JBB : Journal of Bioscience and Bioengineering

            

Journal of Bioscience and Bioengineering vol.123 cover

 



  • Enhanced in-cell folding of reversibly cationized transcription factor using amphipathic peptide
    Publication date: Available online 16 January 2017
    Source:Journal of Bioscience and Bioengineering

    Author(s): Midori Futami, Tomoki Nakano, Mayu Yasunaga, Masahiro Makihara, Takashi Asama, Yoshihisa Hagihara, Yoshihiro Nakajima, Junichiro Futami

    The intracellular delivery of functionally active transcription factor proteins is emerging as a promising technique for artificial regulation of cellular functions. However, in addition to the cell membrane, which acts as a barrier to macromolecules, the aggregation-favored properties of structurally flexible transcription factor proteins limit the application of this method. In-cell folding technique can be used to overcome these issues. This technique solubilizes denatured protein by reversible alkyl-disulfide cationization (S-cationization), and simultaneously endows efficient intracellular delivery and folding to the biologically active conformation in the reducing environment of the cytosol. Because cationized protein is internalized into cells by adsorption-mediated endocytosis, endosomal escape is crucial for this technique. In this study, we utilized a sensitive luciferase reporter gene assay to quantitatively evaluate in-cell folding of the artificial transcription factor GAL4-VP16. Although the cationic moiety of S-cationized protein was slightly affected, co-transduction of amphipathic peptide Endo-PORTER dramatically improved in-cell folding efficiency. Live cell imaging of fluorescent-labeled GAL4-VP16 revealed that some of the proteins diffused into the cytosol and nucleus through co-transduction with Endo-PORTER. Real-time monitoring of light output of luciferase revealed the kinetics of in-cell folding, supporting that endosomal-release assisted by Endo-PORTER was stimulated by endosome acidification. Because this method can transduce proteins uniformly and repeatedly into living cells, S-cationized transcription factor proteins are widely applicable for the artificial regulation of cellular functions.





  • Characterization of particulate matter binding peptides screened from phage display
    Publication date: Available online 13 January 2017
    Source:Journal of Bioscience and Bioengineering

    Author(s): Aw Wei Liang Alvin, Masayoshi Tanaka, Mina Okochi

    Particulate matter (PM), especially particulates with diameters of less than 2.5 μm, can penetrate the alveolar region and increase the risk of respiratory diseases. This has stimulated research efforts to develop detection methods so that counter measures can be taken. In this study, four PM binding peptides were obtained by phage display and binding characteristics of these peptides were investigated using the peptide array. The strongest binding peptide, WQDFGAVRSTRS, displayed a binding property, measured in terms of spot intensity, 11.4 times higher than that of the negative control, AAAAA. Inductively coupled plasma mass spectrometry (ICPMS) analysis of the transition metal compounds in the PM bound to the peptide spots was performed, and two peptides showed higher binding towards Cu and Zn compounds in PM. These results suggest that the screened peptides could serve as an indicator of transition metal compounds, which are related to adverse health effects, contained in PM.





  • Medium optimization based on yeast's elemental composition for glutathione production in Saccharomyces cerevisiae
    Publication date: Available online 12 January 2017
    Source:Journal of Bioscience and Bioengineering

    Author(s): Maximilian Schmacht, Eric Lorenz, Ulf Stahl, Martin Senz

    The production of glutathione (GSH) or GSH enriched yeast is still in the focus of research driven by a high industrial interest. In this study, an optimal growth rate for GSH production via Saccharomyces cerevisiae Sa-07346 was investigated. To further improve the fermentation process in a way that it is independent of lots, the influence of different WMIX medium compositions on biomass and GSH production was studied. Thereby, the fermentation medium was adjusted based on yeast's elemental composition. The resulting chemically defined fermentation medium led to high cell densities in fed-batches. Therefore, it has the potential to be applied successfully for other high cell density yeast fermentation processes. As cysteine is the key component for GSH production, different cysteine addition strategies were studied and finally, a continuous cysteine feeding was applied in the late stage of fermentation. Thereby, a GSH concentration of 1459 ± 57 mg/l was reached by continuously feeding cysteine, which meant an increase to 253% compared to the control without cysteine addition (577 mg/l GSH).





  • Characterization of a novel DyP-type peroxidase from Streptomyces avermitilis
    Publication date: Available online 11 January 2017
    Source:Journal of Bioscience and Bioengineering

    Author(s): Kanako Sugawara, Yuriko Nishihashi, Tomomi Narioka, Toru Yoshida, Mifumi Morita, Yasushi Sugano

    DyP-type peroxidases are a heme peroxidase family with unique properties whose members are widely distributed from prokaryotes to eukaryotes. DyP-type peroxidases are subdivided into class P, I and V based on structure-based sequence alignment. Class V enzymes possess degradation activities for anthraquinone dyes, and include extra sequences compared with class P and I. Class V enzymes are mainly found in fungi, with only two such proteins, AnaPX and DyP2, reported in bacteria. Here, we heterologously expressed, purified and biochemically characterized SaDyP2 protein, predicted to belong to class V. SaDyP2 was purified as a ∼50 kDa enzyme containing a heme cofactor and was found to oxidize the typical peroxidase substrates, ABTS and DMP. SaDyP2 was generally thermostable and exhibited a lower optimal pH, a feature typical of DyP-type peroxidases. It also degraded anthraquinone dyes, a specific substrate of DyP-type peroxidases, although the k cat for SaDyP2 was lower than that for other class V enzymes. The K m value of SaDyP2 for anthraquinone dye was similar to that of other enzymes of this class. Homology modeling revealed that the structure of SaDyP2 best fit that of class V enzymes.





  • Butyrate production under aerobic growth conditions by engineered Escherichia coli
    Publication date: Available online 11 January 2017
    Source:Journal of Bioscience and Bioengineering

    Author(s): Naoya Kataoka, Alisa S. Vangnai, Thunyarat Pongtharangkul, Toshiharu Yakushi, Kazunobu Matsushita

    Butyrate is an important industrial platform chemical. Although several groups have reported butyrate production under oxygen-limited conditions by a native producer, Clostridium tyrobutylicum, and by a metabolically engineered Escherichia coli, efforts to produce butyrate under aerobic growth conditions have met limited success. Here, we constructed a novel butyrate synthetic pathway that functions under aerobic growth conditions in E. coli, by modifying the 1-butanol synthetic pathway reported previously. The pathway consists of phaA (acetyltransferase) and phaB (NADPH-dependent acetoacetyl-CoA reductase) from Ralstonia eutropha, phaJ ((R)-specific enoyl-CoA hydratase) from Aeromonas caviae, ter (trans-enoyl-CoA reductase) from Treponema denticola, and endogenous thioesterase(s) of E. coli. To evaluate the potential of this pathway for butyrate production, culture conditions, including pH, oxygen supply, and concentration of inorganic nitrogen sources, were optimized in a mini-jar fermentor. Under the optimal conditions, butyrate was produced at a concentration of up to 140 mM (12.3 g/L in terms of butyric acid) after 54 h of fed-batch culture.





  • Bacterial community dynamics in a biodenitrification reactor packed with polylactic acid/poly (3-hydroxybutyrate-co-3-hydroxyvalerate) blend as the carbon source and biofilm carrier
    Publication date: Available online 9 January 2017
    Source:Journal of Bioscience and Bioengineering

    Author(s): Tianlei Qiu, Ying Xu, Min Gao, Meilin Han, Xuming Wang

    While heterotrophic denitrification has been widely used for treating such nitrogen-rich wastewater, it requires the use of additional carbon sources. With fluctuations in the nitrate concentration in the influent, controlling the C/N ratio to avoid carbon breakthrough becomes difficult. To overcome this obstacle, solid-phase denitrification (SPD) using biodegradable polymers has been used, where denitrification and carbon source biodegradation depend on microorganisms growing within the reactor. However, the microbial community dynamics in continuous-flow SPD reactors have not been fully elucidated yet. Here, we aimed to study bacterial community dynamics in a biodenitrification reactor packed with a polylactic acid/poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PLA/PHBV) blend as the carbon source and biofilm carrier. A lab-scale denitrifying reactor filled with a PLA/PHBV blend was used. With 85 mg/L of influent NO3–N concentration and a hydraulic retention time (HRT) of 2.5 h, more than 92% of the nitrate was removed. The bacterial community of inoculated activated sludge had the highest species richness in all samples. Bacterial species diversity in the reactor first decreased and then increased to a stable level. Diaphorobacter species were predominant in the reactor after day 24. In total, 178 clones were retrieved from the 16S rRNA gene clone library constructed from the biofilm samples in the reactor at 62 days of operation, and 80.9% of the clones were affiliated with Betaproteobacteria. Of these, 97.2% were classified into phylotypes corresponding to Diaphorobacter nitroreducens strain NA10B with 99% sequence similarity. Diaphorobacter, Rhizobium, Acidovorax, Rubrivivax, Azospira, Thermomonas, and Acidaminobacter constituted the biofilm microflora in the stably running reactor.





  • Acidic pH with coordinated reduction of basic fibroblast growth factor maintains the glioblastoma stem cell-like phenotype in vitro
    Publication date: Available online 4 January 2017
    Source:Journal of Bioscience and Bioengineering

    Author(s): Elizabeth M. Haley, Samantha G. Tilson, Ursula L. Triantafillu, Justin W. Magrath, Yonghyun Kim

    Glioblastoma stem cells (GSCs) are a unique subpopulation of cells within glioblastoma multiforme (GBM) brain tumors that possess the ability to self-renew and differentiate into bulk tumor cells. GSCs are resistant to currently available treatments and are the likely culprit behind tumor relapse in GBM patients. However, GSCs are currently inaccessible to the larger scientific community because obtaining a sufficient number of GSCs remains technically challenging and cost-prohibitive. Thus, the objective of this study was to develop a more efficient GSC culture strategy that results in a higher cell yield of GSCs at a lower cost. We observed that the basic fibroblast growth factor (bFGF) is indispensable in allowing GSCs to retain an optimal stem cell-like phenotype in vitro, but little change was seen in their stemness when grown with lower concentrations of bFGF than the established protocol. Interestingly, a dynamic fluctuation of GSC protein marker expression was observed that corresponded to the changes in the bFGF concentration during the culture period. This suggested that bFGF alone did not control stem cell-like phenotype; rather, it was linked to the fluctuations of both bFGF and media pH. We demonstrated that a high level of stem cell-like phenotype could be retained even when lowering bFGF to 8 ng/mL when the media pH was simultaneously lowered to 6.8. These results provide the proof-of-concept that GSC expansion costs could be lowered to a more economical level and warrant the use of pH- and bFGF-controlled bioprocessing methodologies to more optimally expand GSCs in the future.





  • Importance of sulfide interaction with iron as regulator of the microbial community in biogas reactors and its effect on methanogenesis, volatile fatty acids turnover, and syntrophic long-chain fatty acids degradation
    Publication date: Available online 3 January 2017
    Source:Journal of Bioscience and Bioengineering

    Author(s): Sepehr Shakeri Yekta, Ryan M. Ziels, Annika Björn, Ulf Skyllberg, Jörgen Ejlertsson, Anna Karlsson, Matilda Svedlund, Magnus Willén, Bo H. Svensson

    The inhibitory effects of sulfide on microbial processes during anaerobic digestion have been widely addressed. However, other effects of sulfide are less explored, given that sulfide is a potential sulfur source for microorganisms and its high reactivity triggers a suit of abiotic reactions. We demonstrated that sulfide interaction with Fe regulates the dynamics and activities of microbial community during anaerobic digestion. This was manifested by the S:Fe molar ratio, whose increase adversely influenced the acetoclastic methanogens, Methanosaeta, and turnover of acetate. Dynamics of hydrogenotrophic methanogens, Methanoculleus and Methanobrevibacter, were presumably influenced by sulfide-induced changes in the partial pressure of hydrogen. Interestingly, conversion of the long-chain fatty acid (LCFA), oleate, to methane was enhanced together with the abundance of LCFA-degrading, β-oxidizing Syntrophomonas at an elevated S:Fe molar ratio. The results suggested that sulfur chemical speciation is a controlling factor for microbial community functions in anaerobic digestion processes.





  • Biosynthesis of eight-carbon volatiles from tomato and pepper pomaces by fungi: Trichoderma atroviride and Aspergillus sojae
    Publication date: Available online 3 January 2017
    Source:Journal of Bioscience and Bioengineering

    Author(s): Onur Güneşer, Yonca Karagül Yüceer

    The aim of this study was to investigate the possibility of using tomato and red pepper pomaces for the production eight-carbon volatiles by Trichoderma atroviride and Aspergillus sojae. The fermentation of tomato and pepper pomace-based media by both moulds was conducted in shake flasks and bioreactors. Microbial growth behaviours and fermentation abilities of T. atroviride and A. sojae under both fermentation conditions were followed by microbial counting. The production of flavours from tomato and pepper pomaces by fungal metabolism was determined by gas chromatography–olfactometry, gas chromatography–mass spectrometry and sensory analysis. The results showed that T. atroviride grew faster than A. sojae, and the survival of T. atroviride in the tomato pomace was longer than that of A. sojae. However, T. atroviride grew slower than A. sojae in the pepper pomace. Eight-carbon flavour compounds, including (Z)-1,5-octadien-3-ol, 1-octen-3-ol, (E)-2-octenal and (E)-2-octenol, were produced by T. atroviride and A. sojae from the tomato and pepper pomaces. The highest production levels (265.55 ± 2.79 and 187.47 ± 0.92 μg kg−1) were observed for 1-octen-3-ol in the tomato fermentation by T. atroviride and A. sojae, respectively. The relationships between volatile compounds and their flavour characteristics in tomato and pepper pomaces were analysed using principal component analysis.





  • Evaluation of a newly developed mid-infrared sensor for real-time monitoring of yeast fermentations
    Publication date: Available online 3 January 2017
    Source:Journal of Bioscience and Bioengineering

    Author(s): Robert Schalk, Daniel Geoerg, Jens Staubach, Matthias Raedle, Frank-Juergen Methner, Thomas Beuermann

    A mid-infrared (MIR) sensor using the attenuated total reflection (ATR) technique has been developed for real-time monitoring in biotechnology. The MIR-ATR sensor consists of an IR emitter as light source, a zinc selenide ATR prism as boundary to the process, and four thermopile detectors, each equipped with an optical bandpass filter. The suitability of the sensor for practical application was tested during aerobic batch-fermentations of Saccharomyces cerevisiae by simultaneous monitoring of glucose and ethanol. The performance of the sensor was compared to a commercial Fourier transform mid-infrared (FT-MIR) spectrometer by on-line measurements in a bypass loop. Sensor and spectrometer were calibrated by multiple linear regression (MLR) in order to link the measured absorbance in the transmission ranges of the four optical sensor channels to the analyte concentrations. For reference analysis, high-performance liquid chromatography (HPLC) was applied. Process monitoring using the sensor yielded in standard errors of prediction (SEP) of 6.15 g/L and 1.36 g/L for glucose and ethanol. In the case of the FT-MIR spectrometer the corresponding SEP values were 4.34 g/L and 0.61 g/L, respectively. The advantages of optical multi-channel mid-infrared sensors in comparison to FT-MIR spectrometer setups are the compactness, easy process implementation and lower price.





  • Characterization and functional expression of a rubber degradation gene of a Nocardia degrader from a rubber-processing factory
    Publication date: Available online 3 January 2017
    Source:Journal of Bioscience and Bioengineering

    Author(s): Dao Viet Linh, Nguyen Lan Huong, Michiro Tabata, Shunsuke Imai, Sou Iijima, Daisuke Kasai, To Kim Anh, Masao Fukuda

    A rubber-degrading bacterial consortium named H2DA was obtained from an enrichment culture with natural rubber latex and rubber-processing factory waste in Vietnam. Gel permeation chromatography analysis revealed that only the strain NVL3 degraded synthetic poly(cis-1,4-isoprene) into low-molecular-weight intermediates among the three strains found in the H2DA. The 16S-rRNA gene sequence of NVL3 showed the highest identity with that of Nocardia farcinica DSM 43665T. NVL3 accumulated aldehyde intermediates from synthetic poly(cis-1,4-isoprene) on a rubber-overlay plate as indicated by Schiff's staining. NVL3 also degraded deproteinized natural rubber into low-molecular-weight aldehyde intermediates. A latex-clearing protein (lcp) gene ortholog was identified within the genome sequence of NVL3, and it showed a moderate amino-acid identity (54–75%) with the lcp genes from previously reported rubber degraders. The heterologous expression of the NVL3 lcp in Escherichia coli BL21(DE3) allowed us to purify the 46.8-kDa His-tagged lcp gene product (His-Lcp). His-Lcp degraded synthetic poly(cis-1,4-isoprene) and accumulated aldehyde intermediates from deproteinized natural rubber suggesting the functional expression of the lcp gene from a Nocardia degrader in E. coli. Quantitative reverse transcription PCR analysis indicated the strong transcriptional induction of the lcp gene in NVL3 in the presence of synthetic poly(cis-1,4-isoprene). These results suggest the involvement of the lcp gene in rubber degradation in NVL3.





  • Expression of the alaE gene is positively regulated by the global regulator Lrp in response to intracellular accumulation of l-alanine in Escherichia coli
    Publication date: Available online 3 January 2017
    Source:Journal of Bioscience and Bioengineering

    Author(s): Kohei Ihara, Kazuki Sato, Hatsuhiro Hori, Yumiko Makino, Shuji Shigenobu, Tasuke Ando, Emiko Isogai, Hiroshi Yoneyama

    The alaE gene in Escherichia coli encodes an l-alanine exporter that catalyzes the active export of l-alanine using proton electrochemical potential. In our previous study, alaE expression was shown to increase in the presence of l-alanyl-l-alanine (Ala-Ala). In this study, the global regulator leucine-responsive regulatory protein (Lrp) was identified as an activator of the alaE gene. A promoter less β-galactosidase gene was fused to an alaE upstream region (240 nucleotides). Cells that were lacZ-deficient and harbored this reporter plasmid showed significant induction of β-galactosidase activity (approximately 17-fold) in the presence of 6 mM l-alanine, l-leucine, and Ala-Ala. However, a reporter plasmid possessing a smaller alaE upstream region (180 nucleotides) yielded transformants with strikingly low enzyme activity under the same conditions. In contrast, lrp-deficient cells showed almost no β-galactosidase induction, indicating that Lrp positively regulates alaE expression. We next performed an electrophoretic mobility shift assay (EMSA) and a DNase I footprinting assay using purified hexahistidine-tagged Lrp (Lrp-His). Consequently, we found that Lrp-His binds to the alaE upstream region spanning nucleotide −161 to −83 with a physiologically relevant affinity (apparent KD, 288.7 ± 83.8 nM). Furthermore, the binding affinity of Lrp-His toward its cis-element was increased by l-alanine and l-leucine, but not by Ala-Ala and d-alanine. Based on these results, we concluded that the gene expression of the alaE is regulated by Lrp in response to intracellular levels of l-alanine, which eventually leads to intracellular homeostasis of l-alanine concentrations.





  • Spectrophotometric assay for sensitive detection of glycerol dehydratase activity using aldehyde dehydrogenase
    Publication date: Available online 2 January 2017
    Source:Journal of Bioscience and Bioengineering

    Author(s): Eul-Soo Park, Sunghoon Park, Jong-Shik Shin

    Glycerol dehydratase (GDHt) is a pivotal enzyme for fermentative utilization of glycerol by catalyzing radical-mediated conversion of glycerol into 3-hydroxypropionaldehyde (3-HPA). Precise and sensitive monitoring of cellular GDHt activity during the fermentation process is a prerequisite for reliable metabolic analysis to afford efficient cellular engineering and process optimization. Here we report a new spectrophotometric assay for the sensitive measurement of the GDHt activity with a sub-nanomolar limit of detection (LOD). The assay method employs aldehyde dehydrogenase (ALDH) as a reporter enzyme, so the readout of the GDHt activity is recorded at 340 nm as an increase in UV absorbance which results from NADH generation accompanied by oxidation of 3-HPA to 3-hydroxypropionic acid (3-HP). The GDHt assay was performed under the reaction conditions where the ALDH activity overwhelms the GDHt activity (i.e., 50-fold higher activity of ALDH relative to GDHt activity), affording sensitive detection of GDHt with 360 pM LOD. The ALDH-coupled assay was used to determine kinetic parameters of GDHt for glycerol, leading to K M = 0.73 ± 0.09 mM and k cat = 400 ± 20 s−1 which are in reasonable agreements with the previous reports. Our assay method allowed measurement of even a 104-fold decrease in the cellular GDHt activity during fermentative production of 3-HP, which demonstrates the detection sensitivity much higher than the previous methods.





  • N-terminal SKIK peptide tag markedly improves expression of difficult-to-express proteins in Escherichia coli and Saccharomyces cerevisiae
    Publication date: Available online 2 January 2017
    Source:Journal of Bioscience and Bioengineering

    Author(s): Teruyo Ojima-Kato, Satomi Nagai, Hideo Nakano

    Despite advances in microbial protein expression systems, low production of proteins remains a great concern for some genes. Here we report that the insertion of a short peptide tag, consisting of Ser-Lys-Ile-Lys (SKIK), adjacent to the start codon of genes encoding difficult-to-express proteins can increase protein expression in Escherichia coli and Saccharomyces cerevisiae. Protein expression levels of a mouse monoclonal antibody (mAb), rabbit mAbs obtained from clonal B cells, and an artificially designed peptide were significantly increased simply by the addition of the SKIK tag in E. coli systems. In particular, a ∼30-fold increase in protein production was observed for the mouse mAb, and the artificially designed peptide band became detectable in sodium dodecyl sulfate-poly acrylamide gel electrophoresis after coomassie brilliant blue staining or western blotting on adding the SKIK tag. The tag also increased the expression of tagged proteins in S. cerevisiae and an E. coli cell-free protein synthesis system. Although the mechanism of high protein expression on addition of the tag is unclear, our findings offer great benefits to biotechnology research and industry.





  • Cosynthesis of l-homophenylalanine and 2-phenylethanol by recombinant Saccharomyces cerevisiae expressing aspartate aminotransferase from Escherichia coli BL21(DE3)
    Publication date: January 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 1

    Author(s): Chunhua Luo, Qingmei Lin, Suying Lin, Chun Meng, Hang Wang

    The Escherichia coli aspartate aminotransferase gene was introduced into a high 2-phenylethanol (2-PEA) producing Saccharomyces cerevisiae YS58, and the recombinant strain of S. cerevisiae was utilized for the co-production of 2-PEA and l-homophenylalanine (L-HPA) via a fermentation process. The L-HPA productivity of the recombinant S. cerevisiae improved 78.9% in comparison to the wild-type S. cerevisiae. High yields of 43.7 mM L-HPA and 32.4 mM 2-PEA were achieved. As a result, the coupling of the biosynthesis process for these two products in the recombinant strain led to a more complete and efficient utilization of the substrate, l-phenylalanine.





  • Sake yeast YHR032W/ERC1 haplotype contributes to high S-adenosylmethionine accumulation in sake yeast strains
    Publication date: January 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 1

    Author(s): Muneyoshi Kanai, Tomoko Kawata, Yoshie Yoshida, Yasuko Kita, Takafumi Ogawa, Masaki Mizunuma, Daisuke Watanabe, Hitoshi Shimoi, Akihiro Mizuno, Osamu Yamada, Tsutomu Fujii, Haruyuki Iefuji

    Sake yeasts are ideally suited for sake making, producing higher levels of ethanol, proliferating at lower temperatures, and producing greater levels of various aromatic components and nutrients than laboratory yeasts. To elucidate the mechanism underlying S-adenosylmethionine (SAM) accumulation in sake yeast strains compared with that in laboratory yeast strains, we performed quantitative trait locus (QTL) analysis and identified a significant QTL on chromosome VIII. Of the 165 genes mapped at 49.8 cM from the left-end DNA marker of chromosome VIII, we focused on the YHR032W/ERC1 gene, encoding a member of the multi-drug and toxin extrusion family having antiporter activity and involved in SAM accumulation and ethionine resistance. Expression of the sake yeast ERC1 haplotype (K7ERC1) in a low- and high-copy number plasmid BYΔerc1 resulted in intracellular SAM accumulation, whereas expression of the laboratory yeast ERC1 haplotype (XERC1) did not. Comparison between DNA sequences of K7ERC1 and XERC1 revealed three amino acid substitutions: S51N, V263I, and N545I. Site-directed mutagenesis revealed that the N545I frameshift mutation was responsible for the K7ERC1 phenotype. These results indicate that K7ERC1 contributes to SAM accumulation in sake yeast strains.





  • New alkalophilic β-galactosidase with high activity in alkaline pH region from Teratosphaeria acidotherma AIU BGA-1
    Publication date: January 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 1

    Author(s): Miwa Yamada, Serina Chiba, Yuta Endo, Kimiyasu Isobe

    A β-d-galactosidase exhibiting high activity in the alkaline pH region was purified from Teratosphaeria acidotherma AIU BGA-1, which we previously isolated as a unique fungal producer of three acidophilic and one alkalophilic β-d-galactosidases (Isobe et al., J. Biosci. Bioeng., 116, 171–174, 2013). The enzyme was stable in the pH range 7.5–10.0 and exhibited optimal activity at pH 8.0 and 60°C. The enzyme hydrolyzed 2-nitrophenyl β-d-galactopyranoside, 4-nitrophenyl β-d-galactopyranoside, and lactose, and the K m values were estimated to be 0.349 mM, 0.488 mM, and 701 mM, respectively. Chelating reagents (EDTA and o-phenanthroline) and metals (Cu2+and Ni2+) inhibited the enzyme activity, and Mn2+ was a good activator. The enzyme also exhibited transgalactosylation activity for lactose. The enzyme's molecular mass was estimated to be 180 kDa, and its structure was monomeric. Thus, the enzymatic and physicochemical characteristics of the alkalophilic β-galactosidase in this study clearly differed from those of the previously known alkalophilic β-d-galactosidases.





  • Purification and characterization of xylitol dehydrogenase with l-arabitol dehydrogenase activity from the newly isolated pentose-fermenting yeast Meyerozyma caribbica 5XY2
    Publication date: January 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 1

    Author(s): Wiphat Sukpipat, Hidenobu Komeda, Poonsuk Prasertsan, Yasuhisa Asano

    Meyerozyma caribbica strain 5XY2, which was isolated from an alcohol fermentation starter in Thailand, was found to catabolize l-arabinose as well as d-glucose and d-xylose. The highest production amounts of ethanol from d-glucose, xylitol from d-xylose, and l-arabitol from l-arabinose were 0.45 g/g d-glucose, 0.60 g/g d-xylose, and 0.61 g/g l-arabinose with 21.7 g/L ethanol, 20.2 g/L xylitol, and 30.3 g/l l-arabitol, respectively. The enzyme with l-arabitol dehydrogenase (LAD) activity was purified from the strain and found to exhibit broad specificity to polyols, such as xylitol, d-sorbitol, ribitol, and l-arabitol. Xylitol was the preferred substrate with K m =16.1 mM and k cat/K m =67.0 min−1mM−1, while l-arabitol was also a substrate for the enzyme with K m =31.1 mM and k cat/K m =6.5 min−1 mM−1. Therefore, this enzyme from M. caribbica was named xylitol dehydrogenase (McXDH). McXDH had an optimum temperature and pH at 40°C and 9.5, respectively. The McXDH gene included a coding sequence of 1086 bp encoding a putative 362 amino acid protein of 39 kDa with an apparent homopentamer structure. Native McXDH and recombinant McXDH exhibited relative activities toward l-arabitol of approximately 20% that toward xylitol, suggesting the applicability of this enzyme with the functions of XDH and LAD to the development of pentose-fermenting Saccharomyces cerevisiae.





  • Static and dynamic half-life and lifetime molecular turnover of enzymes
    Publication date: January 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 1

    Author(s): Osato Miyawaki, Tsukasa Kanazawa, Chika Maruyama, Michiko Dozen

    The static half-life of an enzyme is the half-life of a free enzyme not working without substrate and the dynamic half-life is that of an active enzyme working with plenty amount of substrate. These two half-lives were measured and compared for glucoamylase (GA) and β-galactosidase (BG). The dynamic half-life was much longer than the static half-life by one to three orders of magnitude for both enzymes. For BG, the half-life of the enzyme physically entrapped in a membrane reactor was also measured. In this case also, the half-life of BG in the membrane reactor was much longer than the free enzyme without substrate. These results suggest the large difference in stabilities between the free enzyme and the enzyme–substrate complex. This may be related to the natural enzyme metabolism. According to the difference in half-life, the lifetime molecular turnover (LMT), which is the number of product molecules produced by a single molecule of enzyme until it loses its activity completely, was much higher by one to four orders of magnitude for the active enzyme than the free enzyme. The concept of LMT, proposed here, will be important in bioreactor operations with or without immobilization.





  • Trans 18-carbon monoenoic fatty acid has distinct effects from its isomeric cis fatty acid on lipotoxicity and gene expression in Saccharomyces cerevisiae
    Publication date: January 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 1

    Author(s): Toyokazu Nakamura, Vo Thi Anh Nguyet, Sae Kato, Yasuhiro Arii, Toshiharu Akino, Shingo Izawa

    Epidemiological studies have suggested that an excess intake of trans-unsaturated fatty acids increases the risk of coronary heart disease. However, the mechanisms of action of trans-unsaturated fatty acids in eukaryotic cells remain unclear. Since the budding yeast Saccharomyces cerevisiae can grow using fatty acids as the sole carbon source, it is a simple and suitable model organism for understanding the effects of trans-unsaturated fatty acids at the molecular and cellular levels. In this study, we compared the physiological effects of Δ9 cis and trans 18-carbon monoenoic fatty acids (oleic acid and elaidic acid) in yeast cells. The results obtained revealed that the two types have distinct effects on the expression of OLE1, which encodes Δ9 desaturase, and lipotoxicity in are1Δare2Δdga1Δlro1Δ and gat1Δ cells. Our results suggest that cis and trans 18-carbon monoenoic fatty acids exert different physiological effects in the regulation of gene expression and processing of excess fatty acids in yeast.