Baeyer–Villiger oxidation with peracid generated in situ by CaLB-CLEA catalyzed perhydrolysis

Candida antarctica lipase B, immobilized as cross linked enzyme aggregates (CLEAs) was used to mediate the Baeyer–Villiger oxidation of cyclohexanone to ɛ-caprolactone, and the reaction was compared with the one using Novozym^® 435 as catalyst. The conversion was dependent on the initial concentration of cyclohexanone, and was about 90% after 48 h at concentrations of up to 0.25 M but was decreased at higher concentrations. Caprolactone concentrations up to 0.6 M had no effect on the reaction efficiency. Among the cyclic ketones tested, the highest degree of conversion was achieved for cyclopentanone (88%) and the lowest for cyclooctanone (about 2%). The effect of methyl substitution and position of substitution on the cycloketone was studied using methylcyclohexanone and it has shown to influence the conversion efficiency. Both hydrogen peroxide and the reaction by-product acetic acid had a deleterious effect on the stability of the biocatalyst.     

Control of chemoselectivity of microbial reaction with resin adsorbent: enhancement of Baeyer–Villiger oxidation over reduction

Amberlite XAD-7, a hydrophobic polymer, was used to change microbial reaction of ketones from reduction to Baeyer–Villiger (BV) oxidation. Thus, D. magnusii NBRC 4600 and G. reessii NBRC 1112 could catalyze the BV reaction of ketones in the presence of the polymer while reduction of the substrates proceeded, and BV oxidation was scarcely found in the absence of the polymer.     

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).     

Rational design of a lipase to accommodate catalysis of Baeyer–Villiger oxidation with hydrogen peroxide

The mechanism and potential energy surface for the Baeyer-Villiger oxidation of acetone with hydrogen peroxide catalyzed by a Ser105-Ala mutant of Candida antarctica Lipase B has been determined using ab initio and density functional theories. Initial substrate binding has been studied using an automated docking procedure and molecular dynamics simulations. Substrates were found to bind to the active site of the mutant. The activation energy for the first step of the reaction, the nucleophilic attack of hydrogen peroxide on the carbonyl carbon of hydrogen peroxide, was calculated to be 4.4 kcal x mol(-1) at the B3LYP/6-31+G* level. The second step, involving the migration of the alkyl group, was found to be the rate-determining step with a computed activation energy of 19.9 kcal x mol(-1) relative the reactant complex. Both steps were found to be lowered considerably in the reaction catalyzed by the mutated lipase, compared to the uncatalyzed reaction. The first step was lowered by 36.0 kcal x mol(-1) and the second step by 19.5 kcal x mol(-1). The second step of the reaction, the rearrangement step, has a high barrier of 27.7 kcal x mol(-1) relative to the Criegee intermediate. This could lead to an accumulation of the intermediate. It is not clear whether this result is an artifact of the computational procedure, or an indication that further mutations of the active site are required. Figure Second TS (18TS) in the Baeyer-Villiger oxidation in a mutant of CALB. Distances in A     

Studies on the oxidation–reduction systems of the erythrocyte

1. Starvation for 3 days produces a decrease in methaemoglobin-reductase and glutathione-reductase activities, but it does not alter the glucose 6-phosphate-dehydrogenase activity of the rat erythrocyte. 2. The feeding of a protein-free diet for 11 days causes greater changes in the first two enzymes and also a diminution of the third. Under this experimental condition slight decreases in protein and haemoglobin contents were noted. 3. The experimental animals did not show methaemoglobinaemia, probably because the activity of methaemoglobin diaphorase is preserved. 4. The GSH content was not affected but the stability of the tripeptide in the presence of an oxidizing agent was diminished.     

Evolution study of the Baeyer–Villiger monooxygenases enzyme family: Functional importance of the highly conserved residues

Baeyer–Villiger monooxygenases (BVMOs) catalyze the transformation of linear and cyclic ketones into their corresponding esters and lactones by introducing an oxygen atom into a C–C bond. This bioreaction has numerous advantages compared to its chemical version; it does not induce the use of potentially harmful reagents (i.e., green chemistry) and displays significant better enantio- and regio-selectivity.     

Expanding the set of rhodococcal Baeyer–Villiger monooxygenases by high-throughput cloning, expression and substrate screening

To expand the available set of Baeyer-Villiger monooxygenases (BVMOs), we have created expression constructs for producing 22 Type I BVMOs that are present in the genome of Rhodococcus jostii RHA1. Each BVMO has been probed with a large panel of potential substrates. Except for testing their substrate acceptance, also the enantioselectivity of some selected BVMOs was studied. The results provide insight into the biocatalytic potential of this collection of BVMOs and expand the biocatalytic repertoire known for BVMOs. This study also sheds light on the catalytic capacity of this large set of BVMOs that is present in this specific actinomycete. Furthermore, a comparative sequence analysis revealed a new BVMO-typifying sequence motif. This motif represents a useful tool for effective future genome mining efforts.     

Regime analysis of a Baeyer–Villiger bioconversion in a three-phase (air–water–ionic liquid) stirred tank bioreactor

The aim of this work was to conduct a regime analysis on a three-phase (air–water–ionic liquid) stirred tank bioreactor of the Baeyer–Villiger bioconversion process, using [MeBuPyrr][BTA] ionic liquid as the dispersed phase. The regime analysis based on characteristic times of the different mechanisms involved (mixing, mass transfer, reaction) can yield a quantitative estimate of bioreactor performance. The characteristic time obtained for oxygen uptake rate (54 s⁻¹) was among the characteristic times determined for oxygen transfer (13–129 s⁻¹) under different operating conditions, suggesting that the oxygen transfer rate under certain operating conditions could be a limiting step in the bioconversion process. Further enhancement of oxygen transfer rates requires proper selection of the bioreactor operational conditions, and improved design of the ionic liquid used as oxygen transfer vector.     

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.     

Microscale process evaluation of recombinant biocatalyst libraries: application to Baeyer–Villiger monooxygenase catalysed lactone synthesis

Microscale processing techniques are rapidly emerging as a cost- effective means for parallel experimentation and hence the evaluation of large libraries of recombinant biocatalysts. In this work, the potential of an automated microscale process is demonstrated in a linked sequence of operations comprising fermentation, enzyme induction and bioconversion using three whole-cell biocatalysts each expressing cyclohexanone monoxygenase (CHMO). The biocatalysts, Escherichia coli TOP 10 [pQR239], E. coli JM107 and Acinetobacter calcoaceticus NCIMB 9871, were first produced in 96-deep square well fermentations at various carbon source concentrations (10 and 20 g L⁻¹ glycerol). Following induction of CHMO activity biomass concentrations of up to 6 g_DCW L⁻¹ were obtained. Cells from each fermentation were subsequently used for the Baeyer–Villiger oxidation of bicyclo[3.2.0]hept-2-en-6-one, cyclohexanone and cyclopentanone. Each bioconversion was performed at two initial substrate concentrations (0.5 and 1.0 g L⁻¹) in order to simultaneously explore both substrate specificity and inhibition. The microscale process sequences yielded quantitative and reproducible data for each biocatalyst on maximum growth rate, biomass yield, initial rate of lactone formation, specific biocatalyst activity and bioconversion yield. E. coli TOP 10 [pQR239] was demonstrated to be an efficient biocatalyst showing substrate specificities and substrate inhibition effects in line with previous studies. Finally, in order to show that the data obtained with E. coli TOP 10 [pQR239] at microwell scale (1,000 μL) could be related to larger scales of operation, the process was performed in a 2-L stirred-tank bioreactor. Using conditions designed to enable microwell kinetic measurements under none oxygen-limited conditions, the fermentation and bioconversion data obtained at the two scales showed good quantitative agreement. This study therefore confirms the potential of automated microscale experimentation for the whole-process evaluation of recombinant biocatalyst libraries and the specification of pilot and process scale operating conditions.     

Studies on the metabolism of steroids in the foetus. Biosynthesis of 6α-hydroxytestosterone in the human foetal liver

After incubation of testosterone with 105000g microsomes of human foetal liver, 6alpha-hydroxytestosterone was isolated and identified by t.l.c. and g.l.c.-mass spectrometry. This is the first example of 6alpha-hydroxylation of C(19) steroids in the human liver, and the finding is discussed in relation to earlier reports of 6-oxygenated C(19) and C(18) steroids in pregnant women.     

Viability of free and encapsulated Escherichia coli overexpressing cyclopentanone monooxygenase monitored during model Baeyer–Villiger biooxidation by confocal laser scanning microscopy

Baeyer–Villiger biooxidation of 4-methylcyclohexanone–5-methyloxepane-2-one catalysed by recombinant Escherichia coli overexpressing cyclopentanone monooxygenase encapsulated in polyelectrolyte complex capsules was used to investigate effect of substrate conversion on the viability of cells. Confocal laser scanning microscopy (CLSM) was used to assess cell viability using propidium iodide fluorescence marker for necrosis, and flavin autofluorescence to identify living bacteria. Viability of encapsulated cells decreased with increasing substrate concentration from 99 ± 1 to 83 ± 4%, while substrate conversions from decreased 100 to 6 ± 1%. Storage stabilization of encapsulated cells was observed by increased substrate conversion form 68 ± 2 to 96 ± 3%. Measurements by CLSM with standard deviations up to 5% may be regarded as powerful tool for recombinant cell viability determination during Baeyer–Villiger biooxidations.     

Enantioselective Baeyer–Villiger Oxidation of Bicyclo[3.2.0]hept-2-en-6-One with Fungi: Optimization of Biotransformation and Use of TiO2 as Support of Cell Growth

Fungi from Amazonian forest soil (Ecuador) and an Italian factory were screened for Baeyer–Villiger (BV) oxidation of bicyclo [3.2.0]hept-2-en-6-one to 2-oxabicyclo[3.3.0]oct-6-en-3-one (Corey’s lactone). Isolates of Fusarium sp. and F. solani produced the (+)-(1R,5S)-lactone while isolates of Aspergillus terricola and A. amazonicus afforded the (−)-(1S,5R)-lactone. Highest conversions (85% yield and 70% enantiomeric excess) were obtained with A. amazonicus grown in presence of 2.7 mM titanium dioxide.     

Microscopical and Spectroscopic Studies on the Fluorescence of a Daunomycin–aluminum Complex

In this study, the spectroscopic features and microscopical applications of the fluorescent daunomycin-Al3+ complex have been analyzed. In the presence of Al3+, the absorption spectrum of daunomycin showed a deep bathochromic shift and new peaks at 529 and 566nm, whereas the fluorescence emission was considerably modified. The emission of daunomycin alone (peak at 560nm under optimal excitation at 470nm) decreased continuously from 0.5 to 24h after addition of Al3+ ions, and a new emission peak appeared at 580nm (optimal excitation at 530nm). Under the fluorescence microscope using green exciting light, nuclei from chicken blood smears and paraffin sections of rat embryos stained with daunomycin showed a weak emission, which greatly increased after treatment with Al3+ ions. The bright and stable fluorescence of chromatin DNA induced by daunomycin-Al3+ could be a valuable labelling method in fluorescence microscopy and DNA cytochemistry.     

Studies on the mechanism of estrogen biosynthesis in the rat ovary–I

Methods were evaluated for obtaining a reliable, active estrogen synthetase (aromatase) system from the rat ovary for mechanistic studies. Short terrn treatment with luteinizing hormone and follicle stimulating hormone in various combinations did not produce appreciable stimulation, whereas long term treatment (8–16 days) with pregnant mare's serum gonadotropin increased activity in homogenates up to nine fold per mg wet wt of tissue. A similar increase per mg protein was noted in the 105,000_g microsomal fraction where the bulk of the activity was found. Various conditions for preparing and incubating the aromatase were evaluated to obtain optimal enzyme activity. The potencies of six steroids as aromatase inhibitors were compared in the rat ovarian and human placental microsomal systems. In all cases except one the results were comparable.     

Studies on molecular evolution and structural features of double-headed inhibitors of α-amylase and trypsin in plants

Plant seeds usually have high concentrations of proteinase and amylase inhibitors. These inhibitors exhibit a wide range of specificity, stability and oligomeric structure. In this communication, we report analysis of sequences that show statistically significant similarity to the double-headed α-amylase/trypsin inhibitor of ragi (Eleusine coracana). Our aim is to understand their evolutionary and structural features. The 14 sequences of this family that are available in the SWISSPROT database form three evolutionarily distinct branches. The branches relate to enzyme specificities and also probably to the oligomeric state of the proteins and not to the botanical class of the plant from which the enzymes are derived. This suggests that the enzyme specificities of the inhibitors evolved before the divergence of commercially cultivated cereals. The inhibitor sequences have three regions that display periodicity in hydrophobicity. It is likely that this feature reflects extended secondary structure in these segments. One of the most variable regions of the polypeptide corresponds to a loop, which is most probably exposed in the native structure of the inhibitors and is responsible for the inhibitory property.     

Evaluation of the Influence of thiosemicarbazone–triazole hybrids on genes implicated in lipid oxidation and accumulation as potential anti-obesity agents

A series of thiosemicarbazone–triazole hybrids 1a–h are efficiently synthesised and evaluated for their influence on the expression of genes, cpt-1, acc-1 and pgc-1, which are essential in lipid metabolism. The test results show that hybrids 1c and 1g exhibited relatively high influence on the expression of cpt-1 and pgc-1 and suppression of acc-1 as desired.     

Effects of oxidation on redox and cytotoxic properties of copper complex of Aβ1–16 peptide

Abstract

The effect of oxidation on redox and cytotoxic properties of copper complex of amyloid beta (Aβ) peptide was studied by gamma radiolysis. The oxidation of Aβ1–16 and Aβ1–16/Cu(II) complex was carried out using hydroxyl (^?OH) radicals produced by gamma radiolysis and the products were analyzed using mass spectrometry. The presence of Cu(II) was found to enhance the oxidation of Aβ1–16 peptide. The oxidation of residues Asp1, His6, and His13 was enhanced due to their involvement in copper binding. The oxidation of His residues of Aβ1–16 peptide, which are chiefly responsible for copper binding, resulted in altered redox properties and subsequently in higher cytotoxicity of the Aβ1–16 peptide in SH-SY5Y cells.