Improvement of <Emphasis Type="Italic">Aspergillus sulphureus</Emphasis> Endo-β-1,4-Xylanase Expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis> by Codon Optimization and Analysis of the Enzymic Characterization

The gene xynB from Aspergillus sulphureus encoding the endo-β-1,4-xylanase was de novo synthesized by splicing overlap extension polymerase chain reaction according to Pichia pastoris protein’s codon bias. The synthetic DNA and wild-type DNA were placed under the control of a glyceraldehyde-3-phosphate dehydrogenase gene promoter (GAP) in the constitutive expression vector plasmid pGAPzαA and electrotransformed into the P. pastoris X-33 strain, respectively. The transformants screened by Zeocin were able to constitutively secrete the xylanase in YPD liquid medium. The maximum yield of the recombinant xylanase produced by the synthetic DNA was 105 U ml⁻¹, which was about 5-fold higher than that by wild-type DNA under the flask culture at 28 °C for 3 days. The enzyme showed optimal activity at 50 °C and pH 5.0. The residual activity remained above 90% after the recombinant xylanase was pretreated in Na₂HPO₄–citric acid buffer (pH 2.4) for 2 h. The xylanase activity was significantly improved by Zn²⁺. These biochemical characteristics suggest that the recombinant xylanase has a prospective application in feed industry as an additive.     

Characterization of Two New <Emphasis Type="Italic">Endo</Emphasis>-β-1,4-xylanases from <Emphasis Type="Italic">Eupenicillium parvum</Emphasis> 4–14 and Their Applications for Production of Feruloylated Oligosaccharides

Two new endo-1,4-beta-xylanases encoding genes EpXyn1 and EpXyn3 were isolated from mesophilic fungus Eupenicillium parvum 4–14. Based on analysis of catalytic domain and phylogenetic trees, the xylanases EpXYN1 (404 aa) and EpXYN3 (220 aa) belong to glycoside hydrolase (GH) family 10 and 11, respectively. Both EpXYN1 and EpXYN3 were successfully expressed in Pichia pastoris and the recombinant enzymes were characterized using beechwood xylan, birchwood xylan, or oat spelt xylan as substrates, respectively. The optimum temperatures and pH values were 75 °C and 5.5 for EpXYN1, and 55 °C and 5.0 for EpXYN3. EpXYN1 exhibited a high stability at high temperature (65 °C) or at pH values from 8 to 10. EpXYN3 kept over 80% enzymatic activity after treatment at pH values from 3 to 10. The specific activities of EpXYN1 and EpXYN3 were 384.42 and 214.20 U/mg using beechwood xylan as substrate, respectively. EpXYN1 showed lower K_m values and higher specific activities toward different xylans compared to EpXYN3. Thin-layer chromatography analysis indicated that the hydrolysis profiles of xylans or xylo-oligosacharides were different by EpXYN1and EpXYN3. EpXYN3 had a higher efficiency than EpXYN1 in production of feruloylated oligosaccharides (FOs) from de-starched wheat bran. The maximum levels of FOs released by EpXYN1 and EpXYN3 were 11.1 and 14.4 μmol/g, respectively. In conclusion, the two xylanases are potential candidates for various industrial applications.     

Optimization of <Emphasis Type="Italic">α</Emphasis>-Terpineol Production by the Biotransformation of <Emphasis Type="Italic">R</Emphasis>-(+)-Limonene and (−)-β-Pinene

The main objective of this work is to present the optimization of the biotransformation of R-(+)-limonene and (−)-β-pinene aiming at the production of α-terpineol by strains of fungal and yeasts previously isolated by our research group using the methodology of experimental design. New optimized experimental data on α-terpineol production by the biotransformation of R-(+)-limonene and (−)-β-pinene using newly isolated microorganisms are reported in this work. Conversion of about 1,700 mg/L was achieved when R-(+)-limonene was used as substrate and the newly isolated strain 05.01.35 as microorganism at the central point of the experimental design, corresponding to a substrate concentration of 1.75%, mass of inoculum of 2 g, and substrate to ethanol volume ratio of 1:1. The same experimental condition led to higher conversions when (−)-β-pinene was used as substrates and the strains coded as 04.05.08 and 01.04.03 as microorganism. Here, conversions of about 770 mg/L were achieved.     

<Emphasis Type="Italic">β</Emphasis>-Chloro-<Emphasis Type="Italic">α</Emphasis>,<Emphasis Type="Italic">β</Emphasis>-unsaturated carbonyls as convenient precursors of highly substituted benzenes

The Diels–Alder reactions of three β-chloro-α,β-unsaturated carbonyl compounds 1–3 with different dienes were carried out to afford highly functionalized cyclohexenes 4–9, bearing quaternary centers, in good yields. These cycloadducts (CAs) undergo dehydrochlorination with subsequent aromatization in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene DBU to produce new substituted benzenes 11–14. Compound 10 is the product of lactonization and removal of an HCl molecule from compound 7. All products were characterized by NMR, IR, elementary analysis and some of them by MS. Structure assignments of isomers were carried out on the basis of NMR chemical shifts and coupling constants using 1D, 2D and heteronOe NMR techniques.     

An efficient synthesis of <Emphasis Type="Italic">β</Emphasis>-methoxycarbonyl-<Emphasis Type="Italic">γ</Emphasis>-butyrolactones

β-Methoxycarbonyl-γ-butyrolactones bearing a γ-aromatic substituent were prepared via copper-catalyzed reductive aldol addition/lactonization domino reactions of ketones with α,β-unsaturated dicarboxylate esters and a silane under ambient temperature. The reaction has advantage of using readily available reagents, mild conditions and high efficiency.     

Optimization of the Production of Thermostable endo-β-1,4 Mannanases from a Newly Isolated <Emphasis Type="Italic">Aspergillus niger gr</Emphasis> and <Emphasis Type="Italic">Aspergillus flavus gr</Emphasis>

The aim of this work was to establish optimal conditions for the maximum production of endo-β-1,4 mannanases using cheaper sources. Eight thermotolerant fungal strains were isolated from garden soil and compost samples collected in and around the Gulbarga University campus, India. Two strains were selected based on their ability to produce considerable endo-β-1,4 mannanases activity while growing in liquid medium at 37 °C with locust bean gum (LBG) as the only carbon source. They were identified as Aspergillus niger gr and Aspergillus flavus gr. The experiment to evaluate the effect of different carbon sources, nitrogen sources, temperatures and initial pH of the medium on maximal enzyme production was studied. Enzyme productivity was influenced by the type of polysaccharide used as the carbon source. Copra meal defatted with n-hexane showed to be a better substrate than LBG and guar gum for endo-β-1,4 mannanases production by A. niger gr (40.011 U/ml), but for A. flavus gr (33.532 U/ml), the difference was not significant. Endo-β-1,4 mannanases produced from A. niger gr and A. flavus gr have high optimum temperature (65 and 60 °C) and good thermostability in the absence of any stabilizers (maintaining 50% of residual activity for 8 and 6 h, respectively, at 60 °C) and are stable over in a wide pH range. These new strains offer an attractive alternative source of enzymes for the food and feed processing industries.     

10<Emphasis Type="Italic">α</Emphasis>-Hydroxy-<Emphasis Type="Italic">β</Emphasis>-isomorphine,a by-product of the synthesis of 10<Emphasis Type="Italic">α</Emphasis>-hydroxymorphine

Abstract  A new compound was isolated from the reaction mixture after O-demethylation of 6-O-acetyl-10α-acetoxycodeine with boron tribromide. The structure of this compound, 10α-hydroxy-β-isomorphine, was elucidated by spectral data, and its spatial arrangement was deduced from an NOE experiment. Capillary zone electrophoresis was used for separation of morphine and its 10-hydroxy analogues. Graphical abstract        

<Emphasis Type="Italic">N</Emphasis>′-tetrazolylmethoxyl derivatives of <Emphasis Type="Italic">N</Emphasis>-methyldiazene <Emphasis Type="Italic">N</Emphasis>-oxides

A preparation method was developed for previously unknown tetrazole derivatives containing in the 1, 2, and/or 5 positions of the tetrazole ring N-methyldiazene-N-oxide-N′-oxymethyl groups.     

Single-Step Partial Purification of Intracellular <Emphasis Type="Italic">β</Emphasis>-Galactosidase from <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis> Using Microemulsion Droplets

Partial purification of β-galactosidase from the crude extract of Kluyveromyces lactis was carried out using water-in-isooctane microemulsions formed by the anionic surfactant, sodium di-ethylhexyl sulfosuccinate (Aerosol OT). In order to obtain the crude extract, yeast cells of K. lactis were disrupted by a cell disrupter and separated. The purification of β-galactosidase from the extract by a recently developed one-step reversed micellar (i.e., microemulsion-based) extraction method was then tested, by measuring total protein mass and enzyme activity in the product stream and by analyzing its composition using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography. Effects of salt concentration, protein concentration, and pH on the extraction were investigated. Using this approach, a 5.4-fold purification of β-galactosidase was achieved with 96 % total activity recovery, using a feed containing crude extract and 50 mM K-phosphate buffer (pH 7.5) and 50 mM KCl. Gel filtration chromatography showed that the single extraction was successful at removing low molecular weight impurity proteins (molecular weight (MW)?<?42 kDa) from the crude extract.     

Theoretical and experimental NMR studies of the Swern oxidation of methyl 6<Emphasis Type="Italic">α</Emphasis>,7<Emphasis Type="Italic">β</Emphasis>-dihydroxyvouacapan-17<Emphasis Type="Italic">β</Emphasis>-oate

This work describes the synthesis of new derivatives of 6α,7β-dihydroxyvouacapan-17β-oic acid (1) employing the Swern method for the oxidation of C-6 and C-7 of methyl 6α,7β-di-hydroxyvouacapan-17β-oate (2) and the formation of methyl 6,7-dioxovouacapan-17β-oate (3). NMR structural studies associated with theoretical calculations of reaction intermediates and products are also reported. The mixture of methyl 7β-hydroxy-6-oxovouacapan-17β-oate (4; 21%) and methyl 6α-hydroxy-7-oxovouacapan-17β-oate (5; 79%) was the product from the first step of the oxidation of 2. The lower energy of 5, calculated by HF/6-31G* and DFT/BLYP/6-31G* methods, reinforces the thermodynamic control proposed for the reaction. After further Swern oxidation of the mixture of 4 and 5, the isomeric form methyl 6-hydroxy-7-oxovouacap-5-en-17β-oate (6) was obtained. Theoretical calculations indicate a lower energy for the mono-enol 6 than the 1,2-diketo 3 and the other mono-enol methyl 7-hydroxy-6-oxovouacap-7-en-17β-oate (7).     

Synthesis of 20<Emphasis Type="Italic">S</Emphasis>-protopanaxadiol <Emphasis Type="Italic">β</Emphasis>-D-galactopyranosides

20S-Protopanaxadiol (3β,12β,20S-trihydroxydammar-24-ene) 3-, 12-, and 20-O-β-D-galactopyranosides were synthesized for the first time. Condensation of 12β-acetoxy-3β,20S-dihydroxydammar-24-ene (1) and 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosylbromide (α-acetobromogalactose) (2) under Koenigs–Knorr conditions with subsequent removal of the protecting groups resulted in regio- and stereoselective formation of 20S-protopanaxadiol 3-O-β-D-galactopyranoside, an analog of the natural ginsenoside Rh₂. Glycosylation of 12β,20S-dihydroxydammar-24-en-3-one (5) by 2 with subsequent treatment of the reaction products with NaBH₄ in isopropanol and deacetylation with NaOMe gave 20S-protopanaxadiol 12- and 20-O-β-Dgalactopyranosides.     

Spectroscopic and electrical characterization of <Emphasis Type="Italic">α</Emphasis>,<Emphasis Type="Italic">γ</Emphasis>-bisdiphenylene-<Emphasis Type="Italic">β</Emphasis>-phenylallyl radical as an organic semiconductor

The semiconductor properties of the earliest known stable radical, α,γ-bisdiphenylene-β-phenylallyl radical (Koelsch radical, 1^?) were assessed using spectroscopic and electrical techniques. This radical undergoes reversible redox processes, and it has low redox potentials. In addition, 1^? possesses long wavelength absorption bands, owing to the existence of a singly-occupied molecular orbital whose energy level lies between those of the HOMO and LUMO. A spin-coated thin-film of 1^? displays photocurrent and an electron mobility of 6.3 × 10^?7 cm² V^?1 s^?1 on a trially-fabricated organic field effect transistor.     

Allylic oxidation of 19<Emphasis Type="Italic">β</Emphasis>,28-epoxy-a-neo-5<Emphasis Type="Italic">β</Emphasis>-methyl-25-nor-18<Emphasis Type="Italic">α</Emphasis>-olean-9-ene

Allylic oxidation of 19β,28-epoxy-A-neo-5β-methyl-25-nor-18α-olean-9-ene by ozone produced 19β,28-epoxy-A-neo-5β-methyl-25-nor-18α-olean-9-en-1-one.     

Recognition of <Emphasis Type="Italic">R</Emphasis>- and <Emphasis Type="Italic">S</Emphasis>-Isomers of α-Alanine by Chiral Nanotubes

DFT PBE/3ζ study of relative stability of the R- and S-isomers of α-alanine in open carbon nanotubes with chirality indices (5,5), P,M-(5,1) and P,M-(5,2) has shown that the encapsulated molecule of the amino acid notably changes its geometrical and electronic characteristics. Besides, for the clusters α-alanine@nano (5,2) the most stable are practically degenerate in energy adducts S-alanine@P-(5,2) and R-alanine@М-(5,2). All this indicates the ability of chiral nanotubes to recognize encapsulated molecules of the R- and S-isomers.     

Synthesis of <Emphasis Type="Italic">α</Emphasis>-chloropyridine derivatives of <Emphasis Type="Italic">γ</Emphasis>-aminobutyric acid

The sodium salt of N-(6-chloronicotinoyl)-γ-aminobutyric acid, a structural analog of the known nootropic and vasidilating drug picamilon, was synthesized via Schotten–Baumann acylation of γ-aminobutyric acid with 6-chloronicotinoyl chloride and subsequent neutralization of the N-(6-chloronicotinoyl)-γ-aminobutyric acid that was obtained in >60% yield.     

Immobilisation of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> α-amylase and <Emphasis Type="Italic">Aspergillus niger</Emphasis> glucoamylase enzymes as cross-linked enzyme aggregates

Cross-linked enzyme aggregates (CLEA) of Aspergillus oryzea α-amylase (AoAA) and Aspergillus niger glucoamylase (AnGA) were prepared using glutaraldehyde and dextran polyaldehyde as cross-linkers. The maximum activity recoveries for glutaraldehyde cross-linking were 21.8 % and 41.2 %, respectively. The addition of a proteic feeder (bovine serum albumin) exhibited a negative effect on the activity recoveries for both enzymes. Dextran polyaldehyde was used as a cross-linking agent instead of glutaraldehyde to reduce the activity losses. As a result, an activity recovery of 60.0 % was obtained for Aspergillus oryzea α-amylase. On the other hand, no activity recovery was observed for Aspergillus niger glucoamylase due to the latter enzyme’s affinity for dextran.     

Thermophysical properties of <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-dimеthylacetamide mixtures with <Emphasis Type="Italic">n</Emphasis>-butanol

The refraction, dielectric, viscosity, density, data of the binary mixtures of N,N-dimethylacetamide (DMA) with n-butanol at 308.15 and 313.15 K. The measured parameters used to obtain derived properties like Bruggeman factor, molar refraction and excess static dielectric constant, excess inverse relaxation time, excess molar volume and excess viscosity, excess molar refraction. The variation in magnitude with composition and temperature of these quantities has been used to discuss the type, strength and nature of binary interactions. Results confirm that there are strong hydrogen-bond interactions between unlike molecules of DMA+ n-butanol mixtures and that 1: 1 complexes are formed and strength of intermolecular interaction increases with temperature.     

Directed Aminomethylation of Pyrrole,Indole, and Carbazole with <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N′</Emphasis>,<Emphasis Type="Italic">N′</Emphasis>-Tetramethylmethanediamine

Catalytic aminomethylation of pyrrole and indole with N,N,N′,N′-tetramethylmethanediamine in the presence of 5 mol % of ZrOCl₂·8H₂O proceeds selectively at the positions 2, 5 of pyrrole and 1, 3 of indole. Carbazole under the same conditions affords 3-formyl-9-aminomethyl derivative. The reaction in the presence of 5 mol % of K₂CO₃ occurs as monoaminomethylation: for pyrrole at the position 2, for indole at the position 3, and for carbazole at the nitrogen atom of the substrate. Water-soluble 1,1′-(1H-pyrrole-2,5-diyl)bis(N,N-dimethylmethanamine) exhibits a fungistatic activity with respect to phytopathogenic fungi Rhizoctonia solani.