitrile. It was noteworthy that the Ca-alginate-PEG immobilized cells could be reused more than 18 times with little loss of enzyme activity which had shown good operation ability and great application potential.     

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.     

Isomaltulose production using immobilized cells

Three strains of Erwinia rhapontici especially suitable for use in the form of nongrowing immobilized cells were selected by screening strains of cells for high activity and operational stability in an immobilized form. Immobilization in calcium alginate gel pellets was easily the best method of immobilizing E. rhapontici. Much greater operational stabilities were obtained than when other immobilization methods were used. Conditions of operation which optimize the activity, stability, and yield and the ease of operation of the immobilized cell columns working in a steady state are described. These include the effects of substrate concentration, diffusional restrictions and water activity, the concentration of cells immobilized, and the type of reactor used. Thus, the immobilized cells produce about 1500 times their own weight of isomaltulose during one half-life of use (ca. 1 year). Loss of activity was most closely correlated with the volume of substrate processed and so presumably is due to the presence of low concentrations of a cummulative inhibitor in the substrate. Methods for regenerating the activity of the immobilized cells by the periodic administration of nutrients, of forming isomaltulose by continuously supplying nutrients to growing immobilized cells, and of crystallizing isomaltulose from the column eluate are also described.     

Synthesis and lysis of formate by immobilized cells of Escherichia coli

Formate hydrogenlyase (FHL) activity was induced in a strain of Escherichia coli S13 during anaerobic growth in yeast extract-tryptone medium containing 100 mM formate. The cells obtained at the optimum growth phase were immobilized in 2.5% (w/v) agar gel when 50-60% of the whole cell FHL activity was retained. The immobilized FHL system had good storage stability and recycling efficiency. In the lysis of formate, an increase of formate concentration to 1.18M increased QH(2) (initial) value of the immobilized cell, and subsequently cells, hydrogen evolution, in general, ceased after 6 to 8 of incubation, resulting in incomplete lysis of formate. Presence of small amount of glucose (28 mM) was more or less quantitatively lysed with concomitant disappearence of glucose from the medium. Synthesis of formate from hydrogen and bicarbonate solution by the immobilized cells was also characterized. Presence of glucose (10 mM) in 50 mM bicarbonate solution stimulated formate synthesis by immobilized cells. The pH optimum range, K(m), and specific activity of the immobilized cells for the lysis of formate were 6.8-7.2 0.4M, and 66 mL/g cell-h, respectively. The cells could fix hydrogen to the extent of 24.4% (w/w) of its own wet cell mass in a 72-h reaction cycle. Potentiality of the immobilized FHL system for biotechnological exploitation was discussed.     

Secondary metabolite biosynthesis in cultured cells of Catharanthus roseus (L.) G. Don immobilized by adhesion to glass fibres

Summary Suspension-cultured cells of Catharanthus roseus (L.) G. Don were immobilized on glass fibre mats and cultivated in shake flasks. The highly-aggregated immobilized cells exhibited a slower growth rate and accumulated reduced levels of tryptamine and indole alkaloids, represented by catharanthine and ajmalicine, in comparison to cells in suspension. The increased total protein synthesis in immobilized cells suggests a diversion of the primary metabolic flux toward protein biosynthetic pathways and away from other growth processes. In vitro assays for the specific activity of tryptophan decarboxylase (TDC) and tryptophan synthase (TS) suggest that the decreased accumulation of tryptamine in immobilized cells was due to reduced tryptophan biosynthesis. The specific activity of TDC was similar in immobilized and suspension-cultured cells. However, the expression of TS activity in immobilized cells was reduced to less than 25% of the maximum level in suspension-cultured cells. The reduced availability of a free tryptophan pool in immobilized cells is consistent with the reduced TS activity. Reduced tryptamine accumulation, however, was not responsible for the decreased accumulation of indole alkaloids in immobilized cells. Indole alkaloid accumulation increased to a similar level in immobilized and suspension-cultured cells only after the addition of exogenous secolaganin to the culture medium. The addition of tryptophan resulted in increased accumulation of tryptamine, but had no effect on indole alkaloid levels. Reduced biosynthesis of secologanin, the monoterpenoid precursor to indole alkaloids, in immobilized cells is suggested. Immobilization does not appear to alter the activity of indole alkaloid biosynthetic enzymes in our system beyond, and including, strictosidine synthase. Offprint requests to: P. J. Facchini     

Application of immobilized cells ofThermoanaerobium brockii for stereoselective reductions of oxo-acid esters

Summary Cells ofThermoanaerobium brockii were immobilized by entrapment methods as easy-to-handle biocatalyst for stereoselective reductions of oxo-acid esters. Different matrix materials were tested: agarose, k-carrageenan, alginate, polyacrylamide and polyurethanes. The two latter matrices allowed useful lifetimes of the immobilized biocatalysts of more than 2 months at thermophilic operation temperatures (around 65°C). Permeabilization of cells did not improve the catalytic activity. Immobilization of the cells did not enhance the thermostability. Only after a considerable period of operation could the immobilized biocatalysts be fed with medium lacking the complex substrates yeast extract and tryptone. Compared with freely suspended cells, reaction rates were lower. The immobilized system proved to be a relatively stable easy-to-handle biocatalyst, however, the freely suspended cells were superior with respect to flexibility of application and reaction velocity.     

Effect of environmental growth conditions on plasmid stability, plasmid copy number, and catechol 2,3-dioxygenase activity in free and immobilized Escherichia coli cells

In order to better understand the high plasmid stability in immobilized recombinant E. coli cells, the effects of dilution rate on the pTG201 plasmid stability, the copy number, and the catechol 2,3-dioxygenase (encoded by XyIE gene) production were, at first, studied in free E. coli W3101 continuous cultures in minimal media. It was found that decreasing specific growth rate increased the plasmid copy number and the catechol 2,3-dioxygenase activity but the stability decreased. In continuous culture with immobilized cells, an increase was shown in plasmid copy number and catechol 2,3-dioxygenase activity probably due to the distribution of growth in the gel beads. Besides mechanical properties of gel beads which may allow limited cell divisions, the increase in plasmid copy number is involved in enhanced plasmid stability in immobilized cells. In the same way, an experiment conducted in LB medium dealing with competition between pTG201-free and pTG201-containing E. coli B cells was described. It was shown that the competition was not more pronounced in gel bead compared to a free system. The effects of nutritional limitations on pTG201 plasmid stability and catechol 2,3-dioxygenase activity during chemostat cultivations in free and immobilized E. coli B cells were also investigated. It was found that immobilization of cells increased the stability of pTG201 even under glucose, nitrogen, or phosphate limited cultures. However in the case of magnesium depleted culture, pTG201 was shown to be relatively instable and a decrease in viable cell number during the immobilized continuous culture was observed. By contrast to the free system, the catechol 2,3-dioxygenase activity increased in immobilized cells under all culture conditions used.     

Hydrolysis of lactose by immobilized microorganisms.

Cells of Lactobacillus bulgaricus, Escherichia coli, and Kluyveromyces (Saccharomyces) lactis immobilized in polyacrylamide gel beads retained 27 to 61% of the beta-galactosidase activity of intact cells. Optimum temperature and pH and thermostability of these microbial beta-galactosidases were negligibly affected by the immobilization. Km values of beta-galactosidase in immobilized cells of L. bulgaricus, E. coli, and K. lactis toward lactose were 4.2, 5.4, and 30 mM, respectively. Neither inhibition nor activation of beta-galactosidase in immobilized L. bulgaricus and E. coli appeared in the presence of galactose, but remarkable inhibition by galactose was detected in the case of the enzyme of immobilized K. lactis. Glucose inhibited noncompetitively the activity of three species of immobilized microbial cells. These kinetic properties were almost the same as those of free beta-galactosidase extracted from individual microorganisms. The activity of immobilized K. lactis was fairly stable during repeated runs, but those of E. coli and L. bulgaricus decreased gradually. These immobilized microbial cells, when introduced into skim milk, demonstrated high activity for converting lactose to monosaccharides. The flavor of skim milk was hardly affected by treatment with these immobilized cells, although the degree of sweetness was raised considerably.