Applied Biochemistry and Biotechnology (v.170, #5)
Carbon Nanotubes-Based Label-Free Affinity Sensors for Environmental Monitoring by Tapan Sarkar; Yingning Gao; Ashok Mulchandani (1011-1025).
Nanostructures, such as nanowires, nanobelts, nanosprings, and nanotubes, are receiving growing interest as transducer elements of bio/chemical sensors as they provide high sensitivity, multiplexing, small size, and portability. Single-walled carbon nanotubes (SWNTs) are one such class of nanostructure materials that exhibit superior sensing behavior due to its large-surface carbon atoms that are highly responsive to surface adsorption events. Further, their compatibility with modern microfabrication technologies and facile functionalization with molecular recognition elements make them promising candidates for bio/chemical sensors applications. Here, we review recent results on nanosensors based on SWNTs modified with biological receptors such as aptamers, antibodies, and binding proteins, to develop highly sensitive, selective, rapid, and cost-effective label-free chemiresistor/field-effect transistor nanobiosensors for applications in environmental monitoring.
Keywords: 1-D nanostructures; Carbon nanotubes; Label-free biosensor; Chemiresistor; Field-effect transistor; Pathogen; Explosives; Toxins; Nerve agents
Microbial Transformation of Curcumin to Its Derivatives with a Novel Pichia kudriavzevii ZJPH0802 Strain by Weiyu Zhang; Jin Huang; Xingde Wo; Pu Wang (1026-1037).
Curcumin, a polyphenolic compound, has shown a wide range of pharmacological activities and has been widely used as a food additive. However, the clinical use of curcumin is limited to some extent because of its poor water solubility and low bioavailability. To overcome these problems, many approaches have been attempted and structural modification of curcumin by microbial transformation has been proven to be an alternative. In this study, we isolated a novel yeast strain Pichia kudriavzevii ZJPH0802 from a soil sample, which is capable of converting curcumin to its derivatives. The transformed products by this strain were evaluated by HPLC, (+) electrospray ionization (ESI)-MSn, and 1H nuclear magnetic resonance methods. Compared with controls, two new peaks of the transformed broth appeared at retention times of 26 min (I) and 62 min (II) by HPLC analysis. The two transformed products were then further identified by (+) ESI-MSn. The spectrum showed that compound I had an accurate [M+H+NH3]+ ion at m/z 392, [M+H]+ ion at m/z 375, [M+H–H2O]+ ion at m/z 357, and (+) ESI-MS3 spectrum showed that ion at m/z 357 could further form fragment ions at m/z 339, 177, and 163; compound II had an accurate [M+H]+ ion at m/z 373, [M+H–H2O]+ ion at m/z 355, and (+) ESI-MS3 spectrum showed that ion at m/z 355 could further form fragment ions at m/z 219, 179, 177, 163, and 137. These two transformed products thereby were confirmed as hexahydrocurcumin (I) and tetrahydrocurcumin (II).
Keywords: Curcumin; Pichia kudriavzevii ; Biotransformation; Hexahydrocumin; Tetrahydrocurcumin
Rhamnolipids Production by Multi-metal-Resistant and Plant-Growth-Promoting Rhizobacteria by Anil Kumar Singh; Swaranjit Singh Cameotra (1038-1056).
The biosurfactant-producing Pseudomonas aeruginosa A11, with plant-growth-promoting (PGP) and multi-metal-resistant (MMR) features was isolated from the rhizosphere of a wild plant Parthenium hysterophorus. The strain A11 was able to utilize glycerol as a carbon source and produce 4,436.9 mg/L of biosurfactant after 120 h of incubation. The biosurfactants was characterized as rhamnolipids (RLs) by thin layer chromatography, Fourier transform infrared spectroscopy, nuclear magnetic resonance, and liquid chromatography–mass spectrometry analysis. Eight different RLs congeners were detected with RhaRhaC10C10 being most abundant. The purified rhamnolipid, dirhamnolipid, and monorhamnolipid reduced the surface tension of water to 29, 36, and 42 mN/m with critical micelle concentration of 83, 125, and 150 mg/L, respectively. The strain A11 demonstrated resistance against all the metals detected in rhizosphere except Hg and Ni. The strain A11 also possessed plant-growth-promoting features like siderophores, hydrogen cyanide, catalase, ammonia production, and phosphate solubilization. The dirhamnolipids formed crystals upon incubation at 4 °C, thus making separation of dirhamnolipids easy. Biosurfactant-producing ability along with MMR and PGP traits of the strain A11 makes it a potential candidate for application in the bacterial assisted enhancement of phytoremediation of heavy-metal-contaminated sites.
Keywords: Biosurfactant; Rhamnolipids; Multi-metal-resistant; Plant growth promoting; Rhizobacteria
Two-Staged Temperature and Agitation Strategy for the Production of Transglutaminase from a Streptomyces sp. Isolated from Brazilian Soils by Marcela Pavan Bagagli; Hélia Harumi Sato (1057-1065).
Transglutaminase catalyzes the cross-linking reaction between a glutamine residue and a free amine residue of proteins leading to the formation of protein aggregates. In this research, the effects of temperature, agitation, and aeration on the production of transglutaminase in a bench reactor by a newly isolated Streptomyces sp. from Brazilian soils were investigated using a factorial experimental design. The parameters evaluated influenced the enzyme production, and the data showed that the best conditions to enhance cell growth were different from those leading to enhanced transglutaminase production. Thus, a temperature and agitation shift strategy was adopted to increase transglutaminase productivity. The temperature and agitation were first set at 34 °C and 350 rpm, respectively, and after 24 h decreasing to 26 °C and 150 rpm until the end of fermentation. The transglutaminase activity obtained was 2.18 U/mL after 42 h of fermentation, which was twice than that obtained using a constant temperature and agitation fermentation strategy.
Keywords: Fermentation; Enzyme production; Transglutaminase; Streptomyces ; Factorial design
Catalase and Superoxide Dismutase Activities and the Total Protein Content of Protocorm-Like Bodies of Dendrobium Sonia-28 Subjected to Vitrification by Ranjetta Poobathy; Uma Rani Sinniah; Rathinam Xavier; Sreeramanan Subramaniam (1066-1079).
Dendrobium sonia-28 is an important ornamental orchid in the Malaysian flower industry. However, the genus faces both low germination rates and the risk of producing heterozygous progenies. Cryopreservation is currently the favoured long-term storage method for orchids with propagation problems. Vitrification, a frequently used cryopreservation technique, involves the application of pretreatments and cryoprotectants to protect and recover explants during and after storage in liquid nitrogen. However, cryopreservation may cause osmotic injuries and toxicity to cryopreserved explants from the use of highly concentrated additives, and cellular injuries from thawing, devitrification and ice formation. Reactive oxygen species (ROS), occurring during dehydration and cryopreservation, may also cause membrane damage. Plants possess efficient antioxidant systems such as the superoxide dismutase (SOD) and catalase (CAT) enzymes to scavenge ROS during low temperature stress. In this study, protocorm-like bodies (PLBs) of Dendrobium sonia-28 were assayed for the total protein content, and both SOD and CAT activities, at each stage of a vitrification exercise to observe for deleterious stages in the protocol. The results indicated that cryopreserved PLBs of Dendrobium sonia-28 underwent excessive post-thawing oxidative stress due to decreased levels of the CAT enzyme at the post-thawing recovery stage, which contributed to the poor survival rates of the cryopreserved PLBs.
Keywords: Catalase; Protocorm-like bodies; Superoxide dismutase; Total protein content; Vitrification
Production of Microbial Rhamnolipid by Pseudomonas Aeruginosa MM1011 for Ex Situ Enhanced Oil Recovery by Hossein Amani; Markus Michael Müller; Christoph Syldatk; Rudolf Hausmann (1080-1093).
Recently, several investigations have been carried out on the in situ bacteria flooding, but the ex situ biosurfactant production and addition to the sand pack as agents for microbial enhanced oil recovery (MEOR) has little been studied. In order to develop suitable technology for ex situ MEOR processes, it is essential to carry out tests about it. Therefore, this work tries to fill the gap. The intention of this study was to investigate whether the rhamnolipid mix could be produced in high enough quantities for enhanced oil recovery in the laboratory scale and prove its potential use as an effective material for field application. In this work, the ability of Pseudomonas aeruginosa MM1011 to grow and produce rhamnolipid on sunflower as sole carbon source under nitrogen limitation was shown. The production of Rha-C10-C10 and Rha2-C10-C10 was confirmed by thin-layer chromatography and high-performance liquid chromatography analysis. The rhamnolipid mixture obtained was able to reduce the surface and interfacial tension of water to 26 and 2 mN/m, respectively. The critical micelle concentration was 120 mg/L. Maximum rhamnolipid production reached to about 0.7 g/L in a shake flask. The yield of rhamnolipid per biomass (Y RL/x ), rhamnolipid per sunflower oil (Y RL/s ), and the biomass per sunflower oil (Y x/s ) for shake flask were obtained about 0.01, 0.0035, and 0.035 g g−1, respectively. The stability of the rhamnolipid at different salinities, pH and temperature, and also, its emulsifying activity has been investigated. It is an effective surfactant at very low concentrations over a wide range of temperatures, pHs, and salt concentrations, and it also has the ability to emulsify oil, which is essential for enhanced oil recovery. With 120 mg/L rhamnolipid, 27 % of original oil in place was recovered after water flooding from a sand pack. This result not only suggests rhamnolipids as appropriate model biosurfactants for MEOR, but it even shows the potential as a biosurfactant of choice for actual MEOR applications.
Keywords: Biosurfactants; Emulsion index; Enhanced oil recovery; Pseudomonas aeruginosa ; Rhamnolipid
Sulfide-Oxidizing Autotrophic Denitrification: an Evaluation for Nitrogen Removal from Anaerobically Pretreated Domestic Sewage by Theo S. O. Souza; Eugenio Foresti (1094-1103).
Nitrogen removal from effluents of anaerobic reactors using conventional nitrification/denitrification processes depends on the availability of electron donors for denitrification. As sulfide is normally present in such effluents, autotrophic denitrification using sulfide can be an alternative to avoid or reduce the requirements of exogenous organic carbon sources. This study evaluated the application of sulfide-oxidizing autotrophic denitrification to anaerobically pretreated domestic sewage. A denitrifying reactor was fed with a mixture, at different proportions, of anaerobically pretreated (containing sulfides and residual organic matter) and nitrified (containing nitrates) effluents produced by reactors treating synthetic domestic sewage. Autotrophic denitrification was responsible for most of the nitrogen removal and coexisted with heterotrophic activity, resulting in high denitrification efficiencies. Efficient denitrification could be attained at a molar NO3 −/S2− ratio of 2.1, higher than values reported before, and this represents an important parameter for the strategic application of the process. Under the experimental conditions studied, autotrophic denitrification showed to be an efficient and feasible alternative to conventional denitrification using exogenous electron donors.
Keywords: Autotrophic denitrification; Domestic sewage; Electron donor; Nitrogen removal; Posttreatment; Sulfide
Chestnut Shell as Unexploited Source of Fermentable Sugars: Effect of Different Pretreatment Methods on Enzymatic Saccharification by Luisa Maurelli; Elena Ionata; Francesco La Cara; Alessandra Morana (1104-1118).
Chestnut shell (CS) is an agronomic residue mainly used for extraction of antioxidants or as adsorbent of metal ions. It also contains some polysaccharide that has not been considered as potential source of fermentable sugars for biofuel production until now. In this study, the effect of different pretreatment methods on CS was evaluated in order to obtain the greatest conversion of cellulose and xylan into fermentable sugars. Hot acid impregnation, steam explosion (acid-catalysed or not), and aqueous ammonia soaking (AAS) were selected as pretreatments. The pretreated biomass was subjected to saccharification with two enzyme cocktails prepared from commercial preparations, and evaluation of the best pretreatment and enzyme cocktail was based on the yield of fermentable sugars produced. As AAS provided the best result after preliminary experiments, enhancement of sugar production was attempted by changing the concentrations of ammonium hydroxide, enzymes, and CS. The optimal pretreatment condition was 10 % ammonium hydroxide, 70 °C, 22 h with CS at 5 % solid loading. After saccharification of the pretreated CS for 72 h at 50 °C and pH 5.0 with a cocktail containing cellulase (Accellerase 1500), beta-glucosidase (Accellerase BG), and xylanase (Accellerase XY), glucose and xylose yields were 67.8 and 92.7 %, respectively.
Keywords: Aqueous ammonia soaking; Chestnut shell; Cellulose; Enzymatic saccharification; Glucose; Lignin; Pretreatment; Steam explosion
Three-Phase Partitioning as a Rapid and Easy Method for the Purification and Recovery of Catalase from Sweet Potato Tubers (Solanum tuberosum) by Yonca Avcı Duman; Erdem Kaya (1119-1126).
Three-phase partitioning (TPP) was used to purify and recover catalase from potato crude extract. The method consists of ammonium sulfate saturation, t-butanol addition, and adjustment of pH, respectively. The best catalase recovery (262 %) and 14.1-fold purification were seen in the interfacial phase in the presence of 40 % (w/v) ammonium sulfate saturation with 1.0:1.0 crude extract/t-butanol ratio (v/v) at pH 7 in a single step. The sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the enzyme showed comparatively purification and protein molecular weight was nearly found to be 56 kDa. This study shows that TPP is a simple, economical, and quick method for the recovering of catalase and can be used for the purification process.
Keywords: Three-phase partitioning; Recovery; Catalase; Purification
Spectroscopic and In Silico Evaluation of Interaction of DNA with Six Anthraquinone Derivatives by Pritha Ghosh; G. Poornima Devi; R. Priya; A. Amrita; A. Sivaramakrishna; S. Babu; R. Siva (1127-1137).
Anthraquinones consist of several hundreds of derivatives that differ in the nature and positions of substituent groups which are known to have several biological activities including antitumor properties. Interaction of molecules with DNA persists to be an extremely vital parameter while endeavouring to formulate therapeutics. In this study, few anthraquinone derivatives such as 1,2-dihydroxyanthraquinone (alizarin), 1,4-dihydroxyanthraquinone (quinizarin), 1,8-dihydroxyanthraquinone (danthron), 1,2,4-trihydroxyanthraquinone (purpurin), 1,4-diaminoanthraquinone, and 1-methylaminoanthraquinone were analyzed for its possible interaction with calf-thymus DNA through spectroscopy and in silico analysis. Our UV spectroscopic results indicate that all selected anthraquinones interact with DNA probably by external binding. Molar extinction coefficient has been calculated for chosen six anthraquinones. FT-IR results suggest that significant shifts of peaks as well as disappearance of certain characteristic peaks were indicators of the plausible interaction going on due to dye-DNA adduct formation. Among the six dyes used, purpurin showed better results and indicates the relatively strong binding affinity with DNA. Our molecular modeling results also show that purpurin has comparatively higher DNA interaction with a score of −6.18 compared with the ethidium bromide of −5.02 and intercalate the DNA.
Keywords: Anthraquinones; Docking; Dyes; Interaction; Purpurin; Spectroscopy
Visualizing Hepatic Copper Release in Long–Evans Cinnamon Rats Using Single-Photon Emission Computed Tomography by Eric M. Yezdimer; Tomohiro Umemoto; Hiroshi Yamada; Satoshi Makino; Ikuo Tooyama (1138-1150).
The potential utility of an imaging agent for the detection of hepatic copper was investigated in a Wilson’s disease animal model. Solid-phase peptide synthesis was used to construct an imaging agent which consisted of a copper-binding moiety, taken from the prion protein, and a gamma ray-emitting indium radiolabel. Long–Evans Cinnamon (LEC) rats were used for the Wilson’s disease animal model. Our evaluation methodology consisted of administering the indium-labeled agent to both LEC and genetically healthy Long–Evans (LE) cohorts via a tail vein injection and following the pharmacokinetics with single-photon emission computed tomography (SPECT) over the course of an hour. The animals were then sacrificed and their livers necropsied. An additional control agent, lacking the copper-binding moiety, was used to gauge whether any change in the hepatic uptake might be caused by other physiological differences between the two animal models. LEC rats injected with the indium-labeled agent had roughly double the amount of hepatic radioactivity as compared to the healthy control animals. The control agent, without the copper-binding moiety, displayed a hepatic signal similar to that of the control LE animals. Additional intraperitoneal spiking with CuSO4 in C57BL/6 mice also found that the pharmacokinetics of the indium-labeled, prion-based imaging agent is profoundly altered by exposure to in vivo pools of extracellular copper. The described SPECT application with this compound represented a significant improvement over a previous MRI application using the same base peptide sequence.
Keywords: Peptide; Copper; Wilson’s disease; ATP7B; SPECT (single-photon emission computed tomography)
Identification and Quantification of Genetically Modified Moonshade Carnation Lines Using Conventional and TaqMan Real-Time Polymerase Chain Reaction Methods by Peng Li; Junwei Jia; Lan Bai; Aihu Pan; Xueming Tang (1151-1162).
Genetically modified carnation (Dianthus caryophyllus L.) Moonshade was approved for planting and commercialization in several countries from 2004. Developing methods for analyzing Moonshade is necessary for implementing genetically modified organism labeling regulations. In this study, the 5'-transgene integration sequence was isolated using thermal asymmetric interlaced (TAIL)-PCR. Based upon the 5'-transgene integration sequence, conventional and TaqMan real-time PCR assays were established. The relative limit of detection for the conventional PCR assay was 0.05 % for Moonshade using 100 ng total carnation genomic DNA, corresponding to approximately 79 copies of the carnation haploid genome, and the limits of detection and quantification of the TaqMan real-time PCR assay were estimated to be 51 and 254 copies of haploid carnation genomic DNA, respectively. These results are useful for identifying and quantifying Moonshade and its derivatives.
Keywords: Moonshade; Genetically modified organism; Conventional and TaqMan real-time PCR; Thermal asymmetric interlaced-PCR (TAIL-PCR)
An Improved Micropropagation of Arnebia hispidissima (Lehm.) DC. and Assessment of Genetic Fidelity of Micropropagated Plants Using DNA-Based Molecular Markers by Mahendra Phulwaria; Manoj K. Rai; N. S. Shekhawat (1163-1173).
An efficient and improved in vitro propagation method has been developed for Arnebia hispidissima, a medicinally and pharmaceutically important plant species of arid and semiarid regions. Nodal segments (3–4 cm) with two to three nodes obtained from field grown plants were used as explants for shoot proliferation. Murashige and Skoog’s (MS) medium supplemented with cytokinins with or without indole-3-acetic acid (IAA) or naphthalene acetic acid was used for shoot multiplication. Out of different PGRs combinations, MS medium containing 0.5 mg l−1 6-benzylaminopurine and 0.1 mg l−1 IAA was optimal for shoot multiplication. On this medium, explants produced the highest number of shoots (47.50 ± 0.38). About 90 % of shoots rooted ex vitro on sterile soilrite under the greenhouse condition when the base (2–4 mm) of shoots was treated with 300 mg l−1 of indole-3-butyric acid for 5 min. The plantlets were hardened successfully in the greenhouse with 85–90 % survival rate. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were employed to assess the genetic stability of in vitro-regenerated plants of A. hispidissima. Out of 40 (25 RAPD and 15 ISSR) primers screened, 15 RAPD and 7 ISSR primers produced a total number of 111 (77 RAPD and 34 ISSR) reproducible amplicons. The amplified products were monomorphic across all the micropropagated plants and were similar to the mother plant. To the best of our knowledge, it is the first report on the assessment of the genetic fidelity in micropropagated plants of A. hispidissima.
Keywords: Ex vitro rooting; Genetic stability; Molecular markers; Multiplication; Shikonin
Survival of Listeria monocytogenes Cells and the Effect of Extended Frozen Storage (−20 °C) on the Expression of Its Virulence Gene by Rihab Ben Slama; Hanene Miladi; Kamel Chaieb; Amina Bakhrouf (1174-1183).
Listeria monocytogenes is the main bacteria incriminated in contaminating cold storage food and ready-to-eat products. Survival of four L. monocytogenes food isolates was examined during freezing and cold storage. Slices of artisanal cheese were inoculated with Listeria strains and incubated at −20 °C. Their survival rate was recorded during the storage period. Then, transcript levels of four virulence genes (hlyA, iap, fri, and flaA) were evaluated using a semiquantitative reverse transcriptase PCR. Our results revealed that after 6 months of frozen storage, the number of L. monocytogenes cells had declined by 2.04 ± 0.1 log10 colony-forming units (CFU) g−1, 2.52 ± 0.1 log10 CFU g−1, and 2.58 ± 0.1 log10 CFU g−1 for the strains S2, S3, and S4, respectively. Our data revealed that all the studied genes (hlyA, iap, fri, and flaA) were expressed after 6 months of incubation in artisanal cheese at −20 °C, and the transcript level has been affected by the factor freezing, whether for the reference strain ATCC 19115 or for other isolates. The iap gene expression was decreased for the four strains after 6 months of storage, and hlyA expression rate was consistently slightly lower. Transcript levels of fri and flaA genes were consistently higher in L. monocytogenes cells before cold exposure than in stressed cells and were significantly affected by the freezing process.
Keywords: Listeria monocytogenes ; Cheese; Freezing; Virulence genes; RT-PCR
Antimicrobial and Antibiofilm Activity of Designed and Synthesized Antimicrobial Peptide, KABT-AMP by Bency Thankappan; Sivakumar Jeyarajan; Sakaue Hiroaki; Kumarasamy Anbarasu; Kalimuthusamy Natarajaseenivasan; Noriko Fujii (1184-1193).
Lysine-rich peptide, designated as KABT-AMP, was designed and synthesized to supersede the irrational use of chemical antibiotics as standard therapy. KABT-AMP is a 22-amino acid helical cationic peptide (+10) and amphipathic in nature. The antimicrobial kinetics of the peptide was ascertained in the representative strains of gram-positive, gram-negative, and fungal strains, viz., Staphylococcus aureus MTCC 2940, Escherichia coli MTCC 2939, and Candida albicans MTCC 227, respectively. KABT-AMP was synthesized by solid-phase synthesis and purified using reverse-phase high-performance liquid chromatography which resulted in >95 % purity, and matrix-assisted laser desorption/ionization time of flight revealed the mass of the peptide to be 2.8 kDa. KABT-AMP showed significant broad-spectrum antimicrobial activity against the bacterial and fungal strains analyzed in the present study with survivability of 30.8, 30.6, and 31.7 % in E. coli, S. aureus, and C. albicans, respectively, at 6 h. KABT-AMP also demonstrated antibiofilm activity against the tested biofilm forming clinical isolate, Candida tropicalis. The putative membranolytic activity of the peptide was substantiated by electron microscopic analysis. Results reveal that KABT-AMP will exhibit noteworthy antimicrobial activity against multidrug-resistant bacteria and fungus at micromolar concentrations with minimal cytotoxicity and thus could be conceived for biomedical application.
Keywords: Antimicrobial peptides; MDR; Lysine-rich peptide; SEM; Biofilm
Studies on the Microbial Synthesis and Characterization of Polyhydroxyalkanoates Containing 4-Hydroxyvalerate Using γ-Valerolactone by A. R. Muzaiyanah; A. A. Amirul (1194-1215).
In this study, the ability of Cupriavidus sp. USMAA2-4 to synthesize polyhydroxyalkanoates (PHA) containing 4-hydroxyvalerate monomer (4HV) was studied through one-stage cultivation using γ-valerolactone as the carbon precursor. The presence of 4HV monomer unit in the polymer was detected through gas chromatography analysis, proving the capability of this wild strain bacterium to produce poly(3-hydrxybutyrate-co-3-hydroxyvalerate-co-4-hydroxyvalerate) [P(3HB-co-3HV-co-4HV)] terpolymer. Existence of a 4HV monomer unit in the PHA produced was further confirmed through 13C and 1H NMR analysis. P(3HB-co-88 % 3HV-co-1 % 4HV) terpolymer with the highest PHA content of 63 wt% was obtained through combination of 0.14 wt% C of γ-valerolactone with 0.42 wt% C of oleic acid. Various compositions of P(3HB-co-3HV-co-4HV) terpolymer with 3HV and 4HV compositions ranging from 11 to 94 mol% and from 1 to 4 mol%, respectively, were acquired by manipulating γ-valerolactone and oleic acid concentrations. The molecular weight and the thermal and mechanical properties of four different compositions of terpolymers—P(3HB-co-91 % 3HV-co-1 % 4HV), P(3HB-co-55 % 3HV-co-2 % 4HV), P(3HB-co-27 % 3HV-co-2 % 4HV), and P(3HB-co-9 % 3HV-co-1 % 4HV)—were characterized. Among these terpolymers, P(3HB-co-27 % 3HV-co-2 % 4HV) terpolymer with a molecular weight of 5.7 (105 Da) exhibited the highest elongation to break (264 %). The monomer unit compositional distributions of these terpolymers were investigated through acetone–water fractionation analysis. The results suggested that these produced terpolymers had broad 3HV compositional distribution and narrow 4HV compositional distribution.
Keywords: Poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxyvalerate); 4HV monomer unit; Cupriavidus sp. USMAA2-4; γ-Valerolactone; Characterization
Olive Flounder (Paralichthys olivaceus) Cystatin C: Cloning, mRNA Expression, and Enzymatic Characterization of Olive Flounder Cystatin C by Sang Jung Ahn; Hye Jin Bak; Ju Hyeon Park; Jin Young Lee; Na Young Kim; Jin Woo Han; Hyae In Jo; Joon Ki Chung; Hyung Ho Lee (1216-1228).
Cystatins are endogenous inhibitors of mammalian lysosomal cysteine proteinases, such as cathepsins B, L, H, and S. Cystatin C belongs to the type 2 cystatin family. In this study, the 751-bp cystatin C cDNA (PoCystatin C) of olive flounder (Paralichthys olivaceus) was cloned by screening from the olive flounder cDNA library. The mRNA expression of the PoCystatin C gene was examined in various tissues from normal and lipopolysaccharide (LPS)-stimulated olive flounder by RT-PCR and was compared with inflammatory cytokines IL-1β, IL-6, and IL-8. PoCystatin C transcripts ubiquitously existed in all normal and LPS-stimulated tissues that were tested. The recombinant PoCystatin C protein was expressed in Escherichia coli BL21(DE3) in pCold™ TF DNA expression vector as a 70-kDa fusion protein. The protease inhibitory activities of recombinant PoCystatin C toward papain cysteine protease, piscine cathepsins (L, S, K, F, and X), and bovine cathepsin B were measured with the synthetic fluorogenic peptide substrates. PoCystatin C tightly inhibited papain cysteine protease, whereas cathepsins L, S, K, F, X, and B were inhibited with lower affinities. Our results indicate that the P. olivaceus cystatin C is a homolog of mammalian cystatin C due to its sequence, structure, tissue expression, and biochemical activity.
Keywords: Cystatin C; Cathepsin; RT-PCR; Olive flounder; Paralichthys olivaceus ; Reverse zymography
Engineering Plant Alternative Oxidase Function in Mammalian Cells: Substitution of the Motif-like Sequence ENV for QDT Diminishes Catalytic Activity of Arum concinnatum AOX1a Expressed in HeLa Cells by Yusuke Kakizaki; Kikukatsu Ito (1229-1240).
Alternative oxidase (AOX) is a nonproton motive quinol–oxygen oxidoreductase which is a component of the mitochondrial respiratory chain in higher plants. In this study, we have characterized the catalytic activity and regulatory behaviors of Arum concinnatum AOX isoforms, namely AcoAOX1a and AcoAOX1b, and their artificial mutants in HeLa cells. We demonstrated that substitution of the motif-like sequence ENV on the C-terminal half of AcoAOX1a for QDT diminishes its activity and proposed that the innate inactivity of AcoAOX1b in HeLa cells is, at least in part, attributable to its QDT motif. Furthermore, we show that introduction of F130L in the hydrophilic N-terminal extension of AcoAOX1a resulted in greater activity in the presence of pyruvate. This result indicates that functional significance of the N-terminal extension is not particular to the conventional regulatory cysteine. On the basis of these findings, we discuss new insights into the structural integrity of AOX in HeLa cells and the applicability of mammalian cells for functional analysis of this enzyme.
Keywords: Alternative oxidase; HeLa cells; Arum ; Mitochondria; Respiratory chain; Structure–function relationship
Electrophoretic Deposition of Multi-walled Carbon Nanotube on a Stainless Steel Electrode for use in Sediment Microbial Fuel Cells by Tian-shun Song; Peng-Xiao; Xia-yuan Wu; Charles C. Zhou (1241-1250).
Sediment microbial fuel cells (SMFCs) could be used as power sources and one type of new technology for the removal of organic matters in sediments. In order to improve electrode materials and enhance their effect on the performance, we deposited multi-walled carbon nanotube (MWNT) on stainless steel net (SSN). Electrophoretic deposition technique as a method with low cost, process simplicity, and thickness control was used for this electrode modification and produced this novel SSN-MWNT electrode. The performances of SMFCs with SSN-MWNT as electrode were investigated. The results showed that the maximum power density of SMFC with SSN-MWNT cathode was 31.6 mW m−2, which was 3.2 times that of SMFC with an uncoated stainless steel cathode. However, no significant increase in the maximum power density of SMFC with SSN-MWNT anode was detected. Further electrochemical analysis showed that when SSN-MWNT was used as the cathode, the cathodic electrochemical activity and oxygen reduction rate were significantly improved. This study demonstrates that the electrophoretic deposition of carbon nanotubes on conductive substrate can be applied for improving the performance of SMFC.
Keywords: Carbon nanotube; Sediment microbial fuel cell; Stainless steel; Electrophoretic deposition
Erratum to: A Natural Isolate Producing Shikimic Acid: Isolation, Identification, and Culture Condition Optimization by Garima Rawat; Priyanka Tripathi; Firdaus Jahan; R. K. Saxena (1251-1251).