JBB : Journal of Bioscience and Bioengineering

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Journal of Bioscience and Bioengineering vol.118 cover

 

Journal of Bioscience and Bioengineering – Recent Articles

  • Enhanced production of branched-chain amino acids by Gluconacetobacter europaeus with a specific regional deletion in a leucine responsive regulator
    Publication date: December 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 6

    Author(s): Naoki Akasaka , Yuri Ishii , Ryota Hidese , Hisao Sakoda , Shinsuke Fujiwara

    Vinegar with increased amounts of branched-chain amino acids (BCAAs; valine, leucine and isoleucine) is favorable for human health as BCAAs decrease diet-induced obesity and hyperglycemia. To construct Gluconacetobacter europaeus which produces BCAAs, leucine responsive regulator (GeLrp) is focused and two Gelrp mutants were constructed. Wild-type KGMA0119 didn't produce significant amount of valine (0.13 mM) and leucine (0 mM) and strain KGMA7110 which lacks complete Gelrp accumulated valine (0.48 mM) and leucine (0.11 mM) but showed impaired growth, and it was fully restored in the presence of essential amino acids. Strain KGMA7203 was then constructed with a nonsense mutation at codon Trp132 in the Gelrp, which leads a specific deletion at an estimated ligand-sensing region in the C-terminal domain. KGMA7203 produced greater quantities of valine (0.80 mM) and leucine (0.26 mM) and showed the same growth characteristics as KGMA0119. mRNA levels of BCAAs biosynthesis genes (ilvI and ilvC) and probable BCAAs efflux pump (leuE) were determined by quantitative reverse-transcription PCR. Expression rates of ilvI and ilvC in the two Gelrp disruptants were greater than those in KGMA0119. leuE was highly expressed in KGMA7110 only, suggesting that the accumulation in KGMA7110 culture was caused by increased expression of the biosynthesis genes and abnormal enhanced export of amino acids resulting in impaired cell growth. In contrast, KGMA7203 would achieve the high level production through enhanced expression of the biosynthesis genes without enhancing that for the efflux pump. KGMA7203 was considered advantageous for production of vinegar with higher amounts of valine and leucine.





  • Reduction of nitric oxide catalyzed by hydroxylamine oxidoreductase from an anammox bacterium
    Publication date: December 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 6

    Author(s): Tatsuya Irisa , Daisuke Hira , Kenji Furukawa , Takao Fujii

    The hydroxylamine oxidoreductase (HAO) from the anammox bacterium, Candidatus Kuenenia stuttgartiensis has been reported to catalyze the oxidation of hydroxylamine (NH2OH) to nitric oxide (NO) by using bovine cytochrome c as an oxidant. In contrast, we investigated whether the HAO from anammox bacterium strain KSU-1 could catalyze the reduction of NO with reduced benzyl viologen (BVred) and the NO-releasing reagent, NOC 7. The reduction proceeded, resulting in the formation of NH2OH as a product. The oxidation rate of BVred was proportional to the concentration of BVred itself for a short period in each experiment, a situation that was termed quasi-steady state. The analyses of the states at various concentrations of HAO allowed us to determine the rate constant for the catalytic reaction, (2.85 ± 0.19) × 105 M−1 s−1, governing NO reduction by BVred and HAO, which was comparable to that reported for the HAO from the ammonium oxidizer, Nitrosomonas with reduced methyl viologen. These results suggest that the anammox HAO functions to adjust anammox by inter-conversion of NO and NH2OH depending on the redox potential of the physiological electron transfer protein in anammox bacteria.





  • Identification, purification and characterization of a novel collagenolytic serine protease from fig (Ficus carica var. Brown Turkey) latex
    Publication date: December 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 6

    Author(s): Brankica Raskovic , Olga Bozovic , Radivoje Prodanovic , Vesna Niketic , Natalija Polovic

    A novel collagenolytic serine protease was identified and then purified (along with ficin) to apparent homogeneity from the latex of fig (Ficus carica, var. Brown Turkey) by two step chromatographic procedure using gel and covalent chromatography. The enzyme is a monomeric protein of molecular mass of 41 ± 9 kDa as estimated by analytical gel filtration chromatography. It is an acidic protein with a pI value of approximately 5 and optimal activity at pH 8.0–8.5 and temperature 60°C. The enzymatic activity was strongly inhibited by PMSF and Pefabloc SC, indicating that the enzyme is a serine protease. The enzyme showed specificity towards gelatin and collagen (215 GDU/mg and 24.8 CDU/mg, respectively) and non-specific protease activity (0.18 U/mg against casein). The enzyme was stable and retained full activity over a broad range of pH and temperature. The fig latex collagenolytic protease is potentially useful as a non-microbial enzyme with collagenolytic activity for various applications in the fields of biochemistry, biotechnology and medicine.





  • Construction of an efficient Escherichia coli whole-cell biocatalyst for d-mannitol production
    Publication date: December 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 6

    Author(s): Shamlan M.S. Reshamwala , Sandip K. Pagar , Vishal S. Velhal , Vijay M. Maranholakar , Vishal G. Talangkar , Arvind M. Lali

    Mannitol is a six carbon sugar alcohol that finds applications in the pharmaceutical and food industries. A novel Escherichia coli strain capable of converting d-glucose to d-mannitol has been constructed, wherein native mannitol-1-phosphate dehydrogenase (MtlD) and codon-optimized Eimeria tenella mannitol-1-phosphatase (M1Pase) have been overexpressed. Codon-optimized Pseudomonas stutzeri phosphite dehydrogenase (PtxD) was overexpressed for cofactor (NADH) regeneration with the concomitant oxidation of phosphite to phosphate. Whole-cell biotransformation using resting cells in a medium containing d-glucose and equimolar sodium phosphite resulted in d-mannitol yield of 87 mol%. Thus, production of an industrially relevant biochemical without using complex media components and elaborate process control mechanisms has been demonstrated.





  • Extraction of squalene as value-added product from the residual biomass of Schizochytrium mangrovei PQ6 during biodiesel producing process
    Publication date: December 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 6

    Author(s): Minh Hien Hoang , Nguyen Cam Ha , Le Thi Thom , Luu Thi Tam , Hoang Thi Lan Anh , Ngo Thi Hoai Thu , Dang Diem Hong

    Today microalgae represent a viable alternative source of squalene for commercial application. The species Schizochytrium mangrovei, a heterotrophic microalga, has been widely studied and provides a high amount of squalene, polyunsaturated fatty acids and has good profiles for biodiesel production. Our work was aimed at examining the squalene contents in Vietnam's heterotrophic marine microalga S. mangrovei PQ6 biomass and residues of the biodiesel process from this strain. Thin-layer chromatography and high-performance liquid chromatography (HPLC) methods were successfully applied to the determination of squalene in S. mangrovei PQ6. The squalene content and production of S. mangrovei PQ6 reached 33.00 ± 0.02 and 33.04 ± 0.03 mg g−1 of dry cell weight; and 0.992 g L−1 and 1.019 g L−1 in 30 and 150 L bioreactors, respectively after 96 h of fermentation. In addition, squalene was also detected in spent biomass (approximately 80.10 ± 0.03 mg g−1 of spent biomass) from the S. mangrovei PQ6 biodiesel production process. The structure of squalene in residues of the biodiesel process was confirmed from its nuclear magnetic resonance spectra. The results obtained from our work suggest that there is tremendous potential in the exploitation of squalene as a value-added by-product besides biodiesel from S. mangrovei PQ6 to reduce biodiesel price.





  • Factors affecting phenolic acid liberation from rice grains in the sake brewing process
    Publication date: December 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 6

    Author(s): Toshihiko Ito , Nobukazu Suzuki , Airi Nakayama , Masaya Ito , Katsumi Hashizume

    Phenolic acid (ferulic and p-coumaric acid) liberation from rice grains was examined using rice samples containing phenolic acid at different levels, using two sake mash simulated digestion tests to elucidate influencing factors. Phenolic acid levels in a digest made from steamed rice using dialyzed rice koji enzymes were smaller than levels in a rice koji self-digest. Differences in phenolic acid levels among rice samples in the rice koji self-digest were larger than levels in a digest of steamed rice. In the rice koji self-digest, phenolic acid levels in the ingredient rice grains or in the formed digest related to feruloylesterase (FE) activity in the rice koji. Addition of exogenous FE to rice koji self-digestion increased phenolic acid levels, while addition of xylanase (Xyl) showed weak effects. A concerted effect of FE and Xyl was not clearly observed. Addition of ferulic acid to koji made from α-rice grains raised FE activity, but it did not increase the activity of other enzymes. A similar phenomenon was observed in an agar plate culture of koji mold. These results indicated that ferulic acid levels in ingredient rice grains correlate with FE activities of koji, as a resulut, they affect the phenolic acid levels in sake mash.





  • Production of 16.5% v/v ethanol from seagrass seeds
    Publication date: December 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 6

    Author(s): Motoharu Uchida , Tatsuo Miyoshi , Masaki Kaneniwa , Kenji Ishihara , Yutaka Nakashimada , Naoto Urano

    Ethanol fermentation on seeds of seagrass Zostera marina was studied. The seeds were collected from the annual plant colony of Z. marina at Hinase Bay, Okayama. The seeds contained 83.5% carbohydrates including 48.1% crude starch on a dry weight basis, which is comparable to cereals such as wheat flour and corns. The seeds were saccharified with glucoamylase (50°C, 96 h) and 103.4 g/l concentration of glucose juice was obtained. The glucose juice was further fermented (23°C–35°C, 15 days) with Saccharomyces cerevisiae strains NBRC10217T and Kyokai 7-go, and ethanol was obtained at a 65.0 g/l (82.3 ml/l) level by monographic double-fermentation and at a 130.4 g/l (165.1 ml/l) level by parallel double-fermentation. Fermented products of seagrass seeds containing such a high ethanol concentration as the present study have potential to be utilized not only for biofuel but also for foods and beverages in the future. Culturing of seagrass seeds as a crop may enable development of a new marine fermentation industry.





  • Influence of phenolic acids on indole acetic acid production and on the type III secretion system gene transcription in food-associated Pseudomonas fluorescens KM05
    Publication date: December 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 6

    Author(s): Kamila Myszka , Marcin T. Schmidt , Agnieszka K. Olejnik-Schmidt , Katarzyna Leja , Katarzyna Czaczyk

    The purpose of these investigations was to evaluate the reduction capability of phenolic acids (ferulic, chlorogenic, gallic, and p-coumaric acids) on indole acetic acid synthesis by food-associated Pseudomonas fluorescens KM05. Specific genetic primer for the type III secretion system (TTSS) in P. fluorescens KM05 was designed and the influence of phenolic acids on its expression was investigated. In the work the ferulic and chlorogenic acids at the concentration of 0.02 and 0.04 μg/ml affected on bacterial growth pattern and the signal molecules production. The phenolic acids, that were appreciable effective against P. fluorescens KM05 indole acetic acid production, significantly suppressed TTSS gene.





  • Isolation of lactic acid-tolerant Saccharomyces cerevisiae from Cameroonian alcoholic beverage
    Publication date: December 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 6

    Author(s): Ryosuke Kubo , Keisuke Ohta , Shinya Funakawa , Naofumi Kitabatake , Shigeru Araki , Shingo Izawa

    We investigated yeast strains used in Cameroonian microbreweries, and identified a Saccharomyces cerevisiae strain (OCY3) with an excellent capacity for alcoholic fermentation. OCY3 showed higher tolerance to lactic acid and better fermentation performance under acidic conditions than a representative Japanese sake yeast, Kyokai No. 7, and a wine yeast, EC1118.





  • Differentiation of industrial sake yeast strains by a loop-mediated isothermal amplification method that targets the PHO3 gene
    Publication date: December 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 6

    Author(s): Takashi Kuribayashi , Keigo Sato , Daisuke Kasai , Masao Fukuda , Mitsuoki Kaneoke , Ken-ichi Watanabe

    We developed a loop-mediated isothermal amplification method that targets the PHO3 gene for discriminating sake yeast strains. Our data indicate that this assay is simple, rapid, and useful to use for differentiation of specific yeasts in sake mash.





  • Mining biomass-degrading genes through Illumina-based de novo sequencing and metagenomic analysis of free-living bacteria in the gut of the lower termite Coptotermes gestroi harvested in Vietnam
    Publication date: December 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 6

    Author(s): Thi Huyen Do , Thi Thao Nguyen , Thanh Ngoc Nguyen , Quynh Giang Le , Cuong Nguyen , Keitarou Kimura , Nam Hai Truong

    The 5.6 Gb metagenome of free-living microbial flora in the gut of the lower termite Coptotermes gestroi, harvested in Vietnam, was sequenced using Illumina technology. Genes related to biomass degradation were mined for a better understanding of biomass digestion in the termite gut and to identify lignocellulolytic enzymes applicable to biofuel production. The sequencing generated 5.4 Gb of useful reads, containing 125,431 ORFs spanning 78,271,365 bp, 80% of which was derived from bacteria. The 12 most abundant bacterial orders were Spirochaetales, Lactobacillales, Bacteroidales, Clostridiales, Enterobacteriales, Pseudomonades, Synergistales, Desulfovibrionales, Xanthomonadales, Burkholderiales, Bacillales, and Actinomycetales, and 1460 species were estimated. Of more than 12,000 ORFs with predicted functions related to carbohydrate metabolism, 587 encoding hydrolytic enzymes for cellulose, hemicellulose, and pectin were identified. Among them, 316 ORFs were related to cellulose degradation, and included β-glucosidases, 6-phospho-β-glucosidases, licheninases, glucan endo-1,3-β-d-glucosidases, endoglucanases, cellulose 1,4-β-cellobiosidases, glucan 1,3-β-glucosidases, and cellobiose phosphorylases. In addition, 259 ORFs were related to hemicellulose degradation, encoding endo-1,4-β-xylanases, α-galactosidases, α-N-arabinofuranosidases, xylan 1,4-β-xylosidases, arabinan endo-1,5-α-l-arabinosidases, endo-1,4-β-mannanases, and α-glucuronidases. Twelve ORFs encoding pectinesterases and pectate lyases were also obtained. To our knowledge, this is the first successful application of Illumina-based de novo sequencing for the analysis of a free-living bacterial community in the gut of a lower termite C. gestroi and for mining genes related to lignocellulose degradation from the gut bacteria.





  • Electricity generating capacity and performance deterioration of a microbial fuel cell fed with beer brewery wastewater
    Publication date: December 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 6

    Author(s): Emre Oğuz Köroğlu , Bestamin Özkaya , Cenk Denktaş , Mehmet Çakmakci

    This study focused on using beer brewery wastewater (BBW) to evaluate membrane concentrate disposal and production of electricity in microbial fuel cells. In the membrane treatment of BBW, the membrane permeate concentration was 570 ± 30 mg/L corresponding to a chemical oxygen demand (COD) removal efficiency of 75 ± 5%, and the flux values changed between 160 and 40 L/m2-h for all membrane runs. For electricity production from membrane concentrate, the highest current density in the microbial fuel cell (MFC) was observed to be 1950 mA/m2 according to electrode surface area with 36% COD removal efficiency and 2.48% CE with 60% BBW membrane concentrate. The morphologies of the cation exchange membrane and the MFC deterioration were studied using a scanning electron microscope (SEM), attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy, differential scanning calorimetry (DSC), and thermal gravimetric analysis (TGA). A decrease in the thermal stability of the sulfonate (−SO3H) groups was demonstrated and morphological changes were detected in the SEM analysis.





  • Purification of kavalactones from Alpinia zerumbet and their protective actions against hydrogen peroxide-induced cytotoxicity in PC12 cells
    Publication date: December 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 6

    Author(s): Yerra Koteswara Rao , Hui-Nung Shih , Yi-Ching Lee , Wen-Tai Cheng , Hui-Chin Hung , Huang-Chi Wang , Ching Jung Chen , Yew-Min Tzeng , Meng-Jen Lee

    This study found that fruit shells of shell ginger (Alpinia zerumbet) are a rich source of the kavalactones dihydro-5,6-dehydrokavain (DDK) and 5,6-dehydrokavain (DK). The fruit shell extraction with hexane resulted in good purity and higher yields of DDK and DK than did chloroform, ethanol, 10% ethanol, methanol or water. Additionally, this study examined the neuroprotective effects of DDK and DK against H2O2-induced cytotoxicity in PC12 cells and the possible molecular mechanisms involved. 16 h after stimulation with 400 μM H2O2, the viability (MTT reduction) of PC12 cells decreased while membrane damage (LDH release) was noticeably increased. However, pretreatment for 6 h with DDK and DK (1 μM, 5 μM, 10 μM and 50 μM) rescued PC12 cells from H2O2-induced cytotoxicity, as evidenced by decreased LDH release and increased cell viability. DDK and DK inhibit the MAPK family member p38, activate AKT, and reduce caspase-3 activity. DDK also reduced the oxidative status in H2O2-treated PC12 cells. Together, our data indicate that the A. zerumbet constituents, DDK and DK, exert a protective effect against oxidative stress-induced PC12 cell death and that the regulation of p-Akt and the p38 MAPK, and of oxidative states may be involved.





  • Development of industrial yeast strain with improved acid- and thermo-tolerance through evolution under continuous fermentation conditions followed by haploidization and mating
    Publication date: December 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 6

    Author(s): Kanako Mitsumasu , Ze-Shen Liu , Yue-Qin Tang , Takashi Akamatsu , Hisataka Taguchi , Kenji Kida

    Continuous fermentation using the industrial Saccharomyces cerevisiae diploid strain WW was carried out under acidic or high-temperature conditions to achieve acid- or thermo-tolerant mutants. Mutants isolated at pH 2.5 and 41°C showed improved growth and fermentation ability under acidic and elevated temperature conditions. Haploid strains WW17A1 and WW17A4 obtained from the mutated diploid strain WW17A showed better growth and 4.5–6.5% higher ethanol yields at pH 2.7 than the original strains. Haploid strain WW12T4 obtained from mutated diploid strain WW12T showed 1.25–1.50 times and 2.8–4.7 times higher total cell number and cell viability, respectively, than the original strains at 42°C. Strain AT, which had significantly improved acid- and thermo-tolerance, was developed by mating strain WW17A1 with WW12T4. Batch fermentation at 41°C and pH 3.5 showed that the ethanol concentration and yield achieved during fermentation by strain AT were 55.4 g/L and 72.5%, respectively, which were 10 g/L and 13.4% higher than that of the original strain WW. The present study demonstrates that continuous cultivation followed by haploidization and mating is a powerful approach for enhancing the tolerance of industrial strains.





  • Hemicellulase production by Aspergillus niger DSM 26641 in hydrothermal palm oil empty fruit bunch hydrolysate and transcriptome analysis
    Publication date: December 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 6

    Author(s): Christoph Ottenheim , Carl Verdejo , Wolfgang Zimmermann , Jin Chuan Wu

    Palm oil empty fruit bunches (EFB) is an abundant and cheap lignocellulose material in Southeast Asia. Its use as the sole medium for producing lignocellulose-hydrolyzing enzymes would increase its commercial value. A newly isolated Aspergillus niger DSM 26641 was investigated for its capability of producing hemicellulases in EFB hydrolysate obtained by treatment with pressurized hot water (1–20%, w/v) at 120–180°C in a 1 L Parr reactor for 10–60 min. The optimal hydrolysate for the fungal growth and endoxylanase production was obtained when 10% (w/v) of empty fruit bunch was treated at 120°C or 150°C for 10 min, giving an endoxylanase activity of 24.5 mU ml−1 on RBB-Xylan and a saccharification activity of 5 U ml−1 on xylan (DNS assay). When the hydrolysates were produced at higher temperatures, longer treatment times or higher biomass contents, only less than 20% of the above maximal endoxylanase activity was detected, possibly due to the higher carbohydrate concentrations in the medium. Transcriptome analysis showed that 3 endoxylanases (expression levels 59–100%, the highest level was set as 100%), 2 β-xylosidases (4%), 4 side chain-cleaving arabinofuranosidases (1–95%), 1 acetyl xylan esterase (9%) and 2 ferulic acid esterases (0.3–9%) were produced together.





  • Improvement and scale-down of a Trichoderma reesei shake flask protocol to microtiter plates enables high-throughput screening
    Publication date: December 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 6

    Author(s): Heiner Giese , Paulien Kruithof , Kristina Meier , Michaela Sieben , Elena Antonov , Ronald W.J. Hommes , Jochen Büchs

    Nowadays, high-throughput screening is essential for determining the best microbial strains and fermentation conditions. Although microtiter plates allow higher throughput in screening than shake flasks, they do not guarantee sufficient oxygen supply if operated at unsuitable conditions. This is especially the case in viscous fermentations, potentially leading to poor liquid movement and surface growth. Therefore, in this study, two aims were pursued. First, an industrial Trichoderma reesei shake flask protocol is improved with respect to oxygen supply and production. Second, this improved shake flask protocol is scaled down into microtiter plate under consideration of similar oxygen supply. For this purpose, the respiration activity monitoring system (RAMOS) was applied. An approach based on a sulfite system was introduced to ensure equal maximum oxygen transfer capacities (OTRmax) in microtiter plates and shake flasks. OTRmax-values of 250 mL shake flasks and 24-well microtiter plates were determined in a wide range of operating conditions. These sulfite datasets were used to identify operating conditions leading to the same oxygen supply for T. reesei in shake flasks and 24-well microtiter plates. For 24-well microtiter plates, the shake flask OTRmax of 20 mmol/L/h of an industrial protocol was obtained under the following optimal operating conditions: 1 mL filling volume per well, 200 rpm shaking frequency and 50 mm shaking diameter. With these conditions almost identical oxygen transfer rates and product concentrations were measured in both scales. The proposed approach is a fast and accurate means to scale-down established screening procedures into microtiter plates to achieve high-throughput.





  • High-quality green tea leaf production by artificial cultivation under growth chamber conditions considering amino acids profile
    Publication date: December 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 6

    Author(s): Shunsuke Miyauchi , Takayuki Yuki , Hiroshi Fuji , Kunio Kojima , Tsutomu Yonetani , Ayako Tomio , Takeshi Bamba , Eiichiro Fukusaki

    The current study focused on the tea plant (Camellia sinensis) as a target for artificial cultivation because of the variation in its components in response to light conditions. We analyzed its sensory quality by multi-marker profiling using multicomponent data based on metabolomics to optimize the conditions of light and the environment during cultivation. From the analysis of high-quality tea samples ranked in a tea contest, the ranking predictive model was created by the partial least squares (PLS) regression analysis to examine the correlation between the amino-acid content (X variables) and the ranking in the tea contest (Y variables). The predictive model revealed that glutamine, arginine, and theanine were the predominant amino acids present in high-ranking teas. Based on this result, we established a cover-culture condition (i.e., a low-light intensity condition) during the later stage of the culture process and obtained artificially cultured tea samples, which were predicted to be high-quality teas. The aim of the current study was to optimize the light conditions for the cultivation of tea plants by performing data analysis of their sensory qualities through multi-marker profiling in order to facilitate the development of high-quality teas by plant factories.





  • Maintenance of undifferentiated state of human induced pluripotent stem cells through cytoskeleton-driven force acting to secreted fibronectin on a dendrimer-immobilized surface
    Publication date: December 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 6

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

    Understanding of the fundamental mechanisms that govern adhesive properties of human induced pluripotent stem cells (hiPSCs) to culture environments provides surface design strategies for maintaining their undifferentiated state during cell expansion. Polyamidoamine dendrimer surface with first-generation (G1) with dendron structure was used for co-cultures of hiPSCs and SNL feeder cells that formed tightly packed compact hiPSC colonies, similar to those on a conventional gelatin-coated surface. hiPSCs passaged up to 10 times on the G1 surface maintained their undifferentiated state. Immunostaining and reverse transcriptase PCR analysis of fibronectin showed that the secreted fibronectin matrix from feeder cells on the G1 surface contributed to hiPSC attachment. Compared with cells on the gelatin-coated surface, F-actin and paxillin immunostaining revealed a well-organized network of actin stress fibers and focal adhesion formation at cell–substrate sites in hiPSC colonies on the G1 surface. E-cadherin expression levels on these surfaces were almost same, but paxillin and Rac1 expression levels on the G1 surface were significantly higher than those on the gelatin-coated surface. Zyxin showed prominent expression on the G1 surface at sites of focal adhesion and cell–cell contact in colonies, whereas zyxin expression on the gelatin-coated surface was not observed in regions of cell–cell contact. These findings indicate that transduction of mechanical stimuli through actin polymerization at sites of focal adhesion and cell–cell contact results in maintenance of undifferentiated hiPSC colonies on G1 surface. The G1 surface enables a substrate design based on the mechanical cues in the microenvironment from feeder cells to expand undifferentiated hiPSCs in long-term culture.





  • Enzymatic synthesis of 2′-deoxyuridine by whole cell catalyst co-expressing uridine phosphorylase and thymidine phosphorylase through auto-induction system
    Publication date: December 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 6

    Author(s): Shuli Xiong , Yingbin Wang , Xi Wang , Jie Wang , Jie Li , Guiyou Zhang , Rongqing Zhang , Liping Xie , Hongzhong Wang

    Genes encoding uridine phosphorylase (UP) and thymidine phosphorylase (TP) from Escherichia coli K12 were cloned and recombined respectively into plasmids pET-21a(+) and pET-28a(+). The recombinant plasmids BL21/pET21a-UP and BL21/pET28a-TP were co-transformed into E. coli BL21(DE3) to construct highly effective BTU strain (BL21/pET28a-TP, pET21a-UP) overexpressing both of UP and TP. BTU was cultivated in ZYM-Fe-5052 medium for 10 h and used as catalyst to synthesize 2′-deoxyuridine (dUR). It was found to increase the productivity of dUR by 8–9 fold when compared to wild E. coli K12 and E. coli BL21(DE3) strains. A series of experiments were carried out to find out the optimal conditions for synthesis of dUR. At 50°C, with 0.25‰ dry wt./v to catalyze the reaction of 2:1 β-thymidine: uracil (60 mM β-thymidine, 30 mM uracil), the conversion rate of dUR would reach 61.6% at 1 h, which was much higher than the rates obtained by BTU strain cultured in LB medium and induced by IPTG. This result proved co-expression and auto-induction were efficient methods in enhancing the expression quantity and activity of nucleoside phosphorylases, and they also had significant implications for large-scale industrial production of dUR and synthesis of other nucleoside derivatives.





  • Stimulated production of steroids in Inonotus obliquus by host factors from birch
    Publication date: December 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 6

    Author(s): Lian-Xia Wang , Zhen-Ming Lu , Yan Geng , Xiao-Mei Zhang , Guo-Hua Xu , Jin-Song Shi , Zheng-Hong Xu

    Steroids was considered as one of the bioactive components in Inonotus obliquus, while this kind of secondary metabolites are less accumulated in cultured mycelia. In this study, effect of extracts from bark and core of host-related species, birch (Betula platyphylla Suk.), on steroid production of I. obliquus in submerged culture were evaluated. The results showed that all dosages (0.01 and 0.1 g/L) of aqueous extracts and methanol extracts from birch bark and birch core possessed significantly stimulatory effect on steroid production of I. obliquus (P < 0.05). Among the eight extracts, the aqueous extract (0.01 g/L) from birch bark gave the highest steroid production (225.5 ± 8.7 mg/L), which is 97.3% higher than that of the control group. The aqueous extract (0.01 and 0.1 g/L) from birch bark could simultaneously stimulated mycelial growth and steroid content, while the methanol extract from birch bark only elevated the steroid content. High performance liquid chromatography analysis showed that productions of betulin, ergosterol, cholesterol, lanosterol, stigmasterol, and sitosterol in I. obliquus simultaneously increased in the presence of aqueous extract and methanol extract from birch bark. The results presented herein indicate that extracts from birch bark could act as an inducer for steroid biosynthesis of I. obliquus.