Microbiological transformation of steroids

Из 64 исследовавшихся нами штаммов (28 видов) различных микроорганизмов способность к гидроксилированию до положения 11β-стероидной молекулы была найдена только для 19 штаммов (10 видов). Однако ни один из микробов не гидроксилировал молекулу стероида толяко до положения 11б: во всех случаях было отмечено возникновение дальнейших гидроксилированных метаболитов,—преимущественно с гидроксигруппой в положении 11α. Оба эпимера образуются в соотношении, которое зависит от структуры стероидного субстрата, подвергаущегося энзиматическим превращениям. Для прогестерона отношение образующегося 11α-эпимера к 11β-эпимеру составляет в лучшем случае 7∶1, тогда как для стероидов S (Reichstein) отношение обоих эпимеров для штамма Absidia orchidis равно 4∶5. Состав культивационной среды оказывает влияние на накопление микроорганизмами системы необходимых ферментов, а тем самым и на скорость трансформации стероидов, но не на качество и соотношение возникающих метаболитов.     

Microbiological transformation of steroids

Изучение и оценка преобразований 98 свойств штаммов, 24 вид ов и одиннадцать вари антов рода Fusarium в стероидные молекул ы показали два типа реакции:

1. (1)
   Распределение прогестерона побочн ые цепи, в сопровожден ии дегидрирования из стероидных скелет на сайте 1–2. Это приводит к равномерн о сбалансированной с меси о δ-androstene-3, 17-дион и тестостерона в качестве промежуточных продуктов. Конец продуктом распада является testololactone. Это Затем дегидрировани я как промежуточное продукции и конечный продукт на сайте 1–2, порождающи х соответствующие dehydro-производные. Этот ти п преобразования, возникшие в видов Fusarium lateritium, Fusarium и Fusarium solani caucasicum.     
   
   

Microbial transformation of steroids

A systematic study of transformation reactions of Actinomycetes with respect to the progesterone molecule was undertaken. The results obtained, i.e. the types of transformation reactions in different actinomycete species, were evaluated from the point of view of taxonomy. The actinomycetes tested were divided according to the transformation types into three groups: (1) a group of species transforming progesterone in the 16α-position; (2) a group of species transforming progesterone in the β-position; (3) a group of species in which no capacity to transform progesterone into another steroid derivative was established. From the point of view of Actinomycete classification the transformation reactions on the steroid molecule fulfil all the requirements of taxonomic features of Actinomycetes. They appear to be specific properties, independent of strictly cultivation conditions and common to all the strains of individual actinomycete species tested.     

Microbial transformation of steroids

При помощи ?ерменентации со 191 штаммом 94 видов рода Penicillium исследовались превращения прогестерона. Качественный анализ метаболитов при помощи хроматогра?ии на бумаге с различной детекцией при сравнении со стандартными образцами обнаружил З основных типа превращений, а именно, отщепление боковой цепи прогестерона на С₁₇ при образовании. с другой сторны, тестололактона, а с другой стороны, тестостерона в качечестве конечного метаболита и гидроксилирование лрогестерона в положении 11, а также в положении 15 стероидного скелета. У некоторых видов рода Penicillium была установлена неспособность превращать молекулу прогестерона в другое стероидное соединение. Отдельные виды Penicillium были разделены на 5 групп по способности их ?ерментных систем вызывать превращения стероидной молекулы прогестерона.     

Microbiological transformation of steroids

Описана трансформация стероида S (Reichstein) штаммом Absidia orchidis 310 в смеси кортизона, 11-α-эпикортизола и кортизола. Приводится метод аналитической оценки превращения стероида. Было исследовано влияние величины pH среды (оптимум 6,5), температуры (27–30°C), вида и количества растворителя (наиболее удобен метиловый спирт в количестве 2–4 мл. (100 мл среды), числа оборотов при перемешивании в течение ферментации (оптимально 600 об./мин.), далее, влияние аэрации и концентрации исходного стероида на скорость превращения (оптимально до 60мг/100мл среды). При оптимальных условиях выход стероида S, по аналитическим данным, составляет 48,9% кортизола и 43,9% 11α-эпикортизола; кортизон образуется только в микроскопических количествах.     

Molecular detection of alpha-glucosidase inhibitor-producing actinomycetes

In this study, we demonstrate the use of a PCR-based method for the detection of the specific genes involved in natural-product biosynthesis. This method was applied, using specifically designed PCR primers, to the amplification of a gene segment encoding for sedo-heptulose 7-phosphate cyclase, which appears to be involved in the biosynthetic pathways of C7N aminoacyclitol or its keto analogue-containing metabolites, in a variety of actinomycetes species. The sequences of DNA fragments (about 540 bp) obtained from three out of 39 actinomycete strains exhibited a high degree of homology with the sedo-heptulose 7-phosphate cyclase gene, which has been implicated in acarbose biosynthesis. The selective cultivation conditions of this experiment induced the expression of these loci, indicating that the range of C7 aminoacyclitol or its keto analogue-group natural products might be far greater than was previously imagined. Considering that a total of approximately 20 C7 aminoacyclitol metabolites, or its keto analogue-containing metabolites, have been described to date, it appears likely that some of the unknown loci described herein might constitute new classes of C7 aminoacyclitol, or of its keto analogue-containing metabolites. As these metabolites, some of which contain valienamine, are among the most potent antidiabetic agents thus far discovered, the molecular detection of specific metabolite-producing actinomycetes may prove a crucial step in current attempts to expand the scope and diversity of natural-product discovery.     

The transformation and resolution of 8-aza steroids by fungi.

1. When (+/-)-8-aza-d-homo-oestrone 3-methyl ether was added to a culture of Aspergillus ochraceus, the laevo-rotatory enantiomer is reduced to a 17A alcohol. Thus (+/-)-8-aza-d-homo-oestrone 3-methyl ether has been resolved into both its components, the laevo-rotatory enantiomer being obtained by chromic acid oxidation of the alcohol, and the dextro-rotatory enantiomer as residual precursor from the fermentation. 2. Fungi have been found to demethylate the 3-methyl ethers of 8-aza steroids to the corresponding phenols. In particular, 8-aza-d-homo-oestrone 3-methyl ether was demethylated by Aspergillus flavus and 8-aza-d-homo-oestradiol 3-methyl ether by Cunninghamella blakesleeana.     

Rapid detection of `rare' actinomycetes in environmental samples

Natural products continue to be a useful source of new leads for the pharmaceutical industry. Actinomycetes are prolific producers of natural products and one strategy to increase the possibility of discovering novel chemical entities is to screen actinomycetes considered 'rare' in the environment and previously under-represented in natural product screening collections. We describe a method using bacteriophage as a marker to detect these actinomycetes in environmental samples. This method allows samples to be pre-screened for the presence of target actinomycetes before lengthy isolation programmes are undertaken.     

An airlift loop reactor for the transformation of steroids by immobilized cells

Summary The flow behaviour of calcium alginate beads in an airlift reactor (ALR) with external loop was dependent on the airflow rate into and the amount of beads in the reactor. The performance of immobilizedArthrobacter simplex for the ¹-dehydrogenation of hydrocortisone in the ALR compared favourably to that in a stirred tank reactor. The physical stability of the calcium alginate beads was significantly greater in the ALR.     

Recent developments in the fungal transformation of steroids

Steroids constitute a vital part of the active ingredients in pharmaceuticals and intermediates used to produce medicines, and their application in chemical and agrochemical fields is also valued. The complex stereochemistry of steroids requires attention to regio- and stereoselectivity of the reaction during preparation, and therefore, biocatalytic methods are appropriate for their production. This work reviews the recent application of fungi for the transformation of different steroid substrates, new biotransformation techniques, recently characterized reactions, and practical aspects, covering the period from 1990 to 2014. The future prospects of fungal biotechnology and biotransformation in the biopharmaceutical industry are also considered.     

Bacteriolysis by thermophilic actinomycetes

Eighty cultures of thermophilic actinomycetes isolated from Bombay soils were screened for lytic action on heat-killed cells of six test bacteria. The majority possessed lytic properties of varying degrees. Gram-negative bacteria were far more susceptible than gram-positive ones. Acetone-precipitated lytic enzyme systems of two cultures — A 29 (Thermomonospora fusca) and A 60 (Thermoactinomyces vulgaris) proved stable at 70 C for 4 1/2 hr and at 80 C for 50 min but were destroyed in 10 min at 100 C. Optimum activity was at 70 C and at pH 9.0. Lytic enzyme systems from both these cultures partly hydrolysed casein and rapidly liquefied gelatin.     

总状毛霉对4-烯-3-酮甾体的生物转化研究

从土样中筛选到一株能转化甾体的菌株,经形态观察,鉴定为总状毛霉(Mucor racemosus)。首次利用该菌株对4-烯-3-酮类甾体衍生物进行生物转化,目的是合成具有潜在活性的羟基类4-烯-3-酮衍生物。转化条件为27℃,220r/min振荡培养4d。转化产物经乙酸乙酯萃取,用硅胶柱层析法分离,通过红外、质谱和核磁分析确定了甾体转化产物的化学结构。黄体酮生物转化得到的产物是14α-羟基-4-孕甾烯-3,20-二酮和7α,14α-二羟基-4-孕甾烯-3,20-二酮;4-雄烯二酮的转化产物是14α-羟基-雄甾-4-烯-3,17-二酮1、4α,17β-二羟基-雄甾-4-烯-3-酮和6α,17β-二羟基-雄甾-4-烯-3-酮。研究结果表明总状毛霉具有转化甾体的能力,对4-烯-3-酮类甾体进行生物转化的主要产物是14α-羟基甾体衍生物。     

Use of water-organic systems in the enzymatic transformation of steroids

This review surveys existing data on the use of water-organic systems, one of the promising methods of enzymic transformation of steroids. The procedures for reducing the inhibitory effect of solvents on enzymes and microbial cells are discussed. It is shown that hydrophobic capacity of gels and substrates, polarity of solvents, and coefficients of the distribution of reaction components between phases are to be taken into consideration when performing steroid transformation in the presence of organic solvents in order to increase its efficiency.