Enhanced production of ɛ-caprolactone by overexpression of NADPH-regenerating glucose 6-phosphate dehydrogenase in recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> harboring cyclohexanone monooxygenase gene

Whole-cell conversion of cyclohexanone to ɛ-caprolactone was attempted by recombinant Escherichia coli BL21(DE3) expressing cyclohexanone monooxygenase (CHMO) of Acinetobacter calcoaceticus NCIMB 9871. High concentrations of cyclohexanone and ɛ-caprolactone reduced CHMO-mediated bioconversion of cyclohexanone to ɛ-caprolactone in the resting recombinant E. coli cells. Metabolically active cells were employed by adopting a fed-batch culture to improve the production of ɛ-caprolactone from cyclohexanone. A glucose-limited fed-batch Baeyer–Villiger oxidation where a cyclohexanone level was maintained less than 6 g/l resulted in a maximum ɛ-caprolactone concentration of 11.0 g/l. The maximum ɛ-caprolactone concentration was improved further to 15.3 g/l by coexpression of glucose-6-phosphate dehydrogenase, an NADPH-generating enzyme encoded by the zwf gene which corresponded to a 39% enhancement in ɛ-caprolactone concentration compared with the control experiment performed under the same conditions.     

Furfural Derivatives and Phenolic Constituents in Stemona tuberosa Lour

Chemical investigation on the water-soluble constituents of Stemona tuberosa Lour. resulted in the isolation of a previously undescribed furfural derivative namely (S)-5-((R)-hydroxy(5-(hydroxymethyl)furan-2-yl)methyl)-5-methylfuran-2(5H)-one and twenty-five known compounds from the water decoction of the dried root tubers. Their structures were determined by analysis of the extensive spectroscopic data, including 1D/2D NMR, HR-ESI-MS, and ORD, as well as the ECD simulation and comparison. Most of them were phenolic and among them, four compounds were isolated from Stemona plants for the first time. This study uncovers diverse constituents from water decoction of S. tuberosa dedicated for its quality control and allows for the exploitation of chemical markers with potential significance for discrimination of Stemona plants.     

Chemoselectivity issues of the asymmetric interaction between cyclohexanone, β-nitrostyrene,and benzoic acid under 5-aryl prolinate's organocatalysis

4-l -menthyloxycarbonyl 5-aryl prolinates were studied as organocatalysts of a novel three-component reaction of cyclohexanone, benzoic acid, and β-nitrostyrene. The presence of ortho-halogen atom in 5-aryl fragment of the catalyst is favored for driving the formation of chiral 7a-hydroxyoctahydro-2H-indol-2-one scaffold. 5-(o-Chlorophenyl) prolinate selectively afforded 3-phenyl-7a-hydroxyoctahydro-2H-indol-2-one with ee 63%, whereas 5-phenyl prolinate led to conjugation of β-nitrostyrene to cyclohexanone (the Michael adduct). Plausible chlorine effect is accounted for the specific interaction of the 5-aryl prolinate enamine intermediate with β-nitrostyrene in the transition state.     

A comparison of the discriminatory ability and sensitivity of the trigeminal and olfactory systems to chemical stimuli in the tiger salamander

Summary Trigeminal receptors can respond to a wide variety of chemical stimuli, but it is unknown whether these receptors mediate discrimination between chemical stimuli matched for equal perceptual intensity. The present electrophysiological and behavioral experiments address this issue using tiger salamanders, Ambystoma tigrinum, and four compounds (amyl acetate, cyclohexanone, butanol, and d-limonene). In addition, the relative sensitivities of the trigeminaland olfactory systems to these compounds are compared. In electrophysiological cross-adaptation experiments (amyl acetate vs cyclohexanone; butanol vs d-limonene), there was complete cross adaptation such that only concentrations above the background (crossa-dapting) stimulus concentration elicited responses, suggesting that chemical stimuli may stimulate trigeminal receptors nonspecifically. In behavioral experiments (amyl acetate vs cyclohexanone; butanol vs d-limonene), only animals with intact olfactory nerves could discriminate between perceptually equivalent concentrations, that is concentrations that elicited the same level of responding. Both electrophysiologically and behaviorally, the trigeminal system exhibited higher thresholds than the olfactory system. We conclude that trigeminal chemoreceptors, at least in salamanders, are unable to discriminate between these two pairs of compounds when matched for equal perceptual intensity, and that trigeminal chemoreceptors are less sensitive than olfactory receptors.Abbreviations AA amyl acetate - CH cyclohexanone - LI d-limonene - BU butanol - EOG electro-olfactogram - ISI interstim-ulus interval - ONX olfactory nerve cut - ppm parts per million (1 l of compound in vapor phase/1l of air=1 ppm)     

Synthesis of polyfunctional hydroxamic acids for potential use in iron chelation therapy

Two new multidentate N-methylhydroxamic acids were prepared and characterized. β-Cyclodextrin was esterified by treatment with succinic anhydride. The resulting carboxyl groups (14 per cyclodextrin) were converted to the N-hydroxysuccinimide esters and then on to the hydroxamic acids by treatment with N-methylhydroxylamine. Tetracyanoethylation of cyclohexanone followed by hydrolysis of the nitrile and conversion of the carboxylic acid to hydroxamic acid produced a tetrahydroxamic acid derivative of cyclohexanone. Infrared, ¹H NMR, and ¹³C NMR were consistent with the proposed structures. The hydroxamic acids were water soluble and formed the typical red-brown iron complexes. Stability constants (log K) of 29–30 for the iron complexes indicated a strong chelate effect. Animal tests indicated that the two compounds were only weakly effective in removing iron in vivo from iron-overloaded mice. The potency was only 0.1 that of the standard drug desferrioxamine-B.     

Cloning and characterization of a cyclohexanone monooxygenase gene from Arthrobacter sp. L661

Cyclohexanone monooxygenase (CHMO), a type of Baeyer-Villiger oxidation, catalyzes the oxidation of cyclohexanone into ɛ-caprolactone, which has been utilized as a building block in organic synthesis. A bacterium that is capable of growth on cyclohexanone as a sole carbon source was recently isolated and was identified as Arthrobacter sp. L661. The strain is believed to harbor a CHMO gene (chnB), considering the high degradablity of cyclohexanone. In order to characterize the CHMO, a chnB gene was cloned from Arthrobacter sp. L661. The deduced amino acids of the chnB gene evidenced the highest degree of homology (90% identity) with the CHMO of Arthrobacter sp. BP2 (accession no. AY123972). The CHMO of L661 was shown to be functionally expressed in Escherichia coli cells, purified via affinity chromatography, and characterized. The specific activity of the purified enzyme was 24.75 μmol/min/mg protein. The optimum pH was 7.0 and the enzyme maintained over 70% of its activity for up to 24 h in a pH range of 6.0 to 8.0 at 4°C. The CHMO of L661 readily oxidized cyclobutanone and cyclopentanone whereas less activity was detected with those of Arthrobacter sp. BP2, Rhodococcus sp. Phi1, and Rhodococcus sp. Phi2, thereby suggesting that the CHMO of L661 evidenced the different substrate specificities compared with other CHMOs. These results can provide us with useful information for the development of biocatalysts applicable to commercial organic syntheses, especially because only a few CHMO genes have been identified thus far.     

Optimization of chemoenzymatic Baeyer–Villiger oxidation of cyclohexanone to ε-caprolactone using response surface methodology

ε-Caprolactone (ε-CL) has attracted a great deal of attention and a high product concentration is of great significance for reducing production cost. The optimization of ε-CL synthesis through chemoenzymatic Baeyer–Villiger oxidation mediated by immobilized Trichosporon laibacchii lipase was studied using response surface methodology (RSM). The yield of ε-CL was 98.06% with about 1.2 M ε-CL concentration that has a substantial increase mainly due to both better stability of the cross-linked immobilized lipase used and the optimum reaction conditions in which the concentration of cyclohexanone was 1.22 M, the molar ratio of cyclohexanone:urea hydrogen peroxide (UHP) was 1:1.3, and the reaction temperature was 56.5°C. Based on our experimental results, it can be safely concluded that there are three reactions in this reaction system, not just two reactions, in which the third reaction is that the acetic acid formed reacts with UHP to form peracetic acid in situ catalyzed by the immobilized lipase. A quadratic polynomial model based on RSM experimental results was developed and the R² value of the equation is 0.9988, indicating that model can predict the experimental results with high precision. The experimental results also show that the molar ratio of cyclohexanone to UHP has very significant impact on the yield of ε-CL (p < .0006).     

Synthesis of (S)-2-Hydroxy-β-ionone,Employing (S)-3-Hydroxy-2,2-dimethylcyclohexanone as the Chiral Starting Material

(S)-2-Hydroxy-β-ionone of 96% e.e. was synthesized from (S)-3-hydroxy-2,2-dimethyl cyclohexanone, which was easily obtained by the baker’s yeast reduction of 2,2-dimethylcyclohexane-l,3-dione.     

Electroantennogram responses of a parasitic wasp, Microplitis croceipes, to host-related volatile and anthropogenic compounds

The parasitic wasp Microplitis croceipes (Cresson) (Hymenoptera: Braconidae) showed its own characteristic electroantennogram (EAG) response profiles to 13 host‐related (cis‐3‐hexenol, α‐pinene (R)‐(+)‐limonene (S)‐(–)‐limonene, trans‐β‐ocimene (±)‐linalool, (–)‐trans‐caryophyllene, α‐humulene, nerolidol, trans‐nerolidol, cis‐nerolidol, methyl jasmonate and indole) and four anthropogenic (2‐diisopropylaminoethanol, 2,2′‐thiodiethanol, 2‐methyl‐5‐nitroaniline and cyclohexanone) volatile compounds. These profiles were similar between males and females except for 2‐diisopropylaminoethanol, which elicited significantly larger EAG responses in males. Among the compounds tested, cis‐3‐hexenol, linalool and cyclohexanone elicited the largest EAG responses. EAG responses were not influenced by the age of wasps between 1 and 13 days after emergence. EAG responses were dose‐dependent, and highly EAG‐active compounds elicited significant EAG responses with less than 10 μg of the compounds at source. Quantification of compounds released from an odour cartridge indicates that release rate is highly dependent on the chemical nature of stimuli, showing up to 10 000‐fold differences in the amount released between different compounds when the same amount was loaded in the odour cartridge. Wasps having undergone a behavioural training regime to be attracted to either cyclohexanone or methyl jasmonate did not show any differences in EAG responses from those of untrained wasps.     

Enzyme reactions involved in anaerobic cyclohexanol metabolism by a denitrifying Pseudomonas species

The enzymes involved in the anaerobic degration of cyclohexanol were searched for in a denitrifying Pseudomonas species which metabolizes this alicyclic compound to CO₂ anaerobically. All postulated enzyme activities were demonstrated in vitro with sufficient specific activities. Cyclohexanol dehydrogenase catalyzes the oxidation of the substrate to cyclohexanone. Cyclohexanone dehydrogenase oxidizes cyclohexanone to 2-cyclohexenone. 2-Cyclohexenone hydratase and 3-hydroxycyclohexanone dehydrogenase convert 2-cyclohexenone via 3-hydroxycyclohexanone into 1,3-cyclohexanedione. Finally, the dione is cleaved by 1,3-cyclohexanedione hydrolase into 5-oxocaproic acid. Some kinetic and regulatory properties of these enzymes were studied.     

Antilipoxygenase activity of compounds from Hypericum perforatum

The main biologically active constituents of Hypericum species are flavonoids (quercetin, isoquercitrin, hyperoside, rutin), biflavonoids and naphthodianthrones (hypericin, pseudohypericin). Lipoxygenase is the key enzyme in the biosynthesis of leukotriens, which have been postulated to play an important role in the pathophysiology of several inflammatory and allergic diseases. This work deals with the investigation of potential antilipoxygenase activity of different compounds and extracts isolated from Hypericum perforatum L. The highest inhibitory effect was exhibited by flavonoid derivative hyperoside (IC₅₀ 5.768 × 10⁻⁶ M). Acetone and ethanolic extracts caused also an inhibition of lipoxygenase. On the basis of inhibitory effect of compounds tested we assume that the most of them may be involved in the antiinflammatory principles of Hypericum perforatum L.     

Natural Products among Brown Algae: The Case of Cystoseira schiffneri Hamel (Sargassaceae,Phaeophyceae)

A chemotaxonomic study on the marine brown alga Cystoseira schiffneri collected from the Tunisian marine coast allowed us to identify kjellmanianone ( 1 ) and a new isololiolide derivative named schiffnerilolide ( 2 ). The structure elucidation and the assignment of relative configurations of the isolated natural products were based on advanced mass spectrometric and nuclear magnetic resonance techniques. This outcome suggested a close phylogenetic relationship of C. schiffneri with brown algae belonging to genus Sargassum C. Agardh . Molecular characterization using the nuclear small subunit rRNA (SSU rRNA) gene (18S) sequence as genetic marker was made. Pigment analysis showed a significant seasonal change of carotenoids, in particular of fucoxanthin and fucoxanthinol. Also galactolipids, the main constituents of the thylakoid membranes, showed remarkable seasonal changes.     

Rapid conversion of cyclohexenone,cyclohexanone and cyclohexanol to ε-caprolactone by whole cells of Geotrichum candidum CCT 1205

?-Caprolactone (?-CL) was obtained with excellent conversion and short reaction times from the substrates cyclohexenone, cyclohexanone and cyclohexanol using whole cells of Brazilian Geotrichum candidum (CCT 1205). The reactions were monitored over time by gas chromatography, and the intermediates of the one-pot cascade biotransformation involving reductions of C=C and C=O bonds as well as the Baeyer–Villiger oxidation were identified and quantified. The Baeyer–Villiger monooxygenase (BVMO) enzyme was predominant, and all three substrates were completely converted into ?-CL. Furthermore, the whole cells of Geotrichum candidum were recycled and reutilized in the biotransformation of cyclohexanone, producing ?-CL at least six consecutive times without a significant loss of activity, reaction yields or product purity.     

Site of stimulation by mannosyl-P-dolichol of GlcNAc-lipid formation by microsomes of embryonic chick retina

Mannosyl-P-dolichol (man-P-dol) has been shown to stimulate the early reactions of the dolichol pathway, specifically, the biosynthesis of GlcNAc-P-P-dol and GlcNAc-GlcNAc-P-P-dol, and thus may play a regulatory role in glycoprotein biosynthesis. The site of action of man-P-dol has previously been suggested to be the GlcNAc-transferase concerned with the formation of the monoglucosaminyl derivative. Since the concentration of the chitobiosyl compound also increases as a result of the presence of man-P-dol, the immediate site of the activation was reexamined. The effect of man-P-dol on the formation of GlcNAc-GlcNAc-P-P-dol using GlcNAc-P-P-dol synthesizedin situ or added exogenously as the substrate was investigated. In addition, the distribution of radioactivity in the glucosaminyl constituents of the products under the stimulatory conditions was determined. The results of these studies supported the conclusion that the stimulation of GlcNAc-lipid synthesis by man-P-dol is due to the enhanced synthesis of GlcNAc-P-P-dol. It is not a result of the activation of the GlcNAc-transferase catalyzing the attachment of the second GlcNAc residue for the biosynthesis of the chitobiosyl derivative.Abbreviations GlcNAc-P-P-dol N-acetylglucosaminylpyrophosphoryldolichol - GlcNAc-GlcNAc-P-P-dol N-acetylglucosaminyl-N-acetylglucosaminylpyrophosphoryldolichol; - chito N-N-diacetylchitobiose - man-P-dol mannosylphosphoryldolichol - TX-100 triton X-100 - Tes 2-{[tris-(hydroxymethyl)-methyl]-amino}-ethanesulfonic acid     

Elimination of 2-deoxyribose interference in the thiobarbituric acid determination of N-acetylneuraminic acid in tumor cells by pH-dependent extraction with cyclohexanone

The widely used thiobarbituric acid technique for the quantitation of N-acetylneuraminic acid (NANA) was improved to eliminate the interference of the ubiquitous 2-deoxyribose. The 2-deoxyribose chromogen was completely removed by cyclohexanone extraction at pH 5.6–6.0. After readjusting the pH to 1.7–1.9, the chromogen representative of NANA was quantitatively extracted with cyclohexanone. All other aspects of the original technique (L. Warren 1959 J. Biol. Chem. 534, 1971–1975) remained unchanged. The technique has been applied to determine total as well as neuraminidase-susceptible NANA in the preparation of the immunogen (neuraminidase-treated myeloblasts) utilized to stimulate specific immunity in patients with myeloblastic leukemia and certain solid tumors; NANA levels significantly affect immunogenicity. Data obtained from a variety of tumors using pH-dependent extraction as compared to the thiobarbituric acid method, isoamyl alcohol extraction, and ion-exchange purification showed that 2-deoxyribose interference may cause as much a two- to threefold error in the quantitation of NANA.     

Cloning,expression and characterization of a eukaryotic cycloalkanone monooxygenase from <Emphasis Type="Italic">Cylindrocarpon radicicola</Emphasis> ATCC 11011

In this study, we have cloned and characterized a cycloalkanone monooxygenase (CAMO) from the ascomycete Cylindrocarpon radicicola ATCC 11011 (identical to Cylindrocarpon destructans DSM 837). The primary structure of this Baeyer–Villiger monooxygenase (BMVO) revealed 531 residues with around 45% sequence identity to known cyclohexanone monooxygenases. The enzyme was functionally overexpressed in Escherichia coli and investigated with respect to substrate spectrum and kinetic parameters. Substrate specificity studies revealed that a large variety of cycloaliphatic and bicycloaliphatic ketones are converted by this CAMO. A high catalytic efficiency against cyclobutanone was observed and seems to be a particular property of this BVMO. The thus produced butyrolactone derivatives are valuable building blocks for the synthesis of a variety of natural products and bioactive compounds. Furthermore, the enzyme revealed activity against open-chain ketones such as cyclobutyl, cyclopentyl and cyclohexyl methyl ketone which have not been reported to be accepted by typical cyclohexanone monooxygenases. These results suggest that the BVMO from C. radicicola indeed might be rather unique and since no BVMOs originating from eukaryotic organisms have been produced recombinantly so far, this study provides the first example for such an enzyme.     

On the Productivity and Properties of l-Asparaginase from Escherichia coli A–l–3

Oxidation of grayanotoxin (GTX) II with lead (IV) acetate in methanol gave a new derivative, the 1(R)-spiro-3,6(S),14,16-tetra-hydroxy-5-keto derivative. Treatment of GTX-II tetraacetate in acetic acid by using Pb(IV) acetate as an oxidizing agent gave a novel 1,5-seco-GTX derivative, Δ¹⁽¹⁰⁾-1,5-seco-GTX-pentaacetate, together with the 1,5-seco-GTX-1(R) derivative. Oxidation of GTX-II-tetraacetate with Tl(III) acetate in acetic acid or benzene gave the 1,5-seco-GTX-1(S) derivative.     

Facile Synthesis of 1,2: 5,6-Di-O-cyclohexylidene-d-mannitol and 2,3-O-Cyclohexylidene-d-glyceraldehyde

The reaction of cyclohexanone diethyl acetal with d-mannitol yielded quantitatively 1,2: 5,6-di-O-cyclohexylidene-d-mannitol (1) and its isomer (2). From 1, 2,3-O-cyclohexylidene-d-glyceraldehyde (3) was obtained in a quantitative yield without racemization.     

Enzyme cascade converting cyclohexanol into ε-caprolactone coupled with NADPH recycling using surface displayed alcohol dehydrogenase and cyclohexanone monooxygenase on E. coli

The application of enzymes as biocatalysts in industrial processes has great potential due to their outstanding stereo-, regio- and chemoselectivity. Using autodisplay, enzymes can be immobilized on the cell surface of Gram-negative bacteria such as Escherichia coli. In the present study, the surface display of an alcohol dehydrogenase (ADH) and a cyclohexanone monooxygenase (CHMO) on E. coli was investigated. Displaying these enzymes on the surface of E. coli resulted in whole-cell biocatalysts accessible for substrates without further purification. An apparent maximal reaction velocity V_MAX(app) for the oxidation of cyclohexanol with the ADH whole-cell biocatalysts was determined as 59.9 mU ml⁻¹. For the oxidation of cyclohexanone with the CHMO whole-cell biocatalysts a V_MAX(app) of 491 mU ml⁻¹ was obtained. A direct conversion of cyclohexanol to ε-caprolactone, which is a known building block for the valuable biodegradable polymer polycaprolactone, was possible by combining the two whole-cell biocatalysts. Gas chromatography was applied to quantify the yield of ε-caprolactone. 1.12 mM ε-caprolactone was produced using ADH and CHMO displaying whole-cell biocatalysts in a ratio of 1:5 after 4 h in a cell suspension of OD_578nm 10. Furthermore, the reaction cascade as applied provided a self-sufficient regeneration of NADPH for CHMO by the ADH whole-cell biocatalyst.     

Optimization and production of curdlan gum using Bacillus cereus PR3 isolated from rhizosphere of leguminous plant

Curdlan gum is a neutral water-insoluble bacterial exopolysaccharide composed primarily of linear β-(1,3) glycosidic linkages. Recently, there has been increasing interest in the applications of curdlan and its derivatives. Curdlan is found to inhibit tumors and its sulfated derivative possess anti-HIV activity. Curdlan is biodegradable, non-toxic towards human, environment and edible which makes it suitable as drug-delivery vehicles for sustained drug release. The increasing demand for the growing applications of curdlan requires an efficient high yield fermentation production process so as to satisfy the industrial needs. In this perspective, the present work is aimed to screen and isolate an efficient curdlan gum producing bacteria from rhizosphere of ground nut plant using aniline-blue agar. High yielding isolate was selected based on curdlan yield and identified as Bacillus cereus using gas-chromatography fatty acid methyl ester analysis. B. cereus PR3 curdlan gum was characterized using FT-IR spectroscopy, SEM, XRD and TGA. Fermentation time for curdlan production using B. cereus PR3 was optimized. Media constituents like carbon, nitrogen and mineral sources were screened using Plackett–Burman design. Subsequent statistical analysis revealed that Starch, NH₄NO₃, K₂HPO₄, Na₂SO₄, KH₂SO₄ and CaCl₂ were significant media constituents and these concentrations were optimized for enhancement of curdlan production up to 20.88?g/l.