JBB : Journal of Bioscience and Bioengineering

Journal of Bioscience and Bioengineering vol.127 cover

 



  • Maintenance of an undifferentiated state of human-induced pluripotent stem cells through botulinum hemagglutinin-mediated regulation of cell behavior

    Publication date: Available online 17 January 2019

    Source: Journal of Bioscience and Bioengineering

    Author(s): Eri Shuzui, Mee-Hae Kim, Keisuke Azuma, Yukako Fujinaga, Masahiro Kino-oka

    Applications of human induced pluripotent stem cell (hiPSC) culture are impaired by problems with long term maintenance of pluripotency. In this study, we report that exposure to botulinum hemagglutinin (HA), an E-cadherin function-blocking agent, suppressed deviation from an undifferentiated state in hiPSC colonies. Time-lapse imaging of live cells revealed that cells in central regions of colonies moved slowly and underwent a morphological change to a cobblestone-like shape via interaction between contacting cells, forming dense, multiple layers. Staining and migration analysis showed that actin stress fibers and paxillin spots were diminished in colony central regions, and this was associated with alteration of cellular morphology and migratory behavior. However, in culture with HA exposure, cells in the central and peripheral regions of hiPSC colonies were migratory and arranged in loose monolayers, resulting in relatively uniform dispersion of cells in colonies. We also found that a well-organized network of actin stress fibers was of significance in the central and peripheral regions of a colony, resulting in activation of paxillin and E-cadherin expression in hiPSCs. After routine application of HA for serial passages, hiPSCs remained pluripotent and capable of differentiating into all three germ layers. These observations indicate that relaxation of cell–cell junctions by HA induced rearrangements of the cytoskeleton and cell adhesion in hiPSC colonies by promoting migratory behaviors. These results suggest that this simple and readily reproducible culture strategy is a potentially useful tool for improving the robust and scalable maintenance of undifferentiated hiPSC cultures.

  • Acidogenic properties of carbohydrate-rich wasted potato and microbial community analysis: Effect of pH

    Publication date: Available online 14 January 2019

    Source: Journal of Bioscience and Bioengineering

    Author(s): Yan Li, Xiaodong Zhang, Haipeng Xu, Hui Mu, Dongliang Hua, Fuqiang Jin, Guangfan Meng

    Vegetable waste is one of the major organic solid residues available for sustainable biogas production. The aim of this study was to investigate the possibility and optimal controlling strategy for acidogenic fermentation of wasted potato (WP). Three leaching bed reactors (LBRs) were operated at various pH values (6.0, 7.0 and 8.0) with an organic loading rate (OLR) of 6.7 g volatile solid/(L·d) and hydraulic retention time of 6 d. Butyric acid-type fermentation with butyric acid as predominant volatile fatty acid (VFA) was observed with a concentration and proportion (of total VFAs) of butyric acid, which were 7.8 g/L, 49.7 % and 9.6 g/L and 52.2 % at pH 6.0 and 7.0, respectively. Conversely, at pH 8.0, mixed acid-type fermentation was observed with acetic and butyric acid as the major VFAs. Control experiment without pH manipulation didn't perform well in VFAs production at first 6 days and then VFAs concentration increased as pH value was adjusted to 8. It was indicated that the inhibition was caused by high undissociated VFAs concentration due to low pH and the VFAs production could be improved through pH control strategy to regulate the undissociated VFAs concentration. According to the bacterial analysis, the microbial community was diverse and Firmicutes were the most important bacteria at different pH conditions. Therefore, the results suggested that a process of pH control might be feasible for stable and efficient acidogenic fermentation.

  • Utility of a rotation/revolution-type agitator for chondrocyte isolation during preparation of engineered cartilage

    Publication date: Available online 12 January 2019

    Source: Journal of Bioscience and Bioengineering

    Author(s): Akinori Oda, Ryota Takamiya, Rin Kaneko, Haruna Yoshida, Yuta Yanagita, Hatsumi Sekiguchi, Yoshihito Nobe, Kazuaki Muramatsu

    During the manufacture of cell- and tissue-based products, such as engineered cartilage for autologous chondrocyte implantation, maximizing the number of cells isolated from donor tissue substantially improves the productivity of these products. The method used for agitating tissues with digestive fluid and enzymes can considerably affect both the quality and quantity of isolated cells. This study aimed to investigate the effectiveness of a rotation/revolution-type agitator for chondrocyte isolation following the enzymatic digestion of rat costal cartilage. Cartilage tissue cut into 1 mm3-thick sections was equally divided between two groups and placed in 50-mL conical tubes; sections in both groups were digested using 0.1 mg/mL liberase TH (collagenase/thermolysin) at 37 °C for 4 h with either rotation/revolution or conventional orbital agitation method. Compared with using conventional orbital agitator, using the rotation/revolution-type agitator resulted in a significant (>two-fold) increase in the number of isolated cells. In subsequent primary cultures, chondrocytes obtained by rotation/revolution agitation showed superior initial attachment to tissue culture dish on day 1 and 2 compared with those obtained by conventional agitation; however, no differences in cell proliferation or cartilage-related molecule expression patterns were observed between cells derived from either method after 3 days of subculture. These findings suggested that there are no disadvantages to the proposed rotation/revolution agitation method. Rotation/revolution-type agitators are a promising apparatus for preparing chondrocytes for primary cultures and cartilage tissue engineering.

  • Lipid production by Lipomyces starkeyi using sap squeezed from felled old oil palm trunks

    Publication date: Available online 12 January 2019

    Source: Journal of Bioscience and Bioengineering

    Author(s): Ario Betha Juanssilfero, Prihardi Kahar, Rezky Lastinov Amza, Yopi, Kumar Sudesh, Chiaki Ogino, Bambang Prasetya, Akihiko Kondo

    The ability of oleaginous yeast Lipomyces starkeyi to efficiently produce lipids when cultivated on sap extracted from felled oil palm trunk (OPT) as a novel inexpensive renewable carbon source was evaluated. OPT sap was found to contain approximately 98 g/L glucose and 32 g/L fructose. Batch fermentations were performed using three different OPT sap medium conditions: regular sap, enriched sap, and enriched sap at pH 5.0. Under all sap medium conditions, the cell biomass and lipid production achieved were approximately 30 g/L and 60% (w/w), respectively. L. starkeyi tolerated acidified medium (initial pH ≈ 3) and produced considerable amounts of ethanol as well as xylitol as by-products. The fatty acid profile of L. starkeyi was remarkably similar to that of palm oil, one of the most common vegetable oil feedstock used in biodiesel production with oleic acid as the major fatty acid followed by palmitic, stearic and linoleic acids.

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  • Asplenium australasicum) frond extracts on melanoma and human skin'> Anti-melanization effects and inhibitory kinetics of tyrosinase of bird's nest fern (Asplenium australasicum) frond extracts on melanoma and human skin

    Publication date: Available online 11 January 2019

    Source: Journal of Bioscience and Bioengineering

    Author(s): Wen-Wen Zeng, Lih-Shiuh Lai

    Some bioactive properties of p-coumaric acid and fucose-rich polysaccharide in skin health have been studied, including melanogenesis inhibition of the phenolic acid and growth inhibitory effects of the polysaccharide on melanoma. The dermatological benefits of bird's nest fern extracts (BNFE), containing both substantial fucose-rich polysaccharide and p-coumaric acid, like promoting collagen production and growth of fibroblast cell and further improving the elasticity and dryness of human skins have been demonstrated in our previous study. Besides, the anti-melanization effects of various BNFE on B16-F10 melanoma and human skin were first studied here. The promising extracts revealed that the main phenolic acid, p-coumaric acid, in BNFE resulted in suppression against tyrosinase activity from melanogenesis. The inhibitory kinetics on the diphenolase activity indicated that AE40 was a noncompetitive inhibitor of mushroom tyrosinase. On the other hand, the fucose-rich mucilage of BNFE showed pronouncedly suppressing effect on B16-F10 melanoma viability. Clinical trial was performed by recruiting 46 female volunteers and the results indicated that the lotions with 1% of BNFE was non-irritant and reduced effectively the pigmentation on human skin after 7–14 days of continuous application. It was suggested that the fucose-rich mucilage and p-coumaric acid in BNFE may have potential for nutricosmetics and phytotherapy applications as a natural hypopigmenting agent.

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  • Bio-ionic liquid pretreatment and ultrasound-promoted enzymatic hydrolysis of black soybean okara

    Publication date: Available online 9 January 2019

    Source: Journal of Bioscience and Bioengineering

    Author(s): Cheng-An Yu, Chun-Yao Yang

    The effective processing method to produce fermentable sugars and modify the microstructure of black soybean okara using bio-ionic liquid (bio-IL) pretreatment and ultrasound-promoted enzymatic hydrolysis was investigated. The morphology and structural characteristics of okara before and after bio-IL pretreatment and enzymatic hydrolysis under different ultrasonic frequencies were analyzed by field emission scanning electron microscope (FE-SEM), X-ray energy dispersive spectrometer (EDS), and Fourier transform infrared spectroscopy (FTIR). Without pretreatment, the production of total reducing sugar (TRS) under ultrasound (40 kHz/300 W) was 3.4 times of that without ultrasound. Using the bio-IL choline acetate ([Ch][OAc]) in water for the pretreatment of black soybean okara, the TRS production of enzymatic hydrolysis was further increased to 5.2 times of that without ultrasound in 4 h of reaction. The analysis by FTIR and EDS showed that the highly structured matrix of okara was unfolded and broken by the action of combining ultrasound and choline acetate pretreatment, due to which the surface structures with large pores were presented to facilitate the reduction of unfavorable hindrance for enzymatic hydrolysis. The simplified kinetic model was proposed to describe the transport and reaction phenomena of enzymes in a solid–liquid system by using two kinetic parameters to show the impeded behavior of enzyme within the matrix of okara. The combination of bio-IL pretreatment and ultrasound-promoted enzymatic hydrolysis was able to make the efficient structural changes of black soybean okara to enhance the digestion of enzymes, and the okara could be valorized for use in foods.

  • Retraction Notice to “Enhanced production of a self-toxic antibiotic xinghaiamine A from the novel marine-derived species Streptomyces xinghaiensis by resin addition” [Journal of Bioscience and Bioengineering 117 (2) (2014) 200–202]

    Publication date: January 2019

    Source: Journal of Bioscience and Bioengineering, Volume 127, Issue 1

    Author(s): Wen-Ce Jiao, Xinqing Zhao, Yumei Wang, Xiang Geng

  • Systemic method to isolate large bacteriophages for use in biocontrol of a wide-range of pathogenic bacteria

    Publication date: January 2019

    Source: Journal of Bioscience and Bioengineering, Volume 127, Issue 1

    Author(s): Alaaeldin Mohamed Saad, Ahmed Mahrous Soliman, Takeru Kawasaki, Makoto Fujie, Hirofumi Nariya, Tadashi Shimamoto, Takashi Yamada

    Large phages are characterized by genomes around 200 kbp or more. They can infect wide host ranges of bacteria and maintain long-lasting infection. There is no standard method for selective isolation of large phages. In this study, we developed a systemic method to isolate large phages and succeeded in isolating 11 large phages, named Escherichia phage E1∼E11. Electron microscopy observations revealed typical Myoviridae phages with big capsids and long contractile tails. Genome sizes of the isolated phages were determined by pulsed-field gel electrophoresis and found to be in two groups, those around 200 kbp for E1, E2, E5, E6, E7, E9 and E10 phages, and others of approximately 450 kbp for E3, E4, E8 and E11 phages. The isolated large phages had wide host ranges: for example, E9 was effective against Shigella sonnei SH05001, Shigella bydii SH00007, Shigella flexneri SH00006, Salmonella enterica serovar Enteritidis SAL01078 and Escherichia coli C3000 (K-12 derivative), as well as its original host E. coli BL21. Screening of these jumbo phages was performed with non-pathogenic E. coli strains as hosts. Therefore, this method opens a way to isolate jumbo phages infecting wide ranges of pathogenic bacteria in a typical laboratory with standard laboratory strains as the hosts. The isolated large phages will be good candidates for biocontrol of various pathogens.

  • Down-regulation of pyruvate decarboxylase gene of white-rot fungus Phlebia sp. MG-60 modify the metabolism of sugars and productivity of extracellular peroxidase activity

    Publication date: January 2019

    Source: Journal of Bioscience and Bioengineering, Volume 127, Issue 1

    Author(s): Taichi Motoda, Megumi Yamaguchi, Taku Tsuyama, Ichiro Kamei

    Ethanologenic white-rot fungus Phlebia sp. MG-60-P2 produces ethanol directly from several lignocelluloses. Efficient gene silencing methods are needed for metabolic engineering of this fungus for biorefinery use. In this study, we evaluated the effectiveness of RNAi-mediated silencing of the pyruvate decarboxylase gene of Phlebia sp. MG-60-P2 (MGpdc1). The RNAi lines generated showed a variety of suppression levels of ethanol production and MGpdc1 expression, and two selected strains led to different metabolic fluxes, resulting in rapid accumulation of xylitol from xylose. Knockdown lines KD2 and KD10 showed different strength of silencing. The moderate-inhibition line (KD10) showed faster xylitol accumulation from xylose than the severe-inhibition line (KD2). KD2, KD10 and knockout line KO77 showed higher extracellular peroxidase activity than the wild-type. Gene silencing using RNAi for MGpdc1 in the ethanologenic white-rot fungus Phlebia sp. MG-60-P2 is an effective first step in metabolic engineering to produce other chemicals besides ethanol. This high efficiency of transformation and silencing effect will make it possible to cotransform with multiple expression vectors which enhance the minor metabolic pathway or introduce exogenous metabolic reaction in Phlebia sp. MG-60-P2.

  • Comparison of biofunctional activity of Asparagus cochinchinensis (Lour.) Merr. Extract before and after fermentation with Aspergillus oryzae

    Publication date: January 2019

    Source: Journal of Bioscience and Bioengineering, Volume 127, Issue 1

    Author(s): Guey-Horng Wang, Yi-Min Lin, Jong-Tar Kuo, Chia-Pei Lin, Chin-Feng Chang, Min-Chi Hsieh, Chiu-Yu Cheng, Ying-Chien Chung

    Asparagus cochinchinensis root (ACR) is used in traditional Chinese medicine. In this study, ACR was first extracted with 25% ethyl acetate (EA) and then fermented by Aspergillus oryzae to enhance its antioxidant activity and evaluate its potential antityrosinase activity. The physiological activity and cytotoxicity of A. oryzae-fermented ACR extract, along with its antityrosinase activity and effects on melanogenic factor levels in human epidermal melanocytes (HEMs), were analyzed and compared with those of the unfermented extract. The results showed that the physiological activity of the fermented extract in vitro or in cells was significantly higher than that of the unfermented extract. The IC50 values for 2,2-diphenyl-1-picrylhydrazine radical scavenging activity, reducing power, and antityrosinase activity in vitro for the fermented extract were 250.6 ± 32.5, 25.7 ± 3.5, and 50.6 ± 3.1 mg/L, respectively. The fermented extract favored cellular antityrosinase activity with low melanin production in human melanoma cells compared with the unfermented extract. The inhibitory mechanism of melanin synthesis by unfermented extract was independent of the tested melanogenesis-related proteins. However, the inhibitory mechanism of the fermented extract was possibly caused by synergistic inhibition of these proteins. Thus, A. oryzae-fermented ACR extract may be used for developing new health food or cosmetic ingredients.

  • Metabolomics-based profiling of three terminal alkene-producing Jeotgalicoccus spp. during different growth phase

    Publication date: January 2019

    Source: Journal of Bioscience and Bioengineering, Volume 127, Issue 1

    Author(s): Filemon Jalu Nusantara Putra, Sastia Prama Putri, Eiichiro Fukusaki

    Production of terminal alkenes by microbes has gained importance due to its role as a chemical feedstock in commercial industries. Jeotgalicoccus species has been widely unexplored despite being well-known as a natural producer of terminal alkene, catalyzing the one-step fatty acid decarboxylation reaction by OleTJE cytochrome P450. In this study, widely targeted ion-pair LC-MS/MS was used to monitor central carbon metabolism of Jeotgalicoccus halotolerans JCM 5429, Jeotgalicoccus huakuii JCM 8176, and Jeotgalicoccus psychrophilus JCM 5429 at logarithmic and stationary phases. Growth and production profile of terminal alkene, alcohols and organic acids were also measured. Among the three strains used in this study, J. halotolerans and J. psychrophilus showed higher terminal alkene production compared to J. huakuii. All strains achieved maximum terminal alkene production at logarithmic phase and therefore, detailed analysis of the metabolite profiles of the three strains were performed in logarithmic phase. PCA analysis showed that the strains were discriminated based on their ability to produce terminal alkene along PC1 and some of the important metabolites corresponding to this separation is the acetyl-CoA and 2-oxoglutarate. This study is the first report on metabolite profiling of three Jeotgalicoccus spp. in different growth phases. The results from this study can provide a better understanding of the changes that occur in the metabolome level during growth and production of terminal alkene in Jeotgalicoccus species.

  • Microbial community structure in deep natural gas-bearing aquifers subjected to sulfate-containing fluid injection

    Publication date: January 2019

    Source: Journal of Bioscience and Bioengineering, Volume 127, Issue 1

    Author(s): Taiki Katayama, Hideyoshi Yoshioka, Toshiro Yamanaka, Mio Takeuchi, Yoshiyuki Muramoto, Jun Usami, Hidefumi Ikeda, Susumu Sakata

    In the natural gas field located in central Japan, high concentrations of natural gases and iodide ions are dissolved in formation water and commercially produced in deep aquifers. In the iodine recovery process, the produced formation water is amended with sulfate, and this fluid is injected into gas-bearing aquifers, which may lead to infrastructure corrosion by hydrogen sulfide. In this study, we examined the microbial community in aquifers subjected to sulfate-containing fluid injection. Formation water samples were collected from production wells located at different distances from the injection wells. The chemical analysis showed that the injection fluid contained oxygen, nitrate, nitrite and sulfate, in contrast to the formation water, which had previously been shown to be depleted in these components. Sulfur isotopic analysis indicated that sulfate derived from the injection fluid was present in the sample collected from near the injection wells. Quantitative and sequencing analysis of dissimilatory sulfite reductase and 16S rRNA genes revealed that sulfate-reducing bacteria (SRB), sulfur-oxidizing bacteria, and anaerobic methanotrophic archaea (ANME) in the wells located near injection wells were more abundant than those in wells located far from the injection wells, suggesting that fluid injection stimulated these microorganisms through the addition of oxygen, nitrate, nitrite and sulfate to the methane-rich aquifers. The predominant taxa were assigned to the ANME-2 group, its sulfate-reducing partner SEEP-SRB1 cluster and sulfur-oxidizing Epsilonproteobacteria. These results provide important insights for future studies to support the development of natural gas and iodine resources in Japan.

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  • Optimization of a dual-functional biocatalytic system for continuous hydrolysis of lactose in milk

    Publication date: January 2019

    Source: Journal of Bioscience and Bioengineering, Volume 127, Issue 1

    Author(s): Heng Li, Yuting Cao, Shuai Li, Yun Jiang, Jianqi Chen, Zhuofu Wu

    In this study, an amino-functionalized silica matrix encapsulating β-galactosidase was first synthesized in situ, with subsequent covalent anchoring of lysozyme to the outer part of the amino-grafted matrix. Fourier transform infrared (FTIR) spectra verified that β-galactosidase was successfully encapsulated. Meanwhile, the co-immobilized enzymes were demonstrated to retain suitable enzymatic activities and outstanding operational stability during successive reaction cycles. Furthermore, when used for lactose removal from skim milk, the packed-bed column system achieved both a high lactose hydrolysis rate and microbial inactivation ratio during 30 days of continuous operation. Notably, this system exhibited favorable stability during 60 days of continuous hydrolysis of lactose in solution and thus may be appropriate for further development for use in industrial lactose removal from milk.

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  • Development of GFP-based high-throughput screening system for directed evolution of glucose oxidase

    Publication date: January 2019

    Source: Journal of Bioscience and Bioengineering, Volume 127, Issue 1

    Author(s): Gordana Kovačević, Raluca Ostafe, Ana Marija Balaž, Rainer Fischer, Radivoje Prodanović

    Glucose oxidase (GOx) mutants with higher activity or stability have important role in industry and in the development of biosensors and biofuel cells. Discovering these mutants can be time-consuming if appropriate high-throughput screening (HTS) systems are not available. GOx gene libraries were successfully screened and sorted using a HTS system based on GOx activity dependent fluorescent labeling of yeast cells with tyramids and quantification of the amount of expressed enzyme by yeast enhanced green fluorescent protein (yGFP) tagging and flow cytometry. For this purpose, we expressed wild type and a mutant GOx as a chimera with the yGFP to confirm differences in catalytic activity between wild-type and mutant GOx. Fluorescence of yGFP is preserved during expression of chimera, and also after the oxidative enzymatic reaction. We have obtained a 2.5-fold enrichment in population of cells expressing active enzyme, and percentage of enzyme variants with enzymatic mean activity higher than wild type activity was increased to 44% after a single round of GOx gene library sorting. We have found two mutants with 1.3 and 2.3-fold increase in Vmax values compared to the wtGOx. By simultaneous detection of protein expression level and enzyme activity we have increased the likelihood of finding GOx variants with increased activity in a single round of flow cytometry sorting.

  • Comparative study of the insoluble and soluble Ulp1 protease constructs as Carrier free and dependent protein immobilizates

    Publication date: January 2019

    Source: Journal of Bioscience and Bioengineering, Volume 127, Issue 1

    Author(s): Li Jiang, Wenjun Xiao, Xuan Zhou, Weiyu Wang, Jun Fan

    In this study, we analyzed and compared the properties of yeast Ulp1 protease in active inclusion bodies (IBs) as special protein immobilizate, and the soluble Ulp1 via oriented immobilization. Fusion of the N-terminal self-assembling peptide GFIL8 to the Ulp1 increased production of active IBs in Escherichia coli. Attachment of the N-terminal cellulose-binding module facilitated the constructed protein immobilized on the regenerated amorphous cellulose (RAC) with a binding capacity up to about 235 mg protein per gram of RAC. Compared with the immobilized soluble construct, the insoluble Ulp1 showed higher resistance to limited proteolysis with trypsin digestion, lower leaky amount at different storage temperatures, but more rapid decrease in cleavage activity after stored at 4°C for 8 days. The immobilized soluble Ulp1 maintained about 42% initial cleavage activity with repetitive use successively, whereas the aggregated Ulp1 lost its cleavage capacity after cleaving the protein substrate once. Crosslinking of IBs mediated by glutaraldehyde inactivated the Ulp1. Freshly prepared and used IBs showed similar resistance to protease-K digestion, and comparable binding capacity of Congo red and thioflavin T. Taken together, due to different advantages, the Ulp1 constructs as carrier-free and carrier-dependent immobilizates are used under different conditions.

  • Characteristics of refold acid urease immobilized covalently by graphene oxide-chitosan composite beads

    Publication date: January 2019

    Source: Journal of Bioscience and Bioengineering, Volume 127, Issue 1

    Author(s): Liu Yang, Xiaofeng Liu, Nandi Zhou, Yaping Tian

    Bifunctional acid urease can not only remove urea from rice wine, but also effectively remove ethyl carbamate (EC), thereby reducing harmful substances in rice wine. Acid urease from Providencia rettgeri JN-B815 was expressed in Escherichia coli BL21(DE3) as an inclusion body. Subsequently, acid urease was refolded gradually using dilution-ultrafiltration and specific activity of 12.3 U mg−1 was obtained. The acid urease thus obtained was immobilized on graphene oxide/chitosan beads. The recovery of immobilized urease was 77% for the graphene oxide/chitosan composite when coupled for 8 h with 5% glutaraldehyde. Steady-state kinetic analysis showed that the optimum temperature for urease activity was 37°C. Time-dependent thermal inactivation studies showed that the stability of the immobilized urease improved at 37°C and the t1/2 (half-life) of decay in urease activity was 12 h, whereas it was 43 days for soluble and chitosan/graphene oxide immobilized urease at 4°C. A significant change in Km occurred during the immobilization; the Km for urea and EC in the free state were 9.14 mM and 386 mM, respectively, whereas they were 5.28 mM and 180 mM, respectively, in the immobilized state. The values of Vmax for immobilized EC and urea were 1.08 μmol min−1 and 1.16 μmol min−1, respectively, whereas they were 0.782 μmol min−1 and 0.931 μmol min−1, respectively, for soluble EC and urea. Furthermore, 90% of the original activity of graphene oxide/chitosan–urease beads were retained after the beads were used 10 times, indicating excellent reusability.

  • Identification and analysis of binding residues in the CBM68 of pullulanase PulA from Anoxybacillus sp. LM18-11

    Publication date: January 2019

    Source: Journal of Bioscience and Bioengineering, Volume 127, Issue 1

    Author(s): Yan Zeng, Hongchen Zheng, Yingying Shen, Jianyong Xu, Ming Tan, Fang Liu, Hui Song

    Carbohydrate binding module (CBM) as a kind of non-catalytic domain has significant effects on the substrate binding and catalytic properties of glycoside hydrolases. CBM68 of an Anoxybacillus sp. pullulanase (PulA) was identified as a new type of CBM in our previous studies. Then, four key substrate binding amino acid residues (Y14, V91, G92, and R96) were obtained by alanine substitutions in this work. Through kinetic analysis of the mutants, V91A and G92A showed significant reduction both in Km values and kcat values against pullulan. To further identify the changes of substrate affinities of V91A and G92A, devitalized mutants V91A-D413N and G92A-D413N were under measuring by surface plasmon resonance (SPR). Compared with that of PulA-D413N, the substrate affinities of V91A-D413N and G92A-D413N were improved by 1.6-fold and 2.2-fold, respectively. However, as to the product (maltotriose) binding force tested by the isothermal titration calorimetry (ITC), G92A showed higher binding force than that of V91A and PulA by 4.2-fold and 6.2-fold, respectively. That may cause G92A showing significantly lower catalytic efficiency than V91A and PulA. Moreover, four different kinds of amino acids (leucine, serine, glutamic acid and arginine) substitutions for V91 and G92 showed various changes both on the kinetic parameters and enzymatic properties, which demonstrated that V91 and G92 were the critical binding residues in the CBM68. The results of this study made contributed to the rational design for improving the catalytic efficiency of PulA.

  • Mating type switching, formation of diploids, and sporulation in the methylotrophic yeast Ogataea minuta

    Publication date: January 2019

    Source: Journal of Bioscience and Bioengineering, Volume 127, Issue 1

    Author(s): Takehiko Yoko-o, Akiko Komatsuzaki, Erina Yoshihara, Mariko Umemura, Yasunori Chiba

    Ogataea minuta is a methylotrophic yeast that is closely related to Ogataea (Hansenula) polymorpha. Like other methylotrophic yeasts, O. minuta also possesses strongly methanol-inducible genes, such as AOX1. We have focused on O. minuta as a host for the production of therapeutic glycoproteins. However, genetic methods, which are required for the construction of strains by breeding, have not yet been established in this organism. In this study, we investigated the O. minuta mechanisms of mating and sporulation, which would facilitate genetic analysis in this species. Specifically, we determined DNA sequences around the MAT locus in O. minuta strain NBRC 10746, and found that two MAT loci were flanked by a pair of inverted repeat sequences, as reported in O.polymorpha (Maekawa and Kaneko, PLOS Genet., 10, e1004796, 2014). As in O. polymorpha, mating type in O. minuta appears to be switched by inversion of the chromosomal region between the two MAT loci. We successfully obtained O. minuta diploid cells, which showed vegetative growth on rich medium. The size of the diploid cells was 1.3-fold larger than haploid cells of this species. Diploid cells formed ascospores, which contained 2–4 spores, under nutrient starvation conditions. Phenotypes of the resultant haploid cells exhibited Mendelian segregation, indicating that genetic approaches are applicable to O. minuta.

  • Development of novel on-line capillary gas chromatography-based analysis method for volatile organic compounds produced by aerobic fermentation

    Publication date: January 2019

    Source: Journal of Bioscience and Bioengineering, Volume 127, Issue 1

    Author(s): Hyeok-Won Lee, Jung-Ho Park, Hee-Suk Lee, Chun-Suk Kim, Jin-Gyeom Lee, Won-kyo Kim, Kyoung-Hwa Ryu, Jung-Oh Ahn, Eun-Gyo Lee, Seon-Won Kim, Jong-Min Jeon, Yung-Hun Yang, Eui-Sung Choi, Hong-Weon Lee

    Many volatile compounds, such as isoprene, a precursor used in the synthesis of natural rubber, have been produced through fermentation using genetically engineered microorganisms. Despite this biotechnological success, measuring the concentrations of volatile compounds during fermentation is difficult because of their high volatility. In current systems, off-line analytical methods usually lead to product loss, whereas on-line methods raise the production cost due to the requirement of complex devices. Here, we developed a novel on-line gas chromatography (GC)-based system for analyzing the concentration of isoprene with the aim to minimize the cost and requirement for devices as compared to current strategies. In this system, a programmable logic controller is used to combine conventional GC with a syringe pump module (SPM) directly connected to the exhaust pipe of the fermentor, and isoprene-containing samples are continuously pumped from the SPM into the GC using an air cylinder recycle stream. We showed that this novel system enables isoprene analysis during fermentation with convenient equipment and without the requirement of an expensive desorption tube. Furthermore, this system may be extended to the detection of other volatile organic compounds in fermentation or chemical processes.

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  • Analysis of intracellular IgG secretion in Chinese hamster ovary cells to improve IgG production

    Publication date: January 2019

    Source: Journal of Bioscience and Bioengineering, Volume 127, Issue 1

    Author(s): Kohei Kaneyoshi, Keiji Uchiyama, Masayoshi Onitsuka, Noriko Yamano, Yuichi Koga, Takeshi Omasa

    The production of biopharmaceutical immunoglobulin G (IgG) using cultured mammalian cells, especially Chinese hamster ovary (CHO) cells is well established and has been markedly improved through the modification of cells and cell culture engineering technologies. The establishment of high-production cell lines remains a challenge. The intracellular secretion of IgG has been investigated to identify and solve the rate-limiting steps in antibody production. However, strategies that regulate the expression of proteins that are related to antibody secretory pathway have not consistently improved their production. In this study, key features and limitations of the antibody secretion process in recombinant CHO cells were analyzed to develop more efficient approaches for establishing high-production cells. By chase assay with protein translation inhibitors, IgG secretion reached a plateau when at least 20% of IgG remained in the cells. The secretion kinetics and retention ratio of IgG varied between IgG subclasses (two types of IgG1 and an IgG3 subclass). Immunofluorescent microscopy and size exclusion chromatography showed that the remaining intracellular IgG localized mainly within the endoplasmic reticulum (ER) and less with the cis-Golgi network, despite the formation of fully assembled IgG. These results show that remaining intracellular IgG is a target for enhancing antibody secretion, even in high-production CHO cells.