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Addressing the whole-cell system, the cytochrome CYP109B1 from Bacillus subtilis was found to catalyze the oxidation of (+)-valencene (1) yielding nootkatol (2 and 3) and (+)-nootkatone (4).
This study demonstrates that the identification of new P450s capable of producing valuable compounds can basically be achieved by screening of recombinant P450 libraries. BackgroundThe sesquiterpenes are a large class of terpenoid compounds and common constituents of plant essential oils. Schematic experimental setup for the conversion of (+)-valencene (1) by CYP109B1 in a biphasic system. Chemical synthesis of (+)-nootkatone (4) from (+)-valencene (1) has been studied with tert-butyl chromate [5], via copper(I)-mediated oxidation by alkyl hydroperoxides [6] and with surface-functionalized silica supported by metal catalysts such as Co2+ and Mn2+ [7].
In our approach to find new P450s capable to oxidize (+)-valencene (1) regioselectively at allylic C2-position, 125 bacterial P450s from an E. When (+)-valencene (1) was added directly to the cell suspension, the oxidation of the substrate by CYP109B1 proceeded with 270 nM min-1 and after 8 h 6% conversion was achieved. Gas chromatography (GC) analysis of reaction mixtures after the in vivo (+)-valencene (1) biotransformation revealed several new peaks besides the substrate peak (Figure 2). Aqueous-organic two-liquid-phase systems (further referred to as biphasic systems) represent a powerful biotechnological tool for biotransformations of toxic organic compounds [28–30].
It is known that some organic solvents used as a second phase can damage the microbial cells [31]. After addition of 2% DMSO the conversion of (+)-valencene (1) rose significantly in all biphasic systems employed, but was still lower in comparison to the aqueous system where 25% conversion was reached (Table 2).
Generally, the volumetric productivities of the biphasic systems were lower than those of the aqueous system (990 nM min-1). Taking into account both, the reduction of byproduct formation and volumetric productivities, the best performance was observed in the biphasic system comprising 10% dodecane and 2% DMSO.
Conversion of (+)-valencene (1) was carried out in 20 ml volume for the biphasic system comprising 10% dodecane and 2% DMSO.
At a constant cww of 70 g l-1 (corresponding to 18.4 g l-1 cell dry weight (cdw)) higher substrate concentrations resulted, as expected, in higher overall amounts of desired products, however the substrate conversion values decreased (Table 3). ConclusionIt was demonstrated that the identification of new P450s capable of producing valuable compounds can basically be achieved by the screening of recombinant P450 libraries. Batteri della rizosfera (Bacillus subtilis BA 41, Pseudomonas fluorescens PN 53, Pseudomonas spp. L’applicazione MICOSAT F® MO WP comporta una minore suscettibilita della pianta verso turbe parassitarie a seguito della sintesi e traslocazione di sostanze polifenoliche. Il fertilizzante va disciolto in acqua in modo naturale, e distribuito al suolo per fertirrigazione, con manichette forate, con atomizzatori a barre o con palo iniettore (per piccole superfici). Possibile anche la distribuzione per aspersione fogliare (atomizzatori o motopompe munite di lancia). E preferibile usare il fertilizzante biologico MICOSAT F® LEN WP da solo, pur essendo il prodotto compatibile con fertilizzanti, erbicidi, insetticidi e molti fungicidi consentiti nelle produzioni convenzionali e biologiche.
MICOSAT F® MO WP e un fertilizzante di origine naturale che facilita i processi di umificazione e mineralizzazione della sostanza organica. Fertilizzanti naturali con micorrize per piante e colture, risanamento suoli e protezione da parassiti e funghi patogeni. Fertilizzanti naturali > per piante – per agrumi – per ortaggi – per pomodori – per gerani – per limoni – per orto – per fioritura - per fragole - per limoni - vigneto - olivi - cereali - mais.
Biostimolanti > in agricoltura – per piante – per cereali – per olivo – per vigneto – per angurie – dove acquistare – biologico – ortaggi – qualita uva – radicale mais. Department of Pharmacognosy, Faculty of Pharmacy, Shiraz University of Medical Sciences and Health Services, Shiraz, 71345, I. JavaScript is currently disabled, this site works much better if you enable JavaScript in your browser. This chapter describes the cell and colony morphology, cultural and biochemical characteristics of oral microbes that are mainly isolated from supragingival plaques, including gram-positive bacteria of the genera Actinomyces, Bifidobacterium, Lactobacillus, Rothia, Staphylococcus, and Streptococcus, as well as gram-negative bacteria of the genera Leptotrichia and Veillonella. Actinomyces are irregular gram-positive bacilli and are also commonly found anaerobic bacteria in oral samples.
There are differences at the species level regarding oxygen sensitivity, but a primary culture of Actinomyces requires an anaerobic environment. All strains of actinomycetes can ferment glucose and fructose to produce acid, without producing gas.
Actinomycetes are normal members of the oral flora and are the dominant bacteria in dental plaque. Humans’ and other warm-blooded animals’ oral cavity, including subgingival plaque, transparent plaque, and dental calculus, are the main sites of growth.
Bifidobacterium is a genus of bacteria with various forms; nonmotile they are gram-positive, nonsporulating, anaerobic bacilli.
The main terminal acid products in liquid culture medium containing glucose are acetic acid and lactic acid, but a few species also make formic acid and succinic acid. Enter your message here, then click a€?Senda€? button to send to the contact person of this company. Allylic oxidation of the sesquiterpene (+)-valencene (1) provides an attractive route to this sought-after flavoring. However, when the in vivo biooxidation of (+)-valencene (1) with CYP109B1 was carried out in an aqueous milieu, a number of undesired multi-oxygenated products has also been observed accounting for approximately 35% of the total product. The biphasic reaction system described in this work presents an attractive way for the production of (+)-nootkatone (4), as it is safe and can easily be controlled and scaled up.
The parent sesquiterpenes are often readily available and their oxygenation gives derivatives which are sought-after fragrances and flavorings, pharmaceuticals or building blocks for chemical synthesis [1].
However, these synthetic methods are neither safe nor environment-friendly, because they involve toxic heavy metals or peroxides.Biotechnological processes to achieve this oxidation have also been designed.
In this system, the NADH oxidation rate was 64 nmol nmol-1 P450 min-1 and approximately 10% conversion of 200 ?M (+)-valencene (1) was observed, however, only if all three proteins were added to the reaction mixture. This approach allows 1) the use of high overall concentrations of hydrophobic toxic substrates by regulating substrate and product concentrations in the aqueous biocatalyst phase, and 2) simple in situ product recovery into organic phase. In these systems, however, the volumetric productivities were low (? 100 nM min-1) and after 8 h only 2% or less (+)-valencene (1) conversion was achieved in all systems tested. Generally, the addition of an organic phase led to a strong reduction of the generated multi-oxygenated byproducts causing a shift of the product distribution towards the mono-oxygenated compounds nootkatol (2 and 3) and (+)-nootkatone (4) (Table 2).


For example, addition of 10% of isooctane resulted in a 60% loss in original volumetric productivity (400 nM min-1), 20% of isooctane – in an almost 70% loss (Table 2). The high log D for hexadecane implies a larger substrate holding capacity of this solvent, which might explain the significantly reduced volumetric productivity of the whole-cell process for this solvent compared to dodecane.
The influence of cell density and substrate concentration on the yield of nootkatol (2 and 3) and (+)-nootkatone (4) was investigated. In comparison, the concentration of desired products in the aqueous system under the same conditions reached 83 mg l-1. The P450-library used in this study is easy to handle and allows high throughput screening of various substrates.For the biooxidation of (+)-valencene (1) with E. Please, upgrade to a different browser or install Google Chrome Frame to experience this site. Il fertilizzante naturale contribuisce al risanamento biologico dei suoli (ristoppi), apporta maggiore sviluppo dell’apparato radicale, maggiore accesso ai nutrienti del suolo e all’acqua, riduzione della crisi di trapianto, maggiore vigore iniziale e maggiore attecchimento delle piante dopo la loro collocazione a dimora.
Per tutte le colture iniziare le applicazioni dalle prime fasi vegetative e continuare fino alla maturazione dei primi frutti. MICOSAT F® MO WP e indispensabile per instaurare la relazione tra il microbiota in esso contenuto e la pianta ospite tramite il contatto radicale. When they were first discovered, actinomycetes were believed to be fungi or were grouped as “other microorganism.” Recently, numerous studies have shown that actinomycetes have general characteristics common to bacteria and were classified as prokaryotic organisms. The cells are usually rod-shaped, but can occasionally be club-shaped with irregular arrangements including single, paired, chain, clusters, and fence-shaped. Spider-like microcolonies or branched mycelia can form on agar plates after 18–24 h incubation. Other tests, including fermentation of raffinose, xylose, cellobiose, laetrile, ribose, salicin, catalase production, reduction test using nitrate and nitrite, urea hydrolysis, or gelatin, can help identify different species. This species is often involved in eye infections and is occasionally seen in advanced actinomycosis.
These bacteria were first isolated from infant feces and attracted attention because of their important physiological significance to the host organism.
They also appear as long cells with many branches and slightly branching spoon-shaped cells.
Colonies formed on agar plates are convex, creamy or white, glossy, smooth, neat-edged, sticky, and soft.
However, no butyric acid or propionic acid is formed, and no CO2 is generated (with the exception of gluconate degradation). So far, chemical methods to produce (+)-nootkatone (4) from (+)-valencene (1) involve unsafe toxic compounds, whereas several biotechnological approaches applied yield large amounts of undesirable byproducts.
The formation of these byproducts was significantly reduced when aqueous-organic two-liquid-phase systems with four water immiscible organic solvents – isooctane, n-octane, dodecane or hexadecane – were set up, resulting in accumulation of nootkatol (2 and 3) and (+)-nootkatone (4) of up to 97% of the total product. For example, (+)-valencene (1) is found in citrus oils and can be cheaply obtained from oranges. Recent attempts include the manufacturing of (+)-nootkatone (4) with green algae like Chlorella or Euglena [8], fungi such as Aspergillus niger, Fusarium culmorum [9], Mucor sp.
The average expression level of CYP109B1 verified through CO-difference spectra was 160 mg l-1, which corresponds to 7.6 mg g-1 cell wet weight (cww). DMSO increases the solubility of (+)-valencene (1), which is poorly soluble in the aqueous reaction medium.
Interestingly, this phenomena has also been reported for the P450BM3 wild type enzyme and mutants, which accept nootkatone as substrate much better than valencene [25]. Four water-immiscible organic solvents (n-octane, isooctane, dodecane and hexadecane) were chosen to set up biphasic systems and compared to the aqueous system. Presumably, the substrate holding capacity of the organic solvents was too high, preventing its oxidation in the aqueous phase.
Moreover, the amount of byproducts decreased with increasing amounts of organic solvents from 10 to 20%. The highest log D for (+)-valencene under these conditions was determined in isooctane (0.87), presumably also contributing to the low volumetric productivities in addition to the toxicity of this solvent.
Thus, higher cell densities at a constant substrate concentration lead to higher substrate conversion and higher overall amounts of nootkatol (2 and 3) and (+)-nootkatone (4), but lower product yields per g of cells. E fondamentale che questo avvenga per permettere ai microrganismi di instaurare la loro simbiosi con la pianta per poter esprimere appieno le loro potenzialita in termini di incremento dello sviluppo e di difesa dagli stress biotici e abiotici. In the 1984 edition of Bergey’s Manual of Determinative Bacteriology, Actinomyces were included in the group of gram-positive irregular bacilli. The culture atmosphere requires CO2, as the culture grows poorly or not at all under ordinary atmospheric environment. Mature colonies are characteristically less than 2 mm in diameter, raised, white, opaque, and molar-shaped. It can take part in mixed bacteria infections and is one of the pathogenic bacteria in root caries. The mature colonies on the surface of BHI agar measures are less than or equal to 2 mm in diameter. Species that are important human gut bacteria include Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium adolescentis, and Bifidobacterium longum. Bifidobacterium can ferment carbohydrates to produce acid, and generally do not reduce nitrate or produce urease.
In the present work 125 cytochrome P450 enzymes from bacteria were tested for regioselective oxidation of (+)-valencene (1) at allylic C2-position to produce (+)-nootkatone (4) via cis- (2) or trans-nootkatol (3).
The best productivity of 120 mg l-1 of desired products was achieved within 8 h in the system comprising 10% dodecane.
Recently the valencene synthase gene from orange has been cloned and functionally expressed in E.
The organic phase acts as substrate reservoir and allows the accumulation of the mono-oxygenated conversion products, which prevents them from overoxidation. Since natural electron transfer partners for most of these 125 P450s are unknown, PdR and Pdx from P.
Numbered peaks represent cis-nootkatol (2), trans-nootkatol (3), (+)-nootkatone (4) and overoxidation products (5, 6 and 7).
Since in all experiments 70 g cww l-1 were used, the final P450 concentration was approximately 530 ?g ml-1.


In consequence the volumetric productivity could be increased up to 990 nM min-1 and after 8 h 25% of the substrate was converted. Detailed biochemical characterization of CYP109B1 is a topic of our current investigations. A time course of the reaction demonstrated that these byproducts appeared shortly after the two nootkatol-isomers (2 and 3) and nootkatone (4) were formed, which indicates that these compounds could represent products of overoxidation (data not shown). Control experiments showed that none of the organic solvents was oxidized by the host strain.
Although the addition of organic solvents reduced the volumetric productivity of the whole-cell process, the production of nootkatol is promising. The antimicrobial activity of the oil was tested on three strains of Gram positive bacteria (Staphylococcus aureus, S.
Common members of the Actinomyces genus in oral microbiology are Actinomyces israelii, Actinomyces naeslundii, Actinomyces odontolyticus, and Actinomyces viscosus. The main distinguishing characteristic of Actinomyces cells is that the cell wall does not contain DAP and glycine.
This bacterium is mainly a pathogen of the face and neck, and causes lung and abdominal actinomycosis. It is often detected in clinical specimens of periodontitis or infected root canals, but the pathogenesis is unclear.
Their distinct cell morphology can be helpful in differentiating bacteria belonging to this genus. The P450 activity was supported by the co-expression of putidaredoxin reductase (PdR) and putidaredoxin (Pdx) from Pseudomonas putida in Escherichia coli.
Cell free enzymatic reactions for the conversion of (+)-valencene (1) exploiting enzymes from Cichorium intybus L. Expression of PdR and Pdx was verified by sodium dodecyl sulphate polyacrylamid gel electrophoresis (SDS-PAGE) [see Additional file 1]. Furthermore, the fragmentation spectra and molecular weights of these compounds indicated the incorporation of two oxygen atoms into (+)-valencene (1) [see Additional file 2].
The addition of 2% DMSO had no considerable effect on cell viability in aqueous systems and in systems with dodecane or hexadecane, but led to further diminution of cell viability with octane and isooctane (Figure 3). Basically, addition of 10% organic solvent reduced the amount of byproducts to less than 12% of the total product; 20% organic phase resulted in less than 7% byproducts. Considering the accumulation of nootkatol (2 and 3) it should be noted that a bottleneck reaction seems to be the oxidation of nootkatol (2 and 3) to (+)-nootkatone (4).
They are characterized by a relatively high GC content in their DNA, ranging from 57% to 69% (Tm method).
This bacterium can lead to actinomycosis at many locations such as face, neck, chest, abdomen, and eye, and can cause infection of the female genital tract and knee joint empyema.
The important identifying feature of this species is that the colony turns dark red after 2 days of culture on the surface of blood agar under an anaerobic environment. The selective oxidation of (+)-valencene (1) at allylic C2-position yields cis- (2) and trans-nootkatol (3), which can be further oxidized to (+)-nootkatone (4), a high added-value commercial flavoring (Figure 1).
It is detected in oral mixed infections such as gingivitis, periodontitis, and pericoronitis, but the link between A. The percent GC in Bifidobacterium DNA ranges from 55% to 67% when analyzed by the Tm or Bd method. Traditionally nootkatone is extracted from grapefruits, and its price and availability is dependent on the annual harvest, which is restricted to a narrow producing area and very sensitive to weather conditions. Cytochrome P450s belong to a superfamily of heme b-containing proteins that accept a vast number of organic compounds and catalyze an enormous variety of oxidative reactions.
In the screening two enzymes were found to be able to oxidize (+)-valencene (1), namely CYP109B1 from B. Dodecane had the slightest effect on volumetric productivity; about 87% could be retained in the system with 10% dodecane after 8 h conversion (860 nM min-1). For example, CYP109B1 could be optimized for higher affinity and activity towards (+)-valencene (1) and nootkatol (2 and 3) with the goal to achieve an increased productivities of the whole-cell process and higher amounts of (+)-nootkatone (4).
Thus, various approaches for chemical or biotechnological production of (+)-nootkatone have been investigated in the past years (very recently reviewed by Fraatz et al. These reactions include hydroxylation, epoxidation, heteroatom oxidation, C-C bond cleavage via successive hydroxylations and many other complex reactions [22, 23].Interestingly, (+)-nootkatone (4) has been shown to inhibit activity of some human P450s, including CYP2A6 and CYP2C19 [24]. Interestingly, the retained volumetric productivities with hexadecane were significantly lower then those observed in the presence of n-octane, especially at 20% (Table 2), although the viability assay demonstrated that hexadecane is benign for E. The results indicate that the fruits have potential for use as an aromatic antimicrobial agent.
Thus these products were assumed to be products of further oxidation of the two nootkatol isomers (2 and 3), which is in agreement with their MS data.
Indeed, a recent report describes the hydroxylation of (+)-valencene (1) by mutants of the cytochromes P450cam from Pseudomonas putida and P450BM3 from Bacillus megaterium [25]. In order to investigate if the primary oxidation products could be protected from overoxidation, we applied an aqueous-organic two-liquid-phase approach as shown in figure 1. While the reported P450cam-mutants demonstrated quite low activity, the P450BM3-mutants, although being active, had low chemo- and regioselectivity and produced up to six byproducts besides nootkatol (2 and 3) and (+)-nootkatone (4). Another report described a membrane-bound plant P450 monooxygenase from Hyoscyamus muticus, which was able to oxidize (+)-valencene (1) to nootkatol [16].
Because of its high regioselectivity, CYP109B1 was chosen as biocatalyst for further studies. The library contains, for example, the complete P450 complement of Streptomyces coelicolor A3(2), Bacillus subtilis 168, Nocardia farcinica IFM 10152 and Bradyrhizobium japonicum USDA110.
Construction and application of this P450 library has recently been reported with respect to steroid oxidation [26].The actual screening identified CYP109B1 from Bacillus subtilis, which catalyzed the regioselective oxidation of (+)-valencene (1) to nootkatol (2 and 3) and (+)-nootkatone (4).
In order to reduce byproduct-formation biphasic systems with water immiscible organic solvents were applied and the best system was scaled-up. Mozaffarian, The Family of Umbelliferae in Iran-Keys and Distribution, Research Institute of Forests and Rangelands Press, Tehran, 1983, p.



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