JBB : Journal of Bioscience and Bioengineering

Journal of Bioscience and Bioengineering vol.124 cover

 



  • Engineering of global regulators and cell surface properties toward enhancing stress tolerance in Saccharomyces cerevisiae
    Publication date: December 2017
    Source:Journal of Bioscience and Bioengineering, Volume 124, Issue 6

    Author(s): Kouichi Kuroda, Mitsuyoshi Ueda

    Microbial cell factories are subject to various stresses, leading to the reductions of metabolic activity and bioproduction efficiency. Therefore, the development of stress-tolerant microorganisms is important for improving bio-production efficiency. Recently, modifications of cell surface properties and master regulators have been shown to be effective approaches for enhancing stress tolerance. The cell surface is an attractive target owing to its interactions with the environment and its role in transmitting environmental information. Cell surface engineering in yeast has enabled the convenient modification of cell surface properties. Displaying random peptide libraries and subsequent screening can successfully improve stress tolerance. Furthermore, master regulators including transcription factors are also promising target to be engineered because stress tolerance is determined by many cooperative factors and modification of master regulators can simultaneously affect the expression of multiple downstream genes. The key single amino acid mutations in transcription factors have been identified by analyzing tolerant yeasts that were isolated by adaptive evolution under stress conditions. This enabled the reconstruction of stress-tolerant yeast without burdening cells by introducing the identified mutations. Therefore, for the construction of stress-tolerant yeast from any strains, these two approaches are promising alternatives to conventional overexpression and deletion of stress-related genes.





  • Transduction of a Neospora caninum antigen gene into mammalian cells using a modified Bombyx mori nucleopolyhedrovirus for antibody production
    Publication date: December 2017
    Source:Journal of Bioscience and Bioengineering, Volume 124, Issue 6

    Author(s): Tatsuya Kato, Kohei Itagaki, Mai Yoshimoto, Rikito Hiramatsu, Hamizah Suhaimi, Tetsuya Kohsaka, Enoch Y. Park

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) can easily enter and transduce foreign genes into mammalian cells, but these functions are difficult for Bombyx mori nucleopolyhedrovirus (BmNPV). In this study, we investigated the induction of antibody production in mice immunized with an engineered BmNPV. The GP64 of BmNPV (BmGP64) was replaced with the GP64 of AcMNPV (AcGP64); this construct, designated BmNPVΔbgp/AcGP64, displays AcGP64 on the surface of BmNPV. The Neospora caninum antigen (NcSRS2) expression cassette, consisting of the cytomegalovirus immediate-early promoter and NcSRS2 from N. caninum, was inserted into BmNPVΔbgp/AcGP64; this construct was designated BmNPVΔbgp/AcGP64/SRS2. For comparison, AcMNPV/SRS2, which contains the same NcSRS2 expression cassette as for BmNPVΔbgp/AcGP64, was also constructed. NcSRS2 was expressed in HEK293T cells when the engineered BmNPVs were transduced at a multiplicity of infection of 150. BmNPVΔbgp/AcGP64/SRS2 induced the production of NcSRS2-specific antibodies in mice, whereas AcMNPV/SRS2 and the control BmNPV did not. These results suggest that BmNPV prepared from silkworm hemolymph induces the production of antigen-specific antibodies in immunized mice and can be used for antibody production and vaccine development.





  • Activation of cryptic phthoxazolin A production in Streptomyces avermitilis by the disruption of autoregulator-receptor homologue AvaR3
    Publication date: December 2017
    Source:Journal of Bioscience and Bioengineering, Volume 124, Issue 6

    Author(s): Dian Anggraini Suroto, Shigeru Kitani, Kiyoko T. Miyamoto, Yasuko Sakihama, Masayoshi Arai, Haruo Ikeda, Takuya Nihira

    The genomes of actinomycetes encode many cryptic novel/useful bioactive compounds, but access to these cryptic secondary metabolites remains limited. Streptomyces avermitilis predominantly produces three polyketide antibiotics (avermectin, filipin, and oligomycin) but has the potential to produce more secondary metabolites based on the number of cryptic biosynthetic gene clusters. Here, we extensively investigated the metabolite profiles of a gene disruptant of AvaR3 (an autoregulator receptor homologue), which is involved in the pleiotropic regulation of antibiotic production and cell morphology. Unlike the wild-type strain, the avaR3 mutant accumulated compound 3 in the culture. The chemical structure of compound 3 was elucidated on the basis of various spectroscopic analyses, and was identified as phthoxazolin A, a cellulose synthesis inhibitor. Bioassays demonstrated that compound 3 exerts growth inhibitory activity against a broad range of plant pathogenic oomycetes. Moreover, unlike avermectin production, phthoxazolin A (3) production was negatively controlled by avenolide, a new type of autoregulator in streptomycetes, through the function of AvaR3. These results suggest that the genetic manipulation of autoregulator receptor homologues would be a valuable tool for the discovery of cryptic bioactive compounds.





  • Mathematical determination of kinetic parameters for assessing the effect of the organic solvent on the selectivity of peptide synthesis with immobilized α-chymotrypsin
    Publication date: December 2017
    Source:Journal of Bioscience and Bioengineering, Volume 124, Issue 6

    Author(s): Carola Bahamondes, Lorena Wilson, Fanny Guzmán, Andrés Illanes

    The synthesis of the dipeptide N-benzoyl-l-tyrosine-l-argininamide (BTAA) was conducted under kinetic control with N-benzoyl-l-tyrosine ethyl ester as acyl donor and argininamide as nucleophile using immobilized α-chymotrypsin as catalyst. Using a mathematical procedure, the kinetic constants corresponding to the proposed mechanism of peptide synthesis were determined in three different cosolvent media, namely, ethanol, diglyme and acetonitrile. These constants were used for evaluating the selectivity of glyoxyl-agarose immobilized α-chymotrypsin in the synthesis of BTAA by determining the ratios of synthesis to hydrolysis rates.





  • Characterization of a thermostable mutant of Agaricus brasiliensis laccase created by phylogeny-based design
    Publication date: December 2017
    Source:Journal of Bioscience and Bioengineering, Volume 124, Issue 6

    Author(s): Yuhi Hamuro, Katsuya Tajima, Akiko Matsumoto-Akanuma, Sayaka Sakamoto, Ryutaro Furukawa, Akihiko Yamagishi, Naohito Ohno, Satoshi Akanuma

    Laccases are enzymes that oxidize various aromatic compounds, and therefore they have attracted much attention from the standpoints of medical and industrial applications. We previously isolated the cDNA that codes for a laccase isozyme (Lac2a) from the medicinal mushroom Agaricus brasiliensis (Matsumoto-Akanuma et al., Int. J. Med. Mushrooms, 16, 375–393, 2014). In this study, we first attempted heterologous expression of the wild-type laccase using a Pichia pastoris secretory expression system. However, the trial was unsuccessful most likely because the enzyme was too unstable and degraded immediately after production. Therefore, we improved the stability of the laccase by using a phylogeny-based design method. We created a mutant laccase in which sixteen original residues were replaced with those found in the phylogenetically inferred ancestral sequence. The resulting mutant protein was successfully produced using the P. pastoris secretory expression system and then purified. The designed laccase showed catalytic properties similar to those of other fungal laccases. Moreover, the laccase is highly thermally stable at acidic and neutral pH and is also stable at alkaline pH at moderate temperatures. We expect that the laccase will serve as a useful tool for enzymatic polymerization of di-phenolic compounds.





  • Biochemical characterization of Aspergillus oryzae recombinant α-l-rhamnosidase expressed in Pichia pastoris
    Publication date: December 2017
    Source:Journal of Bioscience and Bioengineering, Volume 124, Issue 6

    Author(s): Mai Ishikawa, Yoshihito Shiono, Takuya Koseki

    An α-l-rhamnosidase-encoding gene from Aspergillus oryzae, which belongs to the glycoside hydrolase family 78, was cloned and expressed in Pichia pastoris. SDS-PAGE of the purified recombinant α-l-rhamnosidase protein revealed smeared bands with apparent molecular mass of 90–130 kDa. After N-deglycosylation, the recombinant enzyme showed a molecular mass of 70 kDa. The enzyme exhibited optimal activity at a pH of 5.0 and a temperature of 70 °C. Specific activity of the enzyme was higher toward hesperidin than toward naringin, which consist of α-1,6 and α-1,2 linkages, respectively. The activity was also higher toward hesperidin than toward rutin, which consist of 7-O- and 3-O-glycosyl linkages of flavonoids, respectively. Kinetic analysis of the enzyme showed that the Michaelis constant (K m) was lowest toward rutin, moderate toward naringin, and higher toward p-nitrophenyl-α-l-rhamnopyranoside and hesperidin. Its high catalytic efficiency (k cat/K m) toward rutin was results of its low K m value while its high catalytic efficiency toward hesperidin was results of a considerably high k cat value.





  • Investigation of the Escherichia coli membrane transporters involved in the secretion of d-lactate-based oligomers by loss-of-function screening
    Publication date: December 2017
    Source:Journal of Bioscience and Bioengineering, Volume 124, Issue 6

    Author(s): Camila Utsunomia, Chiaki Hori, Ken'ichiro Matsumoto, Seiichi Taguchi

    d-Lactate (LA)-based oligomers (D-LAOs) are unusual oligoesters consisting of d-LA and d-3-hydroxybutyrate that are produced and secreted by engineered Escherichia coli grown on glucose. The cells heterologously express LA-polymerizing polyhydroxyalkanoate synthase and monomer-supplying enzymes. In this study, we attempted to identify the D-LAO secretion route in E. coli, which is thought to be mediated by intrinsic membrane proteins. To this end, a loss-of-function screening of D-LAO secretion was carried out using 209 single-gene membrane protein deletants, which are involved in the transport of organic compounds. Among the deletants of the outer membrane-associated proteins, ΔompF and ΔompG exhibited diminished D-LAOs secretion and elevated intracellular D-LAO accumulation. When the ompF and ompG expression levels were down- and up-regulated with plasmids harboring these genes, the secreted amounts of the D-LAOs were changed in correspondence with their expression levels. These results suggest that porins mediate D-LAOs transport through the outer membrane. In particular, OmpF is likely to be the major porin involved in the spontaneous secretion of D-LAOs due to the high basal expression of ompF in the parental strain. Among the deletants of the inner membrane-associated proteins, the ΔmngA, ΔargT, ΔmacA, ΔcitA and ΔcpxA strains were selected by the screening. These genes are also candidate transporters related to D-LAO secretion, suggesting the presence of multiple secretion routes across the inner membrane. To the best of our knowledge, this is the first report on the mechanism of the microbial secretion of oligoesters.





  • Highly chemoselective and efficient production of 2,6-difluorobenzamide using Rhodococcus ruber CGMCC3090 resting cells
    Publication date: December 2017
    Source:Journal of Bioscience and Bioengineering, Volume 124, Issue 6

    Author(s): Rui Tang, Yanbing Shen, Min Wang, Ying Zhai, Qian Gao

    Chemoselective biocatalytic hydrolysis of nitriles is a valuable alternative to chemical hydrolysis that operates under harsh conditions. 2,6-Difluorobenzamide (DFBAM) is an essential intermediate derived from synthesis of benzoyl urea insecticide from 2,6-difluorobenzonitrile (DFBN). High yield of DFBAM was achieved, and the method using resting cells of Rhodococcus ruber CGMCC3090 exhibited excellent product specificity. The reaction parameters for DFBAM biosynthesis were also investigated. The resting cells effectively converted DFBN at high concentrations of up to 3.5 mol L−1 without forming acids or other by-products. Therefore, biological production of DFBAM features high yield, chemoselectivity, low cost, and environment friendliness and is more suitable to the industry than chemical synthesis techniques.





  • Negative chemotaxis of Ralstonia pseudosolanacearum to maleate and identification of the maleate chemosensory protein
    Publication date: December 2017
    Source:Journal of Bioscience and Bioengineering, Volume 124, Issue 6

    Author(s): Mattana Tunchai, Akiko Hida, Shota Oku, Yutaka Nakashimada, Toshiyuki Nikata, Takahisa Tajima, Junichi Kato

    Ralstonia pseudosolanacearum Ps29 was repelled by maleate. Screening of a complete collection of Ps29 single-methyl-accepting chemotaxis protein (mcp) gene mutants identified the RSp0303 homolog (McpP) as a chemotaxis sensor mediating negative chemotaxis to maleate. Interestingly, the mcpP-deletion mutant was attracted to maleate, indicating that this bacterium expresses a MCP(s) for both positive and negative chemotaxis to maleate. We constructed a Ps29 derivative (designated POC14) harboring deletions in 14 individual mcp genes, including mcpP, to characterize McpP. Introduction of a plasmid harboring the mcpP gene (pPS16) restored the ability to negatively respond to maleate, confirming that McpP is a MCP for negative chemotaxis to maleate. We thought that maleate might be applied to controlling plant infection by R. pseudosolanacearum. To evaluate this possibility, we measured chemotactic responses of seven other virulent R. pseudosolanacearum strains to maleate. We confirmed that they harbored functional mcpP orthologues, but they showed no chemotactic responses to maleate. Quantitative RT-PCR analysis revealed that these seven R. pseudosolanacearum strains did not show negative chemotaxis to maleate because of negligible transcription of the mcpP genes. We compared the chemotactic responses of POC14 and POC14[pPS16] toward various chemicals and found that McpP senses inorganic phosphate as a chemoattractant.





  • Vinegar production from post-distillation slurry deriving from rice shochu production with the addition of caproic acid-producing bacteria consortium and lactic acid bacterium
    Publication date: December 2017
    Source:Journal of Bioscience and Bioengineering, Volume 124, Issue 6

    Author(s): Hua-Wei Yuan, Li Tan, Hao Chen, Zhao-Yong Sun, Yue-Qin Tang, Kenji Kida

    To establish a zero emission process, the post-distillation slurry of a new type of rice shochu (NTRS) was used for the production of health promoting vinegar. Since the NTRS post-distillation slurry contained caproic acid and lactic acid, the effect of these two organic acids on acetic acid fermentation was first evaluated. Based on these results, Acetobacter aceti CICC 21684 was selected as a suitable strain for subsequent production of vinegar. At the laboratory scale, acetic acid fermentation of the NTRS post-distillation slurry in batch mode resulted in an acetic acid concentration of 41.9 g/L, with an initial ethanol concentration of 40 g/L, and the acetic acid concentration was improved to 44.5 g/L in fed-batch mode. Compared to the NTRS post-distillation slurry, the vinegar product had higher concentrations of free amino acids and inhibition of angiotensin I converting enzyme activity. By controlling the volumetric oxygen transfer coefficient to be similar to that of the laboratory scale production, 45 g/L of acetic acid was obtained at the pilot scale, using a 75-L fermentor with a working volume of 40 L, indicating that vinegar production can be successfully scaled up.





  • Continuous nitrogen removal by a single-stage reactor packed with ring-laced string medium
    Publication date: December 2017
    Source:Journal of Bioscience and Bioengineering, Volume 124, Issue 6

    Author(s): Masahiro Tatara, Shu Ishikawa, Yoshiyuki Ueno

    The efficiency of nitrogen removal by a partial-nitritation/anammox (PNA) reaction was investigated using a packed-bed reactor in which ring-laced strings were used as the supporting medium. A stable population of PNA microorganisms was established from typical activated sludge, after less than two months of acclimation in the packed-bed reactor, by applying a high nitrogen-loading rate (NLR: 0.53 kg/m3/d) and short hydraulic retention time (HRT: 1.8 h). The stability of reactor performance was confirmed in industrial wastewater (IW), demonstrating a nitrogen removal efficiency (NRE) of greater than 77% during 260 days of continuous operation, between 0.19 and 0.53 kg/m3/d of NLR. Partial nitrification was adequately controlled by low-level oxygen supply to the reactor. Pyro-tag sequencing analysis of the biofilm revealed a clear abundance of anammox bacteria in the inner part of the biofilm and ammonium-oxidizing bacteria in the outer part. In the synthetic inorganic medium (SIM), the microbial community structure did not change drastically between the early and late phases of the experiment's continuous operation, which lasted over 200 days. In IW, however, the existence ratio of anammox bacteria decreased to 4% on day 249 of continuous operation. The number of detected operational taxonomic units (OTUs) increased in the IW, implying that the community structure was widely diversified. However, anammox bacteria could propagate sufficiently to catalyze nitrogen removal under this condition because the NRE was stable at approximately 88%.





  • Decolorization of reactive azo dyes using a sequential chemical and activated sludge treatment
    Publication date: December 2017
    Source:Journal of Bioscience and Bioengineering, Volume 124, Issue 6

    Author(s): Ken Meerbergen, Sam Crauwels, Kris A. Willems, Raf Dewil, Jan Van Impe, Lise Appels, Bart Lievens

    Textile wastewater contains high concentrations of organic substances derived from diverse dyes and auxiliary chemicals, some of which are non-biodegradable and/or toxic. Therefore, it is essential that textile wastewater is treated and that these substances are removed before being discharged into the environment. A combination of advanced oxidation processes (AOPs) to obtain partial dye degradation followed by a biological treatment has been suggested as a promising method for cost-effective decolorization of wastewater. The aim of this study was to develop and evaluate a combined method of partial Fenton's oxidation and biological treatment using activated sludge for decolorization of azo dyes, which represent an important group of recalcitrant, toxic textile dyes. Using Reactive Violet 5 (RV5) as a model dye, color removal was significantly higher when the combined Fenton treatment/activated sludge method was used, as opposed to separate application of these treatments. More specifically, pretreatment with Fenton's reagent removed 52.9, 83.9 and 91.3 % of color from a 500 mg l−1 RV5 aqueous solution within 60 min when H2O2 concentrations of 1.0, 1.5, and 2.0 mM were used, respectively. Subsequent biological treatment was found to significantly enhance the chemical treatment, with microbial decolorization removing 70.2 % of the remaining RV5 concentration, on average. Molecular analysis of the microbial community within the activated sludge revealed that exposure to RV5 shifted the community composition from diverse towards a highly-specialized community harboring taxa with azo dye degrading activity, including Trichosporon, Aspergillus and Clostridium species.





  • Stimulation of d- and l-lactate dehydrogenases transcriptional levels in presence of diammonium hydrogen phosphate resulting to enhanced lactic acid production by Lactobacillus strain
    Publication date: December 2017
    Source:Journal of Bioscience and Bioengineering, Volume 124, Issue 6

    Author(s): Mamata Singhvi, Takeshi Zendo, Hiroshi Iida, Digambar Gokhale, Kenji Sonomoto

    The present study revealed the effect of nitrogen sources on lactic acid production and stimulation of d- and l-lactate dehydrogenases (LDH) of parent Lactobacillus lactis NCIM 2368 and its mutant RM2-24 generated after UV mutagenesis. Both the parent and mutant strains were evaluated for d-lactic acid production in control and modified media. The modified media did not show remarkable effect on lactic acid production in case of parent whereas mutant exhibited significant enhancement in d-lactic acid production along with the appearance of l-lactic acid in the broth. Both LDH activities and specific activities were found to be higher in mutant than the parent strain. These results suggested that the diammonium hydrogen phosphate in modified media triggered the expression of LDH genes leading to enhanced lactic acid production. This observation has been proved by studying the expression levels of d- and l-LDH genes of parent and mutant in control and modified media using quantitative RT-PCR technique. In case of mutant, the transcriptional levels of d-LDH and l-LDH increased ∼17 fold and ∼1.38 fold respectively in modified medium compared to the values obtained with control medium. In case of parent, no significant change in transcriptional levels of d- and l-LDH was found when the cells were grown in either control medium or modified medium. This study suggested that the mutant, RM2-24 has l-LDH gene which is expressed in presence of (NH4)2HPO4 resulting in l-lactic acid production. Co-production of l-lactic acid in d-lactic acid fermentation may be detrimental in the PLA production.





  • Intracellular Ca2+ is an essential factor for cell damage induced by unsaturated carbonyl compounds
    Publication date: December 2017
    Source:Journal of Bioscience and Bioengineering, Volume 124, Issue 6

    Author(s): Tsunehito Higashi, Yosuke Mai, Yuichi Mazaki, Soichi Miwa

    The unsaturated carbonyl compounds are known as the environmental pollutants. Acrolein (ACR) and methyl vinyl ketone (MVK) are representative unsaturated carbonyl compounds. ACR is contained in smoke, automobile exhaust, industrial waste, and several foods. MVK is widely used as the industrial chemical. Although ACR and MVK are highly toxic, the molecular mechanism for their cytotoxicity has been unclear. We have previously reported that ACR and MVK are major cytotoxic compounds in the gas phase of cigarette smoke, and protein kinase C (PKC) inhibitor and NADPH oxidases inhibitor partially rescued cells from ACR- or MVK-induced cell death (Noya et al., Toxicology, 314, 1–10, 2013). PKC translocation, which is hallmark for PKC activation, and cell damage were induced by treatment of cultured cells with ACR or MVK. Intracellular Ca2+ chelator completely suppressed ACR- or MVK-induced PKC translocation to the cell membrane and cell damage, while extracellular Ca2+ chelator had no effects on ACR- and MVK-induced cytotoxicity. These results suggest that intracellular Ca2+ is an essential factor for cell damage caused by both PKC-dependent and PKC-independent pathways, and mobilization of Ca2+ from intracellular Ca2+ stores is induced by ACR or MVK.





  • Raft-dependent endocytic movement and intracellular cluster formation during T cell activation triggered by concanavalin A
    Publication date: December 2017
    Source:Journal of Bioscience and Bioengineering, Volume 124, Issue 6

    Author(s): Satomi Yabuuchi, Satoshi Endo, KeangOk Baek, Kunihide Hoshino, Yoshio Tsujino, Mun'delanji C. Vestergaard, Masahiro Takagi

    Certain food ingredients can stimulate the human immune system. A lectin, concanavalin A (ConA), from Canavalia ensiformis (jack bean) is one of the most well-known food-derived immunostimulants and mediates activation of cell-mediated immunity through T cell proliferation. Generally, T cell activation is known to be triggered by the interaction between T cells and antigen-presenting cells (APCs) via a juxtacrine (contact-dependent) signaling pathway. The mechanism has been well characterized and is referred to as formation of the immunological synapse (IS). We were interested in the mechanism behind the T cell activation by food-derived ConA which might be different from that of T cell activation by APCs. The purpose of this study was to characterize T cell activation by ConA with regard to (i) movement of raft domain, (ii) endocytic vesicular transport, (iii) the cytoskeleton (actin and microtubules), and (iv) cholesterol composition. We found that raft-dependent endocytic movement was important for T cell activation by ConA and this movement was dependent on actin, microtubules, and cholesterol. The T cell signaling mechanism triggered by ConA can be defined as endocrine signaling which is distinct from the activation process triggered by interaction between T cells and APCs by juxtacrine signaling. Therefore, we hypothesized that T cell activation by ConA includes both two-dimensional superficial raft movement on the membrane surface along actin filaments and three-dimensional endocytic movement toward the inside of the cell along microtubules. These findings are important for developing new methods for immune stimulation and cancer therapy based on the function of ConA.





  • Development of a rapid immunoassay system: Luminescent detection of antigen-associated antibody-luciferase in the presence of a dye that absorbs light from free antibody-luciferase
    Publication date: December 2017
    Source:Journal of Bioscience and Bioengineering, Volume 124, Issue 6

    Author(s): Akihiro Mori, Teruyo Ojima-Kato, Shingo Fuchi, Shinichi Kaiya, Takaaki Kojima, Hideo Nakano

    In this report, we developed a rapid immunoassay system, designated the bioluminescent interference gathering optical (BINGO) assay, which required no time-consuming washing steps for removal of unbound antibodies. This system employed a luciferase (Luc)-conjugated antibody (LucAb) and a dye that absorbed light from the LucAb. The antigen-associated LucAb was localized by transfer of an antigen to the detector-side of a chamber where a detector photomultiplier tube (PMT) was installed. In contrast, the free LucAb was distributed throughout the solution, and the light emitted by the free LucAb was absorbed by the dye. Therefore, only light from LucAb associated with antigen could be detected by the PMT. The new system could be used to rapidly detect the amount of antigen-antibody-Luc complex by collecting steps, such as centrifugation or magnetic collection of antibody-coated magnetic beads. Proof-of-principle experiments were performed using a model system with streptavidin beads and biotinylated Luc. The feasibility of the system was demonstrated using magnetic beads coated with anti-Escherichia coli O157 antibody, enabling detection of 4 × 103 cells in only 15 min. Thus, this system may have applications in a variety of biomedical fields.





  • Solid-phase analytical derivatization for gas-chromatography–mass-spectrometry-based metabolomics
    Publication date: December 2017
    Source:Journal of Bioscience and Bioengineering, Volume 124, Issue 6

    Author(s): Emi Takeo, Ryoichi Sasano, Shuichi Shimma, Takeshi Bamba, Eiichiro Fukusaki

    A novel derivatization method for gas chromatography/mass spectrometry (GC/MS)-based metabolomics was developed, based on solid-phase analytical derivatization (SPAD) with methoximation followed by trimethylsilylation. This SPAD method realized derivatization on solid phases combining strong anion exchange with strong cation exchange. To omit a sample condensation process, GC/MS injection was performed using a large-volume injection mode. This mode uses a stomach-shaped insert, and enables a large quantity of sample to be vaporized and introduced into the GC/MS system. In the present study, several parameters were investigated for each SPAD step. The optimal derivatization conditions were determined to be 3-min-methoximation with 5 μL of >5% methoxyamine solution, and 10-min-trimethylsilylation with 25 μL of N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA). Derivatized analytes were effectively eluted with 25 μL of n-hexane. The influences of coexisting substances were also investigated. Coexisting saccharides did not significantly affect the derivatization of analytes. Moreover, saccharides were efficiently washed out using 80% (v/v) acetonitrile in water. The influences of coexisting sodium chloride were negated by dilution of the sample solution with water. The developed method enables the derivatization of both anionic and cationic metabolites, and high-throughput sample preparation. The coverage of detectable metabolites for the developed method was similar to that of the conventional method. This is the first report of a SPAD-based human plasma metabolome analysis protocol.





  • Characterization of d-succinylase from Cupriavidus sp. P4-10-C and its application in d-amino acid synthesis
    Publication date: Available online 15 November 2017
    Source:Journal of Bioscience and Bioengineering

    Author(s): Yosuke Sumida, Sachio Iwai, Yoshiaki Nishiya, Shinya Kumagai, Toshihide Yamada, Masayuki Azuma

    d-Amino acids are important building blocks for various compounds, such as pharmaceuticals and agrochemicals. A more cost-effective enzymatic method for d-amino acid production is needed in the industry. We improved a one-pot enzymatic method for d-amino acid production by the dynamic kinetic resolution of N-succinyl amino acids using two enzymes: d-succinylase (DSA) from Cupriavidus sp. P4-10-C, which hydrolyzes N-succinyl-d-amino acids enantioselectively to their corresponding d-amino acid, and N-succinyl amino acid racemase (NSAR, EC.4.2.1.113) from Geobacillus stearothermophilus NCA1503. In this study, DSA and NSAR were purified and their properties were investigated. The optimum temperature of DSA was 50°C and it was stable up to 55°C. The optimum pH of DSA and NSAR was around 7.5. In d-phenylalanine production, the optical purity of product was improved to 91.6% ee from the examination about enzyme concentration. Moreover, 100 mM N-succinyl-dl-tryptophan was converted to d-tryptophan at 81.8% yield with 94.7% ee. This enzymatic method could be useful for the industrial production of various d-amino acids.

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  • Identification of the glucosamine kinase in the chitinolytic pathway of Thermococcus kodakarensis
    Publication date: Available online 14 November 2017
    Source:Journal of Bioscience and Bioengineering

    Author(s): Mehwish Aslam, Naoya Takahashi, Kohei Matsubara, Tadayuki Imanaka, Tamotsu Kanai, Haruyuki Atomi

    Although the chitinolytic pathway of the hyperthermophilic archaeon Thermococcus kodakarensis is well-studied, the genome does not contain genes homologous to previously identified glucosamine kinase genes. As some ADP-dependent glucokinases in the order Thermococcales exhibit phosphorylation activities for both glucose and glucosamine in vitro, the homolog in T. kodakarensis, encoded by TK1110, was selected as a candidate for the missing glucosamine kinase gene. The purified, recombinant TK1110 enzyme exhibited phosphorylation activities for not only glucose but also glucosamine and N-acetylglucosamine. Kinetic analysis indicated that activity towards glucosamine was as significant as that towards glucose. In order to determine the physiological role of TK1110 in the chitinolytic pathway of T. kodakarensis, a gene disruption strain of TK1110 was constructed. When grown in chitin-containing medium, the TK1110 disruption resulted in almost complete impairment in chitin degradation, and a complete loss of chitin-dependent H2 production. As H2 production is tightly linked to cell growth in T. kodakarensis, the present results strongly suggest that TK1110 functions as the glucosamine kinase responsible for the chitin degradation in T. kodakarensis.

    Graphical abstract

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  • Inhibitory effect of the extract of rhizome of Curcuma longa L in gelatinase activity and its effect on human skin
    Publication date: Available online 13 November 2017
    Source:Journal of Bioscience and Bioengineering

    Author(s): Keiko Muta, Shinji Inomata, Tadao Fukuhara, Junko Nomura, Toshio Nishiyama, Yoh-ichi Tagawa, Satoshi Amano

    Exposure to UV radiation to human skin up-regulates the synthesis of matrix metalloproteinase (MMP) family. Gelatinases are member of MMPs which have been suggested to play an important role in photoaging such as wrinkle formation. To inhibit gelatinase activity is regarded to be very important to keep healthy skin and to protect wrinkle formation. On the other hand, anti-photoaging agents are expected to be derived from natural resources, especially plants. Plant extracts having gelatinase-inhibitory effect that might be used as safe anti-photoaging ingredient were widely screened. An extract of rhizomes of Curcuma longa L. showed inhibitory effect of gelatinase activity. Curcuminoids and slight amount of compound, 6,11-dihydroxy-3-(4-hydroxy-3-mthoxyphenethyl)-7-[(E)-4-(4-hydroxy-3-methoxyphenyl)-2-oxo-3-butenyl]-10-methoxy-2-oxabicyclo[6.3.1.]dodeca-1(11),8(12),9-trien-5-yl (E)-3-(4-hydroxy-3-methoxyphenyl)-2-propenoate (curcuminoid D) were isolated as the gelatinase-inhibitory components from methanol extract of rhizomes. The structure of curcuminoid D was determined by means of spectral data including 1H- and 13C-NMR, and IR. Curcumin exerted the enhancing effect on deposition of basement membrane component at dermal-epidermal junction in skin equivalent model. Topical application of cream containing turmeric extract significantly improved facial skin elasticity and decreased the number of gelatinase-positive stratum corneum clusters in human facial skins. These results indicated that turmeric is an effective ingredient to improve skin condition and to prevent skin from photoaging by suppressing activation of gelatinase chronically caused by UV.