Glucose isomerase activity in suspensions of <Emphasis Type="Italic">Arthrobacter nicotianae</Emphasis> cells and adsorption immobilization of the microorganisms on inorganic carriers

Kinetics of monosaccharide isomerization has been studied in suspensions of intact, non-growing Arthrobacter nicotianae cells. Under the conditions of the study, glucose and fructose were isomerized at the same maximum rate of 700 μmol/min per 1 g dried cells, which increased with temperature (the dependence was linear at 60–80°C). The proposed means of adsorption immobilization of A. nicotianae cells involve inorganic carriers differing in macrostructure, chemical nature, and surface characteristics. Biocatalysts obtained by adsorbing the cells of A. nicotianae on carbon-containing foamed ceramics in the coarse of submerged cultivation were relatively stable and retained original activity (catalysis of monosaccharide isomerization) throughout 14 h of use at 70°C. Maximum glucose isomerase activity (2 μmol/min per 1 g) was observed with biocatalysts prepared by adsorption of non-growing A. nicotianae cells to the macroporous carbon-mineral carrier Sapropel and subsequent drying of the cell suspension together with the carrier.     

Biochemical and molecular characterization of a novel high activity creatine amidinohydrolase from Arthrobacter nicotianae strain 02181

A high activity creatine amidinohydrolase (creatinase) from Arthrobacter nicotianae 02181 (a strain newly isolated from soil which may utilize creatinine as the unique organic source) was purified, characterized and the creatinase gene was cloned and analyzed in this study. Cells were cultivated under optimized condition for enzyme yield and creatinase was purified by the DEAE-cellulose and hydroxylapatite (HA) chromatography. The creatinase was found to be a dimmer formed by two identical subunit of 46.4 kDa, and the specific activity of the purified creatinase reached 124.44 U/mg protein, which was about 13 folds of the maximum value ever reported. The enzyme was found to be most active at 37 °C (pH 7.0), and it was found to be relatively stable bellow 45 °C around pH 7.0 by fluorescence spectroscopy and circular dichroism (CD) analysis. The activity of this creatinase could be significantly inhibited by Cu²⁺, Hg²⁺, Fe³⁺and SDS, and it could be improved by Ca²⁺ and NaN₃.The creatinase gene was cloned by the consensus-degenerate hybrid oligonucleotide primers (CODEHOP) PCR and the genome walking method. Nucleotide sequence analysis of this gene revealed an open reading frame (ORF) of 1254 base pair (bp) encoding a 417 amino acid (aa) protein. The primary amino acid sequence alignment search in the database revealed a moderate homology between the deduced amino acid sequence and other creatinase. The sequence has been submitted to Genbank with the accession number EU004199.     

Heterologous expression and characterization of Choline Oxidase from the soil bacterium Arthrobacter nicotianae

In the course of a microbial screening of soil samples for new oxidases, different enrichment strategies were carried out. With choline as the only carbon source, a microorganism was isolated and identified as Arthrobacter nicotianae. From this strain, a gene coding for a choline oxidase was isolated from chromosomal DNA. This gene named codA was cloned in Escherichia coli BL21-Gold and the protein (An_CodA) heterologously overexpressed as a soluble intracellular protein of 59.1 kDa. Basic biochemical characterization of purified protein revealed a pH optimum of 7.4 and activity over a broad temperature range (15–70 °C). Specific activities were determined toward choline chloride (4.70 ± 0.12 U/mg) and the synthetic analogs bis(2-hydroxyethyl)-dimethylammonium chloride (0.05 ± 0.45 × 10^–2 U/mg) and tris-(2-hydroxyethyl)-methylammonium methylsulfate (0.01 ± 0.12 × 10^–2 U/mg). With increasing number of oxidizable groups, a significant decrease in activity was noted. Determination of kinetic parameters in atmorspheric oxygen resulted in K _M = 1.51 ± 0.09 mM and V _max = 42.73 ± 0.42 mU/min for choline chloride and K _M = 4.77 ± 0.76 mM and V _max = 48.40 ± 2.88 mU/min for the reaction intermediate betaine aldehyde respectively. Nuclear magnetic resonance spectroscopic analysis of the products formed during the enzyme reaction with choline chloride showed that in vitro the intermediate betaine aldehyde exists also free in solution.     

Screening of microorganisms having high fumarase activity and their immobilization with carrageenan

Summary To develop an efficient method for continuous production of L-malic acid from fumaric acid using immobilized microbial cells, screening of microorganisms having high fumarase activity was carried out and cultural conditions of selected microorganisms were investigated. As a result of screening microorganisms belonging to the genera Brevibacterium, Proteus, Pseudomonas, and Sarcina were found to produce fumarase in high levels. Among these microorganisms Brevibacterium ammoniagenes, B. flavum, Proteus vulgaris, and Pseudomonas fluorescens were further selected for their high fumarase levels in the cultivation on several media. These 4 microorganisms were entrapped into a k-carrageenan gel lattice, and the resultant immobilized B. flavum showed the highest fumarase activity and operational stability.Cultural conditions for the fumarase formation and the operational stability of fumarase activity of immobilized B. flavum are detailed. Productivity for L-malic acid using immobilized B. flavum with k-carrageenan was 2.3 fold of that using immobilized B. ammoniagenes with polyacrylamide.Presented at the Annual Meeting of the Agricultural Chemical Society of Japan, Nagoya, April 3, 1978     

Hydrogen peroxide yields during the incompatible interaction of tobacco suspension cells inoculated with Phytophthora nicotianae

Rates of H(2)O(2) production by tobacco suspension cells inoculated with zoospores from compatible or incompatible races of the pathogen Phytophthora nicotianae were followed by direct measurement of oxygen evolution from culture supernatants following catalase addition. Rates of HO(2)(*)/O(2)(-) production were compared by following the formation of the formazan of sodium, 3'-[1-[phenylamino-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene-sulfonic acid hydrate. In the incompatible interaction only, both reactive oxygen species (ROS) were produced by the cultured host cells in a minor burst between 0 and 2 h and then in a major burst between 8 and 12 h after inoculation. Absolute levels of H(2)O(2) could not be accurately measured due to its metabolism by host cells, but results are consistent with the majority of H(2)O(2) being formed via dismutation of HO(2)(*)/O(2)(-). The effects of inhibitors of endogenous Cu/Zn superoxide dismutase (diethyldithiocarbamate) and catalase (3-amino-1,2,4-triazole and salicylic acid) were also examined. Yields of ROS in the presence of the inhibitors diphenylene iodonium, allopurinol, and salicylhydroxamic acid suggest that ROS were generated in incompatible host responses by more than one mechanism.     

Kinetics of the isomerization of D-glucose into D-fructose catalyzed by glucose isomerase containing Arthrobacter cells in immobilized and nonimmobilized form

The kinetic properties of immobilized and nonimmobilized glucose isomerase have been investigated. In both cases the kinetics can be described by a modified Michaelis–Menten expression. It appeared that immobilization causes no deactivation. Furthermore, it was shown that the permeability of the cell membrane increases by heat and toluene treatments.     

Influence of inorganic phosphate and immobilization on Claviceps purpurea

Summary The influence of inorganic phosphate and immobilization on cells of Claviceps purpurea strain 1029/N5 producing ergot peptides in shake culture was examined. Immobilization in Ca-alginate beads resulted in a marked reduction of some metabolic activities, i.e. the periods of alkaloid formation and cell growth were prolonged. High concentrations of inorganic phosphate (1 g/l KH₂PO₄) could reduce or stop alkaloid formation both by free and immobilized cells at any time during fermentation. The optimum phosphate concentration for alkaloid production by immobilized cells (about 0.5 mM) was a quarter of that required by free cells. This optimum shift was attributed to (i) the diminished phosphate demand of immobilized cells, due to their reduced metabolic activities, and (ii) the phosphate-dependent morphological behaviour of the biocatalyst. The observed decrease in alkaloid concentrations during later periods of the fermentation supported the idea of alkaloid-degradative enzymes, activated by high phosphate concentrations. Immobilization showed an advantageous influence on this undesirable effect. Offprint requests to: H.J. Rehm     

The kinetics of baculovirus adsorption to insect cells in suspension culture

The influence of various culture parameters on the attachment of a recombinant baculovirus to suspended insect cells was examined under normal culture conditions. These parameters included cell density, multiplicity of infection, and composition of the cell growth medium. It was found that the fractional rate of virus attachment was independent of the multiplicity of infection but dependent on the cell density. A first order mathematical model was used to simulate the adsorption kinetics and predict the efficiency of virus attachment under the various culture conditions. This calculated efficiency of virus attachment was observed to decrease at high cell densities, which was attributed to cell clumping. It was also observed that virus attachment was more efficient in Sf900II serum free medium than it was in IPL-41 serum-supplemented medium. This effect was attributed to the protein in serum which may coat the cells and so inhibit adsorption. A general discussion relating the observations made in-these experiments to the kinetics of recombinant baculovirus adsorption to suspended insect cells is presented.     

Conversion of corticosteroid hormones by immobilized cells of Flavobacterium dehydrogenans,Curvularia lunata and Arthrobacter simplex on ceramic carriers

Different possibilities for converting pregnenolone triacetate to prednisolone using immobilized preparations of Flavobacterium dehydrogenans, Curvularia lunata and Arthrobacter simpelex in a fixed-bed loop reactor were investigated. The effects of the carrier, substrate concentration, pH and temperature on the rate of the substrate conversion were studied also. The biotransformations were performed with a continuous or semicontinuous substrate supply. A convenient pathway for the formation of prednisolone is proposed on the basis of the results obtained. Received: 8 July 1996 / Accepted: 5 August 1996     

Purification, immobilization and characterization of linoleic acid isomerase on modified palygorskite

Linoleic acid isomerase from Lactobacillus delbrueckii subsp. bulgaricus 1.1480 was purified by DEAE ion-exchange chromatography and gel filtration chromatography. An overall 5.1% yield and purification of 93-fold were obtained. The molecular weight of the purified protein was ~41 kDa which was analyzed by SDS-PAGE. The purified enzyme was immobilized on palygorskite modified with 3-aminopropyltriethoxysilane. The immobilized enzyme showed an activity of 82 U/g. The optimal temperature and pH for the activity of the free enzyme were 30 °C and pH 6.5, respectively; whereas those for the immobilized enzyme were 35 °C and pH 7.0, respectively. The immobilized enzyme was more stable than the free enzyme at 30–60 °C, and the operational stability result showed that more than 85% of its initial activity was retained after incubation for 3 h. The K _m and V _max values of the immobilized enzyme were found to be 0.0619 mmol l⁻¹ and 0.147 mmol h⁻¹ mg⁻¹, respectively. The immobilized enzyme had high operational stability and retained high enzymatic activity after seven cycles of reuse at 37 °C.     

Effect of immobilization on kinetic and thermodynamic characteristics of sulfide oxidase from Arthrobacter species

In order to determine the impact of immobilization on biocatalytic efficacy of sulfide oxidase, the kinetic and thermodynamic properties of native and DEAE-cellulose immobilized sulfide oxidase from Arthrobacter species FR-3 were evaluated. Immobilization increased the catalytic efficiency of sulfide oxidase by producing a lower Michaelis-Menten constant (Km) and a higher rate of catalysis (Vmax) at different temperatures. The first-order kinetic analysis of thermal denaturation demonstrated that the values of enthalpy (delta H*d) and entropy (delta S*d) of immobilized sulfide oxidase were lower than the native enzyme, confirming the thermal stabilization of sulfide oxidase by immobilization. The delta H*d and delta S*d of the immobilized enzyme at 35 degrees C were 138.07 kJ/mol and 122.04 J/K/mol, respectively. These results suggest that immobilization made the sulfide oxidase from Arthrobacter sp. FR-3 thermodynamically more efficient for catalysis of sulfide oxidation.     

A simple method for the rapid and economical immobilization of glucose isomerase

The immobilization of glucose isomerase by adsorption on a macroreticular polystyrene sulphonate cation exchanger equilibrated with Ti⁴⁺, Zr⁴⁺, V⁵⁺ ions, followed by alkaline glutaraldehyde-induced crosslinking, is described. Experimental conditions are fixed for a selective and optimal retention of glucose isomerase and for its minimal leaching during subsequent use as a continuous compact glucose isomerase bed reactor, the performance of which is assessed on a laboratory scale for glucose isomerization. Factors influencing the glucose isomerase activity on solid supports, such as ratios of enzyme load - carrier - metal ion concentration, substrate feed concentration, residence period, loss of enzymic activity during storage and use, etc. are studied. The merits and drawbacks of the newly developed glucose isomerase reactor are discussed.     

Streptokinase immobilization in insoluble carriers, and properties of the immobilized protein

Immobilization of streptokinase was performed by bromine cyan-activated cellulose and by aminoethyl cellulose using glutaric aldehyde and N-cyclohexyl-N'-[2-(4-morpholinyl)-ethyl-carbodiimide. The specific activator activity of the immobilized streptokinase is 70-100% of that of free streptokinase. In multiple application of the immobilized protein preparations streptokinase obtained by bromine cyan-activated cellulose is the most stable: it retains more than 40% of initial activity after 10 repeated applications. The immobilized streptokinase is shown to be more thermostable as compared to the soluble one.     

The importance of the morphology and hydrophobicity of different carriers on the immobilization and sugar refinery effluent degradation activity of Phanerochaete chrysosporium

There was no direct correlation between the surface hydrophobicity or hydrophilicity of four solid carriers and the amount of immobilized Phanerochaete chrysosporium. The immobilized biomass was 1.5–1.8 times higher and the fungal degradation activity was 5–8 and 3 times greater in terms of decolorization and phenolics reduction, respectively, with porous carriers than with non-porous carriers. Morphology of the carriers was important and governed the amount of immobilized mycelium and specially the fungal biodegradation activity.     

Synthetic model polymers in the study of protein immobilization on glycidyl methacrylate carriers

The reactive polymer polysuccinimide was aminolysed with the spin label 2,2,6,6-tetramethyl 4-aminopiperidine-N-toxide to obtain on average about 9 unpaired spins/macromolecule. The remaining monomeric units were partially converted (80–99.5 mol%) to the 2-hydroxyethyl aspartamide residues, partly substituted with functional groups modelling side chains of natural proteins: carboxyl, primary alkylamine, imidazole, indole and 4-hydroxyphenyl groups. These model water-soluble polymers were reacted with the macroporous spherical carrier formed by glycidyl methacrylate-ethylene dimethacrylate copolymer and with epoxy-activated Sepharose. The amount of immobilized model compound was estimated by e.s.r. measurement. Reaction kinetics were studied at pH 3.85, 6.7, 8.15 and 9.55 at 4, 37 and 65°C. Flurescent amine was used to visualize the penetration of aqueous reactant solution into the beads. The binding yield of carboxyl-containing models was low at all pH's. Models with amine react easily and the binding yield at various pH values was clearly determined by dissociation. A rather different pH dependence was obtained with imidazole-containing models. The binding of 4-hydroxyphenyl and indole groups proceeded over the whole pH range, being highest on the acid side. Only a minor portion of binding groups accessible to low molecular weight reactants is occupied by the model polymers, even at saturation. The results suggest that with the synthetic carrier the kinetics of immobilization are dominated by diffusion of the reactant into the carrier particle followed by fast sorption on the internal surface which increases its local concentration in the vicinity of the oxirane reactive groups and which is responsible for the rapid onset of immobilization. The effect of sorption is almost absent with epoxy-activated Sepharose.     

Protamine immobilization and heparin adsorption on the protamine-bound cellulose fiber membrane

Immobilization of protamine to the inner lumen of cellulose hollow fibers has been shown useful in preventing both heparin- and protamine-induced complications during an extracorporeal blood circulation procedure. The current study examined the effects of variables on the immobilization of protamine to cyanogen bromide (CNBr)-activated cellulose hollow fibers. The degree of protamine immobilization was controlled by three independent parameters: the amount of CNBr used during the activation process, the duration of the coupling process, and the protamine concentration in the coupling solution. By the adjustment of these parameters, cellulose fibers containing desired amounts of immobilized protamine (ranging from 1 to 20 mg of immobilized protamine per gram of dry fibers) were readily prepared.Heparin adsorption to the protamine-bound cellulose fibers was also examined. The adsorption isotherm followed a Langmuir adsorption model. The amount of heparin adsorbed was dependent on both the heparin concentration in the substrate solution and the protamine loading on the fibers. The Langmuir adsorption constant K was estimated to be 0.37 +/- 0.06 mL/mg, whereas the saturation capacity Q(s) of the protamine-bound fibers increased with increasing the protamine loading.     

Change in the RNA polymerase activity in heart and liver cells during immobilization stress

Immobilization stress produces changes in the activity of polymerase I and II RNA in heart and liver cells. At the beginning of the poststressor reaction of the body the activity of both enzymes is lowered. Later on the activity of polymerase I RNA considerably exceeds the control level, whereas the activity of polymerase II RNA returns to the initial values.