Applied Biochemistry and Biotechnology (v.180, #7)

Titanium-dioxide nanoparticles (TNP) are used in various consumable goods. Evidence has demonstrated the cytotoxicity of TNPs, but exact mechanism is yet to be elucidated. The present study has been aimed at finding out the mechanism of TNP-induced toxicity in biological system. Different doses of anatase-TNPs administrated intravenously to Wistar rats for once a week for 1 month and properties of TH cells, macrophages, cytokines secretion, oxidative damage, apoptotic pathway, and hematological and pathological changes were investigated as downstream events of TNP-mediated cytotoxicity. Result suggests that TNPs induce TH1 and TH2 response as measured by immunophenotyping (interferon gamma (IFN-γ) and interleukin (IL)-4) of TH cells, causing induction of M1 (nitric oxide (NO), nitric oxide synthase (iNOS), NF-kappaB (NF-κB), cyclooxygenase-2 (COX-2), IL-1, IL-6, and TNF-α) and M2 (Arg-1, Ym1) macrophages response. At lower dose, TH1 or M1 response counteracted by TH2 or M2 response, resulting in insignificant oxidative damage. However, with increasing dose of TNPs, the M1 response was increased over M2 response resulting in significant tissue damage. The M1-induced inflammatory response was found to cause DNA and chromosomal damage resulting apoptosis induction via upregulation of Bax/Bcl-2 ratio and subsequent loss of mitochondrial membrane potential and cyto c release in splenocytes. The TNP-led inflammatory response also causes damage at different tissue levels.
Keywords: Inflammation; Macrophage phenotype; Titanium-dioxide nanoparticles; TiO2 ; Spleen; Oxidative stress; Splenocyte; Apoptosis

Toxicology Assessment of a Dual-Function Peptide 5rolGLP-HV in Mice by Zaizhong Ni; Bin Wang; Xiaofeng Ma; Huikun Duan; Pingzhe Jiang; Xiaodan Li; Qian Wei; Xiangzhen Ji; Minggang Li (1276-1285).
5rolGLP-HV had an ideal therapeutic potential in the prevention of hyperglycemia in type 2 diabetes and delay of the thrombosis. The objective of the study was to investigate the toxicology effects of 5rolGLP-HV and guarantee its safety. In acute toxicity test, the mice were orally receiving 5rolGLP-HV at a single dose of 300 mg/kg or 2000 mg/kg. For sub-chronic toxicity study, the mice received 5rolGLP-HV at doses of 800 mg/kg or 1600 mg/kg for 9 weeks. No significant adverse effects were evident in acute and sub-chronic toxicity tests, indicating that the LD50 value is greater than 2000 mg/kg. Although the liver and kidney exhibited a little abnormal in sub-chronic toxicity study, they could recovery to normal after withdrawal 5rolGLP-HV for 2 weeks. In micronucleus assay, the mice received 5rolGLP-HV at doses of 250, 500, or 1000 mg/kg for two consecutive days. The micronucleus numbers and the polychromatic erythrocytes to normochromatic erythrocytes (PCE/NCE) ratios among 5rolGLP-HV groups were within the normal range. Similarly, sperm aberration test demonstrated that 5rolGLP-HV had no teratogenic effect on the mice sperm. In conclusion, the combined results clearly demonstrated the safety of 5rolGLP-HV and support its use as a drug to treat diabetes and thrombosis.
Keywords: 5rolGLP-HV; Toxicology; Acute toxicity; Sub-chronic toxicity; Micronucleus; Sperm aberration

Structural Characterization of a Novel Antioxidant Pigment Produced by a Photochromogenic Microbacterium oxydans Strain by Fatma Meddeb-Mouelhi; Jessica Kelly Moisan; Jodrey Bergeron; Benoit Daoust; Marc Beauregard (1286-1300).
The Microbacteriaceae family, such as Microbacterium, is well known for its ability to produce carotenoid-type pigments, but little has been published on the structure of such pigments. Here, we isolated the yellow pigment that is responsible for the yellowish color of a Microbacterium oxydans strain isolated from a decomposing stump of a resinous tree. The pigment, which is synthesized when the bacterium is grown under light, was purified and characterized using several spectroscopic analyses, such as ultraviolet-visible spectroscopy (UV–Vis), Fourier transform infrared spectroscopy (FTIR), 1H and 13C nuclear magnetic resonance (1H NMR, 13C NMR), and high-resolution mass spectrometry (HRMS). From these analysis, a molecular formula (C27H42O2) and a chemical structure (8-hydroxymethyl-2,4,12-trimethyl-14-(2,6,6-trimethyl-cyclohex-2-enyl)-teradeca-3,7,9,11,13-pentan-2-ol) were deduced. The chemical properties of the pigment, such as aqueous stability at different pH, stability in different organic solvents, and antioxidant capacity, are also reported. Together, these data and previous studies have resulted in the identification of a new antioxidant pigment produced by M. oxydans. To the best of our knowledge, this is the first thorough investigation of this carotenoid-like pigment in the Microbacterium genera.
Keywords: Carotenoid; Spectroscopy; Microbacterium oxydans ; Photochromogenic bacteria; Culture; Wood decay

Detection of Variation in Long-Term Micropropagated Mature Pistachio via DNA-Based Molecular Markers by Hülya Akdemir; Veysel Suzerer; Engin Tilkat; Ahmet Onay; Yelda Ozden Çiftçi (1301-1312).
Determination of genetic stability of in vitro-grown plantlets is needed for safe and large-scale production of mature trees. In this study, genetic variation of long-term micropropagated mature pistachio developed through direct shoot bud regeneration using apical buds (protocol A) and in vitro-derived leaves (protocol B) was assessed via DNA-based molecular markers. Randomly amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), and amplified fragment length polymorphism (AFLP) were employed, and the obtained PIC values from RAPD (0.226), ISSR (0.220), and AFLP (0.241) showed that micropropagation of pistachio for different periods of time resulted in “reasonable polymorphism” among donor plant and its 18 clones. Mantel’s test showed a consistence polymorphism level between marker systems based on similarity matrices. In conclusion, this is the first study on occurrence of genetic variability in long-term micropropagated mature pistachio plantlets. The obtained results clearly indicated that different marker approaches used in this study are reliable for assessing tissue culture-induced variations in long-term cultured pistachio plantlets.
Keywords: Pistacia vera ; Tissue culture-induced variation; RAPD; ISSR; AFLP

The polysaccharides of galactose present in the pectin of the plant cell wall are degraded by endo-β-1,4-galactanases. The filamentous fungus Penicillium purpurogenum, which grows on a number of natural carbon sources, among them sugar beet pulp which contains pectin, has a gene (ppgal1) coding an endo-β-1,4-galactanase (PpGAL1). This enzyme was expressed heterologously in Pichia pastoris. It has a molecular mass of 38 kDa, a pH optimum of 4–4.5, and an optimal temperature of 60 °C. It is 100 % stable for up to 24 h at pH 4–4.5 and 40 °C. These stability properties, which exceed those from other endo-β-1,4-galactanases reported to date, make it particularly suitable for industrial processes requiring acidic conditions and temperatures up to 40 °C. PpGAL1 is, therefore, a potentially effective tool in the food industry and in other biotechnological applications.
Keywords: Endo-β-1,4-galactanase; Penicillium purpurogenum ; Heterologous expression; Pichia pastoris ; Thermostability

Three bacterial isolates, GT2, GT3, and GT7, were isolated from the sludge and water of a circulating cooling system of iron and steel plant by screening on Cr(VI)-containing plates. Three isolates were characterized as the members of the genus Pseudomonas on the basis of phenotypic characteristics and 16S rRNA sequence analysis. All isolates were capable of resisting multiple antibiotics and heavy metals. GT7 was most resistant to Cr(VI), with a minimum inhibitory concentration (MIC) of 6.5 mmol L−1. GT7 displayed varied rates of Cr(VI) reduction in M2 broth, which was dependent on pH, initial Cr(VI) concentration, and inoculating dose. Total chromium analysis revealed that GT7 could remove a part of chromium from the media, and the maximum rate of chromium removal was up to 40.8 %. The Cr(VI) reductase activity of GT7 was mainly associated with the soluble fraction of cell-free extracts and reached optimum at pH 6.0∼8.0. The reductase activity was apparently enhanced by external electron donors and Cu(II), whereas it was seriously inhibited by Hg(II), Cd(II), and Zn(II). The reductase showed a K m of 74 μmol L−1 of Cr(VI) and a V max of 0.86 μmol of Cr(VI) min−1 mg−1 of protein. The results suggested that GT7 could be a promising candidate for in situ bioremediation of Cr(VI).
Keywords: Pseudomonas sp.; Heavy metal resistance; Chromate reduction; Chromium removal; Chromate reductase; Bioremediation

RAPD and ISSR Marker-Based Comparative Evaluation of Genetic Diversity Among Indian Germplasms of Euryale ferox: an Aquatic Food Plant by Hemant Kumar; Priti Priya; Nena Singh; Mukesh Kumar; Binod Kumar Choudhary; Lokendra Kumar; Indu Shekhar Singh; Nitish Kumar (1345-1360).
Euryale ferox Salisbury is an important aquatic food plant cultivated largely in eastern India. E. ferox is a monotypic genus, and breeding programmes have mostly relied on the variability present in the primary gene pool. Knowledge of the genetic structure of the population is limited, and there are very few reports available on the genetic diversity of E. ferox. In this study, comprehensive research on the genetic diversity of 16 germplasms of E. ferox was carried out using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Out of 320 RAPD and 95 ISSR primers screened initially, 61 primers (40 RAPD and 21 ISSR) gave reproducible bands and were selected for further work. Amplification of the 40 RAPD primers gave 533 polymorphic bands with an average of 13.32 polymorphic bands per primer. The percentage of polymorphism ranged from 37.5 to 100, with an average of 88.3 %. The 21 ISSR primers produced 259 bands, of which 214 were polymorphic, with an average of 10.19 polymorphic bands per primer. The percentage of polymorphism using ISSR primers ranged from 50 to 100, with a mean of 82.6 %. Jaccard’s coefficient ranged from 0.45 to 0.69 (RAPD), 0.50 to 0.77 (ISSR) and 0.48 to 0.71 (RAPD and ISSR). Molecular characterization of different germplasms of E. ferox not only is essential for its conservation but also can be used in further breeding programmes.
Keywords: Euryale ferox ; Genetic diversity; RAPD; ISSR

Coagulant Activity of Water-Soluble Moringa oleifera Lectin Is Linked to Lowering of Electrical Resistance and Inhibited by Monosaccharides and Magnesium Ions by Kézia Santana de Moura; Hugo Rafael Chaves da Silva; Leonardo Prezzi Dornelles; Luana Cassandra Breitenbach Barroso Coelho; Thiago Henrique Napoleão; Maria Danielly Lima de Oliveira; Patrícia Maria Guedes Paiva (1361-1371).
Moringa oleifera seeds contain a water-soluble lectin [water-soluble M. oleifera lectin (WSMoL)] that has shown coagulant activity. Magnesium ions are able to interfere with the ability of this lectin to bind carbohydrates. In this study, we performed structural characterization of WSMoL and analyzed its effect on the electrical resistance of a kaolin clay suspension in both presence and absence of monosaccharides (N-acetylglucosamine, glucose, or fructose) and magnesium ions. The coagulant activity of WSMoL was monitored by measuring optical density and electrical resistance over a period of 60 min. Native WSMoL had a molecular mass of 60 kDa and exhibited anionic nature (pI 5.5). In sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), it appeared as three polypeptide bands of 30, 20, and 10 kDa. WSMoL reduced the optical density and electrical resistance of the kaolin suspension, which suggests that suspended particles are destabilized and that this is followed by formation of complexes. The coagulant activity of lectin decreased in the presence of Mg2+ ions and carbohydrates at concentrations that also inhibited hemagglutinating activity. This was most likely due to conformational changes in lectin structure. Our findings suggest that the coagulant activity of WSMoL is enhanced by lowering of electrical resistance of the medium and is impaired by lectin–carbohydrate and lectin–Mg2+ interactions.
Keywords: Coagulant activity; Electric resistance; Kaolin; Lectin; Monosaccharides; Magnesium

This study investigated a system which simultaneously produced electricity and stored energy in the MFC integrated MnO2-modified capacitive bioanode. Compared to the noncapacitive anode, the maximum power density of MFC with MnO2-modified bioanode reached 16.47 W m−3, which was 3.5 times higher than that of the bare anode (4.71 W m−3). During the charging-discharging experiment, the MFC with a capacitance bioanode has a higher average peak current density of 5.06 mA cm−2 and 36 times larger than that with the bare bioanode. With the capacitive electrode, it is possible to let the MFC at the same time for production and storage of renewable electricity. Then two different operations (intermittent operation and continuous operation) of the MFC with a capacitive bioanode were studied to degrade Cr (VI) in cathode chamber. Results showed that the Cr (VI) removal rates of intermittent operation are much higher than that of continuous operation under the same time in the closed circuit state. This is due to the good ability of storing and releasing electron of the biological capacitor with MnO2 modified material. And this study showed that MFC with a capacitive bioanode is better adapted to treat heavy metal pollutants by intermittent mode.
Keywords: MFC; Capacitive bioanode; Intermittent operation; Cr (VI) removal rates

Technical, Economical, and Microbiological Aspects of the Microaerobic Process on H2S Removal for Low Sulfate Concentration Wastewaters by M. R. Sousa; C. J. S. Oliveira; A. C. Lopes; E. R. Rodríguez; G. B. M. Holanda; P. G. C. Landim; P. I. M. Firmino; A. B. Dos Santos (1386-1400).
We studied the feasibility of the microaerobic process, in comparison with the traditional chemical absorption process (NaOH), on H2S removal in order to improve the biogas quality. The experiment consisted of two systems: R1, biogas from an anaerobic reactor was washed in a NaOH solution, and R2, headspace microaeration with atmospheric air in a former anaerobic reactor. The microaeration used for low sulfate concentration wastewater did not affect the anaerobic digestion, but even increased system stability. Methane production in the R2 was 14 % lower compared to R1, due to biogas dilution by the atmospheric air used. The presence of oxygen in the biogas reveals that not all the oxygen was consumed for sulfide oxidation in the liquid phase indicating mass transfer limitations. The reactor was able to rapidly recover its capacity on H2S removal after an operational failure. Bacterial and archaeal richness shifted due to changes in operational parameters, which match with the system functioning. Finally, the microaerobic system seems to be more advantageous for both technical and economical reasons, in which the payback of microaerobic process for H2S removal was 4.7 months.
Keywords: Anaerobic; Biogas desulfurization; DGGE; Hydrogen sulfide; Microaerobic process

Biological Pretreatment of Chicken Feather and Biogas Production from Total Broth by Regina J. Patinvoh; Elisabeth Feuk-Lagerstedt; Magnus Lundin; Ilona Sárvári Horváth; Mohammad J. Taherzadeh (1401-1415).
Chicken feathers are available in large quantities around the world causing environmental challenges. The feathers are composed of keratin that is a recalcitrant protein and is hard to degrade. In this work, chicken feathers were aerobically pretreated for 2–8 days at total solid concentrations of 5, 10, and 20 % by Bacillus sp. C4, a bacterium that produces both α- and β-keratinases. Then, the liquid fraction (feather hydrolysate) as well as the total broth (liquid and solid fraction of pretreated feathers) was used as substrates for biogas production using anaerobic sludge or bacteria granules as inoculum. The biological pretreatment of feather waste was productive; about 75 % of feather was converted to soluble crude protein after 8 days of degradation at initial feather concentration of 5 %. Bacteria granules performed better during anaerobic digestion of untreated feathers, resulting in approximately two times more methane yield (i.e., 199 mlCH4/gVS compared to 105 mlCH4/gVS when sludge was used). Pretreatment improved methane yield by 292 and 105 % when sludge and granules were used on the hydrolysate. Bacteria granules worked effectively on the total broth, yielded 445 mlCH4/gVS methane, which is 124 % more than that obtained with the same type of inoculum from untreated feather.
Keywords: Chicken feather; Pretreatment; Bacillus substilis strain; Keratinase; Biogas production; Mesophilic; Hydrolysate; Total broth; Bacteria granules

Enhanced Butanol Production Through Adding Organic Acids and Neutral Red by Newly Isolated Butanol-Tolerant Bacteria by Cheng Jiang; Guangli Cao; Zhenyu Wang; Ying Li; Jinzhu Song; Hua Cong; Junzheng Zhang; Qian Yang (1416-1427).
As alternative microorganisms for butanol production with high butanol tolerant and productivity are in high demand, one excellent butanol-tolerant bacterium, S10, was isolated and identified as Clostridium acetobutylicum S10. In order to enhance the performance of butanol production, organic acids and neutral red were added during butanol fermentation. Synergistic effects were exhibited in the combinations of organic acids and neutral red to promote butanol production. Consequently, the optimal concentrations of combined acetate, butyrate, and neutral red were determined at sodium acetate 1.61 g/L, sodium butyrate 1.88 g/L, and neutral red 0.79 g/L, respectively, with the butanol yield of 6.09 g/L which was 20.89 % higher than that in control. These results indicated that combination of adding organic acid and neutral red is a potential effective measure to improve butanol production.
Keywords: Butanol; Tolerance; Clostridium acetobutylicum ; Organic acid; Neutral red

Solvent Free Lipase Catalysed Synthesis of Ethyl Laurate: Optimization and Kinetic Studies by Sarita D. Gawas; Sachin V. Jadhav; Virendra K. Rathod (1428-1445).
The current research work represents solvent free enzymatic synthesis of fatty acid ethyl ester; ethyl laurate. Immobilized lipase Fermase CALB™10,000 was used for the synthesis of ethyl laurate from ethanol and lauric acid. The influence of process parameters such as catalyst loading, speed of agitation, mole ratio, molecular sieves and temperature were studied. Fermase CALB™10,000 offered a conversion of 92.46 % at 60 °C in 4 h at optimized condition. The experimental data was best fitted by the Random Bi-Bi model with inhibition of both the substrates i.e. lauric acid and ethanol. The following kinetic parameters were retrieved from the model: Vmax = 1.243 × 103 mmol/min/g enzyme; KA = 0.1283 mmol; KB = 8.51 mmol; KiA = 5.098 mmol; and SSE = 0.0142. The activation energy for the enzymatic esterification was also determined and calculated to be 28.49 kJ/mol. A maximum conversion of 71 % was obtained after 5 successive reuse of Fermase CALB™10,000 lipase.
Keywords: Biocatalysis; Flavour; Lipase; Random Bi-Bi model; Solvent free system

Glutathione (γ-glutamyl-L-cysteinylglycine, GSH) is a pharmaceutical compound often used in food additives and the cosmetics industry. GSH can be produced biologically from L-glutamic acid, L-cysteine, and glycine through an enzymatic process traditionally involving two sequential adenosine triphosphate (ATP)-dependent reactions catalyzed by γ-glutamylcysteine synthetase (γ-GCS or GSHI, EC 6.3.2.2) and GSH synthetase (GS or GSHII, EC 6.3.2.3). Here, we report the enzymatic production of GSH by recombinant cell-free bifunctional γ-glutamylcysteine synthetase/glutathione synthetase (γ-GCS-GS or GshF) coupled with in vitro acetate kinase-based ATP generation. GSH production by an acetate kinase-integrated Escherichia coli Rosetta(DE3) mutant expressing Streptococcus thermophilus GshF reached 18.3 ± 0.1 g l−1 (59.5 ± 0.3 mM) within 3 h, with a molar yield of 0.75 ± 0.00 mol mol−1 added cysteine and a productivity of 6.1 ± 0.0 g l−1 h−1. This is the highest GSH titer reported to date. This newly developed biocatalytic process offers a promising approach for meeting the industrial requirements for GSH production.
Keywords: Acetate kinase; ATP generation; Bifunctional γ-glutamylcysteine synthetase/glutathione synthetase; Glutathione

An extracellular organic solvent-tolerant lipase-producing bacterium was isolated from oil-contaminated soil samples and was identified taxonomically as Pseudomonas stutzeri, from which the lipase was purified and exhibited maximal activity at temperature of 50 °C and pH of 9.0. Meanwhile, the lipase was stable below or at 30 °C and over an alkaline pH range (7.5–11.0). Ca2+ could significantly improve the lipase thermal stability which prompts a promising application in biocatalysis through convenient medium engineering. The lipase demonstrated striking features such as distinct stability to the most tested hydrophilic and hydrophobic solvents (25 %, v/v), and DMSO could activate the lipase dramatically. In the enzyme-catalyzed resolution, lipase ZS04 manifested excellent enantioselective esterification toward the (R)-1-(4-methoxyphenyl)-ethanol (MOPE), a crucial chiral intermediate in pharmaceuticals as well as in other analogs with strict substrate specificity and theoretical highest conversion yield. This strong advantage over other related schemes made lipase ZS04 a promising biocatalyst in organic synthesis and pharmaceutical applications.
Keywords: Organic solvent stability; Lipase; Pseudomonas stutzeri ; Chiral resolution; MOPE