Arthrobacter Simplex Research Articles

Microbiology of activated sludge treating wastewater containing Ni(II) and Cr(VI)

The species of microorganisms dominating a laboratory-scale once-through activated sludge unit treating simulated wastewater with different influent strengths (650 and 1300 mgl−1 COD) and containing 5 to 10 mgl−1 Ni(II) and 1 to 50 mgl−1 Cr(VI) were identified by standard biochemical tests. Within this framework, combinations of metals and influent COD concentrations were tried. Organisms, namely Proteus rettgeri, Acinetobacter sp., Flavobacterium lutescens, Bacillus cereus var. albolactis, Klebsiella sp., Arthrobacter simplex, and Pseudomonas mendocina, were identified at different stages of the experiments.

Strain improvement ofArthrobacter simplex by protoplast fusion

Anl-tryptophan auxotroph and milky mutants were derived from an inducible cholesterol oxidase-producing bacterium,Arthrobacter simplex USA18, via UV-mutagenesis. Protoplasts of these mutants and a constitutive cholesterol oxidase producer, strain US3011, were prepared by growing cells in the presence of ampicillin (20μg ml−1) followed by digestion with lysozyme. Protoplast fusion between tested strains with complementary characteristics was achieved in the presence of 20–40% polyethylene glycol 6000. The fusion frequency was about 1.5–1.7×10−3. The cholesterol oxidase activity of four fusants in a cholesterol-containing medium was 20–60% higher than that of parental strains. This study demonstrated that protoplast fusion is applicable to strain improvement ofArthrobacter strains for enzyme production.

Bioconversion of cholesterol to cholest-4-en-3-one in aqueous/organic solvent two-phase reactors

The bioconversion of cholesterol to cholestenone in an aqueous organic solvent two-phase system using free cells of Arthrobacter simplex U-S-3011 was described. The reactions were carried out in a 500-mL stirred-tank reactor loaded with potassium phosphate buffer and carbon tetrachloride (3:1, v/v). It was found that 93% of added cholesterol was converted to cholestenone within 2.5 h under the following conditions: working volume, 300 ml; temperature, 30°C; agitation speed, 800 rpm; and aeration rate, 100 ml oxygen min −1. The productivity was 12.4 g L −1 h −1. Some factors concerning the two-phase reactors were investigated. It was found that the bioconversion could be inhibited by either a high concentration of substrate or product, and it could be promoted by increasing the agitation speed. However, the enzyme was easily denatured by interface tension. It is confirmed that the water content in a two-phase system is an important parameter for the bioconversion. Under the optimal operation conditions, 94.6% of added cholesterol could be converted to cholestenone within 4 h. The maximum productivity was 26.3 g L −1 h −1.

Microbiology of activated sludge treating wastewater containing Ni(II) and Cr(VI)

The species of microorganisms dominating a laboratory-scale once-through activated sludge unit treating simulated wastewater with different influent strengths (650 and 1300 mgl−1 COD) and containing 5 to 10 mgl−1 Ni(II) and 1 to 50 mgl−1 Cr(VI) were identified by standard biochemical tests. Within this framework, combinations of metals and influent COD concentrations were tried. Organisms, namely Proteus rettgeri, Acinetobacter sp., Flavobacterium lutescens. Bacillus cereus var. albolactis, Klebsiella sp., Arthrobacter simplex, and Pseudomonas mendocina, were identified at different stages of the experiments.

Recent Advances in Studies on Streptomyces Cholesterol Oxidase and Gene Clusters Involved in Steroid Catabolism in Prokaryotes.

Streptomyces cholesterol oxidase, an enzyme on the first step of microbial catabolism of cholesterol, was overproduced in a secretory fashion in Streptomyces lividans with a multi-copy shuttle vector. The gene coding for cholesterol oxidase (choA) was modified and overexpressed in Escherichia coli. The modified choA gene was also used to develop promoter-probe vectors to use in enteric bacteria. Expression of the choA gene in lactic acid bacteria might facilitate the degradation of cholesterol in dairy food. The Streptomyces cholesterol oxidase was unexpectedly found to constitute a novel class of insecticidal proteins that may be useful for controlling pests that are resistant to Bacillus thuringiensis toxins.Furthermore, the Arthrobacter simplex gene (ksdD) encoding 3-ketosteroid-Δ1-dehydrogenase, a key enzyme in the degradation of the steroid nucleus, was cloned and overexpressed in S. lividans using a multi-copy shuttle vector, leading to an about 100-fold overproduction of the enzyme compared to the natural producer. Nucleotide sequence analysis revealed that ksdD is clustered with two more genes possibly involved in steroid catabolism. Upstream of ksdD, a gene (ksdR) encoding a hypothetical regulatory protein that shows similarities to several regulators was found. Adjoining downstream to ksdD in an apparent translational coupling is a gene (ksdI) coding for a protein that would display strong similarities to 3-ketosteroid-Δ5-isomerase. Further sequence analysis downstream of ksdI revealed three ORFs. The deduced protein product of one of these showed significant similarities to the KsdD protein. Thus, genes involved in steroid metabolism seem to be clustered in bacteria.

Use of a genetic algorithm in the development of a synthetic growth medium for Arthrobacter simplex with high hydrocortisone Δ1‐dehydrogenase activity

AbstractThe production process for Arthrobacter simplex high specific hydrocortisone Δ1‐dehydrogenase activity and biomass yield (full experimental plan: 1·5 × 1014 experiments). The specific hydrocortisone Δ1‐dehydrogenase activity was improved by a vector of 4·5 to 1800 U g−1. With the optimized medium composition in a stirred tank reactor for batch production of A. simplex, a specific hydrocortisone Δ1‐dehydrogenase activity of 2000–3000 U g−1 dry biomass was achieved depending on harvest time (previously reported data: 18–37 U g−1).

Purification and characterization of the 3-ketosteroid-delta 1-dehydrogenase of Arthrobacter simplex produced in Streptomyces lividans.

The 3-ketosteroid-delta 1-dehydrogenase (KS1DH) gene of Arthrobacter simplex IFO12069 cloned in Streptomyces lividans was overexpressed, resulting in production of the enzyme both extracellularly and intracellularly. The enzyme was purified by ammonium sulfate fractionation and chromatographies using DEAE-Toyopearl, Butyl-Toyopearl and Toyopearl HW55S from the supernatant of culture broth and cell-free extracts of S. lividans, and both preparations showed the same characteristics. The N-terminal amino acid sequence of both KS1DHs was M-D-W-A-E-E-Y-D, which coincided with the amino acid sequence deduced from the nucleotide sequence. Thus, the extracellular enzyme may derived from leakage of S. lividans cells during cultivation rather than secretion by processing of the signal sequence. The molecular weight of the enzyme was about 55,000, identical with the size deduced from the nucleotide sequence (M(r) 54,329). The optimum conditions for its activity were pH 10.0 and 40 degrees C. The enzyme catalyzed the conversion of several 3-keto-steroids, but those containing 11 alpha- or 11 beta-hydroxyl group were converted at low rates. The amino acid sequence of KS1DH from A. simplex is similar to those of KS1DH of Pseudomonas testosteroni and fumarate reductase from Shewanella putrefaciens.

Modeling membrane filtration of protein and cell suspensions in a vortex flow filtration system

AbstractVortex flow filtration experiments with bovine serum albumin (BSA) solutions and Arthrobacter simplex cell suspensions in a rotary membrane device are modeled in the pressure‐dependent flux and mass‐transfer‐controlled regions. An expression for the concentration‐dependent diffusion coefficients accounting for hydrodynamic and long‐range potential interactions between rigid spherical macromolecules is used for BSA. A shear‐induced diffusion coefficient, strongly concentration‐dependent on the cell concentration, is assumed to describe the diffusion behavior of the cell suspensions. The adjusted model parameters for albumin solutions in the pressure‐dependent region of flux predict the permeation flux behavior for A. Simplex cell suspensions. When the mass‐transfer correlation is calculated using cell diffusion coefficients, based on a volumetric cell fraction that is around half of the average value of the calculated membrane surface fractions, the fluxes are estimated within less than 10.5% relative error. In the mass‐transfer‐controlled region of flux, the volume fractions of cells at the membrane surface are between 0.16 and 0.35, depending on bulk concentration and hydrodynamic conditions.

Molecular cloning, expression in Streptomyces lividans, and analysis of a gene cluster from Arthrobacter simplex encoding 3-ketosteroid-delta 1-dehydrogenase, 3-ketosteroid-delta 5-isomerase and a hypothetical regulatory protein.

The Arthrobacter simplex gene coding for 3-ketosteroid-delta 1-dehydrogenase, a key enzyme in the degradation of the steroid nucleus, was cloned in Streptomyces lividans. Nucleotide sequence analysis revealed that the gene for 3-ketosteroid-delta 1-dehydrogenase (ksdD) is clustered with at least two more genes possibly involved in steroid metabolism. Upstream of ksdD, we found a gene, ksdR, encoding a hypothetical regulatory protein that shows homologies to KdgR, the negative regulator of pectin biodegradation in Erwinia, and GyIR, the activator for glycerol metabolism in Steptomyces. A helix-turn-helix DNA-binding domain can be predicted at similar positions near the N-terminal of KsdR, KdgR and GyIR. ksdl adjoining downstream to ksdD codes for a protein that has strong similarities to 3-ketosteroid-delta 5-isomerases. The highly conserved Tyr and Asp residues are present in the active-centre motif of the enzyme. The translated ksdD gene product was found to be similar to the 3-ketosteroid-delta 1-dehydrogenase of Pseudomonas testosteroni and to the fumarate reductase of Shewanella putrefaciens. A region highly conserved between the two steroid dehydrogenases can be aligned to the active-centre motif of the fumarate reductase. S. lividans strains carrying the ksdD gene overexpressed 3-ketosteroid-delta 1-dehydrogenase. The expression of 3-ketosteroid-delta 5-isomerase, however, was barely detectable in recombinant S. lividans strains carrying the ksdl gene, or in the parental Arthrobacter strain.

Bioconversion of a hydrocortisone derivative in an organic-aqueous two-liquid-phase system

The Δ 1-dehydrogenation of 6-α-methyl-hydrocortisone-21-acetate in an organic-aqueous two-liquid-phase system using free Arthrobacter simplex cells was studied. Chloroform, toluene, and n-octan-1-ol were assayed for steroid solubility and biocompatibility, the latter emerging as the most appropriate solvent. Addition of surfactants (a commercial-grade lecithin, phosphatidylcholine, and phosphatidyl-ethanolamine) led to increased initial reaction rates but did not significantly change the final conversion yields. First-order apparent kinetics was observed in a menadione-saturated system for global substrate concentrations under 10 m m. Maintenance stability was considerably increased when the temperature was lowered from 30 to 25°C. However, operational stability levels were unsufficient for biocatalyst reuse in the tested reactional conditions.

Metabolism ofL-Arginine, Agmatine, and Related Compounds inNocardioides simplex

Nocardioides simplex IFO 12069 (Arthrobacter simplex ATCC 6946) was examined for the degradation of L-ornithine, L-citrulline, L-arginine, D-arginine, agmatine, carbamoylputrescine, and putrescine, and the results were compared with those obtained in a previous study with A. globiformis and Brevibacterium helvolum. D-Arginine as well as L-arginine was degraded via the arginine oxygenase pathway. A part of L-citrulline was probably degraded via the pathway. The L-ornithine degradation via the pathway was obscure. Guanidinobutyrase was induced by agmatine, which was shown to be degraded via a unique pathway. The first step of the pathway converted agmatine to 4-guanidinobutyraldehyde by transferring the amino group to pyruvate. The second step was the NAD-linked dehydrogenation of the aldehyde to 4-guanidinobutyrate, which was then degraded to 4-aminobutyrate. The enzymes for the first and second steps were identified as diaminopropane aminotransferase (EC 2.6.1.-) and aminobutyraldehyde dehydrogenase (EC 1...

GROWTH-INHIBITION OF T47D HUMAN BREAST-CANCER CELLS BY THE NEW ANTIAROMATASE STEROID 3-METHYLENE-1,4-ANDROSTADIEN-17-ONE

The C19 steroid, 3-methylene-1,4-androstadien-17-one (3-methylene-1-dehydro-4-androsten-17-one, abbrev. 3MDA) was synthesized by microbial dehydrogenation with Arthrobacter simplex from the corresponding 1,2-saturated, 3-methylene-4-androsten-17-one. The new steroid induces a Type-1 optical difference spectrum with Cytochrome P450 aromatase from human placentae (K(s) 1.61 muM). Kinetic inhibition studies with placental microsomes show: (i) 3MDA inhibits aromatase irreversibly (k(inact) 0.026 min-1), (ii) the inhibition is time- and concentration-dependent, (iii) it follows pseudo first-order kinetics, and (iv) the inhibition requires the presence of the cofactor NADPH. Human T47D breast tumor cells are able to transform C-14-labeled androstenedione into radioactive estrone/estradiol, as we have established by repetitive HPLC purifications. We have thus clearly demonstrated the presence of an endogenous steroidal androgen-aromatizing enzyme in these breast tumor cells. Besides inhibiting human placental aromatase activity, 3MDA at concentrations of 5-20 muM also significantly decreases the growth of cultured T47D cells as measured by the incorporation of [H-3]thymidine into DNA, but without causing cell death due to cytotoxicity. These findings prompt us to propose 3MDA as a new, mechanism-based suicide substrate of the human aromatase enzyme with potential utility as a 'second generation' anti-breast cancer agent.

Transformation of cholic acid by Arthrobacter simplex

Fermentation of cholic acid with Arthrobacter simplex (IICB 227) under aerobic conditions yielded 3,12-dioxo-23,24-dinorchola-4,6-dienoic acid, 7α-hydroxy-3,12-dioxo-23,24-dinorchol-4-enoic acid, 3α, 7α-dihydroxy-12-oxo-5β-cholan-24-oic acid, 3α,7α-dihydroxy-12-oxo-5β-23, 24-dinorcholan-22-oic acid, 7α,12α-dihydroxy-3-oxo-5β-cholan-24-oic acid, 7α-12α-dihydroxy-3-oxo-4-cholenoic acid, 7α,12α-dihydroxy-3-oxo-23, 24-dinorchol-4-enoic acid, and methyl-3α-7α, 12α-trihydroxy-5β-cholan-24-oate in addition to a new metabolite 2β-hydroxy-3,12-dioxo-23,24-dinorchola-4,6-dienoic acid. Each microbial metabolite was characterized by the application of various spectroscopic methods. The availability of some of the metabolites' enabled complete elucidation of their 13C NMR spectra .

Production of androsta-1,4-diene-3,17-dione from cholesterol using two-step microbial transformation

A novel two-step transformation process for the production of androsta-l by microorganisms-diene-3,17-dione (ADD) from a high concetration of cholesterol by microorganisms is proposed. Cholesterol (20 g/l) was initially converted to cholest-4-en-3-one (cholestenone) by an inducible cholesterol oxidase-producing bacterium, Arthrobacter simplex U-S-A-18. The maximum productivity of cholestenone was 8 g/l per day and the molar conversion rate was 80%. Subsequently, a fine suspension of cholestenone (50 g/l), which was prepared directly from the fermentation broth of A. simplex, was converted to ADD by Mycobacterium sp. NRRL B-3683 in the presence of an androstenone adsorbent, Amberlite XAD-7. The maximum productivity of ADD was 0.91 g/l per day and the molar conversion rate was 35%.

Quinones as External Electron Acceptors in Steroid Dehydrogenation with Entrapped Cells in Organic Medium

A series of quinone-based compounds were tested for their ability to act as external electron acceptors in the 1-dehydrogenation of-α-methyl-hydrocortisone-21-acetate, with polyurethane-entrapped Arthrobacter simplex cells in buffer-saturated n-decan-1-ol. This organic solvent was needed to solubilize the steroid substrate. In aqueous medium, the conversion with free cells virtually stopped after one hour, probably due to substrate limitation. All the tested quinones acted as external electron acceptors, increasing the bioconversion rate. The process kinetics were complex. However, when keeping the concentration of one of the substrates (steroid or quinone) constant and varying that of the other, Michaelis-Menten kinetics provided a reasonably good model for the initial reaction rates, and apparent kinetic constants were estimated. The most effective of the tested external electron acceptors were 2,6-dimethyl-p-benzoquinone and menadione. Mass transfer limitations seemed to appear after some hours of reac...

Activity and stability of an entrapped-cell system for the ?1-dehydrogenation of steroids in organic media

Whole Arthrobacter simplex cells entrapped in kappa-carra-geenan or in two types of polyurethane foam, or adsorbed on silanized Celite, were tested for the Delta(1)-dehydrogenation of hydrocortisone and its derivatives in organic media. Catalytic activity and stability levels were evaluated for the immobilized cells in buffer with 2.5% (vol/vol) methanol, and in three buffer-saturated solvents (n-octan-1-ol, n-decan-1-ol, and chloroform). The addition of glutamate to the immobilization support stabilized the activity of the immobilized cells in the tested organic media. The system with polyurethane (HYPOL6100)-entrapped cells (with coimmobilized glutamate) in n-decan-1-ol provided the highest long-term activity levels. Several factors involved in the polyurethane-entrapment procedure were also studied.

Production of naphthalene- cis-glycol by Pseudomonas putida in the presence of organic solvents

The effect of water-immiscible organic solvents on the conversion of naphthalene to naphthalene-1,2-dihydro-1,2-diol by Pseudomonas putida has been examined. The bacteria were stable in the presence of dodecane, giving a maximum reaction rate (about 1 g g dcw −1 h −1) with naphthalene concentrations greater than 40 g l −1 dodecane. For solvents with log P values less than 3.5, no activity was observed. Immobilization in calcium alginate beads improved the stability of bacteria in the presence of solvents with log P values of 3.5–4. For solvents with log P values greater than 4, there was no activity loss during biotransformations lasting 5 h with either free or immobilized bacteria. The results are compared with those obtained previously for steroid Δ 1-dehydrogenation by Arthrobacter simplex in aqueous/organic reaction mixtures, and some general conclusions are drawn.

Screening of whole-cell immobilization procedures for the Δ1-dehydrogenation of steroids in organic medium

Different whole-cell immobilization methods and conditions were tested for the Δ 1-dehydrogenation of 6-α-methyl-hydrocortisone-21-acetate with Arthrobacter simplex cells, in n-decane-1-ol and chloroform. Among the entrapment methods, polyurethane foams gave the best productivity of n-decane-1-ol, but could not be used in chloroform. Partition and diffusional barriers appeared to be the factors limiting productivity in entrapped-cell systems. The coentrapment of enzyme-stabilizing (glycerol, sucrose, sodium sulfate, monosodium glutamate) or hydrophobic additives did not significantly improve dehydrogenation rates in chloroform, although in some cases higher activities resulted in n-decane-1-ol. Dehydration of κ-carrageenan-immobilized cells lowered the productivity in both solvents. The use of cell adsorption methods on silica-based carriers produced biocatalysts that in some cases were more active in chloroform than the entrapped cells. However, the surface-attached cells appeared to be more sensitive to solvent toxicity, more hydrophilic supports generally giving higher dehydrogenation rates. The estimated partition coefficients for the substrate between several of the tested supports and the two solvents are also presented. High partition ratios followed high measured activities in entrapment matrices but not in Celite (cell adsorption matrix). Activities in chloroform were always very low (less than 1 μmol product per hour and gram of cell dry weight). Polyurethane entrapment with n-decane-1-ol as a reaction medium was the most promising system, with measured dehydrogenation rates up to 40 times higher.

Formation of mixed crystals in microbial conversion of sterols and steroids

The phenomenon of formation of mixed crystals during microbial conversions involving water-insoluble substrate and product was examined through the investigation of two biotransformations: (a) dehydrogenation of hydrocortisone by Arthrobacter simplex and (b) oxidation of cholesterol by Rhodococcus erythropolis. Evidence for the intrinsic ability of hydrocortisone and prednisolone molecules to form mixed crystals from both artificial solutions and microbial conversion systems was provided by x-ray powder diffractometry and differential scanning calorimetry. Mechanical agitation was shown to further promote the bioconversion of mixed crystals from an initial value of 83% prednisolone to an ultimate value of 95%. The solids in the bioconversion system of cholesterol were shown by their x-ray diffractions to be a mixture of single substrate and product crystals. A scheme of the various crystal structures likely to be formed in bioconversions—mixed single crystals, mixed crystals, and coated crystals—was presented. A solubility-based classification of the sterols and steroids with regard to their likelihood to form mixed crystals in bioconversion systems was suggested.

Synthesis of 6-hydroximino-3-oxo steroids, a new class of aromatase inhibitor

(E)-3β,17β-Dihydroxyandrost-4-en-6-one oxime has been converted into (E)-6- hydroximinotestosterone and (E)-6-hydroximinoandrost-4-ene-3,17-dione by chemical methods, and into (E)-6-hydroximinoandrosta-1,4-diene-3,17-dione by biotransformation using Arthrobacter simplex ATCC 6946.