Bioconversion of paper sludge to biofuel by simultaneous saccharification and fermentation using a cellulase of paper sludge origin and thermotolerant Saccharomyces cerevisiae TJ14

<p>Abstract</p> <p>Background</p> <p>Ethanol production from paper sludge (PS) by simultaneous saccharification and fermentation (SSF) is considered to be the most appropriate way to process PS, as it contains negligible lignin. In this study, SSF was conducted using a cellulase produced from PS by the hypercellulase producer, <it>Acremonium cellulolyticus </it>C-1 for PS saccharification, and a thermotolerant ethanol producer <it>Saccharomyces cerevisiae </it>TJ14 for ethanol production. Using cellulase of PS origin minimizes biofuel production costs, because the culture broth containing cellulase can be used directly.</p> <p>Results</p> <p>When 50 g PS organic material (PSOM)/l was used in SSF, the ethanol yield based on PSOM was 23% (g ethanol/g PSOM) and was two times higher than that obtained by a separate hydrolysis and fermentation process. Cellulase activity throughout SSF remained at around 60% of the initial activity. When 50 to 150 g PSOM/l was used in SSF, the ethanol yield was 21% to 23% (g ethanol/g PSOM) at the 500 ml Erlenmeyer flask scale. Ethanol production and theoretical ethanol yield based on initial hexose was 40 g/l and 66.3% (g ethanol/g hexose) at 80 h, respectively, when 161 g/l of PSOM, 15 filter paper units (FPU)/g PSOM, and 20% inoculum were used for SSF, which was confirmed in the 2 l scale experiment. This indicates that PS is a good raw material for bioethanol production.</p> <p>Conclusions</p> <p>Ethanol concentration increased with increasing PSOM concentration. The ethanol yield was stable at PSOM concentrations of up to 150 g/l, but decreased at concentrations higher than 150 g/l because of mass transfer limitations. Based on a 2 l scale experiment, when 1,000 kg PS was used, 3,182 kFPU cellulase was produced from 134.7 kg PS. Produced cellulase was used for SSF with 865.3 kg PS and ethanol production was estimated to be 51.1 kg. Increasing the yeast inoculum or cellulase concentration did not significantly improve the ethanol yield or concentration.</p

Construction of Bordetella pertussis strains with enhanced production of genetically-inactivated Pertussis Toxin and Pertactin by unmarked allelic exchange

<p>Abstract</p> <p>Background</p> <p>Acellular Pertussis vaccines against whooping cough caused by <it>Bordetella pertussis </it>present a much-improved safety profile compared to the original vaccine of killed whole cells. The principal antigen of acellular Pertussis vaccine, Pertussis Toxin (PT), must be chemically inactivated to obtain the corresponding toxoid (PTd). This process, however, results in extensive denaturation of the antigen. The development of acellular Pertussis vaccines containing PTd or recombinant PT (rPT) with inactivated S1, Filamentous Hemagglutinin (FHA), and Pertactin (PRN) has shown that the yield of PRN was limiting, whereas FHA was overproduced. To improve antigen yields and process economics, we have constructed strains of <it>Bordetella pertussis </it>that produce enhanced levels of both rPT and PRN.</p> <p>Results</p> <p>Three recombinant strains of <it>Bordetella pertussis </it>were obtained by homologous recombination using an allelic exchange vector, pSS4245. In the first construct, the segment encoding PT subunit S1 was replaced by two mutations (R9K and E129G) that removed PT toxicity and Bp-WWC strain was obtained. In the second construct, a second copy of the whole cluster of PT structural genes containing the above mutations was inserted elsewhere into the chromosome of Bp-WWC and the Bp-WWD strain was obtained. This strain generated increased amounts of rPT (3.77 ± 0.53 μg/mL) compared to Bp-WWC (2.61 ± 0.16 μg/mL) and wild type strain (2.2 μg/mL). In the third construct, a second copy of the <it>prn </it>gene was inserted into the chromosome of Bp-WWD to obtain Bp-WWE. Strain Bp-WWE produced PRN at 4.18 ± 1.02 μg/mL in the cell extract which was about two-fold higher than Bp-WWC (2.48 ± 0.10 μg/mL) and Bp-WWD (2.31 ± 0.17 μg/mL). Purified PTd from Bp-WWD at 0.8-1.6 μg/well did not show any toxicity against Chinese hamster ovary (CHO) cell whereas purified PT from WT demonstrated a cell clustering endpoint at 2.6 pg/well.</p> <p>Conclusions</p> <p>We have constructed <it>Bordetella pertussis </it>strains expressing increased amounts of the antigens, rPT or rPT and PRN. Expression of the third antigen, FHA was unchanged (always in excess). These strains will be useful for the manufacture of affordable acellular Pertussis vaccines.</p

Puerarin Suppresses Invasion and Vascularization of Endometriosis Tissue Stimulated by 17β-Estradiol

BACKGROUND: Puerarin, a phytoestrogen with a weak estrogenic effect, binds to estrogen receptors, thereby competing with 17β-estradiol (E2) and producing an anti-estrogenic effect. This study was to investigate whether puerarin could suppress the invasion and vascularization of E2-stimulated endometriotic tissue. METHODOLOGY/PRINCIPAL FINDINGS: The endometriotic stromal cells (ESCs) were successfully established and their invasive ability under different treatments was assessed through a Transwell Assay. Simultaneously, matrix metallopeptidase 9 (MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1) were detected by western blotting. Vascularization of endometriotic tissues was observed by chicken chorioallantoic membrane (CAM) assay. The staining of MMP-9, intercellular adhesion molecule 1 (ICAM-1), TIMP-1, and vascular endothelial growth factor (VEGF) in grafted endometriotic tissues was examined using immunohistochemistry analysis. The purity of ESCs in isolated cells was >95%, as determined by the fluoroimmunoassay of vimentin. E2 (10(-8) mol/L) promoted the invasiveness of ESCs by increasing MMP-9 accumulation and decreasing TIMP-1 accumulation. Interestingly, puerarin (10(-9) mol/L) significantly reversed these effects (P<0.01). The CAM assay indicated that puerarin (10(-9) mol/L) also inhibited the angiopoiesis of endometriotic tissue stimulated by the E2 (10(-8) mol/L) treatment (P<0.05). Accordingly, immunohistochemistry showed that the accumulation of MMP-9, ICAM-1, and VEGF was reduced whereas that of TIMP-1 increased in the combination treatment group compared with the E2 treatment group. CONCLUSIONS/SIGNIFICANCE: This study demonstrated that puerarin could suppress the tissue invasion by ESCs and the vascularization of ectopic endometrial tissues stimulated by E2, suggesting that puerarin may be a potential drug for the treatment of endometriosis

Insight on an Arginine Synthesis Metabolon from the Tetrameric Structure of Yeast Acetylglutamate Kinase

N-acetyl-L-glutamate kinase (NAGK) catalyzes the second, generally controlling, step of arginine biosynthesis. In yeasts, NAGK exists either alone or forming a metabolon with N-acetyl-L-glutamate synthase (NAGS), which catalyzes the first step and exists only within the metabolon. Yeast NAGK (yNAGK) has, in addition to the amino acid kinase (AAK) domain found in other NAGKs, a ∼150-residue C-terminal domain of unclear significance belonging to the DUF619 domain family. We deleted this domain, proving that it stabilizes yNAGK, slows catalysis and modulates feed-back inhibition by arginine. We determined the crystal structures of both the DUF619 domain-lacking yNAGK, ligand-free as well as complexed with acetylglutamate or acetylglutamate and arginine, and of complete mature yNAGK. While all other known arginine-inhibitable NAGKs are doughnut-like hexameric trimers of dimers of AAK domains, yNAGK has as central structure a flat tetramer formed by two dimers of AAK domains. These dimers differ from canonical AAK dimers in the −110° rotation of one subunit with respect to the other. In the hexameric enzymes, an N-terminal extension, found in all arginine-inhibitable NAGKs, forms a protruding helix that interlaces the dimers. In yNAGK, however, it conforms a two-helix platform that mediates interdimeric interactions. Arginine appears to freeze an open inactive AAK domain conformation. In the complete yNAGK structure, two pairs of DUF619 domains flank the AAK domain tetramer, providing a mechanism for the DUF619 domain modulatory functions. The DUF619 domain exhibits the histone acetyltransferase fold, resembling the catalytic domain of bacterial NAGS. However, the putative acetyl CoA site is blocked, explaining the lack of NAGS activity of yNAGK. We conclude that the tetrameric architecture is an adaptation to metabolon formation and propose an organization for this metabolon, suggesting that yNAGK may be a good model also for yeast and human NAGSs

Determination of amino acid sequences involved in the processing of the ARG5/ARG6 precursor in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the ARG5/ARG6 locus encodes both acetylglutamate kinase and acetylglutamyl-phosphate reductase, localized in the mitochondria. Genetic analysis, determination of the nucleotide sequence of the ARG5/ARG6 gene and identification of the transcript indicate that it encodes a single translation product containing two enzyme activities. However, analysis of cellular extracts revealed that the activities are completely separable. In this work, we define different domains in the ARG5/ARG6 polypeptide; a mitochondrial target sequence and the acetylglutamate-kinase and acetylglutamyl-phosphate-reductase domains. We show that deletions in the N-terminal end of the protein and point mutations in the junction region between the acetylglutamate-kinase and acetylglutamyl-phosphate-reductase domains lead to the accumulation of large precursor. Our data support the idea that import of the ARG5/ARG6 precursor into the mitochondria is required for its processing into two mature enzymes.Comparative StudyJournal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Characterization of the yeast ARG5,6 gene: determination of the nucleotide sequence, analysis of the control region and of ARG5,6 transcript.

In Saccharomyces cerevisiae, the ARG5,6 gene encodes acetylglutamyl-P reductase and acetylglutamate kinase, two arginine anabolic enzymes which are localized in the mitochondria. The synthesis of both enzymes is co-ordinately controlled by arginine and by three regulatory proteins (ARGRI, ARGRII, and ARGRIII). The ARG5,6 gene was cloned by complementation of an arg5 mutant strain. A subclone containing an EcoRI fragment of about 3.2 kb which complements the arginine requirement was sequenced. This 3163 bp sequence contains only one long open reading frame of 2589 nucleotides encoding a protein of 863 amino acids. The size of this protein is in agreement with the length of the unique transcript determined by Northern hybridization. The measurements of ARG5,6 mRNA under various regulatory conditions show no correlation with the enzyme levels. As in other arginine biosynthetic and catabolic genes, the regulation by arginine through the three ARGR proteins thus involves a post-transcriptional control mechanism. By in vitro mutagenesis we created point mutations and deletions in the 5' non-coding region of the ARG5,6 gene which allowed us to define the primary target of ARGR control. Specific regulation involves two regions: one located between the putative TATA element and the transcriptional initiation site and the second between this site and the first ATG.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Determination of the DNA-binding sequences of ARGR proteins to arginine anabolic and catabolic promoters.

ARGRI, ARGRII, and ARGRIII proteins regulate the expression of arginine anabolic and catabolic genes. The integrity of these three proteins is required to observe the formation of a DNA-protein complex with the different promoters of arginine coregulated genes. A study of deletions and point mutations created in the 5' noncoding region of ARG3, ARG5,6, CAR1, and CAR2 genes shows that at least two regions, called BoxA and BoxB, are required for proper regulation of these genes by arginine and ARGR proteins. By gel retardation assay and DNase I footprinting analysis, we have determined precisely the target of the ARGR proteins. Sequences in and around BoxA are necessary for ARGR binding to these four promoters in vitro, whereas sequences in and around BoxB are clearly protected against DNase I digestion only for CAR1. Sequences present at BoxA and BoxB are well conserved among the four promoters. Moreover, pairing can occur between sequences at BoxA and BoxB which could lead to the creation of secondary structures in ARG3, ARG5,6, CAR1, and CAR2 promoters, favoring the binding of ARGR proteins in vivo.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Differential binding with ERα and ERβ of the phytoestrogen-rich plant Pueraria mirifica

Variations in the estrogenic activity of the phytoestrogen-rich plant, Pueraria mirifica, were determined with yeast estrogen screen (YES) consisting of human estrogen receptors (hER) hERα and hERβ and human transcriptional intermediary factor 2 (hTIF2) or human steroid receptor coactivator 1 (hSRC1), respectively, together with the β-galactosidase expression cassette. Relative estrogenic potency was expressed by determining the β-galactosidase activity (EC50) of the tuber extracts in relation to 17β-estradiol. Twenty-four and 22 of the plant tuber ethanolic extracts interacted with hERα and hERβ, respectively, with a higher relative estrogenic potency with hERβ than with hERα. Antiestrogenic activity of the plant extracts was also determined by incubation of plant extracts with 17β-estradiol prior to YES assay. The plant extracts tested exhibited antiestrogenic activity. Both the estrogenic and the antiestrogenic activity of the tuber extracts were metabolically activated with the rat liver S9-fraction prior to the assay indicating the positive influence of liver enzymes. Correlation analysis between estrogenic potency and the five major isoflavonoid contents within the previously HPLC-analyzed tuberous samples namely puerarin, daidzin, genistin, daidzein, and genistein revealed a negative result