Oxidative Stress Response and Morphological Changes of Blakeslea trispora Induced by Butylated Hydroxytoluene During Carotene Production

The adaptive response of the fungus Blakeslea trispora to the oxidative stress induced by butylated hydroxytoluene (BHT) during carotene production in shake flask culture was investigated. The culture response to oxidative stress was studied by measuring the specific activities of catalase (CAT) and superoxide dismutase (SOD) and the micromorphology of the fungus using a computerized image analysis system. The addition of exogenous BHT to the medium caused changes of the morphology of microorganism from aggregates with large projected area to aggregates with small projected area. This morphological differentiation of the fungus was associated with high oxidative stress as evidenced by remarkable increase of the specific activities of CAT and SOD. The oxidative stress in B. trispora resulted in a fivefold increase of carotene production. The highest concentration of carotenes (125.0 mg/g dry biomass) was obtained in culture grown in medium supplemented with 20 mM of BHT.     

Enhanced Lycopene Content in Blakeslea trispora by Effective Mutation-Screening Method

The zygomycete fungus Blakeslea trispora is usually used as a natural source of lycopene and β-carotene. In this study, the B. trispora (?) strain, a major mating type for lycopene production, was treated with N⁺ ion implantation and N-methyl-N′-nitro-N-nitrosoguanidine (NTG), and further isolated on the screening plates supplemented with lovastatin and crude extracts of trisporic acid (CTA). After several rounds of screening, four mutants with higher yield of lycopene and biomass were isolated. Among these mutants, I5 obtained with N⁺ ion implantation showed a maximum lycopene yield (28.8 mg/g), which was 64 % higher than the parent strain (17.5 mg/g) in the production of lycopene. The results indicated that N⁺ ion implantation is more suitable for B. trispora (?) than NTG treatment, and the addition of lovastatin promoted the generation of positive mutant and CTA amplified the color differences between colonies.     

Mutation Breeding of Lycopene-Producing Strain Blakeslea Trispora by a Novel Atmospheric and Room Temperature Plasma (ARTP)

To improve the fermentation efficiency of lycopene, a plasma jet, driven by an active helium atom supplied with atmospheric and room temperature plasma (ARTP) biological breeding system, was used as a new method to generate mutations in Blakeslea trispora (?). After several rounds of screening, a mutant A5 with high concentration of lycopene and dry biomass was isolated, which showed a maximum lycopene concentration (26.4?±?0.2 mg/g dry biomass) which was 55 % higher than the parent strain (16.9?±?0.3 mg/g dry biomass) in the production of lycopene. Compared with parent strain, B. trispora A5 required less dissolved oxygen (10 % less than that of parent strain) to reach maximum concentration in a 5-L stirred tank reactor batch fermentation.     

Biodegradable Polycaprolactone (PCL) Nanosphere Encapsulating Superoxide Dismutase and Catalase Enzymes

Biodegradable polycaprolactone (PCL) nanosphere encapsulating superoxide dismutase (SOD) and catalase (CAT) were successfully synthesized using double emulsion (w/o/w) solvent evaporation technique. Characterization of the nanosphere using dynamic light scattering, field emission scanning electron microscope, and Fourier transform infrared spectroscopy revealed a spherical-shaped nanosphere in a size range of 812?±?64 nm with moderate protein encapsulation efficiency of 55.42?±?3.7 % and high in vitro protein release. Human skin HaCat cells were used for analyzing antioxidative properties of SOD- and CAT-encapsulated PCL nanospheres. Oxidative stress condition in HaCat cells was optimized with exposure to hydrogen peroxide (H₂O₂; 1 mM) as external stress factor and verified through reactive oxygen species (ROS) analysis using H₂DCFDA dye. PCL nanosphere encapsulating SOD and CAT together indicated better antioxidative defense against H₂O₂-induced oxidative stress in human skin HaCat cells in comparison to PCL encapsulating either SOD or CAT alone as well as against direct supplement of SOD and CAT protein solution. Increase in HaCat cells SOD and CAT activities after treatment hints toward uptake of PCL nanosphere into the human skin HaCat cells. The result signifies the role of PCL-encapsulating SOD and CAT nanosphere in alleviating oxidative stress.     

LIGHT-DEPENDENT CAROTENOID BIOSYNTHESIS IN MUTANT C-6D OF Scenedesmus obliquus

Abstract— Pigment mutant C-6D of the green alga Scenedesmus obliquus exhibits a light-dependent chloroplast differentiation. The changes were analyzed by high performance liquid chromatography (HPLC). Dark-grown cells of mutant C-6D contain only precursors of carotenoids (carotene, neurosporene, lycopene and β-zeacarotene). The lycopene and two neurosporenes were identified to be cw-isomers. After transfer to light lycopene decreases rapidly. Simultaneously the other precursors increase for a short period and then decrease, too.
Using a specific HPLC-system the changes in carotene- and xanthophyll-content were analyzed. During the first 30 min of illumination the amount of β-carotene increases very fast and then levels off. The kinetics of xanthophyll-formation differ remarkably. Whereas lutein is synthesized immediately after onset of illumination, all other xanthophylls are formed with high rates only after a lag-phase of about 30 min.
After illumination in the presence of nicotine no cyclic carotenes and xanthophylls could be detected. Instead of the cw-lycopene these cells contain high amounts of the all -trans lycopene.
The light-regulation of the biosynthetic pathway leading to carotenes and xanthophylls in mutant C-6D is discussed.     

Exogenous Spermidine Alleviates UV-Induced Growth Inhibition of Synechocystis sp. PCC 6803 via Reduction of Hydrogen Peroxide and Malonaldehyde Levels

Effects of exogenously added spermidine (Spd) to UV-treated Synechocystis sp. PCC 6803 cultures on their growth, intracellular pigments, hydrogen peroxide (H₂O₂), malonaldehyde (MDA) contents, and antioxidant enzymes were investigated. Growth inhibition of cells subjected to 1-h UV-A, UV-B, and UV-C irradiation was abolished in culture added with 0.5 mM Spd. Both chlorophyll a and carotenoid contents were decreased under UV radiations in cells grown in BG₁₁ medium. However, the contents of these two pigments were slightly increased under UV radiations in Spd-supplemented cells with the consequence of enhanced oxygen evolution. Intracellular levels of H₂O₂ and MDA generated during 1-h UV irradiation were decreased when the culture medium contained 0.5 mM Spd. The antioxidative enzymes, catalase, and superoxide dismutase had a little or no response towards Spd supplementation under UV irradiation except for some increase in superoxide dismutase activity under UV-C. Total intracellular polyamines were decreased during Spd supplementation under UV stress; however, the cells showed a drastic increase in the amount of Put under this condition. Altogether, exogenous Spd is likely a potential compound that enables Synechocystis cells to cope with UV stress.     

Growth, Osmolyte Concentration and Antioxidant Enzymes in the Leaves of Sesuvium portulacastrum L. Under Salinity Stress

In this study, growth and osmolyte concentration in the leaves of halophyte, Sesuvium portulacastrum, were studied with respect to salinity. Therefore, the changes in shoot growth, leaf tissue water content, osmolyte concentration (proline content, glycine betaine) and antioxidant enzymes [polyphenol oxidase (PPO), superoxide dismutase (SOD) and catalase (CAT)] were investigated. The 30-day old S. portulacastrum plants were subjected to 100, 200, 300, 400, 500 and 600 mM NaCl for 28 days. The plant growth was steadily increased up to 500 mM NaCl stress at 28 days. TWC was higher in 300 mM NaCl treated leaves than that of 600 mM NaCl. Salinity stress induced the accumulation of osmolyte concentration when compared to control during the study period. The antioxidant enzymes PPO, CAT and SOD were increased under salinity.     

High-resolution Orbitrap mass spectrometry for the analysis of carotenoids in tomato fruit: validation and comparative evaluation towards UV–VIS and tandem mass spectrometry

In this study, a generic extraction protocol and full-scan high-resolution Orbitrap-mass spectrometry (MS) detection method were developed, enabling the metabolomic screening for carotenoids in tomato fruit tissue. To this end, the carotenoids lutein, zeaxanthin, α-carotene, β-carotene, and lycopene (representing both xanthofylls and carotenes) were considered. The extraction procedure was optimized by means of a D-optimal design and consisted of a liquid–liquid extraction with methanol/tert-butyl methyl ether (1:1, v/v). The considered compounds were detected by a single-stage Exactive^TM mass spectrometer, operating at a mass resolution of 100,000 full width at half maximum. The validation study demonstrated excellent performance in terms of linearity (R ²?>?0.99), repeatability (CV?≤?10.6 %), within-laboratory reproducibility (CV?≤?12.2 %), and mean corrected recovery (ranging from 85 to 106 %). Additionally, a comparative evaluation towards well-established detection techniques, i.e., tandem mass spectrometry (MS/MS) and ultraviolet-visible spectroscopy (UV–VIS) photodiode array, indicated superior performance of high-resolution Orbitrap-MS with regard to specificity/selectivity and sensitivity (with limits of detection ranging from 1.0 to 3.8 pg μL^?1). As a result, it may be concluded that high-resolution Orbitrap-MS is a suited alternative for UV–VIS or MS/MS in analyzing carotenoids and may offer significant value in carotenoid research because of the metabolomic screening possibilities.

Enzymatic Synthesis of γ-Glutamylmethylamide from Glutamic Acid γ-Methyl Ester and Methylamine Catalyzed by Escherichia coli Having γ-Glutamyltranspeptidase Activity

A new method for the synthesis of γ-glutamylmethylamide is presented. Glutamic acid γ-methyl ester was used as substrate for γ-glutamylmethylamide synthesis catalyzed by Escherichia coli with γ-glutamyltranspeptidase activity. Reaction conditions were optimized by using 300 mM glutamic acid γ-methyl ester and 3,000 mM methylamine at pH 10 and 40 °C. Bioconversion rate of γ-glutamylmethylamide reached 87 % after 10 h. γ-Glutamyltranspeptidase was reversibly inhibited only when glutamic acid γ-methyl ester was above 300 mM.     

Cloning and Characterization of Cold-Adapted α-Amylase from Antarctic <Emphasis Type="Italic">Arthrobacter agilis</Emphasis>

In this study, the gene encoding an α-amylase from a psychrophilic Arthrobacter agilis PAMC 27388 strain was cloned into a pET-28a(+) vector and heterologously expressed in Escherichia coli BL21(DE3). The recombinant α-amylase with a molecular mass of about 80 kDa was purified by using Ni²⁺-NTA affinity chromatography. This recombinant α-amylase exhibited optimal activity at pH 3.0 and 30 °C and was highly stable at varying temperatures (30–60 °C) and within the pH range of 4.0–8.0. Furthermore, α-amylase activity was enhanced in the presence of FeCl₃ (1 mM) and β-mercaptoethanol (5 mM), while CoCl₂ (1 mM), ammonium persulfate (5 mM), SDS (10 %), Triton X-100 (10 %), and urea (1 %) inhibited the enzymatic activity. Importantly, the presence of Ca²⁺ ions and phenylmethylsulfonyl fluoride (PMSF) did not affect enzymatic activity. Thin layer chromatography (TLC) analysis showed that recombinant A. agilis α-amylase hydrolyzed starch, maltotetraose, and maltotriose, producing maltose as the major end product. These results make recombinant A. agilis α-amylase an attractive potential candidate for industrial applications in the textile, paper, detergent, and pharmaceutical industries.     

Preparation and characterization of thermally stable cellulose nanocrystals via a sustainable approach of FeCl<Subscript>3</Subscript>-catalyzed formic acid hydrolysis

Cellulose nanocrystals (CNCs) can be used as building blocks for the production of many renewable and sustainable nanomaterials. In this work, CNCs were produced from bleached eucalyptus kraft pulp with a high yield over 75 % via FeCl₃-catalyzed formic acid (FA) hydrolysis process. It was found that the particle size of resultant CNC products (F-CNC) decreased with the increase of FeCl₃ dosage in FA hydrolysis, and a maximum crystallinity index of about 75 % could be achieved when the dose of FeCl₃ was 0.015 M (i.e. about 7 % based on the weight of starting material). Thermogravimetric analyses revealed that F-CNC exhibited a much higher thermal stability (the decomposition temperature was over 260 °C) than S-CNC prepared by typical sulfuric acid hydrolysis. In the FeCl₃-catalyzed FA hydrolysis process, FA could be easily recovered and reused, and FeCl₃ could be transferred to Fe(OH)₃ as a high value-added product. Thus, the FeCl₃-catalyzed FA hydrolysis process could be sustainable and economically feasible. In addition, F-CNC could be well dispersed in DMSO and its dispersibility in water could be improved by a cationic surface modification.     

Structural and physico-chemical characteristics of tetraethylammonium tetrachloridoferrate(III)

The crystal structure of tetraethylammonium tetrachloridoferrate(III) was determined. The crystals are hexagonal, space group P6₃ mc, a = 8.198(1) Å, b = 8.198(1) Å, c = 13.183(3) Å, V = 767.3(2) Å³, Z = 2. The asymmetric unit of [(C₂H₅)₄N][FeCl₄] consists of half each of a tetraethylammonium cation and a tetrachloridoferrate(III) anion. Tetrachloridoferrate(III) ion adopts almost ideal tetrahedral geometry. Structural characterization of the compound is supplemented by the results of its magnetic susceptibility measurements and electron paramagnetic resonance (EPR) spectra. Magnetic measurements of a powdered [(C₂H₅)₄N][FeCl₄] sample gave a negative Weiss constant of ?1.57, which suggests antiferromagnetic coupling. The susceptibility curve of [(C₂H₅)₄N][FeCl₄] against temperature exhibits a maximum, indicating the presence of antiferromagnetic ordering with a Neel temperature of approximately 2.9 K.     

Factors that may influence the micro-emulsion synthesis of nanosize magnetite particles

The influence of experimental conditions on the formation of nanosize magnetite by micro-emulsion method was investigated using Mössbauer and FT-IR spectroscopies, FE SEM/EDS and TEM. It was found that the concentration of starting chemicals (FeCl₃/FeSO₄), aeration/de-aeration, high alkalinity and γ-irradiation influenced this precipitation process. Rod-like goethite particles ～200–300 nm long and up to 10 nm wide were obtained by destabilization of the micro-emulsions aged 5 to 45 days at RT. Si-containing ferrihydrite was also formed and it retarded the formation of goethite by the dissolution/re-precipitation mechanism. A small amount of Si from a glass flask was dissolved. A very strong influence of γ-irradiation on the formation of nanosize magnetite by micro-emulsion method was observed. γ-irradiation created strong reductive conditions in the micro-emulsions. At an ～460 kGy dose, nanosize magnetite particles sized ～5 to 20 nm and very small amounts of goethite particles were obtained. The EDS measurements on the particles suggested the formation of sub-stoichiometric magnetite (Fe_3?x O₄) and not of maghemite. This finding is in line with the strong reductive conditions induced with γ-irradiation.     

The synthesis of Fe-containing ionic liquid and its catalytic performance for the dehydration of fructose

Four Fe-containing ionic liquids (ILs) were synthesized by coupling of conventional imidazole ionic liquids [C_xmim]Cl (x = 4, 8, 12, 16) with FeCl₃ and were characterized by FT-IR, Raman, ESI–MS and TG. All of the Fe-containing ILs were applied to the conversion of fructose into 5-hydroxymethylfurfural (HMF) in 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) subsequently and the result showed that [C₁₆mim]FeCl₄ exhibited excellent catalytic performance. Then the different reaction parameters with [C₁₆mim]FeCl₄ as catalyst were studied in detail. A 92.8% yield of HMF was obtained with 0.03 g [C₁₆mim]FeCl₄ and 0.1 g fructose in 1.0050 g [Bmim]Cl at 80 °C for 40 min in fructose/[Bmim]Cl solution.     

Improved Production of Poly-γ-Glutamic Acid by Bacillus subtilis D7 Isolated from Doenjang,a Korean Traditional Fermented Food,and Its Antioxidant Activity

The objectives of this study was to improve poly-γ-glutamic acid (γ-PGA) production by Bacillus subtilis D7 isolated from a Korean traditional fermented food and to assess its antioxidant activity for applications in the cosmetics and pharmaceutical industries. Strain D7 produced γ-PGA in the absence of l-glutamic acid, indicating l-glutamic acid-independent production. However, the addition of l-glutamic acid increased γ-PGA production. Several tricarboxylic acid cycle intermediates and amino acids could serve as the metabolic precursors for γ-PGA production, and the addition of pyruvic acid and d-glutamic acid to culture medium improved the yield of γ-PGA markedly. The maximum yield of γ-PGA obtained was 24.93?±?0.64 g/l in improved medium, which was about 5.4-fold higher than the yield obtained in basal medium. γ-PGA was found to have 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (46.8?±?1.5 %), hydroxyl radical scavenging activity (52.0?±?1.8 %), 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonate (ABTS) radical scavenging activity (42.1?±?1.8 %), nitric oxide scavenging activity (35.1?±?1.3 %), reducing power (0.304?±?0.008), and metal chelating activity (91.3?±?3.5 %). These results indicate that γ-PGA has a potential use in the food, cosmetics, and biomedical industries for the development of novel products with radical scavenging activity. As far as we are aware, this is the first report to describe the antioxidant activityof γ-PGA produced by bacteria.     

Layer-by-Layer Self-Assembly Immobilization of Catalases on Wool Fabrics

A new immobilization strategy of catalases on natural fibers was reported in this paper. Catalase (CAT) from Bacillus subtilis was assembled into multiple layers together with poly(diallyldimethylammonium chloride) (PDDA) on wool fabrics via layer-by-layer (LBL) electrostatic self-assembly deposition. The mechanism and structural evaluation of LBL electrostatic self-assembly were studied in terms of scanning electron microscopy (SEM), surface zeta potential, and apparent color depth (K/S). The SEM pictures showed obvious deposits absorbed on the wool surfaces after LBL self-assembly. The surface zeta potential and dyeing depth of CAT/PDDA-assembled wool fabrics presented a regular layer-by-layer alternating trend along with the change of deposited materials, revealing the multilayer structure of the wool fiber immobilized catalases. The V _max values were found to be 2,500?±?238 U/mg protein for the free catalase and 1,000?±?102 U/mg protein for the immobilized catalase. The K _m value of free catalase (11.25?±?2.3 mM) was found to be lower than that of the immobilized catalase (222.2?±?36.5 mM). The immobilized catalase remained high enzymatic activity and showed a measureable amount of reusability, which proved that LBL electrostatic self-assembly deposition is a promising approach to immobilize catalases.     

Study of RP HPLC Retention Behaviours in Analysis of Carotenoids

For determination of selected carotenoids, various types of columns for high-performance liquid chromatography (HPLC) with different properties have been used. The characteristics of the laboratory-used packing material containing monomeric alkyl-bonded phases (C₁₈, C₃₀) and phenyl as well as phenyl-hexyl stationary phases were studied. The retention data of the examined compounds were used to determine the hydrophobicity and silanol activity of stationary phases applied in the study. The presence of the polar and carboxyl groups in the structure of the bonded ligand strongly influences the polarity of the stationary phase. Columns were compared according to methylene selectivity using a series of benzene homologues. The measurements were done using a methanol–water mobile phase. Knowledge of the properties of the applied stationary phase provided the possibility to predict the RP HPLC retention behaviours in analysis of carotenoids including lutein, lycopene and β-carotene. The composition of the mobile phase, the addition of triethylamine and the type of stationary phase had been taken into account in designing the method of carotenoid identification. Also a monolithic column characterised by low hydrodynamic resistance, high porosity and high permeability was applied. The presented results show that the coverage density of the bonded ligands on silica gel packings and length of the linkage strongly influence the carotenoid retention behaviours. In our study, the highest retention parameters for lutein, lycopene and β-carotene were observed for C₃₀ and C₁₈ stationary phase. This effect corresponds with pore size of column packing greater than 100 Å and carbon content higher than 11 %.     

Reaction of carotenoids with CCl<Subscript>3</Subscript>OO<Superscript>.</Superscript> by using pulse radiolysis

The interactions of carotenoids (bixin, β-carotene and lycopene) with CCl₃OO^. in aqueous and i-propylalcohol solution saturated with air have been studied by pulse radiolysis. For bixin and β-carotene reaction products from forming process, absorbing in the region of 650 nm, is observed with concomitant carotenoid bleaching (bixin at 500 nm, β-carotene at 450 nm). Their rate constants from forming process are 1.78 ×10⁸ and 7.8 ×10⁷ mol^-1 · L · s^-1 respectively. However, in the case of lycopene, no such a forming process of reaction as bixin and β-carotene can be observed although there is the bleaching reaction (rate constant 4 ×10⁷ mol^-1 · L · s^-1). The results suggest that the carotenoid radical cation and an additional radical are produced in the case of bixin and β-carotene, whereas lycopene undergoes electron transfer with CCl₃OO^., forming cation radical.     

LC Determination of Malondialdehyde Concentrations in the Human Umbilical Vein Endothelial Cell Culture Medium: Application to the Antioxidant Effect of Vitexin-2″-O-rhamnoside

Vitexin-2″-O-rhamnoside (VOR) presents the leaves of Crataegus pinnatifida Bge. var. major which plays a role in preventing human pathologies related to oxidative stress, but as the principal component in the leaves, little attention has been devoted to its study of antioxidation of VOR. A simple, rapid and sensitive high-performance liquid chromatography method was developed to determine malondialdehyde (MDA) in ECV304 cell culture medium induced by tert-butyl-hydroperoxide (TBHP). The preparation of analyzed samples involved a one-step derivatization with thiobarbituric acid (TBA). The separation was achieved using the Synergi Hydro-RP, a polar endcapped C₁₈ column (250 × 4.6 mm, 4 μm pore size) with a linear gradient elution of acetonitrile and 10 mmol L^?1 of ammonium acetate aqueous solution (pH 6.8). The calibration curve was linear over the ranges 0.0125–1.25 μmol L^?1 MDA (r = 0.9962). The lower limit of quantification of MDA was 0.0125 μmol L^?1. Relative standard deviations of intra-day and inter-day precision were less than 5.2 and 4.4%. The method with high recovery (95.4 ± 1.3%) was successfully applied to the investigation of antioxidation of VOR by determining MDA in ECV304 cell culture medium.