Journal of Bioscience and Bioengineering (JBB) 新着論文

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  • Green synthesis and characterization of iron oxide nanoparticles using Ficus carica (common fig) dried fruit extract

    Publication date: Available online 20 October 2018

    Source: Journal of Bioscience and Bioengineering

    Author(s): Derya Aksu Demirezen, Yalçın Şevki Yıldız, Şeyda Yılmaz, Dilek Demirezen Yılmaz

    Ficus carica (common fig) dried fruit extract was used to synthesize iron oxide nanoparticles in this study. Biomaterials in the common fig dried fruit extract synthesized the iron nanoparticles by reducing the iron precursor salt and then acted as capping and stabilizing agents. The nanoparticles were produced smaller than 20 nm diameters and oxidized due to the high phenolic compound content in the common fig dried fruit extract. Nanoparticles were characterized by energy dispersive X-ray spectroscopy (EDX), transmission electron microscopy (TEM), UV-visible spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS). First, color change and pH reduction occurred immediately due to the iron nanoparticle synthesis. TEM images showed that the nanoparticles were at 9 ± 4 nm diameters and the metallic core-oxide shell form. The nanoparticles were in spherical shapes with a monodisperse distribution. EDX, XRD and FTIR analysis signals showed the iron oxyhydroxide/oxide formation. Absorption peaks were detected at 205 nm and 291 nm due to the iron metallic core hydrolysis products. The intensity-average diameter of nanoparticles was calculated at 475 nm diameter by DLS analysis. Colloid stability was determined as moderate at 20.7 mV.

  • Gene cloning and expression of the l-asparaginase from Bacillus cereus BDRD-ST26 in Bacillus subtilis WB600

    Publication date: Available online 19 October 2018

    Source: Journal of Bioscience and Bioengineering

    Author(s): Yue Feng, Song Liu, Yun Jiao, Yunlong Wang, Miao Wang, Guocheng Du

    l-Asparaginase (ASN; EC 3.5.1.1) shows great commercial value because of its ability to reduce toxic levels of acrylamide in foods. To achieve high-efficiency production of l-asparaginase, an open reading frame of 978 bp encoding asparaginase (BcA) was amplified from Bacillus cereus BDRD-ST26, followed by its expression in Bacillus subtilis WB600, with the highest yield of 374.9 U/ml obtained using an amyE-signal peptide. A four-step purification protocol was used to purify BcA, resulting in a 15.1-fold increase in purification yield, with a specific activity of purified BcA at 550.8 U/mg and accompanied by detection of minimal l-glutaminase activity. Maximum BcA activity was detected at 50°C and pH 9.0 in 20 mM Tris–HCl buffer, with a half-life at 50°C of 17.35 min and a Km and kcat of 9.38 mM and 63.6 s−1, respectively. Compared with untreated potato strips, 72% acrylamide (2.35 mg/kg) was removed from potato strips pretreated with BcA. These results indicated that this novel BcA variant represents a potential candidate for application in the food-processing industry.

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  • Spectroscopic study on the conformation of serum albumin in film state

    Publication date: Available online 16 October 2018

    Source: Journal of Bioscience and Bioengineering

    Author(s): Hironori Yamazoe

    Protein is a promising material for fabricating the biocompatible films used in the biomedical fields and food industry. Previously, we successfully prepared a water-insoluble albumin film possessing native albumin properties such as resistance to cell adhesion and drug-binding ability. Here, I quantitatively investigated the conformation of albumin in a film state using circular dichroism (CD) spectroscopy. The albumin film was prepared by crosslinking albumin with ethylene glycol diglycidyl ether (EGDE). CD measurements of albumin films revealed that approximately 70% of the α-helical structure was retained after film formation. Albumin molecules in the films acquired high stability. The conformation of albumin was completely retained even after heating at 100 °C for 1 h. For comparison, crosslinked albumin film was also prepared using glutaraldehyde (GA). Unlike EGDE-crosslinking, GA-crosslinking induced significant conformational changes in albumin; 46% of the α-helical structure was destroyed in GA-crosslinked albumin films. Cell adhesion studies showed that EGDE-crosslinked albumin film maintained the cell-nonadhesive property inherent in native albumin. This property was lost in GA-crosslinked albumin film, and cells adhesion occurred at a level comparable to that of cell culture dishes. These results indicate that EGDE-crosslinking is a useful method for preparing albumin films in which the native albumin structure and property are retained. The approach described here provides valuable information for creating protein films possessing high functionality.

  • Identification and characterization of multifunctional cationic peptides from traditional Japanese fermented soybean Natto extracts

    Publication date: Available online 16 October 2018

    Source: Journal of Bioscience and Bioengineering

    Author(s): Masayuki Taniguchi, Ryousuke Aida, Kazuki Saito, Akihito Ochiai, Satoshi Takesono, Eiichi Saitoh, Takaaki Tanaka

    In this study, we investigated the lipopolysaccharide (LPS)-neutralizing and angiogenic activities of cationic peptides derived from the traditional Japanese fermented product Natto, which is made by fermenting cooked soybeans using Bacillus subtilis. Initially, we prepared 20 fractions of Natto extracts with various isoelectric points (pI's) using ampholyte-free isoelectric focusing (autofocusing). Cationic peptides were then purified from fractions 19 and 20, whose pH values were greater than 12, using reversed-phase high-performance liquid chromatography, and were identified using matrix-assisted laser/desorption ionization–time-of-flight mass spectroscopy. Among the 13 identified cationic peptides, seven (KFNKYGR, FPFPRPPHQK, GQSSRPQDRHQK, QRFDQRSPQ, ERQFPFPRPPHQK, GEIPRPRPRPQHPE, and EQPRPIPFPRPQPR) had pI's greater than 9.5, positive net charges, and differing molecular weights. These peptides were then chemically synthesized and applied to chromogenic LPS-neutralizing assays using Limulus amebocyte lysates, and 50% effective (neutralizing) concentrations of 2.6–5.5 μM were demonstrated. In addition, tube formation assays in human umbilical vein endothelial cells revealed angiogenic activities for all but one (GEIPRPRPRPQHPE) of these seven cationic peptides, with increases in relative tube lengths of 23–31% in the presence of peptides at 10 μM. Subsequent experiments showed negligible hemolytic activity of these peptides at concentrations of up to 500 μM in mammalian red blood cells. Collectively, these data demonstrate that six cationic peptides from Natto extracts, with the exception of GEIPRPRPRPQHPE, have LPS-neutralizing and angiogenic activities but do not induce hemolysis.

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  • Development of a helical coagulation reactor for harvesting microalgae

    Publication date: Available online 13 October 2018

    Source: Journal of Bioscience and Bioengineering

    Author(s): Haiyang Zhang, Chunhua Liu, Yang Ou, Ting Chen, Lan Yang, Zicheng Hu

    In this study, an innovative helical coagulation reactor (HCR) was developed for harvesting microalgae by sedimentation with polyaluminium chloride (PAC). The effects of construction and hydrodynamic characteristics on harvesting performance were investigated. Results showed that a higher harvesting efficiency, 96.37%, was achieved for the large and compact flocs generated by the HCR, and the settling rate of flocs was substantially influenced by the velocity gradient (G) and the Reynolds number (Re). When the Reynolds number closed to the transition between laminar and turbulent flow (4000), the flocs settled faster (20.51 m h−1), although settling slowed as the Reynolds number increased further because of ruptured flocs. The settling rate of flocs could be further improved to 23.27 m h−1 by a pulse flow field, mainly due to larger and more compact flocs forming in the plug pipe flow. Furthermore, a comparative investigation of a mechanically agitated vessel and the HCR with the same Camp number (Gt) showed that the HCR achieved higher settling rates and a shorter residence time than those with a mechanical agitator. The HCR provided a uniform dissipation of energy and high velocity gradient while avoiding electrical and mechanical energy consumption, suggesting this reactor is an efficient and economic option for microalgae harvesting.

  • Heterologous production of free dihomo-γ-linolenic acid by Aspergillus oryzae and its extracellular release via surfactant supplementation

    Publication date: Available online 13 October 2018

    Source: Journal of Bioscience and Bioengineering

    Author(s): Koichi Tamano, Robert Sidney Cox, Kenji Tsuge, Ai Miura, Ayano Itoh, Jun Ishii, Tomohiro Tamura, Akihiko Kondo, Masayuki Machida

    Free dihomo-γ-linolenic acid (DGLA) and its desaturated form, free arachidonic acid (ARA) are polyunsaturated free fatty acids (FFAs). They are useful raw materials to produce eicosanoid pharmaceuticals. In this study, we aimed at their production by the oleaginous filamentous fungus Aspergillus oryzae via metabolic engineering. Three genes encoding enzymes involved in the synthesis of DGLA and ARA, were isolated from the filamentous fungus Mortierella alpina that produces ARA in a triacylglycerol form. These genes were concatenated to promoters and terminators of highly expressed genes of A. oryzae, and the concatenated DNA fragments were further concatenated with each other to generate a single DNA fragment in the form of a biosynthetic gene cluster. By homologous recombination, the resulting DNA fragment was integrated to the chromosome of the A. oryzae acyl-CoA synthetase gene disruptant whose FFA productivity was enhanced at 9.2-fold more than the wild-type strain. The DNA-integrated disruptant produced free DGLA but did not produce free ARA. Thus, focusing on free DGLA, after removal of the gene for converting DGLA to ARA, the constructed strain produced free DGLA at 145 mg/l for 5 d. Also, by supplementing Triton X-100 surfactant at 1% to the culture, over 80% of free DGLA was released from cells without inhibiting the growth. Consequently, the constructed strain will be useful for attempting production of free DGLA-derived eicosanoids because it bypasses excision of free DGLA from triacylglycerols by lipase. To our knowledge, this is the first report on microbial production of free DGLA and its extracellular release.

  • Fabrication and in vitro evaluation of a packed-bed bioreactor based on an optimum two-stage culture strategy

    Publication date: Available online 12 October 2018

    Source: Journal of Bioscience and Bioengineering

    Author(s): Wei Liu, Dan Hu, Ce Gu, Yan Zhou, Wen-Song Tan

    A packed-bed (PB) bioreactor for bioartificial liver (BAL) was fabricated based on an optimum two-stage culture strategy and evaluated in vitro in this research. Human induced hepatocytes (hiHeps) were first expanded using Cytodex 3 microcarriers and the choice of microcarrier concentration and fetal bovine serum (FBS) content was optimized. Then, the cells expanded under the optimum expansion condition were perfused into a perfusion system containing Fibra-Cel (FC) disks to fabricate a PB bioreactor. Operating parameters including flow rate and seeding density for perfusion culture were optimized, respectively. Results indicated that during suspension culture, rapid cell proliferation and favorable amino acid metabolism were achieved at 3 mg/mL microcarriers combined with 1% FBS. While for the perfusion culture, the most effective flow rate and seeding density were 2 mL/min and 1 × 106 cells/mL, respectively. Under this optimum perfusion condition, hiHeps showed good proliferation ability, high viability, homogeneous distribution, high metabolism activities and efficient albumin secretion as well as high liver-specific genes expression. Therefore, the two-stage culture strategy based on operating parameters optimization provides a new method for the development of PB bioreactors.

  • Continuous production of d-lactic acid from cellobiose in cell recycle fermentation using β-glucosidase-displaying Escherichia coli

    Publication date: Available online 11 October 2018

    Source: Journal of Bioscience and Bioengineering

    Author(s): Yuji Aso, Mikikazu Tsubaki, Bui Hoang Dang Long, Ryo Murakami, Keisuke Nagata, Hirohisa Okano, Ngo Thi Phuong Dung, Hitomi Ohara

    The present study demonstrates continuous production of d-lactic acid from cellobiose in a cell recycle fermentation with a hollow fiber membrane using recombinant Escherichia coli constructed by deleting its pyruvate formate-lyase activating enzyme gene pflA and expressing a heterologous β-glucosidase on its cell surface. The β-glucosidase gene bglC from Thermobifida fusca YX was cloned into a cell surface display vector pGV3, resulting in pGV3-bglC. Recombinant E. coli JM109 harboring the pGV3-bglC showed β-glucosidase activity (18.9 ± 5.7 U/OD600), indicating the cell surface functioning of mutant β-glucosidase. pH-stat cultivation using d-lactic acid producer E. coli BW25113 (ΔpflA) harboring pGV3-bglC in minimum medium with 10 g/L cellobiose in a jar fermentor under anaerobic condition resulted in 5.2 ± 0.1 g/L of d-lactic acid was obtained after 84 h cultivation, indicating that the engineered E. coli produced d-lactic acid directly from cellobiose. For continuous d-lactic acid production, cell recycle fermentation was conducted under anaerobic condition and the culture was continuously ultrafiltrated with a hollow fiber cartridge. The permeate was drawn to the reservoir and a minimum medium containing 10 g/L cellobiose was fed to the fermentor at the same rate (dilution rate, 0.05 h−1). Thus, this system maintained the d-lactic acid production (4.3–5.0 g/L), d-lactic acid production rate (0.22–0.25 g/L/h), and showed no residual cellobiose in the culture during 72 h operation. Interestingly, the d-lactic acid production rate in cell recycle fermentation was more than 3 times higher than that in the batch operation (0.06 ± 0.00 g/L/h).

  • Comparison of sequential derivatization with concurrent methods for GC/MS-based metabolomics

    Publication date: Available online 11 October 2018

    Source: Journal of Bioscience and Bioengineering

    Author(s): Hiromi Miyagawa, Takeshi Bamba

    The gas chromatography/mass spectrometry (GC/MS)-based metabolomics requires a two-step derivatization procedure consisting of oximation and silylation. However, due to the incomplete derivatization and degeneration of the metabolites, good repeatability is difficult to obtain during the batch derivatization, as the time between completing the derivatization process and GC analysis differs from sample to sample. In this research, we successfully obtained good repeatability for the peak areas of 52 selected metabolites by sequential derivatization and interval injection, in which the oximation and silylation times were maintained at constant values. In addition, the derivatization times and amount of reagents employed were varied to confirm that the optimal derivatization conditions differed for the various metabolites. In conventional batch derivatization, six metabolites, viz. glutamine, glutamic acid, histidine, alanine, asparagine, and tryptophan, exhibited fluctuations in their peak areas. Indeed, we found that for all six metabolites these differences originated from the silylation process, while the variations for glutamine and glutamic acid were related to the oximation process.

  • Delta-9 fatty acid desaturase overexpression enhanced lipid production and oleic acid content in Rhodosporidium toruloides for preferable yeast lipid production

    Publication date: Available online 11 October 2018

    Source: Journal of Bioscience and Bioengineering

    Author(s): Yung-Yu Tsai, Takao Ohashi, Chih-Chan Wu, Dolgormaa Bataa, Ryo Misaki, Savitree Limtong, Kazuhito Fujiyama

    The oil plants provide a sufficient source of renewable lipid production for alternative fuel and chemical supplies as an alternative to the depleting fossil source, but the environmental effect from these plants’ cropping is a concern. The high oleic acid (OA; C18:1) content in plant-derived products provide advantages of multiple uses with improved oxidative stability and a wide range of applicable temperature. Here we used a promising lipid producer, the oleaginous yeast Rhodosporidium toruloides, to attempt to obtain an OA-enriched lipid. Saccharomyces cerevisiae OLE1 (ScOLE1) gene encodes Δ9 fatty acid desaturase (Δ9FAD), which is generally known to synthesize palmitoleic acid (POA; C16:1) and OA, but the functions of putative R. toruloides Δ9FAD gene are not well understood. In a complementary test, the RtΔ9FAD gene rescued the survival of an OA-deficient Scole1Δ mutant, and we introduced the RtΔ9FAD gene into R. toruloides strains for the production of OA-enriched lipid. Increasing lipid production was observed in ScOLE1 and genomic RtΔ9FAD gene-overexpressing R. toruloides strains. The ScOLE1 transformant output fivefold more OA content in total amount, with >70% of total lipid. Different enhancing effects from the protein coding sequence and genomic sequence of RtΔ9FAD genes were also observed. Overall, this study resulted in ScOLE1 and RtΔ9FAD gene overexpression in R. toruloides to obtain OA-enriched lipid as a candidate source of designed biodiesel and lipid-related chemicals.

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  • Characterization of a novel exo-chitosanase, an exo-chitobiohydrolase, from Gongronella butleri

    Publication date: Available online 10 October 2018

    Source: Journal of Bioscience and Bioengineering

    Author(s): Kiyohiko Seki, Yasue Nishiyama, Masaru Mitsutomi

    An exo-chitosanase was purified from the culture filtrate of Gongronella butleri NBRC105989 to homogeneity by ammonium sulfate precipitation, followed by column chromatography using CM-Sephadex C-50 and Sephadex G-100. The enzyme comprised a monomeric protein with a molecular weight of approximately 47,000 according to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited optimum activity at pH 4.0, and was stable between pH 5.0 and 11.0. It was most active at 45°C, but was stable at temperatures below 30°C. The enzyme hydrolyzed soluble chitosan and glucosamine (GlcN) oligomers larger than tetramers, but did not hydrolyze N-acetylglucosamine (GlcNAc) oligomers. To clarify the mode of action of the enzyme, we used thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) to investigate the products resulting from the enzyme-catalyzed hydrolysis of chitosan and N1-acetylchitohexaose [(GlcN)5-GlcNAc] with a GlcNAc residue at the reducing end. The results indicated that the enzyme is a novel exo-type chitosanase, exo-chitobiohydrolase, that releases (GlcN)2 from the non-reducing ends of chitosan molecules. Analyses of the hydrolysis products of partially N-acetylated chitooligosaccharides revealed that the enzyme cleaves both GlcN-GlcNAc and GlcNAc-GlcN bonds in addition to GlcN-GlcN bonds in the substrate.

  • 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: Available online 3 October 2018

    Source: Journal of Bioscience and Bioengineering

    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.

  • Enhanced exopolysaccharide production by Cordyceps militaris using repeated batch cultivation

    Publication date: Available online 3 October 2018

    Source: Journal of Bioscience and Bioengineering

    Author(s): Chien-Chang Wang, Jiun-Yan Wu, Chih-Yuan Chang, Shih-Tsung Yu, Yung-Chuan Liu

    Cordyceps militaris exo-polysaccharides (EPS) have been reported to possess many benefits, such as anti-tumor, anti-inflammatory and antioxidant activities. In this study, the production of EPS via cultivation in a bioreactor was investigated. Glucose and yeast extract were determined to be the most suitable carbon and nitrogen sources for EPS production. The appropriate levels of glucose and yeast extract were 40 g/L and 10 g/L, respectively, resulting in EPS production of 1.686 g/L in a submerged culture. In the stirred-tank fermentor, an agitation rate of 150 rpm and aeration rate of 1.5 vvm were the most effective for EPS production. Due to the anchoring of mycelial cells on the wall of fermentor, a repeated batch approach was used. EPS production of C. militaris could be enhanced to a maximum of 5.713 g/L, with a productivity of 476 mg/L/day in the second run. The repeated batch approach was expected to generate higher EPS production, increase EPS yield and productivity and further simplify cultivation operations for bio-industrial application.

  • Effectiveness of cross-linked enzyme aggregates of cellulolytic enzymes in hydrolyzing wheat straw

    Publication date: October 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 4

    Author(s): Shuddhodana, Munishwar N. Gupta, Virendra S. Bisaria

    Development of industrially potent cellulolytic enzymes is one of the greatest challenges faced in lignocellulosic feed-stock based bio-refining. In the current work cross-linked enzyme aggregates (CLEAs) of commercial cellulase mix were successfully prepared and their performance to be used as potential industrial enzymes in terms of stability and wheat straw hydrolysis was evaluated. The CLEAs were more stable compared to native enzymes with half-lives being 2.30-, 1.56-, 3.07- and 1.67-fold higher at 70°C for filter paper activity (FPA), endoglucanase, β-glucosidase and xylanase, respectively. CLEAs retained 77.4% of endoglucanase and 85.9% of xylanase activity after five cycles of hydrolysis of soluble substrates such as carboxymethyl cellulose and xylan, respectively. A maximum saccharification yield of 31.8% by soluble enzymes and 32.9% by CLEAs were obtained when alkali-pretreated wheat straw was subjected to hydrolysis. On repeated batch hydrolysis for five consecutive cycles of 24 h each, the CLEAs showed an overall higher saccharification yield of 43.3% compared to 31.8% with soluble enzymes.

  • Characterization of two fungal lipoxygenases expressed in Aspergillus oryzae

    Publication date: October 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 4

    Author(s): Akiko Sugio, Lars Henrik Østergaard, Kenji Matsui, Shinobu Takagi

    Two fungal lipoxygenase genes were cloned from a rice pathogen, Magnaporthe salvinii, and the take-all fungus, Gaeumannomyces graminis var. tritici, and successfully expressed in Aspergillus oryzae in secreted form. The lipoxygenases expressed, termed MLOX and GLOX, were purified and characterized to evaluate suitability for industrial applications. Both enzymes were active broadly at pH 4–11 and had optimum temperatures around 60 °C, but they were largely different in substrate specificity. Where MLOX was active broadly on arachidonic acid, EPA and DHA, and even on derivatives of fatty acids, such as methyl linoleate or linoleoyl alcohol, GLOX was more specific to linoleic acid and linolenic acid. The most remarkable difference between the two fungal LOXs was the positional and stereo-specificity of oxygenation reactions on polyunsaturated fatty acids. When using linoleic acid as the substrate, the product of MLOX was 9S-hydroperoxy-(E,Z)-octadecadienoic acid (9S(E,Z)-HPODE), on the other hand, the product of GLOX was 13R(E,Z)-HPODE. The enzymes were evaluated for a couple of potential applications and found to be effective on bleaching colored compounds such as carotenoids.

  • Soluble expression of horseradish peroxidase in Escherichia coli and its facile activation

    Publication date: October 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 4

    Author(s): Sushma Chauhan, Taek Jin Kang

    Horseradish peroxidase (HRP) is widely used as a marker enzyme in immunoassays and biosensors, and can possibly be used in industries such as waste water treatments or fine chemical synthesis. Cost-effective production of active HRP is thus very important in the related fields. Also, engineering of HRP for its better performance in the designated application is expected to make the enzyme even more important in several areas of research and industry. One of obstacles to this end and to the large scale production of the enzyme has been its facile expression in a bacterial host. Here we show that HRP could be overexpressed as a soluble form by fusing the enzyme with Escherichia coli phosphoglycerate kinase (PGK). After simple incubation with calcium ion, hemin, and oxidized glutathione, PGK-HRP could be fully activated showing a higher molar specific activity than plant-derived HRP. Our established procedure did not use tedious and inefficient refolding steps that have been used to activate HRP produced as inclusion bodies and thus is superior in its overall yield (>72 mg purified HRP conjugate per L culture) to existing methods. By co-expressing PGK-HRP with ferrochelatase in a special host that permitted the formation of disulfide bonds in the cytoplasm, the activation steps could be simplified even though the resulting specific activity was low.

  • d-Lactate electrochemical biosensor prepared by immobilization of thermostable dye-linked d-lactate dehydrogenase from Candidatus Caldiarchaeum subterraneum

    Publication date: October 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 4

    Author(s): Takenori Satomura, Junji Hayashi, Hiroaki Sakamoto, Takuro Nunoura, Yoshihiro Takaki, Ken Takai, Hideto Takami, Toshihisa Ohshima, Haruhiko Sakuraba, Shin-ichiro Suye

    A stable d-lactate electrochemical sensing system was developed using a dye-linked d-lactate dehydrogenase (Dye-DLDH) from an uncultivated thermophilic archaeon, Candidatus Caldiarchaeum subterraneum. To develop the system, the putative gene encoding the Dye-DLDH from Ca. Caldiarchaeum subterraneum was overexpressed in Escherichia coli, and the expressed product was purified. The recombinant enzyme was a highly thermostable Dye-DLDH that retained full activity after incubation for 10 min at 70°C. The electrode for detection of d-lactate was prepared by immobilizing the thermostable Dye-DLDH and multi-walled carbon nanotube (MWCNT) within Nafion membrane. The electrocatalytic response of the electrode was clearly observed upon exposure to d-lactate. The electrode response to d-lactate was linear within the concentration range of 0.03–2.5 mM, and it showed little reduction in responsiveness after 50 days. This is the first report describing a d-lactate sensing system using a thermostable Dye-DLDH.

  • Characterization of mutants expressing thermostable D1 and D2 polypeptides of photosystem II in the cyanobacterium Synechococcus elongatus PCC 7942

    Publication date: October 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 4

    Author(s): Norihisa Haraguchi, Jun Kaseda, Yasumune Nakayama, Kazuhiro Nagahama, Takahira Ogawa, Masayoshi Matsuoka

    Photosystem II complex embedded in thylakoid membrane performs oxygenic photosynthesis where the reaction center D1/D2 heterodimer accommodates all components of the electron transport chain. To express thermostable D1/D2 heterodimer in a cyanobacterium Synechococcus elongatus PCC 7942, we constructed a series of mutant strains whose psbA1 and psbD1 genes encoding, respectively, the most highly expressed D1 and D2 polypeptides were replaced with those of a thermophilic strain, Thermosynechococcus vulcanus. Because the C-terminal 16 amino acid sequences of D1 polypeptides should be processed prior to maturation but diverge from each other, we also constructed the psbA1ΔC-replaced strain expressing a thermostable D1 polypeptide devoid of the C-terminal extension. The psbA1/psbD1-replaced strain showed decreased growth rate and oxygen evolution rate, suggesting inefficient photosystem II. Immunoblot analyses for thermostable D1, D2 polypeptides revealed that the heterologous D1 protein was absent in thylakoid membrane from any mutant strains with psbA1, psbA1ΔC, and psbA1/psbD1-replacements, whereas the heterologous D2 protein was present in thylakoid membrane as well as purified photosystem II complex from the psbA1/psbD1-replaced strain. In the latter strain, the compensatory expression of psbA3 and psbD2 genes was elevated. These data suggest that heterologous D2 polypeptide could be combined with the host D1 polypeptide to form chimeric D1/D2 heterodimer, whereas heterologous D1 polypeptide even without the C-terminal extension was unable to make complex with the host D2 polypeptide. Since the heterologous D1 could not be detected even in the whole cells of psbA1/psbD1-replaced strain, the rapid degradation of unprocessed or unassembled heterologous D1 was implicated.

  • Development of human-derived hemoglobin–albumin microspheres as oxygen carriers using Shirasu porous glass membrane emulsification

    Publication date: October 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 4

    Author(s): Seiichi Ohta, Kenichiro Hashimoto, Xiaoting Fu, Masamichi Kamihira, Yasuyuki Sakai, Taichi Ito

    Novel hemoglobin-based artificial oxygen carriers are necessary in tissue engineering. We developed human hemoglobin (hHb) and albumin (HSA)-based microspheres using Shirasu porous glass (SPG) membrane emulsification. The obtained microspheres had a uniform size with an average diameter of 15.1 μm measured by optical microscope, which is similar to the diameter of human red blood cells (7–8 μm). The loading amount of hHb in the microspheres was 20 wt%, which is similar to that of red blood cells (33 wt%). The hHb–HSA microspheres showed similar oxygen dissociation behavior and methemoglobin formation resistance to native hHb. Incubation with genetically engineered HeLa cells that shows hypoxia-responsive EGFP expression demonstrated efficient oxygen supply from the microspheres. Our study suggests the utility of hHb–HSA microspheres as oxygen carriers for tissue engineering with a low risk of infectious disease.

    Graphical abstract

    Graphical abstract for this article

  • Production of giant unilamellar vesicles by the water-in-oil emulsion-transfer method without high internal concentrations of sugars

    Publication date: October 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 4

    Author(s): Gakushi Tsuji, Takeshi Sunami, Norikazu Ichihashi

    Giant unilamellar vesicles (GUVs) are large vesicles bounded by a single lipid bilayer, which have been used in various applications as artificial, cell-like compartments. The water-in-oil (w/o) emulsion-transfer method has been attracting attention as a method to prepare GUVs that can efficiently encapsulate macromolecules. For efficient GUV production by this method, non-physiological, high concentrations of sugars are usually required in the inner solution of the GUVs. These sugars limit the utility of the GUVs for a wide range of applications. In this study, we investigated various compositions of the inner and outer solutions to achieve efficient production without high concentrations of sugars through the w/o emulsion-transfer method. Firstly, we adjusted the osmotic pressure and density of the outer solution with NaCl and succeeded in increasing the proportion of GUVs and the absolute number in the prepared liposome population. Secondly, we increased the density of the inner solution with cytochrome c, but the proportion of GUVs and absolute number of vesicles did not increase. Thirdly, we increased the density of the inner and outer solutions with glycerol, which is membrane permeable and can be removed from GUVs, and succeeded in increasing the GUV proportion. These results provide useful information for the efficient preparation of GUVs that enclose a physiologically-relevant environment by the w/o emulsion-transfer method.