JBB : Journal of Bioscience and Bioengineering

JBB Vol. 127表紙  



  • Targeted proteome analysis of microalgae under high-light conditions by optimized protein extraction of photosynthetic organisms

    Publication date: March 2019

    Source: Journal of Bioscience and Bioengineering, Volume 127, Issue 3

    Author(s): Masakazu Toyoshima, Masumi Sakata, Kazuki Ohnishi, Yuma Tokumaru, Yusuke Kato, Ryutaro Tokutsu, Wataru Sakamoto, Jun Minagawa, Fumio Matsuda, Hiroshi Shimizu

    Cell disruption and protein solubilization protocols for the relative quantification of individual subunits in photosystems were developed for photosynthetic organisms including cyanobacterium Synechocystis sp. PCC 6803, green-algae Chlamydomonas reinhardtii, and seed plant Arabidopsis thaliana. The optimal methods for the disruption of Chlamydomonas, Synechocystis, and Arabidopsis cells were sonication, microbeads (Φ approximately 0.1 mm), and large beads (Φ = 5.0 mm), respectively. Extraction of the total proteins exceeded 90% using each optimal cell disruption method. Solubilization efficiency of membrane proteins was improved by the phase transfer surfactant (PTS) method. Ninety seven and 114 proteins from Chlamydomonas and Synechocystis, respectively, including membrane proteins such as photosystem proteins, ATP synthase, and NADH dehydrogenase, were successfully analyzed by nano-liquid chromatography tandem mass spectrometry. These results also indicated the improved efficiency of solubilization and trypsin digestion using PTS buffer. The results of the relative quantitative evaluation of photosystem subunits in Chlamydomonas and Synechocystis grown under high-light conditions were consistent with those of previous studies. Thus, the optimal cell disruption and PTS methods allow for comprehensive relative quantitative proteome analysis of photosynthetic organisms. Additionally, NdhD1 and NdhF1, which are NDH-1 subunit homologs, were increased under high-light conditions, suggesting that the NDH-1L complex, including NdhD1 and NdhF1, is increased under high-light conditions. The relative quantitative proteome analysis of individual subunits indicates the diverse functions of NDH-1 protein.

    Graphical abstract

    Image 1

  • Densitometric quantification for the validation of decolorization of Disperse Orange ERL by lichen Parmelia sp.

    Publication date: March 2019

    Source: Journal of Bioscience and Bioengineering, Volume 127, Issue 3

    Author(s): Ashwini N. Kulkarni, Bhumika N. Bhalkar, Rahul V. Khandare, Mayur B. Kurade, Byong-Hun Jeon, Sanjay P. Govindwar

    Densitometric high performance thin layer chromatography (HPTLC) quantification method was developed to validate the decolorization/biotransformation of Disperse Orange ERL and dye mixture by lichen Parmelia sp. which release several colored compounds during decolorization process, hence unable to use colorimetric estimation. Percent decolorization of Disperse Orange ERL and dye mixture by lichen Parmelia sp. was observed when estimated using developed HPTLC method. Limit of detection and limit of quantification for both dyes in mixture were obtained as 0.3 and 1 μg/μl, respectively. Area of peak of control Disperse Orange ERL was reduced by 43% after 12 h, 71% after 48 h and upto 82% after 72 h of incubation. Precision and repeatability of data elucidated the % relative standard deviation less than 3 for all the values thus indicating statistically acceptable. Biodegradation of dye and mixture was confirmed with Fourier transform infrared spectroscopy analysis, i.e., altered fingerprinting spectral pattern.

    Graphical abstract

    Image 1

  • Role of cell-secreted extracellular matrix formation in aggregate formation and stability of human induced pluripotent stem cells in suspension culture

    Publication date: March 2019

    Source: Journal of Bioscience and Bioengineering, Volume 127, Issue 3

    Author(s): Mee-Hae Kim, Kazuhiro Takeuchi, Masahiro Kino-oka

    Clinical and industrial applications require large quantities of human induced pluripotent stem cells (hiPSCs); however, little is known regarding the mechanisms governing aggregate formation and stability in suspension culture. To address this, we determined differences in growth processes among hiPSC lines in suspension culture. Using an hiPSC aggregate suspension culture system, hiPSCs from different lines formed multicellular aggregates classified as large compact or small loose based on their size and morphology. Time-lapse observation of the growth processes of two different hiPSC lines revealed that the balance between cell division and the extent of subsequent cell death determined the final size and morphology of aggregates. Comparison of the cell survival and death of two hiPSC lines showed that the formation of small, loose aggregates was due to continued cell death during the exponential phase of growth, with apoptotic cells extruded from growing hiPSC aggregates by the concerted contraction of their neighbors. Western blot and immunofluorescent staining revealed that aggregate morphology and proliferative ability relied to a considerable extent upon secretion of the extracellular matrix (ECM). hiPSCs forming large compact and stable aggregates showed enhanced production of collagen type I in suspension culture at 120 h. Furthermore, these aggregates exhibited higher expression of E-cadherin and proliferation marker Ki-67 as compared with levels observed in small and loose aggregates at 120 h. These findings indicated that differences in both aggregate formation and stability in suspension culture among hiPSC lines were caused by differences in ECM secretion capacity.

  • Searching for high-binding peptides to bile acid for inhibition of intestinal cholesterol absorption using principal component analysis

    Publication date: March 2019

    Source: Journal of Bioscience and Bioengineering, Volume 127, Issue 3

    Author(s): Masako Ito, Kazunori Shimizu, Hiroyuki Honda

    We previously proposed a new method for exploring functional peptides using both spot-synthesis peptide libraries and principal component analysis (PCA). Here, we applied these methods to determine if high-binding 6-mer peptides can be used on bile acid for the inhibition of intestinal cholesterol absorption. We used a binding assay of 512 basal 4-mer peptides to bile acid, and from these selected high-binding and low-binding peptides. PCA was performed on data from both these binding groups and many physicochemical variables of the 512 peptides tested, and then through this, the variables were reduced to two principal components (PCs). The peptides were plotted on the PCA chart, and we identified distinct clusters of high- and low-binding regions. These PCA regions were applied to 6-mer random peptides, and we identified 6-mer peptides with high and low binding capacity to bile acid. We confirmed that the average fluorescence intensity of high-binding peptides was 3.0-fold higher than that of low-binding peptides. We succeeded in identifying 6-mer peptides with high and low-binding affinity based on the PCA analysis of 4-mer peptides. These results were compared and discussed with regard to those acquired by our previous computational analysis based on neural networks.

  • Synthesis of polyhydroxyalkanoates through the biodegradation of poly(cis-1,4-isoprene) rubber

    Publication date: March 2019

    Source: Journal of Bioscience and Bioengineering, Volume 127, Issue 3

    Author(s): Rodrigo Andler, Robin Vivod, Alexander Steinbüchel

    The search of alternative substrates for the synthesis of polyhydroxyalkanoates (PHA) has become an important factor in order to decrease the production costs. Therefore, the use of industrial by-products or waste materials as carbon and energy sources for different PHA-producing microorganisms has been evaluated during the last decades. Recombinant strains of Gordonia polyisoprenivorans VH2 harboring plasmid pAK68, which contains phaCAB from Ralstonia eutropha and plasmid pAK71 comprising phaC1 from Pseudomonas aeruginosa were evaluated for PHA production. Cultivations were performed in shake flasks, using different carbon sources under an N-starvation condition. Having in consideration the rubber degrading capability of the actinomycete, poly(cis-1,4-isoprene) was utilized as sole carbon source. After twenty days of cultivation the PHA content was analyzed using GC-MS. In cultures of G. polyisoprenivorans harboring pAK68, the detection of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) monomer units indicated the accumulation of the copolyester poly(3HB-co-3HV). This study proposes a recycling method for rubber waste through its biotransformation into bioplastic.

  • Bio-electrodegradation of 2,4,6-Trichlorophenol by mixed microbial culture in dual chambered microbial fuel cells

    Publication date: March 2019

    Source: Journal of Bioscience and Bioengineering, Volume 127, Issue 3

    Author(s): Nishat Khan, Mohammad Danish Khan, Mohd Yusuf Ansari, Anees Ahmad, Mohammad Zain Khan

    2,4,6-Trichlorophenol (TCP) was bioelectrochemically treated in anodic and cathodic compartments of two identical dual chambered microbial fuel cells MFC-A and MFC-B under anaerobic and aerobic conditions, respectively, and energy was recovered in the form of electricity. It was observed that MFC-B with bio-cathodic treatment of TCP outcompeted the MFC-A with bio-anodic treatment. The maximum power density for MFC-A with bio-anode was found to be 446.76 mW/m2 while for MFC-B with bio-cathode it was 1059.58 mW/m2. The MFC-B consistently showed higher coulombic efficiency, power density and chemical oxygen demand removal efficiency indicating the better performance of the MFC-B as compared to the MFC-A. Scanning electron micrograph also confirmed better accumulation of microbes on the anode of MFC-B and hence its better performance in terms of energy recovery. Some major genera present in the microbial community were quantified using quantitative real-time polymerase chain reaction technique. It also confirmed the dominance of electroactive species in the bio-anodic sludge of MFC-B over the bio-anodic sludge of MFC-A. Cyclic voltammogram also asserted better electrochemical activity of the bio-cathode in the treatment of chlorinated phenol toxicants in MFC-B system. The study shows that MFC can be a viable option in treatment of recalcitrant chemical compounds like TCP with the generation of energy in the form of electrical power.

    Graphical abstract

    Image 1

  • Effects of ammonium and/or sulfide on methane production from acetate or propionate using biochemical methane potential tests

    Publication date: March 2019

    Source: Journal of Bioscience and Bioengineering, Volume 127, Issue 3

    Author(s): Li Tan, Qiu-Shi Cheng, Zhao-Yong Sun, Yue-Qin Tang, Kenji Kida

    The inhibitory effects of ammonium and sulfide on the methane production using acetate or propionate as a carbon source were investigated under different pH and temperature conditions. The methane production rate, duration of the lag phase, and inhibition threshold limit during methane production were estimated using the Gompertz equation and inhibitor mathematical model. The methane production rates at 53°C were 2.3–2.7 times higher than those at 35°C in the case of non-inhibitors. Increasing the NH4+ and/or S2− concentration decreased the methane production rate and increased the duration of the lag phase. For methane fermentation that was not acclimated to high NH4+ concentration, the critical NH4+ concentration beyond which methane fermentation would stop was 4000–5650 mg/L, depending on the pH, temperature, and carbon source. When NH4+ and S2− were coexistent, the critical NH4+ concentration decreased to approximately 3800 mg/L when propionate was used and to approximately 4450 mg/L when acetate was used. However, no synergistic effect of NH4+ and S2− on the methane production rate was found at an NH4+ concentration of < 5000 mg/L and S2− concentration of 50 mg/L.

  • Glucose production from cellulose through biological simultaneous enzyme production and saccharification using recombinant bacteria expressing the β-glucosidase gene

    Publication date: March 2019

    Source: Journal of Bioscience and Bioengineering, Volume 127, Issue 3

    Author(s): Shunsuke Ichikawa, Maiko Ichihara, Toshiyuki Ito, Kazuho Isozaki, Akihiko Kosugi, Shuichi Karita

    Efficient cellulosic biomass saccharification technologies are required to meet biorefinery standards. Biological simultaneous enzyme production and saccharification (BSES), which is glucose production from cellulosic biomass by Clostridium thermocellum, can be a reliable cellulose saccharification technology for biorefineries. However, the current BSES processes require purified β-glucosidase supplementation. In this study, recombinant bacteria expressing the β-glucosidase gene were developed and directly applied to BSES. The engineered Escherichia coli expressing the thermostable β-glucosidase gene from Thermoanaerobacter brockii exhibited 0.5 U/ml of β-glucosidase activities. The signal peptide sequence of lytF gene from Bacillus subtilis was the most appropriate for the β-glucosidase secretion from Brevibacillus choshinensis, and the broth exhibited 0.74 U/ml of β-glucosidase activities. The engineered E. coli and B. choshinensis expressing the thermostable β-glucosidase gene produced 47.4 g/L glucose and 49.4 g/L glucose, respectively. Glucose was produced by the hydrolysis of 100 g/L Avicel cellulose for 10 days through BSES, and the product yield was similar to that obtained through BSES with purified β-glucosidase supplementation. Our findings indicate that the direct supplementation of β-glucosidase using bacterial cells expressing β-glucosidase gene or their broth was applicable to BSES, suggesting the potential of this process as a cost-effective approach to cellulose saccharification.

    Graphical abstract

    Image 1

  • Spatial and daily variations of nitrous oxide emissions from biological reactors in a full-scale activated sludge anoxic/oxic process

    Publication date: March 2019

    Source: Journal of Bioscience and Bioengineering, Volume 127, Issue 3

    Author(s): Sijing Jia, Xiaoqiang Chen, Toshikazu Suenaga, Akihiko Terada, Susumu Ishikawa, Fumitake Nishimura, Shaolan Ding, Taku Fujiwara

    Nitrous oxide (N2O) is an important greenhouse gas that can be emitted from wastewater treatment plants (WWTPs). Such emissions are reportedly process specific and related to operational parameters. This study was conducted to clarify spatial and daily variations of N2O in a full-scale activated sludge anoxic/oxic process that consisted of an anoxic tank and three oxic tanks (oxic-1, oxic-2 and oxic-3), all of which except the final sedimentation tank were fully covered. Higher dissolved N2O (D-N2O) loading and gaseous N2O (G-N2O) emissions were observed for oxic-3 than for the anoxic, oxic-1, and oxic-2 tanks, implying that there was higher N2O production potential via nitrification in the latter stage of the oxic tank. Moreover, the sudden decrease in dissolved oxygen concentration after the peak was found to lead to abrupt production of D-N2O at oxic-3 in the anoxic/oxic process. The increases in AOB amoA, AOB nirK and the following AOB norB gene transcripts at the end of the oxic-2 tank suggested that nitrifier denitrification occurred to produce N2O under low dissolved oxygen conditions when the N2O peak was observed. Additionally, the much lower transcription levels of the two nosZ genes suggested lower N2O consumption. The N2O emission factors ranged from 0.087% to 0.302%, and lower N2O emission factors were observed during summer.

  • Highly efficient deproteinization with an ammonifying bacteria Lysinibacillus fusiformis isolated from brewery spent diatomite

    Publication date: March 2019

    Source: Journal of Bioscience and Bioengineering, Volume 127, Issue 3

    Author(s): Xiaoxi Gong, Weijun Tian, Jie Bai, Kaili Qiao, Jing Zhao, Liang Wang

    To explore a new method for bio-regeneration of high-protein brewery spent diatomite, an ammonifying bacteria (BSD1) was screened out from it and identified as Lysinibacillus fusiformis. The protein degradation characteristics of BSD1 was studied with rice protein as the sole nitrogen source. Maximum protein degradation activity was obtained when BSD1 was inoculated with an inoculum of 5% into a medium with glucose as carbon source and initial pH value of 7.0 and incubated at 30°C for 48 h. In this optimal condition, protein concentration decreased from 156.8 mg/L to 19.2 mg/L, and protein degradation efficiency of BSD1 reached 88%. Free amino acid analysis showed that the content of Phe, Tyr, Pro, Ala, Lys, Thr and His increased in protein degradation process. After degradation, NH4+N concentration producing in medium supernatant reached 232.2 mg/L. These results indicated the strain BSD1 could transform proteins into free amino acids and eventually convert them to ammonium or ammonia. Furthermore, strain BSD1 could also be used for deproteinization of brewery spent diatomite and 51% of proteins in spent diatomite were degraded. After biological deproteinization the specific surface area and total pore volume of diatomite improved obviously. These results illustrated that the application of strain BSD1 for bio-regeneration of high-protein brewery spent diatomite was efficient and feasible.

  • Performance and dynamic characteristics of microbial communities in multi-stage anaerobic reactors treating gibberellin wastewater

    Publication date: March 2019

    Source: Journal of Bioscience and Bioengineering, Volume 127, Issue 3

    Author(s): Erming Ouyang, Yao Lu, Jiating Ouyang, Lele Wang, Xiaohui Wang

    To treat gibberellin (GA) wastewater, a full-scale, multi-stage combined contact process was developed. This whole process employs three anaerobic reactors followed by micro-aerobic, anoxic/aeration and biological oxidation treatment. Pollutant removal results showed that the combined process could remove more than 98% of the chemical oxygen demand (COD), NH3N, and SO42− pollutants because of the different microbial communities in each reactor. 16S rRNA gene sequencing was used to examine the microbial communities in the internal circulation (IC) and in the two up flow anaerobic sludge blanket (UASB) reactors, as well as to investigate the effect of sampling elevation on the microbial community. The results showed that Firmicutes and Euryarchaeota were the most dominant phyla at the bacterial and archaeal levels, respectively. High levels of Synergistaceae_uncultured were detected in IC and UASB1. Chloroflexi_uncultured was the dominant genus of bacterial communities within UASB2, and Methanosaeta was the dominant genus of archaeal communities. Principal coordinates analysis (PCoA) revealed variations among the microbial communities in 9 samples, and Venn analysis showed different operational taxonomic units (OTUs) among samples collected at various elevations within the three anaerobic reactors. However, partial Mantel tests indicated no significant correlation between the microbial community structure and elevation in the three anaerobic reactors.

  • Roles of aging in the production of light-flavored Daqu

    Publication date: March 2019

    Source: Journal of Bioscience and Bioengineering, Volume 127, Issue 3

    Author(s): Guangsen Fan, Zhilei Fu, Baoguo Sun, Yuhang Zhang, Xinlei Wang, Yanqiu Xia, Mingquan Huang, Xiuting Li

    Daqu, a complex starting material used for Baijiu production, contains microorganisms, enzymes, and volatile compounds. An important part of Daqu production is aging, but the physicochemical and microbial changes during aging remain largely unknown. This study characterized aging according to physicochemical parameters, volatile compounds, and microbial communities. Aging was found to aid in the stabilization of the physicochemical parameters. Solid-phase microextraction-gas chromatography-mass spectrometry was used to detect 72 types of volatile compounds, which were predominantly alcohols, esters, aldehydes, alkenes, and alkanes. During aging, these compounds changed considerably, but their structures eventually stabilized. A high-throughput sequencing approach was used to analyze the changing composition of the microbial communities. In general, aging helped to enrich and stabilize the microbial population for making Baijiu. A total of 35 bacteria were detected as prokaryotic; among these, 15 had a diversity abundance ratio of more than 1%. The dominant bacteria were from the genus Pantoea, but these decreased with aging, while bacteria from Lactobacillus and Weissella increased. After aging for 2 months, Pantoea, Lactobacillus and Weissella accounted for 0.4%, 54.0%, and 18.9%, respectively. A total of 12 eukaryotic yeast and fungi were detected, the most abundant of which were Incertae_Sedis_incertae_sedis, Saccharomycopsis, Trichocomaceae_unclassified, Pichia, Tremellales_unclassified, and Galactomyces. During aging, the levels of Trichocomaceae_unclassified, Saccharomycopsis, and Galactomyces initially decreased but then increased. Pichia stayed unchanged as aging progressed. In conclusion, aging led to rebalanced interactions among Daqu microbes and was important in improving Daqu quality and ensuring its stability.

    Graphical abstract

    Image 1

  • Metabolome analysis revealed the knockout of glyoxylate shunt as an effective strategy for improvement of 1-butanol production in transgenic Escherichia coli

    Publication date: March 2019

    Source: Journal of Bioscience and Bioengineering, Volume 127, Issue 3

    Author(s): Katsuaki Nitta, Walter A. Laviña, Sammy Pontrelli, James C. Liao, Sastia P. Putri, Eiichiro Fukusaki

    High 1-butanol titer has been achieved in a transgenic Escherichia coli strain JCL299FT with a heterologous 1-butanol pathway by deleting competing pathways, balancing of cofactor and resolving free CoA imbalance. However, further improvement of 1-butanol production is still possible in the highest producing strain JCL299FT as indicated by the accumulation of acetate, a major undesired by-product during bio-production by microorganisms that competes with 1-butanol production for the available acetyl-CoA and inhibits protein synthesis resulting in poor growth. In this study, liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based metabolome analysis was performed to identify new rate limiting steps in the 1-butanol production pathway of E. coli strain JCL299FT. The results of metabolome analysis showed increased amounts of glyoxylate in JCL299FT compared to the previous highest-producing strain JCL299F. Knocking out aceA successfully decreased the amount of glyoxylate and reduced acetate accumulation, resulting in the increased levels of TCA cycle and 1-butanol pathway metabolites. These observations indicated that there was a redirection of flux from acetate to TCA cycle and 1-butanol producing pathway, which led to better growth of the 1-butanol producing strain. Consequently, 1-butanol production titer was improved by 39% and the production yield was improved by 12% in M9 medium supplemented with yeast extract. This study is the first report of using the knockout of aceA, the first gene in the glyoxylate shunt that encodes isocitrate lyase, as an effective strategy to reduce acetate overflow in 1-butanol producing E. coli.

  • Two NADH-dependent (S)-3-hydroxyacyl-CoA dehydrogenases from polyhydroxyalkanoate-producing Ralstonia eutropha

    Publication date: March 2019

    Source: Journal of Bioscience and Bioengineering, Volume 127, Issue 3

    Author(s): Mutsumi Segawa, Cheng Wen, Izumi Orita, Satoshi Nakamura, Toshiaki Fukui

    Ralstonia eutropha H16 contains both NADH- and NADPH-dependent reduction activities to acetoacetyl-CoA, and the NADPH-dependent activity is mediated by PhaB paralogs with (R)-stereospecificity providing (R)-3-hydroxybutyryl (3HB)-CoA monomer for poly((R)-3-hydroxybutyrate) synthesis. In contrast, the gene encoding the NADH-dependent enzyme has not been identified to date. This study focused on the NADH-dependent dehydrogenase with (S)-stereospecificity in R. eutropha, as the (S)-specific reduction of acetoacetyl-CoA potentially competed with the polyester biosynthesis via (R)-3HB-CoA. The NADH-dependent reduction activity decreased to one-half when the gene for H16_A0282 (PaaH1), one of two homologs of clostridial NADH-3HB-CoA dehydrogenase, was deleted. The enzyme responsible for the remaining activity was partially purified and identified as H16_A0602 (Had) belonging to a different family from PaaH1. Gene disruption analysis elucidated that most of the NADH-dependent activity was mediated by PaaH1 and Had. The kinetic analysis using the recombinant enzymes indicated that PaaH1 and Had were both NADH-dependent 3-hydroxyacyl-CoA dehydrogenases with rather broad substrate specificity to 3-oxoacyl-CoAs of C4 to C8. The deletion of had in the R. eutropha strain previously engineered for biosynthesis of poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) led to decrease in the C6 composition of the copolyester synthesized from soybean oil, suggesting the role of Had in (S)-specific reduction of 3-oxohexanoyl-CoA with reverse β-oxidation direction. Crotonase ((S)-specific enoyl-CoA hydratase) in R. eutropha H16 was also partially purified and identified as H16_A3307.

  • Enhanced production of d-lactate from mixed sugars in Corynebacterium glutamicum by overexpression of glycolytic genes encoding phosphofructokinase and triosephosphate isomerase

    Publication date: March 2019

    Source: Journal of Bioscience and Bioengineering, Volume 127, Issue 3

    Author(s): Yota Tsuge, Naoto Kato, Shogo Yamamoto, Masako Suda, Masayuki Inui

    The use of mixed sugars containing glucose and xylose in lignocellulosic biomass is desirable for the microbial production of chemicals and fuels. We investigated the effect of individual or simultaneous overexpression of glycolytic genes on d-lactate production from a mixture of glucose and xylose by a recombinant xylose-assimilating Corynebacterium glutamicum strain. The individual overexpression of genes encoding phosphofructokinase (PFK) and triosephosphate isomerase (TPI) increased d-lactate production rate by 71% and 34%, respectively, with corresponding increases (2.4- and 1.8-fold) in the glucose consumption; however, the amount of xylose consumed not altered. d-Lactate yield was also increased by 5.5%, but only in the strain overexpressing the gene encoding PFK. In the parent strain and the strains overexpressing the genes encoding PFK or TPI, a reduction in d-lactate production occurred at approximately 900 mM after 32 h. However, the strain that simultaneously overexpressed the genes encoding PFK and TPI continued to produce d-lactate after 32 h, with the eventual production of 1326 mM after production for 80 h in mineral salts medium. Our findings contribute to the cost-effective, large-scale production of d-lactate from mixed sugars.

  • Screening of microorganisms producing a novel protein-asparaginase and characterization of the enzyme derived from Luteimicrobium album

    Publication date: March 2019

    Source: Journal of Bioscience and Bioengineering, Volume 127, Issue 3

    Author(s): Noriko Miwa, Mayu Mitsuhashi, Takayuki Kajiura

    A screening system using enrichment culture has been established with the aim of obtaining a novel enzyme for protein modification that has not been previously reported. This enzyme catalyzes deamidation of the side-chain amide group of asparagine in proteins. Enrichment culture of 390 soil samples was carried out with Z-Asn-Gly as the sole source of nitrogen, and the reaction product, Z-Asp-Gly, was detected in the culture supernatant of 102 strains. Strains with particularly high activity were Leifsonia sp., Luteimicrobium sp., Microbacterium sp., and Agromyces sp., all belonging to the class Actinobacteria. Of these, a protein-asparaginase (PA) was obtained from the culture supernatant of Luteimicrobium album 333B-h1, and its reactivity with different substrates and its basic enzymatic characteristics were investigated. Addition of the enzyme solution resulted in specific deamidation of only the asparagine residue in insulin chain B. The enzyme showed no reactivity with free asparagine or asparagine in low molecular weight peptides. It was demonstrated that the enzyme reacts with various protein substrates. In particular, proteins that have open structures, such as casein or gelatin, were good substrates. The activity and stability of PA at different temperatures and pH values were investigated. It was found that a temperature of 37°C and a roughly neutral pH are optimal conditions for the enzyme.

  • Expression and characterization of silkworm Bombyx mori β-1,2-N-acetylglucosaminyltransferase II, a key enzyme for complex-type N-glycan biosynthesis

    Publication date: March 2019

    Source: Journal of Bioscience and Bioengineering, Volume 127, Issue 3

    Author(s): Takatsugu Miyazaki, Ryunosuke Miyashita, Sota Mori, Tatsuya Kato, Enoch Y. Park

    N-glycans are involved in various physiological functions and their structures diverge among different phyla and kingdoms. Insect cells mainly produce high mannose-type and paucimannose-type glycans but very few mammalian-like complex-type glycans. However, many insects possess genes for proteins homologous to the enzymes involved in complex-type N-glycan synthesis in mammalian cells, and their N-glycosylation pathway is incompletely understood compared with that of mammals. Here, we cloned a candidate gene for β-1,2-N-acetylglucosaminyltransferase II (GnTII), which is a Golgi-localized enzyme involved in a key step in the conversion to complex-type N-glycans, from silkworm Bombyx mori, and the gene was found to be expressed ubiquitously in the larval and pupal stages. In addition, recombinant B. mori GnTII was expressed as a soluble form using a silkworm-B. mori nucleopolyhedrovirus bacmid expression system. The recombinant enzyme exhibited similar pH and temperature dependency and the same substrate specificity as human GnTII, but deglycosylation with peptide:N-glycanase F did not affect its enzymatic activity. Compared with the structure of human GnTII, the amino acid residues involved in catalytic activity and substrate recognition are almost fully conserved in B. mori GnTII, which is consistent with its enzymatic properties. These results raised the possibility of mammalian-like complex-type N-glycan synthesis using the GnTII ortholog in silkworm.

  • Synthesis and characterization of Ogataea thermomethanolica alcohol oxidase immobilized on barium ferrite magnetic microparticles

    Publication date: March 2019

    Source: Journal of Bioscience and Bioengineering, Volume 127, Issue 3

    Author(s): Natthaya Mangkorn, Pattanop Kanokratana, Niran Roongsawang, Apirat Laobuthee, Navadol Laosiripojana, Verawat Champreda

    Alcohol oxidase catalyzes the oxidation of primary alcohols into the corresponding aldehydes, making it a potential biocatalyst in the chemical industry. However, the high production cost and poor operational stability of this enzyme are limitations for industrial application. Immobilization of enzyme onto solid supports is a useful strategy for improving enzyme stability. In this work, alcohol oxidase from the thermotolerant methylotrophic yeast Ogataea thermomethanolica (OthAOX) was covalently immobilized onto barium ferrite (BaFe12O19) magnetic microparticles. Among different conditions tested, the highest immobilization efficiency of 71.0 % and catalytic activity of 34.6 U/g was obtained. Immobilization of OthAOX onto magnetic support was shown by Fourier-Transformed infrared microscopy, scanning electron microscopy and X-ray diffraction. The immobilized OthAOX worked optimally at 55 °C and pH 8.0. Immobilization also improved thermostability, in which >65% of the initial immobilized enzyme activity was retained after 24 h pre-incubation at 45 °C. The immobilized enzyme showed a greater catalytic efficiency for oxidation of methanol and ethanol than free enzyme. The immobilized enzyme could be recovered by magnetization and recycled for at least three consecutive batches, after which 70% activity remained. The properties of the immobilized enzyme suggest its potential industrial application for synthesis of aldehyde.

    Graphical abstract

    Image 1

  • Optimization of the treatment conditions with glycogen synthase kinase-3 inhibitor towards enhancing the proliferation of human induced pluripotent stem cells while maintaining an undifferentiated state under feeder-free conditions

    Publication date: March 2019

    Source: Journal of Bioscience and Bioengineering, Volume 127, Issue 3

    Author(s): Kiyomi Yoda, Yoshitsugu Ohnuki, Hiroshi Kurosawa

    The small molecule inhibitor CHIR99021 (CHIR) is well known as a selective glycogen synthase kinase-3 inhibitor. The purpose of our study was to optimize the conditions of CHIR supplementation that will enhance the proliferation of human induced pluripotent stem cells (hiPSCs) while maintaining their undifferentiated state under feeder-free conditions in adherent cultures for 4 days. Our results revealed that both of the timing and concentration of CHIR affected cell behaviors of hiPSCs, such as colony formation, cell proliferation, and differentiation. The addition of 1–3 μM CHIR to hiPSCs cultures in the late 2-day period of a 4-day cultivation was effective in enhancing cell proliferation. Treatment with 3 μM CHIR significantly enhanced cell proliferation, but led to differentiation of hiPSCs when the treatment was carried out over 4 days. Treatment with higher concentration of CHIR was also conducive to deviating hiPSCs from their undifferentiated state. Treatment with 10 μM CHIR led to decreased expression of pluripotency-associated genes and increased level of mesoderm marker genes, but failed to provided any growth-promoting effect. Interestingly, when treatment with 1 μM CHIR was confined to the late 2-day period of a 4-day cultivation, cell proliferation was enhanced without detectable deviation from the undifferentiated state as evidenced by the expression levels of pluripotency-associated genes, e.g., OCT3/4, NANOG, SOX2, and REX1. Repeated use of 1 μM CHIR in subcultures provided no adverse effect on the proliferation of hiPSCs. Our results indicated that carefully designed CHIR treatment allows for enhanced proliferation of hiPSCs.

  • Influence of graphene oxide and biochar on anaerobic degradation of petroleum hydrocarbons

    Publication date: Available online 12 February 2019

    Source: Journal of Bioscience and Bioengineering

    Author(s): Benru Song, Jingchun Tang, Meinan Zhen, Xiaomei Liu

    The anaerobic degradation of petroleum is an important process in natural environments. So far, few studies have considered the response of the microbial community to nanomaterials during this process. This study explored the potential effects of graphene oxide and biochar on the anaerobic degradation of petroleum hydrocarbons in long-term experiments. Cyclic voltammetry and electrochemical impedance spectroscopy indicated that the addition of carbon-based materials promoted the electrochemical activity of anaerobic cultures that degrade petroleum hydrocarbons. The maximum degradation rates for benzene, toluene, ethylbenzene, and xylene (BTEXs) in the cultures incubated for 10 weeks with graphene oxide (0.02 mg/L) and biochar (20 mg/L) were 76.5% and 77.6%, respectively. The maximum degradation rates of n-alkanes in the cultures incubated for 10 weeks with graphene oxide (2 mg/L) and biochar (100 mg/L) were 70.0% and 77.8%, respectively. The 16S rDNA copy numbers in the treatments with 0.02 mg/L graphene oxide and 20 mg/L biochar were significantly higher than others during the process (P < 0.05). In the 2nd week, the maximum copy numbers of the masD and bamA genes in the treatments with biochar were 349 copies/mL (20 mg/L) and 422 copies/mL (20 mg/L), respectively, and in the treatments with graphene oxide were 289 copies/mL (0 mg/L) and 366 copies/mL (0.02 mg/L). The contents of carbon-based materials had slight effects on the microbial community structure, whereas the culture time had obvious effects. Paracoccus denitrificans, Pseudomonas aeruginosa, and Hydrogenophaga caeni were the dominant microorganisms in the culture systems under all treatments.