JBB : Journal of Bioscience and Bioengineering

            

Journal of Bioscience and Bioengineering vol.122 cover

 



  • Isolation, characterization and molecular three-dimensional structural predictions of metalloprotease from a phytopathogenic fungus, Alternaria solani (Ell. and Mart.) Sor.
    Publication date: August 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 2

    Author(s): Murugesan Chandrasekaran, Raman Chandrasekar, Se-Chul Chun, Muthukrishnan Sathiyabama

    The present study aims at isolation, identification, characterization and prediction of three-dimensional molecular architecture of a proteolytic enzyme from the early blight pathogen, Alternaria solani which are hypothesized to be a marker of phytopathogenicity. Maximum enzyme production by A. solani was observed in Czapex's Dox broth amended with 2% (w/v) casein than other inducer amendments. Results indicate that the enzyme remained highly active in a pH range of 7.0–10.0 and a temperature range of 45–50°C. The enzyme was strongly inhibited by EDTA, whereas phenylmethylsulfonyl fluoride and monovalent cations (Na+, K+) had little effect. Metal ions such as MgSO4, CaCl2, KCl at 10 mM concentration showed a stimulatory effect (>85%) on protease activity. Matrix-assisted laser desorption and ionization time of flight/mass spectrometry analysis of partially purified enzyme revealed the presence of protease belonging to a keratinolytic protein (metalloprotease) of exopeptidase nature. Putative A. solani keratinolytic enzyme (AsK) is made up of 216 amino acid residues with molecular weight (MW) 24.5 kDa, having a molecular formula of C1094H1704N290O342S4. Ramachandran plot analysis of the protein residues falling into the most favored secondary structures was observed at 84.2%. The major protein structural blocks, 2-β-sheets, and 9-α-helices have a greater tendency to be conserved during the evolutionary process than do mere sequences of amino acids. Besides, AsK, model prediction showed the presence of a Zinc atom at helix regions (Helix 3, 6, 7: His57, His130, His169, and Cys123). Thus, it can be concluded that the major proteinases of AsK are divalent cation-requiring metalloproteinases and make them potential targets of protease inhibitors designing.





  • Re-characterization of mono-2-ethylhexyl phthalate hydrolase belonging to the serine hydrolase family
    Publication date: August 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 2

    Author(s): Makoto Iwata, Takuya Imaoka, Takashi Nishiyama, Takao Fujii

    A novel bacterium assimilating di-2-ethylhexyl phthalate as a sole carbon source was isolated, and identified as a Rhodococcus species and the strain was named EG-5. The strain has a mono-2-ethylhexyl phthalate (MEHP) hydrolase (EG-5 MehpH), which exhibits some different enzymatic features when compared with the previously reported MEHP hydrolase (P8219 MehpH) from Gordonia sp. These differences include different pH optimum activity, maximal reaction temperature and heat stability. The K m and V max values of EG-5 MehpH were significantly higher than those of P8219 MehpH. The primary structure of EG-5 MehpH showed the highest sequence identity to that of P8219 MehpH (39%) among hydrolases. The phylogenetic tree suggested that EG-5 MehpH and P8219 MehpH were categorized in different groups of the novel MEHP hydrolase family. Mutation of a conserved R109 residue of EG-5 MehpH to a hydrophobic residue resulted in a dramatic reduction in the V max value towards MEHP without affecting the K m value. These results indicate that this residue may neutralize the negative charge of a carboxylate anion of MEHP, and thus inhibit the catalytic nucleophile from attacking the ester bond. In other words, the R residue blocks inhibition from the carboxylate anion of MEHP. Recently, registered hypothetical proteins exhibiting 98% or 99% identities for EG-5 MehpH or for P8219 MehpH were found from some pathogens belonging to Actinomycetes. The protein may have other activities besides MEHP hydrolysis and function in other physiological reactions in some Actinomycetes.





  • Antibody-based fluorescent and fluorescent ratiometric indicators for detection of phosphotyrosine
    Publication date: August 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 2

    Author(s): Kim Phuong Huynh Nhat, Takayoshi Watanabe, Kensuke Yoshikoshi, Takahiro Hohsaka

    Fluorescent indicators for protein phosphorylation are very important in not only fundamental biology but also biomedical applications. In this study, we developed novel fluorescent and fluorescent ratiometric indicators for detection of phosphotyrosine (pTyr) derivatives. A single-chain antibody variable fragment (scFv) against phosphotyrosine was fluorescent-labeled by incorporation of tetramethylrhodamine (TAMRA)-linked nonnatural amino acid at the N- or C-terminus. The TAMRA-labeled scFv showed fluorescence enhancement upon addition of pTyr-containing peptides based on antigen-dependent fluorescence quenching effect on TAMRA. The TAMRA-labeled scFv was further fused with enhanced green fluorescent protein (EGFP) to generate a double-labeled scFv for pTyr. In the absence of antigen, fluorescence resonance energy transfer (FRET) occurred from EGFP to TAMRA but TAMRA was quenched. The antigen-binding removed the quenching of TAMRA while FRET occurred without altering its efficiency. As a result of the FRET and antigen-dependent fluorescence quenching effect, the double-labeled scFv exhibited fluorescence ratio enhancement upon the antigen-binding. The fluorescent and fluorescent ratiometric indicators obtained in this study will become a novel tool for analysis of protein phosphorylation. Moreover, this strategy utilizes antibody derivatives, and therefore, can be easily applied to other antigen–antibody pairs to generate fluorescent ratiometric indicators for various target molecules.





  • Effective production of Pro–Gly by mutagenesis of l-amino acid ligase
    Publication date: August 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 2

    Author(s): Haruka Kino, Shota Nakajima, Toshinobu Arai, Kuniki Kino

    l-Amino acid ligase (Lal) catalyzes dipeptide synthesis from unprotected l-amino acids by hydrolysis ATP to ADP. Each Lal displays unique substrate specificity, and many different dipeptides can be synthesized by selecting suitable Lal. We have already successfully synthesized Met–Gly selectively by replacing the Pro85 residues of Lal from Bacillus licheniformis (BL00235). From these results, we deduced that the amino acid residue at position 85 had a key role in enzyme activity, and applied these findings to other Lals. When Pro and Gly were used as substrates, TabS from Pseudomonas syringae, synthesized the salt taste enhancing dipeptide Pro–Gly and other three dipeptides (Gly–Pro, Pro–Pro, and Gly–Gly) was hardly synthesized from its substrate specificity. However, the amount of Pro–Gly was low. Therefore, to alter the substrate specificity and increase the amount of Pro–Gly, we selected amino acid residues that might affect the enzyme activity, Ser85 corresponding to Pro85 of BL00235, and His294 on the results from previous studies and the predicted structure of TabS. These residues were replaced with 20 proteogenic amino acids, and Pro–Gly synthesizing reactions were conducted. The S85T and the H294D mutants synthesized more Pro–Gly than wild-type. Furthermore, the S85T/H294D double mutant synthesized considerably more Pro–Gly than the single mutant did. These results showed that the amino acid position 85 of TabS affect the enzyme activity similarly to BL00235. In addition, replacing the amino acid residue positioning around the N-terminal substrate and constructing the double mutant led to increase the amount of Pro–Gly.





  • Pyruvate kinase deletion as an effective phenotype to enhance lysine production in Corynebacterium glutamicum ATCC13032: Redirecting the carbon flow to a precursor metabolite
    Publication date: August 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 2

    Author(s): Masaki Yanase, Tohru Aikoh, Kazunori Sawada, Kotaro Ogura, Takuya Hagiwara, Keita Imai, Masaru Wada, Atsushi Yokota

    Various attempts have been made to enhance lysine production in Corynebacterium glutamicum. Pyruvate kinase (PYK) defect is one of the strategies used to enhance the supply of oxaloacetic acid (OAA), a precursor metabolite for lysine biosynthesis. However, inconsistent effects of this mutation have been reported: positive effects of PYK defect in mutants having phosphoenolpyruvate carboxylase (PEPC) desensitized to feedback inhibition by aspartic acid, while negative effects in simple PYK gene (pyk) knockout mutants. To address these discrepancies, the effects of pyk deletion on lysine yield were investigated with or without the D299N mutation in ppc rendering PEPC desensitization. C. glutamicum ATCC13032 mutant strain P with a feedback inhibition-desensitized aspartokinase was used as the parent strain, producing 9.36 g/L lysine from 100 g/L glucose in a jar fermentor culture. Under these conditions, while the simple mutant D2 with pyk deletion or R2 with the PEPC-desensitization mutation showed marginally increased lysine yield (∼1.1-fold, not significant), the mutant DR2 strain having both mutations showed synergistically increased lysine productivity (1.38-fold, 12.9 g/L). Therefore, the pyk deletion is effective under a PEPC-desensitized background, which ensures enhanced supply of OAA, thus clarifying the discrepancies. A citrate synthase defective mutation (S252C in gltA) further increased the lysine yield in strain DR2 (1.68-fold, 15.7 g/L). Thus, these three mutations coordinately enhanced the lysine yield. Both the malate:quinone oxidoreductase activity and respiration rate were significantly reduced in strains D2 and DR2. Overall, these results provide valuable knowledge for engineering the anaplerotic reaction to increase lysine yield in C. glutamicum.





  • Random sample consensus combined with partial least squares regression (RANSAC-PLS) for microbial metabolomics data mining and phenotype improvement
    Publication date: August 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 2

    Author(s): Shao Thing Teoh, Miki Kitamura, Yasumune Nakayama, Sastia Putri, Yukio Mukai, Eiichiro Fukusaki

    In recent years, the advent of high-throughput omics technology has made possible a new class of strain engineering approaches, based on identification of possible gene targets for phenotype improvement from omic-level comparison of different strains or growth conditions. Metabolomics, with its focus on the omic level closest to the phenotype, lends itself naturally to this semi-rational methodology. When a quantitative phenotype such as growth rate under stress is considered, regression modeling using multivariate techniques such as partial least squares (PLS) is often used to identify metabolites correlated with the target phenotype. However, linear modeling techniques such as PLS require a consistent metabolite-phenotype trend across the samples, which may not be the case when outliers or multiple conflicting trends are present in the data. To address this, we proposed a data-mining strategy that utilizes random sample consensus (RANSAC) to select subsets of samples with consistent trends for construction of better regression models. By applying a combination of RANSAC and PLS (RANSAC-PLS) to a dataset from a previous study (gas chromatography/mass spectrometry metabolomics data and 1-butanol tolerance of 19 yeast mutant strains), new metabolites were indicated to be correlated with tolerance within certain subsets of the samples. The relevance of these metabolites to 1-butanol tolerance were then validated from single-deletion strains of corresponding metabolic genes. The results showed that RANSAC-PLS is a promising strategy to identify unique metabolites that provide additional hints for phenotype improvement, which could not be detected by traditional PLS modeling using the entire dataset.





  • High acetone–butanol–ethanol production in pH-stat co-feeding of acetate and glucose
    Publication date: August 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 2

    Author(s): Ming Gao, Yukihiro Tashiro, Qunhui Wang, Kenji Sakai, Kenji Sonomoto

    We previously reported the metabolic analysis of butanol and acetone production from exogenous acetate by 13C tracer experiments (Gao et al., RSC Adv., 5, 8486–8495, 2015). To clarify the influence of acetate on acetone–butanol–ethanol (ABE) production, we first performed an enzyme assay in Clostridium saccharoperbutylacetonicum N1-4. Acetate addition was found to drastically increase the activities of key enzymes involved in the acetate uptake (phosphate acetyltransferase and CoA transferase), acetone formation (acetoacetate decarboxylase), and butanol formation (butanol dehydrogenase) pathways. Subsequently, supplementation of acetate during acidogenesis and early solventogenesis resulted in a significant increase in ABE production. To establish an efficient ABE production system using acetate as a co-substrate, several shot strategies were investigated in batch culture. Batch cultures with two substrate shots without pH control produced 14.20 g/L butanol and 23.27 g/L ABE with a maximum specific butanol production rate of 0.26 g/(g h). Furthermore, pH-controlled (at pH 5.5) batch cultures with two substrate shots resulted in not only improved acetate consumption but also a further increase in ABE production. Finally, we obtained 15.13 g/L butanol and 24.37 g/L ABE at the high specific butanol production rate of 0.34 g/(g h) using pH-stat co-feeding method. Thus, in this study, we established a high ABE production system using glucose and acetate as co-substrates in a pH-stat co-feeding system with C. saccharoperbutylacetonicum N1-4.





  • Optimization of RNA-based c-di-GMP fluorescent sensors through tuning their structural modules
    Publication date: August 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 2

    Author(s): Saki Inuzuka, Shigeyoshi Matsumura, Yoshiya Ikawa

    Cyclic diguanylate (c-di-GMP) is a second messenger of bacteria and its detection is an important issue in basic and applied microbiology. As c-di-GMP riboswitch ligand-binding domains (aptamer domains) capture c-di-GMP with high affinity and selectivity, they are promising platforms for the development of RNA-based c-di-GMP sensors. We analyzed two previously reported c-di-GMP sensor RNAs derived from the Vc2 riboswitch. We also designed and tested their variants, some of which showed improved properties as RNA-based c-di-GMP sensors.





  • Nitrogen removal from old landfill leachate with SNAP technology using biofix as a biomass carrier
    Publication date: August 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 2

    Author(s): Thanh Tung Vo, Tan Phong Nguyen

    Single-stage nitrogen removal using Anammox and partial nitritation (SNAP) is a novel technology developed in recent years for removing nitrogen. To evaluate the ability of SNAP technology to remove nitrogen in old landfill leachate under the conditions in Vietnam, we conducted a survey with 7 different nitrogen loading rates of 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4 kg-N/m3 day and a concentration from 100 to 700 mg-N/L. The operating conditions were as follows: DO at 1.0–5.3 mg/L, HRT at 12 h, and pH at 7.5–7.8. The biomass carrier was a biofix made from acrylic fiber. The maximum ammonium conversion and nitrogen removal efficiency were approximately 98% and 85%, respectively, at 1.2 kg-N/m3 day. In general, the nitrogen removal efficiency increased and stabilized at the end of each loading rate. The first step showed that SNAP could potentially be applied in real life for removing nitrogen from old landfill leachate.





  • Nitrification resilience and community dynamics of ammonia-oxidizing bacteria with respect to ammonia loading shock in a nitrification reactor treating steel wastewater
    Publication date: August 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 2

    Author(s): Kyungjin Cho, Seung Gu Shin, Joonyeob Lee, Taewoan Koo, Woong Kim, Seokhwan Hwang

    The aim of this study was to investigate the nitrification resilience pattern and examine the key ammonia-oxidizing bacteria (AOB) with respect to ammonia loading shocks (ALSs) in a nitrification bioreactor treating steel wastewater. The perturbation experiments were conducted in a 4-L bioreactor operated in continuous mode with a hydraulic retention time of 10 d. Three sequential ALSs were given to the bioreactor (120, 180 and 180 mg total ammonia nitrogen (TAN)/L. When the first shock was given, the nitrification process completely recovered after 14 d of further operation. However, the resilience duration was significantly reduced to ∼1 d after the second and third ALSs. In the bioreactor, Nitrosomonas aestuarii dominated the other AOB species, Nitrosomonas europaea and N. nitrosa, throughout the process. In addition, the population of N. aestuarii increased with ammonia utilization following each ALS; i.e., this species responded to acute ammonia overloadings by contributing to ammonia oxidation. This finding suggests that N. aestuarii could be exploited to achieve stable nitrification in industrial wastewaters that contain high concentrations of ammonia.





  • Cooperative hydration effect causes thermal unfolding of proteins and water activity plays a key role in protein stability in solutions
    Publication date: August 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 2

    Author(s): Osato Miyawaki, Michiko Dozen, Kaede Hirota

    The protein unfolding process observed in a narrow temperature range was clearly explained by evaluating the small difference in the enthalpy of hydrogen-bonding between amino acid residues and the hydration of amino acid residue separately. In aqueous solutions, the effect of cosolute on the protein stability is primarily dependent on water activity, a w , the role of which has been long neglected in the literature. The effect of a w on protein stability works as a power law so that a small change in a w is amplified substantially through the cooperative hydration effect. In the present approach, the role of hydrophobic interaction stands behind. This affects protein stability indirectly through the change in solution structure caused by the existence of cosolute.





  • Isolation and characterization of wound-induced compounds from the leaves of Citrus hassaku
    Publication date: August 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 2

    Author(s): Tomonori Asai, Tetsuya Matsukawa, Atsushi Ishihara, Shin'ichiro Kajiyama

    Citrus plants are world widely cultivated as horticultural tree crops, and nowadays their pharmacological activities have been well studied. Since research of defense responses in citrus plants have been mainly focused on the post-harvested fruits because of their commercial importance, defense mechanisms during their developmental stages have not been well understood. In the present study, two wound-induced compounds were isolated from leaves of Citrus hassaku, and their structures were elucidated by high-resolution electron spray ionization mass spectra (HRESIMS) and nuclear magnetic resonance (NMR) analyses. One of these compounds was identified as a known flavanone, hesperetin. The other was characterized as a novel furofuran lignan, and was named ‘biscitrusnin-A’. Their antimicrobial activities were also evaluated.





  • Formation of three-dimensional hepatic tissue by the bottom-up method using spheroids
    Publication date: August 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 2

    Author(s): Tatsuya Okudaira, Naoki Amimoto, Hiroshi Mizumoto, Toshihisa Kajiwara

    Liver regenerative medicine has attracted attention as a possible alternative to organ transplantation. To address the challenge of liver regenerative medicine, the development of a construction method has been proposed for liver tissue in vitro with a high cell density and high functionality for transplantation into patients with severe liver failure. In this study, we fabricated highly functional three-dimensional hepatic tissue by a bottom-up method using spheroids. The hepatic tissue was formed by stacking hepatocyte spheroids covered with human umbilical vein endothelial cells (HUVECs). Hepatic tissue constructs were evaluated for cell survival, liver-specific functions, and histologically. As a result, we identified improvements in liver-specific functions (ammonia removal and albumin secretion) and cell survival. In addition, HUVECs were regularly distributed at every 100 μm within the tissue, and live cells were present within the whole tissue construct throughout the culture period. In summary, we successfully fabricated highly functional hepatic tissue by the bottom-up method using HUVEC-covered hepatocyte spheroids.





  • Chondrogenic differentiation of human mesenchymal stem cells on fish scale collagen
    Publication date: August 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 2

    Author(s): Han-Hsiu Hsu, Toshimasa Uemura, Isamu Yamaguchi, Toshiyuki Ikoma, Junzo Tanaka

    Fish collagen has recently been reported to be a novel biomaterial for cell and tissue culture as an alternative to conventional mammalian collagens such as bovine and porcine collagens. Fish collagen could overcome the risk of zoonosis, such as from bovine spongiform encephalopathy. Among fish collagens, tilapia collagen, the denaturing temperature of which is near 37°C, is appropriate for cell and tissue culture. In this study, we investigated chondrogenic differentiation of human mesenchymal stem cells (hMSCs) cultured on tilapia scale collagen fibrils compared with porcine collagen and non-coated dishes. The collagen fibrils were observed using a scanning electronic microscope. Safranin O staining, glycosaminoglycans (GAG) expression, and real-time PCR were examined to evaluate chondrogenesis of hMSCs on each type of collagen fibril. The results showed that hMSCs cultured on tilapia scale collagen showed stronger Safranin O staining and higher GAG expression at day 6. Results of real-time PCR indicated that hMSCs cultured on tilapia collagen showed earlier SOX9 expression on day 4 and higher AGGRECAN and COLLAGEN II expression on day 6 compared with on porcine collagen and non-coated dishes. Furthermore, low mRNA levels of bone gamma-carboxyglutamate, a specific marker of osteogenesis, showed that tilapia collagen fibrils specifically enhanced chondrogenic differentiation of hMSCs in chondrogenic medium, as well as porcine collagen. Accordingly, tilapia scale collagen may provide an appropriate collagen source for hMSC chondrogenesis in vitro.





  • Increased recombinant protein production owing to expanded opportunities for vector integration in high chromosome number Chinese hamster ovary cells
    Publication date: August 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 2

    Author(s): Noriko Yamano, Mai Takahashi, Seyed Mohammad Ali Haghparast, Masayoshi Onitsuka, Toshitaka Kumamoto, Jana Frank, Takeshi Omasa

    Chromosomal instability is a characteristic of Chinese hamster ovary (CHO) cells. Cultures of these cells gradually develop heterogeneity even if established from a single cell clone. We isolated cells containing different numbers of chromosomes from a CHO-DG44-based human granulocyte-macrophage colony stimulating factor (hGM-CSF)-producing cell line and found that high chromosome number cells showed higher hGM-CSF productivity. Therefore, we focused on the relationship between chromosome aneuploidy of CHO cells and high recombinant protein-producing cell lines. Distribution and stability of chromosomes were examined in CHO-DG44 cells, and two cell lines expressing different numbers of chromosomes were isolated from the original CHO-DG44 cell line to investigate the effect of aneuploid cells on recombinant protein production. Both cell lines were stably transfected with a vector that expresses immunoglobulin G3 (IgG3), and specific antibody production rates were compared. Cells containing more than 30 chromosomes had higher specific antibody production rates than those with normal chromosome number. Single cell analysis of enhanced green fluorescent protein (Egfp)-gene transfected cells revealed that increased GFP expression was relative to the number of gene integration sites rather than the difference in chromosome numbers or vector locations. Our results suggest that CHO cells with high numbers of chromosomes contain more sites for vector integration, a characteristic that could be advantageous in biopharmaceutical production.





  • Effects of nozzle type atmospheric dry air plasma on L929 fibroblast cells hybrid poly (ε-caprolactone)/chitosan/poly (ε-caprolactone) scaffolds interactions
    Publication date: August 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 2

    Author(s): Ozan Ozkan, Hilal Turkoglu Sasmazel

    In the study presented here, in order to improve the surface functionality and topography of poly (ε-caprolactone) (PCL)/chitosan/PCL hybrid tissue scaffolds fabricated layer by layer with electrospinning technique, an atmospheric pressure nozzle type plasma surface modification was utilized. The optimization of the plasma process parameters was carried out by monitoring the changes in surface hydrophilicity by using contact angle measurements. SEM, AFM and XPS analyses were utilized to observe the changes in topographical and chemical properties of the modified surfaces. The results showed that applied plasma modification altered the nanotopography and the functionality of the surfaces of the scaffolds. The modification applied for 9 min from a distance of 17 cm was found to provide the possible contact angle value (75.163±0.083) closest to the target value which is the value of tissue culture polystyrene (TCPS) petri dishes (∼49.7°), compared to the unmodified samples (84.46±3.86). In vitro cell culture was carried out by L929 mouse fibroblast cell line in order to examine the effects of plasma surface modification on cell–material interactions. Standard MTT assay showed improved cell viability on/within modified scaffolds confirmed with the observations of the cell attachment and the morphology by means of SEM, fluorescence and confocal imaging. The experiments performed in the study proved the enhanced biocompatibility of the nozzle type dry air plasma modified scaffolds.





  • Effects of macroporous hydroxyapatite carriers on the growth and function of human hepatoblasts derived from fetal hepatocytes
    Publication date: August 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 2

    Author(s): Takaaki Ishii, Hiroshi Saito, Yuji Komizu, Ryuichi Tomoshige, Taku Matsushita

    Improvement of three-dimensional (3D) culture conditions, including substrates for cell growth, is needed for various cell-based applications. In this study, we developed hydroxyapatite (HAp) macroporous carriers having several pore size distributions and tried to obtain the findings about the effective pore sizes for the growth and function of hepatoblasts derived from human fetal hepatocytes. Cellular CYP3A4 activity was significantly enhanced when 20% HAp macroporous carrier was used, reaching 1.49±0.28 pmol/106 cells/min of benzyloxyresorufin-O-dealkylation activity, which is comparable to that of primary human hepatocytes from livers of adult donors. Analysis of the pore size (the radius of curvature) distribution of each HAp carrier using a 3D-electron beam surface roughness analyzer revealed two peaks of pore size distribution at 30–40 μm and 70–80 μm, respectively. Thirty-five percent of the pores in the 20% carrier had a size distribution within 50–80 μm. Especially, pores of 70–80 μm were more abundant in the 20% HAp carrier than in the 10% and 30% HAp carriers. These results suggested that a HAp carrier with the pore size distribution of 50–80 μm might be effective for cell growth and function in human hepatoblasts derived from fetal hepatocytes.





  • Insights into the formation mechanism of chloropropanol fatty acid esters under laboratory-scale deodorization conditions
    Publication date: August 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 2

    Author(s): Katsuhito Hori, Natsuko Hori-Koriyama, Kazunobu Tsumura, Eiichiro Fukusaki, Takeshi Bamba

    Chloropropanol fatty acid esters (CPFAEs) are well-known contaminants in refined oils and fats, and several research groups have studied their formation. However, the results obtained in these studies were not satisfactory because the CPFAEs were not analyzed comprehensively. Thus, in the present study, a comprehensive analysis was performed to obtain new details about CPFAE formation. Each lipid (monopalmitin, dipalmitin, tripalmitin, monoolein, diolein, triolein, and crude palm oil) was heated at 250°C for 90 min, and the CPFAEs were analyzed using supercritical fluid chromatography/tandem mass spectrometry. It was found that CP fatty acid monoesters were formed from monoacylglycerols and diacylglycerols after heating in the presence of a chlorine compound. In addition, CP fatty acid diesters were formed from diacylglycerols and triacylglycerols under the same conditions. In the case of crude palm oil, only CP fatty acid diesters were formed. Therefore, these results indicated that CPFAEs in refined palm oil were formed mainly from triacylglycerols.





  • Concentration of MS2 phage in river water by a combined ferric colloid adsorption and foam separation-based method, with MS2 phage leaching from ferric colloid
    Publication date: August 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 2

    Author(s): Yoshihiro Suzuki, Takumi Kobayashi, Masateru Nishiyama, Tomoya Kono

    The concentration of MS2 phage as a model RNA virus in river water using a combined ferric colloid adsorption and foam separation-based method was examined. The MS2 phage concentrations were determined by the plaque-forming unit (PFU) method and reverse transcription quantitative PCR (RT-qPCR) analysis. When ferric colloid adsorption was performed prior to foam separation, MS2 phage was effectively removed from river water and concentrated in the generated foam within 7 min. The removal efficiency was >99% at the optimum iron and casein concentrations of 5 mg-Fe/L and 10 mg/L, respectively. Furthermore, based on the analysis of the collected ferric colloid dissolved using deferoxamine, the MS2 concentration in the colloid-dissolved solution was 190-fold higher than that found in raw water according to RT-qPCR analysis. This is a novel method for concentrating RNA viruses to facilitate their detection in river water using coagulation and foam separation combined with chelate dissolution of ferric flocs.





  • Ophthalmic acid accumulation in an Escherichia coli mutant lacking the conserved pyridoxal 5′-phosphate-binding protein YggS
    Publication date: Available online 12 July 2016
    Source:Journal of Bioscience and Bioengineering

    Author(s): Tomokazu Ito, Ayako Yamauchi, Hisashi Hemmi, Tohru Yoshimura

    Escherichia coli YggS is a highly conserved pyridoxal 5′-phosphate (PLP)-binding protein whose biochemical function is currently unknown. A previous study with a yggS-deficient E. coli strain (ΔyggS) demonstrated that YggS controls l-Ile- and l-Val-metabolism by modulating 2-ketobutyrate (2-KB), l-2-aminobutyrate (l-2-AB), and/or coenzyme A (CoA) availability in a PLP-dependent fashion. In this study, we found that ΔyggS accumulates an unknown metabolite as judged by amino acid analyses. LC/MS and MS/MS analyses of the compound with propyl chloroformate derivatization, and co-chromatography analysis identified this compound as γ-l-glutamyl-l-2-aminobutyryl-glycine (ophthalmic acid), a glutathione (GSH) analogue in which the l-Cys moiety is replaced by l-2-AB. We also determine the metabolic consequence of the yggS mutation. Absence of YggS initially increases l-2-AB availability, and then causes ophthalmic acid accumulation and CoA limitation in the cell. The expression of a γ-glutamylcysteine synthetase and a glutathione synthetase in a ΔyggS background causes high-level accumulation of ophthalmic acid in the cells (∼1.2 nmol/mg cells) in a minimal synthetic medium. This opens the possibility of a first fermentative production of ophthalmic acid.