• Journal of Microbiology and Biotechnology
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• v.6 no.2
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• pp.142-144
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• 1996

To clone the gene coding for steroid $\Delta^1$-dehydrogenase of Arthrobacter simplex, its genomic library was constructed with a , $\lambda$gt11 expression vector and immunoscreened with antiserum against the enzyme. One positive clone was found to carry a 1.6-kb EcoR I restriction endonuclease fragment of A. simplex DNA. The restriction map of the 1.6-kb EcoR I fragment was determined after cloning of the DNA into pBS vector.

• Microbiology and Biotechnology Letters
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• v.19 no.3
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• pp.242-247
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• 1991

- Since steroid $\Delta^1$-dehydrogenase synthesis has been known to be inducible, the mechanism of the enzyme induction of Arthrobacter simplex IAM 1660 was investigated. Among various steroids tested for inducers, hydrocortisone was the most effective inducer when hydrocortisone was used as a substrate for steroid $\Delta^1$-dehydrogenase. Steroid $\Delta^1$-dehydrogenase synthesis was effectively induced by progesterone, prednisolone and androstenedione, while the enzyme was less induced by cholesterol and not by phytosterols. The results suggest that the presence of 3-keto group and short side chain of steroids are the favorable factors for the induction of the $\Delta^1$-dehydrogenase synthesis. The enzyme was induced at the highest level when hydrocortisone was added at early log phase to the concentration of 0.01% of the culture and the culture was grown for 15 hours.

• Journal of Microbiology and Biotechnology
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• v.4 no.2
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• pp.119-125
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• 1994

Steroid $\Delta^1$-dehydrogenase purified from hydrocortisone-induced cells of Arthrobacter simplex converted various 3-ketosteroids into their corresponding $\Delta^1$-dehydrogenated products. The transformation efficiencies depend upon the chemical structure of the steroids, especially length of the side chain at 17 position and hydroxyl groups at 11 and 17 positions. The Km values for androstenedione, the most favorable substrate examined, and hydrocortisone were 74 ${\mu}M$ and 294 ${\mu}M$, respectively. The optimum temperature and pH of the enzyme reaction were 35$^{\circ}C$ and pH 9, respectively, and the enzyme was relatively stable at the range from 20 to 35$^{\circ}C$ and from pH 5 to 10 after one hour of incubation. The enzyme activity was markedly inhibited in the presence of $Cu^{2+},\;Fe^{3+},\;Hg^{2+},\;Mo^{6+}$ ions, and somewhat inhibited by $Zn^{2+}$ and $Fe^{2+}$. $\alpha,\alpha'$-Dipyridyl that inhibits 9$\alpha$-hydroxylase and accumulates 1,4-androstadiene-3,17-dione from sterols revealed no inhibitory effect on this enzyme. EGTA showed inhibitory effect. $\beta$-Estradiol competitively inhibited the enzyme activity. Chemical modifications of the enzyme were attempted with several reagents. p-Hydroxymer-curibenzoate showed inhibition of the enzyme activity and protection of the substrate. This suggests that cysteine residue may be involved in the active site of the enzyme.

• Journal of Microbiology and Biotechnology
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• v.3 no.3
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• pp.181-187
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• 1993

Steroid $\Delta^1$-dehydrogenase which introduces a double bond into the 1, 2 positions of steroid ring A was purified from Arthrobacter simplex, an excellent biotransformer of hydrocortisone into prednisolone. Hydrocortisone-induced cells were disrupted by vigorous agitation with glass beads, and a solubilized enzyme was obtained after centrifugation at 100, 000$\times$g for 90 minutes. The enzyme was purified 123-fold in three steps of chromatographic procedures with 13% yield. The last step of testosterone-agarose affinity column decisively contributed to the successful purification. The molecular weight of the enzyme was estimated to be 98, 000 by SDS-PAGE and 100, 000 by gel filtration, indicating that this enzyme behaves as a monomer. The enzyme showed demands for artificial electron acceptor, and among the several reagents tested, phenazine methosulfate acted as the most effective electron acceptor. Subcellular distribution of this enzyme was studied by centrifugation experiment. Comparison of the enzyme activities in pelleted membrane and cytosol fractions suggests that the enzyme may be a weakly attached peripheral membrane protein in vivo. But considerable amounts of enzyme was solubilized without any additional treatments for membrane protein.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 1978.10a
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• pp.207.2-207
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• 1978

Arthrobacter simplex (ATCC 6946) was cultured, induced and immobilized in acrylamide polymer. The characteristics of the immobilized whole-cell enayme were studied using hydrocortisone as the substrate. The enzyme activity was increased during the incubation of the gel particle in 0.5％ peptone media. The ennzyme reaction kinetics of the Δ'-dehydrogenase (3-oxosteroid Δ'-oxydo reductase, E. C. 1.3.99.4) foliowed the Michaelis-Menten type. Km and Vm values were different significantly after immobilization of the cell. The optimum pH and temperature were changed, too. Nitrogen sources such as casitone, peptone or tryptone were good media for the enzyme reaction. And there was no need to add cofactors of the enzyme in the pre-sence of energy sources used in the test. The effect of metal ions on the enzyme activity was insignificant. Organic solvents were used increase the substrate concentration and there was no optimum solvent concentration depending on the substrate concentration.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 1979.04a
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• pp.115.4-116
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• 1979

Acetone-dried cells of Arthrobacter simplex were entrapped in several preparations of hydrophilic urethane prepolymers and their steroid converting ability was examined. SeVeral solvents, such as methanol and propylone glycol, wereeffective for the conversion of hydrocortisone to prednisolone. The stability of the immobilized cells during storage and on repeated reactions was also examined. Thisconvenient entrapping method was also applicable for the immobilization of cellular organelles. yeast peroxisomes. The entrapped peroxi-somessh owed the activities of alcohol oxidase and catalase.

• YAKHAK HOEJI
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• v.40 no.2
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• pp.212-217
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• 1996

Streptomyces cattleya is a producer of carbapenem antibiotics, thienamycin and N-acetylthienamycin, which have potent and broad-spectrum antibacterial activities. We stud ied on strain improvement for antibiotic productivity of S. cattleya by transformation technique which employed S.cattleya protoplasts and chromosomal DNAs of glutamic acid producers: Corynebacterium glutamicum and Arthrobacter simplex. 150 Transformant strains were cultured and bioassayed using Bacillus subtilis and Staphylococcus aureus as test organisms. 8.7% of transformants tested showed 1.4~2.6 fold higher productivities than wild type which produced $1.61{\pm}0.67{\mu}g/ml$. The best transformant produced $8.36{\pm}2.84{\mu}g/ml$ carbapenems.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 1979.10a
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• pp.247.2-247
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• 1979

17-케토스데로이드 (안드로스트-4-엔-3, 17-다이온 (AD), 안드로스타-1, 4-다이엔-3, 17-다이온(ADD))는 스테로이드 약물제조의 출발물질로 그 중요성을 인정받고 있다. 이 중요한 17-케토스테로이드를 기질 콜레스테롤로부터 다량 얻기 위하여 미생물을 이용한 방법을 검토하였다. Arthrobacter simplex 균주를 이용하여 이 균주의 전환활성을 증가시키기 위한 배지조성, 저해제의 영향, 계면활성제의 영향, 균액중균의 농도의 영향, 흡착제의 영향 및 식물성기름의 영향등을 검토하였다. 위의 실험결과과 기질농도 0.1％에서 70％ 이상의 전환수율을 얻을 수 있었다.

• YAKHAK HOEJI
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• v.21 no.3
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• pp.167-171
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• 1977

A new assay method for delta-l-dehydrogenated-3-ketoco-rticosteroid in the presence of proteinous material or whole-cell-enzyme and 3-ketocorticosteroid has been developed. This method makes use of the linear relationship between the ratio of absorbances at 265 nm and at 242 nm and the fractional concentration of delta-1-3-ketosteroid. Theoretical values were calculated based on the absorbances of proteinous material at fixed concentrations of the 3-ketosteroid and delta-1-dehydrogenated-3-ketosteroid. The values obtained experimentally showed good agreement with the values obtained experimentally showed good agreement with the values theoretically predicted. The new assay method developed for the steroid mixtiure containing proteinous material is of some practical importance. The use of such assay method enables one to determine the enzyme activity and the rate of enzyme reaction or conversion rather quickly, easily and accurately. By the use of this assay method, the reaction kinetics of whole-cell-enzyme has also been studied. It was found that it followed the simple Michaelis-Menten type enzyme kinetics. Also the reversibility of this reaction with actively metabolizing cell was examined. It was found that delta-l-dehydrogenated-3-ketosteroid could not be hydrogenated reversibly to 3-ketosteroid by this enzyme system.