Δ′-Dehydrogenation of steroids byArthrobacter simplex immobilized in calcium polygalacturonate beads

Summary Arthrobacter simplex ATCC 6946 (viable cells) was immobilized in a calcium polygalacturonate gel. The trapped cells were used for repeated batchwise bioconversion of steroids. Reichstein's compound S and hydrocortisone were dehydrogenated introducing a double bond between C₁ and C₂ of ring A. The products 1-dehydro S and prednisolone, respectively, were identified by high pressure liquid chromatography. Steroid dehydrogenase activity increased in the system when an artificial electron acceptor, such as menadione (vitamin K₃) was present in the reaction mixture. An airlift-type reactor was used to bioconvert up to 90% of substrate in 15 min, under optimal conditions. The gel entrapped cell preparations were used for repeated batch bioconversion during 30 days; 69 batch bioconversions for Reichstein's compound S were performed during 15 days of operation of the reactor. The operational stability of the process and the feasibility of repeated batch bioconversions was shown to be comparable to similar processes.     

Steroid transformations by species of Cephalosporium and other fungi

A total of 58 cultures, tentatively identified as species of the genus Cephalosporium, were screened in flask fermentations for their ability to effect conversions of progesterone (Delta(4)-pregnene-3,20-dione) and Reichstein's Substance S (Delta(4)-pregnene-17alpha,21-diol-3,20-dione). A large number of transformations were observed by means of a series of five paper chromatography systems rated for analysis of steroid compounds ranging in polarity from progesterone to polyhydroxylated steroids. Five different transformation products were selected for isolation and identification. For purposes of recovery, conversions were conducted under submerged conditions in either 4- or 200-liter fermentors in which the broth was agitated and aerated. The steroid substrate was dissolved in acetone and added aseptically to the growing culture in a final concentration of 0.025%. After the conversions were effected, the whole broth was extracted with chloroform, and the transformation products were recovered, either by direct crystallization from solvents or through the use of silica gel columns. It was determined that C. ciferrii 21C converted progesterone to Delta(4)-androstene-3,17-dione. Kendall's Compound F (Delta(4)-pregnene-11beta,17alpha,21-triol-3,20-dione) was converted to its 20beta-ol analogue by Geotrichum sp. 51C (during these studies, a number of cultures were taxonomically reclassified). Cephalosporium sp. 27C formed the Delta(1)-analogue of Reichstein's Substance S, and Cephalosporium sclerotigenum 31C and Verticillium aphidum both converted Substance S to the 6beta-hydroxy derivative. Paecilomyces persicinus 22C converted Substance S to a product believed to be a dihydroxylated derivative.     

Large-Scale Transformation of Steroids by Fungal Spores

Spores of Aspergillus ochraceus and Septomyxa affinis were produced on a large scale by surface sporulation on moist wheat bran and barley. 11alpha-Hydroxylation of progesterone and Reichstein's compound S by spores of A. ochraceus and 1-dehydrogenation of compound S by spores of S. affinis were carried out in 5-liter fermentors. It was shown that, above a certain minimum, increase in aeration and agitation did not significantly affect steroid conversion. The industrial feasibility of the spore process was further demonstrated by 11alpha-hydroxylation of 6alpha-fluoro-16alpha,17alpha-dihydroxypregn-4-ene-3,20-dione in a modified 200-gal stainless-steel vessel with spores of A. ochraceus. Strict aseptic conditions are not necessary, either during harvesting of spores or during steroid transformation.     

Enzymatic breakdown of steroids during 11 alpha- and 11 beta-hydroxylation of the 21-acetate of Reichstein's "substance S" by Tieghemella orchidis (fam. Mucoraceae)]

By-products of the transformation were studied, which were formed in the process of 11alpha and 11beta-hydroxylation of 21-acetate of the Reichstein substance "S" by the culture of Tieghemella orchidis. The following steroid derivatives, known before, were isolated: epihydrocortisone, 6beta-hydroxy derivative of the Reichstein substance "S", hydrocortisone, cortisone. The following steroid derivatives were isolated from the initial chloroform extract of the fermentation medium, and identified: prednisolone, androstendione, testololactone, and 1,2-dehydro derivatives of the Reichstein substance S" and its 21-acetate. The quantitative ratio between the products of transformation depended on the conditions of growth and transformation. These compounds seem to be intermediate metabolites in the course of the steroid destruction via the pathway common for fungi and involving the destruction of D ring of the steroid molecule.     

Preparation and application of polymer-entrapped enzymes and microorganisms in microbial transformation processes with special reference to steroid 11-beta-hydroxylation and delta-dehydrogenation

Fungal cells from Curvularia lunata were entrapped in a crosslinked polyacrylamide gel. The gel-cells obtained as granules were applied in the microbial transformation of Reichstein compound S leading to cortisol through an 11-β-hydroxylation step. Some kinetic studies of this conversion using gel-cells were carried out. In addition, it was shown that gel-cell granules which had lost part of their 11-β-hydroxylase activity on storage could be reactivated yielding preparations with increased activity. From Corynebacterium simplex a steroid dehydrogenase catalyzing the Δ¹- dehydrogenation of cortisol leading to prednisolone was isolated and partially purified. The preparation was entrapped in a crosslinked polyacrylamide gel and the gel-enzyme granules obtained used in steroid dehydrogenation processes.     

L-pyroglutamate spontaneously formed from L-glutamate inhibits growth of the hyperthermophilic archaeon Sulfolobus solfataricus

Identification of physiological and environmental factors that limit efficient growth of hyperthermophiles is important for practical application of these organisms to the production of useful enzymes or metabolites. During fed-batch cultivation of Sulfolobus solfataricus in medium containing L-glutamate, we observed formation of L-pyroglutamic acid (PGA). PGA formed spontaneously from L-glutamate under culture conditions (78 degrees C and pH 3.0), and the PGA formation rate was much higher at an acidic or alkaline pH than at neutral pH. It was also found that PGA is a potent inhibitor of S. solfataricus growth. The cell growth rate was reduced by one-half by the presence of 5.1 mM PGA, and no growth was observed in the presence of 15.5 mM PGA. On the other hand, the inhibitory effect of PGA on cell growth was alleviated by addition of L-glutamate or L-aspartate to the medium. PGA was also produced from the L-glutamate in yeast extract; the PGA content increased to 8.5% (wt/wt) after 80 h of incubation of a yeast extract solution at 78 degrees C and pH 3.0. In medium supplemented with yeast extract, cell growth was optimal in the presence of 3.0 g of yeast extract per liter, and higher yeast extract concentrations resulted in reduced cell yields. The extents of cell growth inhibition at yeast extract concentrations above the optimal concentration were correlated with the PGA concentration in the culture broth. Although other structural analogues of L-glutamate, such as L-methionine sulfoxide, glutaric acid, succinic acid, and L-glutamic acid gamma-methyl ester, also inhibited the growth of S. solfataricus, the greatest cell growth inhibition was observed with PGA. We also observed that unlike other glutamate analogues, N-acetyl-L-glutamate enhanced the growth of S. solfataricus. This compound was stable under cell culture conditions, and replacement of L-glutamate with N-acetyl-L-glutamate in the medium resulted in increased cell density.     

Intestinal microflora and bile acids. In vitro cholic acid transformation by mixed fecal culture of rats.

In vitro cholic acid (CA) transformation by mixed fecal culture was investigated. Concentrations of glucose, peptone, and yeast extract in the medium and the initial pH of the medium markedly affected the CA transformation. Yeast extract enhanced the transformation, whereas high concentrations of glucose and peptone inhibited it. When the initial pH of the medium was below 6.5, CA was converted to 7-keto-deoxycholic acid (7KD), and formation of deoxycholic acid (DC) was not observed. In contrast, with an initial pH of 7.0, about 60% of the CA was converted to 7KD after 3 days of incubation, and then DC gradually formed after 4 days of incubation, following the disappearance of 7KD. The formation of DC in the cultured samples was paralleled in each case by disappearance of 7KD. In pure culture systems, Escherichia coli and some strains of Bacteroides formed 7KD from CA. No DC formation was observed in pure cultures of any of the strains examined.     

Transformation of prednisolone to a 20β-hydroxy prednisolone compound by <Emphasis Type="Italic">Streptomyces roseochromogenes</Emphasis> TS79

Prednisolone represents an important compound in pharmaceutical preparations. To obtain more bioactive prednisolone derivatives, the microbial transformation of prednisolone was carried out. The steroid products were assigned by an interpretation of their spectral data using mass spectrometry and proton nuclear magnetic resonance (¹H NMR) analyses. The product was assigned the chemical structure of 11β, 17α, 20β, 21-tetrahydroxypregna-1,4-diene-3-one (named as 20β-hydroxy prednisolone). The conversion of prednisolone to 20β-hydroxy prednisolone by Streptomyces roseochromogenes TS79 was different from a previous study on the microbial transformation of steroid by this organism, which usually generates a 16α-hydroxy steroid product. The different reaction parameters for maximum conversion of prednisolone were optimized. The analysis revealed that the optimum values of the tested variables were 7.5 mg/ml prednisolone dissolved in DMSO and added to the 24-h pre-culture fermentation culture containing 0.05% MgSO₄ and incubated for 24 h. A conversion of 95.1% of prednisolone was observed, which has the potential to be used in industrial production.     

Degradation of 4-aminobenzenesulfonate by a two-species bacterial coculture

The mutualistic interactions in a 4-aminobenzenesulfonate (sulfanilate) degrading mixed bacterial culture were studied. This coculture consisted of Hydrogenophaga palleronii strain S1 and Agrobacterium radiobacter strain S2. In this coculture only strain S1 desaminated sulfanilate to catechol-4-sulfonate, which did not accumulate in the medium but served as growth substrate for strain S2. During growth in batch culture with sulfanilate as sole source of carbon, energy, nitrogen and sulfur, the relative cell numbers (colony forming units) of both strains were almost constant. None of the strains reached a cell number which was more than threefold higher than the cell number of the second strain. A mineral medium with sulfanilate was inoculated with different relative cell numbers of both strains (relative number of colony forming units S1:S2 2200:1 to 1:500). In all cases, growth was found and the proportion of both strains moved towards an about equal value of about 3:1 (strain S1:strain S2). In contrast to the coculture, strain S1 did not grow in a mineral medium in axenic culture with 4-aminobenzenesulfonate or any other simple organic compound tested. A sterile culture supernatant from strain S2 enabled strain S1 to grow with 4-aminobenzenesulfonate. The same growth promoting effect was found after the addition of a combination of 4-aminobenzoate, biotin and vitamin B₁₂. Strain S1 grew with 4-aminobenzenesulfonate plus the three vitamins with about the same growth rate as the mixed culture in a mineral medium. When (resting) cells of strain S1 were incubated in a pure mineral medium with sulfanilate, up to 30% of the oxidized sulfanilate accumulated as catechol-4-sulfonate in the culture medium. In contrast, only minor amounts of catechol-4-sulfonate accumulated when strain S1 was grown with 4ABS in the presence of the vitamins.     

Growth Factors of Bacterial Origin for the Culture of the Rumen Oligotrich Protozoon, Entodinium caudatum

Attempts were made to develop an artificial medium suitable for axenic culture of Entodinium caudatum. Agnotobiotic cultures of the protozoon were established as stock cultures for testing the suitability of various growth media. A cell-free extract of mixed bacteria isolated from the rumen was shown to contain one or more growth factors for the protozoon when supplied with activated charcoal as a carrier. The medium (CYSE medium), which supported the growth of the protozoon in the presence of 50 μg/ml each of penicillin and chloramphenicol, consisted of activated charcoal (20 mg), heat-treated yeast (Y) (80 mg), 13%β-sitosterol-coated rice starch (S) (120 mg), and cell-free extract of rumen bacteria (1 ml) in 40 ml buffer solution. When culturing the protozoon, the CYSE medium was supplemented daily with 20 mg each of Y and S and half of the medium was replaced with fresh medium once every 5 d. The possible use of this method to establish an axenic culture of E. caudatum is discussed.     

Evidence for a high molecular weight factor(s) in serum which increases alkaline phosphatase specific activity in HeLa.

HeLa (substrain Ho) grown in serum free medium showed an increase in the specific activity of alkaline phosphatase when fetal calf serum (10%) was added to the medium (9.7 nmoles/sec/mg protein to 86.8). Under the same conditions, eight intracellular enzymes showed no increase in activity. Similar results were obtained using a different serum or medium, and with a second strain of HeLa (substrain ATC). For a given set of growth conditions, the effect of serum was dependent on its concentration and required one or more culture generations to develop. The type of isozyme expressed did not change. Neither zinc nor a total serum lipid extract would substitute for serum. The enzyme expressed by HeLaHo was not induced by prednisolone, while that in HeLaATC was. However, for cells grown in excess prednisolone without serum, the specific activity was 25% of that found for cells grown with prednisolone and serum. Cortexolone, an antagonist of prednisolone, was without effect for HeLaHo grown in A3 medium with or without serum. The serum factor had the following characteristics. It was not lost on dialysis, treatment with DNase and RNase, or removal of lipoproteins. It was reduced after heating by 65% and after treatment with Pronase by 82%. The data are interpreted to indicate the presence of a factor (s) in serum, probably a protein, which is involved in stimulating alkaline phosphatase specific activity.     

绿茶粗提物对耻垢分枝杆菌的生长抑制研究

采用乙酸乙酯在中性条件下萃取绿茶,得到含有表没食子儿茶素没食子酸酯(Epigallocatechin gallate,EGCG)的粗提物。通过纸片琼脂扩散法和细菌生长曲线来评价绿茶粗提物对耻垢分枝杆菌的抑菌效果,利用透射电子显微镜(Transmission electron microscopy,TEM)观察其对耻垢分枝杆菌细胞壁结构的影响。结果显示,绿茶粗提物对耻垢分枝杆菌生长产生明显抑制作用,且抑菌作用随着浓度的升高逐渐加强;经绿茶粗提物处理的耻垢分枝杆菌细胞壁呈现膨出、变形等形态学变化。因此,绿茶粗提物具有抑制耻垢分枝杆菌生长的功能,其作用机制可能与其主要成分EGCG影响细胞壁肽聚糖的生物合成有关。     

Optimization of nutritional requirements for gentamicin production by Micromonospora echinospora

Effect of various fermentation media, carbon sources, nitrogen sources, phosphate concentration and culture requirements includes inoculum levels and age were determined on gentamicin production and biomass dry weight production for Micromonospora echinospora, a gentamicin producing strain. Of the substrates tested, starch as a sole carbon source promoted maximal gentamicin production, while maltose promoted maximal growth. Yeast extract as a sole nitrogen source promoted maximal growth, while soyabean meal for gentamicin production. Increasing phosphate concentration enhanced gentamicin production and observed optimum production at 1.2 g/1 (6% v/v) of phosphate having 72 h old inoculum in the medium. Highest gentamicin production was obtained after cultivation with shaking for 120 h in a medium containing starch 0.75% (w/v), soyabean meal 0.5%, K2HPO4 0.12%, CaCO3 0.4%, FeSO4 0.003% and CoCl2 0.0001%. The gentamicin production was 1.2-fold in this medium as compared to basal medium.     

Enhancement of anti-candidal activity of endophytic fungus Phomopsis sp. ED2, isolated from Orthosiphon stamineus Benth, by incorporation of host plant extract in culture medium

This study examined the effect of host extract in the culture medium on anti-candidal activity of Phomopsis sp. ED2, previously isolated from the medicinal herb Orthosiphon stamineus Benth. Interestingly, upon addition of aqueous host extract to the culture medium, the ethyl acetate extract prepared from fermentative broth exhibited moderate anti-candidal activity in a disc diffusion assay. The minimal inhibitory concentration of this extract was 62.5 μg/ml and it only exhibited fungistatic activity against C. albicans. In the time-kill study, a 50% growth reduction of C. albicans was observed at 31.4 h for extract from the culture incorporating host extract. In the bioautography assay, only one single spot (Rf 0.59) developed from the extract exhibited anti-candidal activity. A spot with the a similar Rf was not detected for the crude extract from YES broth without host extract. This indicated that the terpenoid anti-candidal compound was only produced when the host extract was introduced into the medium. The study concluded that the incorporation of aqueous extract of the host plant into the culture medium significantly enhanced the anti-candidal activity of Phomopsis sp. ED2.     

Degradation of 4-aminobenzenesulfonate by a two-species bacterial coculture

The mutualistic interactions in a 4-aminobenzenesulfonate (sulfanilate) degrading mixed bacterial culture were studied. This coculture consisted of Hydrogenophaga palleronii strain S1 and Agrobacterium radiobacter strain S2. In this coculture only strain S1 desaminated sulfanilate to catechol-4-sulfonate, which did not accumulate in the medium but served as growth substrate for strain S2. During growth in batch culture with sulfanilate as sole source of carbon, energy, nitrogen and sulfur, the relative cell numbers (colony forming units) of both strains were almost constant. None of the strains reached a cell number which was more than threefold higher than the cell number of the second strain. A mineral medium with sulfanilate was inoculated with different relative cell numbers of both strains (relative number of colony forming units S1:S2 2200:1 to 1:500). In all cases, growth was found and the proportion of both strains moved towards an about equal value of about 3:1 (strain S1:strain S2). In contrast to the coculture, strain S1 did not grow in a mineral medium in axenic culture with 4-aminobenzenesulfonate or any other simple organic compound tested. A sterile culture supernatant from strain S2 enabled strain S1 to grow with 4-aminobenzenesulfonate. The same growth promoting effect was found after the addition of a combination of 4-aminobenzoate, biotin and vitamin B₁₂. Strain S1 grew with 4-aminobenzenesulfonate plus the three vitamins with about the same growth rate as the mixed culture in a mineral medium. When (resting) cells of strain S1 were incubated in a pure mineral medium with sulfanilate, up to 30% of the oxidized sulfanilate accumulated as catechol-4-sulfonate in the culture medium. In contrast, only minor amounts of catechol-4-sulfonate accumulated when strain S1 was grown with 4ABS in the presence of the vitamins.Abbreviations 4ABS 4-aminobenzenesulfonate - CFU colony forming units - 4CS catechol-4-sulfonate - 4HB 4-hydroxybenzoate     

Diverse secondary metabolites produced by marine-derived fungus Nigrospora sp. MA75 on various culture media

Bioassay-guided isolation of a fungal strain Nigrospora sp. MA75, an endophytic fungus obtained from the marine semi-mangrove plant Pongamia pinnata, which was fermented on three different culture media, resulted in the isolation and identification of seven known compounds, 2, 3, and 5-9, from a medium containing 3.5% NaCl, while a new compound, 2,3-didehydro-19α-hydroxy-14-epicochlioquinone B (10) was obtained from the medium containing 3.5% NaI. In addition, two new griseofulvin derivatives, 6-O-desmethyldechlorogriseofulvin (1) and 6'-hydroxygriseofulvin (4), were isolated and identified from the rice solid medium. Dechlorogriseofulvin (2) and griseofulvin (3) were the major components in fermentation extracts of all these culture media, while compounds 1 and 4, 5 and 6, and 10 were only present in the extract of respective culture medium. The structures of these compounds were elucidated by detailed spectroscopic analysis, and the absolute configuration of 1 was determined by CD measurement. Compounds 9 and 10 exhibited antibacterial activities toward five tested bacterial strains, while compounds 5, 6, and 8 selectively inhibited MRSA, E. coli, and S. epidermidis, and compound 3 showed moderate activity against V. mali and S. solani. Moreover, compound 10 potently inhibited the growth of MCF-7, SW1990, and SMMC7721 tumor cell lines with IC(50) values of 4, 5, and 7 μg/ml, respectively.     

不透明红球菌转化合成α-酮异己酸培养基优化

利用基于统计学的方法对不透明红球菌(Rhodococcus opacus)DSM 43250转化合成α-酮异己酸(KIC)的培养环境进行优化。采用Plackett-Burman(PB)设计筛选获得对KIC产量具有显著影响的关键营养因子:(NH4)2SO4、麦芽膏和NaNO3。通过最陡爬坡实验、Box-Behnken实验设计和SAS软件回归分析建立了KIC产量关于3个关键营养因子的二次多项式模型,并以模型求解确定最佳培养条件(g/L):(NH4)2SO4 0.23,麦芽膏2.42,NaNO3 1.43。在此培养条件下,KIC的理论最高产量为23.98 mg/L,在验证实验中KIC最高产量为23.93 mg/L,比优化前(3.72 mg/L)提高了6.43倍。     

Production of Aflatoxins B1 and G1 by Aspergillus flavus in a Semisynthetic Medium

Isolates of Aspergillus flavus produced 0.2 to 63 mg of aflatoxins B(1) and G(1) per 100 ml in a nutrient solution consisting of 20% sucrose and 2% yeast extract. Various factors influencing the fermentation were studied. The maximal amount of toxin was produced by ATCC culture 15548 in 1-liter flasks containing 100 ml of medium incubated as stationary cultures for 6 days at 25 C.     

Stereospecific Biohydroxylations of Protected Carboxylic Acids with Cunninghamella blakesleeana

Cunninghamella blakesleeana DSM 1906 was found to be an efficient biocatalyst for the biotransformation of cycloalkylcarboxylic acids into hydroxy and oxo derivatives. When cultivated in submerged culture, the fungus grew in pellets. In comparison with malt extract-glucose-peptone-yeast extract medium (medium E), Czapek-Dox medium was found to reduce pellet size. Cycloalkylcarboxylic acids were protected against microbial degradation by chemical transformation into 2-cycloalkyl-1,3-benzoxazoles. The transformations of protected cyclopentyl-, cyclohexyl-, cycloheptyl-, and cyclooctylcarboxylic acids by C. blakesleeana were investigated. The biotransformations were performed in medium E by using an aerated, stirred-tank bioreactor. The transformation of 2-cyclopentyl-1,3-benzoxazole yielded (1S,3S)-3-(benz-1,3-oxazol-2-yl)cyclopentan-1-ol as the main product. The main by-product was (1R)-3-(benz-1,3-oxazol-2-yl)cyclopentan-1-one, and 2-(benz-1,3-oxazol-2-yl)cyclopentan-1-ol was also obtained in small amounts. During the experiment, the enantiomeric excess of the main product increased up to 64%. 2-Cyclohexyl-1,3-benzoxazole was hydroxylated to 4-(benz-1,3-oxazol-2-yl)cyclohexan-1-ol. 2-Cycloheptyl-1,3-benzoxazole and 2-cyclooctyl-1,3-benzoxazole were transformed into several alcohols and ketones, all in low yields (2 to 19%).     

假单胞菌株K9510产肌酐酰氨基水解酶的条件

肌酐酰氨基水解酶是酶法分析血清肌酐浓度的关键酶。本实验室从空气中分离到能分解肌酐的菌株K9510,K9511和K9512,其中K9510菌株初步分类鉴定为假单胞菌（Pseudomonas sp.)。菌株产酶条件优化研究结果表明：菌株在底物或底物类化物的诱导下产酶;混合金属离子溶液对菌株产酶有促进作用,菌株产肌酐酰氨基水解酶是适培养基组成为：肌酐9克,酵母提取物1．5g,麦芽汁0．9g,NH4Cl0.5g,定容1L。适量混合金属离子溶液,用0．1mol/L pH5.5磷酸缓冲液配制.。在250mL三角瓶中装50mL培养基,在250r/min的旋转摇床上35度振荡培养33h,在此条件下菌株产酶量可达1．0u/mL发酵液。