JBB : Journal of Bioscience and Bioengineering

            

Journal of Bioscience and Bioengineering vol.122 cover

 



  • Analyses of chicken sialyltransferases related to O-glycosylation
    Publication date: October 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 4

    Author(s): Shunsuke Kidani, Hidenori Kaneoka, Yuya Okuzaki, Seiya Asai, Yusuke Kojima, Ken-ichi Nishijima, Shinji Iijima

    The chicken β-galactoside α2,3-sialyltransferase 1, 2, and 5 (ST3Gal1, 2, and 5) genes were cloned, and their enzymes were expressed in 293FT cells. ST3Gal1 and 2 exhibited enzymatic activities toward galactose-β1,3-N-acetylgalactosamine and galactose-β1,3-N-acetylglucosamine. ST3Gal5 only exhibited activity toward lactosylceramide. ST3Gal1 and 2 and previously cloned ST3Gal3 and 6 transferred CMP-sialic acid to asialofetuin. Reverse-transcription-quantitative PCR indicated that ST3Gal1 was expressed at higher levels in the trachea, lung, spleen, and magnum, and the strong expression of ST3Gal5 was observed in the spleen, magnum, and small and large intestines. ST3Gal1, 5, and 6 were also expressed in the tubular gland cells of the magnum, which secretes egg-white proteins. ST3Gal1, 5, and 6 were expressed in the egg chorioallantoic membrane, in which influenza viruses are propagated for the production of vaccines.





  • AmyI-1–18, a cationic α-helical antimicrobial octadecapeptide derived from α-amylase in rice, inhibits the translation and folding processes in a protein synthesis system
    Publication date: October 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 4

    Author(s): Masayuki Taniguchi, Akihito Ochiai, Shun Fukuda, Teppei Sato, Eiichi Saitoh, Tetsuo Kato, Takaaki Tanaka

    In our previous study, we used a cell-free rapid translation system (RTS), which is an in vitro protein synthesis system based on Escherichia coli lysate, for evaluating the inhibition of green fluorescent protein (GFP) synthesis by pyrrhocoricin. In this study, using an RTS, we evaluated the inhibition of GFP synthesis by AmyI-1–18, an antimicrobial octadecapeptide. We found that, similarly to pyrrhocoricin, AmyI-1–18 inhibited GFP synthesis in the RTS in a concentration-dependent manner. In addition, the blockage of transcription and translation steps in the RTS was individually estimated using RT-PCR after gene expression to determine the mRNA products and using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine the amounts of GFP expressed from purified mRNA, respectively. The results demonstrated that the inhibition of GFP synthesis by AmyI-1–18 did not occur at the transcription step but rather at the translation step. Furthermore, we assessed the inhibition of DnaK-mediated refolding of chemically denatured luciferase by AmyI-1–18; AmyI-1–18 inhibited the protein folding activity of the ATP-dependent DnaK/DnaJ molecular chaperone system in a concentration-dependent manner. Surface plasmon resonance (SPR) analysis showed that AmyI-1–18 strongly bound to RNA with a K D value of 1.4 × 10−8 M but not to DNA and that AmyI-1–18 specifically bound to DnaK with a K D value of 4.4 × 10−6 M. These SPR analysis results supported the results obtained in both the RTS and the molecular chaperone system. These results demonstrated that both RNA and DnaK are most likely the target of AmyI-1–18 in the protein synthesis system.





  • Screening, identification, and characterization of α-xylosidase from a soil metagenome
    Publication date: October 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 4

    Author(s): Tomohiko Matsuzawa, Nobutada Kimura, Hikaru Suenaga, Katsuro Yaoi

    A novel α-xylosidase, MeXyl31, was isolated and characterized from a soil metagenomic library. The amino acid sequence of MeXyl31 showed a slight homology with other characterized α-xylosidases. The optimal pH and temperature of recombinant MeXyl31 were pH 5.5 and 45°C, respectively. Recombinant MeXyl31 had a higher α-xylosidase activity toward pNP α-d-xylopyranoside than pNP α-d-glucopyranoside, isoprimeverose, and other xyloglucan oligosaccharides. The k cat/K m value toward pNP α-d-xylopyranoside was about 750-fold higher than that of isoprimeverose. MeXyl31 activity was strongly inactivated in the presence of zinc and copper ions. MeXyl31 is the first α-xylosidase isolated from the metagenome and, relative to other xyloglucan oligosaccharides, shows higher activity toward pNP α-d-xylopyranoside.





  • Branched chain amino acids maintain the molecular weight of poly(γ-glutamic acid) of Bacillus licheniformis ATCC 9945 during the fermentation
    Publication date: October 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 4

    Author(s): Hitoshi Mitsunaga, Lena Meissner, Jochen Büchs, Eiichiro Fukusaki

    Poly(γ-glutamic acid) mainly produced by Bacillus spp. is an industrially important compound due to several useful features. Among them, molecular weight is an important characteristic affecting on the physical properties such as viscosities and negative charge densities. However, it is difficult to control the molecular size of PGA since it decreases during fermentation. Previous study reported that PGA produced in the media containing different carbon sources such as glucose and glycerol showed differences in molecular weight. Therefore in this study, the effect of carbon source on the PGA molecular weight was examined; with the aim of developing a strategy to maintain the high molecular weight of PGA during fermentation. Our result showed that the weight average molecular weight (M w ) of PGA of Bacillus licheniformis ATCC 9945 cultivated in the media containing PTS-sugars were higher than the medium containing glycerol (non-PTS). The result of metabolome analysis indicated the possibility of CodY (a global regulator protein) activation in the cells cultivated in the media containing PTS-sugars. To mimic this effect, branched-chain amino acids (BCAAs), which are activators of CodY, were added to a medium containing glycerol. As the result, the M w of PGA in the BCAAs-supplemented media were maintained and high during the early production phase compared to the non BCAAs-supplemented medium. These results indicate that BCAAs can repress the PGA molecular weight reduction during fermentation in B. licheniformis ATCC 9945.





  • Consolidated bioprocessing of poly(lactate-co-3-hydroxybutyrate) from xylan as a sole feedstock by genetically-engineered Escherichia coli
    Publication date: October 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 4

    Author(s): Lucia Salamanca-Cardona, Ryan A. Scheel, Norman Scott Bergey, Arthur J. Stipanovic, Ken'ichiro Matsumoto, Seiichi Taguchi, Christopher T. Nomura

    Consolidated bioprocessing of lignocellulose is an attractive strategy for the sustainable production of petroleum-based alternatives. One of the underutilized sources of carbon in lignocellulose is the hemicellulosic fraction which largely consists of the polysaccharide xylan. In this study, Escherichia coli JW0885 (pyruvate formate lyase activator protein mutant, pflA ) was engineered to express recombinant xylanases and polyhydroxyalkanoate (PHA)-producing enzymes for the biosynthesis of poly(lactate-co-3-hydroxybutyrate) [P(LA-co-3HB)] from xylan as a consolidated bioprocess. The results show that E. coli JW0885 was capable of producing P(LA-co-3HB) when xylan was the only feedstock and different feeding and growth parameters were examined in order to improve upon initial yields. The highest yields of P(LA-co-3HB) copolymer obtained in this study occurred when xylan was added during mid-exponential growth after cells had been grown at high shaking—speeds (290 rpm). The results showed an inverse relationship between total PHA production and LA-monomer incorporation into the copolymer. Proton nuclear magnetic resonance (1H NMR), gel permeation chromatography (GPC), and differential scanning calorimetry (DSC) analyses corroborate that the polymers produced maintain physical properties characteristic of LA-incorporating PHB-based copolymers. The present study achieves the first ever engineering of a consolidated bioprocessing bacterial system for the production of a bioplastic from a hemicelluosic feedstock.





  • Overexpression of ESBP6 improves lactic acid resistance and production in Saccharomyces cerevisiae
    Publication date: October 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 4

    Author(s): Minetaka Sugiyama, Shin-pei Akase, Ryota Nakanishi, Yoshinobu Kaneko, Satoshi Harashima

    Polylactic acid plastics are receiving increasing attention for the control of atmospheric CO2 emissions. Lactic acid, the building block for polylactic acid, is produced by fermentation technology from renewable carbon sources. The yeast Saccharomyces cerevisiae, harboring the lactate dehydrogenases gene (LDH), produces lactic acid at a large scale due to its strong acid resistance, to its simple nutritional requirements and to its ease of genetic engineering. Since improvement of lactic acid resistance is correlated with an increase of lactic acid production under non-neutralizing condition, we isolated a novel gene that enhances lactic acid resistance using a multi-copy yeast genomic DNA library. In this study, we identified the ESBP6 gene, which increases lactic acid resistance when overexpressed and which encodes a protein with similarity to monocarboxylate permeases. Although ESBP6 was not induced in response to lactic acid stress, it caused weak but reproducible sensitivity to lactic acid when disrupted. Furthermore, intracellular pH in the ESBP6 overexpressing strain was higher than that in the wild-type strain under lactic acid stressed condition, suggesting that Esbp6 plays some roles in lactic acid adaptation response. The ESBP6 overexpressing strain carrying the LDH gene induced 20% increase in lactic acid production compared with the wild-type strain carrying the LDH gene under non-neutralizing conditions. These results indicate that overexpression of ESBP6 provides a novel and useful tool to improve lactic acid resistance and lactic acid production in yeast.





  • Fermentative production of 1-propanol from d-glucose, l-rhamnose and glycerol using recombinant Escherichia coli
    Publication date: October 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 4

    Author(s): Mitsuru Matsubara, Nobuyuki Urano, Shohei Yamada, Ai Narutaki, Misaki Fujii, Michihiko Kataoka

    Fermentative production of 1-propanol, which is one of the promising precursors of polypropylene production, from d-glucose, l-rhamnose and glycerol using metabolically engineered Escherichia coli was examined. To confer the ability to produce 1-propanol from 1,2-propanediol (1,2-PD) in recombinant E. coli, a part of the pdu regulon including the diol dehydratase and the propanol dehydrogenase genes together with the adenosylcobalamin (AdoCbl) regeneration enzyme genes of Klebsiella pneumoniae was cloned, and an expression vector for these genes (pRSF_pduCDEGHOQS) was constructed. Recombinant E. coli harboring pRSF_pduCDEGHOQS with 1,2-PD synthetic pathway (pKK_mde) genes, which was constructed in our previous report (Urano et al., Appl. Microbiol. Biotechnol., 99, 2001–2008, 2015), produced 16.1 mM of 1-propanol from d-glucose with a molar yield of 0.36 mol/mol after 72 h cultivation. 29.9 mM of 1-propanol was formed from l-rhamnose with a molar yield of 0.81 mol/mol using E. coli carrying only pRSF_pduCDEGHOQS. In addition, 1-propanol production from glycerol was achieved by addition of the ATP-dependent dihydroxyacetone kinase gene to E. coli harboring pKK_mde and pRSF_pduCDEGOQS. In all cases, 1-propanol production was achieved by adding only a small amount of AdoCbl.





  • Improvement of daptomycin production via increased resistance to decanoic acid in Streptomyces roseosporus
    Publication date: October 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 4

    Author(s): Sung-Kwon Lee, Hong Rip Kim, Ying-Yu Jin, Seung Hwan Yang, Joo-Won Suh

    Daptomycin, a cyclic anionic lipopeptide compound produced by Streptomyces roseosporus, is used to treat skin infections caused by multi-drug resistant gram-positive pathogens. The biosynthesis of daptomycin is initiated by the condensation of decanoic acid (DA, a 10-carbon unit fatty acid) and the N-terminal l-tryptophan. So, the addition of DA to the fermentation medium is essential for increasing daptomycin production. However, increasing of DA concentration in the fermentation medium was not possible due to the high toxicity of DA. The previous studies reported that the cell growth of S. roseosporus was halted from 1 mM DA. In order to improve daptomycin production with increasing DA concentration in the medium, the DA-resistant S. roseosporus was developed via a sequential-adaptation method. The DA-resistant strain (DAR) showed complete resistance to 1 mM DA, and the daptomycin production was increased 1.4-fold (40.5 ± 0.7 mg/L) compared with the wild-type (28.5 ± 0.8 mg/L) at 1 mM DA. Additionally, the initial step of the daptomycin biosynthesis was enhanced by the overexpression of dptE and dptF in DAR. The dptEF overexpression DAR showed 3.9-fold (156.3 ± 8.2 mg/L) increase in the daptomycin production compared with DAR (40.1 ± 2.6 mg/L) at 1 mM DA.





  • Anti-biofilm activity of the metabolites of Streptomyces chrestomyceticus strain ADP4 against Candida albicans
    Publication date: October 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 4

    Author(s): Vartika Srivastava, Ashok K. Dubey

    Candida albicans is a commensal but a significant opportunistic pathogen. It forms biofilms, which protect them against anti-Candida compounds. Therefore, an agent capable of disrupting the Candida biofilms will be useful in the treatment of such infections. The metabolites of Streptomyces chrestomyceticus strain ADP4 displayed strong anti-Candida activity, hence were investigated further for their ability to inhibit biofilm. Strong inhibition of biofilms produced by several reference strains of C. albicans was observed with BIC90 values ranging from 4 to 8 μg/mL. The anti-biofilm activity of ADP4 metabolites appeared to involve membrane disruption and leakage of cellular materials. Also, it effectively inhibited Candida cells from adhering to polystyrene surface and inhibited their conversion to the hyphal state, thereby preventing further development of the biofilm by the adherent cells. This is the first such report on the metabolites produced by any strain of S. chrestomyceticus.





  • Prevention of GABA reduction during dough fermentation using a baker's yeast dal81 mutant
    Publication date: October 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 4

    Author(s): Akira Ando, Toshihide Nakamura

    γ-Aminobutyric acid (GABA) is consumed by yeasts during fermentation. To prevent GABA reduction in bread dough, a baker's yeast mutant AY77 deficient in GABA assimilation was characterized and utilized for wheat dough fermentation. An amber mutation in the DAL81 gene, which codes for a positive regulator of multiple nitrogen degradation pathways, was found in the AY77 strain. The qPCR analyses of genes involved in nitrogen utilization showed that transcriptional levels of the UGA1 and DUR3 genes encoding GABA transaminase and urea transporter, respectively, are severely decreased in the AY77 cells. The AY77 strain cultivated by fed-batch culture using cane molasses exhibited inferior gas production during dough fermentation compared to that of wild-type strain AY13. However, when fed with molasses containing 0.5% ammonium sulfate, the mutant strain exhibited gas production comparable to that of the AY13 strain. In contrast to the AY13 strain, which completely consumed GABA in dough within 5 h, the AY77 strain consumed no GABA under either culture condition. Dough fermentation with the dal81 mutant strain should be useful for suppression of GABA reduction in breads.





  • Isolation and characterization of xylitol-assimilating mutants of recombinant Saccharomyces cerevisiae
    Publication date: October 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 4

    Author(s): Tatsunori Tani, Hisataka Taguchi, Kazuhiro E. Fujimori, Takehiko Sahara, Satoru Ohgiya, Yoichi Kamagata, Takashi Akamatsu

    To clarify the mechanisms of xylitol utilization, three xylitol-assimilating mutants were isolated from recombinant Saccharomyces cerevisiae strains showing highly efficient xylose-utilization. The nucleotide sequences of the mutant genomes were analyzed and compared with those of the wild-type strains and the mutation sites were identified. gal80 mutations were common to all the mutants, and recessive to the wild-type allele. Hence we constructed a gal80Δ mutant and confirmed that the gal80Δ mutant showed a xylitol-assimilation phenotype. When the constructed gal80Δ mutant was crossed with the three isolated mutants, all diploid hybrids showed xylitol assimilation, indicating that the mutations were all located in the GAL80. We analyzed the role of the galactose permease Gal2, controlled by the regulatory protein Gal80, in assimilating xylitol. A gal2Δ gal80Δ double mutant did not show xylitol assimilation, whereas expression of GAL2 under the control of the TDH3 promoter in the GAL80 strain did result in assimilation. These data indicate that Gal2 was needed for xylitol assimilation in the wild-type strain. When the gal80 mutant with an initial cell concentration of A660 = 20 was used for batch fermentation in a complex medium containing 20 g/L xylose or 20 g/L xylitol at pH 5.0 and 30°C under oxygen limitation, the gal80 mutant consumed 100% of the xylose within 12 h, but <30% of the xylitol within 100 h, indicating that xylose reductase is required for xylitol consumption in oxygen-limited conditions.





  • Enhancement of denitrifying phosphorus removal and microbial community of long-term operation in an anaerobic anoxic oxic–biological contact oxidation system
    Publication date: October 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 4

    Author(s): Miao Zhang, Qing Yang, Jianhua Zhang, Cong Wang, Shuying Wang, Yongzhen Peng

    A two-sludge system consisting of anaerobic anoxic oxic–biological contact oxidation (A2/O–BCO) was developed to treat domestic wastewater with a low carbon/nitrogen (COD/TN) ratio (around 3.21) by shortening sludge retention time (SRT) for phosphorus accumulating organisms (PAOs) in the A2/O reactor and prolonging SRT for nitrifiers in the BCO reactor. Specifically, the BCO reactor was composed of three stages in series (N1, N2 and N3), so that simultaneous nitrogen and phosphorus removals by denitrifying PAOs (DNPAOs) was achieved in the A2/O reactor with NO x –N as the electron acceptor from the BCO reactor. Long term operational tests (600 days) were conducted with various operational parameters [e.g., hydraulic retention time (HRTs), nitrate recycling ratio (Rs), volume ratio (Vs)] to examine the denitrifying phosphorus removal performance. The system exhibited the highest removal of TN and PO 4 3 –P at the HRTs of 8 h, Rs of 300% and Vs of 2:4:1. The optimal TN and PO 4 3 –P removals were 80.30% and 96.61% at low COD/TN of 3.21. The species diversity and microbial community examined by the Illumina MiSeq method demonstrated the fact of two-sludge system, and the improved community structure by long-term optimization was prominent comparing with the seed sludge. Additionally, Accumulibacter and Dechloromonas were the dominant functional PAOs with 25.74% in the A2/O reactor, while nitrifiers (including Nitrosomonas and Nitrospira) were gradually enriched with 13.10%, 21.33%, and 31.10% in the three stages of the BCO reactor.





  • Effects of 3,5-dichlorophenol on excess biomass reduction and bacterial community dynamics in activated sludge as revealed by a polyphasic approach
    Publication date: October 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 4

    Author(s): Zen-ichiro Kimura, Yusuke Hirano, Yukiko Matsuzawa, Akira Hiraishi

    The effects of 3,5-dichlorophenol (DCP) on excess sludge reduction and microbial community dynamics were studied using laboratory-scale activated sludge reactors. The addition of 3,5-DCP at an interval of 7–8 days of operation resulted in effective reduction of growing biomass without a significant decrease in substrate removal activity. However, this uncoupling effect completely disappeared after 30 days of operation. Quinone profiling showed that a drastic component shift from ubiquinone-8 (Q-8) to Q-10 as the major homolog took place during this period of operation, suggesting that Q-10-containing bacteria, i.e., Alphaproteobacteria, became predominant at the uncoupler-ineffective stage. This result was supported by PCR-aided denaturating gradient gel electrophoresis and clone library analyses of 16S rRNA genes and fluorescence in situ hybridization. Among the gene clones detected, those corresponding to Brevundimonas predominated at the uncoupler-ineffective stage. The uncoupler-added reactor yielded 3,5-DCP-resistant Pseudomonas strains as the predominant cultivable bacteria and non-3,5-DCP-resistant Brevundimonas strains as the second most abundant isolates These results suggest that the disappearance of the uncoupling function of 3,5-DCP during the long-term operation of the reactor is related to the drastic community change with increasing populations of Alphaproteobacteria. Most of these alphaproteobacteria represented by Brevundimonas are not resistant to 3,5-DCP but, by an unknown mechanism, may support the bioprotection of the microbial community from the uncoupling effect.





  • Physical enrichment of uncultured Accumulibacter and Nitrospira from activated sludge by unlabeled cell sorting technique
    Publication date: October 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 4

    Author(s): Kana Irie, Hirotsugu Fujitani, Satoshi Tsuneda

    It is important to understand the ecology and physiology of microbes in activated sludge of wastewater treatment plants. Recently, molecular based approaches such as 16S rRNA genes and environmental genomics have illuminated black boxes in nutrient removal process and expanded our knowledge. However, most microbes responsible for the removal of phosphate and nitrogen such as Accumulibacter and Nitrospira remain uncultured. This is because optimum methodologies to concentrate these uncultured microbes and to obtain pure cultures have not been established. Here, we report a novel approach for physical enrichment of uncultured Accumulibacter and Nitrospira from microbial communities in activated sludge by a cell sorting system. Two scattering signatures representing forward scatter and side scatter of this system allowed morphological characterization of microbial particles in activated sludge. The distribution and size of microbial particles consisting of single cells, microcolonies, and aggregates depended on the levels of scattering signatures. Next generation sequencer and principal component analysis revealed each microbial population fractionated according to the levels of scattering signatures, resulting that uncultured Accumulibacter and Nitrospira could be sorted as single cells or microcolonies. Finally, quantitative fluorescence in situ hybridization analysis determined optimum fractions to collect sufficiently these target microbes from activated sludge. Consequently, this method would be very useful as an enrichment technique prior to isolation, genomic analysis, and physiological investigation of uncultured bacteria.





  • Enhanced biohydrogen production from corn stover by the combination of Clostridium cellulolyticum and hydrogen fermentation bacteria
    Publication date: October 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 4

    Author(s): Shou-Chi Zhang, Qi-Heng Lai, Yuan Lu, Zhi-Dan Liu, Tian-Min Wang, Chong Zhang, Xin-Hui Xing

    Hydrogen was produced from steam-exploded corn stover by using a combination of the cellulolytic bacterium Clostridium cellulolyticum and non-cellulolytic hydrogen-producing bacteria. The highest hydrogen yield of the co-culture system with C. cellulolyticum and Citrobacter amalonaticus reached 51.9 L H2/kg total solid (TS). The metabolites from the co-culture system were significantly different from those of the mono-culture systems. Formate, which inhibits the growth of C. cellulolyticum, could be consumed by the hydrogen-evolving bacteria, and transformed into hydrogen. Glucose and xylose were released from corn stover via hydrolysis by C. cellulolyticum and were quickly utilized in dark fermentation with the co-cultured hydrogen-producing bacteria. Because the hydrolysis of corn stover by C. cellulolyticum was much slower than the utilization of glucose and xylose by the hydrogen-evolving bacteria, the sugar concentrations were always maintained at low levels, which favored a high hydrogen molar yield.





  • Improved bio-hydrogen production from glucose by adding a specific methane inhibitor to microbial electrolysis cells with a double anode arrangement
    Publication date: October 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 4

    Author(s): Jingnan Zhang, Yanxia Bai, Yaoting Fan, Hongwei Hou

    Improved hydrogen production from glucose was achieved by adding a specific methane inhibitor (such as chloroform) to repress the activity of methanogens in a single-chamber microbial electrolysis cells (MECs) with a double anode arrangement. A maximum hydrogen production of 8.4±0.2 mol H2/mol-G (G represents glucose), a hydrogen production rate of 2.39±0.3 m3 H2/m3/d and a high energy efficiency (relative to the electrical input) of ηE=165±5% had been recorded from 1 g/L glucose at a low dosage of chloroform (5‰, v:v) and an applied voltage of 0.8 V. Almost all of the glucose was removed within 4 h, with 66% of the electrons in intermediates (mainly including acetate and ethanol), and methane gas was not detected in the MECs through 11 batch cycles. The experimental results confirmed that chloroform was an effective methane inhibitor that improved hydrogen production from glucose in the MECs. In addition, the cyclic voltammetry tests demonstrated that the electron transfer in the MECs was mainly due to the biofilm-bound redox compounds rather than soluble electron shuttles.





  • Effects of cultural medium on the formation and antitumor activity of polysaccharides by Cordyceps gunnii
    Publication date: October 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 4

    Author(s): Zhen-Yuan Zhu, Xiao-Cui Liu, Ya-Li Tang, Feng-Ying Dong, Hui-Qing Sun, Lu Chen, Yong-Min Zhang

    The effects of culture medium composition (i.e., carbon and nitrogen sources) on the growth of mycelia, molecular weight distribution and antitumor activity of intracellular polysaccharides (IPS) from Cordyceps gunnii were investigated. Sucrose and peptone were proved to be the best carbon and nitrogen sources for mycelia growth and remarkably improved IPS production. When the sucrose concentration was 2.0%, the mycelium yield reached up to 15.94±1.26 g/L, but with lower IPS yield; whereas the sucrose concentration was 4.5%, IPS yield reached to a maximum of 138.78±3.89 mg/100 mL. The effects of different carbon/nitrogen (C/N) ratios with equal amounts of carbon source matter on the mycelia and IPS formation were optimized. It found that the yield of mycelia and IPS were both reached to the highest at a C/N ratio of 10:3. In addition, the IPS had the highest macro molecular polysaccharide content and antitumor activity when sucrose concentration was 3.5% and the C/N ratio was 10:1.5. Thus, there was a positive correlation between molecular weight distribution and antitumor activity of IPS by C. gunnii.





  • Impact of hydrolysates on monoclonal antibody productivity, purification and quality in Chinese hamster ovary cells
    Publication date: October 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 4

    Author(s): Steven C.L. Ho, Rui Nian, Susanto Woen, Jake Chng, Peiqing Zhang, Yuansheng Yang

    Plant and yeast derived hydrolysates are economical and efficient alternative medium supplements to improve mammalian cell culture performance. We supplemented two commercial Chinese hamster ovary (CHO) culture media with hydrolysates from four different sources, yeast, soybean, Ex-Cell CD (a chemically defined hydrolysate replacement) and wheat to improve the productivity of two cell lines expressing different monoclonal antibodies (mAbs). Yeast, soybean and Ex-Cell CD improved the final mAb titer by increasing the specific productivity (qP) and/or extension of the culture period. Wheat hydrolysates increased peak viable cell density but did not improve productivity. IgG recovery from protein A purification was not compromised for all cultures by adding yeast, soybean and Ex-Cell CD hydrolysates except for one sample from soybean supplemented culture. Adding these three hydrolysates neither increased the amount of host cell protein, DNA or aggregate impurity amounts nor affect their clearance after purification. Profiling of the glycan types revealed that yeast and soybean hydrolysates could affect the distribution of galactosylated glycans. Ex-Cell CD performed the best at maintaining glycan profile compared to the non-supplemented cultures. Overall, yeast performed the best at improving CHO culture growth and productivity without being detrimental to downstream protein A processes but could affect mAb product glycan distribution while Ex-Cell CD yielded lower titers but has less effect on glycosylation. The hydrolysate to use would thus depend on the requirements of each process and our results would provide a good reference for improving culture performance with hydrolysates or related studies.





  • Differentiation of mouse iPS cells is dependent on embryoid body size in microwell chip culture
    Publication date: October 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 4

    Author(s): Daisuke Miyamoto, Kohji Nakazawa

    A microwell chip possessing microwells of several hundred micrometers is a promising platform for generating embryoid bodies (EBs) of stem cells. Here, we investigated the effects of initial EB size on the growth and differentiation of mouse iPS cells in microwell chip culture. We fabricated a chip that contained 195 microwells in a triangular arrangement at a diameter of 600 μm. To evaluate the effect of EB size, four similar conditions were designed with different seeding cell densities of 100, 500, 1000, and 2000 cells/EB. The cells in each microwell gradually aggregated and then spontaneously formed a single EB within 1 d of culture, and EB size increased with further cell proliferation. EB growth was regulated by the initial EB size, and the growth ability of smaller EBs was higher than that of larger EBs. Furthermore, stem cell differentiation also depended on the initial EB size, and the EBs at more than 500 cells/EB promoted hepatic and cardiac differentiations, but the EBs at 100 cells/EB preferred vascular differentiation. These results indicated that the initial EB size was one of the important factors controlling the proliferation and differentiation of stem cells in the microwell chip culture.





  • Colorimetric method to detect ε-poly-l-lysine using glucose oxidase
    Publication date: October 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 4

    Author(s): Kohei Uematsu, Takaaki Ueno, Kazunori Ushimaru, Chitose Maruyama, Yoshimitsu Hamano, Hajime Katano

    We describe a new colorimetric assay method using glucose oxidase (GOx) to detect ε-poly-l-lysine (εPL). This method uses εPL's remarkable effect of promoting the enzymatic reaction of GOx with ferricyanide ion. This reaction reduces ferricyanide ion to ferrocyanide ion, accompanied by a color change from yellow to colorless. In this colorimetric assay, the detection limit of εPL was estimated to be approximately 0.5 mg/L when purified εPL samples were used. εPL has usually been produced by a fermentation process using Streptomyces albulus species. The components of the culture broth showed interference effects against the assay method. However, due to the high sensitivity of the assay method for εPL, εPL could be detected in the culture broth without any pretreatment. The detectable concentration of εPL in the culture broth, c PL,ac, was estimated to be approximately 20 mg/L. By combining the Berlin blue reaction with this method, the c PL,ac was reduced to 10 mg/L. In light of the proposed method's simplicity and sensitivity, it could be useful for screening εPL synthetic enzymes and microorganisms.