JBB : Journal of Bioscience and Bioengineering

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Journal of Bioscience and Bioengineering vol.120 cover

Journal of Bioscience and Bioengineering – Recent Articles

  • Robust expression of heterologous genes by selection marker fusion system in improved Chlamydomonas strains
    Publication date: September 2015
    Source:Journal of Bioscience and Bioengineering, Volume 120, Issue 3

    Author(s): Fantao Kong, Tomohito Yamasaki, Sari Dewi Kurniasih, Liyuan Hou, Xiaobo Li, Nina Ivanova, Shigeru Okada, Takeshi Ohama

    Chlamydomonas is a very attractive candidate plant cell factory. However, its main drawback is the difficulty to find the transformants that robustly express heterologous genes randomly inserted in the nuclear genome. We previously showed that domestic squalene synthase (SQS) gene of Chlamydomonas was much more efficiently overexpressed in a mutant strain [UV-mediated mutant (UVM) 4] than in wild type. In this study, we evaluated the possibility of a new mutant strain, met1, which contains a tag in the maintenance type methyltransferase gene that is expected to play a key role in the maintenance of transcriptional gene silencing. The versatile usefulness of the UVM4 strain to express heterologous genes was also analyzed. We failed to overexpress CrSSL3 cDNA, which is the codon-adjusted squalene synthase-like gene originated from Botryococcus braunii, using the common expression cassette in the wild-type CC-1690 and UVM4 strains. However, we succeeded in isolating western blot-positive transformants through the combinational use of the UVM4 strain and ble2A expression system of which expression cassette bears a fused ORF of the target gene and the antibiotic resistance gene ble via the foot-and-mouth disease virus (FMDV) self-cleaving 2A sequence. It is noteworthy that even with this system, huge deviations in the accumulated protein levels were still observed among the UVM4 transformants.





  • Regulation of 3-hydroxyhexanoate composition in PHBH synthesized by recombinant Cupriavidus necator H16 from plant oil by using butyrate as a co-substrate
    Publication date: September 2015
    Source:Journal of Bioscience and Bioengineering, Volume 120, Issue 3

    Author(s): Shunsuke Sato, Hiroyuki Maruyama, Tetsuya Fujiki, Keiji Matsumoto

    A (R)-3-hydroxyhexanoate (3HH) composition-regulating technology for poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) production was developed using recombinant Cupriavidus necator H16 with butyrate as a co-substrate. A new (R)-3-hydroxyhexanoyl-CoA ((R)-3HH-CoA) synthesis pathway was designed and enhanced by replacing the PHA synthase gene (phaC1) of C. necator by the phaC Ac NSDG (encoding the N149S and D171G mutant of PHA synthase from Aeromonas caviae) and deactivation of the phaA gene (encoding (β-ketothiolase) from C. necator H16 chromosome). The effect of butyrate as co-substrate was assessed in high-cell-density fed-batch cultures of several C. necator mutants, and the 3HH fraction was successfully increased by adding butyrate to the culture. Moreover, overexpression of BktB (encoding the second β-ketothiolase with broad substrate specificity) enhanced the (R)-3HH-CoA synthesis pathway in the phaA deactivated mutant of C. necator by promoting the condensation of acetyl-CoA and butyryl-CoA into 3-ketohexanoyl-CoA. Consequently, PHBH containing 4.2–13.0 mol % 3HH was produced from butyrate and palm kernel oil by the genetically modified C. necator H16 strains.





  • Interaction of carbohydrates with alcohol dehydrogenase: Effect on enzyme activity
    Publication date: September 2015
    Source:Journal of Bioscience and Bioengineering, Volume 120, Issue 3

    Author(s): Swati B. Jadhav, Sandip B. Bankar, Tom Granström, Heikki Ojamo, Rekha S. Singhal, Shrikant A. Survase

    Alcohol dehydrogenase was covalently conjugated with three different oxidized carbohydrates i.e., glucose, starch and pectin. All the carbohydrates inhibited the enzyme. The inhibition was studied with respect to the inhibition rate constant, involvement of thiol groups in the binding, and structural changes in the enzyme. The enzyme activity decreased to half of its original activity at the concentration of 2 mg/mL of pectin, 4 mg/mL of glucose and 10 mg/mL of starch within 10 min at pH 7. This study showed oxidized pectin to be a potent inhibitor of alcohol dehydrogenase followed by glucose and starch. Along with the aldehyde-amino group interaction, thiol groups were also involved in the binding between alcohol dehydrogenase and carbohydrates. The structural changes occurring on binding of alcohol dehydrogenase with oxidized carbohydrates was also confirmed by fluorescence spectrophotometry. Oxidized carbohydrates could thus be used as potential inhibitors of alcohol dehydrogenase.





  • Enhancement of immobilized lipase activity by design of polymer brushes on a hollow fiber membrane
    Publication date: September 2015
    Source:Journal of Bioscience and Bioengineering, Volume 120, Issue 3

    Author(s): Tadashi Okobira, Ai Matsuo, Hikaru Matsumoto, Takanori Tanaka, Kazuya Kai, Chie Minari, Muneharu Goto, Hidetaka Kawakita, Kazuya Uezu

    A polymer brush possessing aminoethanol (AE) functional groups for lipase immobilization was grafted onto a hollow fiber membrane by radiation-induced graft polymerization. Almost the AE groups-grafted polymer brushes unfold through positive charge repulsion between the AE groups, enabling multi-layer immobilization of lipase. The hydroxyl groups in AE can also retain water molecules around hydrophilic part of the lipase. In this study, we controlled the length and density of the polymer brushes consisting of the glycidyl methacrylate (GMA) by changing the concentration of GMA monomer during radiation-induced graft polymerization. Immobilized lipase showed the highest activity on the grafted membrane when 5 wt% of glycidyl methacrylate as monomer for the radiation-induced graft polymerization was used. Consequently high efficiency esterification (approximately 1600 mmol/h/g-membrane) was achieved in five-layer lipase on AE polymer brush than that in monolayer lipase on the polymer brush possessing only hydroxyl groups. Moreover, the polymer brush possessing AE functional groups for lipase immobilization maintained high activity on the reuse for several times.





  • Novel acidophilic β-galactosidase with high activity at extremely acidic pH region from Teratosphaeria acidotherma AIU BGA-1
    Publication date: September 2015
    Source:Journal of Bioscience and Bioengineering, Volume 120, Issue 3

    Author(s): Serina Chiba, Miwa Yamada, Kimiyasu Isobe

    A β-galactosidase exhibiting maximal activity at pH 1.0 was purified from Teratosphaeria acidotherma AIU BGA-1. The enzyme had a molecular mass of 180 kDa and consisted of two heterosubunits of 120 kDa and 66 kDa. The N-terminal amino acid sequence of the large subunit was found to be SPNLQDIVTVDGESY. These physicochemical properties differed from those of other microbial β-galactosidases. At pH values of 1.5 and pH 4.5, the enzyme exhibited its highest activity at temperatures of 70°C and 80°C, respectively. Thus, the enzyme exhibited the lowest optimal pH and highest optimal temperature among the microbial β-galactosidases thus reported. The enzyme retained more than 80% of its original activity in the pH range from 2.0 to 8.0 by incubation at 50°C for 30 min. The enzyme hydrolyzed 4-nitrophenyl-β-d-fucopyranoside, 2-nitrophenyl-β-d-galactopyranoside, and 4-nitrophenyl-β-d-galacto-pyranoside at relative reaction rates of 100, 59, and 24, respectively, at pH 1.5, and its affinity for β-d-galactopyranosides was higher than that for β-d-fucopyranosides. The enzyme also efficiently hydrolyzed lactose in milk and whey from yoghurt at pH 1.5.





  • Sequence modifications and heterologous expression of eukaryotic cytochromes P450 in Escherichia coli
    Publication date: September 2015
    Source:Journal of Bioscience and Bioengineering, Volume 120, Issue 3

    Author(s): Hirofumi Ichinose, Mayumi Hatakeyama, Yukiyo Yamauchi

    The development of a heterologous expression system is often a principal step in biochemical and biotechnological studies on cytochromes P450 (P450s). However, heterologous expression of eukaryotic membrane-bound P450s in Escherichia coli is still a trial-and-error process because theoretical and systematical experimental procedures have not yet been established. In this study, we generated a series of chimeric variants of fungal P450s by replacing their N-terminal domains with the N-terminal domains of other P450s and explored their potentials for heterologous expression in E. coli. Large-scale screening demonstrated that a number of fungal P450s could be expressed when their N-terminal amino acid sequences were replaced with the corresponding domain of CYP5144C1, even when the expression of the non-chimeric sequence was unpromising. Furthermore, a comprehensive screening resulted in the identification of 64 different types of chimeric partners whose N-terminal domains could potentially be used to increase the expression levels of various P450s. These findings will help to elaborate experimental strategies for high-level heterologous expression of a variety of eukaryotic membrane-bound P450s in E. coli.





  • Inactivation of Shiga toxin-producing Escherichia coli O104:H4 using cold atmospheric pressure plasma
    Publication date: September 2015
    Source:Journal of Bioscience and Bioengineering, Volume 120, Issue 3

    Author(s): Matthias Baier, Traute Janßen, Lothar H. Wieler, Jörg Ehlbeck, Dietrich Knorr, Oliver Schlüter

    From cultivation to the end of the post-harvest chain, heat-sensitive fresh produce is exposed to a variety of sources of pathogenic microorganisms. If contaminated, effective gentle means of sanitation are necessary to reduce bacterial pathogen load below their infective dose. The occurrence of rare or new serotypes raises the question of their tenacity to inactivation processes. In this study the antibacterial efficiency of cold plasma by an atmospheric pressure plasma-jet was examined against the Shiga toxin-producing outbreak strain Escherichia coli O104:H4. Argon was transformed into non-thermal plasma at a power input of 8 W and a gas flow of 5 L min−1. Basic tests were performed on polysaccharide gel discs, including the more common E. coli O157:H7 and non-pathogenic E. coli DSM 1116. At 5 mm treatment distance and 105 cfu cm−2 initial bacterial count, plasma reduced E. coli O104:H4 after 60 s by 4.6 ± 0.6 log, E. coli O157:H7 after 45 s by 4.5 ± 0.6 log, and E. coli DSM 1116 after 30 s by 4.4 ± 1.1 log. On the surface of corn salad leaves, gentle plasma application at 17 mm reduced 104 cfu cm−2 of E. coli O104:H4 by 3.3 ± 1.1 log after 2 min, whereas E. coli O157:H7 was inactivated by 3.2 ± 1.1 log after 60 s. In conclusion, plasma treatment has the potential to reduce pathogens such as E. coli O104:H4 on the surface of fresh produce. However, a serotype-specific adaptation of the process parameters is required.





  • Absolute quantitation of glycolytic intermediates reveals thermodynamic shifts in Saccharomyces cerevisiae strains lacking PFK1 or ZWF1 genes
    Publication date: September 2015
    Source:Journal of Bioscience and Bioengineering, Volume 120, Issue 3

    Author(s): Shunsuke Nishino, Nobuyuki Okahashi, Fumio Matsuda, Hiroshi Shimizu

    Internal standard based absolute quantitation of glycolytic intermediates was performed to characterize the thermodynamic states of Saccharomyces cerevisiae metabolism. A mixture of 13C-labeled glycolytic intermediates was prepared via extraction from S. cerevisiae cells cultivated using a synthetic medium containing [U-13C] glucose as the sole carbon source. The 13C-labeled metabolite mixture was used as an internal standard for the analysis of S. cerevisiae cultivated in a medium containing natural glucose. The methodology was employed for the absolute quantitation of glycolytic intermediates of BY4742, pfk1Δ, and zwf1Δ strains of S. cerevisiae. Fructose-1,6-bisphosphate was the most abundant intermediate in the BY4742 strains in the log phase of growth. Estimation of the Gibbs free energy change (ΔG) from the absolute concentration revealed that several reactions, such as those catalyzed by ribose-5-phosphate keto-isomerase and phosphoglucose isomerase, were commonly at near-equilibrium in all three strains. A significant shift in thermodynamic state was also observed for the transketolase–transaldolase reaction, for which ΔG was −6.6 ± 0.5 kJ mol−1 in the BY4742 strain and 5.4 ± 0.3 kJ mol−1 in the zwf1Δ strain.





  • Response of Fe–S cluster assembly machinery of Escherichia coli to mechanical stress in a model of amino-acid crystal fermentation
    Publication date: September 2015
    Source:Journal of Bioscience and Bioengineering, Volume 120, Issue 3

    Author(s): Satoshi Okutani, Takayoshi Iwai, Shintaro Iwatani, Kiyoshi Matsuno, Yasuhiro Takahashi, Toshiharu Hase

    During amino-acid crystal fermentation, mechanical stress on bacterial cells caused by crystal collision often impacts negatively on bacterial growth and amino-acid production. When Escherichia coli cells were cultivated under mechanical stress of polyvinyl chloride particles as a model of the crystal fermentation, activities of iron–sulfur (Fe–S) cluster-containing enzymes were apparently decreased. Based on an assumption that function of Fe–S cluster assembly machinery would be elevated to recover the enzyme activities in such stressed cells, we analyzed levels of various components of Fe–S cluster assembly machinery by western blotting. It was found that the expression of HscA, a chaperon component of the machinery, was up-regulated and that shorter forms of HscA with the N-terminal region truncated were accumulated, suggesting an important role of HscA against the mechanical stress. An overexpression of HscA gene in E. coli cells gave a positive effect on rescue of the stress-induced decrease of the activity of Fe–S cluster-containing enzyme. These results may provide a new strategy to alleviate the mechanical stress during the amino-acid crystal fermentation.





  • Ergothioneine protects Streptomyces coelicolor A3(2) from oxidative stresses
    Publication date: September 2015
    Source:Journal of Bioscience and Bioengineering, Volume 120, Issue 3

    Author(s): Shunsuke Nakajima, Yasuharu Satoh, Kentaro Yanashima, Tomomi Matsui, Tohru Dairi

    Thiol compounds with low-molecular weight, such as glutathione, mycothiol (MSH), bacillithiol, and ergothioneine (ERG), are known to protect microorganisms from oxidative stresses. Mycobacteria and actinobacteria utilize both MSH and ERG. The biological functions of MSH in mycobacteria have been extensively studied by genetic and biochemical studies, which have suggested it has critical roles for detoxification in cells. In contrast, the biological functions of ERG remain ambiguous because its biosynthetic genes were only recently identified in Mycobacterium avium. In this study, we constructed mutants of Streptomyces coelicolor A3(2), in which either the MSH or ERG biosynthetic gene was disrupted, and examined their phenotypes. A mshC (SCO1663)-disruptant completely lost MSH productivity. In contrast, a disruptant of the egtA gene (SCO0910) encoding γ-glutamyl-cysteine synthetase unexpectedly retained reduced productivity of ERG, probably because of the use of l-cysteine instead of γ-glutamyl-cysteine. Both disruptants showed delayed growth at the late logarithmic phase and were more susceptible to hydrogen peroxide and cumene hydroperoxide than the parental strain. Interestingly, the ERG-disruptant, which still kept reduced ERG productivity, was more susceptible. Furthermore, the ERG-disruptant accumulated 5-fold more MSH than the parental strain. In contrast, the amount of ERG was almost the same between the MSH-disruptant and the parental strain. Taken together, our results suggest that ERG is more important than MSH in S. coelicolor A3(2).





  • Omega-3 eicosatetraenoic acid production by molecular breeding of the mutant strain S14 derived from Mortierella alpina 1S-4
    Publication date: September 2015
    Source:Journal of Bioscience and Bioengineering, Volume 120, Issue 3

    Author(s): Tomoyo Okuda, Akinori Ando, Hiroaki Negoro, Hiroshi Kikukawa, Takaiku Sakamoto, Eiji Sakuradani, Sakayu Shimizu, Jun Ogawa

    We investigated the omega-3 eicosatetraenoic acid (ETA) production by molecular breeding of the oleaginous fungus Mortierella alpina, which can slightly accumulate ETA only when cultivated at a low temperature. The endogenous ω3-desaturase gene or the heterologous Saprolegnia diclina Δ17 (sdd17m) desaturase gene were overexpressed in M. alpina S14, a Δ5-desaturation activity-defective mutant derived from M. alpina 1S-4. M. alpina S14 transformants introduced with the endogenous ω3-desaturase gene showed ETA at 42.1% content in the total lipids that was 84.2-fold and 3.2-fold higher than that of the wild-type strain 1S-4 and host strain S14, respectively, when cultivated at 12°C. No accumulation of ETA was observed at 28°C. In contrast, transformants with the heterologous sdd17m gene showed 24.9% of the content of total lipids at 28°C. These results indicated that these M. alpina S14 transformants are promising strains for the production of ETA, which is hard to obtain from natural sources.





  • Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) by recombinant Escherichia coli from glucose
    Publication date: September 2015
    Source:Journal of Bioscience and Bioengineering, Volume 120, Issue 3

    Author(s): Ayaka Hokamura, Izumi Wakida, Yuki Miyahara, Takeharu Tsuge, Hideki Shiratsuchi, Kenji Tanaka, Hiromi Matsusaki

    The polyhydroxyalkanoate (PHA) copolymers consisting of short-chain-length (scl) and medium-chain-length (mcl) monomers have various properties ranging from stiff to flexible depending on the molar fraction of the monomer compositions. It has been reported that PhaG, which is first known as a (R)-3-hydroxyacyl-acyl carrier protein (ACP)-CoA transferase, actually functions as a 3-hydroxyacyl-ACP thioesterase, and the product of PP0763 gene from Pseudomonas putida KT2440 has a (R)-3-hydroxyacyl (3HA)-CoA ligase activity (Wang et al., Appl. Environ. Microbiol., 78, 519–527, 2012). In this study, we found a new (R)-3HA-CoA ligase (the product of PA3924 gene) from Pseudomonas aeruginosa PAO. The PA3924 gene was coexpressed with PHA synthase 1 gene (phaC1 Ps) and phaG Ps gene from Pseudomonas sp. 61-3, and β-ketothiolase gene (phbA Re) and acetoacetyl-CoA reductase gene (phbB Re) from Ralstonia eutropha in Escherichia coli LS5218 at 25°C. As a result, the copolymer containing 94.6 mol% 3-hydroxybutyrate (3HB) and 5.4 mol% mcl-3-hydroxyalkanoates (3HA) consisting of C8, C10, C12 and C14 was synthesized by recombinant E. coli LS5218 from glucose as the sole carbon source. The concentration of P(3HB-co-3HA) (scl-co-mcl-PHA) synthesized by the recombinant E. coli LS5218 harboring phaC1 Ps, phaG Ps, phbAB Re and the PA3924 genes was approximately 7-fold higher than that of the recombinant LS5218 harboring phaC1 Ps, phaG Ps, phbAB Re and the PP0763 genes. The number-average molecular weight of the P(3HB-co-5.4% 3HA) copolymer was 233 × 103, which was relatively high molecular weight. In addition, the physical and the mechanical properties of the copolymer were demonstrated to improve the brittleness of P(3HB) homopolymer.





  • Determination and quantification of kokumi peptide, γ-glutamyl-valyl-glycine, in brewed alcoholic beverages
    Publication date: September 2015
    Source:Journal of Bioscience and Bioengineering, Volume 120, Issue 3

    Author(s): Naohiro Miyamura, Yuko Iida, Motonaka Kuroda, Yumiko Kato, Junko Yamazaki, Toshimi Mizukoshi, Hiroshi Miyano

    Recently, it has been demonstrated that kokumi substances such as glutathione are perceived through the calcium-sensing receptor (CaSR), and screening by CaSR assay and sensory evaluation has shown that γ-glutamyl-valyl-glycine (γ-Glu-Val-Gly) is a potent kokumi peptide. In this study, γ-Glu-Val-Gly contents in various brewed alcoholic beverages were investigated. Contents of γ-Glu-Val-Gly in four brands of wine, four brands of rice wine (sake) and eight brands of beer were analyzed by high performance liquid chromatography-tandem mass spectrometry followed by derivatization with 6-aminoquinoyl-N-hydroxysuccinimidyl-carbamate. The analyses indicated that γ-Glu-Val-Gly was present in all of eight beer samples at concentrations in the range of 0.08–0.18 mg/L, although the peptide was not detected in any wine or rice wine samples. These results suggest that amongst the brewed beverages tested, beer contains γ-Glu-Val-Gly, and that γ-Glu-Val-Gly is widely distributed in beer.





  • Biogas production from chicken manure at different organic loading rates in a mesophilic-thermopilic two stage anaerobic system
    Publication date: September 2015
    Source:Journal of Bioscience and Bioengineering, Volume 120, Issue 3

    Author(s): Kenan Dalkılıc, Aysenur Ugurlu

    This study investigates the biogas production from chicken manure at different organic loading rates (OLRs), in a mesophilic-thermophilic two stage anaerobic system. The system was operated on semi continuous mode under different OLRs [1.9 g volatile solids (VS)/L·d – 4.7 g VS/L·d] and total solid (TS) contents (3.0–8.25%). It was observed that the anaerobic bacteria acclimatized to high total ammonia nitrogen concentration (>3000 mg/L) originated as a result of the degradation of chicken manure. High volatile fatty acid concentrations were tolerated by the system due to high pH in the reactors. The maximum average biogas production rate was found as 554 mL/g VSfeed while feeding 2.2 g VS/L-d (2.3% VS – 3.8% TS) to the system. Average methane content of produced biogas was 74% during the study.





  • Optimization of cell line development in the GS-CHO expression system using a high-throughput, single cell-based clone selection system
    Publication date: September 2015
    Source:Journal of Bioscience and Bioengineering, Volume 120, Issue 3

    Author(s): Tsuyoshi Nakamura, Takeshi Omasa

    Therapeutic antibodies are commonly produced by high-expressing, clonal and recombinant Chinese hamster ovary (CHO) cell lines. Currently, CHO cells dominate as a commercial production host because of their ease of use, established regulatory track record, and safety profile. CHO-K1SV is a suspension, protein-free-adapted CHO-K1-derived cell line employing the glutamine synthetase (GS) gene expression system (GS-CHO expression system). The selection of high-producing mammalian cell lines is a crucial step in process development for the production of therapeutic antibodies. In general, cloning by the limiting dilution method is used to isolate high-producing monoclonal CHO cells. However, the limiting dilution method is time consuming and has a low probability of monoclonality. To minimize the duration and increase the probability of obtaining high-producing clones with high monoclonality, an automated single cell-based clone selector, the ClonePix FL system, is available. In this study, we applied the high-throughput ClonePix FL system for cell line development using CHO-K1SV cells and investigated efficient conditions for single cell-based clone selection. CHO-K1SV cell growth at the pre-picking stage was improved by optimizing the formulation of semi-solid medium. The efficiency of picking and cell growth at the post-picking stage was improved by optimization of the plating time without decreasing the diversity of clones. The conditions for selection, including the medium formulation, were the most important factors for the single cell-based clone selection system to construct a high-producing CHO cell line.





  • Immobilization of feruloyl esterases on magnetic nanoparticles and its potential in production of ferulic acid
    Publication date: September 2015
    Source:Journal of Bioscience and Bioengineering, Volume 120, Issue 3

    Author(s): Fuming He, Song Zhang, Xinli Liu

    Feruloyl esterase plays an indispensable role in hydrolyzing plant cell walls, and ferulic acid will be released as by-product, which has potential applications in food and medicine industry. This study presents immobilization of partially purified feruloyl esterases from the fermentation liquor of recombinant Pichia on magnetic Fe3O4 nanoparticles. Furthermore, the optimal conditions for the immobilization and some characteristics of immobilized enzyme were studied. The optimal immobilization conditions observed were enzyme 0.2 mg (1 mg/mL, 0.2 mL), magnetic Fe3O4 nanoparticles 4 mg, pH 6.0, immobilization time 3 h. The results showed that the optimal reaction temperature of immobilized enzyme was increased from 45°C to 55°C. Thermal stability of the immobilized enzyme also had an improvement, the residual activity retained 80% and 69% after 120 h at 40°C and 50°C, respectively, while free enzymes only showed 45% and 40% remnant activity at the same condition. The immobilized enzyme also exhibited good operational stability and 52.4% of its initial activity was observed during the fifth cycle. In terms of the thermal and operational stability, the immobilized enzyme could be better used in many more applications than the free enzymes. At last the destarched wheat bran was taken as substrate for immobilized and free feruloyl esterases to produce ferulic acid, the maximum ferulic acid yield was 11.2% and 12.3%, respectively, indicating a great potential in industrial application.





  • Effect of kojic acid-grafted-chitosan oligosaccharides as a novel antibacterial agent on cell membrane of gram-positive and gram-negative bacteria
    Publication date: September 2015
    Source:Journal of Bioscience and Bioengineering, Volume 120, Issue 3

    Author(s): Xiaoli Liu, Wenshui Xia, Qixing Jiang, Yanshun Xu, Peipei Yu

    Our work here, for the first time, reported the antibacterial activity of kojic acid-grafted-chitosan oligosaccharides (COS/KA) against three gram-positive and three gram-negative bacteria. Integrity of cell membrane, outer membrane (OM) and inner membrane (IM) permeabilization assay, alkaline phosphatase (ALP) and glucose-6-phosphate dehydrogenase (G6PDH) assay, and SDS-PAGE assay techniques were used to investigate the interactions between COS/KA and bacterial membranes. The antibacterial activity of COS/KA was higher than those of unmodified COS. The electric conductivity of bacteria suspensions increased, followed by increasing of the units of average release for ALP and G6PDH. COS/KA can also rapidly increase the 1-N-phenylanphthylamine (NPN) uptake and the release of β-galactosidase via increasing the permeability of OM and IM in Escherichia coli. SDS-PAGE indicated the content of cellular soluble proteins decreased significantly in COS/KA-treated bacteria. Hence, COS/KA has potential in food industry and biomedical sciences.





  • Efficient enrichment of high-producing recombinant Chinese hamster ovary cells for monoclonal antibody by flow cytometry
    Publication date: September 2015
    Source:Journal of Bioscience and Bioengineering, Volume 120, Issue 3

    Author(s): Takeshi Okumura, Kenji Masuda, Kazuhiko Watanabe, Kenji Miyadai, Koichi Nonaka, Masayuki Yabuta, Takeshi Omasa

    To screen a high-producing recombinant Chinese hamster ovary (CHO) cell from transfected cells is generally laborious and time-consuming. We developed an efficient enrichment strategy for high-producing cell screening using flow cytometry (FCM). A stable pool that had possibly shown a huge variety of monoclonal antibody (mAb) expression levels was prepared by transfection of an expression vector for mAb production to a CHO cell. To enrich high-producing cells derived from a stable pool stained with a fluorescent-labeled antibody that binds to mAb presented on the cell surface, we set the cell size and intracellular density gates based on forward scatter (FSC) and side scatter (SSC), and collected the brightest 5% of fluorescein isothiocyanate (FITC)-positive cells from each group by FCM. The final product concentration in a fed-batch culture of cells sorted without FSC and SSC gates was 1.2–1.3-times higher than that of unsorted cells, whereas that of cells gated by FSC and SSC was 3.4–4.7-fold higher than unsorted cells. Surprisingly, the fraction with the highest final product concentration indicated the smallest value of FSC and SSC, and the middle value of fluorescence intensity among all fractionated cells. Our results showed that our new screening strategy by FCM based on FSC and SSC gates could achieve an efficient enrichment of high-producing cells with the smallest value of FSC and SSC.





  • Vessel network formation in response to intermittent hypoxia is frequency dependent
    Publication date: September 2015
    Source:Journal of Bioscience and Bioengineering, Volume 120, Issue 3

    Author(s): Seema M. Ehsan, Steven C. George

    A combined experimental and mathematical model of intermittent hypoxia (IH) conditioned engineered tissue was used to characterize the effects of IH on the formation of in vitro vascular networks. Results showed that the frequency of hypoxic oscillations has pronounced influence on the vascular response of endothelial cells and fibroblasts.





  • Nuclear localization domains of GATA activator Gln3 are required for transcription of target genes through dephosphorylation in Saccharomyces cerevisiae
    Publication date: August 2015
    Source:Journal of Bioscience and Bioengineering, Volume 120, Issue 2

    Author(s): Minori Numamoto, Shota Tagami, Yusuke Ueda, Yusuke Imabeppu, Yu Sasano, Minetaka Sugiyama, Hiromi Maekawa, Satoshi Harashima

    The GATA transcription activator Gln3 in the budding yeast (Saccharomyces cerevisiae) activates transcription of nitrogen catabolite repression (NCR)-sensitive genes. In cells grown in the presence of preferred nitrogen sources, Gln3 is phosphorylated in a TOR-dependent manner and localizes in the cytoplasm. In cells grown in non-preferred nitrogen medium or treated with rapamycin, Gln3 is dephosphorylated and is transported from the cytoplasm to the nucleus, thereby activating the transcription of NCR-sensitive genes. Caffeine treatment also induces dephosphorylation of Gln3 and its translocation to the nucleus and transcription of NCR-sensitive genes. However, the details of the mechanism by which phosphorylation controls Gln3 localization and transcriptional activity are unknown. Here, we focused on two regions of Gln3 with nuclear localization signal properties (NLS-K, and NLS-C) and one with nuclear export signal (NES). We constructed various mutants for our analyses: gln3 containing point mutations in all potential phosphoacceptor sites (Thr-339, Ser-344, Ser-347, Ser-355, Ser-391) in the NLS and NES regions to produce non-phosphorylatable (alanine) or mimic-phosphorylatable (aspartic acid) residues; and deletion mutants. We found that phosphorylation of Gln3 was impaired in all of these mutations and that the aspartic acid substitution mutants showed drastic reduction of Gln3-mediated transcriptional activity despite the fact that the mutations had no effect on nuclear localization of Gln3. Our observations suggest that these regions are required for transcription of target genes presumably through dephosphorylation.