Immobilization of Arthrobacter simplex in thermally reversible hydrogels: effect of gel hydrophobicity on steroid conversion.

Arthrobacter simplex cells have been immobilized in a series of thermally reversible hydrogels having different gel hydrophobicities. Steroid conversion from hydrocortisone to prednisolone via the delta 1-dehydrogenase system was greatly affected by the relative hydrophobicities of the gel matrices, which were prepared by copolymerizing varying ratios of N-isopropylacrylamide to acrylamide. The characteristics of the immobilized cells, such as optimal temperatures, Km values, and the effects of an added artificial electron acceptor, were largely influenced by the gel matrices and their different lower critical solution temperatures (LCST). The data indicate that the microenvironment of the dehydrogenation system is quite different within the different hydrophilic/hydrophobic gel matrices. The high partitioning of water-insoluble steroids into the hydrophobic regions and the reduced possibility of product inhibition within the more hydrophobic gel matrices may cause the observed higher steroid conversion in these gels. A possible model for immobilized A. simplex cells in such different gel matrices is proposed.     

Application of Urethane Prepolymers to Immobilization of Biocatalysts: Δ1Dehydrogenation of Hydrocortisone by Arthrobacter simplex Cells Entrapped with Urethane Prepolymers

Acetone-dried cells of Arthrobacter simplex having appreciable steroid Δ¹-dehydrogenase activity were immobilized by mixing the cell suspension with water-miscible urethane prepolymers synthesized from toluene diisocyanate and polyether diols. The entrapped cell activity in the transformation of hydrocortisone to prednisolone was affected by the properties of urethane prepolymers, such as the isocyanate group content in prepolymers, the molecular weight of polyether diols and the ethylene oxide content in diols. The addition of 10% of organic solvents, such as methanol and glycols, to the aqueous reaction mixture enhanced the solubility of the substrate greatly and the reaction rate of the immobilized cells. The activity of immobilized cells remained high even in the system containing 30% of methanol, which drastically inhibited the activity of free cells. The presence of an electron acceptor, phenazine methosulfate or 2,6-dichlorophenolindophenol, significantly stimulated the steroid conversion with entrapped cells, as well as free cells. The stability of the cells over repeated reactions was greatly improved by immobilization.     

Purification and characterization of 3-ketosteroid-delta 1-dehydrogenase from Nocardia corallina

The inducible 3-ketosteroid-delta 1-dehydrogenase of Nocardia corallina which catalyzes the introduction of a double bond into the position of carbon 1 and 2 of ring A of 3-ketosteroid has been obtained in four steps with a 50% yield and 360-fold purification. The enzyme is homogeneous as judged by SDS-gel electrophoresis and is a monomeric protein with a molecular weight of 60,500. The isoelectric point of the enzyme is about 3.1. The enzyme contains 1 mol of flavin adenine dinucleotide per mol of protein, and has a typical flavoprotein absorption spectrum with maxima of 458, 362 and 268 nm. The enzyme is very stable in the absence of added cofactors, and catalyzes the dehydrogenation of delta 4-3-ketosteroids in the presence of phenazine methosulfate, which acts as an excellent electron acceptor. Potassium ferricyanide and cytochrome c did not act as electron acceptors. The delta 1-dehydrogenation was also stimulated by molecular oxygen with stoichiometric production of hydrogen peroxide and delta 1,4-3-ketosteroid. The optimum pH is 10 for dehydrogenation using phenazine methosulfate, and is between 8.5 and 10 for the oxidase reaction. The enzyme oxidizes a wide variety of 3-ketosteroids, but not 3 beta-hydroxysteroids. 3-Ketosteroids having an 11 alpha- or 11 beta-hydroxyl group were oxidized at slow rates. The purified enzyme catalyzes efficiently aromatization of the A-ring of 19-nortestosterone and 19-norandrostenedione to produce estradiol and estrone. 19-Hydroxytestosterone, 19-hydroxyandrostenedion and 19-oxotestosterone were converted to the respective phenolic steroids with cleavage of the C10 side-chain. Activities of 3-ketosteroid-delta 4-dehydrogenase, delta 5-3-ketosteroid-4,5-isomerase, 3 beta-hydroxysteroid dehydrogenase and 17 beta-hydroxysteroid dehydrogenase were not observed in the purified preparations. Properties of this novel flavoprotein enzyme are discussed.     

Maintenance and operational stability of immobilized Arthrobacter simplex for the ▵1-dehydrogenation of steroids

The stability of the ?¹-dehydrogenation system of Arthrobacter simplex immobilized in calcium alginate has been studied. A high stability was related to the ability of the cells to utilize a carbon source such as d-glucose or steroid. Inhibition of de novo protein synthesis reduced the ?¹-dehydrogenase [3-oxosteroid: acceptor) ?¹-oxidoreductase, EC 1.3.99.4] stability of the immobilized cells. The operational stability of immobilized cell preparations in the presence of the steroid degradation inhibitor, α,α-dipyridyl, could not be improved significantly by supplementing steroid substrate suspensions with either d-glucose or yeast extract.     

Conversion of disaccharides to 3-ketodisaccharides by nongrowing and immobilized cells ofAgrobacterium tumefaciens

High-performance liquid chromatography was used to estimate 3-ketolactose and 3-ketosucrose in cultures of agrobacteria. The activities of enzymes that convert the disaccharide substrate were evaluated during batch cultivation ofAgrobacterium tumefaciens on sucrose, maltose, and lactose. The highest activity of glucoside 3-dehydrogenase and a slight activity of disaccharide-hydrolyzing enzymes were found in cells grown on lactose. Nongrowing cells converted lactose to 3-ketolactose faster than immobilized cells did. Immobilization of cells into polysaccharide gels did not stabilize the activity of glucoside 3-dehydrogenase. Glutaraldehyde cross-linking of the cell content led to an inactivation of the respiratory chain but Fe³⁺ could be used as an electron acceptor. Cells treated with glutaraldehyde converted lactose faster than nongrowing ones but the activity of glucoside 3-dehydrogenase was not stable.     

Continuous rhamnolipid production using denitrifying Pseudomonas aeruginosa cells in hollow‐fiber bioreactor

Rhamnolipids are high‐value effective biosurfactants produced by Pseudomonas aeruginosa. Large‐scale production of rhamnolipids is still challenging especially under free‐cell aerobic conditions in which the highly foaming nature of the culture broth reduces the productivity of the process. Immobilized systems relying on oxygen as electron acceptor have been previously investigated but oxygen transfer limitation presents difficulties for continuous rhamnolipid production. A coupled system using immobilized cells and nitrate instead of oxygen as electron acceptor taking advantage of the ability of P. aeruginosa to perform nitrate respiration was evaluated. This denitrification‐based immobilized approach based on a hollow‐fiber setup eliminated the transfer limitation problems and was found suitable for continuous rhamnolipid production in a period longer than 1,500 h. It completely eliminated the foaming difficulties related to aerobic systems with a comparable specific productivity of 0.017 g/(g dry cells)‐h and allowed easy recovery of rhamnolipids from the cell‐free medium. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 346–351, 2013     

Interaction of heme and heme-hemopexin with an extracellular oxidant system used to measure cell growth-associated plasma membrane electron transport

Since redox active metals are often transported across membranes into cells in the reduced state, we have investigated whether exogenous ferri-heme or heme bound to hemopexin (HPX), which delivers heme to cells via receptor-mediated endocytosis, interact with a cell growth-associated plasma membrane electron transport (PMET) pathway. PMET reduces the cell-impermeable tetrazolium salt, WST-1, in the presence of the mandatory low potential intermediate electron acceptor, mPMS. In human promyelocytic (HL60) cells, protoheme (iron protoporphyrin IX; 2,4-vinyl), mesoheme (2,4-ethyl) and deuteroheme (2,4-H) inhibited reduction of WST-1/mPMS in a saturable manner supporting interaction with a finite number of high affinity acceptor sites (Kd 221 nM for naturally occurring protoheme). A requirement for the redox-active iron was shown using gallium-protoporphyrin IX (PPIX) and tin-PPIX. Heme-hemopexin, but not apo-hemopexin, also inhibited WST-1 reduction, and copper was required. Importantly, since neither heme nor heme-hemopexin replace mPMS as an intermediate electron acceptor and since inhibition of WST-1/mPMS reduction requires living cells, the experimental evidence supports the view that heme and heme-hemopexin interact with electrons from PMET. We therefore propose that heme and heme-hemopexin are natural substrates for this growth-associated electron transfer across the plasma membrane.     

Transfer of light-induced electron-spin polarization from the intermediary acceptor to the prereduced primary acceptor in the reaction center of photosynthetic bacteria.

In reaction centers and chromatophores of photosynthetic bacteria strong light-induced emissive ESR signals have been found, not only after a flash but also under continuous illumination. The signal, with g = 2.0048 and delta Hpp = 7.6 G, is only present under reducing conditions in material in which the primary acceptor, ubiquinone, U and its associated high-spin ferrous ion are magnetically uncoupled. its amplitude under continuous illumination is strongly dependent on light intensity and on microwave power. The emissive signal is attributed to the prereduced primary acceptor, U-, which becomes polarized through transfer of spin polarization by a magnetic exchange interaction with the photoreduced, spin polarized intermediary acceptor, I-. A kinetic model is presented which explains the observed dependence of emissivity on light intensity and microwave power. Applying this analysis to the light saturation data, a value of the exchange rate between I- and U- of 4.10(8) s-1 is derived, corresponding to an exchange interaction of 3--5 G.     

Ultrastructural demonstration of glucose-6-phosphate dehydrogenase activity in steroid-secreting cells.

The ultrastructural localization of glucose-6-phosphate dehydrogenase (NADP-linked) has been attempted in steroid-secreting cells. Rat adrenocortical cells and newt testicular glandular cells were fixed in an ice-cold mixture of 1% methanol-free formaldehyde and 0.25% glutaraldehyde. Potassium ferricyanide was used as the final electron acceptor. After incubation, the final copper ferrocyanide precipitate is exclusively observed in the hyaloplasm of these cells, provided that an electron carrier (1.0 mM PMS) has been added to the medium in order to by-pass the tissue "diaphorase" (NADPH-ferricyanide reductase) reaction. No precipitate appears in the absence of glucose-6-phosphate (substrate). Incubation in a medium devoid of PMS results in an exclusively mitochondrial reaction; the latter is that of the "diaphorase", which in these cells is mitochondrial. These results prove the importance of utilizing exogenous electron carriers (such as PMS) in coenzyme-linked dehydrogenase cytochemistry. Although polyvinyl alcohol was included in the washing and incubation media, in order to increase their viscosity, problems still exist concerning ultracytochemical localization of this "soluble" enzyme; these problems are discussed in the paper.     

The effect of water-miscible solvents on the Δ1-dehydrogenase activity of free and PAAH-entrapped Arthrobacter simplex

Summary The effect of water-miscible cosolvents on biotransformations of poorly water-soluble substrates by immobilized cells was investigated, using ¹-dehydrogenation of hydrocortisone by Arthrobacter simplex as a model. Criteria for solvent selection on the basis of retention of enzymic activity were postulated and tested. Diols were considered to be the most suitable group of solvents. Substrate solubility increased tenfold in 30% (v/v) ethylene glycol, but reaction rates were significantly slower in such solutions. This was mainly caused by a decrease of oxygen solubility in the presence of the cosolvent and conformational changes imposed on the intracellular enzyme by cosolvent molecules penetrating the cell. The inhibition could be eliminated by the addition of an artificial electron acceptor, phenazine methosulphate (PMS). Reaction rates faster than those for substrate suspensions (no cosolvent added) could thus be achieved. Immobilization of Arthrobacter simplex in cross-linked polyacrylamide hydrazide gave high retentions of activity. PMS exhibited toxic effects on the entrapped cells, leading to reduced activity after extended use.     

Isomaltose formation by free and immobilized dextransucrase

The acceptor reaction of dextransucrase from Leuconostoc mesenteroides NRRL-B512F with glucose as acceptor is of technical interest for isomaltooligosaccharide (IMOs) synthesis. Different experimental conditions were investigated for free and immobilized enzyme. The data for oligosaccharide formation up to a degree of polymerization 4 were correlated with a model developed earlier, and optimal reaction conditions for immobilized dextransucrase design and application were identified for later continuous application. Furthermore, stability was investigated for free and immobilized enzyme including stabilization by sugars.     

Interaction of oxidized and reduced N-methylphenazonium methosulfate (PMS) with Photosystem II

In 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) poisoned chloroplasts, the restoration of the fluorescence induction is presumed to be due to a back reaction of the reduced primary acceptor (Q⁻) and the oxidized primary donor (Z⁺) of Photosystem II. Carbonylcyanide m-chlorophenylhydrazone (CCCP) is known to inhibit this back reaction. The influence of reduced N-methylphenazonium methosulfate (PMS) in the absence of CCCP and of oxidized PMS in the presence of CCCP on the back reaction was investigated and the following results were obtained:

1. (1) Reduced PMS at the concentration of 1 μM inhibits the back reaction as effectively as hydroxylamine, suggesting an electron donating function of reduced PMS for System II.

2. (2) The inhibition of the back reaction by CCCP is regenerated to a high degree by oxidized PMS which led to assume a cyclic System II electron flow catalysed by PMS.

3. (3) At concentrations of reduced PMS higher than 1 μM it is shown that both the fast initial emission and more significantly the variable emission are quenched.

Amino acid transport in membrane vesicles of obligately anaerobic Veillonella alcalescens.

Membrane vesicles of Veillonella alcalescens, grown in the presence of L-lactate and KNO-3, actively transport amino acids under anaerobic conditions in the presence of several electron donors and the electron acceptor nitrate. The highest initial rates of uptake are obtained with L-lactate, followed by reduced nicotinamide adenine dinucleotide, glycerol-1-phosphate, formate, and L-malate.. The membrane vesicles contain the dehydrogenases for these electron donors, and these enzymes are coupled with nitrate reductase. In membrane vesicles from cells, grown in the presence of nitrate, the dehydrogenases are not coupled with fumarate reducatase, and anaerobic transport of amino acids does not occur with fumarate as electron acceptor. Under aerobic conditions none of the physiological electron donors can energize transport. However, a high rate of uptake is observed with the electron donor system ascorbate-phenazine metho-sulfate. This electron donor system also effectively energizes transport under anaerobicconditions in the presence of the electron acceptor nitrate.     

Continuous production of L-phenylalanine by Rhodotorula glutinis immobilized cells using a column reactor

Studies have been conducted on L-phenylalanine (L-Phe) production and phenylalanine ammonia lyase (PAL) stabilization in the presence of several optimum effectors and reducing agents under bioconversion of transcinnamic acid (t-CA) conditions during repeated batch operations. L-Phe production was maximized and reuseability of PAL catalyst was extended to eight consecutive cycles (repeated batches) in the presence of optimum effectors (glutamic acid, polyethylene glycol and glycerol), thioglycolic acid and sparging with nitrogen gas. These best optimum bioconversion conditions desensitize the PAL catalyst to substantially elevated higher substrate t-CA concentrations and inhibit inactivation of PAL enzyme over longer reaction periods compared to the control. The fed batch mode operation of bioconversion of total t-CA (300 mM) to L-Phe was superior (65.2%, conversion), comparing with conventional batch and repeated batch (58.4%, conversion) operations after 120 h. Gamma irradiation process was employed to polymerize and crosslink polyvinyl alcohol (PVA) with N,N'-methylene-bisacrylamide (BIS) agent. The use of immobilized PAL biocatalyst containing cells in PVA-BIS copolymer gel carrier produced by radiation polymerization is obviously advantageous with regards to the yield of L-Phe which was increased in average 1.2-fold when compare to those obtained with free cells during optimum bioconversion process. When comparing the magnitudes of gamma irradiation effects on immobilized entrapped yeast cells in PVA-BIS copolymer gel carrier using scanning electron microscopy it was show that yeast cells were protected and capable to overcome these conditions and had normal shape and other features as free (unirradiated) intact yeast cells. Optimum conditions for continuous production of L-Phe by PVA-BIS copolymer carrier entrapped yeast cells in a packed bed column reactor in recycle fed-batch mode were investigated. Under these optimum conditions L-Phe accumulated to concentration 240.1 mM represts a total conversion yield of 80% (w/w) from (300 mM) t-CA after 84 h of reaction process, which was higher than that obtained after 120 h of reaction, 65.2% (w/w) from (300 mM) t-CA with free cells in fed-batch mode. The results also demonstrated that during about 4 weeks of repeated continuous recycle fed batch mode experiments (using immobilized cells in packed bed reactor), the final production of L-Phe concentrations decreased gradually in eight consecutive runs with no sign of breakage or disintegration of the carrier gel beads.     

NADH production from NAD+ with a formate dehydrogenase system involving immobilized cells of a methylotrophic Arthrobacter strain.

A convenient and economical method of NADH production from NAD+ has been established using a formate dehydrogenase system involving immobilized cells of a methanol-utilizing bacterium. Arthrobacter sp, KM62. Four kinds of cell entrapment were studied. An immobilized cell preparation showing a high NADH production activity was obtained by entrapment in a kappa-carrageenan gel lattice. The NADH-producing activity of the immobilized cells was investigated under various conditions. The NADH-producing activity was evoked on the addition of Triton X-100 to the reaction mixture. The conditions for the continuous production of NADH with an immobilized cell column were also investigated. When a reaction mixture containing 10 mumol (6.63 mg) ml-1 NAD+ was passed through the column (1.2 x 20 cm) containing 1.62 g (as dry weight) of immobilized cells, at a space velocity of 0.125 at 35 degrees C, complete conversion was attained.     

The kinetic relationship between the C-550 absorbance change, the reduction of Q(delta A320) and the variable fluorescence yield change in chloroplasts at room temperature.

The light minus dark difference spectrum and the kinetics of the indicator pigment C-550 have been measured at room temperature in isolate, envelope-free chloroplasts in the presence of 3-(3' ,4'-dichlorophenyl)-1,1-dimethylurea (DCMU). The C-550 spectrum indicates a band shift with peaks at 540 and 550 nm and has an isobestic point at 545 nm. On the assumption of 400 chlorophyll molecules per electron transfer chain the differentaial extinction coefficient delta epsilon (540-550) is calculated to be approximately 5 mM-1 . CM-1. The kinetics of the C-550 absorbance change, occurring upin the onset of continuous illumination, are shown to be biphasic and strictly correlated with the kinetics of the complementary area measured from the fluorescence induction curve under identical cinditions and with those of the absorbance increase at 320 nm due to photoreduction of Q. The lighted-induced change in these three parameters can be described as a function of the variable fluorescence yield change occurring under the same conditions. Such functions are non-linear and reveal a heterogeneous dependence of the variable fluorescence yield on the fraction of closed System II reaction centers. It is concluded that for every molecule of the primary electron acceptor Q of Photosystem II that is photochemically reduced there corresponds an equivalent change in the absorbance of the indicator pigment C-550 and in the size of the complementary area. Ths, C-550 and area are two valid parameters for monitoring the primary photochemical activity of System II at the room temperature.     

Energy-dependent uptake of 4-chlorobenzoate in the coryneform bacterium NTB-1.

The uptake of 4-chlorobenzoate (4-CBA) in intact cells of the coryneform bacterium NTB-1 was investigated. Uptake and metabolism of 4-CBA were observed in cells grown in 4-CBA but not in glucose-grown cells. Under aerobic conditions, uptake of 4-CBA occurred with a high apparent affinity (apparent Kt, 1.7 microM) and a maximal velocity (Vmax) of 5.1 nmol min-1 mg of protein-1. At pH values below 7, the rate of 4-CBA uptake was greatly reduced by nigericin, an ionophore which dissipates the pH gradient across the membrane (delta pH). At higher pH values, inhibition was observed only with valinomycin, an ionophore which collapses the electrical potential across the membrane (delta psi). Under anaerobic conditions, no uptake of 4-CBA was observed unless an alternative electron acceptor was present. With nitrate as the terminal electron acceptor, 4-CBA was rapidly accumulated by the cells to a steady-state level, at which uptake of 4-CBA was balanced by excretion of 4-hydroxybenzoate. The mechanism of energy coupling to 4-CBA transport under anaerobic conditions was further examined by the imposition of an artificial delta psi, delta pH, or both. Uptake of 4-CBA was shown to be coupled to the proton motive force, suggesting a proton symport mechanism. Competition studies with various substrate analogs revealed a very narrow specificity of the 4-CBA uptake system. This is the first report of carrier-mediated transport of halogenated aromatic compounds in bacteria.     

Factors influencing the formation and stability of D-glucoside 3-dehydrogenase activity in cultures of Agrobacterium tumefaciens.

D-glucoside 3-dehydrogenase specific activity in Agrobacterium tumefaciens was maximal towards the end of the exponential growth phase of batch cultures; over 90% of the activity disappeared within the next 15 h. Manganese ions, although essential for growth of the organism, strongly repressed D-glucoside 3-dehydrogenase synthesis in sucrose medium but had little effect when the carbon source was methyl alpha-D-glucoside. D-Glucoside 3-dehydrogenase activity increased linearly with increasing specific growth rate in chemostat cultures limited by carbon, nitrogen, phosphate or manganese when methyl alpha-D-glucoside was the carbon source. High enzyme activity was found with sucrose as carbon source only when the growth medium was manganese-limited. D-Glucoside 3-dehydrogenase activity disappeared from A. tumefaciens incubated in carbon- and nitrogen-free medium or in nitrogen-free medium containing succinate, but on continued incubation the activity returned and was then stable. The recovery of activity could be prevented by chloramphenicol or erythromycin. Bacteria containing the recovered dehydrogenase activity could not convert sucrose to 3-ketosucrose when oxygen acted as the terminal electron acceptor, but produced 3-ketosucrose at the normal rate in the presence of ferricyanide. D-Glucoside 3-dehydrogenase activity disappeared irreversibly from bacteria incubated in nitrogen-free medium containing sucrose. Loss of activity followed first order kinetics in bacteria taken from nitrogen-, phosphate- or manganese-limited chemostat steady states; an accelerating rate of decay occurred in cells grown under carbon-limitation. 8-Hydroxyquinoline, chloramphenicol, erythromycin, 2,4-dinitrophenol and manganese ions could reduce the rate of decay.     

Shortening of life-time of the pair [P680+Pheo-] contributes to general inhibitory effect of dinoseb on electron transfer in PS-II

It is shown that dinoseb, added to subchloroplast photosystem-II (PS-II) preparations from pea at a concentration higher than 5 microM, along with blocking the electron transfer on the acceptor side of PS-II, induces the following effects revealing its capability to have redox interaction with the components of PS-II reaction center (RC)-pheophytin (Pheo) and chlorophyll P680: (1) acceleration of the dark relaxation of absorbance (delta A) and chlorophyll fluorescence (delta F) changes related to photoreduction of Pheo as a result of the photoreaction [P680Pheo] [symbol: see text] [P680Pheo-] that leads to elimination of the delta A and delta F at a concentration of the inhibitor higher than 50 microM; (2) lowering of the maximum level of fluorescence (F) due to a decrease of delta F under the condition when the electron acceptor, QA, is reduced; (3) loss of the described effects of dinoseb and appearance of its capability to donate electron to RC of PS-II in the presence of dithionite which reduces dinoseb in the dark; (4) inhibition of delta A related to photooxidation of P680; (5) activation of delta A related to photooxidation P700 in photosystem-I (PS-I) preparations (a similar effect is observed upon the addition of 0.2 mM methylviologen). It is suggested that redox interaction with the pair [P680+Pheo-] leading to the shortening of its life-time contributes to the general effect of inhibition of electron transfer in PS-II by dinoseb.     

Lipase-mediated conversion of vegetable oils into biodiesel using ethyl acetate as acyl acceptor

Ethyl acetate was explored as an acyl acceptor for immobilized lipase-catalyzed preparation of biodiesel from the crude oils of Jatropha curcas (jatropha), Pongamia pinnata (karanj) and Helianthus annuus (sunflower). The optimum reaction conditions for interesterification of the oils with ethyl acetate were 10% of Novozym-435 (immobilized Candida antarctica lipase B) based on oil weight, ethyl acetate to oil molar ratio of 11:1 and the reaction period of 12h at 50 degrees C. The maximum yield of ethyl esters was 91.3%, 90% and 92.7% with crude jatropha, karanj and sunflower oils, respectively under the above optimum conditions. Reusability of the lipase over repeated cycles in interesterification and ethanolysis was also investigated under standard reaction conditions. The relative activity of lipase could be well maintained over twelve repeated cycles with ethyl acetate while it reached to zero by 6th cycle when ethanol was used as an acyl acceptor.