Improved Properties of Bacillus coagulans β‐Galactosidase through Immobilization

Thermostable β‐galactosidase from Bacillus coagulans RCS3 was purified by successive column chromatography using DEAE‐cellulose and Sephadex G‐50. Immobilization of the purified enzyme was studied with DEAE‐cellulose and calcium alginate. The efficiency of β‐galactosidase retention was 87 % with DEAE‐cellulose (17 mg protein/mL of matrix) and 80 % with calcium alginate (2.2 mg protein/g bead). Comparative studies of immobilization displayed a shift in the optimum temperature from 65 °C to 70 °C provoked by DEAE‐cellulose, although no effect was observed with calcium alginate. The heat inactivation curve revealed an improvement in the stability (t_1/2 of 14.5 h for the immobilized enzyme as compared to 2 h for the free enzyme at 65 °C) in a calcium alginate system. This immobilized enzyme has a wide pH stability range (6.5–11). β‐Galactosidase immobilized by DEAE‐cellulose and calcium alginate allowed a 57 and 70 % lactose hydrolysis, respectively, to be achieved within 48 h after repeated use for twenty times.     

Expression of enzymatically active and correctly targeted strictosidine synthase in transgenic tobacco plants

Strictosidine, a precursor to over 1000 indole alkaloids including the anti-tumor drugs vinblastine, vincristine, and camptothecin, is produced by the condensation of tryptamine and secologanin. Strictosidine synthase, the enzyme responsible for this condensation, is the first committed step in the indole-alkaloid pathway. We have introduced a modified cDNA encoding Strictosidine synthase from Catharanthus roseus (L.) Don. (McKnight et al. 1990, Nucl. Acids Res. 18, 4939) driven by the CaMV 35S promoter into tobacco (Nicotiana tabacum L.). Transgenic tobacco plants expressing this construct had from 3 to 22 times greater strictosidinesynthase activity than C. roseus plants. Ultrastructural immunolocalization demonstrated that strictosidine synthase is a vacuolar protein in C. roseus and is correctly targeted to the vacuole in transgenic tobacco. Immunoblot analysis of strictosidine synthase showed that two distinct forms of the enzyme were produced in transgenic tobacco plants but that only a single form was made in C. roseus. This observation indicates that the second form of the protein is not simply a result of overexpression in tobacco, but may reflect differences in protein processing between tobacco and C. roseus.Abbreviations cDNA complementary DNA - TLC thin-layer chromatography We thank Dr. C.A. Roessner for providing the E. coli strain expressing strictosidine synthase, Dr. J. Balsevich for providing alkaloid standards, and Dr. L. Cloney for assisting with antibody preparation. This work was supported by a National Institutes of Health Biomedical Research Support Grant to T.D.M and by a grant from the US Department of Agriculture, Competitive Research Grants Office (90-37262-5375) to C.L.N.     

Kinetic Traits and Enzyme Form Patterns of (R)‐2‐(2,4‐Dichlorophenoxy)propionate/α‐Ketoglutarate Dioxygenase (RdpA) after Expression in Different Bacterial Strains

The rdpA gene of strains Delftia acidovorans MC1, Rhodoferax sp. P230, and Sphingobium herbicidovorans MH proved to be identical. However, when RdpA [(R)‐2‐(2,4‐dichlorophenoxy)propionate/α‐ketoglutarate dioxygenase] was investigated after purification from the various strains, significant differences in the kinetics and some chemical properties of the enzymes were observed. The preference for substrates ranged in the order (R)‐2‐(2,4‐dichlorophenoxy)propionate (2,4‐DP) > (R)‐2‐(4‐chloro‐2‐methylphenoxy)propionate (MCPP) >> 2,4‐dichlorophenoxyacetate (2,4‐D) ～ 4‐chloro‐2‐methylphenoxyacetate (MCPA), but detailed kinetic investigations revealed significant strain‐dependent differences in the k_cat and K_M values. While the K_M values of RdpA from the various strains were low and their range rather narrow with 2,4‐DP (19–60 μM) and MCPP (35–64 μM), larger differences were observed with phenoxyacetates which were distinctly higher and spanned a wider range with 2,4‐D (237–935 μM) and MCPA (164–510 μM). The lowest K_M values with 2,4‐D and MCPA were found for RdpA originating from strain P230. Investigation of the enzymes from the various sources by 2D gel electrophoresis revealed up to three monomeric enzyme forms which differed in the pI value. The 2D‐patterns were similar with RdpA from strains MC1 and MH, and after heterologous expression of the enzyme in Escherichia coli, but differed significantly from that of strain P230. The presence of enzyme forms and their different composition coincided apparently with the differences observed in the kinetic properties of RdpA in the various strains. The effects are discussed in terms of posttranslational modification of RdpA which appears to be different in extent and kind in the various strains.     

Enzymatic pre‐treatment of microalgae cells for enhanced extraction of proteins

Crude proteins and pigments were extracted from different microalgae strains, both marine and freshwater. The effectiveness of enzymatic pre‐treatment prior to protein extraction was evaluated and compared to conventional techniques, including ultrasonication and high‐pressure water extraction. Enzymatic pre‐treatment was chosen as it could be carried out at mild shear conditions and does not subject the proteins to high temperatures, as with the ultrasonication approach. Using enzymatic pre‐treatment, the extracted proteins yields of all tested microalgae strains were approximately 0.7 mg per mg of dry cell weight. These values were comparable to those achieved using a commercial lytic kit. Ultrasonication was not very effective for proteins extraction from Chlorella sp., and the extracted proteins yields did not exceed 0.4 mg per mg of dry cell weight. For other strains, similar yields were achieved by both treatment methods. The time‐course effect of enzymatic incubation on the proteins extraction efficiency was more evident using laccase compared to lysozyme, which suggested that the former enzyme has a slower rate of cell disruption. The crude extracted proteins were fractionated using an ion exchange resin and were analyzed by the electrophoresis technique. They were further tested for their antioxidant activity, the highest of which was about 60% from Nannochloropsis sp. The total phenolic contents in the selected strains were also determined, with Chlorella sp. showing the highest content reaching 17 mg/g. Lysozyme was also found to enhance the extraction of pigments, with Chlorella sp. showing the highest pigments contents of 16.02, 4.59 and 5.22 mg/g of chlorophyll a, chlorophyll b and total carotenoids, respectively.     

Absolute configuration determination and conformational analysis of (−)‐(3S,6S)‐3α,6β‐diacetoxytropane using vibrational circular dichroism and DFT techniques

The absolute configuration of semisynthetic (?)‐3α,6β‐acetoxytropane 1 , prepared from (?)‐6β‐hydroxyhyoscyamine 2 , has been determined using vibrational circular dichroism (VCD) spectroscopy. The vibrational spectra (IR and VCD) were calculated using DFT at the B3LYP/DGDZVP level of theory for the eight more stable conformers which account for 99.97% of the total relative abundance in the first 10 kcal/mol range. The calculated VCD spectra of all considered conformations showed two distinctive spectral ranges, one between 1300 and 1200 cm^?1, and the other one in the 1150–950 cm^?1 region. When compared with the experimental VCD spectrum, the first spectral region confirmed the calculated conformational preferences, whereas the second region showed little change with conformation, thus allowing the determination of the absolute configuration of 1 as (3S,6S)‐3α,6β‐diacetoxytropane. Also, the bands in the second region showed similarities between 1 and 2 in both the experimental and calculated VCD spectra, suggesting that these bands are mainly related to the absolute configuration of the rigid tropane ring system, since they show conformational independency, no variations with the nature of the substituent, and are composed by closely related vibrational modes. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Improved fruit α‐tocopherol,carotenoid, squalene and phytosterol contents through manipulation of Brassica juncea 3‐HYDROXY‐3‐METHYLGLUTARYL‐COA SYNTHASE1 in transgenic tomato

3‐Hydroxy‐3‐methylglutaryl‐coenzyme A synthase (HMGS) in the mevalonate (MVA) pathway generates isoprenoids including phytosterols. Dietary phytosterols are important because they can lower blood cholesterol levels. Previously, the overexpression of Brassica juncea wild‐type (wt) and mutant (S359A) BjHMGS1 in Arabidopsis up‐regulated several genes in sterol biosynthesis and increased sterol content. Recombinant S359A had earlier displayed a 10‐fold higher in vitro enzyme activity. Furthermore, tobacco HMGS overexpressors (OEs) exhibited improved sterol content, plant growth and seed yield. Increased growth and seed yield in tobacco OE‐S359A over OE‐wtBjHMGS1 coincided with elevations in NtSQS expression and sterol content. Herein, the overexpression of wt and mutant (S359A) BjHMGS1 in a crop plant, tomato (Solanum lycopersicum), caused an accumulation of MVA‐derived squalene and phytosterols, as well as methylerythritol phosphate (MEP)‐derived α‐tocopherol (vitamin E) and carotenoids, which are important to human health as antioxidants. In tomato HMGS‐OE seedlings, genes associated with the biosyntheses of C10, C15 and C20 universal precursors of isoprenoids, phytosterols, brassinosteroids, dolichols, methylerythritol phosphate, carotenoid and vitamin E were up‐regulated. In OE‐S359A tomato fruits, increased squalene and phytosterol contents over OE‐wtBjHMGS1 were attributed to heightened SlHMGR2, SlFPS1, SlSQS and SlCYP710A11 expression. In both tomato OE‐wtBjHMGS1 and OE‐S359A fruits, the up‐regulation of SlGPS and SlGGPPS1 in the MEP pathway that led to α‐tocopherol and carotenoid accumulation indicated cross‐talk between the MVA and MEP pathways. Taken together, the manipulation of BjHMGS1 represents a promising strategy to simultaneously elevate health‐promoting squalene, phytosterols, α‐tocopherol and carotenoids in tomato, an edible fruit.     

Cloning and Tissue Expression of 6-Pyruvoyl Tetrahydropterin Synthase and Xanthine Dehydrogenase from <Emphasis Type="Italic">Poecilia reticulata</Emphasis>

Guppy is a popular ornamental fish owing to its diverse body and fin coloration. More than 40 established color varieties have been selectively bred. The complementary DNAs for 2 enzymes that are involved in the de novo synthesis of pteridines and purines, which are important for the production of color pigments, were cloned from the caudal fin. Two cDNA isoforms for 6-pyruvoyl tetrahydropterin synthase (PTPS), with an open reading frame of 130 and 147 amino acids, respectively, were cloned from the Red Tail variety. The deduced amino acid sequence of the longer isoform shows an overall identity of about 65% to the mammalian PTPS sequences. The cDNA for xanthine dehydrogenase (XDH) was cloned from the Yellow Tail variety, and consists of an open reading frame of 1331 amino acids. Although it shows a higher overall identity to bovine aldehyde oxidase (AO; 54%) than to chicken XDH (51%), it has a NAD-binding domain that is specific to XDHs. Northern blot analysis indicated that both PTPS and XDH messenger RNAs were highly expressed in the liver, but absent in the muscle. In the caudal fins, guppy varieties with a higher proportion of xanthophores and erythrophores showed higher expression of PTPS, while XDH mRNA levels were too low to indicate obvious differential expression among the color guppy varieties. The results implied that high expression of PTPS is correlated with the biosynthesis of pteridines in the erythrophores and xanthophores, while the association between the putative guppy XDH with specific chromatophores is less clear.     

Crystal structure of norcoclaurine‐6‐O‐methyltransferase,a key rate‐limiting step in the synthesis of benzylisoquinoline alkaloids

Growing pharmaceutical interest in benzylisoquinoline alkaloids (BIA) coupled with their chemical complexity make metabolic engineering of microbes to create alternative platforms of production an increasingly attractive proposition. However, precise knowledge of rate‐limiting enzymes and negative feedback inhibition by end‐products of BIA metabolism is of paramount importance for this emerging field of synthetic biology. In this work we report the structural characterization of (S)‐norcoclaurine‐6‐O‐methyltransferase (6OMT), a key rate‐limiting step enzyme involved in the synthesis of reticuline, the final intermediate to be shared between the different end‐products of BIA metabolism, such as morphine, papaverine, berberine and sanguinarine. Four different crystal structures of the enzyme from Thalictrum flavum (Tf 6OMT) were solved: the apoenzyme, the complex with S‐adenosyl‐l ‐homocysteine (SAH), the complexe with SAH and the substrate and the complex with SAH and a feedback inhibitor, sanguinarine. The Tf 6OMT structural study provides a molecular understanding of its substrate specificity, active site structure and reaction mechanism. This study also clarifies the inhibition of Tf 6OMT by previously suggested feedback inhibitors. It reveals its high and time‐dependent sensitivity toward sanguinarine.     

Expression of Glucose‐6‐Phosphate Dehydrogenase and 6‐Phosphogluconate Dehydrogenase in Oxidative Stress Induced by Long‐Term Iron Toxicity in Rat Liver

Reactive oxygen species (ROS) are highly reactive and oxygen‐containing molecules that are derived by metabolic activities or from environmental sources. Toxicity of heavy metals including iron has the ability to generate ROS in all living organisms. The pentose phosphate pathway enzymes, which are glucose 6‐phosphate dehydrogenase and 6‐phosphogluconate dehydrogenase, produce nicotinamide adenine dinucleotide phosphate (NADPH) that enables cells to counterbalance the oxidative stress via the action of the glutathione system. The results presented here have shown that toxic and nontoxic levels of iron have a strong effect on the expression of both genes. While toxic levels of iron exhibited significant changes in enzyme activity, nontoxic levels had no effect on enzymes in rat liver. Our results are the first evidence to elucidate how oxidative stress induced by long‐term iron toxicity affects both enzymes at the enzymatic and molecular level and also to determine any possible correlation between the enzymatic and molecular levels.     

Application of NMR spectroscopy for assignment of the absolute configuration of 8‐tert‐butyl‐2‐hydroxy‐7‐methoxy‐8‐methyl‐9‐oxa‐6‐azaspiro[4.5]dec‐6‐en‐10‐one

In order to assign the absolute configurations of 8‐tert‐butyl‐2‐hydroxy‐7‐methoxy‐8‐methyl‐9‐oxa‐6‐azaspiro[4.5]dec‐6‐en‐10‐one ( 2a , 2b ), their esters ( 5a , 5b , 5c , 5d ) with (R)‐ or (S)‐2‐methoxyphenylacetic acid ( 4a , 4b ) have been synthesized. The absolute configurations of these compounds have been determined on the basis of NOESY correlations between the protons of the tert‐butyl group and the cyclopentane fragment of the molecules. The crucial part of this analysis was assignment of the absolute configuration at C‐5. Additionally, by calculation of the chemical shift anisotropy, δ^RS, for the relevant protons, it was also possible to confirm the absolute configurations at the C‐2 centres of compounds 2a , 2b and 5a , 5b , 5c , 5d . Chirality, 25:422–426, 2013.© 2013 Wiley Periodicals, Inc.     

Synthesis and Evaluation of 6‐Aza‐2′‐deoxyuridine Monophosphate Analogs as Inhibitors of Thymidylate Synthases,and as Substrates or Inhibitors of Thymidine Monophosphate Kinase in Mycobacterium tuberculosis

A series of 5‐substituted analogs of 6‐aza‐2′‐deoxyuridine 5′‐monophosphate, 6‐aza‐dUMP, has been synthesized and evaluated as potential inhibitors of the two mycobacterial thymidylate synthases (i.e., a flavin‐dependent thymidylate synthase, ThyX, and a classical thymidylate synthase, ThyA). Replacement of C(6) of the natural substrate dUMP by a N‐atom in 6‐aza‐dUMP 1a led to a derivative with weak ThyX inhibitory activity (33% inhibition at 50 μM ). Introduction of alkyl and aryl groups at C(5) of 1a resulted in complete loss of inhibitory activity, whereas the attachment of a 3‐(octanamido)prop‐1‐ynyl side chain in derivative 3 retained the weak level of mycobacterial ThyX inhibition (40% inhibition at 50 μM ). None of the synthesized derivatives displayed any significant inhibitory activity against mycobacterial ThyA. The compounds have also been evaluated as potential inhibitors of mycobacterial thymidine monophosphate kinase (TMPKmt). None of the derivatives showed any significant TMPKmt inhibition. However, replacement of C(6) of the natural substrate (dTMP) by a N‐atom furnished 6‐aza‐dTMP ( 1b ), which still was recognized as a substrate by TMPKmt.     

Optimization of Reaction Parameters and Cultivation Conditions for Biocatalytic Hydrogen Transfer Employing Overexpressed ADH‐‘A’ from Rhodococcus ruber DSM 44541 in E. coli

The alcohol dehydrogenase ADH‐‘A’ from Rhodococcus ruber DSM 44541 represents a highly efficient catalyst for biocatalytic hydrogen transfer reactions. Starting from an exceedingly low level of active ADH‐‘A’ in Escherichia coli, the apparent specific activity of ADH‐‘A’ overexpressed in E. coli cells could be drastically enhanced by a factor of 550 by optimizing the host and induction/growth conditions. The influence of reaction parameters like pH, cosubstrate (2‐propanol, acetone) concentration, substrate concentration temperature and additional cofactor on the apparent activity was investigated. In contrast to the purified enzyme, the pH optimum for oxidation and reduction were identical. Due to the employment of whole cells of E. coli/ADH‐‘A’ as catalyst lower operational stability was found.     

Mutation by DNA shuffling of 5‐enolpyruvylshikimate‐3‐phosphate synthase from Malus domestica for improved glyphosate resistance

A new 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS) gene from Malus domestica (MdEPSPS) was cloned and characterized by rapid amplification of cDNA ends to identify an EPSPS gene appropriate for the development of transgenic glyphosate‐tolerant plants. However, wild‐type MdEPSPS is not suitable for the development of transgenic glyphosate‐tolerant plants because of its poor glyphosate resistance. Thus, we performed DNA shuffling on MdEPSPS, and one highly glyphosate‐resistant mutant with mutations in eight amino acids (N63D, N86S, T101A, A187T, D230G, H317R, Y399R and C413A.) was identified after five rounds of DNA shuffling and screening. Among the eight amino acid substitutions on this mutant, only two residue changes (T101A and A187T) were identified by site‐directed mutagenesis as essential and additive in altering glyphosate resistance, which was further confirmed by kinetic analyses. The single‐site A187T mutation has also never been previously reported as an important residue for glyphosate resistance. Furthermore, transgenic rice was used to confirm the potential of MdEPSPS mutant in developing glyphosate‐resistant crops.     

Synthesis and Enzymatic Deprotection of Fully Protected 2′‐5′ Oligoadenylates (2‐5A): Towards a Prodrug Strategy for Short 2‐5A

Fully protected pA2′p5′A2′p5′A trimers 1a and 1b have been prepared as prodrug candidates for a short 2′‐5′ oligoadenylate, 2‐5A, and its 3′‐O‐Me analog, respectively. The kinetics of hog liver carboxyesterase (HLE)‐triggered deprotection in HEPES buffer (pH 7.5) at 37° has been studied. The deprotection of 1a turned out to be very slow, and 2‐5A never appeared in a fully deprotected form. By contrast, a considerable proportion of 1b was converted to the desired 2‐5A trimer, although partial removal of the 3′‐O‐[(acetyloxy)methyl] group prior to exposure of the adjacent phosphodiester linkage resulted in 2′,5′→3′,5′ phosphate migration and release of adenosine as side reactions.     

Utilization of (18‐Crown‐6)‐2,3,11,12‐tetracarboxylic Acid as a Chiral NMR Solvating Agent for Diamines and β‐Amino Acids

The compound (18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid was evaluated as a chiral nuclear magnetic resonance (NMR) solvating agent for a series of diamines and bicyclic β‐amino acids. The amine must be protonated for strong association with the crown ether. An advantage of (18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid over many other crown ethers is that it undergoes a neutralization reaction with neutral amines to form the protonated species needed for binding. Twelve primary diamines in neutral and protonated forms were evaluated. Diamines with aryl and aliphatic groups were examined. Some are atropisomers with equivalent amine groups. Others have two nonequivalent amine groups. Association equilibria for these systems are complex, given the potential formation of 2:1, 1:1, and 1:2 crown‐amine complexes and given the various charged species in solution for mixtures of the crown ether with the neutral amine. The crown ether produced enantiomeric differentiation in the ¹H NMR spectrum of one or more resonances for every diamine substrate. Also, a series of five bicyclic β‐amino acids were examined and (18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid caused enantiomeric differentiation in the ¹H NMR spectrum of three or more resonances of each compound. Chirality 27:708–715, 2015. © 2015 Wiley Periodicals, Inc.     

Synthesis and characterization of high quantum yield and oscillator strength 6‐chloro‐2‐(4‐cynophenyl)‐4‐phenyl quinoline (cl‐CN‐DPQ) organic phosphor for solid‐state lighting

A novel blue luminescent 6‐chloro‐2‐(4‐cynophenyl) substituted diphenyl quinoline (Cl‐CN DPQ) organic phosphor has been synthesized by the acid‐catalyzed Friedlander reaction and then characterized to confirm structural, optical and thermal properties. Structural properties of Cl‐CN‐DPQ were analyzed by Fourier transform infrared (FTIR), nuclear magnetic resonance (NMR) spectroscopy, X‐ray diffraction technique (XRD) and scanning electron microscopy (SEM) and energy dispersive analysis of X‐ray (EDAX) spectroscopy. FTIR spectra confirmed the presence of different functional groups and bond stretching. ¹H–NMR and ¹³C–NMR confirmed the formation of an organic Cl‐CN‐DPQ compound. X‐ray diffraction study provided its crystalline nature. The surface morphology of Cl‐CN‐DPQ was analyzed by SEM, while EDAX spectroscopy revealed the elemental analysis. Differential thermal analysis (TGA/DTA) disclosed its thermal stability up to 250°C. The optical properties of Cl‐CN‐DPQ were investigated by UV–vis absorption and photoluminescence (PL) measurements. Cl‐CN‐DPQ exhibits intense blue emission at 434 nm in a solid‐state crystalline powder with CIE co‐ordinates (0.157, 0.027), when excited at 373 nm. Cl‐CN‐DPQ shows remarkable Stokes shift in the range 14800–5100 cm^?1, which is the characteristic feature of intense light emission. A narrow full width at half‐maximum (FWHM) value of PL spectra in the range 42–48 nm was observed. Oscillator strength, energy band gap, quantum yield, and fluorescence energy yield were also examined using UV–vis absorption and photoluminescence spectra. These results prove its applications towards developing organic luminescence devices and displays, organic phosphor‐based solar cells and displays, organic lasers, chemical sensors and many more.