Amperometric ethanol biosensor based on poly(vinyl alcohol)-multiwalled carbon nanotube-alcohol dehydrogenase biocomposite

A novel amperometric ethanol biosensor was constructed using alcohol dehydrogenase (ADH) physically immobilized within poly(vinyl alcohol)–multiwalled carbon nanotube (PVA–MWCNT) composite obtained by a freezing–thawing process. It comprises a MWCNT conduit, a PVA binder, and an ADH function. The measurement of ethanol is based on the signal produced by β-nicotinamide adenine dinucleotide (NADH), the product of the enzymatic reaction. The homogeneity of the resulting biocomposite film was characterized by atomic force microscopy (AFM). The performance of the PVA–MWCNT–ADH biocomposite modified glassy carbon electrode was evaluated using cyclic voltammetry and amperometry in the presence of NADH and in the presence of ethanol. The ethanol content in standard solutions was determined and a sensitivity of 196 nA mM⁻¹, a linear range up to 1.5 mM, and a response time of about 8 s were obtained. These characteristics allowed its application for direct detection of ethanol in alcoholic beverages: beer, red wine, and spirit.     

The Aspergillus niger carbon catabolite repressor encoding gene, creA

In order to undertake a comparative analysis of carbon catabolite repression in two Aspergillus species, the creA gene has been isolated from A. niger by cross hybridization, using the cloned A. nidulans gene. The A. niger gene has been shown to be functional in A. nidulans by heterologous complementation of the creA204 mutation of A. nidulans. Overall, the genes show 90% sequence similarity (82% identity) at the amino acid (aa) level. There were some striking similarities between the aa sequences encoded by the two fungal creA genes and two genes involved in carbon catabolite repression in Saccharomyces cerevisiae. The zinc-finger regions showed 96% similarity (84% identity) with the zinc-finger region of the MIG1 gene of S. cerevisiae. The CREA protein contains a stretch of 42 aa that is identical in A. niger and A. nidulans, and these show 81% similarity (33% identity) with a region of the S. cerevisiae RGR1 gene.     

Enzyme purification with aqueous two-phase systems: comparison between systems composed of pure polymers and systems composed of crude polymers

The main drawback when using aqueous two-phase systems for macromolecule purification is the high cost of most polymers used. The purification of an enzyme, alcohol dehydrogenase, from a crude extract of Saccharomyces cerevisiae was tested in systems composed of poly(ethylene glycol) and a crude hydroxypropyl starch or Reppal PES 100, a purified fraction of hydroxypropyl starch. Purification factors measured for the enzyme were very similar in both systems (between 0.8 and 1.4 for both systems in the upper phase). However, systems composed of Reppal PES present a greater recovery of enzyme, between 77% and 100% versus 60% and 100%, while systems composed of crude hydroxypropyl starch exhibit a larger Δlog K for the tested ligand, 1.26 versus 0.81.     

Properties of endogenous β-glucosidase of a Saccharomyces cerevisiae strain isolated from Sicilian musts and wines

Three hundred sixty-one yeast strains (80 of which ascribable to Saccharomyces cerevisiae) were isolated from Sicilian musts and wines with the purpose of looking for β-glucosidase (βG, EC 3.2.1.21) activity. Of these, the AL 41 strain had highest endogenous βG activity and was identified as belonging to the species S. cerevisiae by biochemical and molecular methods. This enzyme was subsequently characterized. It had optimum effect at pH 3.5–4.0, whilst optimum temperature was 20 °C, compatible with typical wine-cellar conditions; it was not inhibited by ethanol, at concentrations of 12–14%, or fructose and glucose. The βG was also characterised in terms of the kinetic parameters K_m (2.55 mM) and V_max (1.71 U mg⁻¹ of protein). Finally, it remained stable for at least 35 days in model solutions of must and wine.     

Characteristics of Wine Produced by Mushroom Fermentation

Saccharomyces cerevisiae is the main microorganism used in wine brewing, because this microbe has potent ability to produce alcohol dehydrogenase. We have recently discovered that some genera of mushroom produced alcohol dehydrogenase, and made wine by using a mushroom in place of S. cerevisiae. The highest alcohol concentration in this wine was achieved with Pleurotus ostreatus (2.6 M, 12.2%). In the case of Agaricus blazei, the same alcohol concentration (1.7 M, 8%) was produced under both aerobic and anaerobic conditions. This wine produced by A. blazei contained about 0.68% β-D-glucan, which is known to have a preventive effects against cancer. The wine made by using Flammulina velutipes showed thrombosis-preventing activity, giving a prolonged thrombin clotting time 2.2-fold that of the control. Thus, the wine made by using mushroom seems to be a functional food which can be expected to have preventive effects against cancer and thrombosis     

Characteristics of wine produced by mushroom fermentation

Saccharomyces cerevisiae is the main microorganism used in wine brewing, because this microbe has potent ability to produce alcohol dehydrogenase. We have recently discovered that some genera of mushroom produced alcohol dehydrogenase, and made wine by using a mushroom in place of S. cerevisiae. The highest alcohol concentration in this wine was achieved with Pleurotus ostreatus (2.6 M, 12.2%). In the case of Agaricus blazei, the same alcohol concentration (1.7 M, 8%) was produced under both aerobic and anaerobic conditions. This wine produced by A. blazei contained about 0.68% beta-D-glucan, which is known to have a preventive effects against cancer. The wine made by using Flammulina velutipes showed thrombosis-preventing activity, giving a prolonged thrombin clotting time 2.2-fold that of the control. Thus, the wine made by using mushroom seems to be a functional food which can be expected to have preventive effects against cancer and thrombosis.     

Elevation of glucose 6-phosphate dehydrogenase activity increases xylitol production in recombinant Saccharomyces cerevisiae

To increase the NAD(P)H-dependent xylitol production in recombinant Saccharomyces cerevisiae harboring the xylose reductase gene from Pichia stipitis, the activity of glucose 6-phosphate dehydrogenase (G6PDH) encoded by the ZWF1 gene was amplified to increase the metabolic flux toward the pentose phosphate pathway and NADPH regeneration. Compared with the control strain, the specific G6PDH activity was enhanced approximately 6.0-fold by overexpression of the ZWF1 gene. Amplification in the G6PDH activity clearly improved the NAD(P)H-dependent xylitol production in the recombinant S. cerevisiae strain. With the aid of an elevated G6PDH level, maximum xylitol concentration of 86 g/l was achieved with productivity of 2.0 g/l h in the glucose-limited fed-batch cultivation, corresponding to 25% improvement in volumetric xylitol productivity compared with the recombinant S. cerevisiae strain containing the xylose reductase gene only.     

Utilization of amino acids to enhance glutathione production in Saccharomyces cerevisiae

The effects of amino acids on glutathione (GSH) production by Saccharomyces cerevisiae T65 were investigated in this paper. Cysteine was the most important amino acids, which increased intracellular GSH content greatly but inhibited cell growth at the same time. The suitable amino acids addition strategy was two-step addition: in the first step, cysteine was added after two hours culture to 2 mM and then, the three amino acids (glutamic acid, glycine, and serine) were added after seven hours culture. The optimum concentration of those three key amino acids (10 mM glutamic acid, 10 mM glycine, and 10 mM serine) was obtained by orthogonal matrix method. With the optimum amino acids addition strategy a 1.63% intracellular GSH content was obtained in shake flask culture. Intracellular GSH content was 55.2% higher than the experiments without three amino acids addition. The cell biomass and GSH yield were 9.4 g/L and 153.2 mg/L, respectively. Using this amino acids addition strategy in the fed-batch culture of S. cerevisiae T65, GSH content, the biomass, and GSH yield reached 1.41%, 133 g/L, and 1875 mg/L, respectively, after 44 hours fermentation. GSH yield was about 2.67 times as that of amino acids free.     

Expression of various genes to enhance ubiquinone metabolic pathway in Agrobacterium tumefaciens

Ubiquinone (UQ), a lipid-soluble component, acts as a mobile component of the respiratory chain by playing an essential role in the electron transport system in many organisms, and has been widely used in pharmaceuticals due to its antioxidant property. The biosynthesis of UQ involves 10 sequential reactions brought about by various enzymes. In this study, dps gene, which encodes decaprenyl diphosphate synthase, involved in ubiquinone biosynthesis from Agrobacterium tumefaciens, and coq2 gene of Saccharomyces cerevisiae, ppt1 gene of Schizosaccahromyces pombe and ubiA gene of Escherichia coli, all of them encoding 4-hydroxybenzoate:polyprenyl diphosphate (4-HB:PPP) transferase, were reconfigured into an operon under the control of a single promoter to yield various plasmids including pBIV-dps, pBIV-dpsq, pBIV-dpsp and pBIV-dpsca. The recombinant A. tumefaciens containing dps-ubiC-ubiA gene showed the highest level ubiquinone production than that of the other recombinants and the nonrecombinant bacterium. In an aerobic fed-batch fermentation, A. tumefaciens containing the pBIV-dpsca plasmid produced 25.2 mg of ubiquinone-10 per liter which was 1.68 times higher than that of nonrecombinant type. While in microaerobic fed-batch fermentation, recombinant cell pBIV-dpsca produced 30.8 mg L⁻¹ of ubiquinone-10. Compared to the original A. tumefaciens, the ubiquinone-10 yield and productivities of the recombinant bacterium pBIV-dpsca increased 88.9% and 77.7%, respectively, under microaerobic fed-batch conditions.     

Yeast-immobilized SPV device for koji quality control in sake brewing process

The malted rice, koji, is an indispensable material for the brewing of sake. It saccharifies rice starch and supplies vitamins for the yeast in sake brewing. Since the quality of sake depends strongly on the quality of koji, quality control of koji is very important in the brewing. There are some methods to measure the activity of enzymes and the quantity of vitamins with the quality of koji. None of these methods, however, directly relate to the yeast metabolism. We constructed a sensor system to monitor the yeast metabolism in sake brewing by use of immobilized Saccharomyces cerevisiae and a Surface PhotoVoltage device (SPV). In this system, S. cerevisiae K701 and K9, designed for use in sake brewing by the Brewing Society of Japan, were employed as immobilized microbe. The pH change due to the production of organic acids in sake brewing is measured using the SPV. A linear relationship was observed between decrease in the photocurrent (the metabolism response) and the concentration to less than 60 mM of glucose (r=0.990). Then we measured the koji extract and observed the difference of response between K701 and K9 which corresponded to the productivity of acidic substances by batch test.     

Structural features of the glycogen branching enzyme encoding genes from aspergilli

A maltose binding protein, p78, was purified to homogeneity from Aspergillus nidulans by a single column chromatography step on cross-linked amylose. The partial amino acid sequence was highly homologous to the glycogen branching enzymes (GBEs) of human and yeast, and p78 did show branching enzyme activity. The genomic gene and its cDNA encoding GBE (p78) were isolated from the A. nidulans genomic and cDNA libraries. Furthermore, a cDNA encoding A. oryzae GBE was entirely sequenced. A. nidulans GBE shared overall and significant amino acid sequence identity with GBEs from A. oryzae (83.9%), Saccharomyces cerevisiae (61.1%) and human (63.0%), and with starch branching enzymes from green plants (55–56%).     

Genes encoding multiple drug resistance-like proteins in Aspergillus fumigatus and Aspergillus flavus

Polymerase chain reaction using degenerate primers was used to identify genes encoding proteins of the ATP-binding cassette superfamily in Aspergillus fumigatus and Aspergillus flavus. In A. fumigatus, two genes (AfuMDR1 and AfuMDR2) encoding proteins of the ATP-binding cassette superfamily were identified. One gene (AflMDR1) was isolated from A. flavus and is the apparent homologue to AfuMDR1. AfuMDR1and AflMDR1 encode proteins of molecular weights 148 000 and 143 000, respectively, each containing 12 putative transmembrane regions and two ATP-binding sites. These proteins are arranged in two homologous halves, each half consisting of a hydrophobic region (encoding six putative transmembrane domains) and an ATP-binding site. The AfuMDR1 and AflMDR1-encoded proteins bear a high degree of similarity to the Schizosaccharomyces pombe leptomycin B resistance protein and to human MDR1. The second gene identified in A. fumigatus, AfuMDR2, encodes a protein of molecular weight 85 000, containing four putative transmembrane domains and an ATP binding domain. The encoded protein is similar to those encoded by MDL1 and MDL2, two MDR-like genes of Saccharomyces cerevisiae. Expression of AFUMDR1 in S. cerevisiae conferred increased resistance to the antifungal agent cilofungin (LY121019), an echinocandin B analog.     

Mechanisms of the contractile effect of the alcoholic extract of Aegle marmelos Corr. on isolated guinea pig ileum and tracheal chain

In the present work, the effect of the alcoholic extract of the leaves of Aegle marmelos Corr. on guinea pig isolated ileum and tracheal chain was investigated, as this plant is used traditionally to treat asthma and related afflictions. These effects were investigated using the isolated organ bath method. 1 mg/ml and 2 mg/ml doses of the alcoholic extract of this plant produced a positive relaxant effect in isolated guinea pig ileum and tracheal chain, respectively. In addition, they antagonized the contractions, which are produced by histamine. Because the alcoholic extracts elicited the antagonistic effect against histamine and also relaxed the histamine-induced contractions, it can be concluded that relaxations induced by A. marmelos in both guinea pig ileum and tracheal chain were due to the depression of H₁-receptors. Since we observed a complete relaxation of the guinea pig ileum and tracheal chain produced by the extract, we investigated its antagonistic effect against histamine. These results were due to the presence of one or more anti-histaminic constituents present in the alcoholic extract of this plant, therefore supporting to the traditional use of A. marmelos in asthmatic complaints.     

Construction and characterisation of a BAC library from Arabidopsis halleri: Evaluation of physical mapping based on conserved synteny with Arabidopsis thaliana

We constructed and characterized the first large-insert DNA BAC library for Arabidopsis halleri, a close relative of Arabidopsis thaliana. Double size selection of high molecular weight DNA was performed to increase the average insert size. The BAC library consists of 6128 clones of which 87% have an insert size above 125 kb. Organellar DNA contamination is estimated to 1.4%. The coverage of the library is equivalent to 4.5 times the haploid genome (250 Mb), indicating the library is suitable for almost any application. We explored the possibility of generating a physical map of A. halleri using the high conserved synteny existing between this species and A. thaliana. A set of unigenes separated by 50 kb in a 850 kb region of A. thaliana chromosome II was used to probe the library. The A. halleri BAC clones isolated with these probes were grouped into two contigs. Analysis of BAC-end sequences revealed that the two A. halleri genomic contigs were highly colinear with the A. thaliana genome. Therefore, the exploitation of the conserved synteny existing between the two species will greatly facilitate the construction of a raw full physical map of A. halleri.     

Pilot scale vinification process using immobilized Candida stellata cells and Saccharomyces cerevisiae

Sequential grape juice fermentation first with immobilized Candida stellata and then with an inoculum of Saccharomyces cerevisiae was carried out at pilot scale and under non-sterile conditions in order to evaluate the dynamics of yeast microflora and their influence on the analytical profile of wine. Non-Saccharomyces yeast were adequately controlled while S. cerevisiae wild strains were consistently present after 3 days of fermentation and could compete with the inoculated S. cerevisiae strain. However, the metabolism of immobilized C. stellata cells strongly influenced the analytical profile of wines with a consistent increase in glycerol (70%) and succinic acid content in comparison with values for a S. cerevisiae fermentation control.     

Asparaginase production by a recombinant Pichia pastoris strain harbouring Saccharomyces cerevisiae ASP3 gene

The therapeutic enzyme asparaginase, which is used for the treatment of acute lymphoblastic leukaemia, is industrially produced by the bacteria Escherichia coli or Erwinia crysanthemi. In spite of its effectiveness as a therapeutic agent, the drug causes severe immunological reactions. As asparaginase is also produced by the yeast Saccharomyces cerevisiae, this microorganism could be considered for the production of the enzyme, providing an alternative antitumoral agent. In this study the ASP3 gene, that codes for the periplasmic, nitrogen regulated, asparaginase II from S. cerevisiae, was cloned and expressed in the methylotrophic yeast Pichia pastoris, under the control of the AOX1 gene promoter. Similarly to S. cerevisiae the heterologous enzyme was addressed to the P. pastoris cell periplasmic space. Enzyme yield per dry cell mass reached 800 U g⁻¹, which was seven fold higher than that obtained using a nitrogen de-repressed ure2 dal80 S. cerevisiae strain. High cell density cultures performed with P. pastoris harbouring the ASP3 gene using a 2 l instrumented bioreactor, where biomass concentration reached 107 g l⁻¹, resulted in a dramatic increase in volumetric yield (85,600 U l⁻¹) and global volumetric productivity (1083 U l⁻¹ h⁻¹).     

Effects of alcoholic beverage treatment on spatial learning and fear memory in mice

Although chronic ethanol treatment is known to impair learning and memory, humans commonly consume a range of alcoholic beverages. However, the specific effects of some alcoholic beverages on behavioral performance are largely unknown. The present study compared the effects of a range of alcoholic beverages (plain ethanol solution, red wine, sake and whiskey; with a matched alcohol concentration of 10%) on learning and memory. 6-week-old C57BL6J mice were orally administered alcohol for 7 weeks. The results revealed that red wine treatment exhibited a trend toward improvement of spatial memory and advanced extinction of fear memory. Additionally, red wine treatment significantly increased mRNA levels of brain-derived neurotrophic factor (BDNF) and N-methyl-D-aspartate (NMDA) receptors in mice hippocampus. These results support previous reports that red wine has beneficial effects.     

Quick identification of acetic acid bacteria based on nucleotide sequences of the 16S-23S rDNA internal transcribed spacer region and of the PQQ-dependent alcohol dehydrogenase gene

Acetic acid bacteria (AAB) are well known for oxidizing different ethanol-containing substrates into various types of vinegar. They are also used for production of some biotechnologically important products, such as sorbose and gluconic acids. However, their presence is not always appreciated since certain species also spoil wine, juice, beer and fruits. To be able to follow AAB in all these processes, the species involved must be identified accurately and quickly. Because of inaccuracy and very time-consuming phenotypic analysis of AAB, the application of molecular methods is necessary. Since the pairwise comparison among the 16S rRNA gene sequences of AAB shows very high similarity (up to 99.9%) other DNA-targets should be used. Our previous studies showed that the restriction analysis of 16S–23S rDNA internal transcribed spacer region is a suitable approach for quick affiliation of an acetic acid bacterium to a distinct group of restriction types and also for quick identification of a potentially novel species of acetic acid bacterium (Trcek & Teuber 2002; Trcek 2002). However, with the exception of two conserved genes, encoding tRNA^Ile and tRNA^Ala, the sequences of 16S–23S rDNA are highly divergent among AAB species. For this reason we analyzed in this study a gene encoding PQQ-dependent ADH as a possible DNA-target. First we confirmed the expression of subunit I of PQQ-dependent ADH (AdhA) also in Asaia, the only genus of AAB which exhibits little or no ADH-activity. Further we analyzed the partial sequences of adhA among some representative species of the genera Acetobacter, Gluconobacter and Gluconacetobacter. The conserved and variable regions in these sequences made possible the construction of A. aceti-specific oligonucleotide the specificity of which was confirmed in PCR-reaction using 45 well-defined strains of AAB as DNA-templates. The primer was also successfully used in direct identification of A. aceti from home made cider vinegar as well as for revealing the misclassification of strain IFO 3283 into the species A. aceti.     

拉格啤酒酵母菌物种和株系的快速分子鉴定

近年来的基因组学研究结果已证实拉格啤酒酵母Saccharomyces pastorianus是一个由艾尔啤酒酵母S. cerevisiae和真贝氏酿酒酵母S. eubayanus杂交而成的杂交种,并可根据地域传承和染色体倍性分为两个株系,即I型/Saaz系和II型/Frohberg系。前者主要为异源3倍体,后者则主要为异源4倍体。为了探讨中国啤酒酿造酵母菌的物种和菌系归属,我们根据拉格啤酒酵母及其两个菌系的基因组特性,制定了一套基于IntFR片段种特异性扩增和ITS-RFLP分析的精确但简便易行的拉格啤酒酵母菌物种和株系鉴定新方法,并以酿酒酵母属内相关种的模式或权威菌株和部分酒精及面包酵母为参照,对保藏于中国普通微生物菌种保藏中心（CGMCC）的41株啤酒酿造酵母菌进行了重新鉴定和分型。这些菌株除1株原定名为贝氏酿酒酵母S. bayanus外,其余菌株的原定名均为S. cerevisiae。研究结果确认了S. bayanus菌株鉴定的正确性,但在其余的40株啤酒酵母菌株中,21株属于S. cerevisiae,1株属于葡萄汁酿酒酵母S. uvarum,18株属于S. pastorianus。菌系鉴定和流式细胞测定结果显示在确认的S. pastorianus菌株中,1株为I型/Saaz系,3倍体;17株为II型/Frohberg系,其中9株为4倍体,两株为3倍体,5株介于3倍至4倍体之间。啤酒酵母物种和株系的确认对优化发酵工艺和菌种选育及遗传改造等具有重要意义。     

Purification, N-Terminal Amino Acid Sequence and Partial Characterization of A Cu,Zn Superoxide Dismutase From the Pathogenic Fungus Aspergillusfumiga Tus

A superoxide dismutase (SOD) has been purified to homogeneity from the fungal pathogen Aspergillus fumigatus using a combination of cell homogenization, isoelectric focusing and gel filtration FPLC. The N-terminal amino acid sequence of the purified enzyme demonstrated substantial homology to known Cu, Zn superoxide dismutases for a range of organisms, including Neurospora crassa and Saccharomyces cerevisiae. The enzyme subunit has a pl of 5.9, a relative molecular mass of 19 kDa and a spectral absorbance maximum of 550nm. The non reduced enzyme has a relative molecular mass of 95 kDa. The enzyme remained active after prolonged incubation at 70°C and was pH insensitive in the range 7-11. Potassium cyanide and diethyldithiocarbamate, known Cu, Zn SOD inhibitors, caused inhibition of the purified enzyme at working concentrations of 0.25 mM, whilst sodium azide and o-phenanthroline demonstrated inhibition at higher concentrations (10-30 mM). SOD activity was also detectable in culture filtrate of A. fumigatus. This enzyme may have a potential role as a virulence factor in the avoidance of neutrophil and phagocyte oxidative burst killing mechanisms.