Microbial Baeyer–Villiger oxidation of 5α-steroids using Beauveria bassiana. A stereochemical requirement for the 11α-hydroxylation and the lactonization pathway

Beauveria bassiana KCH 1065, as was recently demonstrated, is unusual amongst fungal biocatalysts in that it converts C₁₉ 3-oxo-4-ene and 3β-hydroxy-5-ene as well as 3β-hydroxy-5α-saturated steroids to 11α-hydroxy ring-D lactones. The Baeyer–Villiger monooxygenase (BVMO) of this strain is distinguished from other enzymes catalyzing BVO of steroidal ketones by the fact that it oxidizes solely substrates with 11α-hydroxyl group. The current study using a series of 5α-saturated steroids (androsterone, 3α-androstanediol and androstanedione) has highlighted that a small change of the steroid structure can result in significant differences of the metabolic fate. It was found that the 3α-stereochemistry of hydroxyl group restricted “normal” binding orientation of the substrate within 11α-hydroxylase and, as a result, androsterone and 3α-androstanediol were converted into a mixture of 7β-, 11α- and 7α-hydroxy derivatives. Hydroxylation of androstanedione occurred only at the 11α-position, indicating that the 3-oxo group limits the alternative binding orientation of the substrate within the hydroxylase. Only androstanedione and 3α-androstanediol were metabolized to hydroxylactones. The study uniquely demonstrated preference for oxidation of equatorial (11α-, 7β-) hydroxyketones by BVMO from B. bassiana. The time course experiments suggested that the activity of 17β-HSD is a factor determining the amount of produced ring-D lactones. The obtained 11α-hydroxylactones underwent further transformations (oxy-red reactions) at C-3. During conversion of androstanedione, a minor dehydrogenation pathway was observed with generation of 11α,17β-dihydroxy-5α-androst-1-en-3-one. The introduction of C1C2 double bond has been recorded in B. bassiana for the first time.     

On the microbial transformation of α,β-unsaturated aryl ketones by the fungus Beauveria bassiana

α,β-unsaturated aryl ketones, 1a–12, have been submitted to the action of the fungus Beauveria bassiana (ATCC 7159) in growing conditions. The saturation of the double bond strictly depends from the substituent α to the carbonyl group. The saturated ketone is then oxidised in a Baeyer-Villiger type reaction. This new oxidative capacity of the fungus has been studied and the adaptability of the micro organism towards structural modifications has been investigated.     

Microbial conversion of steroids III: 11α-hydroxylation by fungal mycelium

Summary A mutant of Aspergillus ochraceus has been developed, which converts progesterone (substrate concentration 40 g/l) to 11-hydroxyprogesterone in high yields (90%). The formation of 6, 11-dihydroxy compound is minimal at high substrate concentrations. The bioconversion rate is also much higher. The various parameters for optimal conversion have been standardised.     

Studies of 11β-hydroxylation by beef adrenal mitochondria

The 11β-hydroxylations of androstenedione (Δ⁴A), 11-deoxycortisol (S) and deoxycorticosterone (DOC) were studied using mitochondria from calf or heifer adrenal tissue. Standard assay conditions were: non-radioactive androstenedione (30.0μM) 11-deoxycortisol (24.5 μM) or deoxycorticosterone (26.0 μM) plus 6.0 × 10⁴ d.p.m. ¹⁴C-steroid, 0.2 mM NADPH, 1.0 mM Mg²⁺ 1.0 mM Ca²⁺ and the mitochondrial fraction equivalent to 20 mg of adrenal tissue in a final vol. of 3ml of 0.1 M HEPES buffer. pH 7.4. Incubations were performed at 37°C for 4min. Product formation under these conditions was identical to product formation measured when the NADPH and Ca²⁺ were replaced with 10mM malate. The 11β-hydroxylation of Δ⁴A showed a requirement for NADPH and oxygen, indicating that the enzyme involved is a mixed-function oxidase. The K_m values for calf adrenal mitochondria were 3.8, 8.5 and 8.0μM for Δ⁴A, S and DOC, respectively. For heifer adrenal mitochondria, the K_m values were 12 and 15μM respectively, for Δ⁴A and S. Competition studies in which equal amounts of two substrates were incubated simultaneously, revealed that Δ⁴A, S and DOC did not compete for the same enzymatic site, but were hydroxylated to the same degree in the presence or absence of each of the other two precursors. The 11β-hydroxylations of S and DOC were stimulated by Mg²⁺ at a concentration of 1.0 mM, while the 11β-hydroxylation of Δ⁴A was inhibited by this concentration of Mg²⁺. In experiments in which the mitochondria were preheated at 50°C for 6 min, the 11β-hydroxylation of Δ⁴A, under standard assay conditions, was 96% of the unheated value, while the 11β-hydroxylation of S and DOC was 77 and 59%, respectively, of the unheated values. These studies indicate that there are three substrate specific 11β-hydroxylases in beef adrenal mitochondria.     

Microbial Baeyer-Villiger oxidation of steroidal ketones using Beauveria bassiana: Presence of an 11α-hydroxyl group essential to generation of D-homo lactones

This paper demonstrates for the first time transformation of a series of 17-oxo steroidal substrates (epiandrosterone, dehydroepiandrosterone, androstenedione) by the most frequently used whole cell biocatalyst, Beauveria bassiana, to 11α-hydroxy-17a-oxa-d-homo-androst-17-one products, in the following sequence of reactions: 11α-hydroxylation and subsequent Baeyer-Villiger oxidation to a ring-D lactone. 11α-Hydroxyprogesterone, the product of the first stage of the progesterone metabolism, was further converted along two routes: hydroxylation to 6β,11α-dihydroxyprogesterone or 17β-acetyl chain degradation leading to 11α-hydroxytestosterone, the main metabolite of the substrate. Part of 11α-hydroxytestosterone underwent a rare reduction to 11α-hydroxy-5β-dihydrotestosterone. The experiments have demonstrated that the Baeyer-Villiger monooxygenase produced by the strain catalyzes solely oxidation of C-20 or C-17 ketones with 11α-hydroxyl group. 17-Oxo steroids, beside the 11α-hydroxylation and Baeyer-Villiger oxidation, also underwent reduction to 17β-alcohols; activity of 17β-hydroxysteroid dehydrogenase (17β-HSD) has significant impact on the amount of the formed ring-D δ-lactone.     

16α-hydroxylation of progesterone by a strain ofactinomyces globosus

В систематическое исследование трансформации свойств Actinomycetes было было установлено, что 17 из 76 видов тестировани е преобразованы прогестерон по 16 га-гидрокси -п рогестерона. Оптимальные условия для этой трансформац ии были изучены этой трансформации б ыли изучены следующие результат ы:

1. (1)
   Оптимальное рН для данного типа трансформации была 6–7. На нижней hydroxylation ценностей была меш ает.     
   
   

Regeneration of the progesterone 11α-hydroxylase of Rhizopus nigricans by the use of periodate

Summary The cell-free progesterone 11-hydroxylase enzyme of Rhizopus nigricans can be directly regenerated by periodate oxidation. This permits action of the enzyme over a period of hours with an activity similar to that in the presence of an NADPH generating system.     

Solvent damage during free cell catalysis and its avoidance: studies of 11α-hydroxylation of progesterone by Aspergillus ochraceus

The solvent tolerance of the progesterone 11α-hydroxylase system of Aspergillus ochraceus has been defined and, given its limited extent for conventional organic solvents, a number of natural oils have been examined. They have been found superior and represent an interesting solvent class for organic reactants of partial polarity. The study emphasizes that solvents for the products of biocatalytic action on organic reactants must often be partially polar and must not interact strongly with cellular lipids.     

Solvent damage during immobilised cell catalysis and its avoidance: studies of 11α-hydroxylation of progesterone by Aspergillus ochraceus

Summary The tolerance towards conventional organic solvents of the progesterone 11-hydroxylase system in alginate immobilised Aspergillus ochraceus has been examined. Though greater than that for the enzyme system in free cells it is still too limited for practical use. Substitution of natural oils gave a more stable catalyst system. The activity versus a free cell catalyst was not attractive in short term use, but may be over the longer term.     

The uptake of Aspergillus ochraceus spores on diatomaceous particles and their use in the 11α-hydroxylation of progesterone

Summary The entrapment of Aspergillus ochraceus spores on to diatomaceous earth particles occurs rapidly, the number of spores entrapped at equilibrium being dependent upon the initial spore:particle ratio. The rate of agitation during spore uptake markedly affected entrapment. Immobilized spores carried out the 11-hydroxylation of progesterone as effectively as free spores.     

The pathogenicity of Beauveria bassiana: what happens after an endophytic phase in plants?

The banana weevil Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae) is a serious constraint to banana (Musa spp.) production throughout the world. The entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) offers a potential weevil management option, but conventional delivery mechanisms have limited its success. As an endophyte, however, B. bassiana can be efficiently delivered to banana planting materials for the potential management of C. sordidus. However, entomopathogens can change morphology and efficacy against their target host when successively sub-cultured on artificial media or when exposed to certain physical and chemical environmental conditions. Whether such changes occur in B. bassiana after an endophytic phase inside a banana plant remains unknown. The primary aim of our study was to evaluate the viability, growth, sporulation and pathogenicity of endophytic B. bassiana. To attain this, two sets of experiments, namely morphological characterization and larval bioassays, were conducted under laboratory conditions. In these experiments, growth and pathogenicity of the wild-type B. bassiana strain G41, obtained originally from banana farms, was compared with the endophytic B. bassiana strain G41, re-isolated from the rhizome of B. bassiana-inoculated banana plants at one month post-inoculation. Morphological characterization, conidial germination, colony growth and sporulation rate was assessed on SDAY media while pathogenicity was determined 15 days after immersing the larvae of C. sordidus in different conidial doses. No differences were observed in colony appearance and growth rate between the endophytic and wild-type strain. Percentage conidial germination for the endophytic strain (91.4-94.0%) was higher than for the wild-type (86.6-89.7%). LD50 equated 1.76 x 10(5) and 0.71 x 10(5) conidia/ml for the wild-type and endophytic B. bassiana strains, respectively, but did not differ between strains. Our study demonstrated that, after an endophytic phase inside the banana plant, B. bassiana retains it morphology and pathogenicity against the banana weevil larvae; and thus can offer protection against the damaging larvae feeding inside the rhizome.     

Microbial C-hydroxylation and β-4-O-methylglucosidation of methyl-benzamide 7-azanorbornane ethers with Beauveria bassiana

N-Substituted 7-azanorbornanes were prepared by acylation of easily accessible 7-azanorbornane hydrochloride. Derivatives possessing an electron-withdrawing docking/protecting group and bearing an aryl methylether were subjected to biotransformation with the fungus Beauveria bassiana ATCC 7159. O-Demethylation and β-4-O-methylglucosidation reactions were observed for the major metabolite in this biotransformation (isolation yields: 6, 30%; 11a, 44%; 11b, 47%; 11c, 14%). C-Hydroxylation on an unfunctionalized carbon was also observed in most of the cases.     

Effect of two-steps substrate addition on steroids 11β-hydroxylation by Curvularia lunata CL-114

In this paper, the effect of substrate 17α-hydroxypregn-4-ene-3,20-dione-21-acetate (RSA, Cortexolone-21-acetate) on the expression of cytochrome P450 and the production of hydrocortisone by Curvularia lunata CL-114 was studied. Meanwhile the effect of pH on the production of hydrocortisone was observed. Based on the effect of substrate RSA two-steps addition on cytochrome P450 expression and hydrocortisone production, a novel fermentation process was established as follows: 0.3 g/L RSA was added for the first time after 16 h of inoculation, followed by the second addition of 0.7 g/L RSA after 8 h later, then pH was regulated to constant 6.5 after another 8 h till the end of fermentation. The results showed that the novel process was much better than the original one on improving the induction of cytochrome P450 and production of hydrocortisone, and the hydrocortisone yield had achieved an improvement of 17.6% higher than that of the original process correspondingly.     

Steroid 11ß-hydroxylation by a fungal microsomal cytochrome P450

The steroid 11ß-hydroxylase activity of the fungus Cochliobolus lunatus was increased about 100-fold by cultivation of mycelia for 4–5 h with 20-hydroxymethyl-1,4-pregnadien-3-one. Cell-free extracts revealed a maximum activity of 45 nmol 11ß-hydroxyprogesterone/h·mg protein in the 100,000 g pellet fraction. The 11ß-hydroxylation was dependent on NADPH. The formation of 11ß-hydroxyprogesterone correlated linearly with the cytochrome P450 concentration. The fungal 11ß-hydroxylase transformed both 21-methyl and 21-hydroxymethyl steroids. The enzyme showed a broader substrate specificity and lower regioselectivity as compared with the adrenal cytochrome P45011ß system. The fungal cytochrome P450 was partially purified to a specific content of 700 pmol P450/mg protein. Western blots showed that polyclonal antibodies against cytochrome P45011 from Rhizopus nigricans cross-react with a 60 kD protein of partially purified fractions. The NADPH-cytochrome c reductase was enriched up to a specific activity of 20 U/mg protein. Polyclonal antibodies against NADPH-cytochrome P450 reductases from Candida maltosa and rat liver cross-reacted with the fungal reductase. It is concluded that the 11ß-hydroxylase of Cochliobolus lunatus represents a microsomal two-component monooxygenase system which is composed of a cytochrome P450 (M_r 60 kD) and a NADPH-cytochrome P450 reductase (M_r 79 kD).     

Effect of Beauveria bassiana and Metarhizium anisopliae on the adult African rice gall midge (AfRGM – Orseolia oryzivora)

Abstract

Rice is an important staple crop whose production is limited by array of insect pests and diseases. African rice gall midge (AfRGM) Orseolia oryzivora Harris & Gagné (Diptera: Cecidomyiidae) is a major insect pest of lowland rice ecology in Africa. Heavy yield losses have been recorded in many farmers’ rice fields. Use of synthetic insecticides has fostered environmental and human health concern that initiates a search for alternative control measures such as Entomopathogenic fungi (EPF) – Beauveria bassiana and Metarhizium anisopliae. The experiment was laid out on completely randomised design (CRD) with three replications. The study showed M. anisopliae IC30 had the greatest control effect on adult AfRGM with 90.58% of non-infested tillers. The percentage of non-infested tiller advantage over the control followed the same trend with M. anisopliae IC30 having the greatest value of 50.72%. Tiller infestation had significant negative correlation with chlorophyll content, leaf breadth and grain number.     

Influence of the position of the double bond in steroid substrates on the efficiency of the proton-transfer reaction by Pseudomonas testosteroni 3-oxo steroid Δ4–Δ5-isomerase

Studies of the proton-transfer reaction by Pseudomonas testosteroni 3-oxo steroid Delta(4)-Delta(5)-isomerase with Delta(5(6))- and Delta(5(10))-steroid substrates demonstrate the importance of the position of the double bond for the efficiency of the isomerization process. Thus 3-oxo-Delta(5(6))-substrates have markedly high k(cat.) values, whereas those of 3-oxo-Delta(5(10))-substrates are very low and their apparent K(m) values approach equilibrium dissociation constants. The first step in the isomerization process is: [Formula: see text] which is governed by the k(-1)/k(+1) ratio and is shown to be very similar for the two classes of substrates (3-oxo-Delta(5(6))- and -Delta(5(10))-steroids). They therefore differ in the steps distal to the initial formation of the Michaelis-Menten complex. The use of the deuterated androst-5(6)-ene-3,17-dione substrate enabled us to calculate individual rate constants k(+1) and k(-1) as well as to determine the apparent rate-limiting step in the isomerization process. With the deuterated oestr-5(10)-ene-3,17-dione substrate, no significant isotope effect was observed suggesting that a different rate-limiting step may be operative in this isomerization process. Data are presented that indicate that under optimal concentrations of the efficient androst-5(6)-ene-3,17-dione substrate, the forward reaction for ES complex formation (as defined by k(+1)) is limited only by diffusion and the apparent K(m) does not approach the equilibrium constant, suggesting that the evolution of this enzyme has proceeded close to ;catalytic perfection'.     

The effect of glucose on 11β- and 19-hydroxylation of reichstein's substance S by Pellicularia filamentosa

Summary Experiments in batch-fermenters have demonstrated that the 11- and 19-hydroxylation of Reichstein's Substance S by Pellicularia filamentosa ceases in the absence of glucose. The effects of glucose consumption rate and growth rate on hydroxylation have been investigated using chemostat cultures. With glucose-limited cultures, increased hydroxylation rates were observed with increased glucose consumption rates. With nitrogen-limited cultures, however, some form of glucose-repression exists. The maximum rate of hydroxylation occurred at a glucose consumption rate at which the culture was just nitrogen-limited. The growth rate had no major importance.     

A form of rabbit liver cytochrome P-450 that catalyzes the 7α-hydroxylation of cholesterol

A form of cytochrome P-450 which comigrates with cytochrome P-450_LM4 (molecular weight, 55000) on SDS-polyacrylamide gel was purified from liver microsomes of cholestyramine-treated rabbits. This form of cytochrome P-450 catalyzed the 7α-hydroxylation of cholesterol with an activity of 37.5 pmol/min per nmol cytochrome P-450 in the reconstituted enzyme system containing cytochrome P-450 and NADPH-cytochrome P-450 reductase. The substrate specificity of this form of cytochrome P-450 was compared with cytochrome P-450_LM4 isolated from phenobarbital- and β-naphthoflavone-treated rabbit liver microsomes. The latter two isoenzymes do not catalyze 7α-hydroxylation of cholesterol, but are more active in O-deethylation of 7-ethoxycoumarin and p-nitrophenetole. Ouchterlony double diffusion revealed cross-reactivity between anti-P-450_LM4 (phenobarbital) IgG and cytochrome P-450 isolated from cholestyramine- or β-naphthoflavone-treated rabbit liver microsomes. A two-dimensional iodinated tryptic peptide fingerprint indicated only minor structural differences among these three cytochrome P-450_LM4 preparations.     

Thermal response and horizontal transmission of cameroonian isolates of the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae – Candidates for microbial controls of the banana root borer Cosmopolites sordidus

Beauveria bassiana and Metarhizium anisopliae are two promising microbial agents for biopesticides development against the banana root borer Cosmopolites sordidus. In this study, germination, mycelial growth, and sporulation of six local Cameroonian isolates of those two species were assessed under seven different thermal conditions (13, 15, 20, 25, 29 33, and 37 °C) to select thermo-tolerant isolates. The Transmission potential of the thermo-tolerant isolates was determined at 25 ± 1 °C by dipping adult weevils in conidial suspensions (3.2 × 10⁸) conidia/ml and mixing these with uninoculated weevils in different proportions (0, 10, 30 and 50%), in groups of 30, and assessing the spread of the mycosis within the group over 35 d of co-incubation. Incubation temperature and isolates significantly affected germination, mycelial growth and conidial production. All isolates had large thermal tolerance ranges (13–33 °C) except MIITAC6.4.2 (20–29 °C). Horizontal transmission resulted in mortality of non-inoculated weevils from 4.63 ± 1.77 to 53.3 ± 11.9%. The isolate BIITAC6.2.2 exhibited high auto-dissemination potential and high conidia yield in cadavers. These results demonstrate the potential use of these isolates for biopesticides development against C. sordidus in Central Africa.     

Optimization and development of antidiabetic phytosomes by the Box–Behnken design

Researchers have extensively reviewed on herbs and natural products for their marked clinical efficacy in some recent years, however, maximum of the newly discovered bioactive constituents offer poor bioavailability due to their large size molecules or to their poor miscibility with oils and lipids, thereby limiting their ability to pass across the lipid-rich outer membranes of the enterocytes of the small intestine. Phytosomes are more bioavailable as compared to herbal extracts owing to their enhanced capacity to cross the bio-membranes and thus reaching the systemic circulation. This study was aimed to investigate the development and optimization of antidiabetic phytosomes using a three-factor, three-level the Box–Behnken design (17 batches). The fruits of Citrullus colocynthis (L.) Momordica balsamina and Momordica dioica were extracted using Soxhlet’s apparatus. The phytochemical fingerprint profile of the combined methanolic extracts was done by using high-performance thin layer chromatography (HPTLC). The polynomial quadratic equation analysis was designed to study the response (entrapment efficiency (EE), % yield) of independent significant factors at different levels. Phytosomes were characterized in terms of drug content, particle size, EE, zeta potential and in vitro dissolution. TEM analysis revealed good stability and a spherical, self-closed structure of phytosomes in complex formulations. Average particle size was found to 450?nm. Total flavonoid content was found to be 10.0?±?0.002?μg/g. Optimized formulation was selected and was prepared using A (1:3), B (60?°C) and C (2.5?h) to give maximum yield and entrapment efficiencies (72% and 92.1?±?5.1%). Phytosomes were found to have antidiabetic activity comparable to metformin in low dose. HPTLC showed the presence of the phyto-constituent quercetin.