Development of transition state analogue inhibitors for <Emphasis Type="Italic">N</Emphasis>-acetylglycosyltransferases bearing <Emphasis Type="SmallCaps">d</Emphasis>psico- or <Emphasis Type="SmallCaps">d</Emphasis>tagatofuranose scaffolds

New potential transition state analogue inhibitors for N-acetylglucosyltransferases (GnTs) were synthesised. These compounds based on psico- and tagatofuranose (structure) scaffold contained a 2-thiophenyl-1-O-diethylphosphate moiety mimicking the proposed model of the transition state of the enzymatic reaction catalysed by N-acetylglucosyltransferases. The synthesised compounds as well as their precursors were fully characterised by NMR, optical rotation and mass techniques. Anomeric configuration of tagatofuranose derivatives was confirmed by X-ray crystallography. Two types of potential human glycosyltransferase (GnTs) inhibitors representing donor UDP-GlcNAc, assigned for biological assays on human GnTs, were prepared.     

Bioconversion of <Emphasis Type="SmallCaps">d</Emphasis>-glucose into <Emphasis Type="SmallCaps">d</Emphasis>-glucosone by Glucose 2-oxidase from <Emphasis Type="Italic">Coriolus versicolor</Emphasis> at Moderate Pressures

Glucose 2-oxidase (pyranose oxidase, pyranose:oxygen-2-oxidoreductase, EC 1.1.3.10) from Coriolus versicolor catalyses the oxidation of d-glucose at carbon 2 in the presence of molecular O₂ producing d-glucosone (2-keto-glucose and d-arabino-2-hexosulose) and H₂O₂. It was used to convert d-glucose into d-glucosone at moderate pressures (i.e. up to 150 bar) with compressed air in a modified commercial batch reactor. Several parameters affecting biocatalysis at moderate pressures were investigated as follows: pressure, [enzyme], [glucose], pH, temperature, nature of fluid and the presence of catalase. Glucose 2-oxidase was purified by immobilized metal affinity chromatography on epoxy-activated Sepharose 6B-IDA-Cu(II) column at pH 6.0. The rate of bioconversion of d-glucose increased with the pressure since an increase in the pressure with compressed air resulted in higher rates of conversion. On the other hand, the presence of catalase increased the rate of reaction which strongly suggests that H₂O₂ acted as inhibitor for this reaction. The rate of bioconversion of d-glucose by glucose 2-oxidase in the presence of either nitrogen or supercritical CO₂ at 110 bar was very low compared with the use of compressed air at the same pressure. The optimum temperature (55°C) and pH (5.0) of d-glucose bioconversion as well as kinetic parameters for this enzyme were determined under moderate pressure. The activation energy (E _a) was 32.08 kJ mol⁻¹ and kinetic parameters (V _max, K _m, K _cat and K _cat/K _m) for this bioconversion were 8.8 U mg⁻¹ protein, 2.95 mM, 30.81 s⁻¹ and 10,444.06 s⁻¹ M⁻¹, respectively. The biomass of C. versicolor as well as the cell-free extract containing glucose 2-oxidase activity were also useful for bioconversion of d-glucose at moderate pressures. The enzyme was apparently stable at moderate pressures since such pressures did not affect significantly the enzyme activity.     

Functionalized Alginate as Immobilization Matrix in Enantioselective <Emphasis Type="SmallCaps">l</Emphasis> (+) Lactic Acid Production by <Emphasis Type="Italic">Lactobacillus delbrucekii</Emphasis>

The imperative role of functionalized natural alginate in immobilization of Lactobacillus delbrucekii (NCIM 2365) cells in production of optically pure L (+) lactic acid was studied. L. delbrucekii cells were immobilized in alginate, succinylated alginate and carrageenan to evaluate the bead stability and selectivity towards production of optically pure L (+) lactic acid. The scanning electron microscopic studies of free and immobilized cells show little morphological changes. The present study highlights the use of functionalized alginate-immobilized L. delbrucekii cells in production of L (+) lactic acid in higher yields (0.93 Yp/s in grams) with an improved enantioselectivity (99%). In addition, they further revealed decreased by-product formation (acetic and propionic acid) when compared to free and other immobilized cell fermentation.     

Production of 6-<Emphasis Type="Italic">O</Emphasis>-<Emphasis Type="SmallCaps">l</Emphasis>-Ascorbyl Palmitate by Immobilized <Emphasis Type="Italic">Candida antarctica</Emphasis> Lipase B

l-ascorbyl palmitate (ASP) is an oil-soluble derivative of ascorbic acid which is used extensively in food, cosmetics industry, and medical hygiene. Enzymatic synthesis of ascorbyl palmitate in tert-butyl alcohol was carried out using indigenously immobilized lipase preparation PyCal with ascorbic acid and palmitic acid as starting material. The developed batch process under optimized reaction conditions resulted in conversion of 90% with relatively shorter reaction time of 6 h. Continuous process in packed bed reactor gave conversion of 50% with space time yield of 15.46 g/L/h which was found to be higher than the reported literature on enzymatic synthesis of ascorbyl palmitate. The immobilized lipase used in the present work showed good reusability. Characterization of formed ascorbyl palmitate was carried out by FTIR, MS/MS, H¹-NMR, and C¹³-NMR. The enzymatic process resulted in selective synthesis of 6-O-l-ascorbyl palmitate with purity of 98.6% and no side product formation. The use of underivatized starting materials, high space time yield of 15.46 g L^?1 h^?1, high recyclability of catalyst, and no by-product formation make the overall process highly efficient and clean in terms of energy consumption and waste generation, respectively. The optimized reaction parameters for ascorbyl palmitate synthesis in the present study can be used as a useful reference for industrial synthesis of fatty acid esters of ascorbic acid by enzymatic route.     

Biotransformation of <Emphasis Type="SmallCaps">l</Emphasis>-tyrosine to Dopamine by a Calcium Alginate Immobilized Mutant Strain of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

The present research work is concerned with the biotransformation of l-tyrosine to dopamine (DA) by calcium alginate entrapped conidiospores of a mutant strain of Aspergillus oryzae. Different strains of A. oryzae were isolated from soil. Out of 13 isolated strains, isolate-2 (I-2) was found to be a better DA producer. The wild-type I-2 was chemically improved by treating it with different concentrations of ethyl methyl sulfonate (EMS). Among seven mutant variants, EMS-6 exhibiting maximal DA activity of 43 μg/ml was selected. The strain was further exposed with l-cysteine HCl to make it resistant against diversion and environmental stress. The conidiospores of selected mutant variant A. oryzae EMS-6 strain were entrapped in calcium alginate beads. Different parameters for immobilization were investigated. The activity was further improved from 44 to 62 μg/ml under optimized conditions (1.5 % sodium alginate, 2 ml inoculum, and 2 mm bead size). The best resistant mutant variable exhibited over threefold increase in DA activity (62 μg/ml) than did wild-type I-2 (21 μg/ml) in the reaction mixture. From the results presented in the study, it was observed that high titers of DA activity in vitro could effectively be achieved by the EMS-induced mutagenesis of filamentous fungus culture used.     

<Emphasis Type="SmallCaps">d</Emphasis>-Amino Acids in Animal Peptides

Summary. Secreted peptides from diverse sources have been found to contain a d-amino acid. From the sequence of cloned mRNAs coding for the precursors of such peptides it could be deduced that in all cases tested so far the d-amino acid in the final product is derived from the corresponding l-amino acid present in the primary product of translation. Enzymes catalyzing such an l- to d-isomerization in peptide linkage have been isolated from the venom of a spider and the skin secretions of frogs. Even though these are completely different proteins, the reaction mechanism is the same, namely a de-protonation/re-protonation of the α-carbon of an amino acid with concomitant inversion of the chirality. Sequences potentially coding for homologues of the frog enzyme are present in the genome of different vertebrate species.     

Extracellular Production and Characterization of Two <Emphasis Type="Italic">Streptomyces</Emphasis><Emphasis Type="SmallCaps">l</Emphasis>-Asparaginases

l-Asparaginase (ASNase) has proved its use in medical and food industries. Sequence-based screening showed the thermophilic Streptomyces strain Streptomyces thermoluteus subsp. fuscus NBRC 14270 (14270 ASNase) to positive against predicted ASNase primary sequences. The 14270 ASNase gene and four l-asparaginase genes from Streptomyces coelicolor, Streptomyces avermitilis, and Streptomyces griseus (SGR ASNase) were expressed in Streptomyces lividans using a hyperexpression vector: pTONA5a. Among those genes, only 14270 ASNase and SGR ASNase were successful for overexpression and detected in culture supernatants without an artificial signal peptide. Comparison of the two Streptomyces enzymes described above demonstrated that 14270 ASNase was superior to SGR ASNase in terms of optimum temperature, thermal stability, and pH stability.     

Stability of <Emphasis Type="Italic">s</Emphasis>-, <Emphasis Type="Italic">p</Emphasis>-, and <Emphasis Type="Italic">d</Emphasis>-element diphosphates in water

The mean atomic Gibbs energies of formation of (Δ _f ? _at ⁰ ) of s-, p-, and d-element diphosphates have been calculated using ion increments of the Gibbs energy (Δ _f G ⁰). The diphosphate hydrolysis kinetics is considered, and a correlation between the Δ _f ? _at ⁰ values and the hydrolysis rate constants is presented.     

Synthesis,crystal structure,and magnetic properties of a 3<Emphasis Type="Italic">d</Emphasis>-3<Emphasis Type="Italic">d</Emphasis> mixed heterometallic coordination polymer

A 3d-3d heterometallic coordination polymer, M(Bpp)(BDC) (I) (M = Co and Zn, Bpp = 1,3-bis(4-pyridyl)propane and H₂BDC = 1,4-benzenedicarboxylic acid), has been prepared by hydrothermal synthesis and structurally characterized by IR, elemental analysis, and single-crystal X-ray diffraction. The Co(II) and Zn(II) ions in I are located indisorder with a molar ratio of 0.35: 0.65. Complex I crystallizes in the orthorhombic space group Pbca and displays a two-dimensional 2-fold interpenetrated structure with (4,4) topology. The coexistence of two metal ions in the structure makes them more thermally stable than thepreviously reported homometallic polymers of Co(II) or Zn(II) ions. Complex I exhibits weaker antiferromagnetism than the pure Co(II) complex owing to the incorporation of Zn(II).     

A Novel 2-Keto-<Emphasis Type="SmallCaps">d</Emphasis>-Gluconic Acid High-Producing Strain <Emphasis Type="Italic">Arthrobacter globiformis</Emphasis> JUIM02

2-Keto-d-gluconic acid (2KGA) is mainly used for industrial production of erythorbic acid, a food antioxidant. In this study, a 2KGA producing strain JUIM02 was firstly identified as Arthrobacter globiformis by morphological observation and 16S rDNA sequencing. The 2KGA synthetic capacity of A. globiformis JUIM02 was evaluated by both fermentation and bioconversion, with 180 g/L dextrose monohydrate as substrates, in shake flasks and 5 L fermenters. For fermentation, 2KGA titer, yield, molar yield, and productivity of JUIM02 reached 159.05 g/L, 0.97 g/g, 90.18%, and 6.63 g/L/h in 24 h. For non-sterile and buffer-free bioconversion by free resting cells (~?3.2 g/L dry cell weight) of JUIM02, these data were 172.96 g/L, 1.06 g/g, 98.07%, and 5.41 g/L/h in 32 h. Moreover, JUIM02 resting cells could be repeatedly used. Resting cells stored at 4 °C within 30 days showed stable bioconversion capacity, with 2KGA titers ≥?171.50 g/L, yields ≥?1.04 g/g, and molar yields ≥?97.24%. The 2KGA synthetic pathway in A. globiformis, which was rarely reported, was also speculated similar to Pseudomonas and verified preliminarily. In conclusion, A. globiformis JUIM02 is a promising 2KGA industrial-producing strain suitable for various production methods and a suitable object for 2KGA metabolism research of A. globiformis.     

New tin(<Emphasis Type="SmallCaps">iv</Emphasis>) <Emphasis Type="Italic">o</Emphasis>-iminosemiquinone complexes

New tin(iv) mono- and bis-o-iminosemiquinone complexes were obtained by the exchange reaction of radical anion lithium salt of 4,6-di-tert-butyl-N-(2,6-diisopropylphenyl)-o-imino-benzoquinone with tin(iv) organochlorides. The compounds synthesized were characterized by EPR spectroscopy and X-ray diffraction analysis. Substituents on the tin atom were found to affect stability of paramagnetic metal derivatives formed.     

Splitting of the <Emphasis Type="Italic">d</Emphasis><Superscript><Emphasis Type="Italic">N</Emphasis></Superscript> atomic states in icosahedral 3<Emphasis Type="Italic">d</Emphasis> metal endofullerenes M@C<Subscript>60</Subscript>

Group-theoretical and quantum-chemical investigations of the spectrum of low-lying excited states have been performed by the ROHF and FCI-RAS (Full CI in Restricted Active Space) methods for 3d metal endofullerenes (MEFs) M@C₆₀ (M =Mn, Cr, and Fe) in different charged states. The major purpose of this study is quantum-chemical verification of the anomalous (“non-Bethe’s”) character of splitting of the d ^N atomic states in an electrostatic field with icosahedral symmetry, predicted previously within the theory of integral invariants theory. The interrelation between the integral invariants theory and the quantumchemical methods applied in this work is considered in detail. Our calculations suggest that the d ^N atomic states in the icosahedral field generated by fullerene C60 (I _h ) on a metal atom (ion) remain non-split for different charged states of the metal and C₆₀. Reasons for this phenomenon and other possible approaches to verification of the prediction are discussed. It is demonstrated that the d ^N states of the encapsulated metal are split in icosahedral 3d MEFs only under very strong compression of these structures.     

<Emphasis Type="Italic">Z</Emphasis>-and <Emphasis Type="Italic">E</Emphasis>-conformers of <Emphasis Type="Italic">N</Emphasis>-chloroacetylcytisine

N-Chloroacetylcytisine was synthesized by acylation of (–)-cytisine. Stable Z- and E-conformers with respect to rotational isomerism around the N-12–CO bond were found in PMR spectra at room temperature. The point at which PMR resonances of the Z- and E-conformers coalesced upon heating was measured. The transition barrier between the conformers was estimated.     

Enhancing the Production of <Emphasis Type="SmallCaps">d</Emphasis>-Mannitol by an Artificial Mutant of <Emphasis Type="Italic">Penicillium</Emphasis> sp. T2-M10

d-Mannitol belongs to a linear polyol with six-carbon and has indispensable usage in medicine and industry. In order to obtain more efficient d-mannitol producer, this study has screened out a stable mutant Penicillium sp. T2-M10 that was isolated from the initial d-mannitol-produced strain Penicillium sp.T2-8 via UV irradiation as well as nitrosoguanidine (NTG) induction. The mutant had a considerable enhancement in yield of d-mannitol based on optimizing fermentation. The production condition was optimized as the PDB medium with 24 g/L glucose for 9 days. The results showed that the production of d-mannitol from the mutant strain T2-M10 increased 125% in contrast with the parental strain. Meanwhile, the fact that d-mannitol is the main product in the mutant simplified the process of purification. Our finding revealed the potential value of the mutant strain Penicillium sp. T2-M10 to be a d-mannitol-producing strain.     

Analysis of the Production Process of Optically Pure <Emphasis Type="SmallCaps">d</Emphasis>-Lactic Acid from Raw Glycerol Using Engineered <Emphasis Type="Italic">Escherichia coli</Emphasis> Strains

Glycerol has become an ideal feedstock for producing fuels and chemicals. Here, five technological schemes for optically pure d-lactic acid production from raw glycerol were designed, simulated, and economically assessed based on five fermentative scenarios using engineered Escherichia coli strains. Fermentative scenarios considered different qualities of glycerol (pure, 98 wt.%, and crude, 85 wt.%) with concentrations ranging from 20 to 60 g/l in the fermentation media, and two fermentation stages were also analyzed. Raw glycerol (60 wt.%) was considered as the feedstock feeding the production process in all cases; then a purification process of raw glycerol up to the required quality was required. Simulation processes were carried out using Aspen Plus, while economic assessments were performed using Aspen Icarus Process Evaluator. D-Lactic acid recovery and purification processes were based on reactive extraction with tri-n-octylamine using dichloromethane as active extractant agent. The use of raw glycerol represents only between 2.4% and 7.8% of the total production costs. Also, the total production costs obtained of D-lactic acid in all cases were lower than its sale price indicating that these processes are potentially profitable. Thus, the best configuration process requires the use of crude glycerol diluted at 40 g/l with total glycerol consumption and with D-lactic acid recovering by reactive extraction. The lowest obtained total production cost was 1.015 US$/kg with a sale price/production cost ratio of 1.53.     

A DFT study of H<Subscript>2</Subscript>CO and HCN adsorptions on 3<Emphasis Type="Italic">d</Emphasis>, 4<Emphasis Type="Italic">d</Emphasis>, and 5<Emphasis Type="Italic">d</Emphasis> transition metal-doped graphene nanosheets

The binding of 3d (Sc, Ti, V), 4d (Y, Zr, Nb), and 5d (La, Hf, Ta) transition metals on graphene nanosheet (TM–GNS) with hydrogen-terminated edges and the adsorption of H₂CO and HCN molecules on the pristine and TM-doped GNSs were theoretically studied using a density functional theory method. The calculation showed that all TM atoms had strong binding with GNS, in which the Ta atom displayed the strongest interaction with GNS. The H₂CO and HCN molecules showed much stronger adsorption on the TM–GNSs than that on the pristine GNS. The H₂CO showed stronger interactions with TM–GNSs than that of HCN, in which the Ta-doping displayed the strongest interactions between the GNS and H₂CO or HCN. The adsorption interactions induced dramatic changes of TM–GNS electronic properties. The results revealed that the adsorption strength and sensor ability of GNS can be greatly improved by introducing appropriate TM dopants. Therefore, TM-doped GNSs are suitable for application in H₂CO and HCN storage and sensor.     

Enhanced <Emphasis Type="SmallCaps">l</Emphasis><Emphasis Type="Italic">-</Emphasis>(+)-Lactic Acid Production by an Adapted Strain of <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> using Corncob Hydrolysate

Corncob is an economic feedstock and more than 20 million tons of corncobs are produced annually in China. Abundant xylose can be potentially converted from the large amount of hemicellulosic materials in corncobs, which makes the crop residue an attractive alternative substrate for a value-added production of a variety of bioproducts. Lactic acid can be used as a precursor for poly-lactic acid production. Although current industrial lactic acid is produced by lactic acid bacteria using enriched medium, production by Rhizopus oryzae is preferred due to its exclusive formation of the l-isomer and a simple nutrition requirement by the fungus. Production of l-(+)-lactic acid by R. oryzae using xylose has been reported; however, its yield and conversion rate are poor compared with that of using glucose. In this study, we report an adapted R. oryzae strain HZS6 that significantly improved efficiency of substrate utilization and enhanced production of l-(+)-lactic acid from corncob hydrolysate. It increased l-(+)-lactic acid final concentration, yield, and volumetric productivity more than twofold compared with its parental strain. The optimized growth and fermentation conditions for Strain HZS6 were defined.     

5<Emphasis Type="Italic">p</Emphasis>-4<Emphasis Type="Italic">d</Emphasis> Emission spectrum with a cascade decay of the 3<Emphasis Type="Italic">d</Emphasis> vacancy in the Xe atom

The 5p-4d emission spectrum, obtained during cascade de-excitation of the initial deep 3d vacancy in the Xe atom, was calculated by directly constructing a decay tree in a stepwise manner. The cascade decay tree was constructed with allowance for all radiative and nonradiative channels of decay for the initial and all intermediate ionic configurations of the cascade. The probabilities of branching and transition energies were calculated in a one-electron approximation with averaging over configurations. The 5p-4d cascade emission spectrum has a complex structure with many components, which reflects the transitions from the wide variety of the many-hole intermediate configurations generated by the cascade. The calculated data agree well with the experiment.     

Production of Oligosaccharide from Alginate Using <Emphasis Type="Italic">Pseudoalteromonas agarovorans</Emphasis>

A marine bacterium was isolated from seawater near the Korean south coast for efficient saccharification from alginate. Based on 16S rDNA sequence, the isolated strain was identified as Pseudoalteromonas agarovorans. Various environmental factors affecting saccharification of alginate using P. agarovorans CHO-12 have been investigated in flask cultures. The optimum concentration of sugar was obtained at 30 rpm and 29 °C. Among various NaCl concentrations, when NaCl concentration was increased from 10 to 30 g/l, the cell concentration sharply increased, while there is no increase at above 40 g/l. The maximum sugar concentration was obtained at 13.8 when 30 g/l of NaCl was used. Yeast extract and corn steep liquor were the best nitrogen source for efficient saccharification. Especially, the sugar concentration of 14.9 g/l was obtained after 3 days of culture using a mixture of 1.0 g/l of yeast extract and 1.5 g/l of corn steep liquor. Scale up was carried out at 50 l of reactor for 3 days using P. agarovorans CHO-12 and Stenotrophomonas maltophilia sp. When S. maltophilia was used, cell concentration was about twofold higher than that of P. agarovorans CHO-12. On the other hand, when P. agarovorans CHO-12 was used, the maximum saccharification rate was obtained, 7.5 g/l/day after 2 days of culture, which was about tenfold higher than that of S. maltophilia.     

Tagatose Production with pH Control in a Stirred Tank Reactor Containing Immobilized <Emphasis Type="SmallCaps">l</Emphasis>-Arabinose Isomerase from <Emphasis Type="Italic">Thermotoga neapolitana</Emphasis>

Chitopearl beads were used as immobilization supports for D-tagatose production from D-galactose by L-arabinose isomerase from Thermotoga neapolitana because chitopearl beads were more stable than alginate beads at temperatures above 60 degrees C. The pH and temperature for the maximum isomerization of galactose were 7.5 and 90 degrees C, respectively. In thermostability experiments, the half-lives of the immobilized enzyme at 70, 75, 80, 85, and 90 degrees C were 388, 106, 54, 36, and 22 h, respectively. The reaction temperature was determined to be 70 degrees C because the enzyme is highly stable up to 70 degrees C during the reaction. When the reaction time, galactose concentration, and temperature were increased, the pH of a mixture containing enzyme and galactose decreased by the Maillard reaction, resulting in decreased tagatose production. With pH control at 7.5, tagatose production (138 g/L) at 70 degrees C in a stirred tank reactor containing immobilized enzyme and 300 g/L galactose increased two times higher, comparing that without pH control.