JBB : Journal of Bioscience and Bioengineering

            

Journal of Bioscience and Bioengineering vol.122 cover

 



  • Enhanced xylitol production: Expression of xylitol dehydrogenase from Gluconobacter oxydans and mixed culture of resting cell
    Publication date: September 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 3

    Author(s): Xiang-Hui Qi, Jing-Fei Zhu, Jun-Hua Yun, Jing Lin, Yi-Lin Qi, Qi Guo, Hong Xu

    Xylitol has numerous applications in food and pharmaceutical industry, and it can be biosynthesized by microorganisms. In the present study, xdh gene, encoding xylitol dehydrogenase (XDH), was cloned from the genome of Gluconobacter oxydans CGMCC 1.49 and overexpressed in Escherichia coli BL21. Sequence analysis revealed that XDH has a TGXXGXXG NAD(H)-binding motif and a YXXXK active site motif, and belongs to the short-chain dehydrogenase/reductase family. And then, the enzymatic properties and kinetic parameter of purified recombinant XDH were investigated. Subsequently, transformations of xylitol from d-xylulose and d-arabitol, respectively, were studied through mixed culture of resting cells of G. oxydans wild-type strain and recombinant strain BL21-xdh. We obtained 28.80 g/L xylitol by mixed culture from 30 g/L d-xylulose in 28 h. The production was increased by more than three times as compared with that of wild-type strain. Furthermore, 25.10 g/L xylitol was produced by the mixed culture from 30 g/L d-arabitol in 30 h with a yield of 0.837 g/g, and the max volumetric productivity of 0.990 g/L h was obtained at 22 h. These contrast to the fact that wild-type strain G. oxydans only produced 8.10 g/L xylitol in 30 h with a yield of 0.270 g/g. To our knowledge, these values are the highest among the reported yields and productivity efficiencies of xylitol from d-arabitol with engineering strains.





  • Inhibitory effect of Cinnamomum osmophloeum Kanehira ethanol extracts on melanin synthesis via repression of tyrosinase expression
    Publication date: September 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 3

    Author(s): Shih-Chieh Lee, Chun-Hao Chen, Chih-Wen Yu, Hsiao Ling Chen, Wei-Tung Huang, Yun-Shiang Chang, Shu-Hsien Hung, Tai-Lin Lee

    Melanin contributes to skin color, and tyrosinase is the enzyme that catalyzes the initial steps of melanin formation. Therefore, tyrosinase inhibitors may contribute to the control of skin hyperpigmentation. The inhibition of tyrosinase activity by Cinnamomum zeylanicum extracts was previously reported. In this report, we test the hypothesis that Cinnamomum osmophloeum Kanehira, an endemic plant to Taiwan, contains compounds that inhibit tyrosinase activity, similar to C. zeylanicum. The cytotoxicity of three sources of C. osmophloeum Kanehira ethanol extracts was measured in B16-F10 cells using a methyl thiazolyl tetrazolium bromide (MTT) assay. At concentrations greater than 21.25 μg/mL, the ethanol extracts were toxic to the cells; therefore, 21.25 μg/mL was selected to test the tyrosinase activities. At this concentration, all three ethanol extracts decreased the melanin content by 50% in IBMX-induced B16-F10 cells. In addition to the melanin content, greater than 20% of the tyrosinase activity was inhibited by these ethanol extracts. The RT-PCR results showed that tyrosinase and transcription factor MITF mRNAs expression were down-regulated. Consistent with the mRNA results, greater than 40% of the human tyrosinase promoter activity was inhibited based on the reporter assay. Furthermore, our results demonstrate that the ethanol extracts protect cells from UV exposure. C. osmophloeum Kanehira neutralized the IBMX-induced increase in melanin content in B16-F10 cells by inhibiting tyrosinase gene expression at the level of transcription. Moreover, the ethanol extracts also partially inhibited UV-induced cell damage and prevented cell death. Taken together, we conclude that C. osmophloeum Kanehira is a potential skin-whitening and protective agent.





  • Improvement of enantioselectivity of the B-type halohydrin hydrogen-halide-lyase from Corynebacterium sp. N-1074
    Publication date: September 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 3

    Author(s): Fumiaki Watanabe, Fujio Yu, Akashi Ohtaki, Yasuaki Yamanaka, Keiichi Noguchi, Masafumi Odaka, Masafumi Yohda

    Halohydrin hydrogen-halide-lyase (H-Lyase) is a bacterial enzyme involved in the degradation of halohydrins. This enzyme catalyzes the intramolecular nucleophilic displacement of a halogen by a vicinal hydroxyl group in halohydrins, producing the corresponding epoxides. The H-Lyases have been classified into A, B and C subtypes based on amino acid sequence similarities. These enzymes have attracted much attention as industrial catalysts in the synthesis of chiral chemicals from prochiral halohydrins. In the present study, we constructed mutants of B-type H-Lyase from Corynebacterium sp. N-1074 (HheB) displaying higher enantioselectivity by structure-based site-directed mutagenesis and random mutagenesis. A triple mutant of HheB exhibited 98.5% enantioselectivity, the highest ever reported, toward (R)-4-chloro-3-hydroxy-butyronitrile production, with the yield reaching approximately two-fold that of the wild-type enzyme. We discuss the structural basis of the high enantioselectivity and productivity of the mutant by comparing the crystal structures of the mutant HheB and the wild-type enzyme in complex with or without the substrate analogue.





  • Salt-induced increase in the yield of enzymatically synthesized phosphatidylinositol and the underlying mechanism
    Publication date: September 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 3

    Author(s): Michiko Muraki, Jasmina Damnjanović, Hideo Nakano, Yugo Iwasaki

    The purpose of this study was to improve the efficiency of enzymatic synthesis of phosphatidylinositol (PI) from phosphatidylcholine (PC) and myo-inositol in a phospholipase D (PLD)-mediated transphosphatidylation. A conventional biphasic reaction system consisting of ethyl acetate and an aqueous buffer afforded PI with a yield of 14 mol%. In contrast, the reaction performed in the presence of high concentration (0.8–4.3 M) of NaCl in the aqueous phase showed improved PI yield in a NaCl concentration-dependent manner. At 4.3 M NaCl, PI yield of as much as 35 mol% was achieved. The increase in the PI yield offered by other tested salts varied; however, we observed that some salts caused inactivation of the enzyme when used at high concentrations. Although NaCl at high concentration increased the apparent hydrolytic activity on aggregated PC, it decreased the activity towards monomeric PC, indicating that high concentration of salt intrinsically inhibits the enzyme. Binding assays revealed that PLD re-localized from the aqueous phase to the solvent–buffer interface, where the enzymatic reaction takes place, in the presence of both, the salt and PC. Hence, we concluded that improvement of the PI synthesis in the presence of salt occurs mainly due to the accumulation of the enzyme at the interface by strengthening the hydrophobic interactions, by which the apparent activation outweighs the salt-induced inhibitory effect. Using this improved system, several PI with defined structures, namely sn-1, 2-dioleoyl-PI, sn-1-palmitoyl-2-oleoyl-PI, and sn-1-stearoyl-2-arachidonoyl-PI, were successfully synthesized with overall yields of 25–37%, and PI isomeric purities of 91–96%.





  • Alleviation of temperature-sensitive secretion defect of Pseudomonas fluorescens ATP-binding cassette (ABC) transporter, TliDEF, by a change of single amino acid in the ABC protein, TliD
    Publication date: September 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 3

    Author(s): Gyeong Tae Eom, Joon Young Oh, Ji Hyun Park, Hye Jin Lim, So Jeong Lee, Eun Young Kim, Ji-Eun Choi, Jonggeon Jegal, Bong Keun Song, Ju-Hyun Yu, Jae Kwang Song

    An ABC transporter, TliDEF, from Pseudomonas fluorescens SIK W1, mediates the secretion of its cognate lipase, TliA, in a temperature-dependent secretion manner; the TliDEF-mediated secretion of TliA was impossible at the temperatures over 33°C. To isolate a mutant TliDEF capable of secreting TliA at 35°C, the mutagenesis of ABC protein (TliD) was performed. The mutated tliD library where a random point mutation was introduced by error-prone PCR was coexpressed with the wild-type tliE, tliF and tliA in Escherichia coli. Among approximately 10,000 colonies of the tliD library, we selected one colony that formed transparent halo on LB-tributyrin plates at 35°C. At the growth temperature of 35°C, the selected mutant TliD showed 1.75 U/ml of the extracellular lipase activity, while the wild-type TliDEF did not show any detectable lipase activity in the culture supernatant of E. coli. Moreover, the mutant TliD also showed higher level of TliA secretion than the wild-type TliDEF at other culture temperatures, 20°C, 25°C and 30°C. The mutant TliD had a single amino acid change (Ser287Pro) in the predicted transmembrane region in the membrane domain of TliD, implying that the corresponding region of TliD was important for causing the temperature-dependent secretion of TliDEF. These results suggested that the property of ABC transporter could be changed by the change of amino acid in the ABC protein.





  • Screening, expression, and characterization of an anti-human oxidized low-density lipoprotein single-chain variable fragment
    Publication date: September 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 3

    Author(s): Miyuki Kumano-Kuramochi, Takashi Fujimura, Shiro Komba, Mari Maeda-Yamamoto, Sachiko Machida

    Increased levels of oxidized low-density lipoprotein (OxLDL) in the blood circulation are correlated with atherosclerosis. Monoclonal antibody-based detection systems have been reported for OxLDL. We identified novel single-chain variable fragments (scFvs) having affinity for human OxLDL and related ligands. We constructed an scFv library from nonimmunized human spleen mRNA. Two types (γ+κ and μ+λ) of scFv phage libraries were enriched by biopanning, and five scFv clones with affinity for OxLDL were identified. The γκ5 scFv, which showed the highest affinity for OxLDL, was cloned into pET-22b(+) and expressed in Escherichia coli BL21(DE3). γκ5, expressed as an inclusion body in BL21(DE3), was refolded and purified. The specificity and sensitivity of γκ5 were analyzed using enzyme-linked immunosorbent assays (ELISAs). The γκ5 scFv showed affinity for OxLDL and acetylated LDL. The sensitivity of γκ5 to low concentrations (1–2 μg/mL) of OxLDL was higher than that to AcLDL and LDL. Finally, we developed a sandwich ELISA using γκ5 and CTLD14 (a lectin-like OxLDL receptor-1 ligand recognition region), which allowed specific detection of OxLDL at a level below 0.1 μg/mL. Our results indicated that the γκ5 scFv was a promising molecule for the detection of modified LDL at very low concentrations.





  • Improvement of lipid content of Chlorella minutissima MCC 5 for biodiesel production
    Publication date: September 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 3

    Author(s): Sourabh Chakraborty, Debabrata Mohanty, Supratim Ghosh, Debabrata Das

    Lipids extracted from microalgae have been considered as a potential source for the production of biodiesel. Enhancement of lipid has the limitations of low biomass productivity. So, the main objective of the present study was to deduce suitable conditions for the improvement of biomass production followed by enhancement of lipid content. After optimization, a strategy for two stage cultivation was utilized where high lipid content was obtained with a high biomass concentration. Optimization of biomass production of Chlorella minutissima MCC 5 was carried out under different intensities of light, temperatures, concentrations of nitrate and phosphate using Taguchi model. A suitable synergy of the four parameters yielded maximum biomass (1.93 g L−1) in airlift reactor. Temperature was found to be relatively effective than other parameters for higher biomass production. Activation energy for the cell growth was determined (47.95 kJ mol−1). Among the various (photo, thermal, nitrate and phosphate) stress conditions studied, nitrate limitation (1 mM) was found to be suitable for the enhancement of lipid resulting highest yield (48.26% w/w). Two stage cultivation of the microalgae yielded a maximum lipid content of 46% w/w with a biomass concentration of 2.2 g L−1. Additionally, FAME analysis exhibited significant increase of oleic acid in the biodiesel. So, C. minutissima MCC 5 cultivated under nitrate stress could be a possible feedstock for biodiesel production.





  • Recovery of laccase from processed Hericium erinaceus (Bull.:Fr) Pers. fruiting bodies in aqueous two-phase system
    Publication date: September 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 3

    Author(s): Devamalini Rajagopalu, Pau Loke Show, Yee Shin Tan, Sekaran Muniandy, Vikineswary Sabaratnam, Tau Chuan Ling

    The feasible use of aqueous two-phase systems (ATPSs) to establish a viable protocol for the recovery of laccase from processed Hericium erinaceus (Bull.:Fr.) Pers. fruiting bodies was evaluated. Cold-stored (4.00±1.00°C) H. erinaceus recorded the highest laccase activities of 2.02±0.04 U/mL among all the processed techniques. The evaluation was carried out in twenty-five ATPSs, which composed of polyethylene glycol (PEG) with various molecular weights and potassium phosphate salt solution to purify the protein from H. erinaceus. Optimum recovery condition was observed in the ATPS which contained 17% (w/w) PEG with a molecular weight of 8000 and 12.2% (w/w) potassium phosphate solution, at a volume ratio (V R) of 1.0. The use of ATPS resulted in one-single primary recovery stage process that produced an overall yield of 99% with a purification factor of 8.03±0.46. The molecular mass of laccases purified from the bottom phase was in the range of 55–66 kDa. The purity of the partitioned laccase was confirmed with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE).





  • Denitrification by Pseudomonas stutzeri coupled with CO2 reduction by Sporomusa ovata with hydrogen as an electron donor assisted by solid-phase humin
    Publication date: September 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 3

    Author(s): Zhixing Xiao, Takanori Awata, Dongdong Zhang, Arata Katayama

    A co-culture system comprising an acetogenic bacterium, Sporomusa ovata DSMZ2662, and a denitrifying bacterium, Pseudomonas stutzeri JCM20778, enabled denitrification using H2 as the sole external electron donor and CO2 as the sole external carbon source. Acetate produced by S. ovata supported the heterotrophic denitrification of P. stutzeri. A nitrogen balance study showed the reduction of nitrate to nitrogen gas without the accumulation of nitrite and nitrous oxide in the co-culture system. S. ovata did not show nitrate reduction to ammonium in the co-culture system. Significant proportions of the consumed H2 were utilized for denitrification: 79.9 ± 4.6% in the co-culture system containing solid-phase humin and 62.9±11.1% in the humin-free co-culture system. The higher utilization efficiency of hydrogen in the humin-containing system was attributed to the higher denitrification activity of P. stutzeri under the acetate deficient conditions. The nitrogen removal rate of the humin-containing co-culture system reached 0.19 kg NO3 –N·m−3·d−1. Stable denitrification activity for 61 days of successive sub-culturing suggested the robustness of this co-culture system. This study provides a novel strategy for the in situ enhancement of microbial denitrification.





  • Comparison of amino and epoxy functionalized SBA-15 used for carbonic anhydrase immobilization
    Publication date: September 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 3

    Author(s): Xiaoyao Fei, Shaoyun Chen, Dai Liu, Chunjie Huang, Yongchun Zhang

    Two functionalized SBA-15 [amine-functionalized SBA-15 (AFS) and epoxy-functionalized SBA-15 (GFS)] with different types of functional groups were synthesized by a hydrothermal process and post functionalized with 3-aminopropyltriethoxysilane (APTES) and 3-glycidyloxypropyltrimethoxysilane (GPTMS), respectively. They were used for the immobilization of carbonic anhydrase (CA). The physicochemical properties of the functionalized SBA-15 were characterized by X-ray powder diffraction (XRD), N2 adsorption–desorption, 13C, 29Si solid-state nuclear magnetic resonance (NMR) spectroscopy, and scanning electron microscopy (SEM). Before and after CA was immobilized on AFS and GFS, the effects of temperature and pH value on the enzyme activity, storage stability, and reusability were investigated using para-nitrophenyl acetate (p-NPA) assay. CA/GFS showed a better performance with respect to storage stability and reusability than CA/AFS. Moreover, the amount of CaCO3 precipitated over CA/AFS was less than that precipitated over CA/GFS, which was almost equal to that precipitated over the free CA. The results indicate that the epoxy group is a more suitable functional group for covalent bonding with CA than the amino group, and GFS is a promising support for CA immobilization.





  • Comparison of electrochemical and microbiological characterization of microbial fuel cells equipped with SPEEK and Nafion membrane electrode assemblies
    Publication date: September 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 3

    Author(s): Kei Suzuki, Rubaba Owen, Joann Mok, Hiroki Mochihara, Takuya Hosokawa, Hiroko Kubota, Hisatoshi Sakamoto, Atsunori Matsuda, Yosuke Tashiro, Hiroyuki Futamata

    Microbial fuel cells equipped with SPEEK-MEA (SPEEK-MFC) and Nafion-MEA (Nafion-MFC) were constructed with organic waste as electron donor and lake sediment as inoculum and were then evaluated comprehensively by electrochemical and microbial analyses. The proton conductivity of SPEEK was several hundreds-fold lower than that of Nafion 117, whereas the oxygen mass and diffusion transfer coefficients of SPEEK were 10-fold lower than those of Nafion 117. It was difficult to predict which was better membrane for MFC based on the feature of membrane. Analyses of polarization curves indicated that the potential of electricity production was similar in both MFCs, as the SPEEK-MFC produced 50–80% of the practical current density generated by the Nafion-MFC. Chronopotentiometry analyses indicated that the Nafion-MEA kept the performance longer than the SPEEK-MEA for long period, whereas performance of both anodes improved on time. Multidimensional scaling analyses based on DGGE profiles revealed the anolytic and biofilm communities of the SPEEK-MFC had developed differently from those of the Nafion-MFC. Clone library analyses indicated that Geobacter spp. represented 6.3% of the biofilm bacterial community in the Nafion-MFC but not detected in the SPEEK-MFC. Interestingly, the clone closely related to Acetobacterium malicum strain HAAP-1, belonging to the homoacetogens, became dominant in both anolytic and biofilm communities of the SPEEK-MFC. It was suggested that the lower proton conductivity of SPEEK-MEA allowed the bacteria closely related to strain HAAP-1 to be dominant specifically in SPEEK-MFC. These results indicated that Nafion-MFC ranked with SPEEK-MFC and that MEAs had strong selective pressure for electricity-producing bacterial community.





  • Genome-wide gene expression analysis of mouse embryonic stem cells exposed to p-dichlorobenzene
    Publication date: September 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 3

    Author(s): Hidenori Tani, Jun-ichi Takeshita, Hiroshi Aoki, Ryosuke Abe, Akinobu Toyoda, Yasunori Endo, Sadaaki Miyamoto, Masashi Gamo, Masaki Torimura

    Because of the limitations of whole animal testing approaches for toxicological assessment, new cell-based assay systems have been widely studied. In this study, we focused on two biological products for toxicological assessment: mouse embryonic stem cells (mESCs) and long noncoding RNAs (lncRNAs). mESCs possess the abilities of self-renewal and differentiation into multiple cell types. LlncRNAs are an important class of pervasive non-protein-coding transcripts involved in the molecular mechanisms associated with responses to chemicals. We exposed mESCs to p-dichlorobenzene (p-DCB) for 1 or 28 days (daily dose), extracted total RNA, and performed deep sequencing analyses. The genome-wide gene expression analysis indicated that mechanisms modulating proteins occurred following acute and chronic exposures, and mechanisms modulating genomic DNA occurred following chronic exposure. Moreover, our results indicate that three novel lncRNAs (Snora41, Gm19947, and Scarna3a) in mESCs respond to p-DCB exposure. We propose that these lncRNAs have the potential to be surrogate indicators of p-DCB responses in mESCs.





  • Variation of the microbial community in thermophilic anaerobic digestion of pig manure mixed with different ratios of rice straw
    Publication date: September 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 3

    Author(s): Sheng Zhou, Marcell Nikolausz, Jining Zhang, Shohei Riya, Akihiko Terada, Masaaki Hosomi

    The effect of pig manure mixed with rice straw on methane yield and the microbial community involved in a thermophilic (55°C) anaerobic digestion process was investigated. Three substrates composed of mixed pig manure and rice straw at different ratios (95:5; 78:22 and 65:35 w/w, which resulted in C/N ratios of 10:1, 20:1 and 30:1) were used for the experiment. The substrate type had a major influence on the total bacterial community, while the methanogens were less affected. The members of the class Clostridia (phylum Firmicutes) were predominant regardless of mixture ratio (C/N ratio), but at species level there was a major difference between the low and high C/N ratio samples. The hydrogenotrophic methanogenic genus of Methanothermobacter was predominant in all samples but higher C/N ratio sequences affiliated to the genus Methanosarcina were also detected. The appearance of Methanosarcina sp. is most likely due to the less inhibition of ammonia during the anaerobic digestion.





  • Kinetics of bisphenol A degradation by Sphingomonas paucimobilis FJ-4
    Publication date: September 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 3

    Author(s): Hitoshi Fujiwara, Satoshi Soda, Masanori Fujita, Michihiko Ike

    A chemostat was operated to characterize degradation of bisphenol A by Sphingomonas paucimobilis FJ-4. The chemostat at 30°C was fed with a medium containing 150 mg L−1 of BPA as the sole carbon and energy source. At the short cell retention time of 8 h, the bacterial cells were washed out from the chemostat. At long cell retention times of 12, 16, 24, and 48 h, steady-states of the bacterial growth on BPA degradation were achieved after a lag time of 16–57 h. A mathematical model was applied to evaluate the BPA degradation ability of strain FJ-4. The maximum specific degradation rate, the half saturation constant, the cell yield, and the specific decay rate were estimated respectively as 0.46 mg-BPA (mg-VSS h)−1, 13.1 mg L−1, 0.39 mg-VSS mg-BPA−1, and 0.0014 h−1.





  • Responses of soil microbial biomass and bacterial community structure to closed-off management (an ecological natural restoration measures): A case study of Dongting Lake wetland, middle China
    Publication date: September 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 3

    Author(s): Juan Dai, Haipeng Wu, Chang Zhang, Guangming Zeng, Jie Liang, Shenglian Guo, Xiaodong Li, Lu Huang, Lunhui Lu, Yujie Yuan

    Soil microbial biomass (SMB) and bacterial community structure, which are critical to global ecosystem and fundamental ecological processes, are sensitive to anthropogenic activities and environmental conditions. In this study, we examined the possible effects of closed-off management (an ecological natural restoration measures, ban on anthropogenic activity, widely employed for many important wetlands) on SMB, soil bacterial community structure and functional marker genes of nitrogen cycling in Dongting Lake wetland. Soil samples were collected from management area (MA) and contrast area (CA: human activities, such as hunting, fishing and draining, are permitted) in November 2013 and April 2014. Soil properties, microbial biomass carbon (MBC), and bacterial community structure were investigated. Comparison of the values of MA and CA showed that SMB and bacterial community diversity of the MA had a significant increase after 7 years closed-off management. The mean value of Shannon–Weiner diversity index of MA and CA respectively were 2.85 and 2.07. The gene copy numbers of 16S rRNA and nosZ of MA were significant higher than those of CA. the gene copy numbers of ammonia-oxidizing archaea (AOA) and nirK of MA were significant lower than those of CA. However, there was no significant change in the gene copy numbers of ammonia-oxidizing bacteria (AOB) and nirS.





  • A simple agroinfiltration method for transient gene expression in plant leaf discs
    Publication date: September 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 3

    Author(s): Kouki Matsuo, Noriho Fukuzawa, Takeshi Matsumura

    In the present study, we developed a simple transient gene expression system based on Agrobacterium-mediated transformation. Vacuum infiltration was applied to leaf discs from Nicotiana benthamiana plants with Agrobacterium suspension solution under conventional vacuum conditions in a needleless plastic syringe. Model proteins, green fluorescent protein, β-glucuronidase, mouse granulocyte-macrophage colony-stimulating factor, and human fibroblast growth factor 1 were successfully expressed in leaf discs within 4 days after infiltration. In addition, the functional evaluation of viral RNA silencing suppressors, Artichoke mottled crinkle virus p19 protein, was also performed. Using this method, the contamination and diffusion of genetically modified bacterium to the environment and important transgenic plants were prevented. This method can be conducted without specialized apparatuses or large amounts of Agrobacterium suspension solutions; thus, the simultaneous evaluation of multiple vectors will be easily possible.





  • Differentiation signalobody: Demonstration of antigen-dependent osteoclast differentiation from a progenitor cell line
    Publication date: September 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 3

    Author(s): Hideto Nakabayashi, Saeko Aoyama, Masahiro Kawahara, Teruyuki Nagamune

    A “cytokine-less” in vitro differentiation method would be promising for cost-effective mass production of cells used for regenerative medicine. In this study, we developed a differentiation signalobody S-RANK, in which the extracellular domain of receptor activator of nuclear factor kappa-B (RANK) is replaced with a single-chain variable fragment (scFv) to attain signaling in response to an inexpensive antigen. A murine macrophage cell line RAW264, which is known to differentiate into an osteoclast by RANK ligand (RANKL), was lentivirally transduced with S-RANK. When the resultant cells were cultured with a specific antigen, the cells differentiated into multinucleated tartrate-resistant acid phosphatase-positive osteoclasts. The differentiation efficiency was almost comparable to those induced by RANKL. In addition, the signaling analysis demonstrated that nuclear factor kappa-B and mitogen-activated protein kinase signaling pathways, which are the major signaling pathways downstream of wild-type RANK, were also activated by S-RANK. These results demonstrate that S-RANK sufficiently mimics signal transduction of wild-type RANK. Differentiation signalobodies may be applied for controlling differentiation of other cell types by using appropriate signaling domains.





  • Calorimetric studies of the growth of anaerobic microbes
    Publication date: September 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 3

    Author(s): Hideo Miyake, Yukiko Maeda, Takashi Ishikawa, Akiyoshi Tanaka

    This article aims to validate the use of calorimetry to measure the growth of anaerobic microbes. It has been difficult to monitor the growth of strict anaerobes while maintaining optimal growth conditions. Traditionally, optical density and ATP concentration are usually used as measures of the growth of anaerobic microbes. However, to take these measurements it is necessary to extract an aliquot of the culture, which can be difficult while maintaining anaerobic conditions. In this study, calorimetry was used to continuously and nondestructively measure the heat generated by the growth of anaerobic microbes as a function of time. Clostridium acetobutylicum, Clostridium beijerinckii, and Clostridium cellulovorans were used as representative anaerobic microbes. Using a multiplex isothermal calorimeter, we observed that peak time (t p) of C. acetobutylicum heat evolution increased as the inoculation rate decreased. This strong correlation between the inoculation rate and t p showed that it was possible to measure the growth rate of anaerobic microbes by calorimetry. Overall, our results showed that there is a very good correlation between heat evolution and optical density/ATP concentration, validating the use of the method.





  • In vitro steroid profiling system for the evaluation of endocrine disruptors
    Publication date: September 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 3

    Author(s): Yosuke Nakano, Toshiyuki Yamashita, Masashi Okuno, Eiichiro Fukusaki, Takeshi Bamba

    Endocrine disruptors (ED) are chemicals that affect various aspects of the endocrine system, often leading to the inhibition of steroidogenesis. Current chemical safety policies that restrict human exposure to such chemicals describe often time-consuming and costly methods for the evaluation of ED effects. We aimed to develop an effective tool for accurate phenotypic chemical toxicology studies. We developed an in vitro ED evaluation system using gas chromatography/mass spectrometry (GC/MS/MS) methods for metabolomic analysis of multi-marker profiles. Accounting for sample preparation and GC/MS/MS conditions, we established a screening method that allowed the simultaneous analysis of 17 steroids with good reproducibility and a linear calibration curve. Moreover, we applied the developed system to H295R human adrenocortical cells exposed to forskolin and prochloraz in accordance with the Organization for Economic Cooperation and Development (OECD) guidelines and observed dose-dependent variations in steroid profiles. While the OECD guidelines include only testosterone and 17β-estradiol, our system enabled a comprehensive and highly sensitive analysis of steroid profile alteration due to ED exposure. The application of our ED evaluation screen could be economical and provide novel insights into the hazards of ED exposure to the endocrine system.





  • Pilot-scale whole-cell biocatalysis for the hydroxylation of cyclosporine derivative, FR901459, at higher concentrations by Lentzea sp. 7887 using soybean flour as a novel substrate dispersant
    Publication date: Available online 18 August 2016
    Source:Journal of Bioscience and Bioengineering

    Author(s): Tetsuya Yabutani, Shiho Shimizu, Hideo Nakano

    Pilot-scale hydroxylation of FR901459, an immunosuppressive cyclosporine derivative, was performed using resting cells of a Gram-positive bacteria Lentzea sp. 7887 (as whole-cell biocatalysts) and soybean flour as a substrate dispersant. Through biocatalysis, FR901459 was hydroxylated at position 9, producing AS1837812, an important intermediate in the production of drug candidates against hepatitis C. Since FR901459 is insoluble in water, the conversion ratio ([moles of AS1837812 produced/moles of FR901459 added]×100) of the biocatalysis decreased under conditions with substrate concentrations higher than 0.615 mM. To increase the concentration of FR901459 for biocatalysis, we screened various materials to effectively disperse FR901459 in the biocatalysis mixture and found that soybean flour was the best substrate dispersant. The addition of soybean flour to the biocatalysis mixture increased the FR901459 concentration in a 3-L reactor up to 3-fold (1.85 mM). Thus, we successfully established a pilot-scale (1-m3) biocatalysis with a 2-fold higher concentration (1.23 mM) of FR901459 using soybean flour as the substrate dispersant and obtained 419 g of AS1837812 at a conversion ratio of 34.5% in a 28-h batch reaction. Soybean flour can be used as a substrate dispersant for various industrial biocatalysis processes because of its low cost, high availability, and low environmental impact.