Applied Biochemistry and Biotechnology (v.171, #7)

Biodegradable Polycaprolactone (PCL) Nanosphere Encapsulating Superoxide Dismutase and Catalase Enzymes by Sushant Singh; Abhay Narayan Singh; Anil Verma; Vikash Kumar Dubey (1545-1558).
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.
Keywords: Catalase; Nanosphere; Oxidative stress; Polycaprolactone; Superoxide dismutase

One-Pot Lipase-Catalyzed Aldol Reaction Combination of In Situ Formed Acetaldehyde by Na Wang; Wei Zhang; Long-Hua Zhou; Qing-Feng Deng; Zong-Bo Xie; Xiao-Qi Yu (1559-1567).
A facile tandem route to α,β-unsaturated aldehydes was developed by combining the two catalytic activities of the same enzyme in a one-pot strategy for the aldol reaction and in situ generation of acetaldehyde. Lipase from Mucor miehei was found to have conventional and promiscuous catalytic activities for the hydrolysis of vinyl acetate and aldol condensation with in situ formed acetaldehyde. The first reaction continuously provided material for the second reaction, which effectively reduced the volatilization loss, oxidation, and polymerization of acetaldehyde, as well as avoided a negative effect on the enzyme of excessive amounts of acetaldehyde. After optimizing the process, several substrates participated in the reaction and provided the target products in moderate to high yields using this single lipase-catalyzed one-pot biotransformation. Figure One-pot tandem approach for the synthesis of α,β-unsaturated aldehydes was developed under the catalysis of lipase from M. miehei (MML), firstly. MML displayed conventional and promiscuous catalytic activities for hydrolysis of vinyl acetate and aldol condensation with in situ formed acetaldehyde.
Keywords: Lipase; Aldol condensation; One pot; Biocatalysis; Acetaldehyde; Promiscuity; In situ

Cytotoxicity Evaluation and Subcellular Location of Titanium Dioxide Nanotubes by Yanli Wang; Keke Sui; Jie Fang; Chenjie Yao; Lulu Yuan; Qiuxia Wu; Minghong Wu (1568-1577).
TiO2 nanotubes (TiO2-NTs) are currently attracting a high interest because the intrinsic properties of TiO2 provide the basis for many outstanding functional features. Herein, we focus on the cytotoxicity and sublocation of TiO2-NTs in neural stem cells (NSCs). The cytotoxicity of TiO2-NTs is investigated using the methyl tetrazolium cytotoxicity and reactive oxygen species assay, the apoptosis assay by flow cytometry. Cell viability assay shows that TiO2-NTs inside cells are nontoxic at the low concentration. A time-dependent relationship is observed, while a dose-dependent relationship is seen only at the concentration higher than 150 μg/ml. The uptake happens shortly after incubation with cells. TiO2-NTs can easily pass through the cell membrane and enter into the cells. The uptake amount is increased with prolonging incubation time and reach to maximum at 48 h. Transmission electron microscopy and confocal is used to study subcellular location of TiO2-NTs. It is found that TiO2-NTs traversed cell membrane and localized in many vesicles (endosomes and lysosomes) and cytoplasm. TiO2-NTs in NSCs firstly disperse or metabolism by lysosomal enzymes and then exocytosis from NSCs. Figure Transmission electron microscopy and confocal are used to study subcellular location of titanium dioxide nanotubes in neural stem cells
Keywords: Cytotoxicity; Subcellular location; Titanium dioxide nanotubes; Neural stem cells

Enhancing the Cellulose-Degrading Activity of Cellulolytic Bacteria CTL-6 (Clostridium thermocellum) by Co-Culture with Non-cellulolytic Bacteria W2-10 (Geobacillus sp.) by Yucai Lü; Ning Li; Xufeng Yuan; Binbin Hua; Jungang Wang; Masaharu Ishii; Yasuo Igarashi; Zongjun Cui (1578-1588).
The effect of a non-cellulolytic bacterium W2-10 (Geobacillus sp.) on the cellulose-degrading activity of a cellulolytic bacterium CTL-6 (Clostridium thermocellum) was determined using cellulose materials (paper and straw) in peptone cellulose solution (PCS) medium under aerobic conditions. The results indicated that in the co-culture, addition of W2-10 resulted in a balanced medium pH, and may provide the required anaerobic environment for CTL-6. Overall, addition of W2-10 was beneficial to CTL-6 growth in the adverse environment of the PCS medium. In co-culture with W2-10, the CTL-6 cellulose degradation efficiency of filter paper and alkaline-treated wheat straw significantly increased up to 72.45 and 37.79 %, respectively. The CMCase activity and biomass of CTL-6 also increased from 0.23 U ml−1 and 45.1 μg ml−1 (DNA content) up to 0.47 U ml−1 and 112.2 μg ml−1, respectively. In addition, co-culture resulted in accumulation of acetate and propionate up to 4.26 and 2.76 mg ml−1. This was a respective increase of 2.58 and 4.45 times, in comparison to the monoculture with CTL-6.
Keywords: Cellulose Degradation; Co-culture; Clostridium thermocellum ; Geobacillus sp.; Bioenergy

A 12-year-old hairy root culture of Cichorium intybus L., a callus culture of the plant as well as roots and leaves of a wild plant of chicory, and roots of two C. intybus L. var. sativum cultivars were examined in respect of their hydroxycinnamate and sesquiterpene lactone compositions and contents. Total phenolics and diphenylpicrylhydrazyl radical scavenging activity of the examined plant tissues were also analyzed. The most active in radical scavenging were extracts from the hairy roots and leaves of chicory. 3,5-Dicaffeoylquinic acid was the major antioxidant present in the hairy roots. Its content in the root biomass reached 5.5 %, calculated on a dry weight basis. 8-Deoxylactucin glucoside (crepidiaside A) was the major sesquiterpene lactone in the hairy roots. Its content reached 1.4 %, calculated on a dry weight basis, and was nearly two orders of magnitude higher than that in the roots of wild chicory plant. The glucosidic derivative of 8-deoxylactucin constituted over 85 % of the total sesquiterpene lactone content in the long-term cultured hairy roots of chicory. Aglycone of this compound was reported to possess anti-inflammatory activity. The qualitative and quantitative analyses of hydroxycinnamates in callus and hairy root cultures of C. intybus were undertaken for the first time.
Keywords: Chicory; Hairy roots; Hydroxycinnamates; Antioxidants; 8-Deoxylactucin glucoside

Isolation and Characterization of Phytotoxic Compounds Produced by Streptomyces sp. AMC 23 from Red Mangrove (Rhizophora mangle) by Eduardo José Crevelin; Sarah Pigato Canova; Itamar Soares Melo; Tiago Domingues Zucchi; Rafael Eduardo da Silva; Luiz Alberto Beraldo Moraes (1602-1616).
Natural products produced by microorganisms have been utilized as sources of new drugs possessing a wide range of agrochemical and pharmacological activities. During our research on Actinomycetes from Brazilian mangroves, the ethyl acetate extract of Streptomyces sp. AMC 23 isolated from the red mangrove (Rhizophora mangle) rhizosphere produced a highly active compound against the microalga Chlorella vulgaris, often used to assess the phytotoxic activity. As a result, the bioassay-guided fractionation led to the isolation of the mixture of the known compounds bafilomycin B1 and bafilomycin B2. The chemical structures of bafilomycin B1 and bafilomycin B2 were established based on their spectroscopic data by infrared (IR), mass spectrometry (MS), 1H nuclear magnetic resonance (NMR), gradient-enhanced heteronuclear multiple quantum coherence (gHMQC), and gradient-enhanced heteronuclear multiple-bond connectivity (gHMBC) as well as comparison with reference data from the literature. Moreover, it was also possible to identify other bafilomycins using non-chromatographic-dependent techniques (Tandem mass spectrometry). Additionally, this is the first report on the phytotoxic activity of bafilomycin B1.
Keywords: Actinomycetes; Mangroves; Chlorella vulgaris ; Macrolides; Phytotoxic activity

Nonthermal Atmospheric Argon Plasma Jet Effects on Escherichia coli Biomacromolecules by Abasalt Hosseinzadeh Colagar; Hamed Memariani; Farshad Sohbatzadeh; Azadeh Valinataj Omran (1617-1629).
Nonthermal atmospheric plasma jet, a promising technology based on ionized gas at low temperatures, can be applied for disinfection of contaminated surfaces. In this study, Escherichia coli cells and their macromolecules were exposed to the nonthermal atmospheric argon plasma jet for different time durations. Total protein, genomic DNA, and malondialdehyde (MDA) levels of E. coli were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining; agarose gel electrophoresis; and measurement of absorbance at 534 nm, respectively. After exposure, the spectroscopic results of liquid samples indicated that the survival reduction of E. coli can reach to 100 % in an exposure time of 600 s. Moreover, inactivation zones of E. coli, DNA degradation, and MDA levels were significantly increased. Additionally, banding patterns of total protein were changed and amino acid concentrations increased following ninhydrin test. The experimental results suggest that the nonthermal plasma could serve as an effective instrument for both sterilizing E. coli and degrading macromolecules from the surface of the objects being sterilized.
Keywords: Nonthermal plasma; Escherichia coli ; Inactivation zones; SDS-PAGE; DNA; Malondialdehyde

Parkin was originally identified as a protein associated with Parkinson's disease. Recently, numerous research studies have suggested that parkin acts as a tumor suppressor. In accordance with these studies, we previously reported that overexpression of parkin in HeLa cells induced growth inhibition. To elucidate possible mechanisms by which parkin may inhibit cell growth, HeLa cells were infected with adenoviruses expressing either the parkin gene or adenovirus alone for 72 h and a total proteomic analysis was performed using 2-D gel electrophoresis followed by LC-MS/MS. We identified three proteins whose expression changed between the two groups: the 40S ribosomal protein SA (RPSA) was downregulated in parkin virus-infected cells, and cytokeratins 8 and 18 exhibited an acid shift in pI value without a change in molecular weight, suggesting that these proteins became phosphorylated in parkin virus-infected cells. The changes in these three proteins were first observed at 60 h postinfection and were most dramatic at 72 h postinfection. Because upregulation of RPSA and dephosphorylation of cytokeratins 8/18 have been linked with tumor progression, these data suggest that parkin may inhibit cell growth, at least in part, by decreasing RPSA expression and inducing phosphorylation of cytokeratin 8/18.
Keywords: Cytokeratin 8; Cytokeratin 18; HeLa cell; Parkin; Proteomic analysis; 40S ribosomal protein SA; Tumor suppressor

Synthesis and Biological Activity of New Resveratrol Derivative and Molecular Docking: Dynamics Studies on NFkB by Babajan Banaganapalli; Chaitanya Mulakayala; Gowsia D; Naveen Mulakayala; Madhusudana Pulaganti; Noor Ahmad Shaik; Anuradha CM; Raja Mohan Rao; Jumana Yousuf Al-Aama; Suresh Kumar Chitta (1639-1657).
Resveratrol (RVS) is a naturally occurring antioxidant, able to display an array of biological activities. In the present investigation, a new derivative of RVS, RVS(a), was synthesized, and its biological activity was determined on U937 cells. It was observed that RVS(a) showed pronounced activity on U937 cells than RVS. RVS(a) is able to induce apoptosis in tumor cell lines through subsequent DNA fragmentation. From the EMSA results, it was evident that RVS(a) was able to suppress the activity of NFkB by interfering its DNA binding ability. Furthermore, the molecular interaction analysis (docking and dynamics) stated that RVS(a) has strong association with the IkB-alpha site of NFkB compared with RVS; this binding nature of RVS(a) might be prevent the NFkB binding ability with DNA. The present findings represent the potential activity of propynyl RVS on U937 cells and signifying it as a one of putative chemotherapeutic drugs against cancer.
Keywords: Resveratrol; RVS(a); EMSA; NFkB; Molecular docking; Dynamics simulations

Enhancement of Human Thyrotropin Synthesis by Sodium Butyrate Addition to Serum-Free CHO Cell Culture by Renata Damiani; Beatriz E. Almeida; João E. Oliveira; Paolo Bartolini; Maria Teresa C. P. Ribela (1658-1672).
The influence of sodium butyrate (NaBu) on the synthesis of recombinant human thyrotropin (r-hTSH) by CHO cells was investigated for the first time. A volumetric productivity of ~10 μg hTSH/mL was repeatedly obtained, with a 3.3-fold increase over a control culture carried out in the absence of NaBu. Since NaBu can induce CHO cell apoptosis and cell growth arrest, the increase in specific productivity was even higher, i.e., ca. 5-fold. Analysis of the N-glycan composition of r-hTSH obtained with the addition of NaBu to the culture medium showed an approximately 12 % increase in the amount of sialic acid, as well as in total carbohydrate, partly due to the increase in the site occupancy from 2.77 to 2.93 glycans per mole of hTSH. The two hormone preparations were characterized by N-glycan structural analysis, which showed that NaBu increased the bi-antennary structures by ca. 13 % while decreasing the tri-antennary structures by approximately the same amount. The in vivo biological activity and pharmacokinetic behavior (clearance) were found to be similar for the two hormone preparations.
Keywords: hTSH; Sodium butyrate; Glycosylation; Biological activity; Pharmacokinetics

Laccase-Catalyzed Oxidative Polymerization of Phenolic Compounds by Xuejiao Sun; Rubing Bai; Ya Zhang; Qiang Wang; Xuerong Fan; Jiugang Yuan; Li Cui; Ping Wang (1673-1680).
Enzymatic polymerization of phenolic compounds (catechol, resorcinol, and hydroquinone) was carried out using laccase. The mechanism of polymerization and the structures of the polymers were evaluated in terms of UV–Vis and Fourier transform infrared spectroscopy. The molecular weights of the produced polyphenols were determined with GPC. The results showed that the phenolic monomers firstly turned into quinone intermediates by laccase catalysis. Through further oxidation, the intermediates formed covalent bonds. Finally, catechol units were linked together with ether bonds, and both resorcinol and hydroquinone units were linked together with C-C bonds. The number-average molecular weights of the polyphenols ranged from 1,000 to 1,400 Da (corresponding to the degree of polymerization that varied from 10 to 12) with a lower polydispersity value of about 1.10, showing selective polymerization of phenolic compounds catalyzed by laccase.
Keywords: Catechol; Resorcinol; Hydroquinone; Laccase; Oxidation; Polymerization

Purification and Characterization of an Ethanol-Tolerant β-Glucosidase from Sporidiobolus pararoseus and Its Potential for Hydrolysis of Wine Aroma Precursors by Milla Alves Baffi; Natália Martin; Thaise Mariá Tobal; Ana Lúcia Ferrarezi; João Henrique Ghilardi Lago; Maurício Boscolo; Eleni Gomes; Roberto Da-Silva (1681-1691).
An extracellular ethanol-tolerant β-glucosidase from Sporidiobolus pararoseus was purified to homogeneity and characterized, and its potential use for the enhancement of wine aroma was investigated. The crude enzymatic extract was purified in four steps (concentration, dialysis, ultrafiltration, and chromatography) with a yield of around 40 % for total activity. The purified enzyme (designated Sp-βgl-P) showed a specific activity of approximately 20.0 U/mg, an estimated molecular mass of 63 kDa after sodium dodecyl sulfate polyacrylamide gel electrophoresis, and isoelectric point of 5.0 by isoelectric focusing. Sp-βgl-P has optimal activity at pH 4.0 and at 55 °C. It was stable in a broad pH range at low temperatures and it was tolerant to ethanol and glucose, indicating suitable properties for winemaking. The hydrolysis of glycosidic terpenes was analyzed by adding Sp-βgl-P directly to the wines. The released terpene compounds were evaluated by gas chromatography/mass spectrometry. The enzymatic treatment significantly increased the amount of free terpenes, suggesting that this enzyme could potentially be applicable in wine aroma improvement.
Keywords: Yeasts; β-Glucosidase; Purification; Wine; Terpenes

Enhanced Lycopene Content in Blakeslea trispora by Effective Mutation-Screening Method by Qiang Wang; Wei Luo; Qiu-ya Gu; Ling-ran Feng; Han-guang Li; Xiao-bin Yu (1692-1700).
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.
Keywords: Lycopene; Blakeslea trispora ; Mutation; Trisporic acid; Lovastatin

One-stage autothermal thermophilic aerobic digestion (ATAD) is effective for the reduction of volatile solids (VSs) and pathogen in sewage sludges. A novel process of combining mesophilic (<35 °C) anaerobic digestion with a thermophilic (55 °C) aerobic digestion process (AN/TAD) occurred in a one-stage digester, which was designed for aeration energy savings. The efficiency of sludge degradation and variation of sludge properties by batch experiments were evaluated for the AN/TAD digester with an effective volume of 23 L for 30 days compared with conventional thermophilic aerobic digestion (TAD). The AN/TAD system can efficiently achieve sludge stabilization on the 16th day with a VS removal rate of 38.1 %. The AN/TAD system was operated at lower ORP values in a digestion period with higher contents of total organic compounds, volatile fatty acids, protein, and polysaccharide in the soluble phase than those of the TAD system, which can rapidly decreased and had low values in the late period of digestion for the AN/TAD system. In the AN/TAD system, intracellular substances had lysis because of initial hydrolytic acidification.
Keywords: Sewage sludge; Anaerobic digestion; Thermophilic aerobic digestion; Biodegradation; Cell lysis

Biodegradation of Crude Oil by a Newly Isolated Strain Rhodococcus sp. JZX-01 by Chen Li; Zheng-Xi Zhou; Xiao-Qiang Jia; Yu Chen; Jiao Liu; Jian-Ping Wen (1715-1725).
A highly efficient oil-degrading bacteria JZX-01 was isolated from the oil-contaminated soil of the seacoast near the Boxi Offshore Oil Field of China. Morphological, physiological, and 16S rDNA gene sequence analyses indicated that JZX-01 was assigned to the genus Rhodococcus sp. This strain decomposed 65.27 ± 5.63 % of the crude oil in 9 days. Gas chromatography–mass spectrometry analysis showed that even the long-chain hydrocarbons (C31–C38) and branched alkanes (pristine and phytane), which were regarded as the stubborn ones, could be degraded. Further study showed that the bacteria still has good oil degradation ability at low temperatures as well as under high salt conditions. Moreover, JZX-01 was found to have a biosurfactant-producing capacity, which significantly favors the surface tension reduction and crude oil degradation. The promising isolated strain Rhodococcus sp. JZX-01 could be further used for the bioremediation of oil-polluted soil or seawater in a wide range of temperatures and high salt conditions.
Keywords: Crude oil; Biodegradation; Rhodococcus sp.; Biosurfactant

Several Clostridium spp. were incubated in a nitrogen-free medium (non-growth medium) containing only butyric acid as a sole precursor for performing butanol production by non-growing cells. Non-growing cells of Clostridium spp., especially Clostridium beijerinckii TISTR 1461, could convert butyric acid to butanol via their sole solventogenic activity. This activity was further enhanced in the presence of glucose as a co-substrate. In addition to glucose, other monosaccharides (i.e., galactose and xylose) and disaccharides (i.e., maltose, sucrose, and lactose) could also be used as a co-substrate with butyric acid. Among the organic acids tested (i.e., formic, acetic, propionic, and butyric acids), only butyric and acetic acids were converted to butanol. This study has shown that it is possible to use the non-growing cells of Clostridium spp. for direct conversion of sugars and organic acids to biobutanol. With this strategy, C. beijerinckii TISTR 1461 produced 12 g/L butanol from 15 g/L glucose and 10 g/L butyric acid with a high butanol yield of 0.68 C-mol/C-mol and a high butanol ratio of 88 %.
Keywords: Butanol; Butyric acid; Clostridium spp.; Nitrogen-free medium; Solventogenesis

Specificity of Glucose Oxidase from Penicillium funiculosum 46.1 Towards Some Redox Mediators by Tatiana Semashko; Raisa Mikhailova; Almira Ramanaviciene; Arunas Ramanavicius (1739-1749).
Glucose oxidase (GOx) from Penicillium funiculosum 46.1 was purified using step-by-step ultrafiltration and it was characterized by spectrophotometric and spectrofluorometric methods. It was shown that spectra of GOx produced by P. funiculosum are typical for flavoproteins. Absorption spectrum has distinct peaks at 380 and 457 nm, excitation spectrum at 373 and 447 nm, and emission spectrum at 530 and 562 nm. The pH correlation of enzyme activity and catalytic characteristics in various buffer systems (phosphate (pH 5.0–9.0), citrate (pH 3.0–5.0), citrate-phosphate (pH 3.0–9.0), and universal (pH 3.0–9.0)) were registered. It was determined that the GOx is the most efficiently interacting with substrate (glucose) in phosphate buffer at pH 7.0 with k cat/K m = 21,825 M−1 s−1. Interaction of several different redox mediators (9,10-phenantroline-5,6-dione, 9,10-phenanthrenequinone, N-methylphenazonium methyl sulfate, ferrocene, ferrocenecarboxylic acid, α-methylferrocenemethanol, ferrocenecarboxaldehyde) with GOx from P. funiculosum was investigated by evaluation of the difference in fluorescence emission intensity of FAD(oxidized) and FADH2(reduced) forms. It was found that 9,10-phenantroline-5,6-dione and 9,10-phenanthrenequinone are the best redox mediators for this type of GOx. Figure ᅟ
Keywords: Glucose oxidase; Penicillium funiculosum ; Redox mediators; Spectrofluorimetry; Biosensors; Biofuel cells

Although the use of lipases as biocatalysts has frequently been proposed, it is yet scarcely being implemented in industrial processes. This is mainly due to the difficulties associated with the discovery and engineering of new enzymes and the lack of versatile screening methods. In this study, we screened the available strategy from a metagenomic pool for the organic solvent-tolerant lipase with enhanced performance for industrial processes. A novel lipase was identified through functional screening from a metagenomic library of activated sludge in an Escherichia coli system. The gene encoding the lipase from the metagenomic pool, metalip1, was sequenced and cloned by PCR. Metalip1 encoding a polypeptide of 316 amino acids had typical residues essential for lipase such as pentapeptide (GXSXGG) and catalytic triad sequences (Ser160, Asp260, and His291). The deduced amino acid sequence of metalip1 showed high similarity to a putative lipase from Pseudomonas sp. CL-61 (80 %, ABC25547). Metalip1 was expressed in E. coli BL21 (DE3) with a his-tag and purified using a Ni-NTA chelating column and characterized. This enzyme showed high expression level and solubility in the heterologous E. coli host. This enzyme was active over broad organic solvents. Among organic solvents examined, dimethyl formamide was the best organic solvent for metalip1. We showed that function-based strategy is an effective method for fishing out an organic solvent-tolerant lipase from the metagenomic library. Also, it revealed high catalytic turnover rates, which make them a very interesting candidate for industrial application.
Keywords: Lipase; Screening; Purification; Organic-tolerant enzyme; Uncultivated microorganism

An extracellular l-asparaginase produced by a protease-deficient isolate, Bacillus aryabhattai ITBHU02, was purified to homogeneity using ammonium sulfate fractionation and subsequent column chromatography on diethylaminoethyl-Sepharose fast flow and Seralose CL-6B. The enzyme was purified 68.9-fold with specific activity of 680.47 U mg−1. The molecular weight of the purified enzyme was approximately 38.8 kDa on SDS-PAGE and 155 kDa on native PAGE gel as well as gel filtration column revealing that the enzyme was a homotetramer. The optimum activity of purified l-asparaginase was achieved at pH 8.5 and temperature 40 °C. Kinetic studies depicted that the K m, V max, and k cat values of the enzyme were 0.257 mM, 1.537 U μg−1, and 993.93 s−1, respectively. Circular dichroism spectroscopy has showed that the enzyme belonged to α + β class of proteins with approximately 74 % α-helices and 12 % β-sheets. BLASTP analysis of N-terminal sequence K-T-I-I-E-A-V-P-E-L-K-K-I-A of purified l-asparaginase had shown maximum similarity with Bacillus megaterium DSM 319. In vitro cytotoxicity assays with HL60 and MOLT-4 cell lines indicated that the l-asparaginase has significant antineoplastic properties.
Keywords: l-Asparaginase; Bacillus aryabhattai ITBHU02; Circular dichroism; Biochemical characterization; Antileukemic activity; N-terminal sequence

Nitrogen Limitation in Neochloris oleoabundans: A Reassessment of Its Effect on Cell Growth and Biochemical Composition by Adriana Garibay-Hernández; Rafael Vazquez-Duhalt; Leobardo Serrano-Carreón; Alfredo Martinez (1775-1791).
The aim of this work was to reassess the effect of nitrogen limitation (from 0 to 1 mM nitrate), on the growth and the biochemical composition of Neochloris oleoabundans cultures, where only the CO2 available in the air was provided. Slight differences in the initial nitrate concentration, even minimal increments of 0.2 mM, significantly modify the microalgal response towards nitrogen limitation. This stress condition reduced cell proliferation, but increased cell mass values due to the simultaneous accumulation of two storage compounds: lipids, which contained up to a 55.9 % of total fatty acids; and carbohydrates, which may be primarily composed by starch. The highest biomass and lipid productivities of 98.24 and 43.24 mg/l/day, respectively, were attained at an initial nitrate concentration of 0.6 mM. The theoretical annual projection, based on these productivities, allowed the estimation of the liquid fuel energy yields, which are comparable or even higher than those calculated for several biomass feedstocks such as corn, oil palm, sugarcane, or even fast growing grasses, confirming the potential of nitrogen-limited N. oleoabundans biomass as an appropriate feedstock for biofuel purposes.
Keywords: Neochloris oleoabundans ; Microalgae; Lipids; Fatty acids; Nitrogen limitation; Biomass; Biofuels

Constitutive Expression of a rhIL-2-HSA Fusion Protein in Pichia pastoris Using Glucose as Carbon Source by Bo Guan; Fengxiang Chen; Jianyong Lei; Yunhua Li; Zuoying Duan; Ruiyu Zhu; Yun Chen; Huazhong Li; Jian Jin (1792-1804).
A constitutive expression vector for rhIL-2-HSA fusion protein production in yeast Pichia pastoris was constructed. The coding gene was placed in frame with the Saccharomyces cerevisiae α-factor secretion signal sequence under the control of the GAP promoter. The recombinant plasmid pGAPZαA-rhIL-2-HSA was integrated into the genome of the P. pastoris GS115. The effect of different carbon sources on rhIL-2-HSA fusion protein expression was evaluated in shaking flask cultures. We found that recombinant P. pastoris grew well and efficiently secreted rhIL-2-HSA fusion protein into the medium when using glucose as carbon source. To achieve higher production, the influence of initial pH and culture temperature was also evaluated. Fed-batch fermentation strategy using glucose as carbon source for constitutive expression of rhIL-2-HSA fusion protein was investigated in 5-L bioreactor and the expression level of rhIL-2-HSA could reach about 250 mg/L after 60-h fermentation. The rhIL-2-HSA fusion protein produced by this constitutive expression system was purified and exhibited a specific bioactivity of 1.040 × 106 IU/mg in vitro. This study described constitutive expression of rhIL-2-HSA fusion protein by P. pastoris and development of a simple high-cell density fermentation strategy for biologically active rhIL-2-HSA fusion protein using glucose as sole carbon source.
Keywords: GAP Promoter; rhIL-2-HSA Fusion Protein; Constitutive Expression; Pichia pastoris ; Fed-Batch Fermentation

Engineering and Kinetic Stabilization of the Therapeutic Enzyme Anabeana variabilis Phenylalanine Ammonia Lyase by Hossein Zarei Jaliani; Safar Farajnia; Seyyed Abolghasem Mohammadi; Abolfazl Barzegar; Saeed Talebi (1805-1818).
Anabeana variabilis phenylalanine ammonia lyase has just recently been discovered and introduced in clinical trials of phenylketonuria enzyme replacement therapy for its outstanding kinetic properties. In the present study, kinetic stabilization of this therapeutically important enzyme has been explored by introduction of a disulfide bond into the structure. Site-directed mutagenesis was performed with quick-change PCR method. Recombinant wild-type and mutated enzymes were expressed in Escherichia coli, and his-tagged proteins were affinity purified. Formation of disulfide bond was confirmed by Ellman’s method, and then chemical unfolding, kinetic behavior, and thermal inactivation of mutated enzyme were compared with the wild type. Based on our results, the Q292C mutation resulted in a significant improvement in kinetic stability and resistance against chemical unfolding of the enzyme while kinetic parameters and pH profile of enzyme activity were remained unaffected. The results of the present study provided an insight towards designing phenylalanine ammonia lyases with higher stability.
Keywords: Disulfide bond; Mutation; Phenylalanine ammonia lyase; Protein stability; Rational engineering

Effect of F68 on Cryopreservation of Mesenchymal Stem Cells Derived from Human Tooth Germ by Ayşegül Doğan; Mehmet Emir Yalvaç; Aysu Yılmaz; Albert Rizvanov; Fikrettin Şahin (1819-1831).
The use of stem-cell-based therapies in regenerative medicine and in the treatment of disorders such as Parkinson, Alzheimer's disease, diabetes, spinal cord injuries, and cancer has been shown to be promising. Among all stem cells, mesenchymal stem cells (MSCs) were reported to have anti-apoptotic, immunomodulatory, and angiogenic effects which are attributed to the restorative capacity of these cells. Human tooth germ stem cells (HTGSCs) having mesenchymal stem cell characteristics have been proven to exert high proliferation and differentiation capacity. Unlike bone-marrow-derived MSCs, HTGSCs can be easily isolated, expanded, and cryopreserved, which makes them an alternative stem cell source. Regardless of their sources, the stem cells are exposed to physical and chemical stresses during cryopreservation, hindering their therapeutic capacity. Amelioration of the side effects of cryopreservation on MSCs seems to be a priority in order to maximize the therapeutic efficacy of these cells. In this study, we tested the effect of Pluronic 188 (F68) on HTGSCs during long-term cryopreservation and repeated freezing and defrosting cycles. Our data revealed that F68 has a protective role on survival and differentiation of HTGSCs in long-term cryopreservation.
Keywords: Cryopreservation; Pluronic; Human tooth germ stem cell

An anaerobic sequencing batch biofilm reactor (AnSBBR—total volume 7.5 L; liquid volume 3.6 L; treated volume per cycle 1.5 L) treated sucrose-based wastewater to produce biohydrogen (at 30 °C). Different applied volumetric organic loads (AVOL of 9.0, 12.0, 13.5, 18.0, and 27.0 kg COD m−3 day−1), which were varied according to the influent concentration (3,600 and 5,400 mg COD L−1) and cycle length (4, 3, and 2 h), have been used to assess the following parameters: productivity and yield of biohydrogen per applied and removed load, reactor stability, and efficiency. The removed organic matter (COD) remained stable and close to 18 % and carbohydrates (sucrose) uptake rate remained between 83 and 97 % during operation. The decrease in removal performance of the reactor with increasing AVOL, by increasing the influent concentration (at constant cycle length) and decreasing the cycle lengths (at constant influent concentrations), resulted in lower conversion efficiencies. Under all conditions, when organic load increased there was a predominance of acetic, propionic, and butyric acid as well as ethanol. The highest concentration of biohydrogen in the biogas (24–25 %) was achieved at conditions with AVOL of 12.0 and 13.5 kg COD m−3 day−1, the highest daily production rate (0.139 mol H2 day−1) was achieved at AVOL of 18.0 kg COD m−3 day−1, and the highest production yields per removed and applied load were 2.83 and 3.04 mol H2 kg SUC−1, respectively, at AVOL of 13.5 kg COD m−3 day−1. The results indicated that the best productivity tends to occur at higher organic loads, as this parameter involves the “biochemical generation” of biogas, and the best yield tends to occur at lower and/or intermediate organic loads, as this parameter involves “biochemical consumption” of the substrate.
Keywords: AnSBBR; Biohydrogen; Agitation; Organic loading; Influent concentration; Cycle length

EvaGreen multiplex real-time polymerase chain reaction (EMRT-PCR) was designed for an assay that can join the advantages of multiplex PCR and real-time PCR to recognize animal genes more quickly in pet foods. EMRT-PCR based on melting temperatures discrimination by using EvaGreen fluorescence dye was developed for the analysis of pork and poultry in pet food. The method combines the use of poultry- and pork-specific primers that amplify small fragments of 12S rRNA and mitochondrial DNA genes. Appropriate mixtures of poultry and pork meat in reference samples were used to develop the assay. Gene yields of poultry and pork were represented in two melting peaks generated simultaneously at temperatures of 80.5 and 87.2 °C, respectively. Based upon the assay results, it has been concluded that EMRT-PCR assay might be an efficient tool for the verification of species origin in pet foods.
Keywords: Real-time PCR; EvaGreen fluorescence dye; Multiplex analysis; Pet food

Growth and fermentation characteristics, biomass composition, lipid characterization and metabolic profiling analysis of two different Schizochytrium sp. strains, the original strain and the industrial adaptive strain, were investigated in the fed-batch fermentation process. The final cell biomass, total lipids content, docosahexanoic acid (DHA) content and DHA productivity of the adaptive strain were much higher than those of the original strain. The metabolic distinctions which extensively existed between these two strains were revealed by the score plot of principal component analysis. In addition, potential biomarkers responsible for discriminating different strains were identified as myo-inositol, histidine, alanine, asparagine, cysteine, and oxalic acid. These findings provided new insights into the industrial strain screening and further improvement of DHA production by Schizochytrium sp.
Keywords: Schizochytrium sp.; Docosahexanoic acid; Biomass composition; Lipid characterization; Metabolic profiling analysis; Fed-batch

Sodium Chloride-Induced Modulation of the Activity and Thermal Stability of Short-Chain Oxidoreductase from the Archaeon Thermococcus sibiricus by Tatiana N. Stekhanova; Ekaterina Y. Bezsudnova; Andrey V. Mardanov; Vadim M. Gumerov; Natalya Artemova; Serguey Y. Kleymenov; Vladimir O. Popov (1877-1889).
Recently, we have studied properties and structural features of the thermostable halotolerant alcohol dehydrogenase from archaeon Thermococcus sibiricus (TsAdh319). In the present work, the effect of sodium chloride on activity and thermostability was explored using circular dichroism, fluorescent spectroscopy, and differential scanning calorimetry. The activity of TsAdh319 increased in the presence of NaCl and remained at the elevated level up to 4 M of NaCl. Sodium chloride at molar concentrations reduced the optimal reaction temperature, increased both Michaelis constant (K m) and k cat values for the substrates tested, decreased affinity for the coenzyme, and stoichiometry of coenzyme binding. No changes were revealed in a secondary or quaternary structure of the protein in the presence of NaCl up to 90 °C. According to differential scanning calorimetry, the irreversible unfolding started around 90 °C, the addition of NaCl decreased T m from 104.2 to 102.2 °C, and reduced ΔH from 438 to 348 kJ/mol. Kinetic studies revealed positive effect of NaCl on the TsAdh319 thermostability. The results are interpreted in regard to TsAdh319 structural data.
Keywords: Archaeal alcohol dehydrogenase; Halotolerance; Thermostability; Salt effect; Coenzyme binding

In this study, biodegradable poly(butylenes succinate) (PBS) fiber mats containing silver nanoparticles (AgNPs) were prepared by the electrospinning process. Small AgNPs (<10 nm) were simply synthesized using polyvinylpyrrolidone as the capping agent as well as the reductant. The morphology of the PBS-AgNPs fiber mats and the distribution of the AgNPs were well characterized by TEM and SEM. The release of Ag from the PBS fiber mats was quantitively determined by ICP. The PBS fiber mats with 0.29 % AgNPs content showed strong antimicrobial activity against both gram-positive Staphylococcus aureus and gram-negative Escherichia coli with the efficacy as high as 99 %. The effective bactericidal activity on E. coli was demonstrated for a short contacting time with the PBS-AgNPs fiber mats. In addition, the long-term release performance of Ag from the fiber mats can keep inhibiting the bacterial growth in the mats over a long period of time.
Keywords: Poly(butylenes succinate); Electrospinning; Silver nanoparticles; Antimicrobial; Fiber mats

Microsporum fulvum IBRL SD3: As Novel Isolate for Chicken Feathers Degradation by I. Darah; A. Nur-Diyana; S. Nurul-Husna; K. Jain; Sheh-Hong Lim (1900-1910).
Keratinous wastes have increasingly become a problem and accumulate in the environment mainly in the form of feathers, generated mainly from a large number of poultry industries. As keratins are very difficult to degrade by general proteases, they pose a major environmental problem. Therefore, microorganisms which would effectively degrade keratins are needed for recycling such wastes. A geophilic dermatophyte, Microsporum fulvum IBRL SD3 which was isolated from a soil sample collected from a chicken feather dumping site using a baiting technique, was capable to produce keratinase significantly. The crude keratinase was able to degrade whole chicken feathers effectively. The end product of the degradation was protein that contained essential amino acids and may have potential application in animal feed production. Thus, M. fulvum could be a novel organism to produce keratinase for chicken feathers degradation.
Keywords: Microsporum fulvum ; Chicken feather; Keratinase; Geophilic dermatophyte; Degradation

An Ancestral Member of the Polysaccharide Lyase Family 2 Displays Endolytic Activity and Magnesium Dependence by D. Wade Abbott; Dallas Thomas; Benjamin Pluvinage; Alisdair B. Boraston (1911-1923).
Polysaccharide lyases (PLs) are enzymes that cleave glycosidic linkages in hexuronate polysaccharides, such as homogalacturonan (HG), using a β-elimination mechanism. Traditionally, PL activities on HG have been associated with catalytic calcium cofactors, unusually high pH optima, and arginine Brønstead bases. Recently, however, PL families that harness transition metal cofactors, utilize lysine and histidine Brønstead bases, and display more neutral pH optima have been described. One such family is PL2, which has members found primarily in phytopathogenic (e.g., Dickeya spp. and Pectobacterium spp.) or enteropathogenic (e.g., Yersinia spp.) bacterial species. PL2 is divided into two major subfamilies that are correlated with either an endolytic or exolytic activity. This study has focused on the activity of a PL2 member, which is not classified within either subfamily and helps to illuminate the origin of enzyme activities within the family. In addition, the role of Mg2+ as a preferential catalytic metal for an intracellular PL2 (PaePL2) is described. The implications for the relationship between catalytic metal selectivity and the cellular location of pectate lyase-mediated catalysis are discussed.
Keywords: Pectin; Polysaccharide lyase; Endolytic; Exolytic; Beta-elimination; Magnesium