JBB : Journal of Bioscience and Bioengineering

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Journal of Bioscience and Bioengineering vol.119 cover

Journal of Bioscience and Bioengineering – Recent Articles

  • Maintenance of an undifferentiated state of human induced pluripotent stem cells through migration-dependent regulation of the balance between cell–cell and cell–substrate interactions
    Publication date: June 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 6

    Author(s): Mee-Hae Kim , Masahiro Kino-oka

    We present an outlook on the current strategies for maintaining and culturing human induced pluripotent stem cells (hiPSCs) in an undifferentiated state without affecting their pluripotency. First, cell structures and function are described in relation to interactions between hiPSCs and their surroundings. Second, the phenomenon of spontaneous deviation from undifferentiated hiPSCs in cultures with feeder cells is addressed, with a summary of current topics that are of particular interest to our studies. The key regulatory factors that can contribute to the growth and maintenance of undifferentiated hiPSCs are also discussed, with a summary of recent work toward a culture strategy to control hiPSC fate through balancing cell–cell and cell–substrate interactions. Finally, we discuss culture process design in our previous studies with respect to maintaining and expanding cultures of undifferentiated and pluripotent hiPSCs. We focus on the regulation of migration-dependent balance between cell–cell and cell–substrate interactions. This review offers novel insights into the regulation and processing of stem cells for research in regenerative medicine.





  • Analyses of chicken sialyltransferases related to N-glycosylation
    Publication date: June 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 6

    Author(s): Yusuke Kojima , Akifumi Mizutani , Yuya Okuzaki , Ken-ichi Nishijima , Hidenori Kaneoka , Takako Sasamoto , Katsuhide Miyake , Shinji Iijima

    Proteins exogenously expressed and deposited in the egg whites of transgenic chickens did not contain terminal sialic acid in their N-glycan. Since this sugar is important for the biological stability of therapeutic proteins, we examined chicken sialyltransferases (STs). Based on homologies in DNA sequences, we cloned and expressed several chicken STs, which appeared to be involved in N-glycosylation in mammals, in 293FT cells. Enzymatic activity was detected with ST3Gal3, ST3Gal6 and ST6Gal1 using galactose-β1,4-N-acetylglucosamine (Galβ1,4GlcNAc) as an acceptor. Using Golgi fractions from the cell-free extracts of chicken organs, α2,3- and/or α2,6-ST activities were detected in the liver and kidney, but were absent in the oviduct cells in which egg-white proteins were produced. This result suggested that the lack of ST activities in oviduct cells mainly caused the lack of sialic acid in the N-glycan of proteins exogenously expressed and deposited in egg white.





  • Characterization of two amine oxidases from Aspergillus carbonarius AIU 205
    Publication date: June 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 6

    Author(s): Asami Sugawara , Daisuke Matsui , Miwa Yamada , Yasuhisa Asano , Kimiyasu Isobe

    We have reported that Aspergillus carbonarius AIU 205, which was isolated by our group, produced three enzymes exhibiting oxidase activity for 4-aminobutanamide (4-ABAD) (J. Biosci. Bioeng., 117, 263–268, 2014). Among three enzymes, characteristics of enzyme I have been revealed, but those of the other two enzymes have not. In this study, we purified enzymes II and III, and compared their characteristics with those of enzyme I. Enzymes II and III also oxidized aliphatic monoamines, aromatic amines, and aliphatic aminoalcohols. In addition, the oxidase activity of both enzymes was strongly inhibited by carbonyl reagents and specific inhibitors for copper-containing amine oxidases. Thus, enzymes II and III were also classified into the copper-containing amine oxidase group (EC 1.4.3.6) along with enzyme I. However, these three enzymes differed from each other in their enzymatic, kinetic, and physicochemical properties. The N-terminal amino acid sequences also differed from each other; that of enzyme I was modified, that of enzyme II was similar to those of peroxisomal copper-containing amine oxidases, and that of enzyme III was similar to those of copper-containing amine oxidases from the genus Aspergillus. Therefore, we concluded that A. carbonarius AIU 205 produced three different types of amine oxidase in the mycelia.





  • Characterization of the linoleic acid Δ9 hydratase catalyzing the first step of polyunsaturated fatty acid saturation metabolism in Lactobacillus plantarum AKU 1009a
    Publication date: June 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 6

    Author(s): Michiki Takeuchi , Shigenobu Kishino , Akiko Hirata , Si-Bum Park , Nahoko Kitamura , Jun Ogawa

    Linoleic acid Δ9 hydratase, which is involved in linoleic acid saturation metabolism of Lactobacillus plantarum AKU 1009a, was cloned, expressed as a his-tagged recombinant enzyme, purified with an affinity column, and characterized. The enzyme required FAD as a cofactor and its activity was enhanced by NADH. The maximal activities for the hydration of linoleic acid and for the dehydration of 10-hydroxy-cis-12-octadecenoic acid (HYA) were observed at 37 °C in buffer at pH 5.5 containing 0.5 M NaCl. Free C16 and C18 fatty acids with cis-9 double bonds and 10-hydroxy fatty acids served as substrates for the hydration and dehydration reactions, respectively. The apparent K m value for linoleic acid was estimated to be 92 μM, with a k cat of 2.6∙10−2 s−1 and a Hill factor of 3.3. The apparent K m value for HYA was estimated to be 98 μM, with a k cat of 1.2∙10−3 s−1.





  • Crystal structure of streptavidin mutant with low immunogenicity
    Publication date: June 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 6

    Author(s): Tatsuya Kawato , Eiichi Mizohata , Tomohiro Meshizuka , Hirofumi Doi , Takeshi Kawamura , Hiroyoshi Matsumura , Kyohei Yumura , Kouhei Tsumoto , Tatsuhiko Kodama , Tsuyoshi Inoue , Akira Sugiyama

    We previously created a low-immunogenic core streptavidin mutant No. 314 (LISA-314) by replacing six amino-acid residues for use as a delivery tool for an antibody multistep pre-targeting process (Yumura et al., Protein Sci., 22, 213–221, 2013). Here, we performed high-resolution X-ray structural analyses of LISA-314 and wild-type streptavidin to investigate the effect of substitutions on the protein function and the three-dimensional structure. LISA-314 forms a tetramer in the same manner as wild-type streptavidin. The binding mode of d-biotin in LISA-314 is also completely identical to that in wild-type streptavidin, and conformational changes were observed mostly at the side chains of substituted sites. Any large conformational changes corresponding to the reduction of B factors around the substituted sites were not observed. These results demonstrated the LISA-314 acquired low immunogenicity without losing structural properties of original wild-type streptavidin.





  • Purification and characterization of a novel NADPH-dependent 2-aminoacetophenone reductase from Arthrobacter sulfureus
    Publication date: June 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 6

    Author(s): Guogang Zhao , Wanru Sun , Jianjun Wang

    A novel 2-aminoacetophenone reductase was purified to homogeneity from Arthrobacter sulfureus BW1010. The enzyme is a monomer with a molecular weight of approximately 60 kDa. Using NADPH as coenzyme, it catalyzes the reduction of ketones, especially amine phenyl ketones, and stereospecifically reduces 2-aminoacetophenone to (S)-2-amino-1-phenylethanol (e.e > 99.8%) with the optimal pH at 7.5.





  • Production and application of a rare disaccharide using sucrose phosphorylase from Leuconostoc mesenteroides
    Publication date: June 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 6

    Author(s): Kenji Morimoto , Akihide Yoshihara , Toshio Furumoto , Goro Takata

    Sucrose phosphorylase (SPase) from Leuconostoc mesenteroides exhibited activity towards eight ketohexoses, which behaved as d-glucosyl acceptors, and α-d-glucose-1-phosphate (G1P), which behaved as a donor. All eight of these ketohexoses were subsequently transformed into the corresponding d-glucosyl-ketohexoses. Of the eight ketohexoses evaluated in the current study, d-allulose behaved as the best substrate for SPase, and the resulting d-glucosyl-d-alluloside product was found to be a non-reducing sugar with a specific optical rotation of [ α ] D 20  + 74.36°. d-Glucosyl-d-alluloside was identified as α-d-glucopyranosyl-(1→2)-β-d-allulofuranoside by NMR analysis. d-Glucosyl-d-alluloside exhibited an inhibitory activity towards an invertase from yeast with a K m value of 50 mM, where it behaved as a competitive inhibitor with a K i value of 9.2 mM. d-Glucosyl-d-alluloside was also successfully produced from sucrose using SPase and d-tagatose 3-epimerase. This process also allowed for the production of G1P from sucrose and d-allulose from d-fructose, which suggested that this method could be used to prepare d-glucosyl-d-alluloside without the need for expensive reagents such as G1P and d-allulose.





  • Faster in vivo clearance of human embryonic kidney than Chinese hamster ovary cell derived protein: Role of glycan mediated clearance
    Publication date: June 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 6

    Author(s): Mengmeng Wang , Tetsuya Ishino , Alison Joyce , Amy Tam , Weili Duan , Laura Lin , William S. Somers , Ronald Kriz , Denise M. O'Hara

    This investigation used in vivo and in vitro tools to study pharmacokinetics and glycosylation of two monomeric antibodies produced either transiently by HEK293 cells or stably by Chinese hamster ovary cells, and demonstrated that higher in vivo clearance of human embryonic kidney antibody was due to higher glycosylation, thus higher mannose receptor mediated uptake.





  • Change in the plasmid copy number in acetic acid Bacteria in response to growth phase and acetic acid Concentration
    Publication date: June 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 6

    Author(s): Naoki Akasaka , Wiwik Astuti , Yuri Ishii , Ryota Hidese , Hisao Sakoda , Shinsuke Fujiwara

    Plasmids pGE1 (2.5 kb), pGE2 (7.2 kb), and pGE3 (5.5 kb) were isolated from Gluconacetobacter europaeus KGMA0119, and sequence analyses revealed they harbored 3, 8, and 4 genes, respectively. Plasmid copy numbers (PCNs) were determined by real-time quantitative PCR at different stages of bacterial growth. When KGMA0119 was cultured in medium containing 0.4% ethanol and 0.5% acetic acid, PCN of pGE1 increased from 7 copies/genome in the logarithmic phase to a maximum of 12 copies/genome at the beginning of the stationary phase, before decreasing to 4 copies/genome in the late stationary phase. PCNs for pGE2 and pGE3 were maintained at 1–3 copies/genome during all phases of growth. Under a higher concentration of ethanol (3.2%) the PCN for pGE1 was slightly lower in all the growth stages, and those of pGE2 and pGE3 were unchanged. In the presence of 1.0% acetic acid, PCNs were higher for pGE1 (10 copies/genome) and pGE3 (6 copies/genome) during the logarithmic phase. Numbers for pGE2 did not change, indicating that pGE1 and pGE3 increase their PCNs in response to acetic acid. Plasmids pBE2 and pBE3 were constructed by ligating linearized pGE2 and pGE3 into pBR322. Both plasmids were replicable in Escherichia coli, Acetobacter pasteurianus and G. europaeus, highlighting their suitability as vectors for acetic acid bacteria.





  • Pichia anomala, a new species of yeast-like endosymbionts and its variation in small brown planthopper (Laodelphax striatellus)
    Publication date: June 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 6

    Author(s): Wei Cao , Zheng Ma , Yolanda H. Chen , Xiaoping Yu

    Yeast-like symbionts (YLS) are endosymbionts that promote the growth of delphacid planthoppers (Hemiptera: Delphacidae), some of which are pests on cultivated rice. Identification and characterization of YLS growth can be helpful for pest control, because it has been demonstrated that there is a variety of YLS in rice planthopper and they affected the planthopper's growth and virulence to plant hosts. So, elucidation of the species of YLS in planthopper is crucial for exploiting a new way to control planthopper. In this study, a new isolated of YLS was obtained from the small brown planthopper, Laodelphax striatellus, which was cultured in vitro, simultaneously identified as Pichia anomala based on its phylogenetic analysis. In order to confirm the existence of P. anomala in the L. striatellus body, we used the denaturing gradient gel electrophoresis (DGGE) to identify the YLS and obtain the specific bands for P. anomala. The quantification and localization of P. anomala in L. striatellus samples were determined by fluorescent in situ hybridization (FISH) using genus-specific 18S rDNA targeted probe. The result confirmed that a certain number of P. anomala exist in L. striatellus's abdomen. Subsequently, the variation and copy number of P. anomala in different L. striatellus instars was measured by using absolute quantitative real-time PCR (qPCR), the results indicated that the new isolated strain was closely related to the developmental process of L. striatellus.





  • Change in enzyme production by gradually drying culture substrate during solid-state fermentation
    Publication date: June 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 6

    Author(s): Kazunari Ito , Katsuya Gomi , Masahiro Kariyama , Tsuyoshi Miyake

    The influence of drying the culture substrate during solid-state fermentation on enzyme production was investigated using a non-airflow box. The drying caused a significant increase in enzyme production, while the mycelium content decreased slightly. This suggests that changes in the water content in the substrate during culture affect enzyme production in fungi.





  • Conductive iron oxides accelerate thermophilic methanogenesis from acetate and propionate
    Publication date: June 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 6

    Author(s): Chihaya Yamada , Souichiro Kato , Yoshiyuki Ueno , Masaharu Ishii , Yasuo Igarashi

    Anaerobic digester is one of the attractive technologies for treatment of organic wastes and wastewater, while continuous development and improvements on their stable operation with efficient organic removal are required. Particles of conductive iron oxides (e.g., magnetite) are known to facilitate microbial interspecies electron transfer (termed as electric syntrophy). Electric syntrophy has been reported to enhance methanogenic degradation of organic acids by mesophilic communities in soil and anaerobic digester. Here we investigated the effects of supplementation of conductive iron oxides (magnetite) on thermophilic methanogenic microbial communities derived from a thermophilic anaerobic digester. Supplementation of magnetite accelerated methanogenesis from acetate and propionate under thermophilic conditions, while supplementation of ferrihydrite also accelerated methanogenesis from propionate. Microbial community analysis revealed that supplementation of magnetite drastically changed bacterial populations in the methanogenic acetate-degrading cultures, in which Tepidoanaerobacter sp. and Coprothermobacter sp. dominated. These results suggest that supplementation of magnetite induce electric syntrophy between organic acid-oxidizing bacteria and methanogenic archaea and accelerate methanogenesis even under thermophilic conditions. Findings from this study would provide a possibility for the achievement of stably operating thermophilic anaerobic digestion systems with high efficiency for removal of organics and generation of CH4.





  • Prospecting cold deserts of north western Himalayas for microbial diversity and plant growth promoting attributes
    Publication date: June 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 6

    Author(s): Ajar Nath Yadav , Shashwati Ghosh Sachan , Priyanka Verma , Anil Kumar Saxena

    Microbial communities in different samples collected from cold deserts of north western Himalayas, India, were analyzed using 16S rRNA gene sequencing and phospholipid fatty acids (PLFA) analysis. A total of 232 bacterial isolates were characterized employing 16S rDNA-Amplified Ribosomal DNA Restriction Analysis with the three restriction endonucleases Alu I, Msp I and Hae III, which led to formation of 29–54 groups for the different sites, adding up to169 groups. 16S rRNA gene based phylogenetic analysis, revealed that 82 distinct species of 31 different genera, belonged to four phyla Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. PLFA profiling was performed for concerned samples which gave an estimate of microbial communities without cultivating the microorganisms. PLFA analysis led to characterization of diverse group of microbes in different samples such as gram-negative, gram-positive bacteria, actinomycetes, cyanobacteria, anaerobic bacteria, sulphate reducing bacteria and fungi. The representative strains were screened for their plant growth promoting attributes, which included production of ammonia, HCN, gibberellic acid, IAA and siderophore; solubilization of phosphorus and activity of ACC deaminase. In vitro antifungal activity assay was performed against Rhizoctonia solani and Macrophomina phaseolina. Cold adapted microorganisms may serve as inoculants for crops growing under cold climatic conditions. To our knowledge, this is the first report for the presence of Arthrobacter nicotianae, Brevundimonas terrae, Paenibacillus tylopili and Pseudomonas cedrina in cold deserts and exhibit multifunctional PGP attributes at low temperatures.





  • Lactic acid production from biomass-derived sugars via co-fermentation of Lactobacillus brevis and Lactobacillus plantarum
    Publication date: June 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 6

    Author(s): Yixing Zhang , Praveen V. Vadlani

    Lignocellulosic biomass is an attractive alternative resource for producing chemicals and fuels. Xylose is the dominating sugar after hydrolysis of hemicellulose in the biomass, but most microorganisms either cannot ferment xylose or have a hierarchical sugar utilization pattern in which glucose is consumed first. To overcome this barrier, Lactobacillus brevis ATCC 367 was selected to produce lactic acid. This strain possesses a relaxed carbon catabolite repression mechanism that can use glucose and xylose simultaneously; however, lactic acid yield was only 0.52 g g−1 from a mixture of glucose and xylose, and 5.1 g L−1 of acetic acid and 8.3 g L−1 of ethanol were also formed during production of lactic acid. The yield was significantly increased and ethanol production was significantly reduced if L. brevis was co-cultivated with Lactobacillus plantarum ATCC 21028. L. plantarum outcompeted L. brevis in glucose consumption, meaning that L. brevis was focused on converting xylose to lactic acid and the by-product, ethanol, was reduced due to less NADH generated in the fermentation system. Sequential co-fermentation of L. brevis and L. plantarum increased lactic acid yield to 0.80 g g−1 from poplar hydrolyzate and increased yield to 0.78 g lactic acid per g of biomass from alkali-treated corn stover with minimum by-product formation. Efficient utilization of both cellulose and hemicellulose components of the biomass will improve overall lactic acid production and enable an economical process to produce biodegradable plastics.





  • Effect of temperature shift on levels of acidic charge variants in IgG monoclonal antibodies in Chinese hamster ovary cell culture
    Publication date: June 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 6

    Author(s): Shohei Kishishita , Tomoko Nishikawa , Yasuharu Shinoda , Hiroaki Nagashima , Hiroshi Okamoto , Shinya Takuma , Hideki Aoyagi

    During the production of therapeutic monoclonal antibodies (mAbs), not only enhancement of mAb productivity but also control of quality attributes is critical. Charge variants, which are among the most important quality attributes, can substantially affect the in vitro and in vivo properties of mAbs. During process development for the production of mAbs in a Chinese hamster ovary cell line, we have observed that an improvement in mAb titer is accompanied by an increase in the content of acidic charge variants. Here, to help maintain comparability among mAbs, we aimed to identify the process parameters that controlled the content of acidic charge variants. First, we used a Plackett–Burman design to identify the effect of selected process parameters on the acidic charge variant content. Eight process parameters were selected by using a failure modes and effects analysis. Among these, temperature shift was identified from the Plackett–Burman design as the factor most influencing the acidic charge variant content. We then investigated in more detail the effects of shift temperature and temperature shift timing on this content. The content decreased with a shift to a lower temperature and with earlier timing of this temperature shift. Our observations suggest that Plackett–Burman designs are advantageous for preliminary screening of bioprocess parameters. We report here for the first time that temperature downshift is beneficial for effective control of the acidic peak variant content.





  • Production of biodiesel from vegetable oil and microalgae by fatty acid extraction and enzymatic esterification
    Publication date: June 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 6

    Author(s): Beatriz Castillo López , Luis Esteban Cerdán , Alfonso Robles Medina , Elvira Navarro López , Lorena Martín Valverde , Estrella Hita Peña , Pedro A. González Moreno , Emilio Molina Grima

    The aim of this work was to obtain biodiesel (methyl esters) from the saponifiable lipids (SLs) fraction of the microalga Nannochloropsis gaditana, whose biomass dry weight contains 12.1 wt% of these lipids. SLs were extracted from the microalga as free fatty acids (FFAs) for subsequent transformation to methyl esters (biodiesel) by enzymatic esterification. Extraction as FFAs rather than as SLs allows them to be obtained with higher purity. Microalgal FFAs were obtained by direct saponification of lipids in the biomass and subsequent extraction-purification with hexane. Esterification of FFAs with methanol was catalysed by lipase Novozym 435 from Candida antarctica. Stability studies of this lipase in the operational conditions showed that the esterification degree (ED) attained with the same batch of lipase remained constant over six reaction cycles (36 h total reaction time). The optimal conditions attained for 4 g of FFAs were 25°C, 200 rpm, methanol/FFA molar ratio of 1.5:1, Novozym 435/FFA ratio of 0.025:1 w/w and 4 h reaction time. In these conditions the ED attained was 92.6%, producing a biodiesel with 83 wt% purity from microalgal FFAs. Several experimental scales were tested (from 4 to 40 g FFAs), and in all cases similar EDs were obtained.





  • Production of biomass and bioactive compounds from adventitious roots by optimization of culturing conditions of Eurycoma longifolia in balloon-type bubble bioreactor system
    Publication date: June 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 6

    Author(s): Tao Lulu , So-Young Park , Rusli Ibrahim , Kee-Yoeup Paek

    The present study aimed to optimize the conditions for the production of adventitious roots from Eurycoma longifolia Jack, an important medicinal woody plant, in bioreactor culture. The effects of the type and concentration of auxin on root growth were studied, as well as the effects of the NH4 +:NO3 ratio on adventitious root growth and the production of phenolics and flavonoids. Approximately 5 g L−1 fresh weight of adventitious roots was inoculated into a 3 L balloon-type bubble bioreactor, which contained 2 L 3/4 MS medium supplemented with 30 g L−1 sucrose and cultures were maintained in the dark for 7 weeks at 24 ± 1°C. Higher concentrations of IBA (7.0 and 9.0 mg L−1) and NAA (5.0 mg L−1) enhanced the biomass and accumulation of total phenolics and flavonoids. The adventitious roots were thin, numerous, and elongated in 3/4 MS medium supplemented with 5.0 and 7.0 mg L−1 IBA, whereas the lateral roots were shorter and thicker with 5.0 mg L−1 NAA compared with IBA treatment. The optimum biomasses of 50.22 g L−1 fresh weight and 4.60 g L−1 dry weight were obtained with an NH4 +:NO3 ratio of 15:30. High phenolic and flavonoid productions (38.59 and 11.27 mg L−1 medium, respectively) were also obtained with a ratio of 15:30. Analysis of the 2,2-diphenyl-1-picrylhydrazyl (DPPH)-scavenging activity indicated higher antioxidant activity with an NH4 +:NO3 ratio of 30:15. These results suggest that balloon-type bubble bioreactor cultures are suitable for the large-scale commercial production of E. longifolia adventitious roots which contain high yield of bioactive compounds.





  • Development of carboxymethyl cellulose nonwoven sheet as a novel hemostatic agent
    Publication date: June 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 6

    Author(s): Seiichi Ohta , Toru Nishiyama , Megumu Sakoda , Kyoko Machioka , Masaya Fuke , Shigetoshi Ichimura , Fuyuki Inagaki , Atsushi Shimizu , Kiyoshi Hasegawa , Norihiro Kokudo , Makoto Kaneko , Yutaka Yatomi , Taichi Ito

    Carboxymethyl cellulose (CMC) is a plant-derived material that has high biocompatibility and water solubility. We developed a CMC nonwoven sheet as a hemostatic agent by carboxymethylating a continuous filament cellulose nonwoven sheet. The CMC nonwoven sheet was able to absorb water and dissolve in it. The rates of absorption and dissolution depended on the degree of carboxymethylation. After dissolving in blood, CMC accelerated clot development (possibly owing to the incorporation of CMC into fibrin fibers) and increased the viscosity of the blood, both of which would contribute to the improved blood clotting of an injured surface. In vivo experiments using a rat tail cutting method showed that a CMC nonwoven sheet shortened the bleeding time of the tail when applied to the cut surface. The hemostatic effect of the CMC nonwoven sheet was almost at the same level as a commercial hemostatic bandage. These results suggest that a CMC nonwoven sheet could be used as a novel sheet-type hemostatic agent.





  • Microbial degradation of linear peptides by strain B-9 of Sphingosinicella and its application in peptide quantification using liquid chromatography-mass spectrometry
    Publication date: June 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 6

    Author(s): Atsushi Miyachi , Fumio Kondo , Miki Kurita , Kiyomi Tsuji , Ken-ichi Harada

    The bacterial strain Sphingosinicella sp. B-9 was originally discovered to have the ability to degrade cyanobacterial cyclic peptides (microcystins), and has three hydrolytic enzymes (MlrA, MlrB, and MlrC). The purpose of this study was to examine in detail the degradation of glucagon/vasoactive intestinal polypeptide (VIP) family peptides by B-9, and to investigate the substrate specificity of B-9 proteases and the possibility of using a B-9 protease as a novel protease for peptide quantification by using a surrogate peptide and mass spectrometry (MS). The effective use of inhibitors revealed the following hydrolytic capability of B-9: One of the B-9 proteases (presumably MlrB) that was not inhibited by ethylenediaminetetraacetic acid (EDTA) cleaved bioactive peptides into medium-sized peptides with broad selectivity, similar to neutral endopeptidase, and another protease that was not inhibited by phenylmethylsulfonyl fluoride (PMSF) corresponded to MlrC and cleaved the resulting medium-sized peptides to smaller peptides or amino acids. The former property was desirable to obtain a suitable surrogate peptide, which was used successfully to quantify peptide using liquid chromatography (LC)-MS. Thus, the present study verified that one of the B-9 proteases has broad cleavage selectivity and cleavage sites, not seen in commercially available proteases, and is applicable to protein and peptide quantification using LC-MS.





  • Protein-poly(amino acid) precipitation stabilizes a therapeutic protein l-asparaginase against physicochemical stress
    Publication date: Available online 13 May 2015
    Source:Journal of Bioscience and Bioengineering

    Author(s): Takuya Maruyama , Shunsuke Izaki , Takaaki Kurinomaru , Kenji Handa , Tomoaki Kimoto , Kentaro Shiraki

    Long-term storage in aqueous solution has been demanded for the practical application of therapeutic proteins. Recently, a precipitation–redissolution method was proposed to prepare salt-dissociable protein–polyelectrolyte complex (PPC). To elucidate the utility of the complex for storage of proteins, we investigated the stress tolerance of PPC precipitates containing l-asparaginase (ASNase) and poly-l-lysine (polyK). PPC precipitate containing ASNase and polyK was prepared by precipitation-redissolution method. The sample was treated to three types of stress, i.e., heat, shaking, and oxidation. The protein concentration, enzyme activity, and CD spectrum of the supernatants of samples were measured after stressed. PPC precipitate consisting of ASNase and polyK showed tolerance against thermal and shaking stress compared to the native solution. In addition, PPC precipitate protected ASNase from inactivation by oxidation. PPC precipitate of ASNase/polyK complex successfully stabilized ASNase against physicochemical stresses. These results suggest that the PPC precipitate has great potential as a storage method in aqueous solution for unstable proteins.