Science.gov

Sample records for fungus whole-cell biocatalysts

  1. Whole-cell biocatalysts for biodiesel fuel production.

    PubMed

    Fukuda, H; Hama, S; Tamalampudi, S; Noda, H

    2008-12-01

    Biodiesel fuel (BDF), which refers to fatty acid alkyl esters, has attracted considerable attention as an environmentally friendly alternative fuel for diesel engines. Alkali catalysis is widely applied for the commercial production of BDF. However, enzymatic transesterification offers considerable advantages, including reducing process operations in biodiesel fuel production and an easy separation of the glycerol byproduct. The high cost of the lipase enzyme is the main obstacle for a commercially feasible enzymatic production of biodiesel fuels. To reduce enzyme associated process costs, the immobilization of fungal mycelium within biomass support particles (BSPs) as well as expression of the lipase enzyme on the surface of yeast cells has been developed to generate whole-cell biocatalysts for industrial applications.

  2. Selection of a whole-cell biocatalyst for methyl parathion biodegradation.

    PubMed

    Yang, Jijian; Liu, Ruihua; Jiang, Hong; Yang, Yao; Qiao, Chuanling

    2012-09-01

    Whole-cell biocatalyst has the potential to become a cost-effective alternative to conventional enzyme methods for solving ecological and energy issues. However, cytosolic-expressing biocatalyst systems are critically disadvantaged due to the low permeability of the cell membrane. To overcome substrate transport barrier, periplasmic secretion and surface display biocatalysts were developed by expressing signal peptides or anchor proteins in Escherichia coli. In this work, six carriers were compared in regard to whole-cell activity of methyl parathion hydrolase (MPH). Our results indicate that the surface display systems yielded one to three times whole-cell activity than the periplasmic secretion systems. Although periplasmic secretion systems showed generally more stable than surface display systems, surface display appeared more suitable for whole-cell biocatalyst. It should note that the applicability of the DsbA/PhoA/AIDA-I leader to MPH expression is shown here for the first time. In addition, the result provided a useful reference for other whole-cell biocatalyst selection.

  3. Use of a whole-cell biocatalyst to produce biodiesel in a water-containing system.

    PubMed

    Jin, Guang; Bierma, Thomas J; Hamaker, Christopher G; Mucha, Raymond; Schola, Valeria; Stewart, Jeb; Wade, Caroline

    2009-01-01

    This study examined the use of a whole-cell biocatalyst to transesterify triglycerides, including high-Free Fatty Acid (FFA) waste greases, in a water-containing system. The whole-cell biocatalyst derived from Rhizopus oryzae (ATCC10260) was grown and reacted at room temperature without immobilization. The effectiveness of improving biodiesel yield through alteration of reaction temperature, additional alcohol, and additional transesterification reaction was also examined. Results showed that whole-cell biocatalyst was able to produce biodiesel with a yield of about 75% for virgin canola oil, 80% for waste vegetable oil and 55% for brown grease with a 72-hr transesterification reaction using no excess methanol. Elevating the reaction temperature to 35 degrees C significantly diminished the yield. An additional dose of methanol with another 24 hours of reaction time or a second 72-hr reaction resulted in biodiesel yield approaching 90% and only 3% residual glycerides (mono-, di- and tri-glycerides). These results suggest that whole-cell biocatalysts are able to transesterify waste oils or greases that are high in FFA and contain water. Brown (trap) grease and similar degraded or complex greases may be good candidates for further whole-cell biocatalyst research.

  4. [Synthesis of diisooctyl adipate catalyzed by lipase-displaying Pichia pastoris whole-cell biocatalysts].

    PubMed

    Zhang, Na; Jin, Zi; Lin, Ying; Zheng, Suiping; Han, Shuangyan

    2013-07-01

    An enzyme-displaying yeast as a whole-cell biocatalyst is an alternative to immobilized enzyme, due to its low-cost preparation and simple recycle course. Here, lipase-displaying Pichia pastoris whole-cell was used as a biocatalyst to synthesize diisooctyl adipate in the non-aqueous system. The maximum productivity of diisooctyl adipate was obtained as 85.0% in a 10 mL reaction system. The yield could be reached as high as 97.8% when the reaction system was scaled up to 200 mL. The purity obtained is 98.2% after vacuum distillation. Thus, the lipase-displaying P. pastoris whole-cell biocatalyst was promising in commercial application for diisooctyl adipate synthesis in non-aqueous phase. PMID:24195369

  5. [Synthesis of diisooctyl adipate catalyzed by lipase-displaying Pichia pastoris whole-cell biocatalysts].

    PubMed

    Zhang, Na; Jin, Zi; Lin, Ying; Zheng, Suiping; Han, Shuangyan

    2013-07-01

    An enzyme-displaying yeast as a whole-cell biocatalyst is an alternative to immobilized enzyme, due to its low-cost preparation and simple recycle course. Here, lipase-displaying Pichia pastoris whole-cell was used as a biocatalyst to synthesize diisooctyl adipate in the non-aqueous system. The maximum productivity of diisooctyl adipate was obtained as 85.0% in a 10 mL reaction system. The yield could be reached as high as 97.8% when the reaction system was scaled up to 200 mL. The purity obtained is 98.2% after vacuum distillation. Thus, the lipase-displaying P. pastoris whole-cell biocatalyst was promising in commercial application for diisooctyl adipate synthesis in non-aqueous phase.

  6. A whole cell biocatalyst for cellulosic ethanol production from dilute acid-pretreated corn stover hydrolyzates.

    PubMed

    Ryu, Seunghyun; Karim, Muhammad Nazmul

    2011-08-01

    In this research, a recombinant whole cell biocatalyst was developed by expressing three cellulases from Clostridium cellulolyticum--endoglucanase (Cel5A), exoglucanase (Cel9E), and β-glucosidase--on the surface of the Escherichia coli LY01. The modified strain is identified as LY01/pRE1H-AEB. The cellulases were displayed on the surface of the cell by fusing with an anchor protein, PgsA. The developed whole cell biocatalyst was used for single-step ethanol fermentation using the phosphoric acid-swollen cellulose (PASC) and the dilute acid-pretreated corn stover. Ethanol production was 3.59 ± 0.15 g/L using 10 g/L of PASC, which corresponds to a theoretical yield of 95.4 ± 0.15%. Ethanol production was 0.30 ± 0.02 g/L when 1 g/L equivalent of glucose in the cellulosic fraction of the dilute sulfuric acid-pretreated corn stover (PCS) was fermented for 84 h. A total of 0.71 ± 0.12 g/L ethanol was produced in 48 h when the PCS was fermented in the simultaneous saccharification and co-fermentation mode using the hemicellulosic (1 g/L of total soluble sugar) and as well as the cellulosic (1 g/L of glucose equivalent) parts of PCS. In a control experiment, 0.48 g/L ethanol was obtained from 1 g/L of hemicellulosic PCS. It was concluded that the whole cell biocatalyst could convert both cellulosic and hemicellulosic substrates into ethanol in a single reactor. The developed C. cellulolyticum-E. coli whole cell biocatalyst also overcame the incompatible temperature problem of the frequently reported fungal-yeast systems. PMID:21519935

  7. Optimization of a whole-cell biocatalyst by employing genetically encoded product sensors inside nanolitre reactors

    NASA Astrophysics Data System (ADS)

    Meyer, Andreas; Pellaux, René; Potot, Sébastien; Becker, Katja; Hohmann, Hans-Peter; Panke, Sven; Held, Martin

    2015-08-01

    Microcompartmentalization offers a high-throughput method for screening large numbers of biocatalysts generated from genetic libraries. Here we present a microcompartmentalization protocol for benchmarking the performance of whole-cell biocatalysts. Gel capsules served as nanolitre reactors (nLRs) for the cultivation and analysis of a library of Bacillus subtilis biocatalysts. The B. subtilis cells, which were co-confined with E. coli sensor cells inside the nLRs, converted the starting material cellobiose into the industrial product vitamin B2. Product formation triggered a sequence of reactions in the sensor cells: (1) conversion of B2 into flavin mononucleotide (FMN), (2) binding of FMN by a RNA riboswitch and (3) self-cleavage of RNA, which resulted in (4) the synthesis of a green fluorescent protein (GFP). The intensity of GFP fluorescence was then used to isolate B. subtilis variants that convert cellobiose into vitamin B2 with elevated efficiency. The underlying design principles of the assay are general and enable the development of similar protocols, which ultimately will speed up the optimization of whole-cell biocatalysts.

  8. Optimization of a whole-cell biocatalyst by employing genetically encoded product sensors inside nanolitre reactors.

    PubMed

    Meyer, Andreas; Pellaux, René; Potot, Sébastien; Becker, Katja; Hohmann, Hans-Peter; Panke, Sven; Held, Martin

    2015-08-01

    Microcompartmentalization offers a high-throughput method for screening large numbers of biocatalysts generated from genetic libraries. Here we present a microcompartmentalization protocol for benchmarking the performance of whole-cell biocatalysts. Gel capsules served as nanolitre reactors (nLRs) for the cultivation and analysis of a library of Bacillus subtilis biocatalysts. The B. subtilis cells, which were co-confined with E. coli sensor cells inside the nLRs, converted the starting material cellobiose into the industrial product vitamin B2. Product formation triggered a sequence of reactions in the sensor cells: (1) conversion of B2 into flavin mononucleotide (FMN), (2) binding of FMN by a RNA riboswitch and (3) self-cleavage of RNA, which resulted in (4) the synthesis of a green fluorescent protein (GFP). The intensity of GFP fluorescence was then used to isolate B. subtilis variants that convert cellobiose into vitamin B2 with elevated efficiency. The underlying design principles of the assay are general and enable the development of similar protocols, which ultimately will speed up the optimization of whole-cell biocatalysts.

  9. Optimization of a whole-cell biocatalyst by employing genetically encoded product sensors inside nanolitre reactors.

    PubMed

    Meyer, Andreas; Pellaux, René; Potot, Sébastien; Becker, Katja; Hohmann, Hans-Peter; Panke, Sven; Held, Martin

    2015-08-01

    Microcompartmentalization offers a high-throughput method for screening large numbers of biocatalysts generated from genetic libraries. Here we present a microcompartmentalization protocol for benchmarking the performance of whole-cell biocatalysts. Gel capsules served as nanolitre reactors (nLRs) for the cultivation and analysis of a library of Bacillus subtilis biocatalysts. The B. subtilis cells, which were co-confined with E. coli sensor cells inside the nLRs, converted the starting material cellobiose into the industrial product vitamin B2. Product formation triggered a sequence of reactions in the sensor cells: (1) conversion of B2 into flavin mononucleotide (FMN), (2) binding of FMN by a RNA riboswitch and (3) self-cleavage of RNA, which resulted in (4) the synthesis of a green fluorescent protein (GFP). The intensity of GFP fluorescence was then used to isolate B. subtilis variants that convert cellobiose into vitamin B2 with elevated efficiency. The underlying design principles of the assay are general and enable the development of similar protocols, which ultimately will speed up the optimization of whole-cell biocatalysts. PMID:26201745

  10. Autodisplay of active sorbitol dehydrogenase (SDH) yields a whole cell biocatalyst for the synthesis of rare sugars.

    PubMed

    Jose, Joachim; von Schwichow, Steffen

    2004-04-01

    Whole cell biocatalysts are attractive technological tools for the regio- and enantioselective synthesis of products, especially from substrates with several identical reactive groups. In the present study, a whole cell biocatalyst for the synthesis of rare sugars from polyalcohols was constructed. For this purpose, sorbitol dehydrogenase (SDH) from Rhodobacter sphaeroides, a member of the short-chain dehydrogenase/reductase (SDR) family, was expressed on the surface of Escherichia coli using Autodisplay. Autodisplay is an efficient surface display system for Gram-negative bacteria and is based on the autotransporter secretion pathway. Transport of SDH to the outer membrane was monitored by SDS-PAGE and Western blotting of different cell fractions. The surface exposure of the enzyme could be verified by immunofluorescence microscopy and fluorescence activated cell sorting (FACS). The activity of whole cells displaying SDH at the surface was determined in an optical test. Specific activities were found to be 12 mU per 3.3 x 10(8) cells for the conversion of D-glucitol (sorbitol) to D-fructose, 7 mU for the conversion D-galactitol to D-tagatose, and 17 mU for the conversion of L-arabitol to L-ribulose. The whole cell biocatalyst obtained by surface display of SDH could also produce D-glucitol from D-fructose (29 mU per 3.3 x 10(8) cells).

  11. Bacterial whole-cell biocatalysts by surface display of enzymes: toward industrial application.

    PubMed

    Schüürmann, Jan; Quehl, Paul; Festel, Gunter; Jose, Joachim

    2014-10-01

    Despite the first report on the bacterial display of a recombinant peptide appeared almost 30 years ago, industrial application of cells with surface-displayed enzymes is still limited. To display an enzyme on the surface of a living cell bears several advantages. First of all, neither the substrate nor the product of the enzymatic reaction needs to cross a membrane barrier. Second, the enzyme being linked to the cell can be separated from the reaction mixture and hence the product by simple centrifugation. Transfer to a new substrate preparation results in multiple cycles of enzymatic conversion. Finally, the anchoring in a matrix, in this case, the cell envelope stabilizes the enzyme and makes it less accessible to proteolytic degradation and material adsorption resulting in continuous higher activities. These advantages in common need to balance some disadvantages before this application can be taken into account for industrial processes, e.g., the exclusion of the enzyme from the cellular metabolome and hence from redox factors or other co-factors that need to be supplied. Therefore, this digest describes the different systems in Gram-positive and Gram-negative bacteria that have been used for the surface display of enzymes so far and focuses on examples among these which are suitable for industrial purposes or for the production of valuable resources, not least in order to encourage a broader application of whole-cell biocatalysts with surface-displayed enzymes.

  12. The Production of Biodiesel from Cottonseed Oil Using Rhizopus oryzae Whole Cell Biocatalysts

    NASA Astrophysics Data System (ADS)

    Athalye, Sneha Kishor

    Biodiesel is an environmentally friendly alternative to fossil fuels which have become increasingly expensive in recent times. An alternate approach to alkaline biodiesel production is needed as catalyst miscibility with the glycerol by-product, generation of large amounts of waste water, and saponification of the feedstock are major disadvantages associated with the process. Lipases are water soluble enzymes which act as catalysts in many lipid based reactions. Reuse of lipases can significantly reduce cost of enzymatic biodiesel production; however retention of lipolytic activity still remains a challenge. Use of microbial cells immobilized on various surfaces like sponge, foam and plastics as biocatalysts instead of extracted enzyme could help overcome this problem. A novel, rigid biomass support with high surface area made from recyclable polyethylene (Bioblok(TM)) was used in this study. Several fungal and bacterial species have been reported to possess appreciable levels of lipase activity. The biomass production and immobilization as well as lipase activity of three different species; Candida rugosa (ATCC #38772), Aspergillus oryzae (ATCC #58299), and Rhizopus oryzae (ATTC #34612) were tested. C. rugosa did not attach well to the support particles while A.oryzae had lower biomass accumulation of 6.1 g (dry cell wt)/L compared to 11.8 g (dry cell wt)/L for R.oryzae. Hence Rhizopus oryzae, fungal specie with cell surface bound lipase was selected for the current study. The study investigated the influence of media composition and growth time of the R.oryzae whole cell biocatalysts, immobilized on the BSPs, for FAME production from cottonseed oil. R.oryzae BSPs grown in basal media supplemented with 1% (w/v) of glucose or oil or both for 48 h, 72 h or 90 h were used in a 36 h transesterification reaction with cottonseed oil and methanol. BSPs grown in both glucose and oil supplemented medium for 72 h had the highest conversion of 22.4% (wt/wt) and a biomass

  13. The Production of Biodiesel from Cottonseed Oil Using Rhizopus oryzae Whole Cell Biocatalysts

    NASA Astrophysics Data System (ADS)

    Athalye, Sneha Kishor

    Biodiesel is an environmentally friendly alternative to fossil fuels which have become increasingly expensive in recent times. An alternate approach to alkaline biodiesel production is needed as catalyst miscibility with the glycerol by-product, generation of large amounts of waste water, and saponification of the feedstock are major disadvantages associated with the process. Lipases are water soluble enzymes which act as catalysts in many lipid based reactions. Reuse of lipases can significantly reduce cost of enzymatic biodiesel production; however retention of lipolytic activity still remains a challenge. Use of microbial cells immobilized on various surfaces like sponge, foam and plastics as biocatalysts instead of extracted enzyme could help overcome this problem. A novel, rigid biomass support with high surface area made from recyclable polyethylene (Bioblok(TM)) was used in this study. Several fungal and bacterial species have been reported to possess appreciable levels of lipase activity. The biomass production and immobilization as well as lipase activity of three different species; Candida rugosa (ATCC #38772), Aspergillus oryzae (ATCC #58299), and Rhizopus oryzae (ATTC #34612) were tested. C. rugosa did not attach well to the support particles while A.oryzae had lower biomass accumulation of 6.1 g (dry cell wt)/L compared to 11.8 g (dry cell wt)/L for R.oryzae. Hence Rhizopus oryzae, fungal specie with cell surface bound lipase was selected for the current study. The study investigated the influence of media composition and growth time of the R.oryzae whole cell biocatalysts, immobilized on the BSPs, for FAME production from cottonseed oil. R.oryzae BSPs grown in basal media supplemented with 1% (w/v) of glucose or oil or both for 48 h, 72 h or 90 h were used in a 36 h transesterification reaction with cottonseed oil and methanol. BSPs grown in both glucose and oil supplemented medium for 72 h had the highest conversion of 22.4% (wt/wt) and a biomass

  14. A robust whole-cell biocatalyst that introduces a thermo- and solvent-tolerant lipase into Aspergillus oryzae cells: characterization and application to enzymatic biodiesel production.

    PubMed

    Adachi, Daisuke; Koh, FookHee; Hama, Shinji; Ogino, Chiaki; Kondo, Akihiko

    2013-05-10

    To develop a robust whole-cell biocatalyst that works well at moderately high temperature (40-50°C) with organic solvents, a thermostable lipase from Geobacillus thermocatenulatus (BTL2) was introduced into an Aspergillus oryzae whole-cell biocatalyst. The lipase-hydrolytic activity of the immobilized A. oryzae (r-BTL) was highest at 50°C and was maintained even after an incubation of 24-h at 60°C. In addition, r-BTL was highly tolerant to 30% (v/v) organic solvents (dimethyl carbonate, ethanol, methanol, 2-propanol or acetone). The attractive characteristics of r-BTL also worked efficiently on palm oil methanolysis, resulting in a nearly 100% conversion at elevated temperature from 40 to 50°C. Moreover, r-BTL catalyzed methanolysis at a high methanol concentration without a significant loss of lipase activity. In particular, when 2 molar equivalents of methanol were added 2 times, a methyl ester content of more than 90% was achieved; the yield was higher than those of conventional whole-cell biocatalyst and commercial Candida antarctica lipase (Novozym 435). On the basis of the results regarding the excellent lipase characteristics and efficient biodiesel production, the developed whole-cell biocatalyst would be a promising biocatalyst in a broad range of applications including biodiesel production. PMID:23608501

  15. Development of a Continuous Bioconversion System Using a Thermophilic Whole-Cell Biocatalyst

    PubMed Central

    Ninh, Pham Huynh; Yokohigashi, Yukako; Okano, Kenji; Omasa, Takeshi; Ohtake, Hisao

    2013-01-01

    The heat treatment of recombinant mesophilic cells having heterologous thermophilic enzymes results in the denaturation of indigenous mesophilic enzymes and the elimination of undesired side reactions; therefore, highly selective whole-cell catalysts comparable to purified enzymes can be readily prepared. However, the thermolysis of host cells leads to the heat-induced leakage of thermophilic enzymes, which are produced as soluble proteins, limiting the exploitation of their excellent stability in repeated and continuous reactions. In this study, Escherichia coli cells having the thermophilic fumarase from Thermus thermophilus (TtFTA) were treated with glutaraldehyde to prevent the heat-induced leakage of the enzyme, and the resulting cells were used as a whole-cell catalyst in repeated and continuous reactions. Interestingly, although electron microscopic observations revealed that the cellular structure of glutaraldehyde-treated E. coli was not apparently changed by the heat treatment, the membrane permeability of the heated cells to relatively small molecules (up to at least 3 kDa) was significantly improved. By applying the glutaraldehyde-treated E. coli having TtFTA to a continuous reactor equipped with a cell-separation membrane filter, the enzymatic hydration of fumarate to malate could be operated for more than 600 min with a molar conversion yield of 60% or higher. PMID:23335777

  16. Short-chain flavor ester synthesis in organic media by an E. coli whole-cell biocatalyst expressing a newly characterized heterologous lipase.

    PubMed

    Brault, Guillaume; Shareck, François; Hurtubise, Yves; Lépine, François; Doucet, Nicolas

    2014-01-01

    Short-chain aliphatic esters are small volatile molecules that produce fruity and pleasant aromas and flavors. Most of these esters are artificially produced or extracted from natural sources at high cost. It is, however, possible to 'naturally' produce these molecules using biocatalysts such as lipases and esterases. A gene coding for a newly uncovered lipase was isolated from a previous metagenomic study and cloned into E. coli BL21 (DE3) for overexpression using the pET16b plasmid. Using this recombinant strain as a whole-cell biocatalyst, short chain esters were efficiently synthesized by transesterification and esterification reactions in organic media. The recombinant lipase (LipIAF5-2) showed good affinity toward glyceryl trioctanoate and the highest conversion yields were obtained for the transesterification of glyceryl triacetate with methanol. Using a simple cetyl-trimethylammonium bromide pretreatment increased the synthetic activity by a six-fold factor and the whole-cell biocatalyst showed the highest activity at 40°C with a relatively high water content of 10% (w/w). The whole-cell biocatalyst showed excellent tolerance to alcohol and short-chain fatty acid denaturation. Substrate affinity was equally effective with all primary alcohols tested as acyl acceptors, with a slight preference for methanol. The best transesterification conversion of 50 mmol glyceryl triacetate into isoamyl acetate (banana fragrance) provided near 100% yield after 24 hours using 10% biocatalyst loading (w/w) in a fluidized bed reactor, allowing recycling of the biocatalyst up to five times. These results show promising potential for an industrial approach aimed at the biosynthesis of short-chain esters, namely for natural flavor and fragrance production in micro-aqueous media. PMID:24670408

  17. Short-chain flavor ester synthesis in organic media by an E. coli whole-cell biocatalyst expressing a newly characterized heterologous lipase.

    PubMed

    Brault, Guillaume; Shareck, François; Hurtubise, Yves; Lépine, François; Doucet, Nicolas

    2014-01-01

    Short-chain aliphatic esters are small volatile molecules that produce fruity and pleasant aromas and flavors. Most of these esters are artificially produced or extracted from natural sources at high cost. It is, however, possible to 'naturally' produce these molecules using biocatalysts such as lipases and esterases. A gene coding for a newly uncovered lipase was isolated from a previous metagenomic study and cloned into E. coli BL21 (DE3) for overexpression using the pET16b plasmid. Using this recombinant strain as a whole-cell biocatalyst, short chain esters were efficiently synthesized by transesterification and esterification reactions in organic media. The recombinant lipase (LipIAF5-2) showed good affinity toward glyceryl trioctanoate and the highest conversion yields were obtained for the transesterification of glyceryl triacetate with methanol. Using a simple cetyl-trimethylammonium bromide pretreatment increased the synthetic activity by a six-fold factor and the whole-cell biocatalyst showed the highest activity at 40°C with a relatively high water content of 10% (w/w). The whole-cell biocatalyst showed excellent tolerance to alcohol and short-chain fatty acid denaturation. Substrate affinity was equally effective with all primary alcohols tested as acyl acceptors, with a slight preference for methanol. The best transesterification conversion of 50 mmol glyceryl triacetate into isoamyl acetate (banana fragrance) provided near 100% yield after 24 hours using 10% biocatalyst loading (w/w) in a fluidized bed reactor, allowing recycling of the biocatalyst up to five times. These results show promising potential for an industrial approach aimed at the biosynthesis of short-chain esters, namely for natural flavor and fragrance production in micro-aqueous media.

  18. Short-Chain Flavor Ester Synthesis in Organic Media by an E. coli Whole-Cell Biocatalyst Expressing a Newly Characterized Heterologous Lipase

    PubMed Central

    Brault, Guillaume; Shareck, François; Hurtubise, Yves; Lépine, François; Doucet, Nicolas

    2014-01-01

    Short-chain aliphatic esters are small volatile molecules that produce fruity and pleasant aromas and flavors. Most of these esters are artificially produced or extracted from natural sources at high cost. It is, however, possible to ‘naturally’ produce these molecules using biocatalysts such as lipases and esterases. A gene coding for a newly uncovered lipase was isolated from a previous metagenomic study and cloned into E. coli BL21 (DE3) for overexpression using the pET16b plasmid. Using this recombinant strain as a whole-cell biocatalyst, short chain esters were efficiently synthesized by transesterification and esterification reactions in organic media. The recombinant lipase (LipIAF5-2) showed good affinity toward glyceryl trioctanoate and the highest conversion yields were obtained for the transesterification of glyceryl triacetate with methanol. Using a simple cetyl-trimethylammonium bromide pretreatment increased the synthetic activity by a six-fold factor and the whole-cell biocatalyst showed the highest activity at 40°C with a relatively high water content of 10% (w/w). The whole-cell biocatalyst showed excellent tolerance to alcohol and short-chain fatty acid denaturation. Substrate affinity was equally effective with all primary alcohols tested as acyl acceptors, with a slight preference for methanol. The best transesterification conversion of 50 mmol glyceryl triacetate into isoamyl acetate (banana fragrance) provided near 100% yield after 24 hours using 10% biocatalyst loading (w/w) in a fluidized bed reactor, allowing recycling of the biocatalyst up to five times. These results show promising potential for an industrial approach aimed at the biosynthesis of short-chain esters, namely for natural flavor and fragrance production in micro-aqueous media. PMID:24670408

  19. Transgalactosylating β-galactosidase from probiotic Lactobacillus plantarum MCC2156: production and permeabilization for use as whole cell biocatalyst.

    PubMed

    Gobinath, Duraiswamy; Prapulla, Siddalingaiya Gurudutt

    2015-09-01

    Key nutritional factors were optimized for the maximum production of transgalactosylating β-galactosidase from Lactobacillus plantarum MCC2156. Galactose, yeast extract, sodium acetate and manganese sulphate were the most important nutrients affecting β-galactosidase production. Maximum β-galactosidase production (3015 miller units) was obtained by culturing L. plantarum in the optimized fermentation medium containing (w/v) galactose (4 %), yeast extract (2 %), sodium acetate (3 %) and manganese sulphate (0.075 %) with an optimum medium pH of 7.0, after 14 h of incubation at 35 °C. Further, permeabilization of L. plantarum cells using various chemical/ solvents for maximum β-galactosidase activity was performed for use as whole cell biocatalyst. Mixture of ethanol: n-butanol was found to effectively permeabilize the cells with maximum β-galactosidase activity under the following optimum conditions; 1: 1 mixture of ethanol (10 %, v/v): n-butanol (30 %, v/v) with a contact time of 10 min at 28 ± 2 °C.

  20. A novel and robust recombinant Pichia pastoris yeast whole cell biocatalyst with intracellular overexpression of a Thermomyces lanuginosus lipase: preparation, characterization and application in biodiesel production.

    PubMed

    Yan, Jinyong; Zheng, Xianliang; Li, Shengying

    2014-01-01

    A novel and robust recombinant Pichia pastoris yeast whole cell catalyst (WCC) with functional intracellular expression of Thermomyces lanuginosus lipase (Tll) was constructed and characterized for biodiesel production from waste cooking oils. This permeabilized WCC was able to convert waste cooking oils to biodiesel with 82% yield within 84 h at 6% dosage whole cells. The WCC showed two fold catalytic activity of 0.73 U/mg DCW compared to its commercial counterpart Lipozyme TLIM (immobilized Tll). Short chain alcohol tolerance of this WCC was significantly improved compared to Lipozyme TLIM. This beneficial property enabled it to catalyze biodiesel production efficiently with one step addition of methanol. The reusability of this biocatalyst retained 78% activity after three batch cycles. This easily prepared and cost-effective WCC showed better catalytic performance than Lipozyme TLIM with respect to biodiesel yield and productivity, thus suggesting a promising cost-effective biocatalyst for biodiesel production.

  1. Bioconversion of l-glutamic acid to α-ketoglutaric acid by an immobilized whole-cell biocatalyst expressing l-amino acid deaminase from Proteus mirabilis.

    PubMed

    Hossain, Gazi Sakir; Li, Jianghua; Shin, Hyun-dong; Chen, Rachel R; Du, Guocheng; Liu, Long; Chen, Jian

    2014-01-01

    The goal of this work was to develop an immobilized whole-cell biocatalytic process for the environment-friendly synthesis of α-ketoglutaric acid (α-KG) from l-glutamic acid. We compared the suitability of Escherichia coli and Bacillus subtilis strains overexpressing Proteus mirabilisl-amino acid deaminase (l-AAD) as potential biocatalysts. Although both recombinant strains were biocatalytically active, the performance of B. subtilis was superior to that of E. coli. With l-glutamic acid as the substrate, α-KG production levels by membranes isolated from B. subtilis and E. coli were 55.3±1.73 and 21.7±0.39μg/mg protein/min, respectively. The maximal conversion ratio of l-glutamic acid to α-KG was 31% (w/w) under the following optimal conditions: 15g/L l-glutamic acid, 20g/L whole-cell biocatalyst, 5mM MgCl2, 40°C, pH 8.0, and 24-h incubation. Immobilization of whole cells with alginate increased the recyclability by an average of 23.33% per cycle. This work established an efficient one-step biotransformation process for the production of α-KG using immobilized whole B. subtilis overexpressing P. mirabilisl-AAD. Compared with traditional multistep chemical synthesis, the biocatalytic process described here has the advantage of reducing environmental pollution and thus has great potential for the large-scale production of α-KG.

  2. Producing biodiesel from cotton seed oil using Rhizopus oryzae ATTC #34612 whole cell biocatalysts: Culture media and cultivation period optimization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effect of culture medium composition and cultivation time on biodiesel production by Rhizopus oryzae ATCC #34612 whole cell catalysts, immobilized on novel rigid polyethylene biomass supports, was investigated. Supplementation of the medium with carbon sources led to higher lipase activity and i...

  3. Releasing intracellular product to prepare whole cell biocatalyst for biosynthesis of Monascus pigments in water-edible oil two-phase system.

    PubMed

    Hu, Minglue; Zhang, Xuehong; Wang, Zhilong

    2016-11-01

    Selective releasing intracellular product in Triton X-100 micelle aqueous solution to prepare whole cell biocatalyst is a novel strategy for biosynthesis of Monascus pigments, in which cell suspension culture exhibits some advantages comparing with the corresponding growing cell submerged culture. In the present work, the nonionic surfactant Triton X-100 was successfully replaced by edible plant oils for releasing intracellular Monascus pigments. High concentration of Monascus pigments (with absorbance nearly 710 AU at 470 nm in the oil phase, normalized to the aqueous phase volume approximately 142 AU) was achieved by cell suspension culture in peanut oil-water two-phase system. Furthermore, the utilization of edible oil as extractant also fulfills the demand for application of Monascus pigments as natural food colorant.

  4. Releasing intracellular product to prepare whole cell biocatalyst for biosynthesis of Monascus pigments in water-edible oil two-phase system.

    PubMed

    Hu, Minglue; Zhang, Xuehong; Wang, Zhilong

    2016-11-01

    Selective releasing intracellular product in Triton X-100 micelle aqueous solution to prepare whole cell biocatalyst is a novel strategy for biosynthesis of Monascus pigments, in which cell suspension culture exhibits some advantages comparing with the corresponding growing cell submerged culture. In the present work, the nonionic surfactant Triton X-100 was successfully replaced by edible plant oils for releasing intracellular Monascus pigments. High concentration of Monascus pigments (with absorbance nearly 710 AU at 470 nm in the oil phase, normalized to the aqueous phase volume approximately 142 AU) was achieved by cell suspension culture in peanut oil-water two-phase system. Furthermore, the utilization of edible oil as extractant also fulfills the demand for application of Monascus pigments as natural food colorant. PMID:27470059

  5. Highly Efficient Synthesis of Optically Pure (S)-1-phenyl-1,2-ethanediol by a Self-Sufficient Whole Cell Biocatalyst

    PubMed Central

    Chen, Xi; Mei, Ting; Cui, Yunfeng; Chen, Qijia; Liu, Xiangtao; Feng, Jinhui; Wu, Qiaqing; Zhu, Dunming

    2015-01-01

    Terminal vicinal diols are important chiral building blocks and intermediates in organic synthesis. Reduction of α-hydroxy ketones provides a straightforward approach to access these important compounds. In this study, it has been found that asymmetric reduction of a series of α-hydroxy aromatic ketones and 1-hydroxy-2-pentanone, catalyzed by Candida magnolia carbonyl reductase (CMCR) with glucose dehydrogenase (GDH) from Bacillus subtilis for cofactor regeneration, afforded 1-aryl-1,2-ethanediols and pentane-1,2-diol, respectively, in up to 99 % ee. In order to evaluate the efficiency of the bioreduction, lyophilized recombinant Escherichia coli whole cells coexpressing CMCR and GDH genes were used as the biocatalyst and α-hydroxy acetophenone as the model substrate, and the reaction conditions, such as pH, cosolvent, the amount of biocatalyst and the presences of a cofactor (i.e., NADP+), were optimized. Under the optimized conditions (pH 6, 16 h), the bioreduction proceeded smoothly at 1.0 m substrate concentration without the external addition of cofactor, and the product (S)-1-phenyl-1,2-ethanediol was isolated with 90 % yield and 99 % ee. This offers a practical biocatalytic method for the preparation of these important vicinal diols. PMID:26478844

  6. Development of a whole-cell biocatalyst/biosensor by display of multiple heterologous proteins on the Escherichia coli cell surface for the detoxification and detection of organophosphates.

    PubMed

    Liu, Ruihua; Yang, Chao; Xu, Yingming; Xu, Ping; Jiang, Hong; Qiao, Chuanling

    2013-08-14

    This paper reports the codisplay of organophosphorus hydrolase (OPH) and methyl parathion hydrolase (MPH)-green fluorescent protein (GFP) fusion on the cell surface of Escherichia coli using the truncated ice nucleation protein (INPNC) and Lpp-OmpA as the anchoring motifs. The surface localization of both OPH and MPH-GFP was demonstrated by cell fractionation, Western blot analysis, protease accessibility experiment, and immunofluorescence microscopy. Anchorage of the foreign proteins on the outer membrane neither inhibits cell growth nor affects cell viability. The recombinant strain can be used as a whole-cell biocatalyst and showed a broader substrate range than strains expressing either OPH or MPH. A mixture of six organophosphorus pesticides (OPs) (0.2 mM each) could be degraded completely within 5 h. The broader substrate specificity in combination with the rapid degradation rate makes the recombinant strain a promising candidate for detoxification of OPs. The fluorescence of surface-displayed GFP is very sensitive to environmental pH change. Because hydrolysis of OPs by OPH or MPH generates protons, the recombinant E. coli could be used as a whole-cell biosensor for the rapid detection of OPs by evaluating fluorescence changes as a function of OP concentrations. PMID:23875606

  7. The effect of cultivation media and washing whole-cell biocatalysts on monoamine oxidase catalyzed oxidative desymmetrization of 3-azabicyclo[3,3,0]octane.

    PubMed

    Ramesh, Hemalata; Zajkoska, Petra; Rebroš, Martin; Woodley, John M

    2016-02-01

    It is well known that washing whole-cells containing enzyme activities after fermentation, but prior to biocatalysis can improve their activity in the subsequent reaction. In this paper, we quantify the impact of both the fermentation media and cell washing on the performance of whole-cell biocatalysis. The results are illustrated using a recombinant monoamine oxidase (expressed in Escherichia coli, used in resting state) for the oxidative desymmetrization of 3-azabicyclo[3,3,0]octane. It was shown that the need for washing biocatalyst prior to use in a reaction is dependent upon growth medium. Unlike cells grown in LB medium, washing of the cells was essential for cells grown on TB medium. With TB media, washing the cells improved the final conversion by approximately a factor of two. Additionally, over 50-fold improvement was achieved in initial activity. A potential reason for this improvement in activity was identified to be the increase in transfer of substrates across the cell membrane as a result of cell washing.

  8. Rules for biocatalyst and reaction engineering to implement effective, NAD(P)H-dependent, whole cell bioreductions.

    PubMed

    Kratzer, Regina; Woodley, John M; Nidetzky, Bernd

    2015-12-01

    Access to chiral alcohols of high optical purity is today frequently provided by the enzymatic reduction of precursor ketones. However, bioreductions are complicated by the need for reducing equivalents in the form of NAD(P)H. The high price and molecular weight of NAD(P)H necessitate in situ recycling of catalytic quantities, which is mostly accomplished by enzymatic oxidation of a cheap co-substrate. The coupled oxidoreduction can be either performed by free enzymes in solution or by whole cells. Reductase selection, the decision between cell-free and whole cell reduction system, coenzyme recycling mode and reaction conditions represent design options that strongly affect bioreduction efficiency. In this paper, each option was critically scrutinized and decision rules formulated based on well-described literature examples. The development chain was visualized as a decision-tree that can be used to identify the most promising route towards the production of a specific chiral alcohol. General methods, applications and bottlenecks in the set-up are presented and key experiments required to "test" for decision-making attributes are defined. The reduction of o-chloroacetophenone to (S)-1-(2-chlorophenyl)ethanol was used as one example to demonstrate all the development steps. Detailed analysis of reported large scale bioreductions identified product isolation as a major bottleneck in process design. PMID:26343336

  9. Scaling-up the synthesis of myristate glucose ester catalyzed by a CALB-displaying Pichia pastoris whole-cell biocatalyst.

    PubMed

    Guo, DongHeng; Jin, Zi; Xu, YanShan; Wang, Ping; Lin, Ying; Han, ShuangYan; Zheng, SuiPing

    2015-01-01

    The novel whole-cell biocatalyst Candida antarctica lipase B displaying-Pichia pastoris (Pp-CALB) is characterized by its low preparation cost and could be an alternative to the commercial immobilized Candida antarctica lipase B (CALB). This study addresses the feasibility of using Pp-CALB in large scale glucose fatty acid esters production. 1,2-O-Isopropylidene-α-D-glucofuranose (IpGlc) was used as the acyl acceptor to overcome the low solubility of glucose in an organic solvent and to avoid the addition of toxic co-solvents. IpGlc significantly improved the Pp-CALB catalyzing esterification efficiency when using long chain fatty acids as the acyl donor. Under the preferred operating conditions (50 °C, 40 g/L molecular sieve dosage and 200 rpm mixing intensity), 60.5% of IpGlc converted to 6-O-myristate-1, 2-O-isopropylidene-α-D-glucofuranose (C14-IpGlc) after a 96-h reaction in a 2-L stirred reactor. In a 5-L pilot scale test, Pp-CALB also showed a similar substrate conversion rate of 55.4% and excellent operational stability. After C14-IpGlc was collected, 70% trifluoroacetic acid was adopted to hydrolyze C14-IpGlc to myristate glucose ester (C14-Glc) with a high yield of 95.3%. In conclusion, Pp-CALB is a powerful biocatalyst available for industrial synthesis, and this study describes an applicable and economical process for the large scale production of myristate glucose ester.

  10. Scaling-up the synthesis of myristate glucose ester catalyzed by a CALB-displaying Pichia pastoris whole-cell biocatalyst.

    PubMed

    Guo, DongHeng; Jin, Zi; Xu, YanShan; Wang, Ping; Lin, Ying; Han, ShuangYan; Zheng, SuiPing

    2015-01-01

    The novel whole-cell biocatalyst Candida antarctica lipase B displaying-Pichia pastoris (Pp-CALB) is characterized by its low preparation cost and could be an alternative to the commercial immobilized Candida antarctica lipase B (CALB). This study addresses the feasibility of using Pp-CALB in large scale glucose fatty acid esters production. 1,2-O-Isopropylidene-α-D-glucofuranose (IpGlc) was used as the acyl acceptor to overcome the low solubility of glucose in an organic solvent and to avoid the addition of toxic co-solvents. IpGlc significantly improved the Pp-CALB catalyzing esterification efficiency when using long chain fatty acids as the acyl donor. Under the preferred operating conditions (50 °C, 40 g/L molecular sieve dosage and 200 rpm mixing intensity), 60.5% of IpGlc converted to 6-O-myristate-1, 2-O-isopropylidene-α-D-glucofuranose (C14-IpGlc) after a 96-h reaction in a 2-L stirred reactor. In a 5-L pilot scale test, Pp-CALB also showed a similar substrate conversion rate of 55.4% and excellent operational stability. After C14-IpGlc was collected, 70% trifluoroacetic acid was adopted to hydrolyze C14-IpGlc to myristate glucose ester (C14-Glc) with a high yield of 95.3%. In conclusion, Pp-CALB is a powerful biocatalyst available for industrial synthesis, and this study describes an applicable and economical process for the large scale production of myristate glucose ester. PMID:26047913

  11. Simultaneously improving stability and specificity of cell surface displayed glucose dehydrogenase mutants to construct whole-cell biocatalyst for glucose biosensor application.

    PubMed

    Liang, Bo; Lang, Qiaolin; Tang, Xiangjiang; Liu, Aihua

    2013-11-01

    The improved stability and substrate specificity of cell surface displayed glucose dehydrogenase (GDH) mutants by replacing four amino acids from Bacillus subtilis by using site-directed mutagenesis was systematically investigated. A series of mutated GDHs including E170R/Q252L, V149K/E170R/Q252L, E170R/Q252L/G259A and V149K/E170R/Q252L/G259A, were fused to the ice nucleation protein for displaying on cell surface of Eschericia coli. Q252L/E170R/V149K, Q252L/E170R/G259A and Q252L/E170R/V149K/G259A variants were found stable at a wide pH range and shown excellent thermostability. Especially, the Q252L/E170R/V149K/G259A mutant showed half-life of ~3.8days at 70 °C. Q252L/E170R/V149K/G259A variant exhibited the narrowest substrate specificity for d-glucose. The whole cell displayed GDH mutant could be cultured in a large scale with excellent enzyme activity and productivity. In addition, a sensitive and stable electrochemical glucose biosensor can be prepared using the GDH-mutant bacteria modified electrode. Thus, the whole cell biocatalysts are promising candidates for exploitation in a wide range of industrial applications. PMID:24012845

  12. Comparative analysis for the production of fatty acid alkyl esterase using whole cell biocatalyst and purified enzyme from Rhizopus oryzae on waste cooking oil (sunflower oil).

    PubMed

    Balasubramaniam, Bharathiraja; Sudalaiyadum Perumal, Ayyappasamy; Jayaraman, Jayamuthunagai; Mani, Jayakumar; Ramanujam, Praveenkumar

    2012-08-01

    The petroleum fuel is nearing the line of extinction. Recent research and technology have provided promising outcomes to rely on biodiesel as the alternative and conventional source of fuel. The use of renewable source - vegetable oil constitutes the main stream of research. In this preliminary study, Waste Cooking Oil (WCO) was used as the substrate for biodiesel production. Lipase enzyme producing fungi Rhizopus oryzae 262 and commercially available pure lipase enzyme were used for comparative study in the production of Fatty Acid Alkyl Esters (FAAE). The whole cell (RO 262) and pure lipase enzyme (PE) were immobilized using calcium alginate beads. Calcium alginate was prepared by optimizing with different molar ratios of calcium chloride and different per cent sodium alginate. Entrapment immobilization was done for whole cell biocatalyst (WCB). PE was also immobilized by entrapment for the transesterification reaction. Seven different solvents - methanol, ethanol, n-propanol, n-butanol, iso-propanol, iso-butanol and iso-amyl alcohol were used as the acyl acceptors. The reaction parameters like temperature (30°C), molar ratio (1:3 - oil:solvent), reaction time (24 h), and amount of enzyme (10% mass ratio to oil) were also optimized for methanol alone. The same parameters were adopted for the other acyl acceptors too. Among the different acyl acceptors - methanol, whose reaction parameters were optimized showed maximum conversion of triglycerides to FAAE-94% with PE and 84% with WCB. On the whole, PE showed better catalytic converting ability with all the acyl acceptor compared to WCB. Gas chromatography analysis (GC) was done to determine the fatty acid composition of WCO (sunflower oil) and FAAE production with different acyl acceptors.

  13. Development of an autofluorescent whole-cell biocatalyst by displaying dual functional moieties on Escherichia coli cell surfaces and construction of a coculture with organophosphate-mineralizing activity .

    PubMed

    Yang, Chao; Zhu, Yaran; Yang, Jijian; Liu, Zheng; Qiao, Chuanling; Mulchandani, Ashok; Chen, Wilfred

    2008-12-01

    Surface display of the active proteins on living cells has enormous potential in the degradation of numerous toxic compounds. Here, we report the codisplay of organophosphorus hydrolase (OPH) and enhanced green fluorescent protein (GFP) on the cell surface of Escherichia coli by use of the truncated ice nucleation protein (INPNC) and Lpp-OmpA fusion systems. The surface localization of both INPNC-OPH and Lpp-OmpA-GFP was demonstrated by Western blot analysis, immunofluorescence microscopy, and a protease accessibility experiment. Anchorage of GFP and OPH on the outer membrane neither inhibits cell growth nor affects cell viability, as shown by growth kinetics of cells and stability of resting cultures. The engineered E. coli can be applied in the form of a whole-cell biocatalyst and can be tracked by fluorescence during bioremediation. This strategy of codisplay should open a new dimension for the display of multiple functional moieties on the surface of a bacterial cell. Furthermore, a coculture comprised of the engineered E. coli and a natural p-nitrophenol (PNP) degrader, Ochrobactrum sp. strain LL-1, was assembled for complete mineralization of organophosphates (OPs) with a PNP substitution. The coculture degraded OPs as well as PNP rapidly. Therefore, the coculture with autofluorescent and mineralizing activities can potentially be applied for bioremediation of OP-contaminated sites. PMID:18952884

  14. One-Step Biosynthesis of α-Keto-γ-Methylthiobutyric Acid from L-Methionine by an Escherichia coli Whole-Cell Biocatalyst Expressing an Engineered L-Amino Acid Deaminase from Proteus vulgaris

    PubMed Central

    Shin, Hyun-dong; Du, Guocheng; Wang, Miao; Liu, Long; Chen, Jian

    2014-01-01

    α-Keto-γ-methylthiobutyric acid (KMTB), a keto derivative of l-methionine, has great potential for use as an alternative to l-methionine in the poultry industry and as an anti-cancer drug. This study developed an environment friendly process for KMTB production from l-methionine by an Escherichia coli whole-cell biocatalyst expressing an engineered l-amino acid deaminase (l-AAD) from Proteus vulgaris. We first overexpressed the P. vulgaris l-AAD in E. coli BL21 (DE3) and further optimized the whole-cell transformation process. The maximal molar conversion ratio of l-methionine to KMTB was 71.2% (mol/mol) under the optimal conditions (70 g/L l-methionine, 20 g/L whole-cell biocatalyst, 5 mM CaCl2, 40°C, 50 mM Tris-HCl [pH 8.0]). Then, error-prone polymerase chain reaction was used to construct P. vulgaris l-AAD mutant libraries. Among approximately 104 mutants, two mutants bearing lysine 104 to arginine and alanine 337 to serine substitutions showed 82.2% and 80.8% molar conversion ratios, respectively. Furthermore, the combination of these mutations enhanced the catalytic activity and molar conversion ratio by 1.3-fold and up to 91.4% with a KMTB concentration of 63.6 g/L. Finally, the effect of immobilization on whole-cell transformation was examined, and the immobilized whole-cell biocatalyst with Ca2+ alginate increased reusability by 41.3% compared to that of free cell production. Compared with the traditional multi-step chemical synthesis, our one-step biocatalytic production of KMTB has an advantage in terms of environmental pollution and thus has great potential for industrial KMTB production. PMID:25531756

  15. One-step biosynthesis of α-keto-γ-methylthiobutyric acid from L-methionine by an Escherichia coli whole-cell biocatalyst expressing an engineered L-amino acid deaminase from Proteus vulgaris.

    PubMed

    Hossain, Gazi Sakir; Li, Jianghua; Shin, Hyun-dong; Du, Guocheng; Wang, Miao; Liu, Long; Chen, Jian

    2014-01-01

    α-Keto-γ-methylthiobutyric acid (KMTB), a keto derivative of l-methionine, has great potential for use as an alternative to l-methionine in the poultry industry and as an anti-cancer drug. This study developed an environment friendly process for KMTB production from l-methionine by an Escherichia coli whole-cell biocatalyst expressing an engineered l-amino acid deaminase (l-AAD) from Proteus vulgaris. We first overexpressed the P. vulgaris l-AAD in E. coli BL21 (DE3) and further optimized the whole-cell transformation process. The maximal molar conversion ratio of l-methionine to KMTB was 71.2% (mol/mol) under the optimal conditions (70 g/L l-methionine, 20 g/L whole-cell biocatalyst, 5 mM CaCl2, 40°C, 50 mM Tris-HCl [pH 8.0]). Then, error-prone polymerase chain reaction was used to construct P. vulgaris l-AAD mutant libraries. Among approximately 104 mutants, two mutants bearing lysine 104 to arginine and alanine 337 to serine substitutions showed 82.2% and 80.8% molar conversion ratios, respectively. Furthermore, the combination of these mutations enhanced the catalytic activity and molar conversion ratio by 1.3-fold and up to 91.4% with a KMTB concentration of 63.6 g/L. Finally, the effect of immobilization on whole-cell transformation was examined, and the immobilized whole-cell biocatalyst with Ca2+ alginate increased reusability by 41.3% compared to that of free cell production. Compared with the traditional multi-step chemical synthesis, our one-step biocatalytic production of KMTB has an advantage in terms of environmental pollution and thus has great potential for industrial KMTB production.

  16. Influence of organic solvents on catalytic behaviors and cell morphology of whole-cell biocatalysts for synthesis of 5'-arabinocytosine laurate.

    PubMed

    Yang, Meiyan; Wu, Hui; Lian, Yan; Li, Xiaofeng; Lai, Furao; Zhao, Guanglei

    2014-01-01

    A whole-cell based method was developed for the regioselective synthesis of arabinocytosine laurate. Among the seven kinds of bacteria strains tested in the acylation reaction, Pseudomonas fluorescens gave the highest productivity and a higher 5'-regioselectivity than 99%. Compared with pure organic solvents, the use of organic solvent mixtures greatly promoted the yield of the whole-cell catalyzed reaction, but showed little influence on the 5'-regioselectivity. Of all the tested solvent mixtures, the best reaction result was found in isopropyl ether/pyridine followed by isopentanol/pyridine. However, the whole-cells showed much lower thermostability in isopropyl ether/pyridine than in THF-pyridine. To better understand the toxic effects of the organic solvents on P. fluorescens whole-cells and growing cells were further examined. Significant influences of organic solvents on the biomass of the cells were found, which differed depending on the type of solvents used. SEM analysis visually revealed the changes in the surface morphology of whole-cells and growing cells cultured in media containing various organic solvents, in terms of surface smoothness, bulges and changed cell sizes. Results demonstrated that organic toxicity to cell structure played an important role in whole-cell mediated catalysis.

  17. Influence of Organic Solvents on Catalytic Behaviors and Cell Morphology of Whole-Cell Biocatalysts for Synthesis of 5′-Arabinocytosine Laurate

    PubMed Central

    Yang, Meiyan; Wu, Hui; Lian, Yan; Li, Xiaofeng; Lai, Furao; Zhao, Guanglei

    2014-01-01

    A whole-cell based method was developed for the regioselective synthesis of arabinocytosine laurate. Among the seven kinds of bacteria strains tested in the acylation reaction, Pseudomonas fluorescens gave the highest productivity and a higher 5′-regioselectivity than 99%. Compared with pure organic solvents, the use of organic solvent mixtures greatly promoted the yield of the whole-cell catalyzed reaction, but showed little influence on the 5′-regioselectivity. Of all the tested solvent mixtures, the best reaction result was found in isopropyl ether/pyridine followed by isopentanol/pyridine. However, the whole-cells showed much lower thermostability in isopropyl ether/pyridine than in THF-pyridine. To better understand the toxic effects of the organic solvents on P. fluorescens whole-cells and growing cells were further examined. Significant influences of organic solvents on the biomass of the cells were found, which differed depending on the type of solvents used. SEM analysis visually revealed the changes in the surface morphology of whole-cells and growing cells cultured in media containing various organic solvents, in terms of surface smoothness, bulges and changed cell sizes. Results demonstrated that organic toxicity to cell structure played an important role in whole-cell mediated catalysis. PMID:25136983

  18. EXPERIMENTAL STUDY OF THE NEW BIOCATALYST METHOD FOR BIODIESEL-FUEL BASED ON THE LIPASE PRODUCTION FUNGUS

    NASA Astrophysics Data System (ADS)

    Hata, Toshiro; Shimada, Miki; Toida, Jinichi

    This paper describes how to develop and evaluate a new biocatalyst method for biodiesel fuel based on the lipase production fungus. This method can convert waste vegetable oil into biodiesel fuel without alkaline waste fluid and byproducts (gly cerine). The main outcomes of this research were: (1) The biodiesel fuel can be manufactured from lipase production fungus (Rhizupus oryzae NBRC 9364). (2) The lipase activity can be enhanced by adding glucose and oil. (3) Phased addition of the methanol enhances the conversion rate of the biodiesel fuel (Maximum conversion rate is 85%). (4) The proposed method can improve vehicle exhaust emission and reduce byproducts (glycerine). We concluded that our proposed methods are effective for the production of biodiesel fuel from waste vegetable oil.

  19. Efficient whole-cell biocatalyst for acetoin production with NAD+ regeneration system through homologous co-expression of 2,3-butanediol dehydrogenase and NADH oxidase in engineered Bacillus subtilis.

    PubMed

    Bao, Teng; Zhang, Xian; Rao, Zhiming; Zhao, Xiaojing; Zhang, Rongzhen; Yang, Taowei; Xu, Zhenghong; Yang, Shangtian

    2014-01-01

    Acetoin (3-hydroxy-2-butanone), an extensively-used food spice and bio-based platform chemical, is usually produced by chemical synthesis methods. With increasingly requirement of food security and environmental protection, bio-fermentation of acetoin by microorganisms has a great promising market. However, through metabolic engineering strategies, the mixed acid-butanediol fermentation metabolizes a certain portion of substrate to the by-products of organic acids such as lactic acid and acetic acid, which causes energy cost and increases the difficulty of product purification in downstream processes. In this work, due to the high efficiency of enzymatic reaction and excellent selectivity, a strategy for efficiently converting 2,3-butandiol to acetoin using whole-cell biocatalyst by engineered Bacillus subtilis is proposed. In this process, NAD+ plays a significant role on 2,3-butanediol and acetoin distribution, so the NADH oxidase and 2,3-butanediol dehydrogenase both from B. subtilis are co-expressed in B. subtilis 168 to construct an NAD+ regeneration system, which forces dramatic decrease of the intracellular NADH concentration (1.6 fold) and NADH/NAD+ ratio (2.2 fold). By optimization of the enzymatic reaction and applying repeated batch conversion, the whole-cell biocatalyst efficiently produced 91.8 g/L acetoin with a productivity of 2.30 g/(L·h), which was the highest record ever reported by biocatalysis. This work indicated that manipulation of the intracellular cofactor levels was more effective than the strategy of enhancing enzyme activity, and the bioprocess for NAD+ regeneration may also be a useful way for improving the productivity of NAD+-dependent chemistry-based products. PMID:25036158

  20. Efficient Whole-Cell Biocatalyst for Acetoin Production with NAD+ Regeneration System through Homologous Co-Expression of 2,3-Butanediol Dehydrogenase and NADH Oxidase in Engineered Bacillus subtilis

    PubMed Central

    Rao, Zhiming; Zhao, Xiaojing; Zhang, Rongzhen; Yang, Taowei; Xu, Zhenghong; Yang, Shangtian

    2014-01-01

    Acetoin (3-hydroxy-2-butanone), an extensively-used food spice and bio-based platform chemical, is usually produced by chemical synthesis methods. With increasingly requirement of food security and environmental protection, bio-fermentation of acetoin by microorganisms has a great promising market. However, through metabolic engineering strategies, the mixed acid-butanediol fermentation metabolizes a certain portion of substrate to the by-products of organic acids such as lactic acid and acetic acid, which causes energy cost and increases the difficulty of product purification in downstream processes. In this work, due to the high efficiency of enzymatic reaction and excellent selectivity, a strategy for efficiently converting 2,3-butandiol to acetoin using whole-cell biocatalyst by engineered Bacillus subtilis is proposed. In this process, NAD+ plays a significant role on 2,3-butanediol and acetoin distribution, so the NADH oxidase and 2,3-butanediol dehydrogenase both from B. subtilis are co-expressed in B. subtilis 168 to construct an NAD+ regeneration system, which forces dramatic decrease of the intracellular NADH concentration (1.6 fold) and NADH/NAD+ ratio (2.2 fold). By optimization of the enzymatic reaction and applying repeated batch conversion, the whole-cell biocatalyst efficiently produced 91.8 g/L acetoin with a productivity of 2.30 g/(L·h), which was the highest record ever reported by biocatalysis. This work indicated that manipulation of the intracellular cofactor levels was more effective than the strategy of enhancing enzyme activity, and the bioprocess for NAD+ regeneration may also be a useful way for improving the productivity of NAD+-dependent chemistry-based products. PMID:25036158

  1. One-step biosynthesis of α-ketoisocaproate from l-leucine by an Escherichia coli whole-cell biocatalyst expressing an l-amino acid deaminase from Proteus vulgaris

    PubMed Central

    Song, Yang; Li, Jianghua; Shin, Hyun-dong; Du, Guocheng; Liu, Long; Chen, Jian

    2015-01-01

    This work aimed to develop a whole-cell biotransformation process for the production of α-ketoisocaproate from L-leucine. A recombinant Escherichia coli strain was constructed by expressing an L-amino acid deaminase from Proteus vulgaris. To enhance α-ketoisocaproate production, the reaction conditions were optimized as follows: whole-cell biocatalyst 0.8 g/L, leucine concentration 13.1 g/L, temperature 35 °C, pH 7.5, and reaction time 20 h. Under the above conditions, the α-ketoisocaproate titer reached 12.7 g/L with a leucine conversion rate of 97.8%. In addition, different leucine feeding strategies were examined to increase the α-ketoisocaproate titer. When 13.1 g/L leucine was added at 2-h intervals (from 0 to 22 h, 12 addition times), the α-ketoisocaproate titer reached 69.1 g/L, while the leucine conversion rate decreased to 50.3%. We have developed an effective process for the biotechnological production of α-ketoisocaproate that is more environmentally friendly than the traditional petrochemical synthesis approach. PMID:26217895

  2. Displaying Lipase B from Candida antarctica in Pichia pastoris Using the Yeast Surface Display Approach: Prospection of a New Anchor and Characterization of the Whole Cell Biocatalyst.

    PubMed

    Moura, Marcelo Victor Holanda; da Silva, Giulia Pontes; Machado, Antônio Carlos de Oliveira; Torres, Fernando Araripe Gonçalves; Freire, Denise Maria Guimarães; Almeida, Rodrigo Volcan

    2015-01-01

    Yeast Surface Display (YSD) is a strategy to anchor proteins on the yeast cell wall which has been employed to increase enzyme stability thus decreasing production costs. Lipase B from Candida antarctica (LipB) is one of the most studied enzymes in the context of industrial biotechnology. This study aimed to assess the biochemical features of this important biocatalyst when immobilized on the cell surface of the methylotrophic yeast Pichia pastoris using the YSD approach. For that purpose, two anchors were tested. The first (Flo9) was identified after a prospection of the P. pastoris genome being related to the family of flocculins similar to Flo1 but significantly smaller. The second is the Protein with Internal Repeats (Pir1) from P. pastoris. An immunolocalization assay showed that both anchor proteins were able to display the reporter protein EGFP in the yeast outer cell wall. LipB was expressed in P. pastoris fused either to Flo9 (FLOLIPB) or Pir1 (PIRLIPB). Both constructions showed hydrolytic activity towards tributyrin (>100 U/mgdcw and >80 U/mgdcw, respectively), optimal hydrolytic activity around 45°C and pH 7.0, higher thermostability at 45°C and stability in organic solvents when compared to a free lipase. PMID:26510006

  3. Displaying Lipase B from Candida antarctica in Pichia pastoris Using the Yeast Surface Display Approach: Prospection of a New Anchor and Characterization of the Whole Cell Biocatalyst

    PubMed Central

    Moura, Marcelo Victor Holanda; da Silva, Giulia Pontes; Machado, Antônio Carlos de Oliveira; Torres, Fernando Araripe Gonçalves; Freire, Denise Maria Guimarães; Almeida, Rodrigo Volcan

    2015-01-01

    Yeast Surface Display (YSD) is a strategy to anchor proteins on the yeast cell wall which has been employed to increase enzyme stability thus decreasing production costs. Lipase B from Candida antarctica (LipB) is one of the most studied enzymes in the context of industrial biotechnology. This study aimed to assess the biochemical features of this important biocatalyst when immobilized on the cell surface of the methylotrophic yeast Pichia pastoris using the YSD approach. For that purpose, two anchors were tested. The first (Flo9) was identified after a prospection of the P. pastoris genome being related to the family of flocculins similar to Flo1 but significantly smaller. The second is the Protein with Internal Repeats (Pir1) from P. pastoris. An immunolocalization assay showed that both anchor proteins were able to display the reporter protein EGFP in the yeast outer cell wall. LipB was expressed in P. pastoris fused either to Flo9 (FLOLIPB) or Pir1 (PIRLIPB). Both constructions showed hydrolytic activity towards tributyrin (>100 U/mgdcw and >80 U/mgdcw, respectively), optimal hydrolytic activity around 45°C and pH 7.0, higher thermostability at 45°C and stability in organic solvents when compared to a free lipase. PMID:26510006

  4. Cell surface display of Yarrowia lipolytica lipase Lip2p using the cell wall protein YlPir1p, its characterization, and application as a whole-cell biocatalyst.

    PubMed

    Yuzbasheva, Evgeniya Y; Yuzbashev, Tigran V; Perkovskaya, Natalia I; Mostova, Elizaveta B; Vybornaya, Tatiana V; Sukhozhenko, Aleksei V; Toropygin, Ilya Y; Sineoky, Sergey P

    2015-04-01

    The Yarrowia lipolytica lipase Lip2p was displayed on the yeast cell surface via N-terminal fusion variant using cell wall protein YlPir1p. The hydrolytic activity of the lipase displayed on Y. lipolytica cells reached 11,900 U/g of dry weight. However, leakage of enzyme from the cell wall was observed. The calculated number of recombinant enzyme displayed on the cell surface corresponds to approximately 6 × 10(5) molecules per cell, which is close to the theoretical maximum (2 × 10(6) molecules/cell). Furthermore, the leaking enzyme was presented as three N-glycosylated proteins, one of which corresponds to the whole hybrid protein. Thus, we attribute the enzyme leakage to the limited space available on the cell surface. Nevertheless, the surface-displayed lipase exhibited greater stability to short-term and long-term temperature treatment than the native enzyme. Cell-bound lipase retained 74 % of its original activity at 60 °C for 5 min of incubation, and 83 % of original activity after incubation at 50 °C during 5 h. Cell-bound lipase had also higher stability in organic solvents and detergents. The developed whole-cell biocatalyst was used for recycling biodiesel synthesis. Two repeated cycles of methanolysis yielded 84.1 and 71.0 % methyl esters after 33- and 45-h reactions, respectively. PMID:25773979

  5. Whole-cell biocomputing

    NASA Technical Reports Server (NTRS)

    Simpson, M. L.; Sayler, G. S.; Fleming, J. T.; Applegate, B.

    2001-01-01

    The ability to manipulate systems on the molecular scale naturally leads to speculation about the rational design of molecular-scale machines. Cells might be the ultimate molecular-scale machines and our ability to engineer them is relatively advanced when compared with our ability to control the synthesis and direct the assembly of man-made materials. Indeed, engineered whole cells deployed in biosensors can be considered one of the practical successes of molecular-scale devices. However, these devices explore only a small portion of cellular functionality. Individual cells or self-organized groups of cells perform extremely complex functions that include sensing, communication, navigation, cooperation and even fabrication of synthetic nanoscopic materials. In natural systems, these capabilities are controlled by complex genetic regulatory circuits, which are only partially understood and not readily accessible for use in engineered systems. Here, we focus on efforts to mimic the functionality of man-made information-processing systems within whole cells.

  6. Whole cell entrapment techniques.

    PubMed

    Trelles, Jorge A; Rivero, Cintia W

    2013-01-01

    Microbial whole cells are efficient, ecological, and low-cost catalysts that have been successfully applied in the pharmaceutical, environmental, and alimentary industries, among others. Microorganism immobilization is a good way to carry out the bioprocess under preparative conditions. The main advantages of this methodology lie in their high operational stability, easy upstream separation and bioprocess scale-up feasibility. Cell entrapment is the most widely used technique for whole cell immobilization. This technique-in which the cells are included within a rigid network-is porous enough to allow the diffusion of substrates and products, protects the selected microorganism from the reaction medium, and has high immobilization efficiency (100 % in most cases). PMID:23934817

  7. Biocatalysts: Beautiful creatures

    SciTech Connect

    Saibi, Walid; Abdeljalil, Salma; Masmoudi, Khaled; Gargouri, Ali

    2012-09-28

    Highlights: Black-Right-Pointing-Pointer Enzymes are vital tools. Black-Right-Pointing-Pointer Bifunctional enzymes. Black-Right-Pointing-Pointer Peculiar biocatalysts. -- Abstract: The chemical industry has come under increasing pressure to make chemical production more eco-friendly and independent to fossil resources. The development of industrial processes based on micro-organisms can especially help to eliminate the use or the generation of hazardous substances and can support the transition from dependence on fossil resources towards real sustainable and eco-safety industrial processes. The biocatalysts are the best solution given by nature that can be used to improve some biotechnological applications. In this research review, we report some peculiar properties of biocatalysts, implicated in a range of metabolic pathways and biotechnological tools.

  8. Construction and characterization of a thermostable whole-cell chitinolytic enzyme using yeast surface display.

    PubMed

    Li, Xiaobo; Jin, Xiaobao; Lu, Xuemei; Chu, Fujiang; Shen, Juan; Ma, Yan; Liu, Manyu; Zhu, Jiayong

    2014-10-01

    To develop a novel yeast whole-cell biocatalyst by yeast surface display technology that can hydrolyze chitin, the chitinaseC gene from Serratia marcescens AS1.1652 strain was cloned and subcloned into the yeast surface display plasmid pYD1, and the recombinant plasmid pYD1/SmchiC was electroporated into Saccharomyces cerevisiae EBY100 cell. Aga2p-SmChiC fusion protein was expressed and anchored on the yeast cell surface by induction with galactose, which was verified by indirect immunofluorescence and Western blotting. The chitinolytic activity of the yeast whole-cell biocatalyst or partially purified enzyme was detected by agar plate clear zone test, SDS-PAGE zymography and dinitrosalicylic acid method. The results showed that the chitinaseC gene from S. marcescens AS1.1652 strain was successfully cloned and expressed on the yeast cell surface, Aga2p-SmChiC fusion protein with molecular weight (67 kDa) was determined. Tests on the effect of temperature and pH on enzyme activity and stability revealed that the yeast whole-cell biocatalyst and partially purified enzyme possessed both thermal stability and activity, and even maintained some activity under acidic and weakly alkaline conditions. The optimum reaction temperature and pH value were set at 52 °C and 5.0, respectively. Yeast surface display technology succeeded in preparing a yeast whole-cell biocatalyst with chitinolytic activity, and the utilization of chitin could benefit from this process of enzyme preparation.

  9. Expression of a Dianthus flavonoid glucosyltransferase in Saccharomyces cerevisiae for whole-cell biocatalysis.

    PubMed

    Werner, Sean R; Morgan, John A

    2009-07-15

    Glycosyltransferases are promising biocatalysts for the synthesis of small molecule glycosides. In this study, Saccharomyces cerevisiae expressing a flavonoid glucosyltransferase (GT) from Dianthus caryophyllus (carnation) was investigated as a whole-cell biocatalyst. Two yeast expression systems were compared using the flavonoid naringenin as a model substrate. Under in vitro conditions, naringenin-7-O-glucoside was formed and a higher specific glucosyl transfer activity was found using a galactose inducible expression system compared to a constitutive expression system. However, S. cerevisiae expressing the GT constitutively was significantly more productive than the galactose inducible system under in vivo conditions. Interestingly, the glycosides were recovered directly from the culture broth and did not accumulate intracellularly. A previously uncharacterized naringenin glycoside formed using the D. caryophyllus GT was identified as naringenin-4'-O-glucoside. It was found that S. cerevisiae cells hydrolyze naringenin-7-O-glucoside during whole-cell biocatalysis, resulting in a low final glycoside titer. When phloretin was added as a substrate to the yeast strain expressing the GT constitutively, the natural product phlorizin was formed. This study demonstrates S. cerevisiae is a promising whole-cell biocatalyst host for the production of valuable glycosides.

  10. Exploiting cell metabolism for biocatalytic whole-cell transamination by recombinant Saccharomyces cerevisiae.

    PubMed

    Weber, Nora; Gorwa-Grauslund, Marie; Carlquist, Magnus

    2014-05-01

    The potential of Saccharomyces cerevisiae for biocatalytic whole-cell transamination was investigated using the kinetic resolution of racemic 1-phenylethylamine (1-PEA) to (R)-1-PEA as a model reaction. As native yeast do not possess any ω-transaminase activity for the reaction, a recombinant yeast biocatalyst was constructed by overexpressing the gene coding for vanillin aminotransferase from Capsicum chinense. The yeast-based biocatalyst could use glucose as the sole co-substrate for the supply of amine acceptor via cell metabolism. In addition, the biocatalyst was functional without addition of the co-factor pyridoxal-5'-phosphate (PLP), which can be explained by a high inherent cellular capacity to sustain PLP-dependent reactions in living cells. In contrast, external PLP supplementation was required when cell viability was low, as it was the case when using pyruvate as a co-substrate. Overall, the results indicate a potential for engineered S. cerevisiae as a biocatalyst for whole-cell transamination and with glucose as the only co-substrate for the supply of amine acceptor and PLP.

  11. Development of whole cell biosensors for the detection of herbicides in drinking water

    SciTech Connect

    Hulme, A.J.

    1990-01-01

    The development of novel whole cell biosensors for the on-line detection of herbicides in drinking water is described. Novel whole cell biosensors were developed using redox mediators to monitor the metabolic activity of whole cells. Photosynthetic microorganisms were chosen as the biocatalyst since many of the commercially available herbicides were known to inhibit the photosynthetic electron transport chain (PETC). The biocatalyst selected for the preliminary investigations was the cyanobacterium Synechococcus. To monitor similar PETC activity in the eukaryotic green alga Chlorella the non-ionic quinones were required. The organism Synechococcus and the mediator potassium ferricyanide were the most appropriate mediator/whole cell combination for the continued development of a whole cell biosensor (WCB). Investigations were undertaken to determine the mechanism by which potassium ferricyanide was able to monitor the photosynthetic activity of Synechococcus. Studies revealed that no PETC components were located on the cytoplasmic membrane, all such activity appeared solely on the intracytoplasmic membrane, potassium ferricyanide did not access the PETC directly, but rather interacted with membrane bound NADPH dehydrogenases, located in the CM. Therefore, any agents known to disturb photosynthetic electron transport should be readily detected as a reduction in current. The sensors were capable of detecting herbicides from the nitriles, ureas, anilides and triazine families at concentrations of 1--3 ppM. All herbicides were readily detected at a concentration of 25 ppB with the nitriles (ioxynil and bromoxynil), the anilide (propanil) and the urea (chlortoluron) readily detected at levels as low as 10 ppB. The sensors were also capable of detecting pentachlorophenol at a concentration of 100 ppB. A procedure was developed which enabled the production of a biocatalyst with a shelf-life of 1--2 months.

  12. Candida parapsilosis: A versatile biocatalyst for organic oxidation-reduction reactions.

    PubMed

    Chadha, Anju; Venkataraman, Sowmyalakshmi; Preetha, Radhakrishnan; Padhi, Santosh Kumar

    2016-10-01

    This review highlights the importance of the biocatalyst, Candida parapsilosis for oxidation and reduction reactions of organic compounds and establishes its versatility to generate a variety of chiral synthons. Appropriately designed reactions using C. parapsilosis effect efficient catalysis of organic transformations such as deracemization, enantioselective reduction of prochiral ketones, imines, and kinetic resolution of racemic alcohols via selective oxidation. This review includes the details of these biotransformations, catalyzed by whole cells (wild type and recombinant strains), purified enzymes (oxidoreductases) and immobilized whole cells of C. parapsilosis. The review presents a bioorganic perspective as it discusses the chemo, regio and stereoselectivity of the biocatalyst along with the structure of the substrates and optical purity of the products. Fermentation scale biocatalysis using whole cells of C. parapsilosis for several biotransformations to synthesize important chiral synthons/industrial chemicals is included. A comparison of C. parapsilosis with other whole cell biocatalysts for biocatalytic deracemization and asymmetric reduction of carbonyl and imine groups in the synthesis of a variety of enantiopure products is presented which will provide a basis for the choice of a biocatalyst for a desired organic transformation. Thus, a wholesome perspective on the present status of C. parapsilosis mediated organic transformations and design of new reactions which can be considered for large scale operations is provided. Taken together, C. parapsilosis can now be considered a 'reagent' for the organic transformations discussed here.

  13. Whole-cell fungal transformation of precursors into dyes

    PubMed Central

    2010-01-01

    Background Chemical methods of producing dyes involve extreme temperatures and unsafe toxic compounds. Application of oxidizing enzymes obtained from fungal species, for example laccase, is an alternative to chemical synthesis of dyes. Laccase can be replaced by fungal biomass acting as a whole-cell biocatalyst with properties comparable to the isolated form of the enzyme. The application of the whole-cell system simplifies the transformation process and reduces the time required for its completion. In the present work, four fungal strains with a well-known ability to produce laccase were tested for oxidation of 17 phenolic and non-phenolic precursors into stable and non-toxic dyes. Results An agar-plate screening test of the organic precursors was carried out using four fungal strains: Trametes versicolor, Fomes fomentarius, Abortiporus biennis, and Cerrena unicolor. Out of 17 precursors, nine were transformed into coloured substances in the presence of actively growing fungal mycelium. The immobilized fungal biomass catalyzed the transformation of 1 mM benzene and naphthalene derivatives in liquid cultures yielding stable and non-toxic products with good dyeing properties. The type of fungal strain had a large influence on the absorbance of the coloured products obtained after 48-hour transformation of the selected precursors, and the most effective was Fomes fomentarius (FF25). Whole-cell transformation of AHBS (3-amino-4-hydroxybenzenesulfonic acid) into a phenoxazinone dye was carried out in four different systems: in aqueous media comprising low amounts of carbon and nitrogen source, in buffer, and in distilled water. Conclusions This study demonstrated the ability of four fungal strains belonging to the ecological type of white rot fungi to transform precursors into dyes. This paper highlights the potential of fungal biomass for replacing isolated enzymes as a cheaper industrial-grade biocatalyst for the synthesis of dyes and other commercially important

  14. Biocatalysts with enhanced inhibitor tolerance

    DOEpatents

    Yang, Shihui; Linger, Jeffrey; Franden, Mary Ann; Pienkos, Philip T.; Zhang, Min

    2015-12-08

    Disclosed herein are biocatalysts for the production of biofuels, including microorganisms that contain genetic modifications conferring tolerance to growth and fermentation inhibitors found in many cellulosic feedstocks. Methods of converting cellulose-containing materials to fuels and chemicals, as well as methods of fermenting sugars to fuels and chemicals, using these biocatalysts are also disclosed.

  15. Monascus kaoliang CBS 302.78 immobilized in polyurethane foam using iso-propanol as co-substrate: Optimized immobilization conditions of a fungus as biocatalyst for the reduction of ketones.

    PubMed

    Quezada, M A; Carballeira, J D; Sinisterra, J V

    2009-03-01

    Monascus kaoliang was selected after a microbial screening as a highly active and selective whole cell catalyst for the reduction of ketones. In the present paper we describe the optimum growing conditions and an interesting immobilization procedure by adsorption in polyurethane foams (PUFs). This methodology is easy to perform and the immobilized catalyst is active, stable and reusable. The use of different co-substrates for cofactor regeneration was also tested and iso-propanol (i-PrOH) was found as the best co-substrate, as it leads to a catalyst reusable for 17 cycles, displaying better NADH regeneration properties than others e.g., glucose (10 cycles) or saccharose (6 cycles). The reduction of different prochiral ketones showed that the ketone reductase activity of this mould follows the Prelog's rule and kinetic experiments demonstrated that the process follows a pseudo-first kinetic order. PMID:19046879

  16. Arming Technology in Yeast—Novel Strategy for Whole-cell Biocatalyst and Protein Engineering

    PubMed Central

    Kuroda, Kouichi; Ueda, Mitsuyoshi

    2013-01-01

    Cell surface display of proteins/peptides, in contrast to the conventional intracellular expression, has many attractive features. This arming technology is especially effective when yeasts are used as a host, because eukaryotic modifications that are often required for functional use can be added to the surface-displayed proteins/peptides. A part of various cell wall or plasma membrane proteins can be genetically fused to the proteins/peptides of interest to be displayed. This technology, leading to the generation of so-called “arming technology”, can be employed for basic and applied research purposes. In this article, we describe various strategies for the construction of arming yeasts, and outline the diverse applications of this technology to industrial processes such as biofuel and chemical productions, pollutant removal, and health-related processes, including oral vaccines. In addition, arming technology is suitable for protein engineering and directed evolution through high-throughput screening that is made possible by the feature that proteins/peptides displayed on cell surface can be directly analyzed using intact cells without concentration and purification. Actually, novel proteins/peptides with improved or developed functions have been created, and development of diagnostic/therapeutic antibodies are likely to benefit from this powerful approach. PMID:24970185

  17. The principles of whole-cell modeling.

    PubMed

    Karr, Jonathan R; Takahashi, Koichi; Funahashi, Akira

    2015-10-01

    Whole-cell models which comprehensively predict how phenotypes emerge from genotype promise to enable rational bioengineering and precision medicine. Here, we outline the key principles of whole-cell modeling which have emerged from our work developing bacterial whole-cell models: single-cellularity; functional, genetic, molecular, and temporal completeness; biophysical realism including temporal dynamics and stochastic variation; species-specificity; and model integration and reproducibility. We also outline the whole-cell model construction process, highlighting existing resources. Numerous challenges remain to achieving fully complete models including developing new experimental tools to more completely characterize cells and developing a strong theoretical understanding of hybrid mathematics. Solving these challenges requires collaboration among computational and experimental biologists, biophysicists, biochemists, applied mathematicians, computer scientists, and software engineers.

  18. The principles of whole-cell modeling.

    PubMed

    Karr, Jonathan R; Takahashi, Koichi; Funahashi, Akira

    2015-10-01

    Whole-cell models which comprehensively predict how phenotypes emerge from genotype promise to enable rational bioengineering and precision medicine. Here, we outline the key principles of whole-cell modeling which have emerged from our work developing bacterial whole-cell models: single-cellularity; functional, genetic, molecular, and temporal completeness; biophysical realism including temporal dynamics and stochastic variation; species-specificity; and model integration and reproducibility. We also outline the whole-cell model construction process, highlighting existing resources. Numerous challenges remain to achieving fully complete models including developing new experimental tools to more completely characterize cells and developing a strong theoretical understanding of hybrid mathematics. Solving these challenges requires collaboration among computational and experimental biologists, biophysicists, biochemists, applied mathematicians, computer scientists, and software engineers. PMID:26115539

  19. Production of flavor esters catalyzed by CALB-displaying Pichia pastoris whole-cells in a batch reactor.

    PubMed

    Jin, Zi; Ntwali, Janvier; Han, Shuang-Yan; Zheng, Sui-Ping; Lin, Ying

    2012-05-31

    Candida antarctica lipase B (CALB) has been employed as an efficient catalyst in the preparation of many flavor esters. A CALB-displaying yeast whole-cell biocatalyst could be an attractive alternative to commercial immobilized CALB because of its low-cost preparation and high enzymatic activity. We investigated the potential application of CALB-displaying Pichia pastoris cells for the production of flavor esters. The optimal conditions for flavor esters synthesis by this biocatalyst were determined in 50-ml shake flasks. Under optimized conditions, the synthesis of 12 kinds flavor esters were scaled up in a 5-l batch stirred reactor. Among these, the mole conversions of 10 exceeded 95% after reactions for 4h. In addition, this biocatalyst showed good tolerance for high substrates concentration and excellent operational stability. Repeated use of the cells in 10 batches resulted in an activity loss of less than 10%. Thus, CALB-displaying P. pastoris whole cells are robust biocatalysts with potential commercial application in the large-scale production of flavor esters in non-aqueous media. PMID:22410080

  20. Biocatalyst development by directed evolution.

    PubMed

    Wang, Meng; Si, Tong; Zhao, Huimin

    2012-07-01

    Biocatalysis has emerged as a great addition to traditional chemical processes for production of bulk chemicals and pharmaceuticals. To overcome the limitations of naturally occurring enzymes, directed evolution has become the most important tool for improving critical traits of biocatalysts such as thermostability, activity, selectivity, and tolerance towards organic solvents for industrial applications. Recent advances in mutant library creation and high-throughput screening have greatly facilitated the engineering of novel and improved biocatalysts. This review provides an update of the recent developments in the use of directed evolution to engineer biocatalysts for practical applications. PMID:22310212

  1. Biocatalyst Development by Directed Evolution

    PubMed Central

    Wang, Meng; Si, Tong; Zhao, Huimin

    2012-01-01

    Biocatalysis has emerged as a great addition to traditional chemical processes for production of bulk chemicals and pharmaceuticals. To overcome the limitations of naturally occurring enzymes, directed evolution has become the most important tool for improving critical traits of biocatalysts such as thermostability, activity, selectivity, and tolerance towards organic solvents for industrial applications. Recent advances in mutant library creation and high-throughput screening have greatly facilitated the engineering of novel and improved biocatalysts. This review provides an update of the recent developments in the use of directed evolution to engineer biocatalysts for practical applications. PMID:22310212

  2. Engineering a pyridoxal 5’-phosphate supply for cadaverine production by using Escherichia coli whole-cell biocatalysis

    PubMed Central

    Ma, Weichao; Cao, Weijia; Zhang, Bowen; Chen, Kequan; Liu, Quanzhen; Li, Yan; Ouyang, Pingkai

    2015-01-01

    Although the routes of de novo pyridoxal 5′-phosphate (PLP) biosynthesis have been well described, studies of the engineering of an intracellular PLP supply are limited, and the effects of cellular PLP levels on PLP-dependent enzyme-based whole-cell biocatalyst activity have not been described. To investigate the effects of PLP cofactor availability on whole-cell biocatalysis, the ribose 5-phosphate (R5P)-dependent pathway genes pdxS and pdxT of Bacillus subtilis were introduced into the lysine decarboxylase (CadA)-overexpressing Escherichia coli strain BL-CadA. This strain was then used as a whole-cell biocatalyst for cadaverine production from L-lysine. Co-expression strategies were evaluated, and the culture medium was optimised to improve the biocatalyst performance. As a result, the intracellular PLP concentration reached 1144 nmol/gDCW, and a specific cadaverine productivity of 25 g/gDCW/h was achieved; these values were 2.4-fold and 2.9-fold higher than those of unmodified BL-CadA, respectively. Additionally, the resulting strain AST3 showed a cadaverine titre (p = 0.143, α = 0.05) similar to that of the BL-CadA strain with the addition of 0.1 mM PLP. These approaches for improving intracellular PLP levels to enhance whole-cell lysine bioconversion activity show great promise for the engineering of a PLP cofactor to optimise whole-cell biocatalysis. PMID:26490441

  3. Engineering NAD+ availability for Escherichia coli whole-cell biocatalysis: a case study for dihydroxyacetone production

    PubMed Central

    2013-01-01

    Background Whole-cell redox biocatalysis has been intensively explored for the production of valuable compounds because excellent selectivity is routinely achieved. Although the cellular cofactor level, redox state and the corresponding enzymatic activity are expected to have major effects on the performance of the biocatalysts, our ability remains limited to predict the outcome upon variation of those factors as well as the relationship among them. Results In order to investigate the effects of cofactor availability on whole-cell redox biocatalysis, we devised recombinant Escherichia coli strains for the production of dihydroxyacetone (DHA) catalyzed by the NAD+-dependent glycerol dehydrogenase (GldA). In this model system, a water-forming NAD+ oxidase (NOX) and a NAD+ transporter (NTT4) were also co-expressed for cofactor regeneration and extracellular NAD+ uptake, respectively. We found that cellular cofactor level, NAD+/NADH ratio and NOX activity were not only strain-dependent, but also growth condition-dependent, leading to significant differences in specific DHA titer among different whole-cell biocatalysts. The host E. coli DH5α had the highest DHA specific titer of 0.81 g/gDCW with the highest NAD+/NADH ratio of 6.7 and NOX activity of 3900 U. The biocatalyst had a higher activity when induced with IPTG at 37°C for 8 h compared with those at 30°C for 8 h and 18 h. When cells were transformed with the ntt4 gene, feeding NAD+ during the cell culture stage increased cellular NAD(H) level by 1.44 fold and DHA specific titer by 1.58 fold to 2.13 g/gDCW. Supplementing NAD+ during the biotransformation stage was also beneficial to cellular NAD(H) level and DHA production, and the highest DHA productivity reached 0.76 g/gDCW/h. Cellular NAD(H) level, NAD+/NADH ratio, and NOX and GldA activity dropped over time during the biotransformation process. Conclusions High NAD+/NADH ratio driving by NOX was very important for DHA production. Once cofactor was

  4. Insoluble protein applications: the use of bacterial inclusion bodies as biocatalysts.

    PubMed

    Hrabárová, Eva; Achbergerová, Lucia; Nahálka, Jozef

    2015-01-01

    Biocatalysis and biotransformations have a broad application in industrial synthetic chemistry. In addition to the whole cell catalysis, purified recombinant enzymes are successfully used for biocatalysis of specific chemical reactions. In this contribution, we report characterization, immobilization, and application of several model target enzymes (D-amino acid oxidase, sialic acid aldolase, maltodextrin phosphorylase, polyphosphate kinase) physiologically aggregated within inclusion bodies (IBs) retaining their biological activity as immobilized biocatalysts.

  5. A Reliable Whole Cell Clamp Technique

    ERIC Educational Resources Information Center

    Li, Chenhong

    2008-01-01

    This article describes a simple whole cell formation technique that the author invented in teaching and experiments. The implementation of the invented technique is a syringe with a hole and slot. With the newly invented technique, novices will shorten their learning curve and veterans will increase their success rate. The invented technique…

  6. Whole-cell based solvent-free system for one-pot production of biodiesel from waste grease.

    PubMed

    Li, Aitao; Ngo, Thao P N; Yan, Jinyong; Tian, Kaiyuan; Li, Zhi

    2012-06-01

    A whole-cell based solvent-free system was developed for efficient conversion of waste grease to biodiesel via one-pot esterification and transesterification. By isolation and screening of lipase-producing strains from soil, Serratia marcescens YXJ-1002 was discovered for the biotransformation of grease to biodiesel. The lipase (SML) from this strain was cloned and expressed in Escherichia coli as an intracellular enzyme, showing 6 times higher whole-cell based hydrolysis activity than that of wild type strain. The recombinant cells were used for biodiesel production from waste grease in one-pot reactions containing no solvent with the addition of methanol in several small portions, and 97% yield of biodiesel (FAME) was achieved under optimized conditions. In addition, the whole-cell biocatalysts showed excellent reusability, retaining 74% productivity after 4 cycles. The developed system, biocatalyst, and process enable the efficient, low-cost, and green production of biodiesel from waste grease, providing with a potential industrial application. PMID:22483351

  7. Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability.

    PubMed

    Gao, Hui; Khera, Eshita; Lee, Jung-Kul; Wen, Fei

    2016-04-22

    We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst immobilization for improved production yield and sustained synthesis. Described herewith is the experimental procedure for the development of such a system consisting of two E. coli strains that express functionally complementary enzymes. Together, these two enzymes can function co-operatively to mediate the regeneration of expensive cofactors for improving the product yield of the bioreaction. In addition, the method of synthesizing an immobilized form of the coupled biocatalytic system by encapsulation of whole cells in calcium alginate beads is reported. As an example, we present the improved biosynthesis of L-xylulose from L-arabinitol by coupling E. coli cells expressing the enzymes L-arabinitol dehydrogenase or NADH oxidase. Under optimal conditions and using an initial concentration of 150 mM L-arabinitol, the maximal L-xylulose yield reached 96%, which is higher than those reported in the literature. The immobilized form of the coupled whole-cell biocatalysts demonstrated good operational stability, maintaining 65% of the yield obtained in the first cycle after 7 cycles of successive re-use, while the free cell system almost completely lost the catalytic activity. Therefore, the methods reported here provides two strategies that could help improve the industrial production of L-xylulose, as well as other value-added compounds requiring the use of cofactors in general.

  8. Innovative approaches for effective selection of lipase-producing microorganisms as whole cell catalysts for biodiesel production.

    PubMed

    Ciudad, Gustavo; Reyes, Isaac; Azócar, Laura; Briones, Reinaldo; Jorquera, Milko; Wick, Lukas Y; Navia, Rodrigo

    2011-07-01

    The high cost of commercial lipases limits their industrial application in the production of biodiesel or fatty acid methyl esters (FAME). This disadvantage has encouraged the search for lipase-producing microorganisms (LPMs) as potential whole cell catalysts for FAME production. The aim of this study, therefore, was to evaluate innovative procedures for easy selection and testing of LPMs as a low-cost whole cell catalyst, based on catalytic performance, methanol tolerance and physico-chemical cell surface properties. The latter (in particular the cell surface hydrophobicity and charge) were analyzed because of their crucial role in microbial adhesion to surfaces and the concomitant increase in cell immobilization and bioavailability of hydrophobic substrates. Biocatalysis experiments performed in the presence of nutrient, rapeseed oil and methanol were an effective tool for studying and identifying, in just two experiments, the capacity of different LPMs as biocatalysts in organic media, as well as the methanol tolerance of the cell and the lipase. This indicates the potential for using live microorganisms for FAME production. Another finding was that the inhibitory effect of methanol is more significant for lipase activity than LPM growth, indicating that the way in which alcohol is supplied to the reaction is a crucial step in FAME production by biocatalysts. According to these results, the application of these innovative assessments should simplify the search for new strains which are able to effectively catalyze the FAME production process.

  9. Application to Photocatalytic H2 Production of a Whole-Cell Reaction by Recombinant Escherichia coli Cells Expressing [FeFe]-Hydrogenase and Maturases Genes.

    PubMed

    Honda, Yuki; Hagiwara, Hidehisa; Ida, Shintaro; Ishihara, Tatsumi

    2016-07-01

    A photocatalytic H2 production system using an inorganic-bio hybrid photocatalyst could contribute to the efficient utilization of solar energy, but would require the development of a new approach for preparing a H2 -forming biocatalyst. In the present study, we constructed a recombinant strain of Escherichia coli expressing the genes encoding the [FeFe]-hydrogenase and relevant maturases from Clostridium acetobutylicum NBRC 13948 for use as a biocatalyst. We investigated the direct application of a whole-cell of the recombinant E. coli. The combination of TiO2 , methylviologen, and the recombinant E. coli formed H2 under light irradiation, demonstrating that whole cells of the recombinant E. coli could be employed for photocatalytic H2 production without any time-consuming and costly manipulations (for example, enzyme purification). This is the first report of the direct application of a whole-cell reaction of recombinant E. coli to photocatalytic H2 production. PMID:27194524

  10. Development and applications of whole cell biosensors for ecotoxicity testing.

    PubMed

    Pasco, Neil F; Weld, Richard J; Hay, Joanne M; Gooneratne, Ravi

    2011-05-01

    Whole cell biosensors are the focus of considerable and increasing interest worldwide as methods for detecting and quantifying environmental toxicity, including biochemical oxygen demand (BOD), heavy metals, antibiotics, pesticides and herbicides. This review follows the development of whole cell biosensors from attempts to utilise changes in cellular metabolism to determine BOD and general toxicity, through the exploitation of unique metabolic pathways to detect specific toxicants, to the increasingly widespread use of genetic engineering to build new, and modify existing, sensing pathways.

  11. RNA Synthesis in Whole Cells and Protoplasts of Centaurea

    PubMed Central

    Kulikowski, Robert R.; Mascarenhas, Joseph P.

    1978-01-01

    Protoplasts enzymically isolated from suspension cultures of Centaurea cyanus L. incorporate radioactive precursors into RNA with kinetics similar to that of whole cells. There are differences, however, in several other aspects of RNA metabolism. The proportion of total RNA that contains poly(A) sequences (25 to 30%) is similar in both freshly isolated protoplasts and whole cells after a 20-minute pulse with [3H]adenosine. After a 4-hour pulse, however, poly(A)-containing RNA makes up 30% of the total RNA in protoplasts whereas it drops to 8% in whole cells. There appears to be a faulty processing of ribosomal precursor into the mature ribosomal species, as the precursor seems to accumulate to higher levels relative to the mature 18S and 25S rRNAs in protoplasts as compared to whole cells. Additional differences are seen in the size distributions of poly(A)-containing RNA, although the length of the poly(A) segment is similar in both protoplasts and whole cells. Within 24 hours protoplasts appear to have resumed a pattern of RNA synthesis similar to that of whole cells. PMID:16660339

  12. Biocatalytic Production of Trehalose from Maltose by Using Whole Cells of Permeabilized Recombinant Escherichia coli.

    PubMed

    Zheng, Zhaojuan; Xu, Ying; Sun, Ye; Mei, Wending; Ouyang, Jia

    2015-01-01

    Trehalose is a non-reducing disaccharide, which can protect proteins, lipid membranes, and cells from desiccation, refrigeration, dehydration, and other harsh environments. Trehalose can be produced by different pathways and trehalose synthase pathway is a convenient, practical, and low-cost pathway for the industrial production of trehalose. In this study, 3 candidate treS genes were screened from genomic databases of Pseudomonas and expressed in Escherichia coli. One of them from P. stutzeri A1501 exhibited the best transformation ability from maltose into trehalose and the least byproduct. Thus, whole cells of this recombinant E. coli were used as biocatalyst for trehalose production. In order to improve the conversion rate of maltose to trehalose, optimization of the permeabilization and biotransformation were carried out. Under optimal conditions, 92.2 g/l trehalose was produced with a high productivity of 23.1 g/(l h). No increase of glucose was detected during the whole course. The biocatalytic process developed in this study might serve as a candidate for the large scale production of trehalose. PMID:26462117

  13. Biocatalytic Production of Trehalose from Maltose by Using Whole Cells of Permeabilized Recombinant Escherichia coli

    PubMed Central

    Sun, Ye; Mei, Wending; Ouyang, Jia

    2015-01-01

    Trehalose is a non-reducing disaccharide, which can protect proteins, lipid membranes, and cells from desiccation, refrigeration, dehydration, and other harsh environments. Trehalose can be produced by different pathways and trehalose synthase pathway is a convenient, practical, and low-cost pathway for the industrial production of trehalose. In this study, 3 candidate treS genes were screened from genomic databases of Pseudomonas and expressed in Escherichia coli. One of them from P. stutzeri A1501 exhibited the best transformation ability from maltose into trehalose and the least byproduct. Thus, whole cells of this recombinant E. coli were used as biocatalyst for trehalose production. In order to improve the conversion rate of maltose to trehalose, optimization of the permeabilization and biotransformation were carried out. Under optimal conditions, 92.2 g/l trehalose was produced with a high productivity of 23.1 g/(l h). No increase of glucose was detected during the whole course. The biocatalytic process developed in this study might serve as a candidate for the large scale production of trehalose. PMID:26462117

  14. Outrunning Nature: Directed Evolution of Superior Biocatalysts

    NASA Astrophysics Data System (ADS)

    Woodyer, Ryan; Chen, Wilfred; Zhao, Huimin

    2004-01-01

    Driven by recent technical advances in genetic engineering and new societal needs, the use of enzymes and microorganisms as catalysts to synthesize chemicals and materials is rapidly expanding. One of the key technical drivers is the development of various directed evolution methods for biocatalyst discovery and optimization. Although it essentially replicates the Darwinian evolutionary processes in a test tube, directed evolution can create biocatalysts with better catalytic performance than Nature's own products within weeks or months rather than eons. In this article, both the technologies and applications of directed evolution in biocatalysis are discussed.

  15. Biocatalysts for biomass deconstruction from environmental genomics.

    PubMed

    Armstrong, Zachary; Mewis, Keith; Strachan, Cameron; Hallam, Steven J

    2015-12-01

    Plant biomass offers a sustainable alternative to the energy and materials produced from fossil fuels. The industrial scale production or biorefining of fermentable sugars and aromatics from plant biomass is currently limited by the lack of cost effective and efficient biocatalysts. One potential solution to this problem is the discovery of biomass deconstructing biocatalysts from uncultivated microbial communities. Here we review recent progress in recovering such biological devices from environmental genomes and consider how this information can be used to build better biorefining ecosystems.

  16. Marine Biocatalysts: Enzymatic Features and Applications

    PubMed Central

    Trincone, Antonio

    2011-01-01

    In several recent reports related to biocatalysis the enormous pool of biodiversity found in marine ecosystems is considered a profitable natural reservoir for acquiring an inventory of useful biocatalysts. These enzymes are characterized by well-known habitat-related features such as salt tolerance, hyperthermostability, barophilicity and cold adaptivity. In addition, their novel chemical and stereochemical characteristics increase the interest of biocatalysis practitioners both in academia and research industry. In this review, starting from the analysis of these featuring habitat-related properties, important examples of marine enzymes in biocatalysis will be reported. Completion of this report is devoted to the analysis of novel chemical and stereochemical biodiversity offered by marine biocatalysts with particular emphasis on current or potential applications of these enzymes in chemical and pharmaceutical fields. The analysis of literature cited here and the many published patent applications concerning the use of marine enzymes supports the view that these biocatalysts are just waiting to be discovered, reflecting the importance of the marine environment. The potential of this habitat should be thoroughly explored and possibly the way to access useful biocatalysts should avoid destructive large-scale collections of marine biomass for enzyme production. These two aspects are day by day increasing in interest and a future increase in the use of marine enzymes in biocatalysis should be expected. PMID:21731544

  17. Recombinant DNA encoding a desulfurization biocatalyst

    DOEpatents

    Rambosek, John; Piddington, Chris S.; Kovacevich, Brian R.; Young, Kevin D.; Denome, Sylvia A.

    1994-01-01

    This invention relates to a recombinant DNA molecule containing a gene or genes which encode a biocatalyst capable of desulfurizing a fossil fuel which contains organic sulfur molecules. For example, the present invention encompasses a recombinant DNA molecule containing a gene or genes of a strain of Rhodococcus rhodochrous.

  18. Recombinant DNA encoding a desulfurization biocatalyst

    DOEpatents

    Rambosek, J.; Piddington, C.S.; Kovacevich, B.R.; Young, K.D.; Denome, S.A.

    1994-10-18

    This invention relates to a recombinant DNA molecule containing a gene or genes which encode a biocatalyst capable of desulfurizing a fossil fuel which contains organic sulfur molecules. For example, the present invention encompasses a recombinant DNA molecule containing a gene or genes of a strain of Rhodococcus rhodochrous. 13 figs.

  19. Diplogelasinospora grovesii IMI 171018 immobilized in polyurethane foam. An efficient biocatalyst for stereoselective reduction of ketones.

    PubMed

    Quezada, M A; Carballeira, J D; Sinisterra, J V

    2012-05-01

    Diplogelasinospora grovesii has been reported as a very active biocatalyst in the reduction of ketones. Along the text, the properties of this filamentous fungus as an immobilized catalyst are described. For this purpose, several immobilization supports as agar and polyurethane foam were tested. Experimental assays were also performed to test different co-substrates for the regeneration of the required enzyme cofactor. The fungus immobilized in polyurethane foam lead to the most stable and active catalyst. This derivative, using i-PrOH as co-substrate, could be reused at least 18 times without appreciable activity loss (>90% activity remains). Kinetic runs experiments shown that the reduction of cyclohexanone, selected as model substrate, followed a pseudo-first kinetic order and that the rate controlling step was the mass transfer through the cell wall. The deactivation kinetic constants were also determined. The reduction of different chiral ketones showed that the ketone reductase activity followed the Prelog's rule. PMID:22424921

  20. Production of phenylpyruvic acid from L-phenylalanine using an L-amino acid deaminase from Proteus mirabilis: comparison of enzymatic and whole-cell biotransformation approaches.

    PubMed

    Hou, Ying; Hossain, Gazi Sakir; Li, Jianghua; Shin, Hyun-Dong; Liu, Long; Du, Guocheng

    2015-10-01

    Phenylpyruvic acid (PPA) is an important organic acid that has a wide range of applications. In this study, the membrane-bound L-amino acid deaminase (L-AAD) gene from Proteus mirabilis KCTC 2566 was expressed in Escherichia coli BL21(DE3) and then the L-AAD was purified. After that, we used the purified enzyme and the recombinant E. coli whole-cell biocatalyst to produce PPA via a one-step biotransformation from L-phenylalanine. L-AAD was solubilized from the membrane and purified 52-fold with an overall yield of 13 %, which corresponded to a specific activity of 0.94 ± 0.01 μmol PPA min(-1)·mg(-1). Then, the biotransformation conditions for the pure enzyme and the whole-cell biocatalyst were optimized. The maximal production was 2.6 ± 0.1 g·L(-1) (specific activity of 1.02 ± 0.02 μmol PPA min(-1)·mg(-1) protein, 86.7 ± 5 % mass conversion rate, and 1.04 g·L(-1)·h(-1) productivity) and 3.3 ± 0.2 g L(-1) (specific activity of 0.013 ± 0.003 μmol PPA min(-1)·mg(-1) protein, 82.5 ± 4 % mass conversion rate, and 0.55 g·L(-1)·h(-1) productivity) for the pure enzyme and whole-cell biocatalyst, respectively. Comparative studies of the enzymatic and whole-cell biotransformation were performed in terms of specific activity, production, conversion, productivity, stability, need of external cofactors, and recycling. We have developed two eco-friendly and efficient approaches for PPA production. The strategy described herein may aid the biotransformational synthesis of other α-keto acids from their corresponding amino acids.

  1. Towards a whole-cell modeling approach for synthetic biology

    NASA Astrophysics Data System (ADS)

    Purcell, Oliver; Jain, Bonny; Karr, Jonathan R.; Covert, Markus W.; Lu, Timothy K.

    2013-06-01

    Despite rapid advances over the last decade, synthetic biology lacks the predictive tools needed to enable rational design. Unlike established engineering disciplines, the engineering of synthetic gene circuits still relies heavily on experimental trial-and-error, a time-consuming and inefficient process that slows down the biological design cycle. This reliance on experimental tuning is because current modeling approaches are unable to make reliable predictions about the in vivo behavior of synthetic circuits. A major reason for this lack of predictability is that current models view circuits in isolation, ignoring the vast number of complex cellular processes that impinge on the dynamics of the synthetic circuit and vice versa. To address this problem, we present a modeling approach for the design of synthetic circuits in the context of cellular networks. Using the recently published whole-cell model of Mycoplasma genitalium, we examined the effect of adding genes into the host genome. We also investigated how codon usage correlates with gene expression and find agreement with existing experimental results. Finally, we successfully implemented a synthetic Goodwin oscillator in the whole-cell model. We provide an updated software framework for the whole-cell model that lays the foundation for the integration of whole-cell models with synthetic gene circuit models. This software framework is made freely available to the community to enable future extensions. We envision that this approach will be critical to transforming the field of synthetic biology into a rational and predictive engineering discipline.

  2. Paired Whole Cell Recordings in Organotypic Hippocampal Slices

    PubMed Central

    Fourie, Chantelle; Kiraly, Marianna

    2014-01-01

    Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology. PMID:25285945

  3. Removal of trace organic contaminants by nitrifying activated sludge and whole-cell and crude enzyme extract of Trametes versicolor.

    PubMed

    Yang, Shufan; Hai, Faisal I; Nghiem, Long D; Roddick, Felicity; Price, William E

    2013-01-01

    The resistance of certain anthropogenic trace organic contaminants (TrOCs) to conventional wastewater treatment and their potential adverse effects on human and ecological health raise significant concerns and have prompted research on their bioremediation by white-rot fungi. This study compared the removal efficiencies of four widespread TrOCs: carbamazepine (CBZ), sulfamethoxazole (SMX), bisphenol A (BPA) and diclofenac (DCF), by nitrifying activated sludge as well as whole-cell and extracellular enzyme (laccase) extract of the white-rot fungus Trametes versicolor. Fungal whole-cell culture removed only BPA and DCF but with high efficiencies (>90%) while the mixed nitrifying culture removed all compounds, although by levels of only 5-40%. Rapid initial sorption on fungal mycelium (44 ± 13% for DCF) was observed; however, biodegradation governed the overall removal. Performance comparison between fungal whole-cell and extracellular extract revealed that, unlike BPA, a catalytic pathway independent of extracellular laccase was responsible for DCF removal. Addition of mediator (1-hydroxybenzotriazole) to extracellular extract improved the removal of SMX which bears an electron donor group, but not that of the resistant compound CBZ. PMID:23508144

  4. Batch conversion of methane to methanol using Methylosinus trichosporium OB3b as biocatalyst.

    PubMed

    Hwang, In Yeub; Hur, Dong Hoon; Lee, Jae Hoon; Park, Chang-Ho; Chang, In Seop; Lee, Jin Won; Lee, Eun Yeol

    2015-03-01

    Recently, methane has attracted much attention as an alternative carbon feedstock since it is the major component of abundant shale and natural gas. In this work, we produced methanol from methane using whole cells of Methylosinus trichosporium OB3b as the biocatalyst. M. trichosporium OB3b was cultured on NMS medium with a supply of 7:3 air/methane ratio at 30°C. The optimal concentrations of various methanol dehydrogenase inhibitors such as potassium phosphate and EDTA were determined to be 100 and 0.5 mM, respectively, for an efficient production of methanol. Sodium formate (40 mM) as a reducing power source was added to enhance the conversion efficiency. A productivity of 49.0 mg/l·h, titer of 0.393 g methanol/l, and conversion of 73.8% (mol methanol/mol methane) were obtained under the optimized batch condition. PMID:25563419

  5. A highly productive, whole-cell DERA chemoenzymatic process for production of key lactonized side-chain intermediates in statin synthesis.

    PubMed

    Ošlaj, Matej; Cluzeau, Jérôme; Orkić, Damir; Kopitar, Gregor; Mrak, Peter; Casar, Zdenko

    2013-01-01

    Employing DERA (2-deoxyribose-5-phosphate aldolase), we developed the first whole-cell biotransformation process for production of chiral lactol intermediates useful for synthesis of optically pure super-statins such as rosuvastatin and pitavastatin. Herein, we report the development of a fed-batch, high-density fermentation with Escherichia coli BL21 (DE3) overexpressing the native E. coli deoC gene. High activity of this biomass allows direct utilization of the fermentation broth as a whole-cell DERA biocatalyst. We further show a highly productive bioconversion processes with this biocatalyst for conversion of 2-substituted acetaldehydes to the corresponding lactols. The process is evaluated in detail for conversion of acetyloxy-acetaldehyde with the first insight into the dynamics of reaction intermediates, side products and enzyme activity, allowing optimization of the feeding strategy of the aldehyde substrates for improved productivities, yields and purities. The resulting process for production of ((2S,4R)-4,6-dihydroxytetrahydro-2H-pyran-2-yl)methyl acetate (acetyloxymethylene-lactol) has a volumetric productivity exceeding 40 g L(-1) h(-1) (up to 50 g L(-1) h(-1)) with >80% yield and >80% chromatographic purity with titers reaching 100 g L(-1). Stereochemical selectivity of DERA allows excellent enantiomeric purities (ee >99.9%), which were demonstrated on downstream advanced intermediates. The presented process is highly cost effective and environmentally friendly. To our knowledge, this is the first asymmetric aldol condensation process achieved with whole-cell DERA catalysis and it simplifies and extends previously developed DERA-catalyzed approaches based on the isolated enzyme. Finally, applicability of the presented process is demonstrated by efficient preparation of a key lactol precursor, which fits directly into the lactone pathway to optically pure super-statins. PMID:23667462

  6. Microbial biocatalyst developments to upgrade fossil fuels.

    PubMed

    Kilbane, John J

    2006-06-01

    Steady increases in the average sulfur content of petroleum and stricter environmental regulations concerning the sulfur content have promoted studies of bioprocessing to upgrade fossil fuels. Bioprocesses can potentially provide a solution to the need for improved and expanded fuel upgrading worldwide, because bioprocesses for fuel upgrading do not require hydrogen and produce far less carbon dioxide than thermochemical processes. Recent advances have demonstrated that biodesulfurization is capable of removing sulfur from hydrotreated diesel to yield a product with an ultra-low sulfur concentration that meets current environmental regulations. However, the technology has not yet progressed beyond laboratory-scale testing, as more efficient biocatalysts are needed. Genetic studies to obtain improved biocatalysts for the selective removal of sulfur and nitrogen from petroleum provide the focus of current research efforts.

  7. Fungus Amongus

    ERIC Educational Resources Information Center

    Wakeley, Deidra

    2005-01-01

    This role-playing simulation is designed to help teach middle level students about the typical lifecycle of a fungus. In this interactive simulation, students assume the roles of fungi, spores, living and dead organisms, bacteria, and rain. As they move around a playing field collecting food and water chips, they discover how the organisms…

  8. Reconstruction of lactate utilization system in Pseudomonas putida KT2440: a novel biocatalyst for l-2-hydroxy-carboxylate production

    PubMed Central

    Wang, Yujiao; Lv, Min; Zhang, Yingxin; Xiao, Xieyue; Jiang, Tianyi; Zhang, Wen; Hu, Chunhui; Gao, Chao; Ma, Cuiqing; Xu, Ping

    2014-01-01

    As an important method for building blocks synthesis, whole cell biocatalysis is hindered by some shortcomings such as unpredictability of reactions, utilization of opportunistic pathogen, and side reactions. Due to its biological and extensively studied genetic background, Pseudomonas putida KT2440 is viewed as a promising host for construction of efficient biocatalysts. After analysis and reconstruction of the lactate utilization system in the P. putida strain, a novel biocatalyst that only exhibited NAD-independent d-lactate dehydrogenase activity was prepared and used in l-2-hydroxy-carboxylates production. Since the side reaction catalyzed by the NAD-independent l-lactate dehydrogenase was eliminated in whole cells of recombinant P. putida KT2440, two important l-2-hydroxy-carboxylates (l-lactate and l-2-hydroxybutyrate) were produced in high yield and high optical purity by kinetic resolution of racemic 2-hydroxy carboxylic acids. The results highlight the promise in biocatalysis by the biotechnologically important organism P. putida KT2440 through genomic analysis and recombination. PMID:25373400

  9. Export of functional Streptomyces coelicolor alditol oxidase to the periplasm or cell surface of Escherichia coli and its application in whole-cell biocatalysis.

    PubMed

    van Bloois, Edwin; Winter, Remko T; Janssen, Dick B; Fraaije, Marco W

    2009-06-01

    Streptomyces coelicolor A3(2) alditol oxidase (AldO) is a soluble monomeric flavoprotein in which the flavin cofactor is covalently linked to the polypeptide chain. AldO displays high reactivity towards different polyols such as xylitol and sorbitol. These characteristics make AldO industrially relevant, but full biotechnological exploitation of this enzyme is at present restricted by laborious and costly purification steps. To eliminate the need for enzyme purification, this study describes a whole-cell AldO biocatalyst system. To this end, we have directed AldO to the periplasm or cell surface of Escherichia coli. For periplasmic export, AldO was fused to endogenous E. coli signal sequences known to direct their passenger proteins into the SecB, signal recognition particle (SRP), or Twin-arginine translocation (Tat) pathway. In addition, AldO was fused to an ice nucleation protein (INP)-based anchoring motif for surface display. The results show that Tat-exported AldO and INP-surface-displayed AldO are active. The Tat-based system was successfully employed in converting xylitol by whole cells, whereas the use of the INP-based system was most likely restricted by lipopolysaccharide LPS in wild-type cells. It is anticipated that these whole-cell systems will be a valuable tool for further biological and industrial exploitation of AldO and other cofactor-containing enzymes. PMID:19224207

  10. Conversion of polyhydroxyalkanoates to methyl crotonate using whole cells.

    PubMed

    Spekreijse, J; Holgueras Ortega, J; Sanders, J P M; Bitter, J H; Scott, E L

    2016-07-01

    Isolated polyhydroxyalkanoates (PHA) can be used to produce biobased bulk chemicals. However, isolation is complex and costly. To circumvent this, whole cells containing PHA may be used. Here, PHA containing 3-hydroxybutyrate and small amounts of 3-hydroxyvalerate was produced from wastewater and used in the conversion of the 3-hydroxybutyrate monomer to methyl crotonate. Due to the increased complexity of whole cell reaction mixtures compared to pure PHA, the effect of 3-hydroxyvalerate content, magnesium salts and water content was studied in order to evaluate the need for downstream processing. A water content up to 20% and the presence of 3-hydroxyvalerate have no influence on the conversion of the 3-hydroxybutyrate to methyl crotonate. The presence of Mg(2+)-ions resulted either in an increased yield or in byproduct formation depending on the counter ion. Overall, it is possible to bypass a major part of the downstream processing of PHA for the production of biobased chemicals. PMID:27023381

  11. Vaccination Against Tuberculosis With Whole-Cell Mycobacterial Vaccines.

    PubMed

    Scriba, Thomas J; Kaufmann, Stefan H E; Henri Lambert, Paul; Sanicas, Melvin; Martin, Carlos; Neyrolles, Olivier

    2016-09-01

    Live attenuated and killed whole-cell vaccines (WCVs) offer promising vaccination strategies against tuberculosis. A number of WCV candidates, based on recombinant bacillus Calmette-Guerin (BCG), attenuated Mycobacterium tuberculosis, or related mycobacterial species are in various stages of preclinical or clinical development. In this review, we discuss the vaccine candidates and key factors shaping the development pathway for live and killed WCVs and provide an update on progress. PMID:27247343

  12. Whole-cell Patch-clamp Recordings in Brain Slices.

    PubMed

    Segev, Amir; Garcia-Oscos, Francisco; Kourrich, Saïd

    2016-01-01

    Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices, a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular, when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals

  13. Whole-Cell Bioreporters for the Detection of Bioavailable Metals

    NASA Astrophysics Data System (ADS)

    Hynninen, Anu; Virta, Marko

    Whole-cell bioreporters are living microorganisms that produce a specific, quantifiable output in response to target chemicals. Typically, whole-cell bioreporters combine a sensor element for the substance of interest and a reporter element coding for an easily detectable protein. The sensor element is responsible for recognizing the presence of an analyte. In the case of metal bioreporters, the sensor element consists of a DNA promoter region for a metal-binding transcription factor fused to a promoterless reporter gene that encodes a signal-producing protein. In this review, we provide an overview of specific whole-cell bioreporters for heavy metals. Because the sensing of metals by bioreporter microorganisms is usually based on heavy metal resistance/homeostasis mechanisms, the basis of these mechanisms will also be discussed. The goal here is not to present a comprehensive summary of individual metal-specific bioreporters that have been constructed, but rather to express views on the theory and applications of metal-specific bioreporters and identify some directions for future research and development.

  14. Whole-cell-based biosensors for environmental biomonitoring and application.

    PubMed

    Gu, Man Bock; Mitchell, Robert J; Kim, Byoung Chan

    2004-01-01

    A variety of whole-cell-based biosensors has been developed using numerous native and recombinant biosensing cells. The use of reporter genes, for example bacterial luciferase and gfp, to monitor gene expression is discussed in terms of each reporters' benefits and disadvantages, including their possible use on-line, their sensitivity, the need for extra substrate, etc. All biosensing cells in use can be classified into two groups in terms of their biosensing mechanisms--constitutive expression and stress- or chemical-specific inducible expression. In this review several examples of each are presented and discussed. The use of recombinant whole-cell biosensors in the field requires three components--biosensing cells, a measurement device, and a signal-transducing apparatus, the last two depending on the first and the final applications of the system. The use of different immobilization techniques in several studies to maintain the cells and their viability is also discussed, in particular their use in the development of both high-throughput and chip-based biosensing systems. Finally the application of whole-cell-based biosensors to different environmental media, such as water, soil, and atmospheric monitoring is discussed; particular attention is given to their use for detection of various stressors, including dioxins, endocrine-disrupting chemicals, and ionizing radiation.

  15. Engineered Escherichia coli with Periplasmic Carbonic Anhydrase as a Biocatalyst for CO2 Sequestration

    PubMed Central

    Jo, Byung Hoon; Kim, Im Gyu; Seo, Jeong Hyun; Kang, Dong Gyun

    2013-01-01

    Carbonic anhydrase is an enzyme that reversibly catalyzes the hydration of carbon dioxide (CO2). It has been suggested recently that this remarkably fast enzyme can be used for sequestration of CO2, a major greenhouse gas, making this a promising alternative for chemical CO2 mitigation. To promote the economical use of enzymes, we engineered the carbonic anhydrase from Neisseria gonorrhoeae (ngCA) in the periplasm of Escherichia coli, thereby creating a bacterial whole-cell catalyst. We then investigated the application of this system to CO2 sequestration by mineral carbonation, a process with the potential to store large quantities of CO2. ngCA was highly expressed in the periplasm of E. coli in a soluble form, and the recombinant bacterial cell displayed the distinct ability to hydrate CO2 compared with its cytoplasmic ngCA counterpart and previously reported whole-cell CA systems. The expression of ngCA in the periplasm of E. coli greatly accelerated the rate of calcium carbonate (CaCO3) formation and exerted a striking impact on the maximal amount of CaCO3 produced under conditions of relatively low pH. It was also shown that the thermal stability of the periplasmic enzyme was significantly improved. These results demonstrate that the engineered bacterial cell with periplasmic ngCA can successfully serve as an efficient biocatalyst for CO2 sequestration. PMID:23974145

  16. Directed evolution: tailoring biocatalysts for industrial applications.

    PubMed

    Kumar, Ashwani; Singh, Suren

    2013-12-01

    Current challenges and promises of white biotechnology encourage protein engineers to use a directed evolution approach to generate novel and useful biocatalysts for various sets of applications. Different methods of enzyme engineering have been used in the past in an attempt to produce enzymes with improved functions and properties. Recent advancement in the field of random mutagenesis, screening, selection and computational design increased the versatility and the rapid development of enzymes under strong selection pressure with directed evolution experiments. Techniques of directed evolution improve enzymes fitness without understanding them in great detail and clearly demonstrate its future role in adapting enzymes for use in industry. Despite significant advances to date regarding biocatalyst improvement, there still remains a need to improve mutagenesis strategies and development of easy screening and selection tools without significant human intervention. This review covers fundamental and major development of directed evolution techniques, and highlights the advances in mutagenesis, screening and selection methods with examples of enzymes developed by using these approaches. Several commonly used methods for creating molecular diversity with their advantages and disadvantages including some recently used strategies are also discussed.

  17. Detection of Organic Compounds with Whole-Cell Bioluminescent Bioassays

    PubMed Central

    Xu, Tingting; Close, Dan; Smartt, Abby; Ripp, Steven

    2015-01-01

    Natural and manmade organic chemicals are widely deposited across a diverse range of ecosystems including air, surface water, groundwater, wastewater, soil, sediment, and marine environments. Some organic compounds, despite their industrial values, are toxic to living organisms and pose significant health risks to humans and wildlife. Detection and monitoring of these organic pollutants in environmental matrices therefore is of great interest and need for remediation and health risk assessment. Although these detections have traditionally been performed using analytical chemical approaches that offer highly sensitive and specific identification of target compounds, these methods require specialized equipment and trained operators, and fail to describe potential bioavailable effects on living organisms. Alternatively, the integration of bioluminescent systems into whole-cell bioreporters presents a new capacity for organic compound detection. These bioreporters are constructed by incorporating reporter genes into catabolic or signaling pathways that are present within living cells and emit a bioluminescent signal that can be detected upon exposure to target chemicals. Although relatively less specific compared to analytical methods, bioluminescent bioassays are more cost-effective, more rapid, can be scaled to higher throughput, and can be designed to report not only the presence but also the bioavailability of target substances. This chapter reviews available bacterial and eukaryotic whole-cell bioreporters for sensing organic pollutants and their applications in a variety of sample matrices. PMID:25084996

  18. Continuous glutamate production using an immobilized whole-cell system

    SciTech Connect

    Kim, H.S.; Ryu, D.D.Y.

    1982-10-01

    For the purpose of saving the energy and raw materials required in a glutamate fermentation, an immobilized whole-cell system was prepared and its performance in a continuous reactor system was evaluated. Corynebacterium glutamicum (a mutant strain of ATCC 13058) whole cell was immobilized in k-carrageenan matrix and the gel structure was strengthened by treatment with a hardening agent. The effective diffusivities of carrageenan gel for glucose and oxygen were formed to decrease significantly with an increase in carrageenan concentration, while the gel strength showed an increasing trend. Based on the physical and chemical properties of carrageenan gel, the immobilized method was improved and the operation of the continuous reactor system was partially optimized. In an air-stirred fermentor, the continuous production of glutamate was carried out. The effect of the dilution rate of glutamate production and operation stability was investigated. The performance of the continuous wbole-cell reactor system was evaluated by measuring glutamate productivity for a period of 30 days; it was found to be far superior to the performance of convention batch reactor systems using free cells.

  19. Whole-cell, multicolor superresolution imaging using volumetric multifocus microscopy

    PubMed Central

    Hajj, Bassam; Wisniewski, Jan; El Beheiry, Mohamed; Chen, Jiji; Revyakin, Andrey; Wu, Carl; Dahan, Maxime

    2014-01-01

    Single molecule-based superresolution imaging has become an essential tool in modern cell biology. Because of the limited depth of field of optical imaging systems, one of the major challenges in superresolution imaging resides in capturing the 3D nanoscale morphology of the whole cell. Despite many previous attempts to extend the application of photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) techniques into three dimensions, effective localization depths do not typically exceed 1.2 µm. Thus, 3D imaging of whole cells (or even large organelles) still demands sequential acquisition at different axial positions and, therefore, suffers from the combined effects of out-of-focus molecule activation (increased background) and bleaching (loss of detections). Here, we present the use of multifocus microscopy for volumetric multicolor superresolution imaging. By simultaneously imaging nine different focal planes, the multifocus microscope instantaneously captures the distribution of single molecules (either fluorescent proteins or synthetic dyes) throughout an ∼4-µm-deep volume, with lateral and axial localization precisions of ∼20 and 50 nm, respectively. The capabilities of multifocus microscopy to rapidly image the 3D organization of intracellular structures are illustrated by superresolution imaging of the mammalian mitochondrial network and yeast microtubules during cell division. PMID:25422417

  20. Detection of organic compounds with whole-cell bioluminescent bioassays.

    PubMed

    Xu, Tingting; Close, Dan; Smartt, Abby; Ripp, Steven; Sayler, Gary

    2014-01-01

    Natural and manmade organic chemicals are widely deposited across a diverse range of ecosystems including air, surface water, groundwater, wastewater, soil, sediment, and marine environments. Some organic compounds, despite their industrial values, are toxic to living organisms and pose significant health risks to humans and wildlife. Detection and monitoring of these organic pollutants in environmental matrices therefore is of great interest and need for remediation and health risk assessment. Although these detections have traditionally been performed using analytical chemical approaches that offer highly sensitive and specific identification of target compounds, these methods require specialized equipment and trained operators, and fail to describe potential bioavailable effects on living organisms. Alternatively, the integration of bioluminescent systems into whole-cell bioreporters presents a new capacity for organic compound detection. These bioreporters are constructed by incorporating reporter genes into catabolic or signaling pathways that are present within living cells and emit a bioluminescent signal that can be detected upon exposure to target chemicals. Although relatively less specific compared to analytical methods, bioluminescent bioassays are more cost-effective, more rapid, can be scaled to higher throughput, and can be designed to report not only the presence but also the bioavailability of target substances. This chapter reviews available bacterial and eukaryotic whole-cell bioreporters for sensing organic pollutants and their applications in a variety of sample matrices.

  1. Whole-cell biocatalytic production of variously substituted β-aryl- and β-heteroaryl-β-amino acids.

    PubMed

    Ratnayake, Nishanka Dilini; Theisen, Chelsea; Walter, Tyler; Walker, Kevin D

    2016-01-10

    Biologically-active β-peptides and pharmaceuticals that contain key β-amino acids are emerging as target therapeutics; thus, synthetic strategies to make substituted, enantiopure β-amino acids are increasing. Here, we use whole-cell Escherichia coli (OD600 ∼ 35) engineered to express a Pantoea agglomerans phenylalanine aminomutase (PaPAM) biocatalyst. In either 5 mL, 100mL, or 1L of M9 minimal medium containing α-phenylalanine (20mM), the cells produced ∼ 1.4 mg mL(-1) of β-phenylalanine in each volume. Representative pilot-scale 5-mL cultures, fermentation reactions converted 18 variously substituted α-arylalanines to their (S)-β-aryl-β-amino acids in vivo and were not toxic to cells at mid- to late-stage growth. The β-aryl-β-amino acids made ranged from 0.043 mg (p-nitro-β-phenylalanine, 4% converted yield) to 1.2mg (m-bromo-β-phenylalanine, 96% converted yield) over 6h in 5 mL. The substituted β-amino acids made herein can be used in redox and Stille-coupling reactions to make synthetic building blocks, or as bioisosteres in drug design.

  2. A novel convenient procedure for extractive work-up of whole-cell biotransformations using de-emulsifying hydrolases.

    PubMed

    Jörg, Gerhard; Leppchen, Kathrin; Daussmann, Thomas; Bertau, Martin

    2004-08-20

    Extractive work-up of whole-cell biotransformations generally suffers from the formation of stable gels and slimes upon addition of the organic solvent to the cell suspension and the cell-free solution, respectively. This problem has been overcome by enzymatic lysis of emulsifying agents present in the medium through addition of hydrolases. Of these agents, proteases have exhibited the most powerful de-emulsifying activity. Enzyme treatment of cell-free culture media of Saccharomyces cerevisiae with pronase E drastically reduced phase separation time (t(p)) from 1 week to 30 min without significantly affecting product integrity. Yeast cell suspensions were de-emulsified best with protease N-01, where phase separation was complete after 1 h. As was exemplified with cell-free culture media of Lactobacillus kefir, wherein addition of pronase E or protease N-01 reduced t(p) from 1 week to 2 h each, this practical, ready-to-use method is appropriate for both fungal and bacterial biocatalysts.

  3. Archaeal Enzymes and Applications in Industrial Biocatalysts.

    PubMed

    Littlechild, Jennifer A

    2015-01-01

    Archaeal enzymes are playing an important role in industrial biotechnology. Many representatives of organisms living in "extreme" conditions, the so-called Extremophiles, belong to the archaeal kingdom of life. This paper will review studies carried by the Exeter group and others regarding archaeal enzymes that have important applications in commercial biocatalysis. Some of these biocatalysts are already being used in large scale industrial processes for the production of optically pure drug intermediates and amino acids and their analogues. Other enzymes have been characterised at laboratory scale regarding their substrate specificity and properties for potential industrial application. The increasing availability of DNA sequences from new archaeal species and metagenomes will provide a continuing resource to identify new enzymes of commercial interest using both bioinformatics and screening approaches.

  4. Archaeal Enzymes and Applications in Industrial Biocatalysts

    PubMed Central

    Littlechild, Jennifer A.

    2015-01-01

    Archaeal enzymes are playing an important role in industrial biotechnology. Many representatives of organisms living in “extreme” conditions, the so-called Extremophiles, belong to the archaeal kingdom of life. This paper will review studies carried by the Exeter group and others regarding archaeal enzymes that have important applications in commercial biocatalysis. Some of these biocatalysts are already being used in large scale industrial processes for the production of optically pure drug intermediates and amino acids and their analogues. Other enzymes have been characterised at laboratory scale regarding their substrate specificity and properties for potential industrial application. The increasing availability of DNA sequences from new archaeal species and metagenomes will provide a continuing resource to identify new enzymes of commercial interest using both bioinformatics and screening approaches. PMID:26494981

  5. Hydrogen production by immobilized whole cells of Clostridium butyricum

    NASA Astrophysics Data System (ADS)

    Suzuki, S.; Karube, I.

    Immobilized microbial cells were used in a batch system in an attempt to achieve continuous hydrogen production from glucose and waste waters. Clostridium butyricum IFO 3847 was immobilized in polyacrylamide gel and continuously produced hydrogen from glucose. The hydrogen producing bacteria were then immobilized in 2% agar gel and showed continuous hydrogen production from an alcohol factory's waste waters. The hydrogen production rate became constant above BOD 1500 ppm when performed with a batch system. The immobilized whole cells continuously produced hydrogen over a 20 day period, producing about 6 ml/min/kg wet gels. Hydrogen production by bacteria immobilized in acetylcellulose filters was six times higher than that by cells entrapped in agar gels.

  6. Electron microscopy of whole cells in liquid with nanometer resolution

    PubMed Central

    de Jonge, N.; Peckys, D. B.; Kremers, G. J.; Piston, D. W.

    2009-01-01

    Single gold-tagged epidermal growth factor (EGF) molecules bound to cellular EGF receptors of fixed fibroblast cells were imaged in liquid with a scanning transmission electron microscope (STEM). The cells were placed in buffer solution in a microfluidic device with electron transparent windows inside the vacuum of the electron microscope. A spatial resolution of 4 nm and a pixel dwell time of 20 μs were obtained. The liquid layer was sufficiently thick to contain the cells with a thickness of 7 ± 1 μm. The experimental findings are consistent with a theoretical calculation. Liquid STEM is a unique approach for imaging single molecules in whole cells with significantly improved resolution and imaging speed over existing methods. PMID:19164524

  7. Electrochemical As(III) whole-cell based biochip sensor.

    PubMed

    Cortés-Salazar, Fernando; Beggah, Siham; van der Meer, Jan Roelof; Girault, Hubert H

    2013-09-15

    The development of a whole-cell based sensor for arsenite detection coupling biological engineering and electrochemical techniques is presented. This strategy takes advantage of the natural Escherichia coli resistance mechanism against toxic arsenic species, such as arsenite, which consists of the selective intracellular recognition of arsenite and its pumping out from the cell. A whole-cell based biosensor can be produced by coupling the intracellular recognition of arsenite to the generation of an electrochemical signal. Hereto, E. coli was equipped with a genetic circuit in which synthesis of beta-galactosidase is under control of the arsenite-derepressable arsR-promoter. The E. coli reporter strain was filled in a microchip containing 16 independent electrochemical cells (i.e. two-electrode cell), which was then employed for analysis of tap and groundwater samples. The developed arsenic-sensitive electrochemical biochip is easy to use and outperforms state-of-the-art bacterial bioreporters assays specifically in its simplicity and response time, while keeping a very good limit of detection in tap water, i.e. 0.8ppb. Additionally, a very good linear response in the ranges of concentration tested (0.94ppb to 3.75ppb, R(2)=0.9975 and 3.75 ppb to 30ppb, R(2)=0.9991) was obtained, complying perfectly with the acceptable arsenic concentration limits defined by the World Health Organization for drinking water samples (i.e. 10ppb). Therefore, the proposed assay provides a very good alternative for the portable quantification of As (III) in water as corroborated by the analysis of natural groundwater samples from Swiss mountains, which showed a very good agreement with the results obtained by atomic absorption spectroscopy. PMID:23584229

  8. Comparison of toxicities of acellular pertussis vaccine with whole cell pertussis vaccine in experimental animals.

    PubMed

    Sato, Y; Sato, H

    1991-01-01

    There is no suitable animal model for pertussis encephalopathy in humans. In this study, we have compared the toxicity of acellular pertussis vaccine with whole cell pertussis vaccine in mice or guinea pigs. Two lots of acellular and two lots of whole cell vaccine produced in different countries were assayed in the test. 1. There was no statistical difference in mouse protective potency between these acellular or whole cell pertussis vaccines. 2. There were no differences in chemical ingredients between acellular and whole cell pertussis vaccines except for protein nitrogen content. The protein nitrogen content of whole cell vaccine was at least three times higher than that of the acellular product. 3. Anti-PT antibody productivity of the acellular vaccine was higher than that of the whole cell vaccine. 4. Anti-agglutinogen antibody productivity of the whole cell vaccine was higher than that of the acellular vaccine. 5. There was no pyrogenic activity with the acellular vaccine, but high pyrogenicity was seen with whole cell vaccine. 6. There was high body-weight decreasing toxicity in mice and guinea pigs by the whole cell vaccine. 7. The mice died when they received whole cell pertussis vaccine iv, but no deaths occurred in the mice which received acellular pertussis vaccine. PMID:1778317

  9. Genetically modified whole-cell bioreporters for environmental assessment

    PubMed Central

    Xu, Tingting; Close, Dan M.; Sayler, Gary S.; Ripp, Steven

    2015-01-01

    Living whole-cell bioreporters serve as environmental biosentinels that survey their ecosystems for harmful pollutants and chemical toxicants, and in the process act as human and other higher animal proxies to pre-alert for unfavorable, damaging, or toxic conditions. Endowed with bioluminescent, fluorescent, or colorimetric signaling elements, bioreporters can provide a fast, easily measured link to chemical contaminant presence, bioavailability, and toxicity relative to a living system. Though well tested in the confines of the laboratory, real-world applications of bioreporters are limited. In this review, we will consider bioreporter technologies that have evolved from the laboratory towards true environmental applications, and discuss their merits as well as crucial advancements that still require adoption for more widespread utilization. Although the vast majority of environmental monitoring strategies rely upon bioreporters constructed from bacteria, we will also examine environmental biosensing through the use of less conventional eukaryotic-based bioreporters, whose chemical signaling capacity facilitates a more human-relevant link to toxicity and health-related consequences. PMID:26594130

  10. Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

    PubMed Central

    Foehring, Robert C.; Guan, Dongxu; Toleman, Tara; Cantrell, Angela R.

    2011-01-01

    We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex. Because of the lack of specific pharmacological agents for these channels, we have taken a genetic approach to manipulating channel expression. We use an organotypic culture preparation (16) in order to maintain cell morphology and the laminar pattern of cortex. We typically isolate acute neocortical slices at postnatal days 8-10 and maintain the slices in culture for 3-7 days. This allows us to study neurons at a similar age to those in our work with acute slices and minimizes the development of exuberant excitatory connections in the slice. We record from visually-identified pyramidal neurons in layers II/III or V using infrared illumination (IR-) and differential interference contrast microscopy (DIC) with whole cell patch clamp in current- or voltage-clamp. We use biolistic (Gene gun) transfection of wild type or mutant potassium channel DNA to manipulate expression of the channels to study their function. The transfected cells are easily identified by epifluorescence microscopy after co-transfection with cDNA for green fluorescent protein (GFP). We compare recordings of transfected cells to adjacent, untransfected neurons in the same layer from the same slice. PMID:21673642

  11. Whole Cell Vaccines — Past Progress and Future Strategies

    PubMed Central

    Keenan, Bridget P.; Jaffee, Elizabeth M.

    2012-01-01

    Cancer vaccines have shown success in curing tumors in pre-clinical models. Accumulating evidence also supports their ability to induce immune responses in patients. In many cases, these responses correlate with improved clinical outcomes. However, cancer vaccines have not yet demonstrated their true potential in clinical trials. This is likely due to the difficulty in mounting a significant antitumor response in patients with advanced disease because of preexisting tolerance mechanisms that are actively turning off immune recognition in cancer patients. This review will examine the recent progress being made in the design and implementation of whole cell cancer vaccines, one vaccine approach that simultaneously targets multiple tumor antigens to activate the immune response. These vaccines have been shown to induce antigen specific T cell responses. Pre-clinical studies evaluating these vaccines given in sequence with other agents and cancer treatment modalities support the use of immunomodulating doses of chemotherapy and radiation, as well as immune modulating pathway targeted monoclonal antibodies, to enhance the efficacy of cancer vaccines. Based on emerging pre-clinical data, clinical trials are currently exploring the use of combinatorial immune based therapies for the treatment of cancer. PMID:22595050

  12. Whole Cell Modeling: From Single Cells to Colonies

    PubMed Central

    Cole, John A.

    2015-01-01

    A great deal of research over the last several years has focused on how the inherent randomness in movements and reactivity of biomolecules can give rise to unexpected large-scale differences in the behavior of otherwise identical cells. Our own research has approached this problem from two vantage points – a microscopic kinetic view of the individual molecules (nucleic acids, proteins, etc.) diffusing and interacting in a crowded cellular environment; and a broader systems-level view of how enzyme variability can give rise to well-defined metabolic phenotypes. The former led to the development of the Lattice Microbes software – a GPU-accelerated stochastic simulator for reaction-diffusion processes in models of whole cells; the latter to the development of a method we call population flux balance analysis (FBA). The first part of this article reviews the Lattice Microbes methodology, and two recent technical advances that extend the capabilities of Lattice Microbes to enable simulations of larger organisms and colonies. The second part of this article focuses on our recent population FBA study of Escherichia coli, which predicted variability in the usage of different metabolic pathways resulting from heterogeneity in protein expression. Finally, we discuss exciting early work using a new hybrid methodology that integrates FBA with spatially resolved kinetic simulations to study how cells compete and cooperate within dense colonies and consortia. PMID:26989262

  13. An Approach for Lactulose Production Using the CotX-Mediated Spore-Displayed β-Galactosidase as a Biocatalyst.

    PubMed

    Wang, He; Yang, Ruijin; Hua, Xiao; Zhang, Wenbin; Zhao, Wei

    2016-07-28

    Currently, enzymatic synthesis of lactulose, a synthetic prebiotic disaccharide, is commonly performed with glycosyl hydrolases. In this work, a new type of lactulose-producing biocatalyst was developed by displaying β-galactosidase from Bacillus stearothermophilus IAM11001 (Bs-β-Gal) on the surface of Bacillus subtilis 168 spores. Localization of β-Gal on the spore surface as a fusion to CotX was verified by western blot analysis, immunofluorescence microscopy, and flow cytometry. The optimum pH and temperature for the resulting spore-displayed β-Gal was 6.0 and 75°C, respectively. Under optimal conditions, it showed maximum activity of 0.42 U/mg spores (dry weight). Moreover, the spore-displayed CotX-β-Gal was employed as a whole cell biocatalyst to produce lactulose, yielding 8.8 g/l from 200 g/l lactose and 100 g/l fructose. Reusability tests showed that the spore-displayed CotX-β-Gal retained around 30.3% of its initial activity after eight successive conversion cycles. These results suggest that the CotX-mediated spore-displayed β-Gal may provide a promising strategy for lactulose production.

  14. Understanding biocatalyst inhibition by carboxylic acids

    PubMed Central

    Jarboe, Laura R.; Royce, Liam A.; Liu, Ping

    2013-01-01

    Carboxylic acids are an attractive biorenewable chemical in terms of their flexibility and usage as precursors for a variety of industrial chemicals. It has been demonstrated that such carboxylic acids can be fermentatively produced using engineered microbes, such as Escherichia coli and Saccharomyces cerevisiae. However, like many other attractive biorenewable fuels and chemicals, carboxylic acids become inhibitory to these microbes at concentrations below the desired yield and titer. In fact, their potency as microbial inhibitors is highlighted by the fact that many of these carboxylic acids are routinely used as food preservatives. This review highlights the current knowledge regarding the impact that saturated, straight-chain carboxylic acids, such as hexanoic, octanoic, decanoic, and lauric acids can have on E. coli and S. cerevisiae, with the goal of identifying metabolic engineering strategies to increase robustness. Key effects of these carboxylic acids include damage to the cell membrane and a decrease of the microbial internal pH. Certain changes in cell membrane properties, such as composition, fluidity, integrity, and hydrophobicity, and intracellular pH are often associated with increased tolerance. The availability of appropriate exporters, such as Pdr12, can also increase tolerance. The effect on metabolic processes, such as maintaining appropriate respiratory function, regulation of Lrp activity and inhibition of production of key metabolites such as methionine, are also considered. Understanding the mechanisms of biocatalyst inhibition by these desirable products can aid in the engineering of robust strains with improved industrial performance. PMID:24027566

  15. Utilization of biocatalysts in cellulose waste minimization

    SciTech Connect

    Woodward, J.; Evans, B.R.

    1996-09-01

    Cellulose, a polymer of glucose, is the principal component of biomass and, therefore, a major source of waste that is either buried or burned. Examples of biomass waste include agricultural crop residues, forestry products, and municipal wastes. Recycling of this waste is important for energy conservation as well as waste minimization and there is some probability that in the future biomass could become a major energy source and replace fossil fuels that are currently used for fuels and chemicals production. It has been estimated that in the United States, between 100-450 million dry tons of agricultural waste are produced annually, approximately 6 million dry tons of animal waste, and of the 190 million tons of municipal solid waste (MSW) generated annually, approximately two-thirds is cellulosic in nature and over one-third is paper waste. Interestingly, more than 70% of MSW is landfilled or burned, however landfill space is becoming increasingly scarce. On a smaller scale, important cellulosic products such as cellulose acetate also present waste problems; an estimated 43 thousand tons of cellulose ester waste are generated annually in the United States. Biocatalysts could be used in cellulose waste minimization and this chapter describes their characteristics and potential in bioconversion and bioremediation processes.

  16. Engineered yeast whole-cell biocatalyst for direct degradation of alginate from macroalgae and production of non-commercialized useful monosaccharide from alginate.

    PubMed

    Takagi, Toshiyuki; Yokoi, Takahiro; Shibata, Toshiyuki; Morisaka, Hironobu; Kuroda, Kouichi; Ueda, Mitsuyoshi

    2016-02-01

    Alginate is a major component of brown macroalgae. In macroalgae, an endolytic alginate lyase first degrades alginate into oligosaccharides. These oligosaccharides are further broken down into monosaccharides by an exolytic alginate lyase. In this study, genes encoding various alginate lyases derived from alginate-assimilating marine bacterium Saccharophagus degradans were isolated, and their enzymes were displayed using the yeast cell surface display system. Alg7A-, Alg7D-, and Alg18J-displaying yeasts showed endolytic alginate lyase activity. On the other hand, Alg7K-displaying yeast showed exolytic alginate lyase activity. Alg7A, Alg7D, Alg7K, and Alg18J, when displayed on yeast cell surface, demonstrated both polyguluronate lyase and polymannuronate lyase activities. Additionally, polyguluronic acid could be much easily degraded by Alg7A, Alg7K, and Alg7D than polymannuronic acid. In contrast, polymannuronic acid could be much easily degraded by Alg18J than polyguluronic acid. We further constructed yeasts co-displaying endolytic and exolytic alginate lyases. Degradation efficiency by the co-displaying yeasts were significantly higher than single alginate lyase-displaying yeasts. Alg7A/Alg7K co-displaying yeast had maximum alginate degrading activity, with production of 1.98 g/L of reducing sugars in a 60-min reaction. This system developed, along with our findings, will contribute to the efficient utilization and production of useful and non-commercialized monosaccharides from alginate by Saccharomyces cerevisiae. PMID:26490549

  17. Aquifer Microbial Diversity Represented by Whole Cell and Dissolved DNA

    NASA Astrophysics Data System (ADS)

    Shields, M. S.; Briggs, B.

    2004-12-01

    Microbial diversity measurements of aquifers are faced with two major sources of bias. These are a reflection of molecular (caused by amplification, labeling and extraction dissimilarities) and sampling aspects. Sampling of aquifers represents a considerable and often insurmountable bias since almost all evaluations rely on water samples where free living bacteria will predominate. This highly problematic when one desires to know the metagenomic potential of zone of well influence. We present a method designed to avoid cellular collection biases imposed by the difficulty in collecting attached biomass. Dissolved DNA (d-DNA) and whole bacterial cells were concentrated from Snake River Plain aquifer water. 96% of dDNA was recovered from DNA spiked surrogates using an anion-exchange membrane method. Approximately 2,000 ng DNA was recovered per liter of water. Total DNA turnover rate was measured at 49.3 ng/ml/day, at DNA concentrations above 1 mg/l, indicating that aquifer dDNA represents a dynamic steady state balanced between dDNA release and native DNA degradation activities. 16s, dual labeled T-RFLP analysis was used analyze the genomic complexity of whole bacterial cells collected via filtration versus d-DNA sources of bacterial DNA from 16 l of water. Highly similar T-RFLP profiles (with two restriction endonucleases) were obtained for both dDNA (200 nm filtrate) and the whole cells harvested from the same filter. This demonstrates that dDNA was an accurate reflection of the total bacterial community found within the well volume. We present evaluations of the anion-exchange system with respect to DNA recovery parameters, and demonstrate the suitability of this DNA template for PCR amplification reactions.

  18. Whole-cell bioluminescent bioreporter sensing of foodborne toxicants

    NASA Astrophysics Data System (ADS)

    Ripp, Steve A.; Applegate, Bruce M.; Simpson, Michael L.; Sayler, Gary S.

    2001-03-01

    The presence of biologically derived toxins in foods is of utmost significance to food safety and human health concerns. Biologically active amines, referred to as biogenic amines, serve as a noteworthy example, having been implicated as the causative agent in numerous food poisoning episodes. Of the various biogenic amines encountered, histamine, putrescine, cadaverine, tyramine, tryptamine, beta-phenylethylamine, spermine, and spermidine are considered to be the most significant, and can be used as hygienic-quality indicators of food. Biogenic amines can be monitored using whole-cell bioluminescent bioreporters, which represent a family of genetically engineered microorganisms that generate visible light in response to specific chemical or physical agents in their environment. The light response occurs due to transcriptional activation of a genetically incorporated lux cassette, and can be measured using standard photomultiplier devices. We have successfully engineered a lux-based bioreporter capable of detecting and monitoring the biogenic amine beta-phenylethylamine. This research represents a biologically-based sensor technology that can be readily integrated into Hazard Analysis Critical Control Point programs to provide a rugged monitoring regime that can be uniformly applied for field-based and in-house laboratory quality control analyses. Since the bioreporter and biosensing elements are completely self-contained within the sensor design, this system provides ease of use, with operational capabilities realized by simply combining the food sample with the bioreporter and allowing the sensor to process the ensuing bioluminescent signal and communicate the results. The application of this technology to the critically important issue of food safety and hygienic quality represents a novel method for detecting, monitoring, and preventing biologically active toxins in food commodities.

  19. Biotransformation of acetoin to 2,3-butanediol: Assessment of plant and microbial biocatalysts.

    PubMed

    Javidnia, Katayoun; Faghih-Mirzaei, Ehsan; Miri, Ramin; Attarroshan, Mahshid; Zomorodian, Kamiar

    2016-07-01

    2,3-Butanediol (2,3-BD) is a valuable bulk chemical owing to its extensive application in chemical and pharmaceutical industry with diverse applications in drug, cosmetics and food products. In the present study, the biotransformation of acetoin to 2,3-BD by five plant species (Brassica oleracea, Brassica rapa, Daucuscarota, Pastinaca sativa, and Raphnussativus) and five microorganisms (Aspergillusfoetidus, Penicillumcitrinum, Saccharomyces carlbergensis, Pichiafermentans, and Rhodotrulaglutinis) was investigated as a method for the production of 2,3-BD, which can serve as an alternative to the common pentoses and hexoses fermentation by microorganisms. The produced 2,3-BD stereoisomers were characterized and their total conversion yields were determined. The results showed that the examined plants can be used as a green factory for the production of all 2,3-BD stereoisomers, except B. rapa. In microorganisms, P. fermentans and S. carlbergensis produced (-)-2R,3R and mesobutanediol, while P. citrinum produced (+)-2S,3S and mesobutanediol. R. glutinis and A. foetidus produced all three isomers. In conclusion, efficient whole-cell biocatalysts from plants and microorganisms were determined in the bioconversion of acetoin to 2,3-BD. The profile of produced stereoisomers demonstrated that microorganisms produce more specific stereoisomers. PMID:27651816

  20. Biotransformation of acetoin to 2,3-butanediol: Assessment of plant and microbial biocatalysts.

    PubMed

    Javidnia, Katayoun; Faghih-Mirzaei, Ehsan; Miri, Ramin; Attarroshan, Mahshid; Zomorodian, Kamiar

    2016-07-01

    2,3-Butanediol (2,3-BD) is a valuable bulk chemical owing to its extensive application in chemical and pharmaceutical industry with diverse applications in drug, cosmetics and food products. In the present study, the biotransformation of acetoin to 2,3-BD by five plant species (Brassica oleracea, Brassica rapa, Daucuscarota, Pastinaca sativa, and Raphnussativus) and five microorganisms (Aspergillusfoetidus, Penicillumcitrinum, Saccharomyces carlbergensis, Pichiafermentans, and Rhodotrulaglutinis) was investigated as a method for the production of 2,3-BD, which can serve as an alternative to the common pentoses and hexoses fermentation by microorganisms. The produced 2,3-BD stereoisomers were characterized and their total conversion yields were determined. The results showed that the examined plants can be used as a green factory for the production of all 2,3-BD stereoisomers, except B. rapa. In microorganisms, P. fermentans and S. carlbergensis produced (-)-2R,3R and mesobutanediol, while P. citrinum produced (+)-2S,3S and mesobutanediol. R. glutinis and A. foetidus produced all three isomers. In conclusion, efficient whole-cell biocatalysts from plants and microorganisms were determined in the bioconversion of acetoin to 2,3-BD. The profile of produced stereoisomers demonstrated that microorganisms produce more specific stereoisomers.

  1. Biotransformation of acetoin to 2,3-butanediol: Assessment of plant and microbial biocatalysts

    PubMed Central

    Javidnia, Katayoun; Faghih-Mirzaei, Ehsan; Miri, Ramin; Attarroshan, Mahshid; Zomorodian, Kamiar

    2016-01-01

    2,3-Butanediol (2,3-BD) is a valuable bulk chemical owing to its extensive application in chemical and pharmaceutical industry with diverse applications in drug, cosmetics and food products. In the present study, the biotransformation of acetoin to 2,3-BD by five plant species (Brassica oleracea, Brassica rapa, Daucuscarota, Pastinaca sativa, and Raphnussativus) and five microorganisms (Aspergillusfoetidus, Penicillumcitrinum, Saccharomyces carlbergensis, Pichiafermentans, and Rhodotrulaglutinis) was investigated as a method for the production of 2,3-BD, which can serve as an alternative to the common pentoses and hexoses fermentation by microorganisms. The produced 2,3-BD stereoisomers were characterized and their total conversion yields were determined. The results showed that the examined plants can be used as a green factory for the production of all 2,3-BD stereoisomers, except B. rapa. In microorganisms, P. fermentans and S. carlbergensis produced (–)-2R,3R and mesobutanediol, while P. citrinum produced (+)-2S,3S and mesobutanediol. R. glutinis and A. foetidus produced all three isomers. In conclusion, efficient whole-cell biocatalysts from plants and microorganisms were determined in the bioconversion of acetoin to 2,3-BD. The profile of produced stereoisomers demonstrated that microorganisms produce more specific stereoisomers.

  2. Biotransformation of acetoin to 2,3-butanediol: Assessment of plant and microbial biocatalysts

    PubMed Central

    Javidnia, Katayoun; Faghih-Mirzaei, Ehsan; Miri, Ramin; Attarroshan, Mahshid; Zomorodian, Kamiar

    2016-01-01

    2,3-Butanediol (2,3-BD) is a valuable bulk chemical owing to its extensive application in chemical and pharmaceutical industry with diverse applications in drug, cosmetics and food products. In the present study, the biotransformation of acetoin to 2,3-BD by five plant species (Brassica oleracea, Brassica rapa, Daucuscarota, Pastinaca sativa, and Raphnussativus) and five microorganisms (Aspergillusfoetidus, Penicillumcitrinum, Saccharomyces carlbergensis, Pichiafermentans, and Rhodotrulaglutinis) was investigated as a method for the production of 2,3-BD, which can serve as an alternative to the common pentoses and hexoses fermentation by microorganisms. The produced 2,3-BD stereoisomers were characterized and their total conversion yields were determined. The results showed that the examined plants can be used as a green factory for the production of all 2,3-BD stereoisomers, except B. rapa. In microorganisms, P. fermentans and S. carlbergensis produced (–)-2R,3R and mesobutanediol, while P. citrinum produced (+)-2S,3S and mesobutanediol. R. glutinis and A. foetidus produced all three isomers. In conclusion, efficient whole-cell biocatalysts from plants and microorganisms were determined in the bioconversion of acetoin to 2,3-BD. The profile of produced stereoisomers demonstrated that microorganisms produce more specific stereoisomers. PMID:27651816

  3. Lactobacillus casei as a biocatalyst for biofuel production.

    PubMed

    Vinay-Lara, Elena; Wang, Song; Bai, Lina; Phrommao, Ekkarat; Broadbent, Jeff R; Steele, James L

    2016-09-01

    Microbial fermentation of sugars from plant biomass to alcohols represents an alternative to petroleum-based fuels. The optimal biocatalyst for such fermentations needs to overcome hurdles such as high concentrations of alcohols and toxic compounds. Lactic acid bacteria, especially lactobacilli, have high innate alcohol tolerance and are remarkably adaptive to harsh environments. This study assessed the potential of five Lactobacillus casei strains as biocatalysts for alcohol production. L. casei 12A was selected based upon its innate alcohol tolerance, high transformation efficiency and ability to utilize plant-derived carbohydrates. A 12A derivative engineered to produce ethanol (L. casei E1) was compared to two other bacterial biocatalysts. Maximal growth rate, maximal optical density and ethanol production were determined under conditions similar to those present during alcohol production from lignocellulosic feedstocks. L. casei E1 exhibited higher innate alcohol tolerance, better growth in the presence of corn stover hydrolysate stressors, and resulted in higher ethanol yields. PMID:27312380

  4. Apparatus for the production of gel beads containing a biocatalyst

    DOEpatents

    Scott, C.D.; Scott, T.C.; Davison, B.H.

    1998-03-19

    An apparatus is described for the large-scale and continuous production of gel beads containing a biocatalyst. The apparatus is a columnar system based on the chemical cross-linking of hydrocolloidal gels that contain and immobilize a biocatalyst, the biocatalyst being a microorganism or an enzyme. Hydrocolloidal gels, such as alginate, carrageenan, and a mixture of bone gelatin and modified alginate, provide immobilization matrices that can be used to entrap and retain the biocatalyst while allowing effective contact with substrates and release of products. Such immobilized biocatalysts are generally formulated into small spheres or beads that have high concentrations of the biocatalyst within the gel matrix. The columnar system includes a gel dispersion nozzle submerged in a heated non-interacting liquid, typically an organic liquid, that is immiscible with water to allow efficient formation of spherical gel droplets, the non-interacting liquid having a specific gravity that is less than water so that the gel droplets will fall through the liquid by the force of gravity. The heated non-interacting liquid is in direct contact with a chilled upflowing non-interacting liquid that will provide sufficient residence time for the gel droplets as they fall through the liquid so that they will be cooled below the gelling temperature and form solid spheres. The upflowing non-interacting liquid is in direct contact with an upflowing temperature-controlled aqueous solution containing the necessary chemicals for cross-linking or fixing of the gel beads to add the necessary stability. The flow rates of the two liquid streams can be varied to control the proper residence time in each liquid section to accommodate the production of gel beads of differing settling velocities. A valve is provided for continuous removal of the stabilized gel beads from the bottom of the column. 1 fig.

  5. Apparatus for the production of gel beads containing a biocatalyst

    DOEpatents

    Scott, Charles D.; Scott, Timothy C.; Davison, Brian H.

    1998-01-01

    An apparatus for the large-scale and continuous production of gel beads containing a biocatalyst. The apparatus is a columnar system based on the chemical cross-linking of hydrocolloidal gels that contain and immobilize a biocatalyst, the biocatalyst being a microorganism or an enzyme. Hydrocolloidal gels, such as alginate, carrageenan, and a mixture of bone gelatin and modified alginate, provide immobilization matrices that can be used to entrap and retain the biocatalyst while allowing effective contact with substrates and release of products. Such immobilized biocatalysts are generally formulated into small spheres or beads that have high concentrations of the biocatalyst within the gel matrix. The columnar system includes a gel dispersion nozzle submerged in a heated non-interacting liquid, typically an organic liquid, that is immiscible with water to allow efficient formation of spherical gel droplets, the non-interacting liquid having a specific gravity that is less than water so that the gel droplets will fall through the liquid by the force of gravity. The heated non-interacting liquid is in direct contact with a chilled upflowing non-interacting liquid that will provide sufficient residence time for the gel droplets as they fall through the liquid so that they will be cooled below the gelling temperature and form solid spheres. The upflowing non-interacting liquid is in direct contact with an upflowing temperature-controlled aqueous solution containing the necessary chemicals for cross-linking or fixing of the gel beads to add the necessary stability. The flow rates of the two liquid streams can be varied to control the proper residence time in each liquid section to accommodate the production of gel beads of differing settling velocities. A valve is provided for continuous removal of the stabilized gel beads from the bottom of the column.

  6. Summary of the DREAM8 Parameter Estimation Challenge: Toward Parameter Identification for Whole-Cell Models

    PubMed Central

    Karr, Jonathan R.; Williams, Alex H.; Zucker, Jeremy D.; Raue, Andreas; Steiert, Bernhard; Timmer, Jens; Kreutz, Clemens; Wilkinson, Simon; Allgood, Brandon A.; Bot, Brian M.; Hoff, Bruce R.; Kellen, Michael R.; Covert, Markus W.; Stolovitzky, Gustavo A.; Meyer, Pablo

    2015-01-01

    Whole-cell models that explicitly represent all cellular components at the molecular level have the potential to predict phenotype from genotype. However, even for simple bacteria, whole-cell models will contain thousands of parameters, many of which are poorly characterized or unknown. New algorithms are needed to estimate these parameters and enable researchers to build increasingly comprehensive models. We organized the Dialogue for Reverse Engineering Assessments and Methods (DREAM) 8 Whole-Cell Parameter Estimation Challenge to develop new parameter estimation algorithms for whole-cell models. We asked participants to identify a subset of parameters of a whole-cell model given the model’s structure and in silico “experimental” data. Here we describe the challenge, the best performing methods, and new insights into the identifiability of whole-cell models. We also describe several valuable lessons we learned toward improving future challenges. Going forward, we believe that collaborative efforts supported by inexpensive cloud computing have the potential to solve whole-cell model parameter estimation. PMID:26020786

  7. Reversible covalent immobilization of Trametes villosa laccase onto thiolsulfinate-agarose: An insoluble biocatalyst with potential for decoloring recalcitrant dyes.

    PubMed

    Gioia, Larissa; Rodríguez-Couto, Susana; Menéndez, María Del Pilar; Manta, Carmen; Ovsejevi, Karen

    2015-01-01

    The development of a solid-phase biocatalyst based on the reversible covalent immobilization of laccase onto thiol-reactive supports (thiolsulfinate-agarose [TSI-agarose]) was performed. To achieve this goal, laccase-producing strains isolated from Eucalyptus globulus were screened and white rot fungus Trametes villosa was selected as the best strain for enzyme production. Reduction of disulfide bonds and introduction of "de novo" thiol groups in partially purified laccase were assessed to perform its reversible covalent immobilization onto thiol-reactive supports (TSI-agarose). Only the thiolation process dramatically improved the immobilization yield, from 0% for the native and reduced enzyme to 60% for the thiolated enzyme. Mild conditions for the immobilization process (pH 7.5 and 4°C) allowed the achievement of nearly 100% of coupling efficiency when low loads were applied. The kinetic parameters, pH, and thermal stabilities for the immobilized biocatalyst were similar to those for the native enzyme. After the first use and three consecutives reuses, the insoluble derivative kept more than 80% of its initial capacity for decolorizing Remazol Brilliant Blue R, showing its suitability for color removal from textile industrial effluents. The possibility of reusing the support was demonstrated by the reversibility of enzyme-support binding. PMID:25196324

  8. Evaluation of a ceftiofur-washed whole cell Streptococcus suis bacterin in pigs

    PubMed Central

    2004-01-01

    Abstract The efficacy of currently available washed whole cell Streptococcus suis bacterins is generally poor. We developed and tested the efficacy of a novel ceftiofur-washed whole cell bacterin. Sixty-six, 2-week-old specific pathogen free (SPF) pigs were randomly divided into 5 groups. Three groups were vaccinated 28 and 14 d prior to challenge. The 3 ceftiofur-washed whole cell bacterins each contained 1 of 3 different adjuvants (Montanide ISA 25, Montanide ISA 50, and Saponin). Pigs exhibiting severe central nervous system disease or severe joint swelling and lameness were euthanized immediately and necropsied. All remaining pigs were necropsied at 14 d post inoculation. The ceftiofur-washed whole cell S. suis bacterin with Montanide ISA 50 adjuvant significantly (P < 0.05) reduced bacteremia, meningitis, pneumonia, and mortality associated with S. suis challenge. Further work on this novel approach to bacterin production is warranted. PMID:15352553

  9. Integrated lipase production and in situ biodiesel synthesis in a recombinant Pichia pastoris yeast: an efficient dual biocatalytic system composed of cell free enzymes and whole cell catalysts

    PubMed Central

    2014-01-01

    Background Lipase-catalyzed biotransformation of acylglycerides or fatty acids into biodiesel via immobilized enzymes or whole cell catalysts has been considered as one of the most promising methods to produce renewable and environmentally friendly alternative liquid fuels, thus being extensively studied so far. In all previously pursued approaches, however, lipase enzymes are prepared in an independent process separated from enzymatic biodiesel production, which would unavoidably increase the cost and energy consumption during industrial manufacture of this cost-sensitive energy product. Therefore, there is an urgent need to develop novel cost-effective biocatalysts and biocatalytic processes with genuine industrial feasibility. Result Inspired by the consolidated bioprocessing of lignocellulose to generate bioethanol, an integrated process with coupled lipase production and in situ biodiesel synthesis in a recombinant P. pastoris yeast was developed in this study. The novel and efficient dual biocatalytic system based on Thermomyces lanuginosus lipase took advantage of both cell free enzymes and whole cell catalysts. The extracellular and intracellular lipases of growing yeast cells were simultaneously utilized to produce biodiesel from waste cooking oils in situ and in one pot. This integrated system effectively achieved 58% and 72% biodiesel yield via concurrent esterified-transesterified methanolysis and stepwise hydrolysis-esterification at 3:1 molar ratio between methanol and waste cooking oils, respectively. Further increasing the molar ratio of methanol to waste cooking oils to 6:1 led to an 87% biodiesel yield using the stepwise strategy. Both water tolerance and methanol tolerance of this novel system were found to be significantly improved compared to previous non-integrated biodiesel production processes using separately prepared immobilized enzymes or whole cell catalysts. Conclusion We have proposed a new concept of integrated biodiesel production

  10. Cyanobacteria as photosynthetic biocatalysts: a systems biology perspective.

    PubMed

    Gudmundsson, Steinn; Nogales, Juan

    2015-01-01

    The increasing need to replace oil-based products and to address global climate change concerns has triggered considerable interest in photosynthetic microorganisms. Cyanobacteria, in particular, have great potential as biocatalysts for fuels and fine-chemicals. During the last few years the biotechnological applications of cyanobacteria have experienced an unprecedented increase and the use of these photosynthetic organisms for chemical production is becoming a tangible reality. However, the field is still immature and many concerns about the economic feasibility of the biotechnological potential of cyanobacteria remain. In this review we describe recent successes in biofuel and fine-chemical production using cyanobacteria. We discuss the role of the photosynthetic metabolism and highlight the need for systems-level metabolic optimization in order to achieve the true potential of cyanobacterial biocatalysts.

  11. [Advance in the bioavailability monitoring of heavy metal based on microbial whole-cell sensor].

    PubMed

    Hou, Qi-Hui; Ma, An-Shou; Zhuang, Xiu-Liang; Zhuang, Guo-Qiang

    2013-01-01

    Microbial whole-cell biosensor is an excellent tool to assess the bioavailability of heavy metal in soil and water. However, the traditional physicochemical instruments are applied to detect the total metal. Furthermore, microbial whole-cell biosensor is simple, rapid and economical in manipulating, and is thus a highly qualified candidate for emergency detection of pollution incidents. The biological component of microbial whole-cell biosensor mostly consists of metalloregulatory proteins and reporter genes. In detail, metalloregulatory proteins mainly include the MerR family, ArsR family and RS family, and reporter genes mainly include gfp, lux and luc. Metalloregulatory protein and reporter gene are related to the sensitivity, specificity and properties in monitoring. The bioavailability of heavy metals is alterable under different conditions, influenced by pH, chelate and detection methods and so on. Increasing the accumulation of intracellular heavy metal, modifying the metalloregulatory proteins and optimizing the detecting conditions are important for improving the sensitivity, specificity and accuracy of the microbial whole-cell biosensor. The future direction of microbial whole-cell biosensor is to realize the monitoring of pollutions in situ and on line.

  12. [Advance in the bioavailability monitoring of heavy metal based on microbial whole-cell sensor].

    PubMed

    Hou, Qi-Hui; Ma, An-Shou; Zhuang, Xiu-Liang; Zhuang, Guo-Qiang

    2013-01-01

    Microbial whole-cell biosensor is an excellent tool to assess the bioavailability of heavy metal in soil and water. However, the traditional physicochemical instruments are applied to detect the total metal. Furthermore, microbial whole-cell biosensor is simple, rapid and economical in manipulating, and is thus a highly qualified candidate for emergency detection of pollution incidents. The biological component of microbial whole-cell biosensor mostly consists of metalloregulatory proteins and reporter genes. In detail, metalloregulatory proteins mainly include the MerR family, ArsR family and RS family, and reporter genes mainly include gfp, lux and luc. Metalloregulatory protein and reporter gene are related to the sensitivity, specificity and properties in monitoring. The bioavailability of heavy metals is alterable under different conditions, influenced by pH, chelate and detection methods and so on. Increasing the accumulation of intracellular heavy metal, modifying the metalloregulatory proteins and optimizing the detecting conditions are important for improving the sensitivity, specificity and accuracy of the microbial whole-cell biosensor. The future direction of microbial whole-cell biosensor is to realize the monitoring of pollutions in situ and on line. PMID:23487961

  13. [Effect of sequential biocatalyst addition on Anammox process].

    PubMed

    Tang, Chongjian; Zheng, Ping; Chen, Jianwei

    2011-01-01

    Anaerobic ammonium oxidation (Anammox) process is a high-rate nitrogen removal technology that has been applied in sludge dewatering effluents treatment with nitrogen removal rate as high as 9.5 kg/(m x d). However, due to the slow growth rate of the autotrophic Anammox bacteria and the susceptivity to environmental conditions, the start-up of Anammox process is very long; the operation is unstable; and the nitrogen removal from organic-containing and/or toxicant-containing ammonium-rich wastewaters using Anammox process becomes difficult. Thus, the application of this high-rate process is significantly limited. In this paper, a newly-developed Anammox process with sequential biocatalyst (Anammox biomass) addition was established based on the procedure in fermentation engineering. We introduced the Anammox process with sequential biocatalyst addition on start-up, stable operation and the treatment of organic-containing and toxicant-containing ammonium-rich wastewaters. Results show that supplementing high-activity Anammox biomass into reactors will increase the amount of as well as the ratio of Anammox bacteria. Thus, the innovative Anammox process with sequential biocatalyst addition not only accelerates the start-up course, but also enhances the stability of Anammox process. Furthermore, it overcomes the drawbacks of wastewaters containing high organic content and toxic substances. Therefore, the application of Anammox process may be further enlarged. PMID:21553484

  14. Prospecting for Novel Biocatalysts in a Soil Metagenome

    PubMed Central

    Voget, S.; Leggewie, C.; Uesbeck, A.; Raasch, C.; Jaeger, K.-E.; Streit, W. R.

    2003-01-01

    The metagenomes of complex microbial communities are rich sources of novel biocatalysts. We exploited the metagenome of a mixed microbial population for isolation of more than 15 different genes encoding novel biocatalysts by using a combined cultivation and direct cloning strategy. A 16S rRNA sequence analysis revealed the presence of hitherto uncultured microbes closely related to the genera Pseudomonas, Agrobacterium, Xanthomonas, Microbulbifer, and Janthinobacterium. Total genomic DNA from this bacterial community was used to construct cosmid DNA libraries, which were functionally searched for novel enzymes of biotechnological value. Our searches in combination with cosmid sequencing resulted in identification of four clones encoding 12 putative agarase genes, most of which were organized in clusters consisting of two or three genes. Interestingly, nine of these agarase genes probably originated from gene duplications. Furthermore, we identified by DNA sequencing several other biocatalyst-encoding genes, including genes encoding a putative stereoselective amidase (amiA), two cellulases (gnuB and uvs080), an α-amylase (amyA), a 1,4-α-glucan branching enzyme (amyB), and two pectate lyases (pelA and uvs119). Also, a conserved cluster of two lipase genes was identified, which was linked to genes encoding a type I secretion system. The novel gene aguB was overexpressed in Escherichia coli, and the enzyme activities were determined. Finally, we describe more than 162 kb of DNA sequence that provides a strong platform for further characterization of this microbial consortium. PMID:14532085

  15. In Vivo Whole-Cell Patch-Clamp Recording in the Zebrafish Brain.

    PubMed

    Zhang, Rong-Wei; Du, Jiu-Lin

    2016-01-01

    Zebrafish (Danio rerio) is a newly emerged vertebrate animal model with a conserved gross architecture of the brain and a rich repertoire of behaviors. Due to the optical transparency and structural simplicity of its brain, larval zebrafish has become an ideal in vivo model for dissecting neural mechanisms of brain functions at a whole-brain scale based on a strategy that spans scales from synapses, neurons, and circuits to behaviors. Whole-cell patch-clamp recording is an indispensable approach for studying synaptic and circuit mechanisms of brain functions. Due to the small size of neurons in the zebrafish brain, it is challenging to get whole-cell recordings from these cells. Here, we describe a protocol for obtaining in vivo whole-cell patch-clamp recordings from neurons in larval zebrafish. PMID:27464815

  16. Environmental sensing of heavy metals through whole cell microbial biosensors: a synthetic biology approach.

    PubMed

    Bereza-Malcolm, Lara Tess; Mann, Gülay; Franks, Ashley Edwin

    2015-05-15

    Whole cell microbial biosensors are offering an alternative means for rapid, on-site heavy metal detection. Based in microorganisms, biosensing constructs are designed and constructed to produce both qualitative and quantitative outputs in response to heavy metal ions. Previous microbial biosensors designs are focused on single-input constructs; however, development of multiplexed systems is resulting in more flexible designs. The movement of microbial biosensors from laboratory based designs toward on-site, functioning heavy metal detectors has been hindered by the toxic nature of heavy metals, along with the lack of specificity of heavy metals promoter elements. Applying a synthetic biology approach with alternative microbial chassis may increase the robustness of microbial biosensors and mitigate these issues. Before full applications are achieved, further consideration has to be made regarding the risk and regulations of whole cell microbial biosensor use in the environment. To this end, a standard framework for future whole cell microbial biosensor design and use is proposed.

  17. Whole-Cell Fluorescent Biosensors for Bioavailability and Biodegradation of Polychlorinated Biphenyls

    PubMed Central

    Liu, Xuemei; Germaine, Kieran J.; Ryan, David; Dowling, David N.

    2010-01-01

    Whole-cell microbial biosensors are one of the newest molecular tools used in environmental monitoring. Such biosensors are constructed through fusing a reporter gene such as lux, gfp or lacZ, to a responsive promoter. There have been many reports of the applications of biosensors, particularly their use in assaying pollutant toxicity and bioavailability. This paper reviews the basic concepts behind the construction of whole-cell microbial biosensors for pollutant monitoring, and describes the applications of two such biosensors for detecting the bioavailability and biodegradation of Polychlorinated Biphenyls (PCBs). PMID:22205873

  18. Thermophilic Gram-Positive Biocatalysts for Biomass Conversion to Ethanol

    SciTech Connect

    Shanmugam, K.T.; Ingram, L.O.; Maupin-Furlow, J.A.; Preston, J.F.; Aldrich, H.C.

    2003-12-01

    Production of energy from renewable sources is receiving increased attention due to the finite nature of fossil fuels and the environmental impact associated with the continued large scale use of fossil energy sources. Biomass, a CO2-neutral abundant resource, is an attractive alternate source of energy. Biomass-derived sugars, such as glucose, xylose, and other minor sugars, can be readily fermented to fuel ethanol and commodity chemicals. Extracellular cellulases produced by fungi are commercially developed for depolymerization of cellulose in biomass to glucose for fermentation by appropriate biocatalysts in a simultaneous saccharification and fermentation (SSF) process. Due to the differences in the optimum conditions for the activity of the fungal cellulases and the growth and fermentation characteristics of the current industrial biocatalysts, SSF of cellulose is envisioned at conditions that are not optimal for the fungal cellulase activity leading to higher than required cost of cellulase in SSF. We have isolated bacterial biocatalysts whose growth and fermentation requirements match the optimum conditions for commercial fungal cellulase activity (pH 5.0 and 50 deg. C). These isolates fermented both glucose and xylose, major components of cellulose and hemicellulose, respectively, to L(+)-lactic acid. Xylose was metabolized through the pentose-phosphate pathway by these organisms as evidenced by the fermentation profile and analysis of the fermentation products of 13C1-xylose by NMR. As expected for the metabolism of xylose by the pentose-phosphate pathway, 13C-lactate accounted for more than 90% of the total 13C-labeled products. All three strains fermented crystalline cellulose to lactic acid with the addition of fungal cellulase (Spezyme CE) (SSF) at an optimum of about 10 FPU/g cellulose. These isolates also fermented cellulose and sugar cane bagasse hemicellulose acid hydrolysate simultaneously. Based on fatty acid profile and 16S rRNA sequence, these

  19. Efficient xylose fermentation by the brown rot fungus Neolentinus lepideus.

    PubMed

    Okamoto, Kenji; Kanawaku, Ryuichi; Masumoto, Masaru; Yanase, Hideshi

    2012-02-10

    The efficient production of bioethanol on an industrial scale requires the use of renewable lignocellulosic biomass as a starting material. A limiting factor in developing efficient processes is identifying microorganisms that are able to effectively ferment xylose, the major pentose sugar found in hemicellulose, and break down carbohydrate polymers without pre-treatment steps. Here, a basidiomycete brown rot fungus was isolated as a new biocatalyst with unprecedented fermentability, as it was capable of converting not only the 6-carbon sugars constituting cellulose, but also the major 5-carbon sugar xylose in hemicelluloses, to ethanol. The fungus was identified as Neolentinus lepideus and was capable of assimilating and fermenting xylose to ethanol in yields of 0.30, 0.33, and 0.34 g of ethanol per g of xylose consumed under aerobic, oxygen-limited, and anaerobic conditions, respectively. A small amount of xylitol was detected as the major by-product of xylose metabolism. N. lepideus produced ethanol from glucose, mannose, galactose, cellobiose, maltose, and lactose with yields ranging from 0.34 to 0.38 g ethanol per g sugar consumed, and also exhibited relatively favorable conversion of non-pretreated starch, xylan, and wheat bran. These results suggest that N. lepideus is a promising candidate for cost-effective and environmentally friendly ethanol production from lignocellulosic biomass. To our knowledge, this is the first report on efficient ethanol fermentation from various carbohydrates, including xylose, by a naturally occurring brown rot fungus.

  20. Fatty acid hydration activity of a recombinant Escherichia coli-based biocatalyst is improved through targeting the oleate hydratase into the periplasm.

    PubMed

    Jung, Sang-Min; Seo, Joo-Hyun; Lee, Jung-Hoo; Park, Jin-Byung; Seo, Jin-Ho

    2015-12-01

    Whole-cell biotransformation of fatty acids can be influenced by the activities of catalytic enzymes and by the efficiency of substrate transport into host cells. Here, we improved fatty acid hydration activity of the recombinant Escherichia coli expressing an oleate hydratase of Stenotrophomonas maltophilia by targeting the catalytic enzyme into the periplasm instead of the cytoplasm. Recombinant E. coli producing OhyA in the periplasm under guidance of the PelB signal sequence (E. coli OhyA_PP) exhibited significantly greater hydration activity with oleic acid and linoleic acid compared to a recombinant E. coli producing OhyA in the cytoplasm (E. coli OhyA_CS). For example, the oleate double bond hydration rate of E. coli OhyA_PP was >400 μmol/g dry cells/min (400 U/g dry cells), which is >10-fold higher than that of E. coli OhyA_CS. As the specific activities of the enzymes targeted into the cytoplasm and periplasm were comparable, we assumed that targeting OhyA into the periplasm could accelerate fatty acid transport to the catalytic enzymes by skipping the major mass transport barrier of the cytoplasmic membrane. Our results will contribute to the development of whole-cell biocatalysts for fatty acid biotransformation.

  1. Glycan-specific whole cell affinity chromatography: a versatile microbial adhesion platform

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have constructed a C-glycoside ketohydrazide affinity chromatography resin that interacts with viable whole-cell microbial populations with biologically appropriate stereo-specificity in a carbohydrate-defined manner. It readily allows for the quantification, selection, and manipulation of target...

  2. Electrophoresis and isoelectric focusing of whole cell and membrane proteins from the extremely halophilic archaebacteria

    NASA Technical Reports Server (NTRS)

    Stan-Lotter, Helga; Lang, Frank J., Jr.; Hochstein, Lawrence I.

    1989-01-01

    The subunits from two purified halobacterial membrane enzymes (ATPase and nitrate reductase) behaved differently with respect to isoelectric focusing, silver staining and interaction with ampholytes. Differential behavior was also observed in whole cell proteins from Halobacterium saccharovorum regarding resolution in two-dimensional gels and silver staining. It is proposed that these differences reflect the existence of two classes of halobacterial proteins.

  3. Whole-cell antigenicity data support sequence-based kinetoplastid taxonomy.

    PubMed

    Couvreur, Bernard

    2013-04-01

    Early immunological data, obtained by immunodiffusion and immunoelectrophoresis, on the whole-cell antigenicity of kinetoplastid protozoa were retrieved and used to construct a dendrogram of antigenic distances. Remarkably, they supported the same taxonomic conclusions as analyses based on DNA and protein sequence data.

  4. Comparison of Three Whole-Cell Pertussis Vaccines in the Baboon Model of Pertussis

    PubMed Central

    Warfel, Jason M.; Zimmerman, Lindsey I.

    2015-01-01

    Pertussis is a highly contagious respiratory illness caused by the bacterial pathogen Bordetella pertussis. Pertussis rates in the United States have escalated since the 1990s and reached a 50-year high of 48,000 cases in 2012. While this pertussis resurgence is not completely understood, we previously showed that the current acellular pertussis vaccines do not prevent colonization or transmission following challenge. In contrast, a whole-cell pertussis vaccine accelerated the rate of clearance compared to rates in unvaccinated animals and animals treated with the acellular vaccine. In order to understand if these results are generalizable, we used our baboon model to compare immunity from whole-cell vaccines from three different manufacturers that are approved outside the United States. We found that, compared to clearance rates with no vaccine and with an acellular pertussis vaccine, immunization with any of the three whole-cell vaccines significantly accelerated the clearance of B. pertussis following challenge. Whole-cell vaccination also significantly reduced the total nasopharyngeal B. pertussis burden, suggesting that these vaccines reduce the opportunity for pertussis transmission. Meanwhile, there was no difference in either the duration or in B. pertussis burden between unvaccinated and acellular-pertussis-vaccinated animals, while previously infected animals were not colonized following reinfection. We also determined that transcription of the gene encoding interleukin-17 (IL-17) was increased in whole-cell-vaccinated and previously infected animals but not in acellular-pertussis-vaccinated animals following challenge. Together with our previous findings, these data are consistent with a role for Th17 responses in the clearance of B. pertussis infection. PMID:26561389

  5. Thermostable Enzymes as Biocatalysts in the Biofuel Industry

    PubMed Central

    Yeoman, Carl J.; Han, Yejun; Dodd, Dylan; Schroeder, Charles M.; Mackie, Roderick I.

    2015-01-01

    Lignocellulose is the most abundant carbohydrate source in nature and represents an ideal renewable energy source. Thermostable enzymes that hydrolyze lignocellulose to its component sugars have significant advantages for improving the conversion rate of biomass over their mesophilic counterparts. We review here the recent literature on the development and use of thermostable enzymes for the depolymerization of lignocellulosic feedstocks for biofuel production. Furthermore, we discuss the protein structure, mechanisms of thermostability, and specific strategies that can be used to improve the thermal stability of lignocellulosic biocatalysts. PMID:20359453

  6. Recent Developments in Chemical Synthesis with Biocatalysts in Ionic Liquids.

    PubMed

    Potdar, Mahesh K; Kelso, Geoffrey F; Schwarz, Lachlan; Zhang, Chunfang; Hearn, Milton T W

    2015-01-01

    Over the past decade, a variety of ionic liquids have emerged as greener solvents for use in the chemical manufacturing industries. Their unique properties have attracted the interest of chemists worldwide to employ them as replacement for conventional solvents in a diverse range of chemical transformations including biotransformations. Biocatalysts are often regarded as green catalysts compared to conventional chemical catalysts in organic synthesis owing to their properties of low toxicity, biodegradability, excellent selectivity and good catalytic performance under mild reaction conditions. Similarly, a selected number of specific ionic liquids can be considered as greener solvents superior to organic solvents owing to their negligible vapor pressure, low flammability, low toxicity and ability to dissolve a wide range of organic and biological substances, including proteins. A combination of biocatalysts and ionic liquids thus appears to be a logical and promising opportunity for industrial use as an alternative to conventional organic chemistry processes employing organic solvents. This article provides an overview of recent developments in this field with special emphasis on the application of more sustainable enzyme-catalyzed reactions and separation processes employing ionic liquids, driven by advances in fundamental knowledge, process optimization and industrial deployment. PMID:26389873

  7. Recent Developments in Chemical Synthesis with Biocatalysts in Ionic Liquids.

    PubMed

    Potdar, Mahesh K; Kelso, Geoffrey F; Schwarz, Lachlan; Zhang, Chunfang; Hearn, Milton T W

    2015-09-15

    Over the past decade, a variety of ionic liquids have emerged as greener solvents for use in the chemical manufacturing industries. Their unique properties have attracted the interest of chemists worldwide to employ them as replacement for conventional solvents in a diverse range of chemical transformations including biotransformations. Biocatalysts are often regarded as green catalysts compared to conventional chemical catalysts in organic synthesis owing to their properties of low toxicity, biodegradability, excellent selectivity and good catalytic performance under mild reaction conditions. Similarly, a selected number of specific ionic liquids can be considered as greener solvents superior to organic solvents owing to their negligible vapor pressure, low flammability, low toxicity and ability to dissolve a wide range of organic and biological substances, including proteins. A combination of biocatalysts and ionic liquids thus appears to be a logical and promising opportunity for industrial use as an alternative to conventional organic chemistry processes employing organic solvents. This article provides an overview of recent developments in this field with special emphasis on the application of more sustainable enzyme-catalyzed reactions and separation processes employing ionic liquids, driven by advances in fundamental knowledge, process optimization and industrial deployment.

  8. Engineering Cytochrome P450 Biocatalysts for Biotechnology, Medicine, and Bioremediation

    PubMed Central

    Kumar, Santosh

    2009-01-01

    Importance of the field: Cytochrome P450 enzymes comprise a superfamily of heme monooxygenases that are of considerable interest for the: 1) synthesis of novel drugs and drug metabolites, 2) targeted cancer gene therapy, 3) biosensor design, and 4) bioremediation. However, their applications are limited because cytochrome P450, especially mammalian P450 enzymes, show a low turnover rate and stability, and require a complex source of electrons through cytochrome P450 reductase and NADPH. Areas covered in this review: In this review, we discuss the recent progress towards the use of P450 enzymes in a variety of above-mentioned applications. We also present alternate and cost-effective ways to perform P450-mediated reaction, especially using peroxides. Furthermore, we expand upon the current progress in P450 engineering approaches describing several recent examples that are utilized to enhance heterologous expression, stability, catalytic efficiency, and utilization of alternate oxidants. What the reader will gain: The review will provide a comprehensive knowledge in the design of P450 biocatalysts for potentially practical purposes. Finally, we provide a prospective on the future aspects of P450 engineering and its applications in biotechnology, medicine, and bioremediation. Take home message: Because of its wide applications, academic and pharmaceutical researchers, environmental scientists, and health care providers are expected to gain current knowledge and future prospects of the practical use of P450 biocatalysts. PMID:20064075

  9. Phase II trial of whole-cell pertussis vaccine vs an acellular vaccine containing agglutinogens.

    PubMed

    Miller, E; Ashworth, L A; Robinson, A; Waight, P A; Irons, L I

    1991-01-12

    An acellular pertussis vaccine containing agglutinogens 2 and 3, pertussis toxin, and filamentous haemagglutinin was developed by the Centre for Applied Microbiology and Research in the UK. 188 infants were entered into a randomised blind trial and received either the acellular or a whole-cell vaccine, combined with diphtheria and tetanus toxoids, in a 3, 5, and 8-10 month schedule. Local reactions were similar in the two groups but significantly fewer infants had systemic symptoms after the acellular vaccine. Mean log-antibody titres to the agglutinogen and toxin components were higher with the acellular than with the whole-cell vaccine. Persistence of antibodies one year after the third dose was also better in the acellular group. PMID:1670725

  10. Whole-Cell-Catalyzed Multiple Regio- and Stereoselective Functionalizations in Cascade Reactions Enabled by Directed Evolution.

    PubMed

    Li, Aitao; Ilie, Adriana; Sun, Zhoutong; Lonsdale, Richard; Xu, Jian-He; Reetz, Manfred T

    2016-09-19

    Biocatalytic cascade reactions using isolated stereoselective enzymes or whole cells in one-pot processes lead to value-added chiral products in a single workup. The concept has been restricted mainly to starting materials and intermediate products that are accepted by the respective wild-type enzymes. In the present study, we exploited directed evolution as a means to create E. coli whole cells for regio- and stereoselective cascade sequences that are not possible using man-made catalysts. The approach is illustrated using P450-BM3 in combination with appropriate alcohol dehydrogenases as catalysts in either two-, three-, or four-step cascade reactions starting from cyclohexane, cyclohexanol, or cyclohexanone, respectively, leading to either (R,R)-, (S,S)-, or meso-cyclohexane-1,2-diol. The one-pot conversion of cyclohexane into (R)- or (S)-2-hydroxycyclohexanone in the absence of ADH is also described. PMID:27573978

  11. An evaluation of serious neurological disorders following immunization: a comparison of whole-cell pertussis and acellular pertussis vaccines.

    PubMed

    Geier, David A; Geier, Mark R

    2004-08-01

    Serious neurological disorders reported following whole-cell pertussis in comparison to acellular pertussis vaccines were evaluated. The Vaccine Adverse Events Reporting System (VAERS) was analyzed for Emergency Department (ED) visits, life-threatening reactions, hospitalizations, disabilities, deaths, seizures, infantile spasms, encephalitis/encephalopathy, autism, Sudden Infant Death Syndrome (SIDS) and speech disorders reported with an initial onset of symptoms within 3 days following whole-cell pertussis and acellular pertussis vaccines among those residing in the US from 1997 to 1999. Controls were employed to evaluate potential biases in VAERS. Evaluations as to whether whole-cell and acellular vaccines were administered to populations of similar age and sex were undertaken because these factors might influence the study's results. Statistical increases were observed for all events examined following whole-cell pertussis vaccination in comparison to acellular pertussis vaccination, excepting cerebellar ataxia. Reporting biases were minimal in VAERS, and whole-cell and acellular pertussis vaccines were administered to populations of similar age and sex. Biologic mechanisms for the increased reactogenicity of whole-cell pertussis vaccines may stem from the fact that whole-cell pertussis vaccines contain 3,000 different proteins, whereas DTaP contains two to five proteins. Whole-cell pertussis vaccine contains known neurotoxins including: endotoxin, pertussis toxin and adenylate cyclase. Our results, and conclusions by the US Institute of Medicine, suggest an association between serious neurological disorders and whole-cell pertussis immunization. In light of the presence of a safer and at least equally efficacious acellular pertussis vaccine alternative, the Japanese and US switch to using acellular pertussis vaccine seems well justified. Other countries using whole-cell pertussis-containing vaccines should consider following suite in the near future.

  12. Biological production of acetaldehyde from ethanol using non-growing Pichia pastoris whole cells

    SciTech Connect

    Chiang, Heien-Kun; Foutch, G.L.; Fish, W.W.

    1991-12-31

    Acetaldehyde has been produced biologically using whole-cell Pichia Pass in a semibatch fermentor. Ethanol and air were fed continuously, and the product, acetaldehyde, was removed by the air stream. Operation of the reactor exceeded 100 h, maintaining high alcohol oxidase activity. Low cell-mass concentration (9.9 g/L) minimized product inhibition. Ethanol concentration in the broth, oxygen concentration in the air, and pH were evaluated for their effects on the fermentation process.

  13. Fungus-growing ants.

    PubMed

    Weber, N A

    1966-08-01

    Fungus-growing ants (Attini) are in reality unique fungus-culturing insects.There are several hundred species in some dozen genera, of which Acromyrmex and Atta are the conspicuous leaf-cutters. The center of their activities is the fungus garden, which is also the site of the queen and brood. The garden, in most species, is made from fresh green leaves or other vegetal material. The ants forage for this, forming distinct trails to the vegetation that is being harvested. The cut leaves or other substrate are brought into the nest and prepared for the fungus. Fresh leaves and flowers are cut into pieces a millimeter or two in diameter; the ants form them into a pulpy mass by pinching them with the mandibles and adding saliva. Anal droplets are deposited on the pieces, which are then forced into place in the garden. Planting of the fungus is accomplished by an ant's picking up tufts of the adjacent mycelium and dotting the surface of the new substrate with it. The combination of salivary and anal secretions, together with the constant care given by the ants, facilitates the growth of the ant fungus only, despite constant possibilities for contamination. When the ants are removed, alien fungi and other organisms flourish. A mature nest of Atta Sexdens may consist of 2000 chambers, some temporarily empty, some with refuse, and the remainder with fungus gardens. Thousands of kilograms of fresh leaves will have been used. A young laboratory colony of Atta cephalotes will use 1 kilogram of fresh leaves for one garden. The attines are the chief agents for introducing organic matter into the soil in tropical rain forests; this matter becomes the nucleus for a host of other organisms, including nematodes and arthropods, after it is discarded by the ants. One ant species cultures a yeast; all others grow a mycelium. In the higher species the mycelium forms clusters of inflated hyphae. Mycologists accept as valid two names for confirmed fruiting stages: Leucocoprinus ( or

  14. Yeast cell surface display for lipase whole cell catalyst and its applications

    SciTech Connect

    Liu, Yun; Zhang, Rui; Lian, Zhongshuai; Wang, Shihui; Wright, Aaron T.

    2014-08-01

    The cell surface display technique allows for the expression of target proteins or peptides on the microbial cell surface by fusing an appropriate protein as an anchoring motif. Yeast display systems, such as Pichia pastoris, Yarowia lipolytica and Saccharomyces cerevisiae, are ideal, alternative and extensive display systems with the advantage of simple genetic manipulation and post-translational modification of expressed heterologous proteins. Engineered yeasts show high performance characteristics and variant utilizations. Herein, we comprehensively summarize the variant factors affecting lipase whole cell catalyst activity and display efficiency, including the structure and size of target proteins, screening anchor proteins, type and chain length of linkers, and the appropriate matching rules among the above-mentioned display units. Furthermore, we also address novel approaches to enhance stability and activity of recombinant lipases, such as VHb gene co-expression, multi-enzyme co-display technique, and the micro-environmental interference and self-assembly techniques. Finally, we represent the variety of applications of whole cell surface displayed lipases on yeast cells in non-aqueous phases, including synthesis of esters, PUFA enrichment, resolution of chiral drugs, organic synthesis and biofuels. We demonstrate that the lipase surface display technique is a powerful tool for functionalizing yeasts to serve as whole cell catalysts, and increasing interest is providing an impetus for broad application of this technique.

  15. Applications and Mechanisms of Ionic Liquids in Whole-Cell Biotransformation

    PubMed Central

    Fan, Lin-Lin; Li, Hong-Ji; Chen, Qi-He

    2014-01-01

    Ionic liquids (ILs), entirely composed of cations and anions, are liquid solvents at room temperature. They are interesting due to their low vapor pressure, high polarity and thermostability, and also for the possibility to fine-tune their physicochemical properties through modification of the chemical structures of their cations or anions. In recent years, ILs have been widely used in biotechnological fields involving whole-cell biotransformations of biodiesel or biomass, and organic compound synthesis with cells. Research studies in these fields have increased from the past decades and compared to the typical solvents, ILs are the most promising alternative solvents for cell biotransformations. However, there are increasing limitations and new challenges in whole-cell biotransformations with ILs. There is little understanding of the mechanisms of ILs’ interactions with cells, and much remains to be clarified. Further investigations are required to overcome the drawbacks of their applications and to broaden their application spectrum. This work mainly reviews the applications of ILs in whole-cell biotransformations, and the possible mechanisms of ILs in microbial cell biotransformation are proposed and discussed. PMID:25007820

  16. Defining an additivity framework for mixture research in inducible whole-cell biosensors

    PubMed Central

    Martin-Betancor, K.; Ritz, C.; Fernández-Piñas, F.; Leganés, F.; Rodea-Palomares, I.

    2015-01-01

    A novel additivity framework for mixture effect modelling in the context of whole cell inducible biosensors has been mathematically developed and implemented in R. The proposed method is a multivariate extension of the effective dose (EDp) concept. Specifically, the extension accounts for differential maximal effects among analytes and response inhibition beyond the maximum permissive concentrations. This allows a multivariate extension of Loewe additivity, enabling direct application in a biphasic dose-response framework. The proposed additivity definition was validated, and its applicability illustrated by studying the response of the cyanobacterial biosensor Synechococcus elongatus PCC 7942 pBG2120 to binary mixtures of Zn, Cu, Cd, Ag, Co and Hg. The novel method allowed by the first time to model complete dose-response profiles of an inducible whole cell biosensor to mixtures. In addition, the approach also allowed identification and quantification of departures from additivity (interactions) among analytes. The biosensor was found to respond in a near additive way to heavy metal mixtures except when Hg, Co and Ag were present, in which case strong interactions occurred. The method is a useful contribution for the whole cell biosensors discipline and related areas allowing to perform appropriate assessment of mixture effects in non-monotonic dose-response frameworks PMID:26606975

  17. Target-Based Identification of Whole-Cell Active Inhibitors of Biotin Biosynthesis in Mycobacterium tuberculosis

    PubMed Central

    Park, Sae Woong; Casalena, Dominick; Wilson, Daniel; Dai, Ran; Nag, Partha; Liu, Feng; Boyce, Jim P.; Bittker, Joshua; Schreiber, Stuart; Finzel, Barry C.; Schnappinger, Dirk; Aldrich, Courtney C.

    2014-01-01

    SUMMARY Biotin biosynthesis is essential for survival and persistence of Mycobacterium tuberculosis (Mtb) in vivo. The aminotransferase BioA, which catalyzes the antepenultimate step in the biotin pathway, has been established as a promising target due to its vulnerability to chemical inhibition. We performed high-throughput screening (HTS) employing a fluorescence displacement assay and identified a diverse set of potent inhibitors including many diversity-oriented synthesis (DOS) scaffolds. To efficiently select only hits targeting biotin biosynthesis, we then deployed a whole-cell counter-screen in either biotin-free and biotin-containing medium against wild-type Mtb and in parallel with isogenic bioA Mtb strains that possess differential levels of BioA expression. This counter-screen proved crucial to filter out compounds whose whole-cell activity was off-target as well as identify hits with weak, but measurable whole-cell activity in BioA-depleted strains. Several of the most promising hits were co-crystallized with BioA to provide a framework for future structure-based drug design efforts. PMID:25556942

  18. Defining an additivity framework for mixture research in inducible whole-cell biosensors

    NASA Astrophysics Data System (ADS)

    Martin-Betancor, K.; Ritz, C.; Fernández-Piñas, F.; Leganés, F.; Rodea-Palomares, I.

    2015-11-01

    A novel additivity framework for mixture effect modelling in the context of whole cell inducible biosensors has been mathematically developed and implemented in R. The proposed method is a multivariate extension of the effective dose (EDp) concept. Specifically, the extension accounts for differential maximal effects among analytes and response inhibition beyond the maximum permissive concentrations. This allows a multivariate extension of Loewe additivity, enabling direct application in a biphasic dose-response framework. The proposed additivity definition was validated, and its applicability illustrated by studying the response of the cyanobacterial biosensor Synechococcus elongatus PCC 7942 pBG2120 to binary mixtures of Zn, Cu, Cd, Ag, Co and Hg. The novel method allowed by the first time to model complete dose-response profiles of an inducible whole cell biosensor to mixtures. In addition, the approach also allowed identification and quantification of departures from additivity (interactions) among analytes. The biosensor was found to respond in a near additive way to heavy metal mixtures except when Hg, Co and Ag were present, in which case strong interactions occurred. The method is a useful contribution for the whole cell biosensors discipline and related areas allowing to perform appropriate assessment of mixture effects in non-monotonic dose-response frameworks

  19. Defining an additivity framework for mixture research in inducible whole-cell biosensors.

    PubMed

    Martin-Betancor, K; Ritz, C; Fernández-Piñas, F; Leganés, F; Rodea-Palomares, I

    2015-01-01

    A novel additivity framework for mixture effect modelling in the context of whole cell inducible biosensors has been mathematically developed and implemented in R. The proposed method is a multivariate extension of the effective dose (EDp) concept. Specifically, the extension accounts for differential maximal effects among analytes and response inhibition beyond the maximum permissive concentrations. This allows a multivariate extension of Loewe additivity, enabling direct application in a biphasic dose-response framework. The proposed additivity definition was validated, and its applicability illustrated by studying the response of the cyanobacterial biosensor Synechococcus elongatus PCC 7942 pBG2120 to binary mixtures of Zn, Cu, Cd, Ag, Co and Hg. The novel method allowed by the first time to model complete dose-response profiles of an inducible whole cell biosensor to mixtures. In addition, the approach also allowed identification and quantification of departures from additivity (interactions) among analytes. The biosensor was found to respond in a near additive way to heavy metal mixtures except when Hg, Co and Ag were present, in which case strong interactions occurred. The method is a useful contribution for the whole cell biosensors discipline and related areas allowing to perform appropriate assessment of mixture effects in non-monotonic dose-response frameworks. PMID:26606975

  20. Construction of a dual fluorescence whole-cell biosensor to detect N-acyl homoserine lactones.

    PubMed

    Deng, Xuemei; Zhuang, Guoqiang; Ma, Anzhou; Yu, Qing; Zhuang, Xuliang

    2014-02-01

    Detection of N-acyl homoserine lactones (AHLs) is useful for understanding quorum sensing (QS) behaviors, including biofilm formation, virulence and metabolism. For detecting AHLs and indicating the host cells in situ, we constructed the plasmid pUCGMA2T(1-4) to make a dual fluorescent whole-cell biosensor based on the AhlIR AHL system of Pseudomonas syringae pv. syringae B728a. The plasmid contains three components: constitutively expressed P(npatII::gfp) for indicating host cells, P(ahlI::mcherry) that produces red fluorescence in response to AHL, and the ahlR gene that encodes an AHL regulatory protein. Meanwhile, two copies of T(1-4) (four tandem copies of a transcriptional terminator) were added into the plasmid to reduce background. The results showed that when the plasmid was placed into Escherichia coli, the dual fluorescence whole-cell biosensor was able to respond with red fluorescence within 6 hr to 5 x 10(-8)-1 x 10(-5) mol/L of 3OC6-HSL. Bright green fluorescence indicated the host cells. Furthermore, when the plasmid was transferred to wildtype Pseudomonas PhTA125 (an AHL-producing bacterium), it also showed both green and red fluorescence. This result demonstrates that this plasmid can be used to construct whole-cell indicators that can indicate the AHL response and spatial behaviors of microbes in a microenvironmental niche.

  1. Electrical properties of polyaniline nanofibre synthesized with biocatalyst

    NASA Astrophysics Data System (ADS)

    Kim, Byoung-Kye; Kim, Yong Hwan; Won, Keehoon; Chang, Hyunju; Choi, Youngmin; Kong, Ki-jeong; Rhyu, Beoyong Whan; Kim, Ju-Jin; Lee, Jeong-O.

    2005-08-01

    Polyaniline (PANI) nanofibres were synthesized using a biocatalyst (recombinant Coprinus cinereus peroxidase) instead of toxic chemical oxidants. Relatively uniform nanofibres with 50-100 nm diameter were easily obtained with this method, and the doping state of the PANI nanofibre could be controlled either with 1N camphorsulfonic acid (CSA) or with 30% NH4OH. Doped (or dedoped) PANI nanofibres were deposited on pre-patterned Au electrodes for electrical characterization. Completely dedoped PANI behaves as an insulator, while a larger current, by more than four orders of magnitude, was observed from doped PANI nanofibres. A weak p-type gate effect was observed for PANI nanofibre devices as well. As one could expect from the easy doping nature of PANI, PANI nanofibre devices show high sensitivity toward dedoping (NH3) gases, thereby demonstrating the possibility of using enzyme-synthesized PANI nanofibre devices as sensitive chemical sensors.

  2. Principles, techniques, and applications of biocatalyst immobilization for industrial application.

    PubMed

    Eş, Ismail; Vieira, José Daniel Gonçalves; Amaral, André Corrêa

    2015-03-01

    Immobilization is one of the most effective and powerful tools used in industry, which has been studied and improved since the last century. Various immobilization techniques and support materials have been used on both laboratory and industrial scale. Each immobilization technique is applicable for a specific production mostly depending on the cost and sensibility of process. Compared to free biocatalyst systems, immobilization techniques often offer better stability, increased activity and selectivity, higher resistance, improved separation and purification, reuse of enzymes, and consequently more efficient process. Recently, many reviews have been published about immobilization systems; however, most of them have focused on a specific application or not emphasized in details. This review focuses on most commonly used techniques in industry with many recent applications including using bioreactor systems for industrial production. It is also aimed to emphasize the advantages and disadvantages of the immobilization techniques and how these systems improve process productivity compared to non-immobilized systems.

  3. Earthworm Is a Versatile and Sustainable Biocatalyst for Organic Synthesis

    PubMed Central

    Guan, Zhi; Chen, Yan-Li; Yuan, Yi; Song, Jian; Yang, Da-Cheng; Xue, Yang; He, Yan-Hong

    2014-01-01

    A crude extract of earthworms was used as an eco-friendly, environmentally benign, and easily accessible biocatalyst for various organic synthesis including the asymmetric direct aldol and Mannich reactions, Henry and Biginelli reactions, direct three-component aza-Diels-Alder reactions for the synthesis of isoquinuclidines, and domino reactions for the synthesis of coumarins. Most of these reactions have never before seen in nature, and moderate to good enantioselectivities in aldol and Mannich reactions were obtained with this earthworm catalyst. The products can be obtained in preparatively useful yields, and the procedure does not require any additional cofactors or special equipment. This work provides an example of a practical way to use sustainable catalysts from nature. PMID:25148527

  4. Extremozymes--biocatalysts with unique properties from extremophilic microorganisms.

    PubMed

    Elleuche, Skander; Schröder, Carola; Sahm, Kerstin; Antranikian, Garabed

    2014-10-01

    Extremozymes are enzymes derived from extremophilic microorganisms that are able to withstand harsh conditions in industrial processes that were long thought to be destructive to proteins. Heat-stable and solvent-tolerant biocatalysts are valuable tools for processes in which for example hardly decomposable polymers need to be liquefied and degraded, while cold-active enzymes are of relevance for food and detergent industries. Extremophilic microorganisms are a rich source of naturally tailored enzymes, which are more superior over their mesophilic counterparts for applications at extreme conditions. Especially lignocellulolytic, amylolytic, and other biomass processing extremozymes with unique properties are widely distributed in thermophilic prokaryotes and are of high potential for versatile industrial processes.

  5. Exploring the Mechanism of Biocatalyst Inhibition in Microbial Desulfurization

    PubMed Central

    Abin-Fuentes, Andres; Mohamed, Magdy El-Said; Wang, Daniel I. C.

    2013-01-01

    Microbial desulfurization, or biodesulfurization (BDS), of fuels is a promising technology because it can desulfurize compounds that are recalcitrant to the current standard technology in the oil industry. One of the obstacles to the commercialization of BDS is the reduction in biocatalyst activity concomitant with the accumulation of the end product, 2-hydroxybiphenyl (HBP), during the process. BDS experiments were performed by incubating Rhodococcus erythropolis IGTS8 resting-cell suspensions with hexadecane at 0.50 (vol/vol) containing 10 mM dibenzothiophene. The resin Dowex Optipore SD-2 was added to the BDS experiments at resin concentrations of 0, 10, or 50 g resin/liter total volume. The HBP concentration within the cytoplasm was estimated to decrease from 1,100 to 260 μM with increasing resin concentration. Despite this finding, productivity did not increase with the resin concentration. This led us to focus on the susceptibility of the desulfurization enzymes toward HBP. Dose-response experiments were performed to identify major inhibitory interactions in the most common BDS pathway, the 4S pathway. HBP was responsible for three of the four major inhibitory interactions identified. The concentrations of HBP that led to a 50% reduction in the enzymes' activities (IC50s) for DszA, DszB, and DszC were measured to be 60 ± 5 μM, 110 ± 10 μM, and 50 ± 5 μM, respectively. The fact that the IC50s for HBP are all significantly lower than the cytoplasmic HBP concentration suggests that the inhibition of the desulfurization enzymes by HBP is responsible for the observed reduction in biocatalyst activity concomitant with HBP generation. PMID:24096431

  6. OxaD: A Versatile Indolic Nitrone Synthase from the Marine-Derived Fungus Penicillium oxalicum F30.

    PubMed

    Newmister, Sean A; Gober, Claire M; Romminger, Stelamar; Yu, Fengan; Tripathi, Ashootosh; Parra, Lizbeth Lorena L; Williams, Robert M; Berlinck, Roberto G S; Joullié, Madeleine M; Sherman, David H

    2016-09-01

    Indole alkaloids are a diverse class of natural products known for their wide range of biological activities and complex chemical structures. Rarely observed in this class are indolic nitrones, such as avrainvillamide and waikialoid, which possess potent bioactivities. Herein the oxa gene cluster from the marine-derived fungus Penicillium oxalicum F30 is described along with the characterization of OxaD, a flavin-dependent oxidase that generates roquefortine L, a nitrone-bearing intermediate in the biosynthesis of oxaline. Nitrone functionality in roquefortine L was confirmed by spectroscopic methods and 1,3-dipolar cycloaddition with methyl acrylate. OxaD is a versatile biocatalyst that converts an array of semisynthetic roquefortine C derivatives bearing indoline systems to their respective nitrones. This work describes the first implementation of a nitrone synthase as a biocatalyst and establishes a novel platform for late-stage diversification of a range of complex natural products. PMID:27505044

  7. Performance of a Cyanobacteria Whole Cell-Based Fluorescence Biosensor for Heavy Metal and Pesticide Detection

    PubMed Central

    Shing, Wong Ling; Heng, Lee Yook; Surif, Salmijah

    2013-01-01

    Whole cell biosensors always face the challenge of low stability of biological components and short storage life. This paper reports the effects of poly(2-hydroxyethyl methacrylate) (pHEMA) immobilization on a whole cell fluorescence biosensor for the detection of heavy metals (Cu, Pb, Cd), and pesticides (dichlorophenoxyacetic acid (2,4-D), and chlorpyrifos). The biosensor was produced by entrapping the cyanobacterium Anabaena torulosa on a cellulose membrane, followed by applying a layer of pHEMA, and attaching it to a well. The well was then fixed to an optical probe which was connected to a fluorescence spectrophotometer and an electronic reader. The optimization of the biosensor using several factors such as amount of HEMA and drying temperature were undertaken. The detection limits of biosensor without pHEMA for Cu, Cd, Pb, 2,4-D and chlorpyrifos were 1.195, 0.027, 0.0100, 0.025 and 0.025 μg/L respectively. The presence of pHEMA increased the limits of detection to 1.410, 0.250, 0.500, 0.235 and 0.117 μg/L respectively. pHEMA is known to enhance the reproducibility of the biosensor with average relative standard deviation (RSD) of ±1.76% for all the pollutants tested, 48% better than the biosensor without pHEMA (RSD = ±3.73%). In storability test with Cu 5 μg/L, the biosensor with pHEMA performed 11.5% better than the test without pHEMA on day-10 and 5.2% better on day-25. pHEMA is therefore a good candidate to be used in whole cell biosensors as it increases reproducibility and enhances biosensor storability. PMID:23673679

  8. Performance of a cyanobacteria whole cell-based fluorescence biosensor for heavy metal and pesticide detection.

    PubMed

    Shing, Wong Ling; Heng, Lee Yook; Surif, Salmijah

    2013-05-14

    Whole cell biosensors always face the challenge of low stability of biological components and short storage life. This paper reports the effects of poly(2-hydroxyethyl methacrylate) (pHEMA) immobilization on a whole cell fluorescence biosensor for the detection of heavy metals (Cu, Pb, Cd), and pesticides (dichlorophenoxyacetic acid (2,4-D), and chlorpyrifos). The biosensor was produced by entrapping the cyanobacterium Anabaena torulosa on a cellulose membrane, followed by applying a layer of pHEMA, and attaching it to a well. The well was then fixed to an optical probe which was connected to a fluorescence spectrophotometer and an electronic reader. The optimization of the biosensor using several factors such as amount of HEMA and drying temperature were undertaken. The detection limits of biosensor without pHEMA for Cu, Cd, Pb, 2,4-D and chlorpyrifos were 1.195, 0.027, 0.0100, 0.025 and 0.025 µg/L respectively. The presence of pHEMA increased the limits of detection to 1.410, 0.250, 0.500, 0.235 and 0.117 µg/L respectively. pHEMA is known to enhance the reproducibility of the biosensor with average relative standard deviation (RSD) of ±1.76% for all the pollutants tested, 48% better than the biosensor without pHEMA (RSD = ±3.73%). In storability test with Cu 5 µg/L, the biosensor with pHEMA performed 11.5% better than the test without pHEMA on day-10 and 5.2% better on day-25. pHEMA is therefore a good candidate to be used in whole cell biosensors as it increases reproducibility and enhances biosensor storability.

  9. Nondestructive monitoring of carotenogenesis in Haematococcus pluvialis via whole-cell optical density spectra.

    PubMed

    Solovchenko, Alexei; Aflalo, Claude; Lukyanov, Alexander; Boussiba, Sammy

    2013-05-01

    We investigated the feasibility of rapid, nondestructive assay of carotenoid-to-chlorophyll (Car/Chl) ratio and total carotenoids (Car) in cell suspensions of the carotenogenic chlorophyte Haematococcus pluvialis Flotow under stressful conditions. Whole-cell spectra are characterized by variable nonlinear contributions of Car and chlorophylls (Chl), with a strong influence of Car packaging and sieve effect inherent to stressed H. pluvialis cells. Nevertheless, nondestructive assay of Car/Chl in the range of 0.55-31.2 (Car content up to 188 mg L(-1); 5.4 % of the cell dry weight) turned to be achievable with a simple spectrophotometer lacking an integrating sphere upon deposition of the cells on glass fiber filters. The scattering-corrected optical density (OD) in the blue-green region of the whole-cell spectrum, normalized to that in the red maximum of Chl absorption (OD500/OD678), was tightly related (r (2) = 0.96) with the Car/Chl ratio found in extracts. Some features such as the amplitude and position of the minimum of the normalized first-derivative OD whole-cell spectra also exhibited a strong (r (2) > 0.90) nonlinear correlation with Car/Chl. These spectral indices were also tightly related with Car, but the slope of the relationship varied with the stressor intensity. The importance of calibration over the widest possible range of pigment contents and a correct choice of biomass load per filter are emphasized. The advantages and limitations of nondestructive monitoring of carotenogenesis in H. pluvialis are discussed in view of its possible application in optical sensors for laboratory cultivation and mass production systems of the algae.

  10. Whole-cell and single channel K+ and Cl- currents in epithelial cells of frog skin

    PubMed Central

    1991-01-01

    Whole-cell and single channel currents were studied in cells from frog (R. pipiens and R. catesbiana) skin epithelium, isolated by collagenase and trypsin treatment, and kept in primary cultures up to three days. Whole-cell currents did not exhibit any significant time-dependent kinetics under any ionic conditions used. With an external K gluconate Ringer solution the currents showed slight inward rectification with a reversal potential near zero and an average conductance of 5 nS at reversal. Ionic substitution of the external medium showed that most of the cell conductance was due to K and that very little, if any, Na conductance was present. This confirmed that most cells originate from inner epithelial layers and contain membranes with basolateral properties. At voltages more positive than 20 mV outward currents were larger with K in the medium than with Na or N-methyl-D-glucamine. Such behavior is indicative of a multi-ion transport mechanism. Whole-cell K current was inhibited by external Ba and quinidine. Blockade by Ba was strongly voltage dependent, while that by quinidine was not. In the presence of high external Cl, a component of outward current that was inhibited by the anion channel blocker diphenylamine-2-carboxylate (DPC) appeared in 70% of the cells. This component was strongly outwardly rectifying and reversed at a potential expected for a Cl current. At the single channel level the event most frequently observed in the cell-attached configuration was a K channel with the following characteristics: inward-rectifying I-V relation with a conductance (with 112.5 mM K in the pipette) of 44 pS at the reversal potential, one open and at least two closed states, and open probability that increased with depolarization. Quinidine blocked by binding in the open state and decreasing mean open time. Several observations suggest that this channel is responsible for most of the whole-cell current observed in high external K, and for the K conductance of the

  11. Whole-cell and single channel K+ and Cl- currents in epithelial cells of frog skin.

    PubMed

    García-Díaz, J F

    1991-07-01

    Whole-cell and single channel currents were studied in cells from frog (R. pipiens and R. catesbiana) skin epithelium, isolated by collagenase and trypsin treatment, and kept in primary cultures up to three days. Whole-cell currents did not exhibit any significant time-dependent kinetics under any ionic conditions used. With an external K gluconate Ringer solution the currents showed slight inward rectification with a reversal potential near zero and an average conductance of 5 nS at reversal. Ionic substitution of the external medium showed that most of the cell conductance was due to K and that very little, if any, Na conductance was present. This confirmed that most cells originate from inner epithelial layers and contain membranes with basolateral properties. At voltages more positive than 20 mV outward currents were larger with K in the medium than with Na or N-methyl-D-glucamine. Such behavior is indicative of a multi-ion transport mechanism. Whole-cell K current was inhibited by external Ba and quinidine. Blockade by Ba was strongly voltage dependent, while that by quinidine was not. In the presence of high external Cl, a component of outward current that was inhibited by the anion channel blocker diphenylamine-2-carboxylate (DPC) appeared in 70% of the cells. This component was strongly outwardly rectifying and reversed at a potential expected for a Cl current. At the single channel level the event most frequently observed in the cell-attached configuration was a K channel with the following characteristics: inward-rectifying I-V relation with a conductance (with 112.5 mM K in the pipette) of 44 pS at the reversal potential, one open and at least two closed states, and open probability that increased with depolarization. Quinidine blocked by binding in the open state and decreasing mean open time. Several observations suggest that this channel is responsible for most of the whole-cell current observed in high external K, and for the K conductance of the

  12. Whole-Cell Mediated 11β-Hydroxylation on the Basic Limonoid Skeleton by Cunninghamella echinulata.

    PubMed

    Haldar, Saikat; Mulani, Fayaj A; Aarthy, Thiagarayaselvam; Thulasiram, Hirekodathakallu V

    2015-06-19

    Regio- and stereoselective 11β-hydroxylation was achieved on the basic limonoid skeleton through microbial transformation. Whole cells of Cunninghamella echinulata efficiently converted basic limonoids such as epoxyazadiradione, azadiradione, and gedunin to their 11β-hydroxy analogues as the sole metabolite. Fermentation conditions affecting the efficiency (96%) of biotransformation including substrate concentration, incubation period, pH, and temperature were optimized. The position and stereochemistry of hydroxyl functionality on the isolated metabolites were established through extensive spectroscopic and spectrometric studies (1D, 2D NMR, ESI-MS, and MS/MS). PMID:25985231

  13. Highly Enantioselective Production of (R)-Halohydrins with Whole Cells of Rhodotorula rubra KCh 82 Culture

    PubMed Central

    Janeczko, Tomasz; Dymarska, Monika; Kostrzewa-Susłow, Edyta

    2014-01-01

    Biotransformation of ten α-haloacetophenones in the growing culture of the strain Rhodotorula rubra KCh 82 has been carried out. Nine of the substrates underwent an effective enantioselective reduction to the respective (R)-alcohols according to Prelog’s rule, with the exception of 2-chloro-1,2-diphenylethan-1-one that was not transformed by this strain. The expected reduction proceeded without dehalogenation, leading to the respective (R)-halohydrins in high yields. The use of this biocatalyst yielded (R)-2-bromo-1-phenyl-ethan-1-ol (enantiomeric excess (ee) = 97%) and its derivatives: 4'-Bromo- (ee = 99%); 4'-Chloro- (ee > 99%); 4'-Methoxy- (ee = 96%); 3'-Methoxy- (ee = 93%); 2'-Methoxy- (ee = 98%). There were also obtained and characterized 2,4'-dichloro-, 2,2',4'-trichloro- and 2-chloro-4'-fluoro-phenyetan-1-ol with >99% of enantiomeric excesses. PMID:25486054

  14. Highly enantioselective production of (R)-halohydrins with whole cells of Rhodotorula rubra KCh 82 culture.

    PubMed

    Janeczko, Tomasz; Dymarska, Monika; Kostrzewa-Susłow, Edyta

    2014-12-04

    Biotransformation of ten α-haloacetophenones in the growing culture of the strain Rhodotorula rubra KCh 82 has been carried out. Nine of the substrates underwent an effective enantioselective reduction to the respective (R)-alcohols according to Prelog's rule, with the exception of 2-chloro-1,2-diphenylethan-1-one that was not transformed by this strain. The expected reduction proceeded without dehalogenation, leading to the respective (R)-halohydrins in high yields. The use of this biocatalyst yielded (R)-2-bromo-1-phenyl-ethan-1-ol (enantiomeric excess (ee) = 97%) and its derivatives: 4'-Bromo- (ee = 99%); 4'-Chloro- (ee > 99%); 4'-Methoxy- (ee = 96%); 3'-Methoxy- (ee = 93%); 2'-Methoxy- (ee = 98%). There were also obtained and characterized 2,4'-dichloro-, 2,2',4'-trichloro- and 2-chloro-4'-fluoro-phenyetan-1-ol with >99% of enantiomeric excesses.

  15. Isotype and antigen specificity of pertussis agglutinins following whole-cell pertussis vaccination and infection with Bordetella pertussis.

    PubMed Central

    Mink, C M; O'Brien, C H; Wassilak, S; Deforest, A; Meade, B D

    1994-01-01

    Elevated agglutinin titers have been shown to correlate with protection from disease following whole-cell pertussis vaccination, but the isotype and antigen specificity of human agglutinating antibodies is unknown. In 13 immunoassays, immunoglobulin G antifimbria antibodies had the strongest correlation with agglutinin titers following culture-proven infection with Bordetella pertussis (R' = 0.79; P < 0.0001) and following whole-cell pertussis vaccination (R' = 0.87, P < 0.0001). PMID:7509316

  16. Chimeric self-sufficient P450cam-RhFRed biocatalysts with broad substrate scope

    PubMed Central

    Robin, Aélig; Köhler, Valentin; Jones, Alison; Ali, Afruja; Kelly, Paul P; O'Reilly, Elaine; Turner, Nicholas J

    2011-01-01

    Summary A high-throughput screening protocol for evaluating chimeric, self-sufficient P450 biocatalysts and their mutants against a panel of substrates was developed, leading to the identification of a number of novel biooxidation activities. PMID:22238522

  17. Marine hydrocarbonoclastic bacteria as whole-cell biosensors for n-alkanes.

    PubMed

    Sevilla, Emma; Yuste, Luis; Rojo, Fernando

    2015-07-01

    Whole-cell biosensors offer potentially useful, cost-effective systems for the in-situ monitoring of seawater for hydrocarbons derived from accidental spills. The present work compares the performance of a biosensor system for the detection of alkanes in seawater, hosted in either Escherichia coli (commonly employed in whole-cell biosensors but not optimized for alkane assimilation) or different marine bacteria specialized in assimilating alkanes. The sensor system was based on the Pseudomonas putida AlkS regulatory protein and the PalkB promoter fused to a gene encoding the green fluorescent protein. While the E. coli sensor provided the fastest response to pure alkanes (25-fold induction after 2 h under the conditions used), a sensor based on Alcanivorax borkumensis was slower, requiring 3-4 h to reach similar induction values. However, the A. borkumensis sensor showed a fourfold lower detection threshold for octane (0.5 μM), and was also better at sensing the alkanes present in petrol. At petrol concentrations of 0.0125%, the A. borkumensis sensor rendered a sevenfold induction, while E. coli sensor showed no response. We discuss possible explanations to this behaviour in terms of the cellular adaptations to alkane uptake and the basal fluorescence produced by each bacterial strain, which was lowest for A. borkumensis. PMID:25874658

  18. Whole-Cell Biocatalysis for Producing Ginsenoside Rd from Rb1 Using Lactobacillus rhamnosus GG.

    PubMed

    Ku, Seockmo; You, Hyun Ju; Park, Myeong Soo; Ji, Geun Eog

    2016-07-28

    Ginsenosides are the major active ingredients in ginseng used for human therapeutic plant medicines. One of the most well-known probiotic bacteria among the various strains on the functional food market is Lactobacillus rhamnosus GG. Biocatalytic methods using probiotic enzymes for producing deglycosylated ginsenosides such as Rd have a growing significance in the functional food industry. The addition of 2% cellobiose (w/v) to glucose-free de Man-Rogosa-Sharpe broths notably induced β-glucosidase production from L. rhamnosus GG. Enzyme production and activity were optimized at a pH, temperature, and cellobiose concentration of 6.0, 40°C, and 2% (w/v), respectively. Under these controlled conditions, β-glucosidase production in L. rhamnosus GG was enhanced by 25-fold. Additionally, whole-cell homogenates showed the highest β-glucosidase activity when compared with disrupted cell suspensions; the cell disruption step significantly decreased the β-glucosidase activity. Based on the optimized enzyme conditions, whole-cell L. rhamnosus GG was successfully used to convert ginsenoside Rb1 into Rd.

  19. Water pollutant monitoring by a whole cell array through lens-free detection on CCD.

    PubMed

    Tsai, Hsieh-Fu; Tsai, Yi-Ching; Yagur-Kroll, Sharon; Palevsky, Noa; Belkin, Shimshon; Cheng, Ji-Yen

    2015-03-21

    Environmental contamination has become a serious problem to human and environmental health, as exposure to a wide range of possible contaminants continuously increases due to industrial and agricultural activities. Whole cell sensors have been proposed as a powerful tool to detect class-specific toxicants based upon their biological activity and bioavailability. We demonstrated a robust toxicant detection platform based on a bioluminescence whole cell sensor array biochip (LumiChip). LumiChip harbors an integrated temperature control and a 16-member sensor array, as well as a simple but highly efficient luminescence collection setup. On LumiChip, samples were infused in an oxygen-permeable microfluidic flow channel to reach the sensor array. Time-lapse changes in bioluminescence emitted by the array members were measured on a single window-removed linear charge-coupled device (CCD) commonly used in commercial industrial process control or in barcode readers. Removal of the protective window on the linear CCD allowed lens-free direct interfacing of LumiChip to the CCD surface for measurement with high light collection efficiency. Bioluminescence induced by simulated contamination events was detected within 15 to 45 minutes. The portable LumiSense system utilizing the linear CCD in combination with the miniaturized LumiChip is a promising potential platform for on-site environmental monitoring of toxicant contamination.

  20. Whole-Cell Biocatalysis for Producing Ginsenoside Rd from Rb1 Using Lactobacillus rhamnosus GG.

    PubMed

    Ku, Seockmo; You, Hyun Ju; Park, Myeong Soo; Ji, Geun Eog

    2016-07-28

    Ginsenosides are the major active ingredients in ginseng used for human therapeutic plant medicines. One of the most well-known probiotic bacteria among the various strains on the functional food market is Lactobacillus rhamnosus GG. Biocatalytic methods using probiotic enzymes for producing deglycosylated ginsenosides such as Rd have a growing significance in the functional food industry. The addition of 2% cellobiose (w/v) to glucose-free de Man-Rogosa-Sharpe broths notably induced β-glucosidase production from L. rhamnosus GG. Enzyme production and activity were optimized at a pH, temperature, and cellobiose concentration of 6.0, 40°C, and 2% (w/v), respectively. Under these controlled conditions, β-glucosidase production in L. rhamnosus GG was enhanced by 25-fold. Additionally, whole-cell homogenates showed the highest β-glucosidase activity when compared with disrupted cell suspensions; the cell disruption step significantly decreased the β-glucosidase activity. Based on the optimized enzyme conditions, whole-cell L. rhamnosus GG was successfully used to convert ginsenoside Rb1 into Rd. PMID:27012233

  1. Marine hydrocarbonoclastic bacteria as whole-cell biosensors for n-alkanes

    PubMed Central

    Sevilla, Emma; Yuste, Luis; Rojo, Fernando

    2015-01-01

    Whole-cell biosensors offer potentially useful, cost-effective systems for the in-situ monitoring of seawater for hydrocarbons derived from accidental spills. The present work compares the performance of a biosensor system for the detection of alkanes in seawater, hosted in either Escherichia coli (commonly employed in whole-cell biosensors but not optimized for alkane assimilation) or different marine bacteria specialized in assimilating alkanes. The sensor system was based on the Pseudomonas putida AlkS regulatory protein and the PalkB promoter fused to a gene encoding the green fluorescent protein. While the E. coli sensor provided the fastest response to pure alkanes (25-fold induction after 2 h under the conditions used), a sensor based on Alcanivorax borkumensis was slower, requiring 3–4 h to reach similar induction values. However, the A. borkumensis sensor showed a fourfold lower detection threshold for octane (0.5 μM), and was also better at sensing the alkanes present in petrol. At petrol concentrations of 0.0125%, the A. borkumensis sensor rendered a sevenfold induction, while E. coli sensor showed no response. We discuss possible explanations to this behaviour in terms of the cellular adaptations to alkane uptake and the basal fluorescence produced by each bacterial strain, which was lowest for A. borkumensis. PMID:25874658

  2. Whole Cell Model of Actin Diffusion and Reaction based on Single Molecule Speckle Microscopy Measurements

    NASA Astrophysics Data System (ADS)

    McMillen, Laura; Vavylonis, Dimitrios; Vavylonis Group Team

    It is debated whether transport of actin across the cell by diffusion alone is sufficiently fast to account for the rapid reorganization of actin filaments at the leading edge of motile cells. In order to investigate this question, we created a 3D model of the whole cell that includes reaction and diffusion of actin using a particle Monte Carlo method. For the lamellipodium of the simulated cell we use the model by Smith et al. Biophys. J 104:247 (2013), which includes two diffuse pools of actin, one which is slowly diffusing and the other which diffuses more quickly, as well as a pool of filamentous actin undergoing retrograde flow towards the cell center. We adjusted this model to fit a circular geometry around the whole cell. We also consider actin in the cell center which is either diffusing or in stationary filamentous form, representing cortical actin or actin in stress fibers. The local rates of polymerization and the lifetime distributions of polymerized actin were estimated from single molecule speckle microscopy experiments by the group of N. Watanabe. With this model we are able to simulate prior experiments that monitored the redistribution of actin after photoactivation or fluorescence recovery after photobleaching in various parts of the cell. We find that transport by diffusion is sufficient to fit these data, without the need for an active transport mechanism, however significant concentration gradients may develop at steady state.

  3. Whole cell tracking through the optimal control of geometric evolution laws

    NASA Astrophysics Data System (ADS)

    Blazakis, Konstantinos N.; Madzvamuse, Anotida; Reyes-Aldasoro, Constantino Carlos; Styles, Vanessa; Venkataraman, Chandrasekhar

    2015-09-01

    Cell tracking algorithms which automate and systematise the analysis of time lapse image data sets of cells are an indispensable tool in the modelling and understanding of cellular phenomena. In this study we present a theoretical framework and an algorithm for whole cell tracking. Within this work we consider that "tracking" is equivalent to a dynamic reconstruction of the whole cell data (morphologies) from static image data sets. The novelty of our work is that the tracking algorithm is driven by a model for the motion of the cell. This model may be regarded as a simplification of a recently developed physically meaningful model for cell motility. The resulting problem is the optimal control of a geometric evolution law and we discuss the formulation and numerical approximation of the optimal control problem. The overall goal of this work is to design a framework for cell tracking within which the recovered data reflects the physics of the forward model. A number of numerical simulations are presented that illustrate the applicability of our approach.

  4. Marine hydrocarbonoclastic bacteria as whole-cell biosensors for n-alkanes.

    PubMed

    Sevilla, Emma; Yuste, Luis; Rojo, Fernando

    2015-07-01

    Whole-cell biosensors offer potentially useful, cost-effective systems for the in-situ monitoring of seawater for hydrocarbons derived from accidental spills. The present work compares the performance of a biosensor system for the detection of alkanes in seawater, hosted in either Escherichia coli (commonly employed in whole-cell biosensors but not optimized for alkane assimilation) or different marine bacteria specialized in assimilating alkanes. The sensor system was based on the Pseudomonas putida AlkS regulatory protein and the PalkB promoter fused to a gene encoding the green fluorescent protein. While the E. coli sensor provided the fastest response to pure alkanes (25-fold induction after 2 h under the conditions used), a sensor based on Alcanivorax borkumensis was slower, requiring 3-4 h to reach similar induction values. However, the A. borkumensis sensor showed a fourfold lower detection threshold for octane (0.5 μM), and was also better at sensing the alkanes present in petrol. At petrol concentrations of 0.0125%, the A. borkumensis sensor rendered a sevenfold induction, while E. coli sensor showed no response. We discuss possible explanations to this behaviour in terms of the cellular adaptations to alkane uptake and the basal fluorescence produced by each bacterial strain, which was lowest for A. borkumensis.

  5. Industrial Acetogenic Biocatalysts: A Comparative Metabolic and Genomic Analysis.

    PubMed

    Bengelsdorf, Frank R; Poehlein, Anja; Linder, Sonja; Erz, Catarina; Hummel, Tim; Hoffmeister, Sabrina; Daniel, Rolf; Dürre, Peter

    2016-01-01

    Synthesis gas (syngas) fermentation by anaerobic acetogenic bacteria employing the Wood-Ljungdahl pathway is a bioprocess for production of biofuels and biocommodities. The major fermentation products of the most relevant biocatalytic strains (Clostridium ljungdahlii, C. autoethanogenum, C. ragsdalei, and C. coskatii) are acetic acid and ethanol. A comparative metabolic and genomic analysis using the mentioned biocatalysts might offer targets for metabolic engineering and thus improve the production of compounds apart from ethanol. Autotrophic growth and product formation of the four wild type (WT) strains were compared in uncontrolled batch experiments. The genomes of C. ragsdalei and C. coskatii were sequenced and the genome sequences of all four biocatalytic strains analyzed in comparative manner. Growth and product spectra (acetate, ethanol, 2,3-butanediol) of C. autoethanogenum, C. ljungdahlii, and C. ragsdalei were rather similar. In contrast, C. coskatii produced significantly less ethanol and its genome sequence lacks two genes encoding aldehyde:ferredoxin oxidoreductases (AOR). Comparative genome sequence analysis of the four WT strains revealed high average nucleotide identity (ANI) of C. ljungdahlii and C. autoethanogenum (99.3%) and C. coskatii (98.3%). In contrast, C. ljungdahlii WT and C. ragsdalei WT showed an ANI-based similarity of only 95.8%. Additionally, recombinant C. ljungdahlii strains were constructed that harbor an artificial acetone synthesis operon (ASO) consisting of the following genes: adc, ctfA, ctfB, and thlA (encoding acetoacetate decarboxylase, acetoacetyl-CoA:acetate/butyrate:CoA-transferase subunits A and B, and thiolase) under the control of thlA promoter (P thlA ) from C. acetobutylicum or native pta-ack promoter (P pta-ack ) from C. ljungdahlii. Respective recombinant strains produced 2-propanol rather than acetone, due to the presence of a NADPH-dependent primary-secondary alcohol dehydrogenase that converts acetone to 2

  6. Industrial Acetogenic Biocatalysts: A Comparative Metabolic and Genomic Analysis

    PubMed Central

    Bengelsdorf, Frank R.; Poehlein, Anja; Linder, Sonja; Erz, Catarina; Hummel, Tim; Hoffmeister, Sabrina; Daniel, Rolf; Dürre, Peter

    2016-01-01

    Synthesis gas (syngas) fermentation by anaerobic acetogenic bacteria employing the Wood–Ljungdahl pathway is a bioprocess for production of biofuels and biocommodities. The major fermentation products of the most relevant biocatalytic strains (Clostridium ljungdahlii, C. autoethanogenum, C. ragsdalei, and C. coskatii) are acetic acid and ethanol. A comparative metabolic and genomic analysis using the mentioned biocatalysts might offer targets for metabolic engineering and thus improve the production of compounds apart from ethanol. Autotrophic growth and product formation of the four wild type (WT) strains were compared in uncontrolled batch experiments. The genomes of C. ragsdalei and C. coskatii were sequenced and the genome sequences of all four biocatalytic strains analyzed in comparative manner. Growth and product spectra (acetate, ethanol, 2,3-butanediol) of C. autoethanogenum, C. ljungdahlii, and C. ragsdalei were rather similar. In contrast, C. coskatii produced significantly less ethanol and its genome sequence lacks two genes encoding aldehyde:ferredoxin oxidoreductases (AOR). Comparative genome sequence analysis of the four WT strains revealed high average nucleotide identity (ANI) of C. ljungdahlii and C. autoethanogenum (99.3%) and C. coskatii (98.3%). In contrast, C. ljungdahlii WT and C. ragsdalei WT showed an ANI-based similarity of only 95.8%. Additionally, recombinant C. ljungdahlii strains were constructed that harbor an artificial acetone synthesis operon (ASO) consisting of the following genes: adc, ctfA, ctfB, and thlA (encoding acetoacetate decarboxylase, acetoacetyl-CoA:acetate/butyrate:CoA-transferase subunits A and B, and thiolase) under the control of thlA promoter (PthlA) from C. acetobutylicum or native pta-ack promoter (Ppta-ack) from C. ljungdahlii. Respective recombinant strains produced 2-propanol rather than acetone, due to the presence of a NADPH-dependent primary-secondary alcohol dehydrogenase that converts acetone to 2

  7. Industrial Acetogenic Biocatalysts: A Comparative Metabolic and Genomic Analysis.

    PubMed

    Bengelsdorf, Frank R; Poehlein, Anja; Linder, Sonja; Erz, Catarina; Hummel, Tim; Hoffmeister, Sabrina; Daniel, Rolf; Dürre, Peter

    2016-01-01

    Synthesis gas (syngas) fermentation by anaerobic acetogenic bacteria employing the Wood-Ljungdahl pathway is a bioprocess for production of biofuels and biocommodities. The major fermentation products of the most relevant biocatalytic strains (Clostridium ljungdahlii, C. autoethanogenum, C. ragsdalei, and C. coskatii) are acetic acid and ethanol. A comparative metabolic and genomic analysis using the mentioned biocatalysts might offer targets for metabolic engineering and thus improve the production of compounds apart from ethanol. Autotrophic growth and product formation of the four wild type (WT) strains were compared in uncontrolled batch experiments. The genomes of C. ragsdalei and C. coskatii were sequenced and the genome sequences of all four biocatalytic strains analyzed in comparative manner. Growth and product spectra (acetate, ethanol, 2,3-butanediol) of C. autoethanogenum, C. ljungdahlii, and C. ragsdalei were rather similar. In contrast, C. coskatii produced significantly less ethanol and its genome sequence lacks two genes encoding aldehyde:ferredoxin oxidoreductases (AOR). Comparative genome sequence analysis of the four WT strains revealed high average nucleotide identity (ANI) of C. ljungdahlii and C. autoethanogenum (99.3%) and C. coskatii (98.3%). In contrast, C. ljungdahlii WT and C. ragsdalei WT showed an ANI-based similarity of only 95.8%. Additionally, recombinant C. ljungdahlii strains were constructed that harbor an artificial acetone synthesis operon (ASO) consisting of the following genes: adc, ctfA, ctfB, and thlA (encoding acetoacetate decarboxylase, acetoacetyl-CoA:acetate/butyrate:CoA-transferase subunits A and B, and thiolase) under the control of thlA promoter (P thlA ) from C. acetobutylicum or native pta-ack promoter (P pta-ack ) from C. ljungdahlii. Respective recombinant strains produced 2-propanol rather than acetone, due to the presence of a NADPH-dependent primary-secondary alcohol dehydrogenase that converts acetone to 2

  8. Starch Biocatalyst Based on α-Amylase-Mg/Al-Layered Double Hydroxide Nanohybrids.

    PubMed

    Bruna, Felipe; Pereira, Marita G; Polizeli, Maria de Lourdes T M; Valim, João B

    2015-08-26

    The design of new biocatalysts through the immobilization of enzymes, improving their stability and reuse, plays a major role in the development of sustainable methodologies toward the so-called green chemistry. In this work, α-amylase (AAM) biocatalyst based on Mg3Al-layered double-hydroxide (LDH) matrix was successfully developed with the adsorption method. The adsorption process was studied and optimized as a function of time and enzyme concentration. The biocatalyst was characterized, and the mechanism of interaction between AAM and LDH, as well as the immobilization effects on the catalytic activity, was elucidated. The adsorption process was fast and irreversible, thus yielding a stable biohybrid material. The immobilized AAM partially retained its enzymatic activity, and the biocatalyst rapidly hydrolyzed starch in an aqueous solution with enhanced efficiency at intermediate loading values of ca. 50 mg/g of AAM/LDH. Multiple attachments through electrostatic interactions affected the conformation of the immobilized enzyme on the LDH surface. The biocatalyst was successfully stored in its dry form, retaining 100% of its catalytic activity. The results reveal the potential usefulness of a LDH compound as a support of α-amylase for the hydrolysis of starch that may be applied in industrial and pharmaceutical processes as a simple, environmentally friendly, and low-cost biocatalyst.

  9. Biocatalyst Engineering by Assembly of Fatty Acid Transport and Oxidation Activities for In Vivo Application of Cytochrome P-450BM-3 Monooxygenase

    PubMed Central

    Schneider, Silke; Wubbolts, Marcel G.; Sanglard, Dominique; Witholt, Bernard

    1998-01-01

    The application of whole cells containing cytochrome P-450BM-3 monooxygenase [EC 1.14.14.1] for the bioconversion of long-chain saturated fatty acids to ω-1, ω-2, and ω-3 hydroxy fatty acids was investigated. We utilized pentadecanoic acid and studied its conversion to a mixture of 12-, 13-, and 14-hydroxypentadecanoic acids by this monooxygenase. For this purpose, Escherichia coli recombinants containing plasmid pCYP102 producing the fatty acid monooxygenase cytochrome P-450BM-3 were used. To overcome inefficient uptake of pentadecanoic acid by intact E. coli cells, we made use of a cloned fatty acid uptake system from Pseudomonas oleovorans which, in contrast to the common FadL fatty acid uptake system of E. coli, does not require coupling by FadD (acyl-coenzyme A synthetase) of the imported fatty acid to coenzyme A. This system from P. oleovorans is encoded by a gene carried by plasmid pGEc47, which has been shown to effect facilitated uptake of oleic acid in E. coli W3110 (M. Nieboer, Ph.D. thesis, University of Groningen, Groningen, The Netherlands, 1996). By using a double recombinant of E. coli K27, which is a fadD mutant and therefore unable to consume substrates or products via the β-oxidation cycle, a twofold increase in productivity was achieved. Applying cytochrome P-450BM-3 monooxygenase as a biocatalyst in whole cells does not require the exogenous addition of the costly cofactor NADPH. In combination with the coenzyme A-independent fatty acid uptake system from P. oleovorans, cytochrome P-450BM-3 recombinants appear to be useful alternatives to the enzymatic approach for the bioconversion of long-chain fatty acids to subterminal hydroxylated fatty acids. PMID:9758800

  10. Biocatalyst engineering by assembly of fatty acid transport and oxidation activities for In vivo application of cytochrome P-450BM-3 monooxygenase.

    PubMed

    Schneider, S; Wubbolts, M G; Sanglard, D; Witholt, B

    1998-10-01

    The application of whole cells containing cytochrome P-450BM-3 monooxygenase [EC 1.14.14.1] for the bioconversion of long-chain saturated fatty acids to omega-1, omega-2, and omega-3 hydroxy fatty acids was investigated. We utilized pentadecanoic acid and studied its conversion to a mixture of 12-, 13-, and 14-hydroxypentadecanoic acids by this monooxygenase. For this purpose, Escherichia coli recombinants containing plasmid pCYP102 producing the fatty acid monooxygenase cytochrome P-450BM-3 were used. To overcome inefficient uptake of pentadecanoic acid by intact E. coli cells, we made use of a cloned fatty acid uptake system from Pseudomonas oleovorans which, in contrast to the common FadL fatty acid uptake system of E. coli, does not require coupling by FadD (acyl-coenzyme A synthetase) of the imported fatty acid to coenzyme A. This system from P. oleovorans is encoded by a gene carried by plasmid pGEc47, which has been shown to effect facilitated uptake of oleic acid in E. coli W3110 (M. Nieboer, Ph.D. thesis, University of Groningen, Groningen, The Netherlands, 1996). By using a double recombinant of E. coli K27, which is a fadD mutant and therefore unable to consume substrates or products via the beta-oxidation cycle, a twofold increase in productivity was achieved. Applying cytochrome P-450BM-3 monooxygenase as a biocatalyst in whole cells does not require the exogenous addition of the costly cofactor NADPH. In combination with the coenzyme A-independent fatty acid uptake system from P. oleovorans, cytochrome P-450BM-3 recombinants appear to be useful alternatives to the enzymatic approach for the bioconversion of long-chain fatty acids to subterminal hydroxylated fatty acids.

  11. [Novel Immobilized Biocatalyst for Microbiological Synthesis of Pharmaceutical Steroids].

    PubMed

    Andryushina, V A; Karpova, N V; Druzhinina, A V; Stytsenko, T S; Podorozhko, E A; Ryabev, A N; Lozinskii, V I

    2015-01-01

    The steroid-transforming activity of free and immobilized cells of Pimelobacter simplex VKPM As-1632 entrapped in an operationally stable macroporous polyvinyl alcohol cryogel was studied. It was shown that the macroporous matrix of the carrier did not create any diffusional limitations for steroid access to the cells or the removal of the transformation products from them. The optimal conditions for the hydrocortisone 1,2-dehydration into prednisolone by free and immobilized cells were elucidated. The immobilized biocatalyst was obtained in a granulated form and used in 32 successive cycles of steroid transformation. The average cycle duration was 45 min, and the prednisolone yield of during the first 20 cycles was 98%. It was established that the immobilized cells of the actinobacteria P. simplex retained high steroid-transforming activity over all of the transformation cycles. The physicochemical and diffusion characteristics of the polyvinyl alcohol gels and its granules were determined, and their high stability during repeated cycles of steroid transformation was shown. The results indicated that P. simplex immobilized cells represent an effective catalyst suitable for multiple use. Biomass consumption decreased upon its use, and product isolation, as well as culture storage, was much easier. PMID:26596083

  12. Use of immobilised biocatalysts in the processing of cheese whey.

    PubMed

    Kosseva, Maria R; Panesar, Parmjit S; Kaur, Gurpreet; Kennedy, John F

    2009-12-01

    Food processing industry operations need to comply with increasingly more stringent environmental regulations related to the disposal or utilisation of by-products and wastes. These include growing restrictions on land spraying with agro-industrial wastes, and on disposal within landfill operations, and the requirements to produce end products that are stabilised and hygienic. Much of the material generated as wastes by the dairy processing industries contains components that could be utilised as substrates and nutrients in a variety of microbial/enzymatic processes, to give rise to added-value products. A good example of a waste that has received considerable attention as a source of added-value products is cheese whey. The carbohydrate reservoir of lactose (4-5%) in whey and the presence of other essential nutrients make it a good natural medium for the growth of microorganisms and a potential substrate for bioprocessing through microbial fermentation. Immobilised cell and enzyme technology has also been applied to whey bioconversion processes to improve the economics of such processes. This review focuses upon the elaboration of a range of immobilisation techniques that have been applied to produce valuable whey-based products. A comprehensive literature survey is also provided to illustrate numerous immobilisation procedures with particular emphasis upon lactose hydrolysis, and ethanol and lactic acid production using immobilised biocatalysts.

  13. Heavy metal whole-cell biosensors using eukaryotic microorganisms: an updated critical review.

    PubMed

    Gutiérrez, Juan C; Amaro, Francisco; Martín-González, Ana

    2015-01-01

    This review analyzes the advantages and disadvantages of using eukaryotic microorganisms to design whole-cell biosensors (WCBs) for monitoring environmental heavy metal pollution in soil or aquatic habitats. Basic considerations for designing a eukaryotic WCB are also shown. A comparative analysis of the promoter genes used to design WCBs is carried out, and the sensitivity and reproducibility of the main reporter genes used is also reviewed. Three main eukaryotic taxonomic groups are considered: yeasts, microalgae, and ciliated protozoa. Models that have been widely analyzed as potential WCBs are the Saccharomyces cerevisiae model among yeasts, the Tetrahymena thermophila model for ciliates and Chlamydomonas model for microalgae. The advantages and disadvantages of each microbial group are discussed, and a ranking of sensitivity to the same type of metal pollutant from reported eukaryotic WCBs is also shown. General conclusions and possible future developments of eukaryotic WCBs are reported.

  14. Heavy metal whole-cell biosensors using eukaryotic microorganisms: an updated critical review

    PubMed Central

    Gutiérrez, Juan C.; Amaro, Francisco; Martín-González, Ana

    2015-01-01

    This review analyzes the advantages and disadvantages of using eukaryotic microorganisms to design whole-cell biosensors (WCBs) for monitoring environmental heavy metal pollution in soil or aquatic habitats. Basic considerations for designing a eukaryotic WCB are also shown. A comparative analysis of the promoter genes used to design WCBs is carried out, and the sensitivity and reproducibility of the main reporter genes used is also reviewed. Three main eukaryotic taxonomic groups are considered: yeasts, microalgae, and ciliated protozoa. Models that have been widely analyzed as potential WCBs are the Saccharomyces cerevisiae model among yeasts, the Tetrahymena thermophila model for ciliates and Chlamydomonas model for microalgae. The advantages and disadvantages of each microbial group are discussed, and a ranking of sensitivity to the same type of metal pollutant from reported eukaryotic WCBs is also shown. General conclusions and possible future developments of eukaryotic WCBs are reported. PMID:25750637

  15. Whole-cell-analysis of live cardiomyocytes using wide-field interferometric phase microscopy

    PubMed Central

    Shaked, Natan T.; Satterwhite, Lisa L.; Bursac, Nenad; Wax, Adam

    2010-01-01

    We apply wide-field interferometric microscopy techniques to acquire quantitative phase profiles of ventricular cardiomyocytes in vitro during their rapid contraction with high temporal and spatial resolution. The whole-cell phase profiles are analyzed to yield valuable quantitative parameters characterizing the cell dynamics, without the need to decouple thickness from refractive index differences. Our experimental results verify that these new parameters can be used with wide field interferometric microscopy to discriminate the modulation of cardiomyocyte contraction dynamics due to temperature variation. To demonstrate the necessity of the proposed numerical analysis for cardiomyocytes, we present confocal dual-fluorescence-channel microscopy results which show that the rapid motion of the cell organelles during contraction preclude assuming a homogenous refractive index over the entire cell contents, or using multiple-exposure or scanning microscopy. PMID:21258502

  16. Whole-cell bioconversion of vanillin to vanillic acid by Streptomyces viridosporus

    SciTech Connect

    Pometto, A.L. III; Crawford, D.L.

    1983-05-01

    A two-step batch fermentation-bioconversion of vanillin (4-hydroxy-3-methoxybenzaldehyde) to vanillic acid (4-hydroxy-3-methoxybenzoic acid) was developed, utilizing whole cells of Streptomyces viridosporus T7A. In the first step, cells were grown in a yeast extract-vanillin medium under conditions where cells produced an aromatic aldehyde oxidase. In the second step, vanillin was incubated with the active cells and was quantitatively oxidized to vanillic acid which accumulated in the growth medium. Vanillic acid was readily recovered from the spent medium by a combination of acid precipitation and ether extraction at greater than or equal to96% molar yield and upon recrystallization from glacial acetic acid was obtained in greater than or equal to99% purity.

  17. Whole-cell phase contrast imaging at the nanoscale using Fresnel Coherent Diffractive Imaging Tomography

    NASA Astrophysics Data System (ADS)

    Jones, Michael W. M.; van Riessen, Grant A.; Abbey, Brian; Putkunz, Corey T.; Junker, Mark D.; Balaur, Eugeniu; Vine, David J.; McNulty, Ian; Chen, Bo; Arhatari, Benedicta D.; Frankland, Sarah; Nugent, Keith A.; Tilley, Leann; Peele, Andrew G.

    2013-07-01

    X-ray tomography can provide structural information of whole cells in close to their native state. Radiation-induced damage, however, imposes a practical limit to image resolution, and as such, a choice between damage, image contrast, and image resolution must be made. New coherent diffractive imaging techniques, such Fresnel Coherent Diffractive Imaging (FCDI), allows quantitative phase information with exceptional dose efficiency, high contrast, and nano-scale resolution. Here we present three-dimensional quantitative images of a whole eukaryotic cell by FCDI at a spatial resolution below 70 nm with sufficient phase contrast to distinguish major cellular components. From our data, we estimate that the minimum dose required for a similar resolution is close to that predicted by the Rose criterion, considerably below accepted estimates of the maximum dose a frozen-hydrated cell can tolerate. Based on the dose efficiency, contrast, and resolution achieved, we expect this technique will find immediate applications in tomographic cellular characterisation.

  18. Whole-cell bioconversion of vanillin to vanillic acid by Streptomyces viridosporus.

    PubMed Central

    Pometto, A L; Crawford, D L

    1983-01-01

    A two-step batch fermentation-bioconversion of vanillin (4-hydroxy-3-methoxybenzaldehyde) to vanillic acid (4-hydroxy-3-methoxybenzoic acid) was developed, utilizing whole cells of Streptomyces viridosporus T7A. In the first step, cells were grown in a yeast extract-vanillin medium under conditions where cells produced an aromatic aldehyde oxidase. In the second step, vanillin was incubated with the active cells and was quantitatively oxidized to vanillic acid which accumulated in the growth medium. Vanillic acid was readily recovered from the spent medium by a combination of acid precipitation and ether extraction at greater than or equal to 96% molar yield and upon recrystallization from glacial acetic acid was obtained in greater than or equal to 99% purity. PMID:6870241

  19. Whole Cell Screen for Inhibitors of pH Homeostasis in Mycobacterium tuberculosis

    PubMed Central

    Darby, Crystal M.; Ingólfsson, Helgi I.; Jiang, Xiuju; Shen, Chun; Sun, Mingna; Zhao, Nan; Burns, Kristin; Liu, Gang; Ehrt, Sabine; Warren, J. David; Anderson, Olaf S.; Brickner, Steven J.; Nathan, Carl

    2013-01-01

    Bacterial pathogens like Mycobacterium tuberculosis (Mtb) encounter acidic microenvironments in the host and must maintain their acid-base homeostasis to survive. A genetic screen identified two Mtb strains that cannot control intrabacterial pH (pHIB) in an acidic environment; infection with either strain led to severe attenuation in mice. To search for additional proteins that Mtb requires to survive at low pH, we introduced a whole-cell screen for compounds that disrupt pHIB, along with counter-screens that identify ionophores and membrane perturbors. Application of these methods to a natural product library identified four compounds of interest, one of which may inhibit novel pathway(s). This approach yields compounds that may lead to the identification of pathways that allow Mtb to survive in acidic environments, a setting in which Mtb is resistant to most of the drugs currently used to treat tuberculosis. PMID:23935911

  20. Immobilization of Bacillus acidocaldarius whole-cell rhodanese in polysaccharide and insolubilized gelatin gels

    SciTech Connect

    De Riso, L.; Alteriis, E. de; Parascandola, P. |; La Cara, F.; Sada, A.

    1996-04-01

    The presence of rhodanese activity has been investigated in two strains of thermophilic eubacteria and two strains of extremophiles. Bacillus acidocaldarius, a thermoacidophilic eubacterium, showed the highest levels of enzyme activity. Whole cells, previously subjected to one cycle of freeze-thawing, were immobilized by entrapment in the polysaccharide matrices Ca-alginate, {kappa}-carrageenan and chitosan, and in an insolubilized gelatin gel. The results obtained with the different immobilizates in terms of activity yield, possibility of regeneration and operative stability were evaluated with the aim of setting up a continuous system. This was achieved with a system consisting of B. acidocaldarius cells entrapped in an insolubilized gelatin matrix. The latter, in the form of a thin membrane, was employed in a custom-conceived reactor operating as a plug flow reactor. 21 refs., 3 figs., 2 tabs.

  1. Advanced Materials for the Recognition and Capture of Whole Cells and Microorganisms.

    PubMed

    Bole, Amanda L; Manesiotis, Panagiotis

    2016-07-01

    Selective cell recognition and capture has recently attracted significant interest due to its potential importance for clinical, diagnostic, environmental, and security applications. Current methods for cell isolation from complex samples are largely dependent on cell size and density, with limited application scope as many of the target cells do not exhibit appreciable differences in this respect. The most recent and forthcoming developments in the area of selective recognition and capture of whole cells, based on natural receptors, as well as synthetic materials utilising physical and chemical properties of the target cell or microorganism, are highlighted. Particular focus is given to the development of cell complementary surfaces using the cells themselves as templating agents, by means of molecular imprinting, and their combination with sensing platforms for rapid cell detection in complex media. The benefits and challenges of each approach are discussed and a perspective of the future of this research area is given.

  2. Whole cell cryo-electron tomography suggests mitochondria divide by budding.

    PubMed

    Hu, Guo-Bin

    2014-08-01

    Eukaryotes rely on mitochondrial division to guarantee that each new generation of cells acquires an adequate number of mitochondria. Mitochondrial division has long been thought to occur by binary fission and, more recently, evidence has supported the idea that binary fission is mediated by dynamin-related protein (Drp1) and the endoplasmic reticulum. However, studies to date have depended on fluorescence microscopy and conventional electron microscopy. Here, we utilize whole cell cryo-electron tomography to visualize mitochondrial division in frozen hydrated intact HeLa cells. We observe a large number of relatively small mitochondria protruding from and connected to large mitochondria or mitochondrial networks. Therefore, this study provides evidence that mitochondria divide by budding. PMID:24870811

  3. Copper sulfate improves pullulan production by bioconversion using whole cells of Aureobasidium pullulans as the catalyst.

    PubMed

    Wang, Dahui; Ju, Xiaomin; Zhang, Gaochuan; Wang, Donghua; Wei, Gongyuan

    2016-10-01

    The effects of mineral salts on pullulan production by bioconversion using whole cells of Aureobasidium pullulans CCTCC M 2012259 as the catalyst were investigated. Copper sulfate (CuSO4) improved pullulan production by 36.2% and 42.3% when added at the optimum concentration of 0.2mg/L to the bioconversion broth or seed medium, respectively, as compared with controls without CuSO4 addition. Pullulan production was further enhanced when CuSO4 was added to both seed medium and bioconversion broth simultaneously. In order to probe the mechanism of CuSO4 improvement, cell viability, membrane integrity, intracellular adenosine triphosphate (ATP) levels and the activities of key enzymes involved in pullulan biosynthesis were determined. As a result, CuSO4 increased the activities of key biosynthetic enzymes, maintained intracellular ATP at a higher level, and accelerated the rate of pullulan secretion, all of which contributed to improved pullulan production by bioconversion.

  4. Human xanthine oxidase recombinant in E. coli: A whole cell catalyst for preparative drug metabolite synthesis.

    PubMed

    Ferreira Antunes, Márcia; Eggimann, Fabian Kurt; Kittelmann, Matthias; Lütz, Stephan; Hanlon, Steven P; Wirz, Beat; Bachler, Thorsten; Winkler, Margit

    2016-10-10

    Human xanthine oxidoreductase (XOR), which is responsible for the final steps of the purine metabolism pathway and involved in oxidative drug metabolism, was successfully expressed in Escherichia coli BL21(DE3) Gold. Recombinant human (rh) XOR yielded higher productivity with the gene sequence optimized for expression in E.coli than with the native gene sequence. Induction of XOR expression with lactose or IPTG resulted in complete loss of activity whereas shake flasks cultures using media rather poor in nutrients resulted in functional XOR expression in the stationary phase. LB medium was used for a 25L fermentation in fed-batch mode, which led to a 5 fold increase of the enzyme productivity when compared to cultivation in shake flasks. Quinazoline was used as a substrate on the semi-preparative scale using an optimized whole cell biotransformation protocol, yielding 73mg of the isolated product, 4-quinazolinone, from 104mg of starting material.

  5. Production of cytidine 5'-diphosphorylcholine with high utilization of ATP by whole cells of Saccharomyces cerevisiae.

    PubMed

    Tang, Jiapeng; Chen, Yong; Chen, Xiaochun; Yao, Yuelan; Ying, Hanjie; Xiong, Jian; Bai, Jianxin

    2010-11-01

    Cytidine 5'-diphosphorylcholine (CDP-choline) was produced using a high efficiency ATP regeneration system and the Kennedy pathway in whole cells of Saccharomyces cerevisiae As 2.398. Out of eight variables, KH(2)PO(4), glycerol and (NH(4))(2)SO(4) were considered to be the most significant factors by response surface methodology including a Plackett-Burman design, path of steepest accent and central composite design. The optimum levels of the three variables were 20.13g/L KH(2)PO(4), 12.35g/L glycerol and 0.49g/L (NH(4))(2)SO(4), respectively. Energy utilization efficiency increased from 10.59% to 16.72% and choline chloride conversion yields increased from 12.35% to 42.78%. A high efficiency ATP regeneration system improves CDP-choline production.

  6. Whole Cell-SELEX Aptamers for Highly Specific Fluorescence Molecular Imaging of Carcinomas In Vivo

    PubMed Central

    He, Xiaoxiao; Guo, Qiuping; Wang, Kemin; Ye, Xiaosheng; Tang, Jinlu

    2013-01-01

    Background Carcinomas make up the majority of cancers. Their accurate and specific diagnoses are of great significance for the improvement of patients' curability. Methodology/Principal Findings In this paper, we report an effectual example of the in vivo fluorescence molecular imaging of carcinomas with extremely high specificity based on whole cell-SELEX aptamers. Firstly, S6, an aptamer against A549 lung carcinoma cells, was adopted and labeled with Cy5 to serve as a molecular imaging probe. Flow cytometry assays revealed that Cy5-S6 could not only specifically label in vitro cultured A549 cells in buffer, but also successfully achieve the detection of ex vivo cultured target cells in serum. When applied to in vivo imaging, Cy5-S6 was demonstrated to possess high specificity in identifying A549 carcinoma through a systematic comparison investigation. Particularly, after Cy5-S6 was intravenously injected into nude mice which were simultaneously grafted with A549 lung carcinoma and Tca8113 tongue carcinoma, a much longer retention time of Cy5-S6 in A549 tumor was observed and a clear targeted cancer imaging result was presented. On this basis, to further promote the application to imaging other carcinomas, LS2 and ZY8, which are two aptamers selected by our group against Bel-7404 and SMMC-7721 liver carcinoma cells respectively, were tested in a similar way, both in vitro and in vivo. Results showed that these aptamers were even effective in differentiating liver carcinomas of different subtypes in the same body. Conclusions/Significance This work might greatly advance the application of whole cell-SELEX aptamers to carcinomas-related in vivo researches. PMID:23950940

  7. Fragment-Based Whole Cell Screen Delivers Hits against M. tuberculosis and Non-tuberculous Mycobacteria

    PubMed Central

    Moreira, Wilfried; Lim, Jia Jie; Yeo, Si Ying; Ramanujulu, Pondy M.; Dymock, Brian W.; Dick, Thomas

    2016-01-01

    Reactive multi-target ‘fragment drugs’ represent critical components of current tuberculosis regimens. These compounds, such as pyrazinamide, are old synthetic antimycobacterials that are activated inside Mycobacterium tuberculosis bacilli and are smaller than the usual drug-like, single-target molecules. Based on the success of small ‘dirty’ drugs in the chemotherapy of tuberculosis, we suggested previously that fragment-based whole cell screens should be introduced in our current antimycobacterial drug discovery efforts. Here, we carried out such a screen and characterized bactericidal activity, selectivity and spectrum of hits we obtained. A library of 1725 fragments was tested at a single concentration for growth inhibitory activity against M. bovis BCG as screening strain and 38 of 116 primary hits were confirmed in dose response analyses to be active against virulent M. tuberculosis. Bacterial kill experiments showed that most hits displayed bactericidal activity at their minimal inhibitory concentration. Cytotoxicity assays established that a large proportion of hits displayed a favorable selectivity index for mammalian cells. Importantly, one third of M. tuberculosis active fragments were also active against M. abscessus and M. avium, two emerging non-tuberculous mycobacterial (NTM) pathogens, opening the opportunity to develop broad spectrum antimycobacterials. Activity determination against Gram positive (Staphylococcus aureus) and Gram negative (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa) bacteria, as well as fungi (Candida albicans, Cryptococcus neoformans) showed only a small overlap indicating a generally narrow spectrum of these novel antimicrobial hits for mycobacteria. In conclusion, we carried out the first fragment-based whole cell screen against bacteria and identified a substantial number of hits with excellent physicochemical properties and dual activity against M. tuberculosis and NTM

  8. Fragment-Based Whole Cell Screen Delivers Hits against M. tuberculosis and Non-tuberculous Mycobacteria.

    PubMed

    Moreira, Wilfried; Lim, Jia Jie; Yeo, Si Ying; Ramanujulu, Pondy M; Dymock, Brian W; Dick, Thomas

    2016-01-01

    Reactive multi-target 'fragment drugs' represent critical components of current tuberculosis regimens. These compounds, such as pyrazinamide, are old synthetic antimycobacterials that are activated inside Mycobacterium tuberculosis bacilli and are smaller than the usual drug-like, single-target molecules. Based on the success of small 'dirty' drugs in the chemotherapy of tuberculosis, we suggested previously that fragment-based whole cell screens should be introduced in our current antimycobacterial drug discovery efforts. Here, we carried out such a screen and characterized bactericidal activity, selectivity and spectrum of hits we obtained. A library of 1725 fragments was tested at a single concentration for growth inhibitory activity against M. bovis BCG as screening strain and 38 of 116 primary hits were confirmed in dose response analyses to be active against virulent M. tuberculosis. Bacterial kill experiments showed that most hits displayed bactericidal activity at their minimal inhibitory concentration. Cytotoxicity assays established that a large proportion of hits displayed a favorable selectivity index for mammalian cells. Importantly, one third of M. tuberculosis active fragments were also active against M. abscessus and M. avium, two emerging non-tuberculous mycobacterial (NTM) pathogens, opening the opportunity to develop broad spectrum antimycobacterials. Activity determination against Gram positive (Staphylococcus aureus) and Gram negative (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa) bacteria, as well as fungi (Candida albicans, Cryptococcus neoformans) showed only a small overlap indicating a generally narrow spectrum of these novel antimicrobial hits for mycobacteria. In conclusion, we carried out the first fragment-based whole cell screen against bacteria and identified a substantial number of hits with excellent physicochemical properties and dual activity against M. tuberculosis and NTM pathogens

  9. Fragment-Based Whole Cell Screen Delivers Hits against M. tuberculosis and Non-tuberculous Mycobacteria

    PubMed Central

    Moreira, Wilfried; Lim, Jia Jie; Yeo, Si Ying; Ramanujulu, Pondy M.; Dymock, Brian W.; Dick, Thomas

    2016-01-01

    Reactive multi-target ‘fragment drugs’ represent critical components of current tuberculosis regimens. These compounds, such as pyrazinamide, are old synthetic antimycobacterials that are activated inside Mycobacterium tuberculosis bacilli and are smaller than the usual drug-like, single-target molecules. Based on the success of small ‘dirty’ drugs in the chemotherapy of tuberculosis, we suggested previously that fragment-based whole cell screens should be introduced in our current antimycobacterial drug discovery efforts. Here, we carried out such a screen and characterized bactericidal activity, selectivity and spectrum of hits we obtained. A library of 1725 fragments was tested at a single concentration for growth inhibitory activity against M. bovis BCG as screening strain and 38 of 116 primary hits were confirmed in dose response analyses to be active against virulent M. tuberculosis. Bacterial kill experiments showed that most hits displayed bactericidal activity at their minimal inhibitory concentration. Cytotoxicity assays established that a large proportion of hits displayed a favorable selectivity index for mammalian cells. Importantly, one third of M. tuberculosis active fragments were also active against M. abscessus and M. avium, two emerging non-tuberculous mycobacterial (NTM) pathogens, opening the opportunity to develop broad spectrum antimycobacterials. Activity determination against Gram positive (Staphylococcus aureus) and Gram negative (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa) bacteria, as well as fungi (Candida albicans, Cryptococcus neoformans) showed only a small overlap indicating a generally narrow spectrum of these novel antimicrobial hits for mycobacteria. In conclusion, we carried out the first fragment-based whole cell screen against bacteria and identified a substantial number of hits with excellent physicochemical properties and dual activity against M. tuberculosis and NTM

  10. A computational framework for particle and whole cell tracking applied to a real biological dataset.

    PubMed

    Yang, Feng Wei; Venkataraman, Chandrasekhar; Styles, Vanessa; Kuttenberger, Verena; Horn, Elias; von Guttenberg, Zeno; Madzvamuse, Anotida

    2016-05-24

    Cell tracking is becoming increasingly important in cell biology as it provides a valuable tool for analysing experimental data and hence furthering our understanding of dynamic cellular phenomena. The advent of high-throughput, high-resolution microscopy and imaging techniques means that a wealth of large data is routinely generated in many laboratories. Due to the sheer magnitude of the data involved manual tracking is often cumbersome and the development of computer algorithms for automated cell tracking is thus highly desirable. In this work, we describe two approaches for automated cell tracking. Firstly, we consider particle tracking. We propose a few segmentation techniques for the detection of cells migrating in a non-uniform background, centroids of the segmented cells are then calculated and linked from frame to frame via a nearest-neighbour approach. Secondly, we consider the problem of whole cell tracking in which one wishes to reconstruct in time whole cell morphologies. Our approach is based on fitting a mathematical model to the experimental imaging data with the goal being that the physics encoded in the model is reflected in the reconstructed data. The resulting mathematical problem involves the optimal control of a phase-field formulation of a geometric evolution law. Efficient approximation of this challenging optimal control problem is achieved via advanced numerical methods for the solution of semilinear parabolic partial differential equations (PDEs) coupled with parallelisation and adaptive resolution techniques. Along with a detailed description of our algorithms, a number of simulation results are reported on. We focus on illustrating the effectivity of our approaches by applying the algorithms to the tracking of migrating cells in a dataset which reflects many of the challenges typically encountered in microscopy data.

  11. Killed but metabolically active Leishmania infantum as a novel whole-cell vaccine for visceral leishmaniasis.

    PubMed

    Bruhn, Kevin W; Birnbaum, Ron; Haskell, Jacquelyn; Vanchinathan, Veena; Greger, Stephanie; Narayan, Rupa; Chang, Pei-Lin; Tran, Thu Anh; Hickerson, Suzanne M; Beverley, Stephen M; Wilson, Mary E; Craft, Noah

    2012-04-01

    There are currently no effective vaccines for visceral leishmaniasis, the second most deadly parasitic infection in the world. Here, we describe a novel whole-cell vaccine approach using Leishmania infantum chagasi promastigotes treated with the psoralen compound amotosalen (S-59) and low doses of UV A radiation. This treatment generates permanent, covalent DNA cross-links within parasites and results in Leishmania organisms termed killed but metabolically active (KBMA). In this report, we characterize the in vitro growth characteristics of both KBMA L. major and KBMA L. infantum chagasi. Concentrations of S-59 that generate optimally attenuated parasites were identified. Like live L. infantum chagasi, KBMA L. infantum chagasi parasites were able to initially enter liver cells in vivo after intravenous infection. However, whereas live L. infantum chagasi infection leads to hepatosplenomegaly in mice after 6 months, KBMA L. infantum chagasi parasites were undetectable in the organs of mice at this time point. In vitro, KBMA L. infantum chagasi retained the ability to enter macrophages and induce nitric oxide production. These characteristics of KBMA L. infantum chagasi correlated with the ability to prophylactically protect mice via subcutaneous vaccination at levels similar to vaccination with live, virulent organisms. Splenocytes from mice vaccinated with either live L. infantum chagasi or KBMA L. infantum chagasi displayed similar cytokine patterns in vitro. These results suggest that KBMA technology is a potentially safe and effective novel vaccine strategy against the intracellular protozoan L. infantum chagasi. This approach may represent a new method for whole-cell vaccination against other complex intracellular pathogens.

  12. Phospholipid-templated silica nanocapsules as efficient polyenzymatic biocatalysts.

    PubMed

    Phuoc, Lai Truong; Laveille, Paco; Chamouleau, Françoise; Renard, Gilbert; Drone, Jullien; Coq, Bernard; Fajula, François; Galarneau, Anne

    2010-09-28

    Solid polyenzymatic biocatalysts have been designed by combining two immobilized enzymes, the first one allowing the in situ generation of H(2)O(2) from air and the second one performing an oxidation reaction. The in situ H(2)O(2) generation system is based on the reaction of glucose with air using a glucose oxidase (GOx). The optimization of the encapsulation of GOx into phospholipids-templated silica capsules (NPS) was performed. A bienzymatic system made of GOx and horseradish peroxidase (HRP) was studied. Optimal conditions for the activity of the GOx/HRP bienzymatic system have been determined for both homogeneous and heterogeneous conditions. The encapsulation in NPS materials increases the stability of both enzymes. The performance of the encapsulated bienzymatic GOx/HRP system in the model reaction of 4-aminoantipyridine with phenol is similar when the enzymes are immobilized separately in two NPS or coencapsulated in the same NPS. An excess of peroxidase compared to GOx ([HRP]/[GOx] = 5-10) is necessary to obtain the optimal activity. To show the potentiality of bienzymatic systems in real applications, HRP has been replaced by hemoglobin, which is known for its ability to oxidize polycyclic aromatic hydrocarbons (PAH) pollutants through a pseudoperoxidase pathway. A larger excess of Hb compared to GOx ([Hb]/[GOx] = 1000) was necessary to obtain the maximum PAH removal, as Hb is not a real peroxidase as HRP but a hemoprotein with some pseudoperoxidase activity. In opposite to real enzymes, the immobilization of Hb by adsorption in mesoporous silica is preferable as its encapsulation. Therefore, the bienzymatic system made of GOx encapsulated in NPS and Hb adsorbed in mesoporous silica has been used for the removal of 11 PAH from water. This heterogeneous bienzymatic system allows 64% of PAH removal from water using simple air as oxidant.

  13. Protein engineering and de novo designing of a biocatalyst.

    PubMed

    Kaushik, Mahima; Sinha, Prashant; Jaiswal, Pragya; Mahendru, Swati; Roy, Kapil; Kukreti, Shrikant

    2016-10-01

    Proteins as a biomolecule have been recognized as a "molecule with manifold biological functions". The functions not only include the structural, regulatory and transportation processes inside the body but also its capacity as an extremely specific catalyst for various biochemical reactions. Nature has been quite admirably using proteins as biocatalysts which are known as enzymes. Properties like higher reaction rate, good specificity, faster kinetics, production of lesser by-products and their non-hazardous nature make enzymes the most suitable targets for a process chemist to exploit. At the same time, limitations like a narrow range of substrates, requirement of coenzymes, lesser stability, smaller shelf-life, along with difficulties in procuring these enzymes, make this biocatalysis field quite challenging. For exploiting a broad range of applications related to therapeutics, biosensors, biotechnology, nanotechnology etc., de novo designing of proteins is of utmost importance. Enzymes with altered, specific and modified properties might be designed by utilizing the prior knowledge of structure and function of a protein with the help of computational modeling. Various protein engineering techniques like directed evolution, rational designing and immobilization strategies etc. have already been extensively used to address some of the issues. This review aims to update the repertoire of the advancements in the field of protein engineering, which can help in laying some guiding principles about designing, modifying and altering their usage for commercial industrial purposes. This possibility of effective and novel designing of peptides and proteins might further facilitate our understanding about the structure, function and folding patterns along with their inter-relationships. Copyright © 2016 John Wiley & Sons, Ltd.

  14. Advanced Synthesis of Conductive Polyaniline Using Laccase as Biocatalyst

    PubMed Central

    de Salas, Felipe; Pardo, Isabel; Salavagione, Horacio J.; Aza, Pablo; Amougi, Eleni; Vind, Jesper; Martínez, Angel T.; Camarero, Susana

    2016-01-01

    Polyaniline is a conductive polymer with distinctive optical and electrical properties. Its enzymatic synthesis is an environmentally friendly alternative to the use of harsh oxidants and extremely acidic conditions. 7D5L, a high-redox potential laccase developed in our lab, is the biocatalyst of choice for the synthesis of green polyaniline (emeraldine salt) due to its superior ability to oxidize aniline and kinetic stability at the required polymerization conditions (pH 3 and presence of anionic surfactants) as compared with other fungal laccases. Doses as low as 7.6 nM of 7D5L catalyze the polymerization of 15 mM aniline (in 24 h, room temperature, 7% yield) in the presence of different anionic surfactants used as doping templates to provide linear and water-soluble polymers. Aniline polymerization was monitored by the increase of the polaron absorption band at 800 nm (typical for emeraldine salt). Best polymerization results were obtained with 5 mM sodium dodecylbenzenesulfonate (SDBS) as template. At fixed conditions (15 mM aniline and 5mM SDBS), polymerization rates obtained with 7D5L were 2.5-fold the rates obtained with commercial Trametes villosa laccase. Moreover, polyaniline yield was notably boosted to 75% by rising 7D5L amount to 0.15 μM, obtaining 1g of green polyaniline in 1L-reaction volume. The green polymer obtained with the selected system (7D5L/SDBS) holds excellent electrochemical and electro-conductive properties displayed in water-dispersible nanofibers, which is advantageous for the nanomaterial to be readily cast into uniform films for different applications. PMID:27741301

  15. White biotechnology: State of the art strategies for the development of biocatalysts for biorefining.

    PubMed

    Heux, S; Meynial-Salles, I; O'Donohue, M J; Dumon, C

    2015-12-01

    White biotechnology is a term that is now often used to describe the implementation of biotechnology in the industrial sphere. Biocatalysts (enzymes and microorganisms) are the key tools of white biotechnology, which is considered to be one of the key technological drivers for the growing bioeconomy. Biocatalysts are already present in sectors such as the chemical and agro-food industries, and are used to manufacture products as diverse as antibiotics, paper pulp, bread or advanced polymers. This review proposes an original and global overview of highly complementary fields of biotechnology at both enzyme and microorganism level. A certain number of state of the art approaches that are now being used to improve the industrial fitness of biocatalysts particularly focused on the biorefinery sector are presented. The first part deals with the technologies that underpin the development of industrial biocatalysts, notably the discovery of new enzymes and enzyme improvement using directed evolution techniques. The second part describes the toolbox available by the cell engineer to shape the metabolism of microorganisms. And finally the last part focuses on the 'omic' technologies that are vital for understanding and guide microbial engineering toward more efficient microbial biocatalysts. Altogether, these techniques and strategies will undoubtedly help to achieve the challenging task of developing consolidated bioprocessing (i.e. CBP) readily available for industrial purpose. PMID:26303096

  16. White biotechnology: State of the art strategies for the development of biocatalysts for biorefining.

    PubMed

    Heux, S; Meynial-Salles, I; O'Donohue, M J; Dumon, C

    2015-12-01

    White biotechnology is a term that is now often used to describe the implementation of biotechnology in the industrial sphere. Biocatalysts (enzymes and microorganisms) are the key tools of white biotechnology, which is considered to be one of the key technological drivers for the growing bioeconomy. Biocatalysts are already present in sectors such as the chemical and agro-food industries, and are used to manufacture products as diverse as antibiotics, paper pulp, bread or advanced polymers. This review proposes an original and global overview of highly complementary fields of biotechnology at both enzyme and microorganism level. A certain number of state of the art approaches that are now being used to improve the industrial fitness of biocatalysts particularly focused on the biorefinery sector are presented. The first part deals with the technologies that underpin the development of industrial biocatalysts, notably the discovery of new enzymes and enzyme improvement using directed evolution techniques. The second part describes the toolbox available by the cell engineer to shape the metabolism of microorganisms. And finally the last part focuses on the 'omic' technologies that are vital for understanding and guide microbial engineering toward more efficient microbial biocatalysts. Altogether, these techniques and strategies will undoubtedly help to achieve the challenging task of developing consolidated bioprocessing (i.e. CBP) readily available for industrial purpose.

  17. Surface complexation of neptunium (V) onto whole cells and cell componets of Shewanella alga

    SciTech Connect

    Reed, Donald Timothy; Deo, Randhir P; Rittmann, Bruce E; Songkasiri, Warinthorn

    2008-01-01

    We systematically quantified surface complexation of neptunium(V) onto whole cells of Shewanella alga strain BrY and onto cell wall and extracellular polymeric substances (EPS) of S. alga. We first performed acid and base titrations and used the mathematical model FITEQL with constant-capacitance surface-complexation to determine the concentrations and deprotonation constants of specific surface functional groups. Deprotonation constants most likely corresponded to a carboxyl site associated with amino acids (pK{sub a} {approx} 2.4), a carboxyl group not associated with amino acids (pK{sub a} {approx} 5), a phosphoryl site (pK{sub a} {approx} 7.2), and an amine site (pK{sub a} > 10). We then carried out batch sorption experiments with Np(V) and each of the S. alga components at different pHs. Results show that solution pH influenced the speciation of Np(V) and each of the surface functional groups. We used the speciation sub-model of the biogeochemical model CCBATCH to compute the stability constants for Np(V) complexation to each surface functional group. The stability constants were similar for each functional group on S. alga bacterial whole cells, cell walls, and EPS, and they explain the complicated sorption patterns when they are combined with the aqueous-phase speciation of Np(V). For pH < 8, NpO{sub 2}{sup +} was the dominant form of Np(V), and its log K values for the low-pK{sub a} carboxyl, other carboxyl, and phosphoryl groups were 1.75, 1.75, and 2.5 to 3.1, respectively. For pH greater than 8, the key surface ligand was amine >XNH3+, which complexed with NpO{sub 2}(CO{sub 3}){sub 3}{sup 5-}. The log K for NpO{sub 2}(CO{sub 3}){sub 3}{sup 5-} complexed onto the amine groups was 3.1 to 3.6. All of the log K values are similar to those of Np(V) complexes with aqueous carboxyl and N-containing carboxyl ligands. These results point towards the important role of surface complexation in defining key actinide-microbiological interactions in the subsurface.

  18. [Major results of research and development of heterogenous biocatalysts for regenerated water clearing from harmful admixture].

    PubMed

    2012-01-01

    The biological method of clearing atmospheric condensate in pressurized habitats exploits filters with a heterogenic biocatalyst produced by way of immobilizing harmless for human, animal and plant microoganisms on water-insoluble solid carrier--foam polyvinyl-formal (FPVF), and a hydrogen peroxide biofilter containing triacetate cellulose-immobilized catalase. Experience of forming an immobilized bacterial association as a polyenzyme system is particularly promising for development of advanced biotechnologies. Biocatalysts with expanded applicability can be manufactured using a FPVF-immobilized associative bacterial culture composed of Paracoccus denitrificans, Pseudomonas esterophilus and Methilopila capsulata. In aerobic condition at room temperature the heterogenic biocatalyst is capable to transform harmful organics in atmospheric condensate, e.g. methyl amine, ethyl acetate, acetic acid, ethanol and acetone into the end-products, i.e. carbon dioxide and water. Ammonia is consumed by 3 cultures as a source of nitrogen. PMID:23116037

  19. An Efficient, Recyclable, and Stable Immobilized Biocatalyst Based on Bioinspired Microcapsules-in-Hydrogel Scaffolds.

    PubMed

    Zhang, Shaohua; Jiang, Zhongyi; Shi, Jiafu; Wang, Xueyan; Han, Pingping; Qian, Weilun

    2016-09-28

    Design and preparation of high-performance immobilized biocatalysts with exquisite structures and elucidation of their profound structure-performance relationship are highly desired for green and sustainable biotransformation processes. Learning from nature has been recognized as a shortcut to achieve such an impressive goal. Loose connective tissue, which is composed of hierarchically organized cells by extracellular matrix (ECM) and is recognized as an efficient catalytic system to ensure the ordered proceeding of metabolism, may offer an ideal prototype for preparing immobilized biocatalysts with high catalytic activity, recyclability, and stability. Inspired by the hierarchical structure of loose connective tissue, we prepared an immobilized biocatalyst enabled by microcapsules-in-hydrogel (MCH) scaffolds via biomimetic mineralization in agarose hydrogel. In brief, the in situ synthesized hybrid microcapsules encapsulated with glucose oxidase (GOD) are hierarchically organized by the fibrous framework of agarose hydrogel, where the fibers are intercalated into the capsule wall. The as-prepared immobilized biocatalyst shows structure-dependent catalytic performance. The porous hydrogel permits free diffusion of glucose molecules (diffusion coefficient: ∼6 × 10(-6) cm(2) s(-1), close to that in water) and retains the enzyme activity as much as possible after immobilization (initial reaction rate: 1.5 × 10(-2) mM min(-1)). The monolithic macroscale of agarose hydrogel facilitates the easy recycling of the immobilized biocatalyst (only by using tweezers), which contributes to the nonactivity decline during the recycling test. The fiber-intercalating structure elevates the mechanical stability of the in situ synthesized hybrid microcapsules, which inhibits the leaching and enhances the stability of the encapsulated GOD, achieving immobilization efficiency of ∼95%. This study will, therefore, provide a generic method for the hierarchical organization of (bio

  20. Apparatus and method for the production of gel beads containing a biocatalyst

    DOEpatents

    Scott, C.D.; Scott, T.C.; Davison, B.H.

    1998-01-27

    An apparatus and method are disclosed for the large-scale and continuous production of gel beads containing a biocatalyst. The apparatus is a columnar system based on the chemical cross-linking of hydrocolloidal gels that contain and immobilize a biocatalyst, the biocatalyst being a microorganism or an enzyme. Hydrocolloidal gels, such as alginate, carrageenan, and a mixture of bone gelatin and modified alginate, provide immobilization matrices that can be used to entrap and retain the biocatalyst while allowing effective contact with substrates and release of products. Such immobilized biocatalysts are generally formulated into small spheres or beads that have high concentrations of the biocatalyst within the gel matrix. The columnar system includes a gel dispersion nozzle submerged in a heated non-interacting liquid, typically an organic liquid, that is immiscible with water to allow efficient formation of spherical gel droplets, the non-interacting liquid having a specific gravity that is less than water so that the gel droplets will fall through the liquid by the force of gravity. The heated non-interacting liquid is in direct contact with a chilled upflowing non-interacting liquid that will provide sufficient residence time for the gel droplets as they fall through the liquid so that they will be cooled below the gelling temperature and form solid spheres. The upflowing non-interacting liquid is in direct contact with an upflowing temperature-controlled aqueous solution containing the necessary chemicals for cross-linking or fixing of the gel beads to add the necessary stability. The flow rates of the two liquid streams can be varied to control the proper residence time in each liquid section to accommodate the production of gel beads of differing settling velocities. A valve is provided for continuous removal of the stabilized gel beads from the bottom of the column. 1 fig.

  1. Apparatus and method for the production of gel beads containing a biocatalyst

    DOEpatents

    Scott, Charles D.; Scott, Timothy C.; Davison, Brian H.

    1998-01-01

    An apparatus and method for the large-scale and continuous production of gel beads containing a biocatalyst. The apparatus is a columnar system based on the chemical cross-linking of hydrocolloidal gels that contain and immobilize a biocatalyst, the biocatalyst being a microorganism or an enzyme. Hydrocolloidal gels, such as alginate, carrageenan, and a mixture of bone gelatin and modified alginate, provide immobilization matrices that can be used to entrap and retain the biocatalyst while allowing effective contact with substrates and release of products. Such immobilized biocatalysts are generally formulated into small spheres or beads that have high concentrations of the biocatalyst within the gel matrix. The columnar system includes a gel dispersion nozzle submerged in a heated non-interacting liquid, typically an organic liquid, that is immiscible with water to allow efficient formation of spherical gel droplets, the non-interacting liquid having a specific gravity that is less than water so that the gel droplets will fall through the liquid by the force of gravity. The heated non-interacting liquid is in direct contact with a chilled upflowing non-interacting liquid that will provide sufficient residence time for the gel droplets as they fall through the liquid so that they will be cooled below the gelling temperature and form solid spheres. The upflowing non-interacting liquid is in direct contact with an upflowing temperature-controlled aqueous solution containing the necessary chemicals for cross-linking or fixing of the gel beads to add the necessary stability. The flow rates of the two liquid streams can be varied to control the proper residence time in each liquid section to accommodate the production of gel beads of differing settling velocities. A valve is provided for continuous removal of the stabilized gel beads from the bottom of the column.

  2. An Efficient, Recyclable, and Stable Immobilized Biocatalyst Based on Bioinspired Microcapsules-in-Hydrogel Scaffolds.

    PubMed

    Zhang, Shaohua; Jiang, Zhongyi; Shi, Jiafu; Wang, Xueyan; Han, Pingping; Qian, Weilun

    2016-09-28

    Design and preparation of high-performance immobilized biocatalysts with exquisite structures and elucidation of their profound structure-performance relationship are highly desired for green and sustainable biotransformation processes. Learning from nature has been recognized as a shortcut to achieve such an impressive goal. Loose connective tissue, which is composed of hierarchically organized cells by extracellular matrix (ECM) and is recognized as an efficient catalytic system to ensure the ordered proceeding of metabolism, may offer an ideal prototype for preparing immobilized biocatalysts with high catalytic activity, recyclability, and stability. Inspired by the hierarchical structure of loose connective tissue, we prepared an immobilized biocatalyst enabled by microcapsules-in-hydrogel (MCH) scaffolds via biomimetic mineralization in agarose hydrogel. In brief, the in situ synthesized hybrid microcapsules encapsulated with glucose oxidase (GOD) are hierarchically organized by the fibrous framework of agarose hydrogel, where the fibers are intercalated into the capsule wall. The as-prepared immobilized biocatalyst shows structure-dependent catalytic performance. The porous hydrogel permits free diffusion of glucose molecules (diffusion coefficient: ∼6 × 10(-6) cm(2) s(-1), close to that in water) and retains the enzyme activity as much as possible after immobilization (initial reaction rate: 1.5 × 10(-2) mM min(-1)). The monolithic macroscale of agarose hydrogel facilitates the easy recycling of the immobilized biocatalyst (only by using tweezers), which contributes to the nonactivity decline during the recycling test. The fiber-intercalating structure elevates the mechanical stability of the in situ synthesized hybrid microcapsules, which inhibits the leaching and enhances the stability of the encapsulated GOD, achieving immobilization efficiency of ∼95%. This study will, therefore, provide a generic method for the hierarchical organization of (bio

  3. Sputtering deposition of magnetic Ni nanoparticles directly onto an enzyme surface: a novel method to obtain a magnetic biocatalyst.

    PubMed

    Bussamara, Roberta; Eberhardt, Dario; Feil, Adriano F; Migowski, Pedro; Wender, Heberton; de Moraes, Diogo P; Machado, Giovanna; Papaléo, Ricardo M; Teixeira, Sérgio R; Dupont, Jairton

    2013-02-14

    A simple one-step method based on the sputtering deposition of Ni nanoparticles (NP) has been developed for the production of magnetic biocatalysts, avoiding the complications and drawbacks of methods based on chemical functionalisation or coating of magnetic NP. This new technique provided high levels of recovery, reusability and catalytic activity for the lipase-Ni biocatalyst.

  4. Biocatalysts and methods for conversion of hemicellulose hydrolysates to biobased products

    SciTech Connect

    Preston, James F

    2015-03-31

    The invention relates to processes and biocatalysts for producing ethanol and other useful products from biomass and/or other materials. Initial processing of lignocellulosic biomass frequently yields methylglucuronoxylose (MeGAX) and related products which are resistant to further processing by common biocatalysts. Strains of Enterobacter asburiae are shown to be useful in bioprocessing of MeGAX and other materials into useful bioproducts such as ethanol, acetate, lactate, and many others. Genetic engineering may be used to enhance production of desired bioproducts.

  5. A UAV-Mounted Whole Cell Biosensor System for Environmental Monitoring Applications.

    PubMed

    Lu, Yi; Macias, Dominique; Dean, Zachary S; Kreger, Nicole R; Wong, Pak Kin

    2015-12-01

    This study reports the development of a portable whole cell biosensor system for environmental monitoring applications, such as air quality control, water pollution monitoring, and radiation leakage detection. The system consists of a lightweight mechanical housing, a temperature regulating system, and a microfluidic bacterial inoculation channel. The overall system, which is less than 200 g, serves as a portable incubator for cell inoculation and can be mounted on an unmanned aerial vehicle for monitoring remote and unreachable locations. The feedback control system maintains the inoculation temperature within 0.05 °C. The large surface-to-volume ratio of the polydimethylsiloxane microchannel facilitates effective gas exchange for rapid bacterial growth. Molecular dynamic simulation shows effective diffusion of major gas pollutants in PDMS toward gas sensing applications. By optimizing the design, we demonstrate the operation of the system in ambient temperatures from 5 °C to 32 °C and rapid bacterial growth in microchannels compared to standard bacterial culture techniques.

  6. Toxicity assessment and modelling of Moringa oleifera seeds in water purification by whole cell bioreporter.

    PubMed

    Al-Anizi, Ali Adnan; Hellyer, Maria Theresa; Zhang, Dayi

    2014-06-01

    Moringa oleifera has been used as a coagulation reagent for drinking water purification, especially in developing countries such as Malawi. This research revealed the cytoxicity and genotoxicity of M. oleifera by Acinetobacter bioreporter. The results indicated that significant cytoxicity effects were observed when the powdered M. oleifera seeds concentration is from 1 to 50 mg/L. Through direct contact, ethanolic-water extraction and hexane extraction, the toxic effects of hydrophobic and hydrophilic components in M. oleifera seeds were distinguished. It suggested that the hydrophobic lipids contributed to the dominant cytoxicity, consequently resulting in the dominant genotoxicity in the water-soluble fraction due to limited dissolution when the M. oleifera seeds granule concentration was from 10 to 1000 mg/L. Based on cytoxicity and genotoxicity model, the LC50 and LC90 of M. oleifera seeds were 8.5 mg/L and 300 mg/L respectively and their genotoxicity was equivalent to 8.3 mg mitomycin C per 1.0 g dry M. oleifera seed. The toxicity of M. oleifera has also remarkable synergistic effects, suggesting whole cell bioreporter as an appropriate and complementary tool to chemical analysis for environmental toxicity assessment.

  7. Gold nanoparticle uptake in whole cells in liquid examined by environmental scanning electron microscopy.

    PubMed

    Peckys, Diana B; de Jonge, Niels

    2014-02-01

    The size of gold nanoparticles (AuNPs) can influence various aspects of their cellular uptake. Light microscopy is not capable of resolving most AuNPs, while electron microscopy (EM) is not practically capable of acquiring the necessary statistical data from many cells and the results may suffer from various artifacts. Here, we demonstrate the use of a fast EM method for obtaining high-resolution data from a much larger population of cells than is usually feasible with conventional EM. A549 (human lung carcinoma) cells were subjected to uptake protocols with 10, 15, or 30 nm diameter AuNPs with adsorbed serum proteins. After 20 min, 24 h, or 45 h, the cells were fixed and imaged in whole in a thin layer of liquid water with environmental scanning electron microscopy equipped with a scanning transmission electron microscopy detector. The fast preparation and imaging of 145 whole cells in liquid allowed collection of nanoscale data within an exceptionally small amount of time of ~80 h. Analysis of 1,041 AuNP-filled vesicles showed that the long-term AuNP storing lysosomes increased their average size by 80 nm when AuNPs with 30 nm diameter were uptaken, compared to lysosomes of cells incubated with AuNPs of 10 and 15 nm diameter.

  8. Substoichiometric hydroxynonenylation of a single protein recapitulates whole-cell-stimulated antioxidant response.

    PubMed

    Parvez, Saba; Fu, Yuan; Li, Jiayang; Long, Marcus J C; Lin, Hong-Yu; Lee, Dustin K; Hu, Gene S; Aye, Yimon

    2015-01-14

    Lipid-derived electrophiles (LDEs) that can directly modify proteins have emerged as important small-molecule cues in cellular decision-making. However, because these diffusible LDEs can modify many targets [e.g., >700 cysteines are modified by the well-known LDE 4-hydroxynonenal (HNE)], establishing the functional consequences of LDE modification on individual targets remains devilishly difficult. Whether LDE modifications on a single protein are biologically sufficient to activate discrete redox signaling response downstream also remains untested. Herein, using T-REX (targetable reactive electrophiles and oxidants), an approach aimed at selectively flipping a single redox switch in cells at a precise time, we show that a modest level (∼34%) of HNEylation on a single target is sufficient to elicit the pharmaceutically important antioxidant response element (ARE) activation, and the resultant strength of ARE induction recapitulates that observed from whole-cell electrophilic perturbation. These data provide the first evidence that single-target LDE modifications are important individual events in mammalian physiology.

  9. Whole-cell Gluconobacter oxydans biosensor for 2-phenylethanol biooxidation monitoring.

    PubMed

    Schenkmayerová, Andrea; Bertóková, Anikó; Sefčovičová, Jana; Stefuca, Vladimír; Bučko, Marek; Vikartovská, Alica; Gemeiner, Peter; Tkáč, Ján; Katrlík, Jaroslav

    2015-01-01

    A microbial biosensor for 2-phenylethanol (2-PE) based on the bacteria Gluconobacter oxydans was developed and applied in monitoring of a biotechnological process. The cells of G. oxydans were immobilized within a disposable polyelectrolyte complex gel membrane consisting of sodium alginate, cellulose sulphate and poly(methylene-co-guanidine) attached onto a miniaturized Clark oxygen electrode, forming whole cell amperometric biosensor. Measured changes in oxygen concentration were proportional to changes in 2-PE concentration. The biosensor sensitivity was 864 nA mM(-1) (RSD=6%), a detection limit of 1 μM, and the biosensor response towards 2-PE was linear in the range 0.02-0.70 mM. The biosensor preserved 93% of its initial sensitivity after 7h of continuous operation and exhibited excellent storage stability with loss of only 6% of initial sensitivity within two months, when stored at 4°C. The developed system was designed and successfully used for an off-line monitoring of whole course of 2-PE biooxidation process producing phenylacetic acid (PA) as industrially valuable aromatic compound. The biosensor measurement did not require the use of hazardous organic solvent. The biosensor response to 2-PE was not affected by interferences from PA and phenylacetaldehyde at concentrations present in real samples during the biotransformation and the results were in a very good agreement with those obtained via gas chromatography.

  10. Whole-cell imaging at nanometer resolutions using fast and slow focused helium ions.

    PubMed

    Chen, Xiao; Udalagama, Chammika N B; Chen, Ce-Belle; Bettiol, Andrew A; Pickard, Daniel S; Venkatesan, T; Watt, Frank

    2011-10-01

    Observations of the interior structure of cells and subcellular organelles are important steps in unraveling organelle functions. Microscopy using helium ions can play a major role in both surface and subcellular imaging because it can provide subnanometer resolutions at the cell surface for slow helium ions, and fast helium ions can penetrate cells without a significant loss of resolution. Slow (e.g., 10-50 keV) helium ion beams can now be focused to subnanometer dimensions (∼0.25 nm), and keV helium ion microscopy can be used to image the surfaces of cells at high resolutions. Because of the ease of neutralizing the sample charge using a flood electron beam, surface charging effects are minimal and therefore cell surfaces can be imaged without the need for a conducting metallic coating. Fast (MeV) helium ions maintain a straight path as they pass through a cell. Along the ion trajectory, the helium ion undergoes multiple electron collisions, and for each collision a small amount of energy is lost to the scattered electron. By measuring the total energy loss of each MeV helium ion as it passes through the cell, we can construct an energy-loss image that is representative of the mass distribution of the cell. This work paves the way to use ions for whole-cell investigations at nanometer resolutions through structural, elemental (via nuclear elastic backscattering), and fluorescence (via ion induced fluorescence) imaging.

  11. Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

    PubMed Central

    Booker, Sam A.; Song, Jie; Vida, Imre

    2014-01-01

    GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons. PMID:25350149

  12. A whole-cell electrochemical biosensing system based on bacterial inward electron flow for fumarate quantification.

    PubMed

    Si, Rong-Wei; Zhai, Dan-Dan; Liao, Zhi-Hong; Gao, Lu; Yong, Yang-Chun

    2015-06-15

    Fumarate is of great importance as it is an oncometabolite as well as food spoilage indicator. However, cost-effective and fast quantification method for fumarate is lacking although it is urgently required. This work developed an electrochemical whole-cell biosensing system for fumarate quantification. A sensitive inwards electric output (electron flow from electrode into bacteria) responded to fumarate in Shewanella oneidensis MR-1 was characterized, and an electrochemical fumarate biosensing system was developed without genetic engineering. The biosensing system delivered symmetric current peak immediately upon fumarate addition, where the peak area increased in proportion to the increasing fumarate concentration with a wide range of 2 μM-10 mM (R(2)=0.9997). The limit of detection (LOD) and the limit of quantification (LOQ) are 0.83 μM and 1.2 μM, respectively. This biosensing system displayed remarkable specificity to fumarate against other possible interferences. It was also successfully applied to samples of apple juice and kidney tissue. This study added new dimension to electrochemical biosensor design, and provide a simple, cost-effective, fast and robust tool for fumarate quantification.

  13. Evaluation of bioavailable arsenic and remediation performance using a whole-cell bioreporter.

    PubMed

    Yoon, Youngdae; Kim, Sunghoon; Chae, Yooeun; Jeong, Seung-Woo; An, Youn-Joo

    2016-03-15

    The traditional method of evaluating the effects of soil contaminants on living organisms by measuring the total amount of contaminant has been largely inadequate, in part because testing contamination levels is hindered in real samples. Here we report a novel strategy for testing arsenic (As) bioavailability in soil samples by direct (in vivo) and indirect (in vitro) measurement using an Escherichia coli-based whole-cell bioreporter (WCB). The WCB was used to test As-amended Landwirtschaftliche Untersuchungs und Forschungsanstalt soils as well as field soils collected from a smelter area under remediation in order to evaluate the efficiency of bioavailable As removal. The percentage of bioavailable As in amended and field soils was 5.8% (range: 4.9%-7.6%) and 0.6% (0.08%-1.09%) of total As, respectively. In contaminated soils, total As was decreased, whereas bioavailable As was slightly increased after soil washing. These results emphasize the importance of considering ecotoxicological aspects of soil remediation; to this end, the WCB is a useful tool for evaluating the efficiency of soil remediation by assessing bioavailability along with the total amount of contaminant present. PMID:26780137

  14. Changing from whole-cell to acellular pertussis vaccines would trade superior tolerability for inferior protection.

    PubMed

    Herzog, Christian

    2015-01-01

    Notifications of infant deaths, assumed to be related to the introduction of new pentavalent DTwP-Hib-HBV childhood vaccines, caused, during 2008-2010 in few Asian countries, temporary interruptions of the respective vaccination programs. The sudden appearance of fatal cases was due to increased awareness/publicity and improved safety monitoring/reporting in countries with relatively high background infant mortalities. WHO investigations could not establish any causal relationships and vaccinations were again resumed. Recently, questions were raised in one concerned country as to why not to change to less reactogenic acellular pertussis (aP)-containing vaccines that are available in private practice and are generally perceived as 'better'. For resource-poor countries, the financial impacts render such a switch impossible and would also not be supported by external funding. Furthermore, it would be a disservice to the children, as in recent years evidence of inferior long-term efficacy of aP vaccines has accumulated. This report summarizes current knowledge on comparative whole-cell pertussis (wP) and aP vaccine performance, outlines the new July 2014 WHO guidance on the choice of pertussis vaccines and presents recent data on outbreak protection, antibody waning, long-term protection, wP-priming, pathogen adaptation, transmission and herd immunity.

  15. Identification of different subsets of lung cells using Raman microspectroscopy and whole cell nucleus isolation.

    PubMed

    Pijanka, Jacek K; Stone, Nicholas; Rutter, Abigail V; Forsyth, Nicholas; Sockalingum, Ganesh D; Yang, Ying; Sulé-Suso, Josep

    2013-09-01

    Raman spectroscopy has been widely used to study its possible clinical application in cancer diagnosis. However, in order to make it into clinical practice, it is important that this technique is able not only to identify cancer cells from their normal counterparts, but also from the array of cells present in human tissues. To this purpose, we used Raman spectroscopy to assess whether this technique was able to differentiate not only between lung cancer cells and lung epithelial cells but also from lung fibroblasts. Furthermore, we studied whether the differences were due to cell lineage (epithelial versus fibroblast) or to different proliferative characteristics of cells, and where in the cell compartment these differences might reside. To answer these questions we studied cell cytoplasm, cell nucleus and isolated whole cell nuclei. Our data suggests that Raman spectroscopy can differentiate between lung cancer, lung epithelial cells and lung fibroblasts. More important, it can also differentiate between 2 cells from the same lineage (fibroblast) but with one of them rendered immortal and with an increased proliferative activity. Finally, it seems that the main spectral differences reside in the cell nucleus and that the study of isolated nuclei strengthens the differences between cells.

  16. Nanoscale imaging of whole cells using a liquid enclosure and a scanning transmission electron microscope.

    PubMed

    Peckys, Diana B; Veith, Gabriel M; Joy, David C; de Jonge, Niels

    2009-12-14

    Nanoscale imaging techniques are needed to investigate cellular function at the level of individual proteins and to study the interaction of nanomaterials with biological systems. We imaged whole fixed cells in liquid state with a scanning transmission electron microscope (STEM) using a micrometer-sized liquid enclosure with electron transparent windows providing a wet specimen environment. Wet-STEM images were obtained of fixed E. coli bacteria labeled with gold nanoparticles attached to surface membrane proteins. Mammalian cells (COS7) were incubated with gold-tagged epidermal growth factor and fixed. STEM imaging of these cells resulted in a resolution of 3 nm for the gold nanoparticles. The wet-STEM method has several advantages over conventional imaging techniques. Most important is the capability to image whole fixed cells in a wet environment with nanometer resolution, which can be used, e.g., to map individual protein distributions in/on whole cells. The sample preparation is compatible with that used for fluorescent microscopy on fixed cells for experiments involving nanoparticles. Thirdly, the system is rather simple and involves only minimal new equipment in an electron microscopy (EM) laboratory.

  17. Transcription of host-substituted simian virus 40 DNA in whole cells and extracts.

    PubMed Central

    Kuff, E L; Ferdinand, F J; Khoury, G

    1978-01-01

    Viral transcriptional complexes were extracted from the nuclei of monkey kidney cells infected with wild-type simian virus 40 (SV40) or a variant strain containing a high proportion of host-substituted DNA molecules. The RNAs synthesized by these complexes in an in vitro system were analyzed for their content of SV40 and host sequences by a technique of sequential hybridization to plaque-purified and substituted viral DNAs. The relative labeling of the two types of sequences was commensurate with their proportion in the viral DNA (about 20% host). The substituted virus contains both reiterated and unique types of cellular sequences, and both kinds appeared to be transcribed. Transcripts of the substituted sequences formed a much smaller proportion of the virus related RNA recovered from intact infected cells, suggesting that host sequence transcripts are synthesized but rapidly degraded in the whole cell. The alternative, that transcription of these sequences is artificially enhanced in the in vitro system, cannot be rigorously excluded. We compared the self-annealing of viral RNAs from nuclear extracts of cells infected with wild-type and substituted viruses; transcripts labeled both in vivo and in vitro showed a two- to threefold-higher level of self-annealing in the case of the variant than in the case of wild type SV40. PMID:202741

  18. Yeast dual-affinity biobricks: Progress towards renewable whole-cell biosensors.

    PubMed

    Venkatesh, A G; Sun, Alexander; Brickner, Howard; Looney, David; Hall, Drew A; Aronoff-Spencer, Eliah

    2015-08-15

    Point-of-care (POC) diagnostic biosensors offer a promising solution to improve healthcare, not only in developed parts of the world, but also in resource limited areas that lack adequate medical infrastructure and trained technicians. However, in remote and resource limited settings, cost and storage of traditional POC immunoassays often limit actual deployment. Synthetically engineered biological components ("BioBricks") provide an avenue to reduce costs and simplify assay procedures. In this article, the design and development of an ultra-low cost, whole-cell "renewable" capture reagent for use in POC diagnostic applications is described. Yeast cells were genetically modified to display both single chain variable fragment (scFv) antibodies and gold-binding peptide (GBP) on their surfaces for simple one step enrichment and surface functionalization. Electrochemical impedance spectroscopy (EIS) and fluorescent imaging were used to verify and characterize the binding of cells to gold electrodes. A complete electrochemical detection assay was then performed on screen-printed electrodes fixed with yeast displaying scFv directed to Salmonella outer membrane protein D (OmpD). Electrochemical assays were optimized and cross-validated with established fluorescence techniques. Nanomolar detection limits were observed for both formats. PMID:25863344

  19. X-rays Reveal the Internal Structure of Keratin Bundles in Whole Cells.

    PubMed

    Hémonnot, Clément Y J; Reinhardt, Juliane; Saldanha, Oliva; Patommel, Jens; Graceffa, Rita; Weinhausen, Britta; Burghammer, Manfred; Schroer, Christian G; Köster, Sarah

    2016-03-22

    In recent years, X-ray imaging of biological cells has emerged as a complementary alternative to fluorescence and electron microscopy. Different techniques were established and successfully applied to macromolecular assemblies and structures in cells. However, while the resolution is reaching the nanometer scale, the dose is increasing. It is essential to develop strategies to overcome or reduce radiation damage. Here we approach this intrinsic problem by combing two different X-ray techniques, namely ptychography and nanodiffraction, in one experiment and on the same sample. We acquire low dose ptychography overview images of whole cells at a resolution of 65 nm. We subsequently record high-resolution nanodiffraction data from regions of interest. By comparing images from the two modalities, we can exclude strong effects of radiation damage on the specimen. From the diffraction data we retrieve quantitative structural information from intracellular bundles of keratin intermediate filaments such as a filament radius of 5 nm, hexagonal geometric arrangement with an interfilament distance of 14 nm and bundle diameters on the order of 70 nm. Thus, we present an appealing combined approach to answer a broad range of questions in soft-matter physics, biophysics and biology.

  20. A UAV-Mounted Whole Cell Biosensor System for Environmental Monitoring Applications.

    PubMed

    Lu, Yi; Macias, Dominique; Dean, Zachary S; Kreger, Nicole R; Wong, Pak Kin

    2015-12-01

    This study reports the development of a portable whole cell biosensor system for environmental monitoring applications, such as air quality control, water pollution monitoring, and radiation leakage detection. The system consists of a lightweight mechanical housing, a temperature regulating system, and a microfluidic bacterial inoculation channel. The overall system, which is less than 200 g, serves as a portable incubator for cell inoculation and can be mounted on an unmanned aerial vehicle for monitoring remote and unreachable locations. The feedback control system maintains the inoculation temperature within 0.05 °C. The large surface-to-volume ratio of the polydimethylsiloxane microchannel facilitates effective gas exchange for rapid bacterial growth. Molecular dynamic simulation shows effective diffusion of major gas pollutants in PDMS toward gas sensing applications. By optimizing the design, we demonstrate the operation of the system in ambient temperatures from 5 °C to 32 °C and rapid bacterial growth in microchannels compared to standard bacterial culture techniques. PMID:26584498

  1. Use of Tunable Whole-Cell Bioreporters to Assess Bioavailable Cadmium and Remediation Performance in Soils

    PubMed Central

    Yoon, Youngdae; Kim, Sunghoon; Chae, Yooeun; Kang, Yerin; Lee, Youngshim; Jeong, Seung-Woo; An, Youn-Joo

    2016-01-01

    It is important to have tools to measure the bioavailability to assess the risks of pollutants because the bioavailability is defined as the portions of pollutants showing the biological effects on living organisms. This study described the construction of tunable Escherichia coli whole-cell bioreporter (WCB) using the promoter region of zinc-inducible operon and its application on contaminated soils. It was verified that this WCB system showed specific and sensitive responses to cadmium rather than zinc in the experimental conditions. It was inferred that Cd(II) associates stronger with ZntR, a regulatory protein of zinc-inducible operon, than other metal ions. Moreover, the expression of reporter genes, egfp and mcherry, were proportional to the concentration of cadmium, thereby being a quantitative sensor to monitor bioavailable cadmium. The capability to determine bioavailable cadmium was verified with Cd(II) amended LUFA soils, and then the applicability on environmental systems was investigated with field soils collected from smelter area in Korea before and after soil-washing. The total amount of cadmium was decreased after soil washing, while the bioavailability was increased. Consequently, it would be valuable to have tools to assess bioavailability and the effectiveness of soil remediation should be evaluated in the aspect of bioavailability as well as removal efficiency. PMID:27171374

  2. Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology.

    PubMed Central

    Amann, R I; Krumholz, L; Stahl, D A

    1990-01-01

    Fluorescent-dye-conjugated oligonucleotides were used to classify 14 Fibrobacter strains by fluorescence microscopy. On the basis of partial 16S rRNA sequences of six Fibrobacter strains, four hybridization probes were designed to discriminate between the species Fibrobacter succinogenes and Fibrobacter intestinalis and to identify F. succinogenes subsp. succinogenes. After in situ hybridization to whole cells of the six sequenced strains, epifluorescence microscopy confirmed probe specificity. The four probes were then used to make presumptive species and subspecies assignments of eight additional Fibrobacter strains not previously characterized by comparative sequencing. These assignments were confirmed by comparative sequencing of the 16S rRNA target regions from the additional organisms. Single-mismatch discrimination between certain probe and nontarget sequences was demonstrated, and fluorescent intensity was shown to be enhanced by hybridization to multiple probes of the same specificity. The direct detection of F. intestinalis in mouse cecum samples demonstrated the application of this technique to the characterization of complex natural samples. Images FIG. 3a-3c FIG. 3d-3e FIG. 5 PMID:1688842

  3. Nanoscale Imaging of Whole Cells Using a Liquid Enclosure and a Scanning Transmission Electron Microscope

    PubMed Central

    Peckys, Diana B.; Veith, Gabriel M.; Joy, David C.; de Jonge, Niels

    2009-01-01

    Nanoscale imaging techniques are needed to investigate cellular function at the level of individual proteins and to study the interaction of nanomaterials with biological systems. We imaged whole fixed cells in liquid state with a scanning transmission electron microscope (STEM) using a micrometer-sized liquid enclosure with electron transparent windows providing a wet specimen environment. Wet-STEM images were obtained of fixed E. coli bacteria labeled with gold nanoparticles attached to surface membrane proteins. Mammalian cells (COS7) were incubated with gold-tagged epidermal growth factor and fixed. STEM imaging of these cells resulted in a resolution of 3 nm for the gold nanoparticles. The wet-STEM method has several advantages over conventional imaging techniques. Most important is the capability to image whole fixed cells in a wet environment with nanometer resolution, which can be used, e.g., to map individual protein distributions in/on whole cells. The sample preparation is compatible with that used for fluorescent microscopy on fixed cells for experiments involving nanoparticles. Thirdly, the system is rather simple and involves only minimal new equipment in an electron microscopy (EM) laboratory. PMID:20020038

  4. Whole Cell Target Engagement Identifies Novel Inhibitors of Mycobacterium tuberculosis Decaprenylphosphoryl-β-d-ribose Oxidase.

    PubMed

    Batt, Sarah M; Cacho Izquierdo, Monica; Castro Pichel, Julia; Stubbs, Christopher J; Vela-Glez Del Peral, Laura; Pérez-Herrán, Esther; Dhar, Neeraj; Mouzon, Bernadette; Rees, Mike; Hutchinson, Jonathan P; Young, Robert J; McKinney, John D; Barros Aguirre, David; Ballell, Lluis; Besra, Gurdyal S; Argyrou, Argyrides

    2015-12-11

    We have targeted the Mycobacterium tuberculosis decaprenylphosphoryl-β-d-ribose oxidase (Mt-DprE1) for potential chemotherapeutic intervention of tuberculosis. A multicopy suppression strategy that overexpressed Mt-DprE1 in M. bovis BCG was used to profile the publically available GlaxoSmithKline antimycobacterial compound set, and one compound (GSK710) was identified that showed an 8-fold higher minimum inhibitory concentration relative to the control strain. Analogues of GSK710 show a clear relationship between whole cell potency and in vitro activity using an enzymatic assay employing recombinant Mt-DprE1, with binding affinity measured by fluorescence quenching of the flavin cofactor of the enzyme. M. bovis BCG spontaneous resistant mutants to GSK710 and a closely related analogue were isolated and sequencing of ten such mutants revealed a single point mutation at two sites, E221Q or G248S within DprE1, providing further evidence that DprE1 is the main target of these compounds. Finally, time-lapse microscopy experiments showed that exposure of M. tuberculosis to a compound of this series arrests bacterial growth rapidly followed by a slower cytolysis phase. PMID:27623058

  5. Waning vaccine immunity in teenagers primed with whole cell and acellular pertussis vaccine: recent epidemiology.

    PubMed

    Sheridan, Sarah L; Frith, Katie; Snelling, Thomas L; Grimwood, Keith; McIntyre, Peter B; Lambert, Stephen B

    2014-09-01

    The recent epidemics of pertussis (whooping cough) in parts of the USA and Australia have led to the largest numbers of annual cases reported in over half a century. These epidemics demonstrated a new pattern, with particularly high rates of disease among pre-adolescents and early adolescents. These high rates of pertussis coincided with the first cohorts vaccinated with purely acellular pertussis vaccine, which replaced whole-cell pertussis (wP) vaccine in the later 1990s in the USA and Australia. Studies undertaken during these epidemics provide new evidence of more rapid waning of acellular pertussis-containing vaccines and longer-term protection from effective wP-containing vaccines. There is evidence that receiving wP as at least the first dose of pertussis-containing vaccine provides greater and more long-lived protection, irrespective of the nature of subsequent doses. This evidence will be reviewed together with the immunobiology associated with both vaccines, and the implications for pertussis control discussed. PMID:25093268

  6. Toward a Whole-Cell Model of Ribosome Biogenesis: Kinetic Modeling of SSU Assembly

    PubMed Central

    Earnest, Tyler M.; Lai, Jonathan; Chen, Ke; Hallock, Michael J.; Williamson, James R.; Luthey-Schulten, Zaida

    2015-01-01

    Central to all life is the assembly of the ribosome: a coordinated process involving the hierarchical association of ribosomal proteins to the RNAs forming the small and large ribosomal subunits. The process is further complicated by effects arising from the intracellular heterogeneous environment and the location of ribosomal operons within the cell. We provide a simplified model of ribosome biogenesis in slow-growing Escherichia coli. Kinetic models of in vitro small-subunit reconstitution at the level of individual protein/ribosomal RNA interactions are developed for two temperature regimes. The model at low temperatures predicts the existence of a novel 5′→3′→central assembly pathway, which we investigate further using molecular dynamics. The high-temperature assembly network is incorporated into a model of in vivo ribosome biogenesis in slow-growing E. coli. The model, described in terms of reaction-diffusion master equations, contains 1336 reactions and 251 species that dynamically couple transcription and translation to ribosome assembly. We use the Lattice Microbes software package to simulate the stochastic production of mRNA, proteins, and ribosome intermediates over a full cell cycle of 120 min. The whole-cell model captures the correct growth rate of ribosomes, predicts the localization of early assembly intermediates to the nucleoid region, and reproduces the known assembly timescales for the small subunit with no modifications made to the embedded in vitro assembly network. PMID:26333594

  7. Whole cell based electrical impedance sensing approach for a rapid nanotoxicity assay

    NASA Astrophysics Data System (ADS)

    Hondroulis, Evangelia; Liu, Chang; Li, Chen-Zhong

    2010-08-01

    A whole cell based biosensor for rapid real-time testing of human and environmental toxicity of nanoscale materials is reported. Recent studies measuring nanoparticle cytotoxicity in vitro provide a final measurement of toxicity to a cell culture overlooking the ongoing cytotoxic effects of the nanoparticles over the desired timeframe. An array biosensor capable of performing multiple cytotoxicity assays simultaneously was designed to address the need for a consistent method to measure real-time assessments of toxicity. The impedimetric response of human lung fibroblasts (CCL-153) and rainbow trout gill epithelial cells (RTgill-W1) when exposed to gold and silver nanoparticles (AuNPs, AgNPs), single walled carbon nanotubes (SWCNTs) and cadmium oxide (CdO) was tested. Exposure to CdO particles exhibited the fastest rate of cytotoxicity and demonstrated the biosensor's ability to monitor toxicity instantaneously in real time. Advantages of the present method include shorter run times, easier usage, and multi-sample analysis leading to a method that can monitor the kinetic effects of nanoparticle toxicity continuously over a desired timeframe.

  8. Whole-cell phase contrast imaging at the nanoscale using Fresnel Coherent Diffractive Imaging Tomography

    PubMed Central

    Jones, Michael W. M.; van Riessen, Grant A.; Abbey, Brian; Putkunz, Corey T.; Junker, Mark D.; Balaur, Eugeniu; Vine, David J.; McNulty, Ian; Chen, Bo; Arhatari, Benedicta D.; Frankland, Sarah; Nugent, Keith A.; Tilley, Leann; Peele, Andrew G.

    2013-01-01

    X-ray tomography can provide structural information of whole cells in close to their native state. Radiation-induced damage, however, imposes a practical limit to image resolution, and as such, a choice between damage, image contrast, and image resolution must be made. New coherent diffractive imaging techniques, such Fresnel Coherent Diffractive Imaging (FCDI), allows quantitative phase information with exceptional dose efficiency, high contrast, and nano-scale resolution. Here we present three-dimensional quantitative images of a whole eukaryotic cell by FCDI at a spatial resolution below 70 nm with sufficient phase contrast to distinguish major cellular components. From our data, we estimate that the minimum dose required for a similar resolution is close to that predicted by the Rose criterion, considerably below accepted estimates of the maximum dose a frozen-hydrated cell can tolerate. Based on the dose efficiency, contrast, and resolution achieved, we expect this technique will find immediate applications in tomographic cellular characterisation. PMID:23887204

  9. Copper sulfate improves pullulan production by bioconversion using whole cells of Aureobasidium pullulans as the catalyst.

    PubMed

    Wang, Dahui; Ju, Xiaomin; Zhang, Gaochuan; Wang, Donghua; Wei, Gongyuan

    2016-10-01

    The effects of mineral salts on pullulan production by bioconversion using whole cells of Aureobasidium pullulans CCTCC M 2012259 as the catalyst were investigated. Copper sulfate (CuSO4) improved pullulan production by 36.2% and 42.3% when added at the optimum concentration of 0.2mg/L to the bioconversion broth or seed medium, respectively, as compared with controls without CuSO4 addition. Pullulan production was further enhanced when CuSO4 was added to both seed medium and bioconversion broth simultaneously. In order to probe the mechanism of CuSO4 improvement, cell viability, membrane integrity, intracellular adenosine triphosphate (ATP) levels and the activities of key enzymes involved in pullulan biosynthesis were determined. As a result, CuSO4 increased the activities of key biosynthetic enzymes, maintained intracellular ATP at a higher level, and accelerated the rate of pullulan secretion, all of which contributed to improved pullulan production by bioconversion. PMID:27312631

  10. Classifying compound mechanism of action for linking whole cell phenotypes to molecular targets.

    PubMed

    Bourne, Christina R; Wakeham, Nancy; Bunce, Richard A; Nammalwar, Baskar; Berlin, K Darrell; Barrow, William W

    2012-04-01

    Drug development programs have proven successful when performed at a whole cell level, thus incorporating solubility and permeability into the primary screen. However, linking those results to the target within the cell has been a major setback. The Phenotype Microarray system, marketed and sold by Biolog, seeks to address this need by assessing the phenotype in combination with a variety of chemicals with known mechanism of action (MOA). We have evaluated this system for usefulness in deducing the MOA for three test compounds. To achieve this, we constructed a database with 21 known antimicrobials, which served as a comparison for grouping our unknown MOA compounds. Pearson correlation and Ward linkage calculations were used to generate a dendrogram that produced clustering largely by known MOA, although there were exceptions. Of the three unknown compounds, one was definitively placed as an antifolate. The second and third compounds' MOA were not clearly identified, likely because the unique MOA was not represented within the database. The availability of the database generated in this report for Staphylococcus aureus ATCC 29213 will increase the accessibility of this technique to other investigators. From our analysis, the Phenotype Microarray system can group compounds with clear MOA, but the distinction of unique or broadly acting MOA at this time is less clear. PMID:22434711

  11. Nanoscale imaging of whole cells using a liquid enclosure and a scanning transmission electron microscopy

    SciTech Connect

    De Jonge, Niels; Peckys, Diana B; Veith, Gabriel M; Joy, David Charles

    2009-01-01

    Nanoscale imaging techniques are needed to investigate cellular function at the level of individual proteins and to study the interaction of nanomaterials with biological systems. We imaged whole fixed cells in liquid state with a scanning transmission electron microscope (STEM) using a micrometer-sized liquid enclosure with electron transparent windows providing a wet specimen environment. Wet-STEM images were obtained of fixed E. coli bacteria labeled with gold nanoparticles attached to surface membrane proteins. Mammalian cells (COS7) were incubated with gold-tagged epidermal growth factor and fixed. STEM imaging of these cells resulted in a resolution of 3 nm for the gold nanoparticles. The wet-STEM method has several advantages over conventional imaging techniques. Most important is the capability to image whole fixed cells in a wet environment with nanometer resolution, which can be used, e.g., to map individual protein distributions in/on whole cells. The sample preparation is compatible with that used for fluorescent microscopy on fixed cells for experiments involving nanoparticles. Thirdly, the system is rather simple and involves only minimal new equipment in an electron microscopy (EM) laboratory.

  12. Identification of Yeast V-ATPase Mutants by Western Blots Analysis of Whole Cell Lysates

    NASA Astrophysics Data System (ADS)

    Parra-Belky, Karlett

    2002-11-01

    A biochemistry laboratory was designed for an undergraduate course to help students better understand the link between molecular engineering and biochemistry. Students identified unknown yeast strains with high specificity using SDS-PAGE and Western blot analysis of whole cell lysates. This problem-solving exercise is a common application of biochemistry in biotechnology research. Three different strains were used: a wild-type and two mutants for the proton pump vacuolar ATPase (V-ATPase). V-ATPases are multisubunit enzymes and the mutants used were deletion mutants; each lacked one structural gene of the complex. After three, three-hour labs, mutant strains were easily identified by the students and distinguished from wild-type cells analyzing the pattern of SDS-PAGE distribution of proteins. Identifying different subunits of one multimeric protein allowed for discussion of the structure and function of this metabolic enzyme, which captured the interest of the students. The experiment can be adapted to other multimeric protein complexes and shows improvement of the described methodology over previous reports, perhaps because the problem and its solution are representative of the type of techniques currently used in research labs.

  13. Glycan-specific whole cell affinity chromatography: A versatile microbial adhesion platform

    PubMed Central

    Van Tassell, Maxwell L.; Price, Neil P.J.; Miller, Michael J.

    2014-01-01

    We have sought a universal platform for elucidating and exploiting specificity of glycan-mediated adhesion by potentially uncharacterized microorganisms. Several techniques exist to explore microbial interactions with carbohydrate structures. Many are unsuitable for investigating specific mechanisms or uncharacterized organisms, requiring pure cultures, labeling techniques, expensive equipment, or other limitations such as questionable stability, stereospecificity, or scalability. We have adapted an affinity chromatography resin as a model to overcome these drawbacks, among others. It readily allows for the quantification, selection, and manipulation of target organisms based on interactions with glycan ligands. To maximize its utility as a selective screening method, we have constructed the tool such that it:•Promotes whole-cell interactions using viable, unaltered cells.•Provides robust spatial interactions with target glycans, presented with controlled stereo-specificity, for high affinity/avidity interactions that reflect a complex in vivo matrix.•Has the ability to utilize any reducing glycan, is quick, efficient, safe, and affordable to construct, and is scalable and reusable for multiple applications. PMID:26150959

  14. Using Multiple Whole-Cell Recordings to Study Spike-Timing-Dependent Plasticity in Acute Neocortical Slices.

    PubMed

    Lalanne, Txomin; Abrahamsson, Therese; Sjöström, P Jesper

    2016-01-01

    This protocol provides a method for quadruple whole-cell recording to study synaptic plasticity of neocortical connections, with a special focus on spike-timing-dependent plasticity (STDP). It also describes how to morphologically identify recorded cells from two-photon laser-scanning microscopy (2PLSM) stacks. PMID:27250948

  15. Forced co-expression of IL-21 and IL-7 in whole-cell cancer vaccines promotes antitumor immunity.

    PubMed

    Gu, Yang-Zhuo; Fan, Chuan-Wen; Lu, Ran; Shao, Bin; Sang, Ya-Xiong; Huang, Qiao-Rong; Li, Xue; Meng, Wen-Tong; Mo, Xian-Ming; Wei, Yu-Quan

    2016-01-01

    Genetic modification of whole-cell cancer vaccines to augment their efficacies has a history of over two and a half decades. Various genes and gene combinations, targeting different aspects of immune responses have been tested in pursuit of potent adjuvant effects. Here we show that co-expression of two cytokine members of the common cytokine receptor γ-chain family, IL-21 and IL-7, in whole-cell cancer vaccines boosts antitumor immunity in a CD4(+) and CD8(+) T cell-dependent fashion. It also generates effective immune memory. The vaccine-elicited short-term effects positively correlated with enhanced infiltration of CD4(+) and CD8(+) effector T cells, and the long-term effects positively correlated with enhanced infiltration of effector memory T cells, especially CD8(+) effector memory T cells. Preliminary data suggested that the vaccine exhibited good safety profile in murine models. Taken together, the combination of IL-21 and IL-7 possesses potent adjuvant efficacy in whole-cell vaccines. This finding warrants future development of IL-21 and IL-7 co-expressing whole-cell cancer vaccines and their relevant combinatorial regimens. PMID:27571893

  16. Forced co-expression of IL-21 and IL-7 in whole-cell cancer vaccines promotes antitumor immunity

    PubMed Central

    Gu, Yang-Zhuo; Fan, Chuan-Wen; Lu, Ran; Shao, Bin; Sang, Ya-Xiong; Huang, Qiao-Rong; Li, Xue; Meng, Wen-Tong; Mo, Xian-Ming; Wei, Yu-Quan

    2016-01-01

    Genetic modification of whole-cell cancer vaccines to augment their efficacies has a history of over two and a half decades. Various genes and gene combinations, targeting different aspects of immune responses have been tested in pursuit of potent adjuvant effects. Here we show that co-expression of two cytokine members of the common cytokine receptor γ-chain family, IL-21 and IL-7, in whole-cell cancer vaccines boosts antitumor immunity in a CD4+ and CD8+ T cell-dependent fashion. It also generates effective immune memory. The vaccine-elicited short-term effects positively correlated with enhanced infiltration of CD4+ and CD8+ effector T cells, and the long-term effects positively correlated with enhanced infiltration of effector memory T cells, especially CD8+ effector memory T cells. Preliminary data suggested that the vaccine exhibited good safety profile in murine models. Taken together, the combination of IL-21 and IL-7 possesses potent adjuvant efficacy in whole-cell vaccines. This finding warrants future development of IL-21 and IL-7 co-expressing whole-cell cancer vaccines and their relevant combinatorial regimens. PMID:27571893

  17. RAINBOW TROUT ANDROGEN RECEPTOR ALPHA AND THE HUMAN ANDROGEN RECEPTOR: COMPARISONS IN THE COS WHOLE CELL BINDING ASSAY

    EPA Science Inventory

    RAINBOW TROUT ANDROGEN RECEPTOR ALPHA AND HUMAN ANDROGEN RECEPTOR: COMPARISONS IN THE COS WHOLE CELL BINDING ASSAY.
    MC Cardon, PC Hartig,LE Gray, Jr. and VS Wilson.
    U.S. EPA, ORD, NHEERL, RTD, Research Triangle Park, NC, USA.
    Typically, in vitro hazard assessments for ...

  18. RAINBOW TROUT ANDROGEN RECEPTOR ALPHA AND THE HUMAN ANDROGEN RECEPTOR: COMPARISONS IN THE COS WHOLE CELL BINDING ASSAY

    EPA Science Inventory

    Rainbow Trout Androgen Receptor Alpha And Human Androgen Receptor: Comparisons in the COS Whole Cell Binding Assay
    Mary C. Cardon, L. Earl Gray, Jr. and Vickie S. Wilson
    U.S. Environmental Protection Agency, ORD, NHEERL, Reproductive Toxicology Division, Research Triangle...

  19. Whole-cell MALDI-TOF MS: a new tool to assess the multifaceted activation of macrophages.

    PubMed

    Ouedraogo, Richard; Daumas, Aurélie; Ghigo, Eric; Capo, Christian; Mege, Jean-Louis; Textoris, Julien

    2012-10-22

    Whole-cell MALDI-TOF MS is routinely used to identify bacterial species in clinical samples. This technique has also proven to allow identification of intact mammalian cells, including macrophages. Here, we wondered whether this approach enabled the assessment human macrophages plasticity. The whole-cell MALDI-TOF spectra of macrophages stimulated with IFN-γ and IL-4, two inducers of M1 and M2 macrophage polarisation, consisted of peaks ranging from 2 to 12 kDa. The spectra of unstimulated and stimulated macrophages were clearly different. The fingerprints induced by the M1 agonists, IFN-γ, TNF, LPS and LPS+IFN-γ, and the M2 agonists, IL-4, TGF-β1 and IL-10, were specific and readily identifiable. Thus, whole-cell MALDI-TOF MS was able to characterise M1 and M2 macrophage subtypes. In addition, the fingerprints induced by extracellular (group B Streptococcus, Staphylococcus aureus) or intracellular (BCG, Orientia tsutsugamushi, Coxiella burnetii) bacteria were bacterium-specific. The whole-cell MALDI-TOF MS fingerprints therefore revealed the multifaceted activation of human macrophages. This approach opened a new avenue of studies to assess the immune response in the clinical setting, by monitoring the various activation patterns of immune cells in pathological conditions.

  20. Stereoselective Bioreduction of α-Azido Ketones by Whole Cells of Marine-Derived Fungi.

    PubMed

    Rocha, Lenilson C; Seleghim, Mirna H R; Comasseto, João V; Sette, Lara D; Porto, André L M

    2015-12-01

    Seven strains of marine-derived fungi (Aspergillus sclerotiorum CBMAI 849, Cladosporium cladosporioides CBMAI 857, Penicillium raistrickii CBMAI 931, Penicillium citrinum CBMA 1186, Mucor racemosus CBMAI 847, Beauveria felina CBMAI 738, and Penicillium oxalicum CBMAI 1185) and terrestrial fungus Penicillium chrysogenum CBMA1199 were screened as catalysts for the asymmetric reduction of α-keto azides 5-8 to their corresponding β-azidophenylethanols 9-12. The marine fungi showed Prelog and anti-Prelog selectivities to the reduction α-keto azides 5-8. The fungi A. sclerotiorum CBMAI 849, C. cladosporioides CBMAI 857, P. raistrickii CBMAI 931, and P. citrinum CBMA 1186 catalyzed the reduction of azido ketone 6 to the corresponding (R)-2-azido-1-(4-methoxyphenyl)ethanol (10) with good conversions (68-100 %) and excellent enantiomeric excesses (>99 % ee) according to Prelog rule.

  1. Voltage clamp limitations of dual whole-cell gap junction current and voltage recordings. I. Conductance measurements.

    PubMed

    Veenstra, R D

    2001-05-01

    Previous correction methods for series access resistance errors in the dual whole-cell configuration did not take into account the effect of nonzero resting potentials (E(rest)) and junctional reversal potentials (E(rev)). Dual whole-cell currents were modeled according to resistor-circuit analysis and two correction formulas for the measurement of junctional currents (I(j)) were assessed. The equations for I(j), derived from Kirchoff's law before and after baseline subtraction of the nonjunctional current, were assessed for accuracy under a variety of whole-cell patch-clamp recording conditions. Both equations accurately correct for dual whole-cell voltage-clamp errors provided that the cellular parameters are included in the nonbaseline subtracted I(j) derivations. Junctional conductance (g(j)) estimates are most reliable at high junctional resistance (R(j)) values and minimize the need for corrective methods based on electrode series and cellular input resistances (R(el) and R(in)). In the "open-cell" configuration, low R(j) values relative to R(in) are required for accurate g(j) estimates. These methods provide the basis for accurate quantitative measurements of junctional resistance (or conductance) of gap junction channels or connexin hemichannels in the dual whole-cell or open-cell configurations. Revaluation of V(j)-dependent gating of rat connexin40 g(j) produced nearly identical Boltzmann fits to previously published data. Continuous g(j)-V(j) curves generated by variable slope V(j) ramps provide for more accurate fits and assessment of the time-dependence of the half-inactivation voltage and net gating charge movement. PMID:11325726

  2. HCN Producing Bacteria Enable Sensing Of Non-Bioavailable Hg Species by the Whole Cell Biosensor

    NASA Astrophysics Data System (ADS)

    Horvat, M.; Rijavec, T.; Koron, N.; Lapanje, A.

    2015-12-01

    Bacteria play an important role in Hg transformation reactions. The production of cyanide (HCN) and other secondary metabolites seems to be key elements involved in these transformations. Current hypotheses link the role of HCN production to growth inhibition of nonHCN producing competitor organisms (role of an antimicrobial agent). Our past investigations showed that HCN production did not correlate with antimicrobial activity and since pK value of HCN is very high (pK = 9,21), it can be expected that most of the produced HCN is removed from the microenvironment. This way, the expected inhibitory concentrations can hardly be reached. Accordingly, we proposed a new concept, where the ability of complexation of transient metals by HCN served as a regulation process for the accessibility of micro-elements. In our study, we focused on the presence of HCN producing bacteria and carried it out in the Hg contaminated environment connected to the Idrija Mercury Mine, Slovenia. We characterised the isolates according to the presence of Hg resistance (HgR), level of HCN production and genetic similarities. In laboratory setups, using our merR whole cell based biosensor, we determined the transformation of low bioavailable Hg0 and HgS forms into bioavailable Hg by these HCN producing bacteria. We observed that HgR strains producing HCN had the highest impact on increased Hg bioavailability. In the proposed ecological strategy HgR HCN producing bacteria increase their competitive edge over non-HgR competitors through the increase of Hg toxicity. Due to their activity, Hg is made available to other organisms as well and thus enters into the ecosystem. Finally, using some of the characteristics of bacteria (e.g. Hg resistance genetic elements), we developed a fully automated sensing approach, combining biosensorics and mechatronics, to measure the bioavailability of Hg in situ.

  3. Resonant dielectrophoresis and electrohydrodynamics for high-sensitivity impedance detection of whole-cell bacteria.

    PubMed

    Couniot, Numa; Francis, Laurent A; Flandre, Denis

    2015-08-01

    We present the co-integration of CMOS-compatible Al/Al2O3 interdigitated microelectrodes (IDEs) with an electrokinetic-driven macroelectrode for sensitive detection of whole-cell bacteria in a microfluidic channel. Two frequency ranges applied to the macroelectrode were identified to notably increase the bacterial coverage of the impedimetric sensor per unit time. Around 10 kHz, the bacterial cells were directed towards the IDE center thanks to AC electroosmosis (AC-EO) and the sensor capacitance linearly increased, achieving a limit of detection (LoD) of 3.5 × 10(5) CFU mL(-1) after an incubation time of 20 min with Staphylococcus epidermidis. At 63 MHz precisely, a resonance effect due to the device was found to dramatically increase the trapping of S. epidermidis on the sensor periphery, due to the combined actions of short-range contactless dielectrophoresis (cDEP) and long-range Joule heating electrothermal (J-ET) flow. Thanks to a flow-based method, the bacterial cells were redirected towards the sensor center and an LoD of 10(5) CFU mL(-1) was achieved within 20 min of incubation, which is almost two orders of magnitude better than the impedimetric sensor alone. Analytical models and 2D simulations using the Maxwell stress tensor (MST) provide a comprehensive analysis of the experimental results, especially about the spectral balance between cDEP, AC-EO and J-ET accounting for the 33-nm thick insulating layer atop the electrodes. Electrode CMOS compatibility confers portability, miniaturization and affordability capabilities for building point-of-care (PoC) diagnostic tests in a lab-on-a-chip (LoC).

  4. Incompatibility of lyophilized inactivated polio vaccine with liquid pentavalent whole-cell-pertussis-containing vaccine.

    PubMed

    Kraan, Heleen; Ten Have, Rimko; van der Maas, Larissa; Kersten, Gideon; Amorij, Jean-Pierre

    2016-08-31

    A hexavalent vaccine containing diphtheria toxoid, tetanus toxoid, whole cell pertussis, Haemophilius influenza type B, hepatitis B and inactivated polio vaccine (IPV) may: (i) increase the efficiency of vaccination campaigns, (ii) reduce the number of injections thereby reducing needlestick injuries, and (iii) ensure better protection against pertussis as compared to vaccines containing acellular pertussis antigens. An approach to obtain a hexavalent vaccine might be reconstituting lyophilized polio vaccine (IPV-LYO) with liquid pentavalent vaccine just before intramuscular delivery. The potential limitations of this approach were investigated including thermostability of IPV as measured by D-antigen ELISA and rat potency, the compatibility of fluid and lyophilized IPV in combination with thimerosal and thimerosal containing hexavalent vaccine. The rat potency of polio type 3 in IPV-LYO was 2 to 3-fold lower than standardized on the D-antigen content, suggesting an alteration of the polio type 3 D-antigen particle by lyophilization. Type 1 and 2 had unaffected antigenicity/immunogenicity ratios. Alteration of type 3 D-antigen could be detected by showing reduced thermostability at 45°C compared to type 3 in non-lyophilized liquid controls. Reconstituting IPV-LYO in the presence of thimerosal (TM) resulted in a fast temperature dependent loss of polio type 1-3 D-antigen. The presence of 0.005% TM reduced the D-antigen content by ∼20% (polio type 2/3) and ∼60% (polio type 1) in 6h at 25°C, which are WHO open vial policy conditions. At 37°C, D-antigen was diminished even faster, suggesting that very fast, i.e., immediately after preparation, intramuscular delivery of the conceived hexavalent vaccine would not be a feasible option. Use of the TM-scavenger, l-cysteine, to bind TM (or mercury containing TM degradation products), resulted in a hexavalent vaccine mixture in which polio D-antigen was more stable.

  5. Electropermeabilization and fluorescent tracer exchange: the role of whole-cell capacitance.

    PubMed

    Sukhorukov, V L; Djuzenova, C S; Frank, H; Arnold, W M; Zimmermann, U

    1995-11-01

    Transmembrane crossing of charged fluorescent tracers such as propidium iodide (PI) and carboxyfluorescein+ (CF) can be used to quantitate membrane permeabilization. Murine myeloma Sp2/0-Ag14 cells were loaded with CF (0.1 fmol/cell) before electropulsation (0.5-3.0 kV/cm, 40 microseconds) in medium containing 25-50 micrograms/ml PI at 21-23 degrees C. Cytograms of PI vs. CF fluorescence showed three readily distinguishable subpopulations: 1) intact living cells with CF but without PI (these form > 95% of the prepulsed population), 2) transiently electropermeabilized but resealed cells showing both CF and low-level PI fluorescence, and 3) permanently permeabilized cells without CF but with very high PI fluorescence. Despite the ready influx of PI, the efflux of CF from transiently permeabilized cells was negligible and was insensitive to pulse parameters; however, electrically killed cells (subpopulation 3) lost all CF fluorescence and probably lost their cytoplasm. This difference in transmembrane passage of the dyes is best explained by binding of intracellular CF to macromolecules (and/or organelles). In isotonic "pulse medium," the membranes resealed after electropulsing with a time constant (tau R) of about 2 min. In 150 mOsm medium, resealing was faster (typically tau R approximately 0.5 min). The population distribution of PI uptake [coefficient of variation (CV) > 40%] was very broad and could not be accounted for by the radius dependence of pulse-induced voltage (CVradius approximately 10%). The variability in PI uptake could be explained if the electrical energy of the charged membrane, which depends on the whole-cell capacitance (Cc), was taken into account. Evaluation of the Cc values with single-cell resolution was based on measurement of the electrical charging time constant of the plasma membrane by electrorotation. PMID:8582245

  6. Scavenging ROS dramatically increase NMDA receptor whole-cell currents in painted turtle cortical neurons.

    PubMed

    Dukoff, David James; Hogg, David William; Hawrysh, Peter John; Buck, Leslie Thomas

    2014-09-15

    Oxygen deprivation triggers excitotoxic cell death in mammal neurons through excessive calcium loading via over-activation of N-methyl-d-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. This does not occur in the western painted turtle, which overwinters for months without oxygen. Neurological damage is avoided through anoxia-mediated decreases in NMDA and AMPA receptor currents that are dependent upon a modest rise in intracellular Ca(2+) concentrations ([Ca(2+)]i) originating from mitochondria. Anoxia also blocks mitochondrial reactive oxygen species (ROS) generation, which is another potential signaling mechanism to regulate glutamate receptors. To assess the effects of decreased intracellular [ROS] on NMDA and AMPA receptor currents, we scavenged ROS with N-2-mercaptopropionylglycine (MPG) or N-acetylcysteine (NAC). Unlike anoxia, ROS scavengers increased NMDA receptor whole-cell currents by 100%, while hydrogen peroxide decreased currents. AMPA receptor currents and [Ca(2+)]i concentrations were unaffected by ROS manipulation. Because decreases in [ROS] increased NMDA receptor currents, we next asked whether mitochondrial Ca(2+) release prevents receptor potentiation during anoxia. Normoxic activation of mitochondrial ATP-sensitive potassium (mKATP) channels with diazoxide decreased NMDA receptor currents and was unaffected by subsequent ROS scavenging. Diazoxide application following ROS scavenging did not rescue scavenger-mediated increases in NMDA receptor currents. Fluorescent measurement of [Ca(2+)]i and ROS levels demonstrated that [Ca(2+)]i increases before ROS decreases. We conclude that decreases in ROS concentration are not linked to anoxia-mediated decreases in NMDA/AMPA receptor currents but are rather associated with an increase in NMDA receptor currents that is prevented during anoxia by mitochondrial Ca(2+) release.

  7. Resonant dielectrophoresis and electrohydrodynamics for high-sensitivity impedance detection of whole-cell bacteria.

    PubMed

    Couniot, Numa; Francis, Laurent A; Flandre, Denis

    2015-08-01

    We present the co-integration of CMOS-compatible Al/Al2O3 interdigitated microelectrodes (IDEs) with an electrokinetic-driven macroelectrode for sensitive detection of whole-cell bacteria in a microfluidic channel. Two frequency ranges applied to the macroelectrode were identified to notably increase the bacterial coverage of the impedimetric sensor per unit time. Around 10 kHz, the bacterial cells were directed towards the IDE center thanks to AC electroosmosis (AC-EO) and the sensor capacitance linearly increased, achieving a limit of detection (LoD) of 3.5 × 10(5) CFU mL(-1) after an incubation time of 20 min with Staphylococcus epidermidis. At 63 MHz precisely, a resonance effect due to the device was found to dramatically increase the trapping of S. epidermidis on the sensor periphery, due to the combined actions of short-range contactless dielectrophoresis (cDEP) and long-range Joule heating electrothermal (J-ET) flow. Thanks to a flow-based method, the bacterial cells were redirected towards the sensor center and an LoD of 10(5) CFU mL(-1) was achieved within 20 min of incubation, which is almost two orders of magnitude better than the impedimetric sensor alone. Analytical models and 2D simulations using the Maxwell stress tensor (MST) provide a comprehensive analysis of the experimental results, especially about the spectral balance between cDEP, AC-EO and J-ET accounting for the 33-nm thick insulating layer atop the electrodes. Electrode CMOS compatibility confers portability, miniaturization and affordability capabilities for building point-of-care (PoC) diagnostic tests in a lab-on-a-chip (LoC). PMID:26120099

  8. Incompatibility of lyophilized inactivated polio vaccine with liquid pentavalent whole-cell-pertussis-containing vaccine.

    PubMed

    Kraan, Heleen; Ten Have, Rimko; van der Maas, Larissa; Kersten, Gideon; Amorij, Jean-Pierre

    2016-08-31

    A hexavalent vaccine containing diphtheria toxoid, tetanus toxoid, whole cell pertussis, Haemophilius influenza type B, hepatitis B and inactivated polio vaccine (IPV) may: (i) increase the efficiency of vaccination campaigns, (ii) reduce the number of injections thereby reducing needlestick injuries, and (iii) ensure better protection against pertussis as compared to vaccines containing acellular pertussis antigens. An approach to obtain a hexavalent vaccine might be reconstituting lyophilized polio vaccine (IPV-LYO) with liquid pentavalent vaccine just before intramuscular delivery. The potential limitations of this approach were investigated including thermostability of IPV as measured by D-antigen ELISA and rat potency, the compatibility of fluid and lyophilized IPV in combination with thimerosal and thimerosal containing hexavalent vaccine. The rat potency of polio type 3 in IPV-LYO was 2 to 3-fold lower than standardized on the D-antigen content, suggesting an alteration of the polio type 3 D-antigen particle by lyophilization. Type 1 and 2 had unaffected antigenicity/immunogenicity ratios. Alteration of type 3 D-antigen could be detected by showing reduced thermostability at 45°C compared to type 3 in non-lyophilized liquid controls. Reconstituting IPV-LYO in the presence of thimerosal (TM) resulted in a fast temperature dependent loss of polio type 1-3 D-antigen. The presence of 0.005% TM reduced the D-antigen content by ∼20% (polio type 2/3) and ∼60% (polio type 1) in 6h at 25°C, which are WHO open vial policy conditions. At 37°C, D-antigen was diminished even faster, suggesting that very fast, i.e., immediately after preparation, intramuscular delivery of the conceived hexavalent vaccine would not be a feasible option. Use of the TM-scavenger, l-cysteine, to bind TM (or mercury containing TM degradation products), resulted in a hexavalent vaccine mixture in which polio D-antigen was more stable. PMID:27470209

  9. Different T cell memory in preadolescents after whole-cell or acellular pertussis vaccination.

    PubMed

    Smits, Kaatje; Pottier, Gaelle; Smet, Julie; Dirix, Violette; Vermeulen, Françoise; De Schutter, Iris; Carollo, Maria; Locht, Camille; Ausiello, Clara Maria; Mascart, Françoise

    2013-12-17

    To better understand vaccine-induced protection and its potential failure in light of recent whooping cough resurgence, we evaluated quantity as well as quality of memory T cell responses in B. pertussis-vaccinated preadolescent children. Using a technique based on flow cytometry to detect proliferation, cytokine production and phenotype of antigen-specific cells, we evaluated residual T cell memory in a cohort of preadolescents who received a whole-cell pertussis (wP; n=11) or an acellular pertussis vaccine (aP; n=13) during infancy, and with a median of 4 years elapsed from the last pertussis booster vaccine, which was aP for all children. We demonstrated that B. pertussis-specific memory T cells are detectable in the majority of preadolescent children several years after vaccination. CD4(+) and CD8(+) T cell proliferation in response to pertussis toxin and/or filamentous hemagglutinin was detected in 79% and 60% of the children respectively, and interferon-γ or tumor necrosis factor-α producing CD4(+) T cells were detected in 65% and 53% of the children respectively. Phenotyping of the responding cells showed that the majority of antigen-specific cells, whether defined by proliferation or cytokine production, were CD45RA(-)CCR7(-) effector memory T cells. Although the time since the last booster vaccine was significantly longer for wP-compared to aP-vaccinated children, their proliferation capacity in response to antigenic stimulation was comparable, and more children had a detectable cytokine response after wP- compared to aP-vaccination. This study supports at the immunological level recent epidemiological studies indicating that infant vaccination with wP induces longer lasting immunity than vaccination with aP-vaccines. PMID:24176499

  10. Quantification of Enterococcus italicus in traditional Italian cheeses by fluorescence whole-cell hybridization.

    PubMed

    Fornasari, Maria Emanuela; Rossetti, Lia; Remagni, Chiara; Giraffa, Giorgio

    2008-08-01

    The objective of this work was to investigate the spread of Enterococcus italicus in cheese. For this purpose, a fluorescence whole-cell hybridization protocol (FWCH) with a 16S rRNA probe was optimized to evaluate the presence and abundance of this organism in artisanal Italian cheeses. The FWCH method avoided the quantification problems using classical plate count techniques related to the well-known difficulties to cultivate E. italicus in selective enterococci media. After probe and FWCH optimization, 10 commercially available Italian semi-hard cheeses made with raw ewe or cow milk without starter addition were analyzed. All of them were subjected to FWCH experiments and six of them gave positive results with the probe, i.e. the E. italicus content was >4 log cells/g according to the detection limit of FWCH. Counts showed that E. italicus was present at levels ranging from 5.91+/-0.17 to 7.34+/-0.14 log cells/g; such levels were similar to, or even higher than, the total enterococci counted from the corresponding cheeses using kanamycin aesculin azide agar. The overall reliability of the FWCH method was tested by species-specific PCR. The positive amplification of the expected 323 bp fragment from both a cheese matrix and cell bulks of cheese samples containing high loads of this organism (as determined by FWCH counts) and the successful isolation of E. italicus strains from the above cheeses provided definitive proof of both probe specificity and the presence of this organism in cheeses. Although there is very little available quantitative data on the incidence of E. italicus in cheese, or its role in product quality, this study showed a wide diffusion of this organism in artisanal cheeses, where secondary non-starter lactic acid bacterial microflora, which enterococci belong to, may become dominant during ripening. PMID:18562146

  11. Kefir immobilized on corn grains as biocatalyst for lactic acid fermentation and sourdough bread making.

    PubMed

    Plessas, Stavros; Alexopoulos, Athanasios; Bekatorou, Argyro; Bezirtzoglou, Eugenia

    2012-12-01

    The natural mixed culture kefir was immobilized on boiled corn grains to produce an efficient biocatalyst for lactic acid fermentation with direct applications in food production, such as sourdough bread making. The immobilized biocatalyst was initially evaluated for its efficiency for lactic acid production by fermentation of cheese whey at various temperatures. The immobilized cells increased the fermentation rate and enhanced lactic acid production compared to free kefir cells. Maximum lactic acid yield (68.8 g/100 g) and lactic acid productivity (12.6 g/L per day) were obtained during fermentation by immobilized cells at 37 °C. The immobilized biocatalyst was then assessed as culture for sourdough bread making. The produced sourdough breads had satisfactory specific loaf volumes and good sensory characteristics. Specifically, bread made by addition of 60% w/w sourdough containing kefir immobilized on corn was more resistant regarding mould spoilage (appearance during the 11(th) day), probably due to higher lactic acid produced (2.86 g/Kg of bread) compared to the control samples. The sourdough breads made with the immobilized biocatalyst had aroma profiles similar to that of the control samples as shown by headspace SPME GC-MS analysis.

  12. Enzyme-based inverse opals: a facile and promising platform for fabrication of biocatalysts.

    PubMed

    Jiang, Yanjun; Cui, Cuicui; Huang, Yan; Zhang, Xu; Gao, Jing

    2014-05-28

    A facile and promising approach was developed to fabricate enzyme-based 3D-ordered macroporous biocatalysts (enzyme-based inverse opals) by using the colloidal crystal templating method. Horseradish peroxidase- and amylase-based inverse opals were prepared, which verified that this method is suitable for various enzymes. PMID:24722982

  13. Countercurrent multistage fluidized bed reactor for immobilized biocatalysts: II. Operation of a laboratory-scale reactor.

    PubMed

    Vos, H J; Zomerdijk, M; Groen, D J; Luyben, K C

    1990-08-01

    In Part I of this series,(1) we derived a model and made simulations for a multistage fluidized bed reactor (MFBR). It was concluded that the MFBR can be an attractive alternative for a fixed bed reactor when operated with a deactivating biocatalyst. In Part II of this series, the design of a laboratory-scale MFBR and its evaluation to investigate the practical feasibility of this reactor type, will be described. Experiments with a duration as long as 10 days were carried out successfully using immobilized glucose isomerase as a model reaction system. The results predicted by the model are in good agreement with the measured glucose concentration and biocatalyst activity gradients, indicating perfect mixing of the particles in the reactor compartments.The diameters of the biocatalyst particles used in the experiments showed a large spread, with the largest being 1.7 times the smallest. Therefore, an additional check was carried out, to make sure that the particles were not segregating according to size. Particles withdrawn from the reactor compartments were investigated using an image analyzer. Histograms of particle size distribution do not indicate segregation and it is concluded that the particles used have been mixed completely within the compartments. As a result, transport of biocatalyst is nearly plug flow. PMID:18595091

  14. Enzyme-based inverse opals: a facile and promising platform for fabrication of biocatalysts.

    PubMed

    Jiang, Yanjun; Cui, Cuicui; Huang, Yan; Zhang, Xu; Gao, Jing

    2014-05-28

    A facile and promising approach was developed to fabricate enzyme-based 3D-ordered macroporous biocatalysts (enzyme-based inverse opals) by using the colloidal crystal templating method. Horseradish peroxidase- and amylase-based inverse opals were prepared, which verified that this method is suitable for various enzymes.

  15. Reductive amination by recombinant Escherichia coli: whole cell biotransformation of 2-keto-3-methylvalerate to L-isoleucine.

    PubMed

    Lorenz, Elisabeth; Klatte, Stephanie; Wendisch, Volker F

    2013-11-01

    A whole cell biotransformation system for reductive amination has been studied in recombinant Escherichia coli cells. Reductive amination of 2-keto-3-methylvalerate to L-isoleucine by a two-enzyme-cascade was achieved by overproduction of endogenous L-alanine dependent transaminase AvtA and heterologous L-alanine dehydrogenase from Bacillus subtilis in recombinant E. coli. Up to 100 mM L-isoleucine were produced from 100 mM 2-keto-3-methylvalerate and 100 mM ammonium sulfate. Regeneration of NADH as cofactor in the whole cell system was driven by glucose catabolism. The effects of defined gene deletions in the central carbon metabolism on biotransformation were tested. Strains lacking the NuoG subunit of NADH:ubiquinone oxidoreductase (complex I) or aceA encoding the glyoxylate cycle enzyme isocitrate lyase exhibited increased biotransformation rates.

  16. Whole-cell kinetics of trichloroethylene degradation by phenol hydroxylase in a Ralstonia eutropha JMP134 derivative

    SciTech Connect

    Ayoubi, P.J.; Harker, A.R.

    1998-11-01

    The rate, progress, and limits of trichloroethylene (TCE) degradation by Ralstonia eutropha AEK301/pYK3021 whole cells were examined in the absence of aromatic induction. At TCE concentrations up to 800 {micro}M, degradation rates were sustained until TCE was no longer detectable. The K{sub s} and V{sub max} for TCE degradation by AEK301/pYK3021 whole cells were determined to be 630 {micro}M and 22.6 nmol/min/mg of total protein, respectively. The sustained linear rates of TCE degradation by AEK301/pYK3021 up to a concentration of 800 {micro}M TCE suggest that solvent effects are limited during the degradation of TCE and that this construct is little affected by the formation of toxic intermediates at the TCE levels and assay duration tested. TCE degradation by this strain is subject to carbon catabolite repression.

  17. Whole-cell kinetics of trichloroethylene degradation by phenol hydroxylase in a ralstonia eutropha JMP134 derivative

    PubMed

    Ayoubi; Harker

    1998-11-01

    The rate, progress, and limits of trichloroethylene (TCE) degradation by Ralstonia eutropha AEK301/pYK3021 whole cells were examined in the absence of aromatic induction. At TCE concentrations up to 800 &mgr;M, degradation rates were sustained until TCE was no longer detectable. The Ks and Vmax for TCE degradation by AEK301/pYK3021 whole cells were determined to be 630 &mgr;M and 22.6 nmol/min/mg of total protein, respectively. The sustained linear rates of TCE degradation by AEK301/pYK3021 up to a concentration of 800 &mgr;M TCE suggest that solvent effects are limited during the degradation of TCE and that this construct is little affected by the formation of toxic intermediates at the TCE levels and assay duration tested. TCE degradation by this strain is subject to carbon catabolite repression. PMID:9797289

  18. Whole-Cell Kinetics of Trichloroethylene Degradation by Phenol Hydroxylase in a Ralstonia eutropha JMP134 Derivative

    PubMed Central

    Ayoubi, Patricia J.; Harker, Alan R.

    1998-01-01

    The rate, progress, and limits of trichloroethylene (TCE) degradation by Ralstonia eutropha AEK301/pYK3021 whole cells were examined in the absence of aromatic induction. At TCE concentrations up to 800 μM, degradation rates were sustained until TCE was no longer detectable. The Ks and Vmax for TCE degradation by AEK301/pYK3021 whole cells were determined to be 630 μM and 22.6 nmol/min/mg of total protein, respectively. The sustained linear rates of TCE degradation by AEK301/pYK3021 up to a concentration of 800 μM TCE suggest that solvent effects are limited during the degradation of TCE and that this construct is little affected by the formation of toxic intermediates at the TCE levels and assay duration tested. TCE degradation by this strain is subject to carbon catabolite repression. PMID:9797289

  19. Resistance of spheroplasts and whole cells of Pseudomonas aeruginosa to bactericidal activity of various biocides: evidence of the membrane implication.

    PubMed

    Guérin-Méchin, Laurence; Leveau, Jean-Yves; Dubois-Brissonnet, Florence

    2004-01-01

    To emphasise the role of outer and inner membranes in the resistance of Pseudomonas aeruginosa to bactericidal activity of various disinfectants, spheroplasts and whole cells were compared. Spheroplasts are more sensitive than whole cells to quaternary ammonium compounds such as didecyl dimethyl ammonium bromide (DDAB) and C16-benzalkonium chloride. The outer membrane acts as a barrier to prevent these disinfectants from entering the cell. It seems to have no influence on activities of smaller molecules such as C12, C14-benzalkonium chlorides and sodium dichloroisocyanurate. For tri-sodium phosphate, the presence of outer membrane emphasized the action of the molecule. Moreover, resistance of DDAB-adapted spheroplasts to bactericidal activity of DDAB is higher than the resistance of non-adapted spheroplasts. This suggests that the inner membrane could also play a role in resistance to DDAB. PMID:15160607

  20. Establishment of the Dual Whole Cell Recording Patch Clamp Configuration for the Measurement of Gap Junction Conductance.

    PubMed

    Veenstra, Richard D

    2016-01-01

    The development of the patch clamp technique has enabled investigators to directly measure gap junction conductance between isolated pairs of small cells with resolution to the single channel level. The dual patch clamp recording technique requires specialized equipment and the acquired skill to reliably establish gigaohm seals and the whole cell recording configuration with high efficiency. This chapter describes the equipment needed and methods required to achieve accurate measurement of macroscopic and single gap junction channel conductances. Inherent limitations with the dual whole cell recording technique and methods to correct for series access resistance errors are defined as well as basic procedures to determine the essential electrical parameters necessary to evaluate the accuracy of gap junction conductance measurements using this approach. PMID:27207298

  1. Alginate immobilization of Escherichia coli MTCC 1652 whole cells for bioconversion of glycyrrhizinic acid and into 18-beta glycyrrhetinic acid.

    PubMed

    Ahmad, M; Panda, B P

    2013-12-15

    Microbial biotransformation of Glycyrrhizinic acid (GL) into 18-beta Glycyrrhetinic Acid (GA) was achieved using Escherichia coli MTCC 1652 whole cell. The E. coli whole cell was immobilized by entrapment method within calcium alginate beads using cell suspension of equal volume with sodium alginate 8%. The pH of solution, reaction volume and % of GL were optimized during the immobilization procedure and optimum pH 6.5, reaction volume of 4 mL and at 3% GL concentration for 12 h of incubation time showed highest concentration of GA (72.649 microg mL(-1)) with 76% bioconversion of GL to GA. Under optimized condition the immobilized cell produces 58.663 microg per mL of GA in licorice root extract containing 95.118 microg of GL per mL of the extract with 61% conversion at 12 h.

  2. Limitations of the whole cell patch clamp technique in the control of intracellular concentrations.

    PubMed Central

    Mathias, R T; Cohen, I S; Oliva, C

    1990-01-01

    Recent experimental studies (Pusch and Neher, 1988) and theoretical studies (Oliva et al., 1988) have found that the pipette tip is a significant barrier to diffusion in the whole cell patch clamp configuration. In this paper, we extend the theoretical analysis of fluxes between the pipette and cell to include transmembrane fluxes. The general conclusions are: (a) within the pipette, ion fluxes are driven primarily by diffusion rather than voltage gradients. (b) At steady state there is a concentration difference between the bulk pipette and intracellular solution that is described by delta c = jRp/Dp, where delta c = 1 mM for a flux, j = 1 fmol/s, through a pipette of resistance, Rp = 1 M omega, filled with a solution of resistivity, p = 100 omega --cm, given a solute diffusion coefficient, D = 10(-5) cm2/s. (c) The time to steady state is always accelerated by membrane transport, regardless of the direction of transport. We apply our analysis to the measurement of transport by the Na/K pump and Na/Ca exchanger in cells from the ventricles of mammalian heart. We find that the binding curve for intracellular Na+ to the Na/K pump will appear significantly less steep and more linear if one does not correct for the concentration difference between intracellular and pipette Na+. Similar shifts in the binding curve for extracellular Na+ to the Na/Ca exchanger can occur due to depletion of intracellular Ca(+)+ when the exchanger is stimulated. Lastly, in Appendix we analyze the effects of mobile and fixed intracellular buffers on the movement of Ca(+)+ between the pipette and cell. Fixed buffers greatly slow the time for equilibration of pipette and intracellular Ca(+)+. Mobile buffers act like a shuttle system, as they carry Ca(+)+ from pipette to cell then diffuse back when they are empty. Vigorous transport by the Na/Ca exchanger depletes mobile buffered calcium, thus stimulating diffusion from the pipette to match the rate of Ca(+)+ transport. Moreover, we find that

  3. A sensitive whole-cell biosensor for the simultaneous detection of a broad-spectrum of toxic heavy metal ions.

    PubMed

    Cerminati, S; Soncini, F C; Checa, S K

    2015-04-01

    Bacterial biosensors are simple, cost-effective and efficient analytical tools for detecting bioavailable heavy metals in the environment. This work presents the design, construction and calibration of a novel whole-cell fluorescent biosensory device that, simultaneously and with high sensitivity, reports the presence of toxic mercury, lead, cadmium and/or gold ions in aqueous samples. This bio-reporter can be easily applied as an immediate alerting tool for detecting the presence of harmful pollutants in drinking water. PMID:25730473

  4. A sensitive whole-cell biosensor for the simultaneous detection of a broad-spectrum of toxic heavy metal ions.

    PubMed

    Cerminati, S; Soncini, F C; Checa, S K

    2015-04-01

    Bacterial biosensors are simple, cost-effective and efficient analytical tools for detecting bioavailable heavy metals in the environment. This work presents the design, construction and calibration of a novel whole-cell fluorescent biosensory device that, simultaneously and with high sensitivity, reports the presence of toxic mercury, lead, cadmium and/or gold ions in aqueous samples. This bio-reporter can be easily applied as an immediate alerting tool for detecting the presence of harmful pollutants in drinking water.

  5. Equipotent generation of protective antitumor immunity by various methods of dendritic cell loading with whole cell tumor antigens.

    PubMed

    Lambert, L A; Gibson, G R; Maloney, M; Barth, R J

    2001-01-01

    Multiple clinically applicable methods have been used to induce dendritic cells (DCs) to express whole cell tumor antigens, including pulsing DCs with tumor lysate, and mixing DCs with apoptotic or live tumor cells. Herein we demonstrate, using two different tumor systems, that these methods are equipotent inducers of systemic antitumor immunity. Furthermore, tumor lysate pulsed DC vaccines generate more potent antitumor immunity than immunization with irradiated tumor cells plus the classic adjuvant, Corynebacterium parvum. PMID:11394500

  6. High-Resolution 3D Imaging and Quantification of Gold Nanoparticles in a Whole Cell Using Scanning Transmission Ion Microscopy

    PubMed Central

    Chen, Xiao; Chen, Ce-Belle; Udalagama, Chammika N.B.; Ren, Minqin; Fong, Kah Ee; Yung, Lin Yue Lanry; Giorgia, Pastorin; Bettiol, Andrew Anthony; Watt, Frank

    2013-01-01

    Increasing interest in the use of nanoparticles (NPs) to elucidate the function of nanometer-sized assemblies of macromolecules and organelles within cells, and to develop biomedical applications such as drug delivery, labeling, diagnostic sensing, and heat treatment of cancer cells has prompted investigations into novel techniques that can image NPs within whole cells and tissue at high resolution. Using fast ions focused to nanodimensions, we show that gold NPs (AuNPs) inside whole cells can be imaged at high resolution, and the precise location of the particles and the number of particles can be quantified. High-resolution density information of the cell can be generated using scanning transmission ion microscopy, enhanced contrast for AuNPs can be achieved using forward scattering transmission ion microscopy, and depth information can be generated from elastically backscattered ions (Rutherford backscattering spectrometry). These techniques and associated instrumentation are at an early stage of technical development, but we believe there are no physical constraints that will prevent whole-cell three-dimensional imaging at <10 nm resolution. PMID:23561518

  7. Adjuvant effect of DEAE-dextran and tetanus toxoid on whole cell heat inactivated phenol preserved typhoid vaccine.

    PubMed

    Kaistha, J; Sokhey, J; Singh, S; Kumar, S; John, P C; Sharma, N C

    1996-10-01

    Active mouse protection test (AMPT) and enzyme linked immunosorbent assay (ELISA) were used to determine the immunogenicity of whole cell typhoid vaccine when administered in conjunction with either tetanus toxoid (TT) or DEAE-Dextran (DD). Immunization of mice with whole cell typhoid vaccine showed enhanced potency either when administered in conjunction with TT or DD and values were statistically significant (p < 0.05) in comparison to conventional or standard typhoid vaccines. For ELISA, the mice were immunized with 2 different schedules, one in which a single dose of 0.25 ml subcutaneously (s/c) was administered and in another two doses of 0.25 ml each s/c, 14 days apart. In case of single dose schedule of immunization D vaccine (Whole cell typhoid + 5 mg/ml DD) showed significant increase of immune response (3.201 log10) as compared to plain vaccine (2.550 log10). Two dose schedule further increased the titres to 3.856 log10. DD adjuvanted vaccine showed higher potency by AMPT as compared to the TT adjuvanted vaccine or plain vaccine. The present study clearly demonstrates that a single dose of 0.25 ml which is equivalent to half of the conventionally used single human dose of typhoid vaccine adjuvanted with DD can significantly improve the immunogenicity of the vaccine.

  8. Optimization of the whole-cell catalytic activity of recombinant Escherichia coli cells with surface-immobilized organophosphorus hydrolase.

    PubMed

    Zhang, Hongxing; Li, Qianqian; Ye, Ting; Zhang, Zhen; Li, Lin

    2013-04-01

    Previous studies have verified the feasibility of using Escherichia coli systems that display organophosphorous hydrolase (OPH) on the cell surface as whole-cell catalysts. However, the inefficient display of the enzyme on cell surfaces remains unaddressed. In the present study, multiple optimization experiments on full-length and truncated ice nucleation protein anchors, E. coli host cells, culture media, and culture conditions were performed to optimize whole-cell OPH enzymatic activity. The results show that apart from the dramatic effect of isopropyl-beta-D-thiogalactoside concentration and culture temperature, the coordination between the anchor protein, culture media, and host cells is essential for highly efficient OPH display. Under optimal conditions, namely, culturing in M9 medium, 20 degrees C induction temperature, 0.1 mmol l(-1) IPTG, and 100 micromol I(-1) Co2+, the engineered E. coli strain MB109-406 that expresses the fusion enzyme lnaK-N-OPH exhibited a whole-cell OPH activity of 0.62 U mg(-1) x cell d.wt. This result is much higher than that of several currently available OPH-displaying systems, which shows the potential of the current system for further large-scale industrial or environmental applications. PMID:24620599

  9. Utilization of Whole-Cell MALDI-TOF Mass Spectrometry to Differentiate Burkholderia pseudomallei Wild-Type and Constructed Mutants.

    PubMed

    Niyompanich, Suthamat; Srisanga, Kitima; Jaresitthikunchai, Janthima; Roytrakul, Sittiruk; Tungpradabkul, Sumalee

    2015-01-01

    Whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (whole-cell MALDI-TOF MS) has been widely adopted as a useful technology in the identification and typing of microorganisms. This study employed the whole-cell MALDI-TOF MS to identify and differentiate wild-type and mutants containing constructed single gene mutations of Burkholderia pseudomallei, a pathogenic bacterium causing melioidosis disease in both humans and animals. Candidate biomarkers for the B. pseudomallei mutants, including rpoS, ppk, and bpsI isolates, were determined. Taxon-specific and clinical isolate-specific biomarkers of B. pseudomallei were consistently found and conserved across all average mass spectra. Cluster analysis of MALDI spectra of all isolates exhibited separate distribution. A total of twelve potential mass peaks discriminating between wild-type and mutant isolates were identified using ClinProTools analysis. Two peaks (m/z 2721 and 2748 Da) were specific for the rpoS isolate, three (m/z 3150, 3378, and 7994 Da) for ppk, and seven (m/z 3420, 3520, 3587, 3688, 4623, 4708, and 5450 Da) for bpsI. Our findings demonstrated that the rapid, accurate, and reproducible mass profiling technology could have new implications in laboratory-based rapid differentiation of extensive libraries of genetically altered bacteria. PMID:26656930

  10. Inhibition of methanogenesis in salt marsh sediments and whole-cell suspensions of methanogenic bacteria by nitrogen oxides.

    PubMed Central

    Balderston, W L; Payne, W J

    1976-01-01

    Hydrogen-dependent evolution of methane from salt marsh sediments and whole-cell suspensions of Methanobacterium thermoautotrophicum and Methanobacterium fornicicum ceased or decreased after the introduction of nitrate, nitrite, nitric oxide, or nitrous oxide. Sulfite had a similar effect on methanogenesis in the whole-cell suspensions. In salt marsh sediments, nitrous oxide was the strongest inhibitor, followed by nitric oxide, nitrite, and nitrate in decreasing order of inhibition. In whole-cell suspensions, nitric oxide was the strongest inhibitor, followed by nitrous oxide, nitrite, and nitrate. Consideration of the results from experiments using an indicator of oxidation potential, along with the reversed order of effectiveness of the nitrogen oxides in relation to their degree of reduction ,suggests that the inhibitory effect observed was not due to a redox change. Evidence is also presented that suggests that the decrease in the rate of methane production in the presence of oxides of nitrogen was not attributable to competition for methane-producing substrates. PMID:970945

  11. [Biosynthesis of indigo and indirubin by whole-cell catalyst designed by combination of protein engineering and metabolic engineering].

    PubMed

    Li, Yang; Zhu, Junge; Wang, Jianjun; Xia, Huanzhang; Wu, Sheng

    2016-01-01

    The phenylacetone monooxygenase, isolated from Thermobifida fusca, mainly catalyzes Baeyer-Villiger oxidation reaction towards aromatic compounds. Met446 plays a vital role in catalytic promiscuity, based on the structure and function of phenylacetone monooxygenase. Mutation in Met446 locus can offer enzyme new catalytic feature to activate C-H bond, oxidizing indole to finally generate indigo and indirubin, but the yield was only 1.89 mg/L. In order to further improve the biosynthesis efficiency of the whole-cell catalyst, metabolic engineering was applied to change glucose metabolism pathway of Escherichia coli. Blocking glucose isomerase gene pgi led to pentose phosphate pathway instead of the glycolytic pathway to become the major metabolic pathways of glucose, which provided more cofactor NADPH needed in enzymatic oxidation of indole. Engineering the host E. coli led to synthesis of indigo and indirubin efficiency further increased to 25 mg/L. Combination of protein and metabolic engineering to design efficient whole-cell catalysts not only improves the synthesis of indigo and indirubin, but also provides a novel strategy for whole-cell catalyst development. PMID:27363197

  12. [Biosynthesis of indigo and indirubin by whole-cell catalyst designed by combination of protein engineering and metabolic engineering].

    PubMed

    Li, Yang; Zhu, Junge; Wang, Jianjun; Xia, Huanzhang; Wu, Sheng

    2016-01-01

    The phenylacetone monooxygenase, isolated from Thermobifida fusca, mainly catalyzes Baeyer-Villiger oxidation reaction towards aromatic compounds. Met446 plays a vital role in catalytic promiscuity, based on the structure and function of phenylacetone monooxygenase. Mutation in Met446 locus can offer enzyme new catalytic feature to activate C-H bond, oxidizing indole to finally generate indigo and indirubin, but the yield was only 1.89 mg/L. In order to further improve the biosynthesis efficiency of the whole-cell catalyst, metabolic engineering was applied to change glucose metabolism pathway of Escherichia coli. Blocking glucose isomerase gene pgi led to pentose phosphate pathway instead of the glycolytic pathway to become the major metabolic pathways of glucose, which provided more cofactor NADPH needed in enzymatic oxidation of indole. Engineering the host E. coli led to synthesis of indigo and indirubin efficiency further increased to 25 mg/L. Combination of protein and metabolic engineering to design efficient whole-cell catalysts not only improves the synthesis of indigo and indirubin, but also provides a novel strategy for whole-cell catalyst development.

  13. Process development and modeling of fluidized-bed reactor with coimmobilized biocatalyst for fuel ethanol production

    NASA Astrophysics Data System (ADS)

    Sun, May Yongmei

    This research focuses on two steps of commercial fuel ethanol production processes: the hydrolysis starch process and the fermentation process. The goal of this research is to evaluate the performance of co-immobilized biocatalysts in a fluidized bed reactor with emphasis on economic and engineering aspects and to develop a predictive mathematical model for this system. The productivity of an FBR is higher than productivity of a traditional batch reactor or CSTR. Fluidized beds offer great advantages over packed beds for immobilized cells when small particles are used or when the reactant feed contains suspended solids. Plugging problems, excessive pressure drops (and thus attrition), or crushing risks may be avoided. No mechanical stirring is required as mixing occurs due to the natural turbulence in the fluidized process. Both enzyme and microorganism are immobilized in one catalyst bead which is called co-immobilization. Inside this biocatalyst matrix, starch is hydrolyzed by the enzyme glucoamylase to form glucose and then converted to ethanol and carbon dioxide by microorganisms. Two biocatalysts were evaluated: (1) co-immobilized yeast strain Saccharomyces cerevisiae and glucoamylase. (2) co-immobilized Zymomonas mobilis and glucoamylase. A co-immobilized biocatalyst accomplishes the simultaneous saccharification and fermentation (SSF process). When compared to a two-step process involving separate saccharification and fermentation stages, the SSF process has productivity values twice that given by the pre-saccharified process when the time required for pre-saccharification (15--25 h) was taken into account. The SSF process should also save capital cost. The information about productivity, fermentation yield, concentration profiles along the bed, ethanol inhibition, et al., was obtained from the experimental data. For the yeast system, experimental results showed that: no apparent decrease of productivity occurred after two and half months, the productivity

  14. Metagenomics-Guided Mining of Commercially Useful Biocatalysts from Marine Microorganisms.

    PubMed

    Uria, A R; Zilda, D S

    2016-01-01

    Marine microorganisms are a rich reservoir of highly diverse and unique biocatalysts that offer potential applications in food, pharmaceutical, fuel, and cosmetic industries. The fact that only less than 1% of microbes in any marine habitats can be cultured under standard laboratory conditions has hampered access to their extraordinary biocatalytic potential. Metagenomics has recently emerged as a powerful and well-established tool to investigate the vast majority of hidden uncultured microbial diversity for the discovery of novel industrially relevant enzymes from different types of environmental samples, such as seawater, marine sediment, and symbiotic microbial consortia. We discuss here in this review about approaches and methods in metagenomics that have been used and can potentially be used to mine commercially useful biocatalysts from uncultured marine microbes. PMID:27452163

  15. Development of biocatalysts for production of commodity chemicals from lignocellulosic biomass.

    PubMed

    Adsul, M G; Singhvi, M S; Gaikaiwari, S A; Gokhale, D V

    2011-03-01

    Lignocellulosic biomass is recognized as potential sustainable source for production of power, biofuels and variety of commodity chemicals which would potentially add economic value to biomass. Recalcitrance nature of biomass is largely responsible for the high cost of its conversion. Therefore, it is necessary to introduce some cost effective pretreatment processes to make the biomass polysaccharides easily amenable to enzymatic attack to release mixed fermentable sugars. Advancement in systemic biology can provide new tools for the development of such biocatalysts for sustainable production of commodity chemicals from biomass. Integration of functional genomics and system biology approaches may generate efficient microbial systems with new metabolic routes for production of commodity chemicals. This paper provides an overview of the challenges that are faced by the processes converting lignocellulosic biomass to commodity chemicals. The critical factors involved in engineering new microbial biocatalysts are also discussed with more emphasis on commodity chemicals. PMID:21277771

  16. Copper-binding peptides from human prion protein and newly designed peroxidative biocatalysts.

    PubMed

    Kagenishi, Tomoko; Yokawa, Ken; Kadono, Takashi; Uezu, Kazuya; Kawano, Tomonori

    2011-01-01

    A previous work suggested that peptides from the histidine-containing copper-binding motifs in human prion protein (PrP) function as peroxidase-like biocatalysts catalyzing the generation of superoxide anion radicals in the presence of neurotransmitters (aromatic monoamines) and phenolics such as tyrosine and tyrosyl residues on proteins. In this study, using various phenolic substrates, the phenol-dependent superoxide-generating activities of PrP-derived peptide sequences were compared. Among the peptides tested, the GGGTH pentapeptide was shown to be the most active catalyst for phenol-dependent reactions. Based on these results, we designed a series of oligoglycyl-histidines as novel peroxidative biocatalysts, and their catalytic performances including kinetics, heat tolerance, and freezing tolerance were analysed. PMID:21630593

  17. Metagenomics-Guided Mining of Commercially Useful Biocatalysts from Marine Microorganisms.

    PubMed

    Uria, A R; Zilda, D S

    2016-01-01

    Marine microorganisms are a rich reservoir of highly diverse and unique biocatalysts that offer potential applications in food, pharmaceutical, fuel, and cosmetic industries. The fact that only less than 1% of microbes in any marine habitats can be cultured under standard laboratory conditions has hampered access to their extraordinary biocatalytic potential. Metagenomics has recently emerged as a powerful and well-established tool to investigate the vast majority of hidden uncultured microbial diversity for the discovery of novel industrially relevant enzymes from different types of environmental samples, such as seawater, marine sediment, and symbiotic microbial consortia. We discuss here in this review about approaches and methods in metagenomics that have been used and can potentially be used to mine commercially useful biocatalysts from uncultured marine microbes.

  18. Enzymes in Food Processing: A Condensed Overview on Strategies for Better Biocatalysts

    PubMed Central

    Fernandes, Pedro

    2010-01-01

    Food and feed is possibly the area where processing anchored in biological agents has the deepest roots. Despite this, process improvement or design and implementation of novel approaches has been consistently performed, and more so in recent years, where significant advances in enzyme engineering and biocatalyst design have fastened the pace of such developments. This paper aims to provide an updated and succinct overview on the applications of enzymes in the food sector, and of progresses made, namely, within the scope of tapping for more efficient biocatalysts, through screening, structural modification, and immobilization of enzymes. Targeted improvements aim at enzymes with enhanced thermal and operational stability, improved specific activity, modification of pH-activity profiles, and increased product specificity, among others. This has been mostly achieved through protein engineering and enzyme immobilization, along with improvements in screening. The latter has been considerably improved due to the implementation of high-throughput techniques, and due to developments in protein expression and microbial cell culture. Expanding screening to relatively unexplored environments (marine, temperature extreme environments) has also contributed to the identification and development of more efficient biocatalysts. Technological aspects are considered, but economic aspects are also briefly addressed. PMID:21048872

  19. Integrated (Meta) Genomic and Synthetic Biology Approaches to Develop New Biocatalysts

    PubMed Central

    Parages, María L.; Gutiérrez-Barranquero, José A.; Reen, F. Jerry; Dobson, Alan D.W.; O’Gara, Fergal

    2016-01-01

    In recent years, the marine environment has been the subject of increasing attention from biotechnological and pharmaceutical industries as a valuable and promising source of novel bioactive compounds. Marine biodiscovery programmes have begun to reveal the extent of novel compounds encoded within the enormous bacterial richness and diversity of the marine ecosystem. A combination of unique physicochemical properties and spatial niche-specific substrates, in wide-ranging and extreme habitats, underscores the potential of the marine environment to deliver on functionally novel biocatalytic activities. With the growing need for green alternatives to industrial processes, and the unique transformations which nature is capable of performing, marine biocatalysts have the potential to markedly improve current industrial pipelines. Furthermore, biocatalysts are known to possess chiral selectivity and specificity, a key focus of pharmaceutical drug design. In this review, we discuss how the explosion in genomics based sequence analysis, allied with parallel developments in synthetic and molecular biology, have the potential to fast-track the discovery and subsequent improvement of a new generation of marine biocatalysts. PMID:27007381

  20. [Application of Whole-cell Biosensor ADP1_pWHlux for Acute Toxicity Detection in Water Environment].

    PubMed

    Tang, Hui; Song, Yi-zhi; Jiang, Bo; Chen, Guang-yu; Jia, Jian-li; Zhang, Xu; Li, Guang-he

    2015-10-01

    A whole-cell biosensor acinetobacter ADP1_pWHlux was constructed by genetic engineering for detecting acute toxicity, so as to overcome the harsh application conditions when detecting acute toxicity using natural luminescent bacteria or whole-cell biosensor constructed by model microorganisms as the host cell. Detection methods, detection sensitivity and detection range of acinetobacter ADP1_pWHlux were studied. The results showed that the luminescence of ADP1_pWHlux was inhibited by acute poison, poison dose and inhibition of luminescence exhibit dose-response relationship. ADPL_pWHlux was respond to 4 mg x L(-1) HgCl2 within 5 min. The detection limit for HgCl2 was 0.04 mg x L(-1). The detectable effects for indicators of Be2+, Ba2+, Cu2+, Ni2+ in standards for drinking water quality were obvious. The detection range of Be2+, Ba2+, Cu2+ were 0.025-250 mg x L(-1), the detection range of Ni2+, was 0.0025-250 mg x L(-1), the detection limit of Pb2+, BrO3(-) , ClO2(-) were 0.002 5 mg x L(-1), the detection limit of ClO3(-) was 0.025 mg x L(-1). The whole-cell biosensor ADPl_pWHlux detection method has been applied to evaluate acute toxicity in water environment of Qinghe river in Beijing, indicating the established method can be used to detect contaminated water samples. PMID:26841625

  1. [Construction and properties of a microbial whole-cell sensor CB10 for the bioavailability detection of Cr6+].

    PubMed

    Hou, Qi-Hui; Ma, An-Zhou; Zhuang, Xu-Liang; Zhuang, Guo-Qiang

    2013-03-01

    A microbial whole-cell biosensor CB10 for the bioavailability assessing of Cr6+ was constructed by molecular biotechnology. The regulatory gene and promoter of CB10 was from the chromium resistance system of plasmid pMOL28 from Cupriavidus metallidurans CH34, and the reporter gene of CB10 was luc which was derived from Photinus pyralis. Finally, its response characteristic was discussed under different incubation conditions e. g. pH and temperature. The results showed that a microbial whole-cell biosensor CB10 had been successfully constructed which could respond to Cr6+ within 30 min, with a LOD for Cr6+ of 2 micromol x L(-1). When the incubation concentration of Cr6+ was between 20 micromol x L(-1) and 200 micromol x L(-1), the luc activity of the CB10 biosensor was in linear correlation with the concentration of Cr6+. When the concentration of heavy metal was in the range of 10-50 micromol x L(-1), the response of CB10 was relatively more specific. Moreover, high concentrations of Pb2+, Mn2+ and Sb2+ could also induce CB10. By analyzing the response characteristic of CB10 biosensor, we could draw the conclusion that 15-30 degrees C and pH 4-7 were appropriate for CB10, and 30 degrees C and pH 7 were the optimal conditions for the incubation of the CB10 biosensor. The microbial whole-cell biosensor CB10 for the detection of Cr6+ was fast-responding, specific, sensitive and stable under various conditions. In prospective, it could be used in the fast detection of Cr6+ in water and assessment of the bioavailability of Cr6+ in soil.

  2. [Construction and properties of a microbial whole-cell sensor CB10 for the bioavailability detection of Cr6+].

    PubMed

    Hou, Qi-Hui; Ma, An-Zhou; Zhuang, Xu-Liang; Zhuang, Guo-Qiang

    2013-03-01

    A microbial whole-cell biosensor CB10 for the bioavailability assessing of Cr6+ was constructed by molecular biotechnology. The regulatory gene and promoter of CB10 was from the chromium resistance system of plasmid pMOL28 from Cupriavidus metallidurans CH34, and the reporter gene of CB10 was luc which was derived from Photinus pyralis. Finally, its response characteristic was discussed under different incubation conditions e. g. pH and temperature. The results showed that a microbial whole-cell biosensor CB10 had been successfully constructed which could respond to Cr6+ within 30 min, with a LOD for Cr6+ of 2 micromol x L(-1). When the incubation concentration of Cr6+ was between 20 micromol x L(-1) and 200 micromol x L(-1), the luc activity of the CB10 biosensor was in linear correlation with the concentration of Cr6+. When the concentration of heavy metal was in the range of 10-50 micromol x L(-1), the response of CB10 was relatively more specific. Moreover, high concentrations of Pb2+, Mn2+ and Sb2+ could also induce CB10. By analyzing the response characteristic of CB10 biosensor, we could draw the conclusion that 15-30 degrees C and pH 4-7 were appropriate for CB10, and 30 degrees C and pH 7 were the optimal conditions for the incubation of the CB10 biosensor. The microbial whole-cell biosensor CB10 for the detection of Cr6+ was fast-responding, specific, sensitive and stable under various conditions. In prospective, it could be used in the fast detection of Cr6+ in water and assessment of the bioavailability of Cr6+ in soil. PMID:23745432

  3. [Application of Whole-cell Biosensor ADP1_pWHlux for Acute Toxicity Detection in Water Environment].

    PubMed

    Tang, Hui; Song, Yi-zhi; Jiang, Bo; Chen, Guang-yu; Jia, Jian-li; Zhang, Xu; Li, Guang-he

    2015-10-01

    A whole-cell biosensor acinetobacter ADP1_pWHlux was constructed by genetic engineering for detecting acute toxicity, so as to overcome the harsh application conditions when detecting acute toxicity using natural luminescent bacteria or whole-cell biosensor constructed by model microorganisms as the host cell. Detection methods, detection sensitivity and detection range of acinetobacter ADP1_pWHlux were studied. The results showed that the luminescence of ADP1_pWHlux was inhibited by acute poison, poison dose and inhibition of luminescence exhibit dose-response relationship. ADPL_pWHlux was respond to 4 mg x L(-1) HgCl2 within 5 min. The detection limit for HgCl2 was 0.04 mg x L(-1). The detectable effects for indicators of Be2+, Ba2+, Cu2+, Ni2+ in standards for drinking water quality were obvious. The detection range of Be2+, Ba2+, Cu2+ were 0.025-250 mg x L(-1), the detection range of Ni2+, was 0.0025-250 mg x L(-1), the detection limit of Pb2+, BrO3(-) , ClO2(-) were 0.002 5 mg x L(-1), the detection limit of ClO3(-) was 0.025 mg x L(-1). The whole-cell biosensor ADPl_pWHlux detection method has been applied to evaluate acute toxicity in water environment of Qinghe river in Beijing, indicating the established method can be used to detect contaminated water samples.

  4. Whole-Cell Patch-Clamp Recording of Mouse and Rat Inner Hair Cells in the Intact Organ of Corti.

    PubMed

    Goutman, Juan D; Pyott, Sonja J

    2016-01-01

    Whole-cell patch clamping is a widely applied method to record currents across the entire membrane of a cell. This protocol describes application of this method to record currents from the sensory inner hair cells in the intact auditory sensory epithelium, the organ of Corti, isolated from rats or mice. This protocol particularly outlines the basic equipment required, provides instructions for the preparation of solutions and small equipment items, and methodology for recording voltage-activated and evoked synaptic currents from the inner hair cells.

  5. Countercurrent multistage fluidized bed reactor for immobilized biocatalysts: III. Hydrodynamic aspects.

    PubMed

    Vos, H J; van Houwelingen, C; Zomerdijk, M; Luyben, K C

    1990-08-01

    In Parts I and II of this series we described the modelling, design, and operation of a multistage fluidized bed reactor (MFBR) for immobilized biocatalysts. This article deals with those aspects of the MFBR which are different from single-stage fluidized beds which are operated in batch mode with respect to the solids. The semicontinuous transport of the particles requires perfect mixing of the particles in the reactor compartments, because particles are mainly transported from the bottom of these compartments. A large spread in the physical properties of the biocatalyst particles, especially of both size and density, may cause the particles to segregate into layers with different diameter and/or density. This affects the efficient use of the biocatalyst. The properties of the particles are dependent on the immobilization method. The suitability of different methods for possible future application in the MFBR is therefore compared. Because of segregation, successful use of a biofilm catalyst with a nonuniform thickness of the biofilm is doubtful. Experiments in a small scale reactor (+/- 0.1 m diameter) demonstrated that perfect particle mixing is possible using commercially available biocatalyst particles of uniform density. Co-immobilization of the biocatalyst with glass powder in a gel is a simple and effective method of increasing gel density. High density particles allow high liquid flow rates, and thus an improved external mass transfer can be achieved.The distributor plates, which separate the reactor compartments, must allow unhindered transport of particles. Therefore, the holes in these plates must have a diameter of at least 4.5 times that of the largest particles which are present in the particle mixture used. Furthermore, the plates must be designed such that, when scaling-up the reactor, a uniform liquid distribution over the cross-sectional area of the reactor occurs. Large-scale experiments were not carried out, but published correlations, indicate

  6. Biotransformation of (-)-α-Pinene by Whole Cells of White Rot Fungi, Ceriporia sp. ZLY-2010 and Stereum hirsutum.

    PubMed

    Lee, Su-Yeon; Kim, Seon-Hong; Hong, Chang-Young; Kim, Ho-Young; Ryu, Sun-Hwa; Choi, In-Gyu

    2015-09-01

    Two white rot fungi, Ceriporia sp. ZLY-2010 (CER) and Stereum hirsutum (STH) were used as biocatalysts for the biotransformation of (-)-α-pinene. After 96 hr, CER converted the bicyclic monoterpene hydrocarbon (-)-α-pinene into α-terpineol (yield, 0.05 g/L), a monocyclic monoterpene alcohol, in addition to, other minor products. Using STH, verbenone was identified as the major biotransformed product, and minor products were myrtenol, camphor, and isopinocarveol. We did not observe any inhibitory effects of substrate or transformed products on mycelial growth of the fungi. The activities of fungal manganese-dependent peroxidase and laccase were monitored for 15 days to determine the enzymatic pathways related to the biotransformation of (-)-α-pinene. We concluded that a complex of enzymes, including intra- and extracellular enzymes, were involved in terpenoid biotransformation by white rot fungi. PMID:26539046

  7. Biotransformation of (-)-α-Pinene by Whole Cells of White Rot Fungi, Ceriporia sp. ZLY-2010 and Stereum hirsutum

    PubMed Central

    Lee, Su-Yeon; Kim, Seon-Hong; Hong, Chang-Young; Kim, Ho-Young; Ryu, Sun-Hwa

    2015-01-01

    Two white rot fungi, Ceriporia sp. ZLY-2010 (CER) and Stereum hirsutum (STH) were used as biocatalysts for the biotransformation of (-)-α-pinene. After 96 hr, CER converted the bicyclic monoterpene hydrocarbon (-)-α-pinene into α-terpineol (yield, 0.05 g/L), a monocyclic monoterpene alcohol, in addition to, other minor products. Using STH, verbenone was identified as the major biotransformed product, and minor products were myrtenol, camphor, and isopinocarveol. We did not observe any inhibitory effects of substrate or transformed products on mycelial growth of the fungi. The activities of fungal manganese-dependent peroxidase and laccase were monitored for 15 days to determine the enzymatic pathways related to the biotransformation of (-)-α-pinene. We concluded that a complex of enzymes, including intra- and extracellular enzymes, were involved in terpenoid biotransformation by white rot fungi. PMID:26539046

  8. Importance of solute partitioning in biphasic oxidation of benzyl alcohol by free and immobilized whole cells of Pichia pastoris

    SciTech Connect

    Kawakami, Koei; Nakahara, Takehiko . Dept. of Chemical Engineering)

    1994-04-25

    Using free and immobilized whole cells of Pichia pastoris, the biocatalytic oxidation of benzyl alcohol was investigated in different two-phase systems. This reaction was strongly influenced by both the substrate and product inhibitions, and the production rate of benzaldehyde in the aqueous system became maximum at the initial substrate concentration of ca. 29 g/L with the aldehyde formation less than 4 to 5 g/L even after a longer reaction period. The reaction rates in the two-liquid phase systems were predominantly determined by the partitioning behaviors of the substrate and the product between the two phases rather than by enzyme deactivation by the organic solvents. In the two-liquid phase systems, consequently, the organic solvent acted as a reservoir to reduce these inhibitory effects, and it was essential to select the organic solvent providing the optimal partitioning of the substrate into the aqueous phase as well as the preferential extraction of the product into the organic phase. The whole cells immobilized in a mixed matrix composed of silicone polymer and Ca alginate gel worked well in the xylene and decane media, providing comparable activities with the free cells. The production rate of aldehyde was also influenced by the solute partitioning into the hydrophilic alginate phase where the cells existed.

  9. Preliminary characterization of voltage-activated whole-cell currents in developing human vestibular hair cells and calyx afferent terminals.

    PubMed

    Lim, Rebecca; Drury, Hannah R; Camp, Aaron J; Tadros, Melissa A; Callister, Robert J; Brichta, Alan M

    2014-10-01

    We present preliminary functional data from human vestibular hair cells and primary afferent calyx terminals during fetal development. Whole-cell recordings were obtained from hair cells or calyx terminals in semi-intact cristae prepared from human fetuses aged between 11 and 18 weeks gestation (WG). During early fetal development (11-14 WG), hair cells expressed whole-cell conductances that were qualitatively similar but quantitatively smaller than those observed previously in mature rodent type II hair cells. As development progressed (15-18 WG), peak outward conductances increased in putative type II hair cells but did not reach amplitudes observed in adult human hair cells. Type I hair cells express a specific low-voltage activating conductance, G K,L. A similar current was first observed at 15 WG but remained relatively small, even at 18 WG. The presence of a "collapsing" tail current indicates a maturing type I hair cell phenotype and suggests the presence of a surrounding calyx afferent terminal. We were also able to record from calyx afferent terminals in 15-18 WG cristae. In voltage clamp, these terminals exhibited fast inactivating inward as well as slower outward conductances, and in current clamp, discharged a single action potential during depolarizing steps. Together, these data suggest the major functional characteristics of type I and type II hair cells and calyx terminals are present by 18 WG. Our study also describes a new preparation for the functional investigation of key events that occur during maturation of human vestibular organs.

  10. Detergent Induction of HEK 293A Cell Membrane Permeability Measured under Quiescent and Superfusion Conditions Using Whole Cell Patch Clamp

    PubMed Central

    2015-01-01

    Detergents have several biological applications but present cytotoxicity concerns, since they can solubilize cell membranes. Using the IonFlux 16, an ensemble whole cell planar patch clamp, we observed that anionic sodium dodecyl sulfate (SDS), cationic cetyltrimethylammonium bromide (CTAB), and cationic, fluorescent octadecyl rhodamine B (ORB) increased the membrane permeability of cells substantially within a second of exposure, under superfusion conditions. Increased permeability was irreversible for 15 min. At subsolubilizing detergent concentrations, patched cells showed increased membrane currents that reached a steady state and were intact when imaged using fluorescence microscopy. SDS solubilized cells at concentrations of 2 mM (2× CMC), while CTAB did not solubilize cells even at concentrations of 10 mM (1000× CMC). The relative activity for plasma membrane current induction was 1:20:14 for SDS, CTAB, and ORB, respectively. Under quiescent conditions, the relative ratio of lipid to detergent in cell membranes at the onset of membrane permeability was 1:7:5 for SDS, CTAB, and ORB, respectively. The partition constants (K) for SDS, CTAB, and ORB were 23000, 55000, and 39000 M–1, respectively. Combining the whole cell patch clamp data and XTT viability data, SDS ≤ 0.2 mM and CTAB and ORB ≤ 1 mM induced cell membrane permeability without causing acute toxicity. PMID:24548291

  11. Bacterial cell wall composition and the influence of antibiotics by cell-wall and whole-cell NMR.

    PubMed

    Romaniuk, Joseph A H; Cegelski, Lynette

    2015-10-01

    The ability to characterize bacterial cell-wall composition and structure is crucial to understanding the function of the bacterial cell wall, determining drug modes of action and developing new-generation therapeutics. Solid-state NMR has emerged as a powerful tool to quantify chemical composition and to map cell-wall architecture in bacteria and plants, even in the context of unperturbed intact whole cells. In this review, we discuss solid-state NMR approaches to define peptidoglycan composition and to characterize the modes of action of old and new antibiotics, focusing on examples in Staphylococcus aureus. We provide perspectives regarding the selected NMR strategies as we describe the exciting and still-developing cell-wall and whole-cell NMR toolkit. We also discuss specific discoveries regarding the modes of action of vancomycin analogues, including oritavancin, and briefly address the reconsideration of the killing action of β-lactam antibiotics. In such chemical genetics approaches, there is still much to be learned from perturbations enacted by cell-wall assembly inhibitors, and solid-state NMR approaches are poised to address questions of cell-wall composition and assembly in S. aureus and other organisms.

  12. Effects of cicletanine on whole-cell currents of single smooth muscle cells from the guinea-pig portal vein.

    PubMed Central

    Noack, T.; Deitmer, P.

    1993-01-01

    1. Smooth muscle cells of the guinea-pig portal vein were dispersed by enzymatic treatment and recordings of membrane currents were made in the whole-cell mode by the patch-clamp technique. The effects of extracellular application of cicletanine-hydrochloride on the whole-cell currents of isolated smooth muscle cells from the guinea-pig portal vein were studied in solutions containing a normal concentration of calcium (2.5 mM). 2. Cicletanine, 10 to 100 microM, reduced the voltage-dependent inward calcium current with an IC50 of 250 microM. These effects of cicletanine were reversible. 3. The action of cicletanine on calcium currents can be interpreted as a decrease of the availability of calcium channels but not by an alteration of the time course or voltage-dependency of inactivation. 4. The control calcium current was enhanced by application of Bay K 8644. On this enhanced inward current, cicletanine also exerted inhibitory effects which were not use-dependent. 5. Cicletanine, 1 to 100 microM, did not enhance outward potassium currents. 6. It is concluded that at least one component of the vasorelaxant effects of cicletanine is produced by inhibition of calcium currents. PMID:7684299

  13. TolC plays a crucial role in immune protection conferred by Edwardsiella tarda whole-cell vaccines

    PubMed Central

    Wang, Chao; Peng, Bo; Li, Hui; Peng, Xuan-xian

    2016-01-01

    Although vaccines developed from live organisms have better efficacy than those developed from dead organisms, the mechanisms underlying this differential efficacy remain unexplored. In this study, we combined sub-immunoproteomics with immune challenge to investigate the action of the outer membrane proteome in the immune protection conferred by four Edwardsiella tarda whole-cell vaccines prepared via different treatments and to identify protective immunogens that play a key role in this immune protection. Thirteen spots representing five outer membrane proteins and one cytoplasmic protein were identified, and it was found that their abundance was altered in relation with the immune protective abilities of the four vaccines. Among these proteins, TolC and OmpA were found to be the key immunogens conferring the first and second highest degrees of protection, respectively. TolC was detected in the two effective vaccines (live and inactivated-30-F). The total antiserum and anti-OmpA titers were higher for the two effective vaccines than for the two ineffective vaccines (inactivated-80-F and inactivated-100). Further evidence demonstrated that the live and inactivated-30-F vaccines demonstrated stronger abilities to induce CD8+ and CD4+ T cell differentiation than the other two evaluated vaccines. Our results indicate that the outer membrane proteome changes dramatically following different treatments, which contributes to the effectiveness of whole-cell vaccines. PMID:27406266

  14. Evaluation of colors in green mutants isolated from purple bacteria as a host for colorimetric whole-cell biosensors.

    PubMed

    Yoshida, Kazuyuki; Yoshioka, Daiki; Inoue, Koichi; Takaichi, Shinichi; Maeda, Isamu

    2007-10-01

    The change in carotenoid-based bacterial color from yellow to red can be applied to whole-cell biosensors. We generated several green mutants to emphasize the color change in such biosensors. The blue-green crtI-deleted mutant, Rhodopseudomonas palustris no.711, accumulated the colorless carotenoid precursor, phytoene. Green Rhodovulum sulfidophilum M31 accumulated neurosporene, a downstream product of phytoene. Another green mutant, Rhodobacter sphaeroides Ga, accumulated neurosporene and chloroxanthin, which are both downstream products of phytoene. All green mutants accumulated bacteriochlorophyll a. Photosynthetic membrane obtained from the green mutants all exhibited decreased absorption of wavelength range at 510-570 nm. Therefore, these indicate that the greenish bacterial colors were mainly caused by the existence of bacteriochlorophyll a and the changes in carotenoid composition in photosynthetic membrane. The colors of the green mutants and their wild-type strains were plotted in the CIE-L*a*b* color space, and the color difference (DeltaE*ab) values between a green mutant and its wild type were calculated. DeltaE*ab values were higher in the green mutants than in Rdv. sulfidophilum CDM2, the yellowish host strain of reported biosensors. These data indicate that change in bacterial color from green to red is more distinguishable than that from yellow to red as a reporter signal of carotenoid-based whole-cell biosensors.

  15. Bacterial cell wall composition and the influence of antibiotics by cell-wall and whole-cell NMR

    PubMed Central

    Romaniuk, Joseph A. H.; Cegelski, Lynette

    2015-01-01

    The ability to characterize bacterial cell-wall composition and structure is crucial to understanding the function of the bacterial cell wall, determining drug modes of action and developing new-generation therapeutics. Solid-state NMR has emerged as a powerful tool to quantify chemical composition and to map cell-wall architecture in bacteria and plants, even in the context of unperturbed intact whole cells. In this review, we discuss solid-state NMR approaches to define peptidoglycan composition and to characterize the modes of action of old and new antibiotics, focusing on examples in Staphylococcus aureus. We provide perspectives regarding the selected NMR strategies as we describe the exciting and still-developing cell-wall and whole-cell NMR toolkit. We also discuss specific discoveries regarding the modes of action of vancomycin analogues, including oritavancin, and briefly address the reconsideration of the killing action of β-lactam antibiotics. In such chemical genetics approaches, there is still much to be learned from perturbations enacted by cell-wall assembly inhibitors, and solid-state NMR approaches are poised to address questions of cell-wall composition and assembly in S. aureus and other organisms. PMID:26370936

  16. Detergent induction of HEK 293A cell membrane permeability measured under quiescent and superfusion conditions using whole cell patch clamp.

    PubMed

    Vaidyanathan, Sriram; Orr, Bradford G; Banaszak Holl, Mark M

    2014-02-27

    Detergents have several biological applications but present cytotoxicity concerns, since they can solubilize cell membranes. Using the IonFlux 16, an ensemble whole cell planar patch clamp, we observed that anionic sodium dodecyl sulfate (SDS), cationic cetyltrimethylammonium bromide (CTAB), and cationic, fluorescent octadecyl rhodamine B (ORB) increased the membrane permeability of cells substantially within a second of exposure, under superfusion conditions. Increased permeability was irreversible for 15 min. At subsolubilizing detergent concentrations, patched cells showed increased membrane currents that reached a steady state and were intact when imaged using fluorescence microscopy. SDS solubilized cells at concentrations of 2 mM (2× CMC), while CTAB did not solubilize cells even at concentrations of 10 mM (1000× CMC). The relative activity for plasma membrane current induction was 1:20:14 for SDS, CTAB, and ORB, respectively. Under quiescent conditions, the relative ratio of lipid to detergent in cell membranes at the onset of membrane permeability was 1:7:5 for SDS, CTAB, and ORB, respectively. The partition constants (K) for SDS, CTAB, and ORB were 23000, 55000, and 39000 M(-1), respectively. Combining the whole cell patch clamp data and XTT viability data, SDS ≤ 0.2 mM and CTAB and ORB ≤ 1 mM induced cell membrane permeability without causing acute toxicity.

  17. ASSESSMENT OF MARKER PROTEINS IDENTIFIED IN WHOLE CELL EXTRACTS FOR BACTERIAL SPECIATION USING LIQUID CHROMATOGRAPHY ELECTROSPRAY IONIZATION TANDEM MASS SPECTROMETRY

    PubMed Central

    Kooken, Jennifer; Fox, Karen; Fox, Alvin; Wunschel, David

    2014-01-01

    Staphylococcal strains (CoNS) were speciated in this study. Digests of proteins released from whole cells were converted to tryptic peptides for analysis. Liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI MS/MS, Orbitrap) was employed for peptide analysis. Data analysis was performed employing the open-source software X!Tandem which uses sequenced genomes to generate a virtual peptide database for comparison to experimental data. The search database was modified to include the genomes of the 11 Staphylococcus species most commonly isolated from man. The number of total peptides matching each protein along with the number of peptides specifically matching to the homologue (or homologues) for strains of the same species were assesed. Any peptides not matching to the species examined were considered conflict peptides. The proteins typically identified with the largest percentage of sequence coverage, number of matched peptides and number of peptides corresponding to only the correct species were elongation factor Tu (EF Tu) and enolase (Enol). Additional proteins with consistently observed peptides as well as peptides matching only homologues from the same species were citrate synthase (CS) and 1-pyrroline-5-carboxylate dehydrogenase (1P5CD). Protein markers, previously identified from gel slices, (aconitate hydratase and oxoglutarate dehydrogenase) were found to provide low confidence scores when employing whole cell digests. The methodological approach described here provides a simple yet elegant way of identification of staphylococci. However, perhaps more importantly the technology should be applicable universally for identification of any bacterial species. PMID:23994725

  18. A pulmonary fungus ball produced by Cladosporium cladosporioides.

    PubMed

    Kwon-Chung, K J; Schwartz, I S; Rybak, B J

    1975-10-01

    A case of pulmonary fungus ball produced by Cladosporium cladosporioides is reported. The fungus occupied a cavity in the upper lobe of right lung. Invasion of the cavitary wall or adjacent pulmonary tissue by the fungus was not observed.

  19. Surface Complexation of Neptunium(V) onto Whole Cells and Cell Components of Shewanella alga: Modeling and Experimental Study

    SciTech Connect

    Deo, Randhir P.; Songkasiri, Warinthorn; Rittmann, Bruce E.; Reed, Donald T.

    2012-04-30

    We systematically quantified surface complexation of Np(V) onto whole cells, cell wall, and extracellular polymeric substances (EPS) of Shewanella alga strain BrY. We first performed acid and base titrations and used the mathematical model FITEQL to estimate the concentrations and deprotonation constants of specific surface functional groups. Deprotonation constants most likely corresponded to a carboxyl group not associated with amino acids (pK{sub a} {approx} 5), a phosphoryl site (pK{sub a} {approx} 7.2), and an amine site (pK{sub a} > 10). We then carried out batch sorption experiments with Np(V) and each of the S. alga components as a function of pH. Since significant Np(V) sorption was observed on S. alga whole cells and its components in the pH range 2-5, we assumed the existence of a fourth site: a low-pK{sub a} carboxyl site (pK{sub a} {approx} 2.4) that is associated with amino acids. We used the SPECIATE submodel of the biogeochemical model CCBATCH to compute the stability constants for Np(V) complexation to each surface functional group. The stability constants were similar for each functional group on S. alga bacterial whole cells, cell walls, and EPS, and they explain the complicated sorption patterns when they are combined with the aqueous-phase speciation of Np(V). For pH < 8, the aquo NpO{sub 2}{sup +} species was the dominant form of Np(V), and its log K values for the low-pK{sub a} carboxyl, mid-pK{sub a} carboxyl, and phosphoryl groups were 1.8, 1.8, and 2.5-3.1, respectively. For pH greater than 8, the key surface ligand was amine > XNH{sub 3}{sup +}, which complexed with NpO{sub 2}(CO{sub 3}){sub 3}{sup 5-}. The log K for NpO{sub 2}(CO{sub 3}){sub 3}{sup 5-} complexed onto the amine groups was 3.1-3.9. All of the log K values are similar to those of Np(V) complexes with aqueous carboxyl and N-containing carboxyl ligands. These results help quantify the role of surface complexation in defining actinide-microbiological interactions in the

  20. Surface complexation of Neptunium(V) onto whole cells and cell components of Shewanella alga: modeling and experimental study.

    PubMed

    Deo, Randhir P; Songkasiri, Warinthorn; Rittmann, Bruce E; Reed, Donald T

    2010-07-01

    We systematically quantified surface complexation of Np(V) onto whole cells, cell wall, and extracellular polymeric substances (EPS) of Shewanella alga strain BrY. We first performed acid and base titrations and used the mathematical model FITEQL to estimate the concentrations and deprotonation constants of specific surface functional groups. Deprotonation constants most likely corresponded to a carboxyl group not associated with amino acids (pK(a) approximately 5), a phosphoryl site (pK(a) approximately 7.2), and an amine site (pK(a) > 10). We then carried out batch sorption experiments with Np(V) and each of the S. alga components as a function of pH. Since significant Np(V) sorption was observed on S. alga whole cells and its components in the pH range 2-5, we assumed the existence of a fourth site: a low-pK(a) carboxyl site (pK(a) approximately 2.4) that is associated with amino acids. We used the SPECIATE submodel of the biogeochemical model CCBATCH to compute the stability constants for Np(V) complexation to each surface functional group. The stability constants were similar for each functional group on S. alga bacterial whole cells, cell walls, and EPS, and they explain the complicated sorption patterns when they are combined with the aqueous-phase speciation of Np(V). For pH < 8, the aquo NpO(2)(+) species was the dominant form of Np(V), and its log K values for the low-pK(a) carboxyl, mid-pK(a) carboxyl, and phosphoryl groups were 1.8, 1.8, and 2.5-3.1, respectively. For pH greater than 8, the key surface ligand was amine >XNH(3)(+), which complexed with NpO(2)(CO(3))(3)(5-). The log K for NpO(2)(CO(3))(3)(5-) complexed onto the amine groups was 3.1-3.9. All of the log K values are similar to those of Np(V) complexes with aqueous carboxyl and N-containing carboxyl ligands. These results help quantify the role of surface complexation in defining actinide-microbiological interactions in the subsurface.

  1. Biological lignocellulose solubilization: Comparative evaluation of biocatalysts and enhancement via cotreatment

    DOE PAGES

    Paye, Julie M. D.; Guseva, Anna; Hammer, Sarah K.; Gjersing, Erica; Davis, Mark F.; Davison, Brian H.; Olstad, Jessica; Donohoe, Bryon S.; Nguyen, Thanh Yen; Wyman, Charles E.; et al

    2016-01-12

    Feedstock recalcitrance is the most important barrier impeding cost-effective production of cellulosic biofuels. Pioneer commercial cellulosic ethanol facilities employ thermochemical pretreatment and addition of fungal cellulase, reflecting the main research emphasis in the field. However, it has been suggested that it may be possible to process cellulosic biomass without thermochemical pretreatment using thermophilic, cellulolytic bacteria. Thus, to further explore this idea, we examine the ability of various biocatalysts to solubilize autoclaved but otherwise unpretreated cellulosic biomass under controlled but not industrial conditions.

  2. Highly selective solar-driven methanol from CO2 by a photocatalyst/biocatalyst integrated system.

    PubMed

    Yadav, Rajesh K; Oh, Gyu Hwan; Park, No-Joong; Kumar, Abhishek; Kong, Ki-jeong; Baeg, Jin-Ook

    2014-12-01

    The successful development of a photocatalyst/biocatalyst integrated system that carries out selective methanol production from CO2 is reported herein. The fine-tuned system was derived from a judicious combination of graphene-based visible light active photocatalyst (CCG-IP) and sequentially coupled enzymes. The covalent attachment of isatin-porphyrin (IP) chromophore to chemically converted graphene (CCG) afforded newly developed CCG-IP photocatalyst for this research endeavor. The current work represents a new benchmark for carrying out highly selective methanol formation from CO2 in an environmentally benign manner.

  3. Comparison of immune responses to a killed bivalent whole cell oral cholera vaccine between endemic and less endemic settings.

    PubMed

    Desai, Sachin N; Akalu, Zenebe; Teferi, Mekonnen; Manna, Byomkesh; Teshome, Samuel; Park, Ju Yeon; Yang, Jae Seung; Kim, Deok Ryun; Kanungo, Suman; Digilio, Laura

    2016-02-01

    Studies on safety, immunogenicity and efficacy of the killed, bivalent whole cell oral cholera vaccine (Shanchol) have been conducted in historically endemic settings of Asia. Recent cholera vaccination campaigns in Haiti and Guinea have also demonstrated favourable immunogenicity and effectiveness in nonendemic outbreak settings. We performed a secondary analysis, comparing immune responses of Shanchol from two randomised controlled trials performed in an endemic and a less endemic area (Addis Ababa) during a nonoutbreak setting. While Shanchol may offer some degree of immediate protection in primed populations living in cholera endemic areas, as well as being highly immunogenic in less endemic settings, understanding the characteristics of immune responses in each of these areas is vital in determining ideal dosing strategies that offer the greatest public health impact to populations from areas with varying degrees of cholera endemicity.

  4. Long-term human serum antibody responses after immunization with whole-cell pertussis vaccine in France.

    PubMed Central

    Grimprel, E; Bégué, P; Anjak, I; Njamkepo, E; François, P; Guiso, N

    1996-01-01

    Three hundred sixty children were tested for pertussis serology 0.5 to 1.58 months after complete whole-cell pertussis vaccination. An immunoblot assay was used to detect serum antibodies to pertussis toxin, filamentous hemagglutinin, adenylate cyclase-hemolysin, and pertactin, and agglutination was used for detection of anti-agglutinogen antibodies. Antibodies against pertussis toxin, pertactin, and agglutinogens decreased rapidly after vaccination but increased secondarily, suggesting exposure to infected persons. In contrast, anti-filamentous hemagglutinin antibodies persisted and anti-adenylate cyclase-hemolysin antibodies increased continuously, suggesting either cross-reaction with non-Bordetella antigens or exposure to Bordetella isolates expressing these two antigens, including Bordetella pertussis. These data suggest that unrecognized pertussis is common in France despite massive and sustained immunization in infants and that vaccinated children become susceptible to infection more than 6 years after their last vaccination. PMID:8770511

  5. The cell mediated and humoral immune response to vaccination with acellular and whole cell pertussis vaccine in adult humans.

    PubMed

    Petersen, J W; Ibsen, P H; Bentzon, M W; Capiau, C; Heron, I

    1991-10-01

    The cell mediated immune response (CMI) against pertussis antigens following vaccination with the traditional Danish whole cell pertussis vaccine (WC-P) and the Japanese acellular pertussis vaccine (A-PV) JNIH-3 was studied in four adult human volunteers. Vaccination with the A-PV induced an in vitro proliferative response of peripheral blood lymphocytes to pertussis toxin (PT) subunits S2-S4, S3-S4 and S5 and the filamentous hemagglutinin (FHA), and a better serological response to native PT, detoxified PT (dPT) and FHA than the WC-PV. The induced CMI and serological response were followed over a period of 17 weeks, and were not seen to decline during this period. Further, an in vitro proliferative response to Bordetella pertussis agglutinogen 2 and 3 were demonstrated using lymphocytes from recently and not-so-recently pertussis-vaccinated adults. PMID:1797049

  6. Global whole-cell FTICR mass spectrometric proteomics analysis of the heat shock response in the radioresistant bacterium Deinococcus radiodurans

    SciTech Connect

    Schmid, Amy K.; Lipton, Mary S.; Mottaz, Heather M.; Monroe, Matthew E.; Smith, Richard D.; Lidstrom, Mary E.

    2005-05-01

    Despite intense interest in the response to radiation in D. radiodurans, little is known about how the organism responds to other stress factors. Our previous studies indicated that D. radiodurans mounts a regulated protective response to heat shock, and that expression of the groESL and dnaKJ operons are induced in response to elevated temperature. In order to gain greater insight into the heat shock response of D. radiodurans on a more global scale, we undertook the study reported here. Using whole-cell semiquantitative mass spectrometric proteomics integrated with global transcriptome microarray analyses, we have determined a core set of highly induced heat shock genes whose expression correlates well at the transcriptional and translational levels. In addition, we observed that the higher the absolute expression of a given gene at physiological conditions, the better the quantitative correlation between RNA and protein expression levels.

  7. Pseudomonas fluorescens HK44: Lessons Learned from a Model Whole-Cell Bioreporter with a Broad Application History

    PubMed Central

    Trögl, Josef; Chauhan, Archana; Ripp, Steven; Layton, Alice C.; Kuncová, Gabriela; Sayler, Gary S.

    2012-01-01

    Initially described in 1990, Pseudomonas fluorescens HK44 served as the first whole-cell bioreporter genetically endowed with a bioluminescent (luxCDABE) phenotype directly linked to a catabolic (naphthalene degradative) pathway. HK44 was the first genetically engineered microorganism to be released in the field to monitor bioremediation potential. Subsequent to that release, strain HK44 had been introduced into other solids (soils, sands), liquid (water, wastewater), and volatile environments. In these matrices, it has functioned as one of the best characterized chemically-responsive environmental bioreporters and as a model organism for understanding bacterial colonization and transport, cell immobilization strategies, and the kinetics of cellular bioluminescent emission. This review summarizes the characteristics of P. fluorescens HK44 and the extensive range of its applications with special focus on the monitoring of bioremediation processes and biosensing of environmental pollution. PMID:22438725

  8. Characterization of Clostridia by Gas Chromatography: Differentiation of Species by Trimethylsilyl Derivatives of Whole-Cell Hydrolysates

    PubMed Central

    Farshy, David C.; Moss, C. Wayne

    1970-01-01

    Trimethylsilyl (TMS) derivatives prepared from whole-cell hydrolysates of 36 strains, representing 10 species of Clostridium were examined by gas-liquid chromatography (GLC). The TMS profile of each species contained a group of peaks which characterized the species. Variation among strains within a species was much lower than variation between species. Some of the closely related clostridia could be differentiated by comparing their TMS profiles. Strains of Clostridium botulinum were distinguished from C. sporogenes on the basis of the ratio of two GLC peaks which corresponded to arabinose and glucose. A peak with a retention time identical to that of mannose was present in all C. bifermentans strains but was absent in those of C. sordellii. PMID:4318575

  9. Transesterification of used edible and non-edible oils to alkyl esters by Aspergillus sp. as a whole cell catalyst.

    PubMed

    Prakash, Ranjana; Aulakh, Satnam S

    2011-12-01

    Aspergillus sp. (MTCC 5436), isolated from contaminated clarified butter was used as a whole cell catalyst for transesterification of oils from different sources. The strain was observed to be tolerant and grow in 90% oil as carbon source. Oils of Jathropa, karanj and spent cottonseed were used as carbon sources in the study. The product, alkyl ester, was characterized and quantified using (1) H-NMR. The strain was observed to facilitate transesterification in an oil:minimal medium with the ratio of 70:30 resulting in a 98% conversion of oil to ethyl esters within 48 h at 28 °C and 120 rpm. The physico-chemical characteristics of the ethyl ester (>98%) at 70% oil as carbon source were similar to the standards specified for biodiesel as per standards of American Society for Testing and Materials (ASTM) and Bureau of Indian Standards (BIS), India.

  10. Characterization of Aeromonas strains isolated from Indian foods using rpoD gene sequencing and whole cell protein analysis.

    PubMed

    Nagar, Vandan; Shashidhar, Ravindranath; Bandekar, Jayant R

    2013-04-01

    Aeromonas are responsible for causing gastroenteritis and extra-intestinal infections in humans. Twenty-two Aeromonas strains isolated from different food sources were re-identified up to species level using rpoD gene sequence analysis. Biochemical tests and 16S rRNA gene sequencing were insufficient to identify Aeromonas till species level. However, incorporation of additional biochemical tests lead to correct identification of 95.5 % strains up to species level. The 16S rRNA gene sequencing was useful to identify Aeromonas isolates at the genus level only. Sequences of the rpoD gene showed greater discriminatory power than 16S rRNA gene and provided conclusive discrimination of the strains for which the phenotypic species identification was uncertain. All these 22 strains were accurately identified up to species level by rpoD gene as A. salmonicida (6), A. veronii bv. veronii (4), A. caviae (3), A. hydrophila (2), A. veronii bv. sobria (2), A. jandaei (1), A. trota (1), A. sobria (1), A. allosaccharophila (1) and A. bivalvium (1). All these strains were also characterized using whole cell protein (WCP) analysis by gradient SDS-PAGE and showed different whole cell protein (WCP) profile [22-28 polypeptide bands (~10 to >97 kDa)], indicating high genetic diversity. The present work emphasizes the use of molecular methods such as rpoD gene sequencing along with comprehensive biochemical tests for the rapid and accurate identification of Aeromonas isolates till species level. The WCP profile can be subsequently used to characterize Aeromonas isolates below species level.

  11. Pneumococcal whole-cell vaccine: optimization of cell growth of unencapsulated Streptococcus pneumoniae in bioreactor using animal-free medium.

    PubMed

    Liberman, C; Takagi, M; Cabrera-Crespo, J; Sbrogio-Almeida, M E; Dias, W O; Leite, L C C; Gonçalves, V M

    2008-11-01

    The high cost of the available pneumococcal conjugated vaccines has been an obstacle in implementing vaccination programs for children in developing countries. As an alternative, Malley et al. proposed a vaccine consisting of inactivated whole-cells of unencapsulated S. pneumoniae, which provides serotype-independent protection and involves lower production costs. Although the pneumococcus has been extensively studied, little research has focused on its large-scale culture, thus implying a lack of knowledge of process parameters, which in turn are essential for its successful industrial production. The strain Rx1Al- eryR was originally cultured in Todd-Hewitt medium (THY), which is normally used for pneumococcus isolation, but is unsuitable for human vaccine preparations. The purposes of this study were to compare the strains Rx1Al- eryR and kanR, develop a new medium, and generate new data parameters for scaling-up the process. In static flasks, cell densities were higher for eryR than kanR. In contrast, the optical density (OD) of the former decreased immediately after reaching the stationary phase, and the OD of the latter remained stable. The strain Rx1Al- kanR was cultivated in bioreactors with medium based on either acid-hydrolyzed casein (AHC) or enzymatically hydrolyzed soybean meal (EHS). Biomass production in EHS was 2.5 times higher than in AHC, and about ten times higher than in THY. The process developed for growing the strain Rx1Al- kanR in pH-controlled bioreactors was shown to be satisfactory to this fastidious bacterium. The new culture conditions using this animal-free medium may allow the production of the pneumococcal whole-cell vaccine.

  12. Stability enhancement of an atomic force microscope for long-term force measurement including cantilever modification for whole cell deformation.

    PubMed

    Weafer, P P; McGarry, J P; van Es, M H; Kilpatrick, J I; Ronan, W; Nolan, D R; Jarvis, S P

    2012-09-01

    Atomic force microscopy (AFM) is widely used in the study of both morphology and mechanical properties of living cells under physiologically relevant conditions. However, quantitative experiments on timescales of minutes to hours are generally limited by thermal drift in the instrument, particularly in the vertical (z) direction. In addition, we demonstrate the necessity to remove all air-liquid interfaces within the system for measurements in liquid environments, which may otherwise result in perturbations in the measured deflection. These effects severely limit the use of AFM as a practical tool for the study of long-term cell behavior, where precise knowledge of the tip-sample distance is a crucial requirement. Here we present a readily implementable, cost effective method of minimizing z-drift and liquid instabilities by utilizing active temperature control combined with a customized fluid cell system. Long-term whole cell mechanical measurements were performed using this stabilized AFM by attaching a large sphere to a cantilever in order to approximate a parallel plate system. An extensive examination of the effects of sphere attachment on AFM data is presented. Profiling of cantilever bending during substrate indentation revealed that the optical lever assumption of free ended cantilevering is inappropriate when sphere constraining occurs, which applies an additional torque to the cantilevers "free" end. Here we present the steps required to accurately determine force-indentation measurements for such a scenario. Combining these readily implementable modifications, we demonstrate the ability to investigate long-term whole cell mechanics by performing strain controlled cyclic deformation of single osteoblasts.

  13. Whole-Cell Electrical Activity Under Direct Mechanical Stimulus by AFM Cantilever Using Planar Patch Clamp Chip Approach.

    PubMed

    Upadhye, Kalpesh V; Candiello, Joseph E; Davidson, Lance A; Lin, Hai

    2011-06-01

    Patch clamp is a powerful tool for studying the properties of ion-channels and cellular membrane. In recent years, planar patch clamp chips have been fabricated from various materials including glass, quartz, silicon, silicon nitride, polydimethyl-siloxane (PDMS), and silicon dioxide. Planar patch clamps have made automation of patch clamp recordings possible. However, most planar patch clamp chips have limitations when used in combination with other techniques. Furthermore, the fabrication methods used are often expensive and require specialized equipments. An improved design as well as fabrication and characterization of a silicon-based planar patch clamp chip are described in this report. Fabrication involves true batch fabrication processes that can be performed in most common microfabrication facilities using well established MEMS techniques. Our planar patch clamp chips can form giga-ohm seals with the cell plasma membrane with success rate comparable to existing patch clamp techniques. The chip permits whole-cell voltage clamp recordings on variety of cell types including Chinese Hamster Ovary (CHO) cells and pheochromocytoma (PC12) cells, for times longer than most available patch clamp chips. When combined with a custom microfluidics chamber, we demonstrate that it is possible to perfuse the extra-cellular as well as intra-cellular buffers. The chamber design allows integration of planar patch clamp with atomic force microscope (AFM). Using our planar patch clamp chip and microfluidics chamber, we have recorded whole-cell mechanosensitive (MS) currents produced by directly stimulating human keratinocyte (HaCaT) cells using an AFM cantilever. Our results reveal the spatial distribution of MS ion channels and temporal details of the responses from MS channels. The results show that planar patch clamp chips have great potential for multi-parametric high throughput studies of ion channel proteins. PMID:22174731

  14. Knockdown of ASIC1 and epithelial sodium channel subunits inhibits glioblastoma whole cell current and cell migration.

    PubMed

    Kapoor, Niren; Bartoszewski, Rafal; Qadri, Yawar J; Bebok, Zsuzsanna; Bubien, James K; Fuller, Catherine M; Benos, Dale J

    2009-09-01

    High grade gliomas such as glioblastoma multiforme express multiple members of the epithelial sodium channel (ENaC)/Degenerin family, characteristically displaying a basally active amiloride-sensitive cation current not seen in normal human astrocytes or lower grade gliomas. Using quantitative real time PCR, we have shown higher expression of ASIC1, alphaENaC, and gammaENaC in D54-MG human glioblastoma multiforme cells compared with primary human astrocytes. We hypothesize that this glioma current is mediated by a hybrid channel composed of a mixture of ENaC and acid-sensing ion channel (ASIC) subunits. To test this hypothesis we made dominant negative cDNAs for ASIC1, alphaENaC, gammaENaC, and deltaENaC. D54-MG cells transfected with the dominant negative constructs for ASIC1, alphaENaC, or gammaENaC showed reduced protein expression and a significant reduction in the amiloride-sensitive whole cell current as compared with untransfected D54-MG cells. Knocking down alphaENaC or gammaENaC also abolished the high P(K)(+)/P(Na)(+) of D54-MG cells. Knocking down deltaENaC in D54-MG cells reduced deltaENaC protein expression but had no effect on either the whole cell current or K(+) permeability. Using co-immunoprecipitation we show interactions between ASIC1, alphaENaC, and gammaENaC, consistent with these subunits interacting with each other to form an ion channel in glioma cells. We also found a significant inhibition of D54-MG cell migration after ASIC1, alphaENaC, or gammaENaC knockdown, consistent with the hypothesis that ENaC/Degenerin subunits play an important role in glioma cell biology. PMID:19561078

  15. An Inactivated Antibiotic-Exposed Whole-Cell Vaccine Enhances Bactericidal Activities Against Multidrug-Resistant Acinetobacter baumannii

    PubMed Central

    Shu, Meng-Hooi; MatRahim, NorAziyah; NorAmdan, NurAsyura; Pang, Sui-Ping; Hashim, Sharina H.; Phoon, Wai-Hong; AbuBakar, Sazaly

    2016-01-01

    Vaccination may be an alternative treatment for infection with multidrug-resistance (MDR) Acinetobacter baumannii. The study reported here evaluated the bactericidal antibody responses following immunization of mice using an inactivated whole-cell vaccine derived from antibiotic-exposed MDR A. baumannii (I-M28-47-114). Mice inoculated with I-M28-47 (non-antibiotic-exposed control) and I-M28-47-114 showed a high IgG antibody response by day 5 post-inoculation. Sera from mice inoculated with I-M28-47-114 collected on day 30 resulted in 80.7 ± 12.0% complement-mediated bacteriolysis in vitro of the test MDR A. baumannii treated with imipenem, which was a higher level of bacteriolysis over sera from mice inoculated with I-M28-47. Macrophage-like U937 cells eliminated 49.3 ± 11.6% of the test MDR A. baumannii treated with imipenem when opsonized with sera from mice inoculated with I-M28-47-114, which was a higher level of elimination than observed for test MDR A. baumannii opsonized with sera from mice inoculated with I-M28-47. These results suggest that vaccination with I-M28-47-114 stimulated antibody responses capable of mounting high bactericidal killing of MDR A. baumannii. Therefore, the inactivated antibiotic-exposed whole-cell vaccine (I-M28-47-114) has potential for development as a candidate vaccine for broad clearance and protection against MDR A. baumannii infections. PMID:26923424

  16. Whole-cell hydroxylation of n-octane by Escherichia coli strains expressing the CYP153A6 operon.

    PubMed

    Gudiminchi, Rama Krishna; Randall, Charlene; Opperman, Diederik J; Olaofe, Oluwafemi A; Harrison, Susan T L; Albertyn, Jacobus; Smit, Martha S

    2012-12-01

    CYP153A6 is a well-studied terminal alkane hydroxylase which has previously been expressed in Pseudomonas putida and Escherichia coli by using the pCom8 plasmid. In this study, CYP153A6 was successfully expressed in E. coli BL21(DE3) by cloning the complete operon from Mycobacterium sp. HXN-1500, also encoding the ferredoxin reductase and ferredoxin, into pET28b(+). LB medium with IPTG as well as auto-induction medium was used to express the proteins under the T7 promoter. A maximum concentration of 1.85 μM of active CYP153A6 was obtained when using auto-induction medium, while with IPTG induction of LB cultures, the P450 concentration peaked at 0.6-0.8 μM. Since more biomass was produced in auto-induction medium, the specific P450 content was often almost the same, 0.5-1.0 μmol P450 g (DCW)⁻¹, for both methods. Analytical scale whole-cell biotransformations of n-octane were conducted with resting cells, and it was found that high P450 content in biomass did not necessarily result in high octanol production. Whole cells from LB cultures induced with IPTG gave higher specific and volumetric octanol formation rates than biomass from auto-induction medium. A maximum of 8.7 g octanol L (BRM)⁻¹ was obtained within 24 h (0.34 g L (BRM)⁻¹  h⁻¹) with IPTG-induced cells containing only 0.20 μmol P450 g (DCW)⁻¹, when glucose (22 g L (BRM)⁻¹) was added for cofactor regeneration.

  17. Acute reversal of phospholamban inhibition facilitates the rhythmic whole-cell propagating calcium waves in isolated ventricular myocytes.

    PubMed

    Chan, Yi-Hsin; Tsai, Wei-Chung; Song, Zhen; Ko, Christopher Y; Qu, Zhilin; Weiss, James N; Lin, Shien-Fong; Chen, Peng-Sheng; Jones, Larry R; Chen, Zhenhui

    2015-03-01

    Phospholamban (PLB) inhibits the activity of cardiac sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a). Phosphorylation of PLB during sympathetic activation reverses SERCA2a inhibition, increasing SR Ca(2+) uptake. However, sympathetic activation also modulates multiple other intracellular targets in ventricular myocytes (VMs), making it impossible to determine the specific effects of the reversal of PLB inhibition on the spontaneous SR Ca(2+) release. Therefore, it remains unclear how PLB regulates rhythmic activity in VMs. Here, we used the Fab fragment of 2D12, a monoclonal anti-PLB antibody, to test how acute reversal of PLB inhibition affects the spontaneous SR Ca(2+) release in normal VMs. Ca(2+) sparks and spontaneous Ca(2+) waves (SCWs) were recorded in the line-scan mode of confocal microscopy using the Ca(2+) fluorescent dye Fluo-4 in isolated permeabilized mouse VMs. Fab, which reverses PLB inhibition, significantly increased the frequency, amplitude, and spatial/temporal spread of Ca(2+) sparks in VMs exposed to 50 nM free [Ca(2+)]. At physiological diastolic free [Ca(2+)] (100-200 nM), Fab facilitated the formation of whole-cell propagating SCWs. At higher free [Ca(2+)], Fab increased the frequency and velocity, but decreased the decay time of the SCWs. cAMP had little additional effect on the frequency or morphology of Ca(2+) sparks or SCWs after Fab addition. These findings were complemented by computer simulations. In conclusion, acute reversal of PLB inhibition alone significantly increased the spontaneous SR Ca(2+) release, leading to the facilitation and organization of whole-cell propagating SCWs in normal VMs. PLB thus plays a key role in subcellular Ca(2+) dynamics and rhythmic activity of VMs.

  18. Formulation and characterization of an immobilized laccase biocatalyst and its application to eliminate organic micropollutants in wastewater.

    PubMed

    Nair, Rakesh R; Demarche, Philippe; Agathos, Spiros N

    2013-09-25

    Over the past decades, water pollution by trace organic compounds (ng L(-1)) has become one of the key environmental issues for developed countries. To date there is no effective and sustainable remediation strategy available. Laccases from white rot fungi were found particularly attractive for the removal of some micropollutants such as the plasticizer bisphenol A (BPA), the anti-inflammatory drug diclofenac (DF) and the steroidal hormone 17-α-ethinylestradiol (EE2). Laccase immobilization is a prerequisite for their use in continuous water treatment processes. In this study, laccase from Coriolopsis gallica was immobilized on mesoporous silica spheres in a two-step adsorption-crosslinking process. The initial laccase activity, crosslinker (glutaraldehyde) concentration and extra protein (albumin) concentration were varied following a central composite experimental design and optimized with respect to the immobilization yield, activity and thermal stability of the biocatalysts. After a multi-objective optimization of the biocatalyst formulation, a maximum biocatalyst activity of 383 Ug(-1), determined with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) at pH 4.5, was obtained. Biocatalyst particles were physically characterized by means of scanning electron microscopy, Brunauer-Emmett-Teller surface area and Barrett-Joyner-Halenda pore size analyses revealing few modifications of the surface area and structure during/after the immobilization procedure. The biocatalyst showed a significantly higher thermostability than the free enzyme with a half-life of 31.5 hours and 3.9 hours compared to 6.1 hours and 0.6 hours at 55°C and 75°C respectively. The biocatalyst was able to eliminate in a continuously stirred membrane reactor more than 95% of BPA 10 μM and EE2 10 μM and 70% of DF 10 μM when treated individually and more than 90% when treated as a mixture in aqueous buffered solution (pH 5) for more than 60 reactor volumes. In real wastewater conditions (pH 7

  19. Formulation and characterization of an immobilized laccase biocatalyst and its application to eliminate organic micropollutants in wastewater.

    PubMed

    Nair, Rakesh R; Demarche, Philippe; Agathos, Spiros N

    2013-09-25

    Over the past decades, water pollution by trace organic compounds (ng L(-1)) has become one of the key environmental issues for developed countries. To date there is no effective and sustainable remediation strategy available. Laccases from white rot fungi were found particularly attractive for the removal of some micropollutants such as the plasticizer bisphenol A (BPA), the anti-inflammatory drug diclofenac (DF) and the steroidal hormone 17-α-ethinylestradiol (EE2). Laccase immobilization is a prerequisite for their use in continuous water treatment processes. In this study, laccase from Coriolopsis gallica was immobilized on mesoporous silica spheres in a two-step adsorption-crosslinking process. The initial laccase activity, crosslinker (glutaraldehyde) concentration and extra protein (albumin) concentration were varied following a central composite experimental design and optimized with respect to the immobilization yield, activity and thermal stability of the biocatalysts. After a multi-objective optimization of the biocatalyst formulation, a maximum biocatalyst activity of 383 Ug(-1), determined with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) at pH 4.5, was obtained. Biocatalyst particles were physically characterized by means of scanning electron microscopy, Brunauer-Emmett-Teller surface area and Barrett-Joyner-Halenda pore size analyses revealing few modifications of the surface area and structure during/after the immobilization procedure. The biocatalyst showed a significantly higher thermostability than the free enzyme with a half-life of 31.5 hours and 3.9 hours compared to 6.1 hours and 0.6 hours at 55°C and 75°C respectively. The biocatalyst was able to eliminate in a continuously stirred membrane reactor more than 95% of BPA 10 μM and EE2 10 μM and 70% of DF 10 μM when treated individually and more than 90% when treated as a mixture in aqueous buffered solution (pH 5) for more than 60 reactor volumes. In real wastewater conditions (pH 7

  20. Are Lipases Still Important Biocatalysts? A Study of Scientific Publications and Patents for Technological Forecasting

    PubMed Central

    Daiha, Karina de Godoy; Angeli, Renata; de Oliveira, Sabrina Dias; Almeida, Rodrigo Volcan

    2015-01-01

    The great potential of lipases is known since 1930 when the work of J. B. S. Haldane was published. After eighty-five years of studies and developments, are lipases still important biocatalysts? For answering this question the present work investigated the technological development of four important industrial sectors where lipases are applied: production of detergent formulations; organic synthesis, focusing on kinetic resolution, production of biodiesel, and production of food and feed products. The analysis was made based on research publications and patent applications, working as scientific and technological indicators, respectively. Their evolution, interaction, the major players of each sector and the main subject matters disclosed in patent documents were discussed. Applying the concept of technology life cycle, S-curves were built by plotting cumulative patent data over time to monitor the attractiveness of each technology for investment. The results lead to a conclusion that the use of lipases as biocatalysts is still a relevant topic for the industrial sector, but developments are still needed for lipase biocatalysis to reach its full potential, which are expected to be achieved within the third, and present, wave of biocatalysis. PMID:26111144

  1. Improvement of Biocatalysts for Industrial and Environmental Purposes by Saturation Mutagenesis

    PubMed Central

    Valetti, Francesca; Gilardi, Gianfranco

    2013-01-01

    Laboratory evolution techniques are becoming increasingly widespread among protein engineers for the development of novel and designed biocatalysts. The palette of different approaches ranges from complete randomized strategies to rational and structure-guided mutagenesis, with a wide variety of costs, impacts, drawbacks and relevance to biotechnology. A technique that convincingly compromises the extremes of fully randomized vs. rational mutagenesis, with a high benefit/cost ratio, is saturation mutagenesis. Here we will present and discuss this approach in its many facets, also tackling the issue of randomization, statistical evaluation of library completeness and throughput efficiency of screening methods. Successful recent applications covering different classes of enzymes will be presented referring to the literature and to research lines pursued in our group. The focus is put on saturation mutagenesis as a tool for designing novel biocatalysts specifically relevant to production of fine chemicals for improving bulk enzymes for industry and engineering technical enzymes involved in treatment of waste, detoxification and production of clean energy from renewable sources. PMID:24970191

  2. Are Lipases Still Important Biocatalysts? A Study of Scientific Publications and Patents for Technological Forecasting.

    PubMed

    Daiha, Karina de Godoy; Angeli, Renata; de Oliveira, Sabrina Dias; Almeida, Rodrigo Volcan

    2015-01-01

    The great potential of lipases is known since 1930 when the work of J. B. S. Haldane was published. After eighty-five years of studies and developments, are lipases still important biocatalysts? For answering this question the present work investigated the technological development of four important industrial sectors where lipases are applied: production of detergent formulations; organic synthesis, focusing on kinetic resolution, production of biodiesel, and production of food and feed products. The analysis was made based on research publications and patent applications, working as scientific and technological indicators, respectively. Their evolution, interaction, the major players of each sector and the main subject matters disclosed in patent documents were discussed. Applying the concept of technology life cycle, S-curves were built by plotting cumulative patent data over time to monitor the attractiveness of each technology for investment. The results lead to a conclusion that the use of lipases as biocatalysts is still a relevant topic for the industrial sector, but developments are still needed for lipase biocatalysis to reach its full potential, which are expected to be achieved within the third, and present, wave of biocatalysis. PMID:26111144

  3. Stabilization by multipoint covalent attachment of a biocatalyst with polygalacturonase activity used for juice clarification.

    PubMed

    Ramírez Tapias, Yuly A; Rivero, Cintia W; Gallego, Fernando López; Guisán, José M; Trelles, Jorge A

    2016-10-01

    Derivatized-agarose supports are suitable for enzyme immobilization by different methods, taking advantage of different physical, chemical and biological conditions of the protein and the support. In this study, agarose particles were modified with MANAE, PEI and glyoxyl groups and evaluated to stabilize polygalacturonase from Streptomyces halstedii ATCC 10897. A new immobilized biocatalyst was developed using glyoxyl-agarose as support; it exhibited high performance in degrading polygalacturonic acid and releasing oligogalacturonides. Maximal enzyme activity was detected at 5h of reaction using 0.05g/mL of immobilized biocatalyst, which released 3mg/mL of reducing sugars and allowed the highest product yield conversion and increased stability. These results are very favorable for pectin degradation with reusability up to 18 successive reactions (90h) and application in juice clarification. Plum (4.7°Bx) and grape (10.6°Bx) juices were successfully clarified, increasing reducing sugars content and markedly decreasing turbidity and viscosity. PMID:27132847

  4. An (R)‐Imine Reductase Biocatalyst for the Asymmetric Reduction of Cyclic Imines

    PubMed Central

    Hussain, Shahed; Leipold, Friedemann; Man, Henry; Wells, Elizabeth; France, Scott P.; Mulholland, Keith R.; Grogan, Gideon

    2015-01-01

    Abstract Although the range of biocatalysts available for the synthesis of enantiomerically pure chiral amines continues to expand, few existing methods provide access to secondary amines. To address this shortcoming, we have over‐expressed the gene for an (R)‐imine reductase [(R)‐IRED] from Streptomyces sp. GF3587 in Escherichia coli to create a recombinant whole‐cell biocatalyst for the asymmetric reduction of prochiral imines. The (R)‐IRED was screened against a panel of cyclic imines and two iminium ions and was shown to possess high catalytic activity and enantioselectivity. Preparative‐scale synthesis of the alkaloid (R)‐coniine (90 % yield; 99 % ee) from the imine precursor was performed on a gram‐scale. A homology model of the enzyme active site, based on the structure of a closely related (R)‐IRED from Streptomyces kanamyceticus, was constructed and used to identify potential amino acids as targets for mutagenesis. PMID:27547270

  5. Biocatalyst Enhancement

    EPA Science Inventory

    The increasing availability of enzyme collections has assisted attempts by pharmaceutical producers to adopt green chemistry approaches to manufacturing. A joint effort between an enzyme producer and a pharmaceutical manufacturer has been enhanced over the past three years by ena...

  6. Adaptive immune response to whole cell pertussis vaccine reflects vaccine quality: A possible complementation to the Pertussis Serological Potency test.

    PubMed

    Hoonakker, M E; Verhagen, L M; van der Maas, L; Metz, B; Uittenbogaard, J P; van de Waterbeemd, B; van Els, C A C M; van Eden, W; Hendriksen, C F M; Sloots, A; Han, W G H

    2016-08-17

    Whole cell Bordetella pertussis (wP) vaccines are still used in many countries to protect against the respiratory disease pertussis. The potency of whole-cell pertussis vaccine lots is determined by an intracerebral challenge test (the Kendrick test). This test is criticized due to lack of immunological relevance of the read-out after an intracerebral challenge with B. pertussis. The alternative in vivo test, which assesses specific antibody levels in serum after wP vaccination, is the Pertussis Serological Potency test (PSPT). Although the PSPT focuses on a parameter that contributes to protection, the protective immune mechanisms after wP vaccination includes more elements than specific antibody responses only. In this study, additional parameters were investigated, i.e. circulating pro-inflammatory cytokines, antibody specificity and T helper cell responses and it was evaluated whether they can be used as complementary readout parameters in the PSPT to assess wP lot quality. By deliberate manipulation of the vaccine preparation procedure, a panel of high, intermediate and low quality wP vaccines were made. The results revealed that these vaccines induced similar IL-6 and IP10 levels in serum 4h after vaccination (innate responses) and similar antibody levels directed against the entire bacterium. In contrast, the induced antibody specificity to distinct wP antigens differed after vaccination with high, intermediate and low quality wP vaccines. In addition, the magnitude of wP-induced Th cell responses (Th17, Th1 and Th2) was reduced after vaccination with a wP vaccine of low quality. T cell responses and antibody specificity are therefore correlates of qualitative differences in the investigated vaccines, while the current parameter of the PSPT alone was not sensitive enough to distinguish between vaccines of different qualities. This study demonstrates that assessment of the magnitude of Th cell responses and the antigen specificity of antibodies induced by w

  7. Adaptive immune response to whole cell pertussis vaccine reflects vaccine quality: A possible complementation to the Pertussis Serological Potency test.

    PubMed

    Hoonakker, M E; Verhagen, L M; van der Maas, L; Metz, B; Uittenbogaard, J P; van de Waterbeemd, B; van Els, C A C M; van Eden, W; Hendriksen, C F M; Sloots, A; Han, W G H

    2016-08-17

    Whole cell Bordetella pertussis (wP) vaccines are still used in many countries to protect against the respiratory disease pertussis. The potency of whole-cell pertussis vaccine lots is determined by an intracerebral challenge test (the Kendrick test). This test is criticized due to lack of immunological relevance of the read-out after an intracerebral challenge with B. pertussis. The alternative in vivo test, which assesses specific antibody levels in serum after wP vaccination, is the Pertussis Serological Potency test (PSPT). Although the PSPT focuses on a parameter that contributes to protection, the protective immune mechanisms after wP vaccination includes more elements than specific antibody responses only. In this study, additional parameters were investigated, i.e. circulating pro-inflammatory cytokines, antibody specificity and T helper cell responses and it was evaluated whether they can be used as complementary readout parameters in the PSPT to assess wP lot quality. By deliberate manipulation of the vaccine preparation procedure, a panel of high, intermediate and low quality wP vaccines were made. The results revealed that these vaccines induced similar IL-6 and IP10 levels in serum 4h after vaccination (innate responses) and similar antibody levels directed against the entire bacterium. In contrast, the induced antibody specificity to distinct wP antigens differed after vaccination with high, intermediate and low quality wP vaccines. In addition, the magnitude of wP-induced Th cell responses (Th17, Th1 and Th2) was reduced after vaccination with a wP vaccine of low quality. T cell responses and antibody specificity are therefore correlates of qualitative differences in the investigated vaccines, while the current parameter of the PSPT alone was not sensitive enough to distinguish between vaccines of different qualities. This study demonstrates that assessment of the magnitude of Th cell responses and the antigen specificity of antibodies induced by w

  8. A Temporal Examination of the Planktonic and Biofilm Proteome of Whole Cell Pseudomonas aeruginosa PAO1 Using Quantitative Mass Spectrometry*

    PubMed Central

    Park, Amber J.; Murphy, Kathleen; Krieger, Jonathan R.; Brewer, Dyanne; Taylor, Paul; Habash, Marc; Khursigara, Cezar M.

    2014-01-01

    Chronic polymicrobial lung infections are the chief complication in patients with cystic fibrosis. The dominant pathogen in late-stage disease is Pseudomonas aeruginosa, which forms recalcitrant, structured communities known as biofilms. Many aspects of biofilm biology are poorly understood; consequently, effective treatment of these infections is limited, and cystic fibrosis remains fatal. Here we combined in-solution protein digestion of triplicate growth-matched samples with a high-performance mass spectrometry platform to provide the most comprehensive proteomic dataset known to date for whole cell P. aeruginosa PAO1 grown in biofilm cultures. Our analysis included protein–protein interaction networks and PseudoCAP functional information for unique and significantly modulated proteins at three different time points. Secondary analysis of a subgroup of proteins using extracted ion currents validated the spectral counting data of 1884 high-confidence proteins. In this paper we demonstrate a greater representation of proteins related to metabolism, DNA stability, and molecular activity in planktonically grown P. aeruginosa PAO1. In addition, several virulence-related proteins were increased during planktonic growth, including multiple proteins encoded by the pyoverdine locus, uncharacterized proteins with sequence similarity to mammalian cell entry protein, and a member of the hemagglutinin family of adhesins, HecA. Conversely, biofilm samples contained an uncharacterized protein with sequence similarity to an adhesion protein with self-association characteristics (AidA). Increased levels of several phenazine biosynthetic proteins, an uncharacterized protein with sequence similarity to a metallo-beta-lactamase, and lower levels of the drug target gyrA support the putative characteristics of in situ P. aeruginosa infections, including competitive fitness and antibiotic resistance. This quantitative whole cell approach advances the existing P. aeruginosa

  9. Entomology: A Bee Farming a Fungus.

    PubMed

    Oldroyd, Benjamin P; Aanen, Duur K

    2015-11-16

    Farming is done not only by humans, but also by some ant, beetle and termite species. With the discovery of a stingless bee farming a fungus that provides benefits to its larvae, bees can be added to this list.

  10. Plant lipases: biocatalyst aqueous environment in relation to optimal catalytic activity in lipase-catalyzed synthesis reactions.

    PubMed

    Caro, Yanis; Pina, Michel; Turon, Fabrice; Guilbert, Stephane; Mougeot, Estelle; Fetsch, David V; Attwool, Philip; Graille, Jean

    2002-03-20

    Adsorption and desorption isotherms of two commercial enzyme preparations of papain and bromelain were determined with a Dynamic Vapor System. The Guggenheim-Anderson-deBoer (GAB) modeling of the obtained sorption isotherms allowed the definition of different levels of hydration of those samples. Afterward, these enzyme preparations were used as biocatalysts in water and solvent-free esterification and alcoholysis reactions. The evolution of the obtained fatty acid ester level as a function of the initial hydration level of the biocatalyst, i.e., thermodynamic water activity (a(w)) and water content, was studied. The results show an important correlation between the initial hydration level of the biocatalyst and its catalytic activity during the lipase-catalyzed synthesis reactions. Thus, the Carica papaya lipase (crude papain preparation) catalytic activity is highly dependent on the biocatalyst hydration state. The optimized synthesis reaction yield is obtained when the a(w) value of the enzyme preparation is stabilized at 0.22, which corresponds to 2% water content. This optimal level of hydration occurs on the linear part of the biocatalyst's sorption isotherm, where the water molecules can form a mono- or multiple layer with the protein network. The synthesis reaction yield decreases when the a(w) of the preparation is higher than 0.22, because the excess water molecules modify the system equilibrium leading to the reverse and competitive reaction, i.e., hydrolysis. These results show also that an optimal storage condition for the highly hydrophilic crude papain preparation is a relative humidity strictly lower than 70% to avoid an irreversible structural transition leading to a useless biocatalyst. Concerning the bromelain preparation, no effect of the hydration level on the catalytic activity during esterification reactions was observed. This biocatalyst has too weak a catalytic activity which makes it difficult to observe any differences. Furthermore, the

  11. Whole Cell Formaldehyde Cross-Linking Simplifies Purification of Mitochondrial Nucleoids and Associated Proteins Involved in Mitochondrial Gene Expression

    PubMed Central

    Rajala, Nina; Hensen, Fenna; Wessels, Hans J. C. T.; Ives, Daniel; Gloerich, Jolein; Spelbrink, Johannes N.

    2015-01-01

    Mitochondrial DNA/protein complexes (nucleoids) appear as discrete entities inside the mitochondrial network when observed by live-cell imaging and immunofluorescence. This somewhat trivial observation in recent years has spurred research towards isolation of these complexes and the identification of nucleoid-associated proteins. Here we show that whole cell formaldehyde crosslinking combined with affinity purification and tandem mass-spectrometry provides a simple and reproducible method to identify potential nucleoid associated proteins. The method avoids spurious mitochondrial isolation and subsequent multifarious nucleoid enrichment protocols and can be implemented to allow for label-free quantification (LFQ) by mass-spectrometry. Using expression of a Flag-tagged Twinkle helicase and appropriate controls we show that this method identifies many previously identified nucleoid associated proteins. Using LFQ to compare HEK293 cells with and without mtDNA, but both expressing Twinkle-FLAG, identifies many proteins that are reduced or absent in the absence of mtDNA. This set not only includes established mtDNA maintenance proteins but also many proteins involved in mitochondrial RNA metabolism and translation and therefore represents what can be considered an mtDNA gene expression proteome. Our data provides a very valuable resource for both basic mitochondrial researchers as well as clinical geneticists working to identify novel disease genes on the basis of exome sequence data. PMID:25695250

  12. Whole-cell biosensor for label-free detection of GPCR-mediated drug responses in personal cell lines.

    PubMed

    Hillger, Julia M; Schoop, Jeffison; Boomsma, Dorret I; Slagboom, P Eline; IJzerman, Adriaan P; Heitman, Laura H

    2015-12-15

    Deciphering how genetic variation in drug targets such as G protein-coupled receptors (GPCRs) affects drug response is essential for precision medicine. GPCR signaling is traditionally investigated in artificial cell lines which do not provide sufficient physiological context. Patient-derived cell lines such as lymphoblastoid cell lines (LCLs) could represent the ideal cellular model system. Here we describe a novel label-free, whole-cell biosensor method for characterizing GPCR-mediated drug responses in LCLs. Generally, such biosensor technology is deemed only compatible with adherent cell lines. We optimized and applied the methodology to study cellular adhesion properties as well as GPCR drug responses in LCLs, which are suspension cells. Coating the detector surface with the extracellular matrix protein fibronectin resulted in cell adherence and allowed detection of cellular responses. A prototypical GPCR present on these cells, i.e. the cannabinoid receptor 2 (CB2), was selected for pharmacological characterization. Receptor activation with the agonist JWH133, blockade by antagonist AM630 as well as downstream signaling inhibition by PTX could be monitored sensitively and receptor-specifically. Potencies and effects were comparable between LCLs of two genetically unrelated individuals, providing the proof-of-principle that this biosensor technology can be applied to LCLs, despite their suspension cell nature, in order to serve as an in vitro model system for the evaluation of individual genetic influences on GPCR-mediated drug responses.

  13. Correlative Fluorescence Microscopy and Scanning Transmission Electron Microscopy of Quantum Dot Labeled Proteins in Whole Cells in Liquid

    PubMed Central

    Dukes, Madeline J.; Peckys, Diana B.; de Jonge, Niels

    2010-01-01

    Correlative fluorescence microscopy and transmission electron microscopy (TEM) is a state-of-the-art microscopy methodology to study cellular function, combining the functionality of light microscopy with the high resolution of electron microscopy. However, this technique involves complex sample preparation procedures due to its need for either thin sections or frozen samples for TEM imaging. Here, we introduce a novel correlative approach capable of imaging whole eukaryotic cells in liquid with fluorescence microscopy and with scanning transmission electron microscopy (STEM); there is no additional sample preparation necessary for the electron microscopy. Quantum dots (QDs) were bound to epidermal growth factor (EGF) receptors of COS7 fibroblast cells. Fixed whole cells in saline water were imaged with fluorescence microscopy and subsequently with STEM. The STEM images were correlated with fluorescence images of the same cellular regions. QDs of dimensions 7 × 12 nm were visible in a 5 μm thick layer of saline water, consistent with calculations. A spatial resolution of 3 nm was achieved on the QDs. PMID:20550177

  14. Dithiocarbamate fungicide thiram detection: comparison of bioluminescent and fluorescent whole-cell bioassays based on hsp22 stress promoter induction.

    PubMed

    Mandon, C A; Diaz-Latoud, C; Arrigo, A-P; Blum, L J

    2006-07-13

    Detection of toxic substances interfering with endocrine system is one of the major preoccupations of the European community. A whole-cell bioassay for pollution detection based on stress induction has been designed. Well characterized toxicants, cadmium chloride and thiram (a dithiocarbamate fungicide), were used to optimize the detection conditions such as time-course conditions, cell line and reporter gene to be used. HeLa cells containing the firefly luciferase (luc) reporter gene under the control of the Drosophila melanogaster hsp22 promoter were compared to liver cells (HepG2) containing the same stress gene promoter fused either to the luc or the EGFP (Enhanced-Green Fluorescent Protein) gene. The sensitivity of the obtained bioassay was found to be enhanced by the concomitant use of liver cells and EGFP reporter gene. The detection limits of the toxicants were then lowered from 1 to 0.1 microM and from 1 to 0.01 microM for CdCl(2) and thiram, respectively.

  15. Biodegradation of methyl parathion by whole cells of marine-derived fungi Aspergillus sydowii and Penicillium decaturense.

    PubMed

    Alvarenga, Natália; Birolli, Willian G; Seleghim, Mirna H R; Porto, André L M

    2014-12-01

    Seven marine fungi strains (Aspergillus sydowii CBMAI 934, A. sydowii CBMAI 935, A. sydowii CBMAI 1241, Penicillium decaturense CBMAI 1234, Penicillium raistrickii CBMAI 931, P. raistrickii CBMAI 1235, and Trichoderma sp. CBMAI 932) were screened by their growth in the presence of methyl parathion (MP) in a solid culture medium. The strains with best growth were A. sydowii CBMAI 935 and P. decaturense CBMAI 1234. Biodegradation reactions were performed in 10, 20 and 30d in a malt extract liquid medium containing commercial MP and whole cells of A. sydowii CBMAI 935 and P. decaturense CBMAI 1234. In 20d, A. sydowii CBMAI 935 was able to degrade all pesticide, whereas P. decaturense CBMAI 1234 promoted a complete degradation in 30d. A. sydowii CBMAI 935 and P. decaturense CBMAI 1234 could degrade the product of the MP enzymatic hydrolysis, p-nitrophenol, on average of 51 and 40% respectively. Both strains used MP as a sole source of carbon and provided satisfactory results. Metabolites detected in the medium showed that the presumable reaction pathway occurred through the activation of MP to its more toxic form, methyl paraoxon, which was further degraded to p-nitrophenol.

  16. Correlative fluorescence microscopy and scanning transmission electron microscopy of quantum-dot-labeled proteins in whole cells in liquid.

    PubMed

    Dukes, Madeline J; Peckys, Diana B; de Jonge, Niels

    2010-07-27

    Correlative fluorescence microscopy and transmission electron microscopy (TEM) is a state-of-the-art microscopy methodology to study cellular function, combining the functionality of light microscopy with the high resolution of electron microscopy. However, this technique involves complex sample preparation procedures due to its need for either thin sections or frozen samples for TEM imaging. Here, we introduce a novel correlative approach capable of imaging whole eukaryotic cells in liquid with fluorescence microscopy and with scanning transmission electron microscopy (STEM); there is no additional sample preparation necessary for the electron microscopy. Quantum dots (QDs) were bound to epidermal growth factor (EGF) receptors of COS7 fibroblast cells. Fixed whole cells in saline water were imaged with fluorescence microscopy and subsequently with STEM. The STEM images were correlated with fluorescence images of the same cellular regions. QDs of dimensions 7x12 nm were visible in a 5 microm thick layer of saline water, consistent with calculations. A spatial resolution of 3 nm was achieved on the QDs.

  17. Whole-cell imaging of the budding yeast Saccharomyces cerevisiae by high-voltage scanning transmission electron tomography.

    PubMed

    Murata, Kazuyoshi; Esaki, Masatoshi; Ogura, Teru; Arai, Shigeo; Yamamoto, Yuta; Tanaka, Nobuo

    2014-11-01

    Electron tomography using a high-voltage electron microscope (HVEM) provides three-dimensional information about cellular components in sections thicker than 1 μm, although in bright-field mode image degradation caused by multiple inelastic scattering of transmitted electrons limit the attainable resolution. Scanning transmission electron microscopy (STEM) is believed to give enhanced contrast and resolution compared to conventional transmission electron microscopy (CTEM). Samples up to 1 μm in thickness have been analyzed with an intermediate-voltage electron microscope because inelastic scattering is not a critical limitation, and probe broadening can be minimized. Here, we employed STEM at 1 MeV high-voltage to extend the useful specimen thickness for electron tomography, which we demonstrate by a seamless tomographic reconstruction of a whole, budding Saccharomyces cerevisiae yeast cell, which is ~3 μm in thickness. High-voltage STEM tomography, especially in the bright-field mode, demonstrated sufficiently enhanced contrast and intensity, compared to CTEM tomography, to permit segmentation of major organelles in the whole cell. STEM imaging also reduced specimen shrinkage during tilt-series acquisition. The fidelity of structural preservation was limited by cytoplasmic extraction, and the spatial resolution was limited by the relatively large convergence angle of the scanning probe. However, the new technique has potential to solve longstanding problems of image blurring in biological specimens beyond 1 μm in thickness, and may facilitate new research in cellular structural biology.

  18. The Effect of Electron Beam Irradiation in Environmental Scanning Transmission Electron Microscopy of Whole Cells in Liquid.

    PubMed

    Hermannsdörfer, Justus; Tinnemann, Verena; Peckys, Diana B; de Jonge, Niels

    2016-06-01

    Whole cells can be studied in their native liquid environment using electron microscopy, and unique information about the locations and stoichiometry of individual membrane proteins can be obtained from many cells thus taking cell heterogeneity into account. Of key importance for the further development of this microscopy technology is knowledge about the effect of electron beam radiation on the samples under investigation. We used environmental scanning electron microscopy (ESEM) with scanning transmission electron microscopy (STEM) detection to examine the effect of radiation for whole fixed COS7 fibroblasts in liquid. The main observation was the localization of nanoparticle labels attached to epidermal growth factor receptors (EGFRs). It was found that the relative distances between the labels remained mostly unchanged (<1.5%) for electron doses ranging from the undamaged native state at 10 e-/Å2 toward 103 e-/Å2. This dose range was sufficient to determine the EGFR locations with nanometer resolution and to distinguish between monomers and dimers. Various different forms of radiation damage became visible at higher doses, including severe dislocation, and the dissolution of labels. PMID:27137077

  19. In vitro innate immune cell based models to assess whole cell Bordetella pertussis vaccine quality: a proof of principle.

    PubMed

    Hoonakker, M E; Verhagen, L M; Hendriksen, C F M; van Els, C A C M; Vandebriel, R J; Sloots, A; Han, W G H

    2015-03-01

    Lot release testing of vaccines is primarily based on animal models that are costly, time-consuming and sometimes of questionable relevance. In order to reduce animal use, functional in vitro assays are being explored as an alternative approach for the current lot release testing paradigm. In this study, we present an evaluation of APC platforms assessing innate immune activation by whole cell Bordetella pertussis (wP) vaccines. Primary monocytes, monocyte-derived DC (moDC) and human monocyte/DC cell lines (MonoMac6 and MUTZ-3) were compared for their capacity to respond to wP vaccines of varying quality. To produce such vaccines, the production process of wP was manipulated, resulting in wP vaccines covering a range of in vivo potencies. The responses of MUTZ-3 cells and primary monocytes to these vaccines were marginal and these models were therefore considered inappropriate. Importantly, moDC and MonoMac6 cells responded to the wP vaccines and discriminated between vaccines of varying quality, although slight variations in the responses to wP vaccines of similar quality were also observed. This study provides a proof of principle for the use of in vitro APC platforms as part of a new strategy to assess wP vaccine lot consistency, though careful standardisation of assay conditions is necessary.

  20. The Effect of Electron Beam Irradiation in Environmental Scanning Transmission Electron Microscopy of Whole Cells in Liquid.

    PubMed

    Hermannsdörfer, Justus; Tinnemann, Verena; Peckys, Diana B; de Jonge, Niels

    2016-06-01

    Whole cells can be studied in their native liquid environment using electron microscopy, and unique information about the locations and stoichiometry of individual membrane proteins can be obtained from many cells thus taking cell heterogeneity into account. Of key importance for the further development of this microscopy technology is knowledge about the effect of electron beam radiation on the samples under investigation. We used environmental scanning electron microscopy (ESEM) with scanning transmission electron microscopy (STEM) detection to examine the effect of radiation for whole fixed COS7 fibroblasts in liquid. The main observation was the localization of nanoparticle labels attached to epidermal growth factor receptors (EGFRs). It was found that the relative distances between the labels remained mostly unchanged (<1.5%) for electron doses ranging from the undamaged native state at 10 e-/Å2 toward 103 e-/Å2. This dose range was sufficient to determine the EGFR locations with nanometer resolution and to distinguish between monomers and dimers. Various different forms of radiation damage became visible at higher doses, including severe dislocation, and the dissolution of labels.

  1. A chromosomally based tod-luxCDABE whole-cell reporter for benzene, toluene, ethybenzene, and xylene (BTEX) sensing

    SciTech Connect

    Applegate, B.M.; Kehrmeyer, S.R.; Sayler, G.S.

    1998-07-01

    A tod-luxCDABE fusion was constructed and introduced into the chromosome of Pseudomonas putida F1, yielding the strain TVA8. This strain was used to examine the induction of the tod operon when exposed to benzene, toluene, ethylbenzene, and xylene (BTEX) compounds and aqueous solutions of JP-4 jet fuel constituents. Since this system contained the complete lux cassette (luxCDABE), bacterial bioluminescence in response to putative chemical inducers of the tod operon was measured on-line in whole cells without added aldehyde substrate. There was an increasing response to toluene concentrations from 30 {micro}g/liter to 50 mg/liter, which began to saturate at higher concentrations. The detection limit was 30 {micro}g/liter. There was a significant light response to benzene, m- and p-xylenes, phenol, and water-soluble JP-4 jet fuel components, but there was no bioluminescence response upon exposure to o-xylene. The transposon insertion was stable and had no negative effect on cell growth.

  2. Enzymatic and whole-cell synthesis of lactate-containing polyesters: toward the complete biological production of polylactate.

    PubMed

    Matsumoto, Ken'ichiro; Taguchi, Seiichi

    2010-01-01

    The importance of polylactic acid, a representative bio-based polyester, has been established on a worldwide scale in response to emerging global environmental problems such as green house gas emission and limited petroleum consumption. The current methods for generating this bio-based polymer involve biological synthesis and lactic acid (LA) fermentation, followed by chemical ring-opening polymerization. Among the research community working on polyhydroxyalkanoate polyesters, the prospect of direct biological synthesis of LA into a polymeric form is very attractive from the academic and industrial perspectives. In 2008, this challenge was met for the first time by the discovery of an "LA-polymerizing enzyme". Using this novel enzyme, the metabolic engineering approach outlined here provided an entirely new, single organism generation of the polymer. This is a major breakthrough in the field. In this review, we provide an overview of the whole-cell synthesis of LA-containing polyesters in comparison with conventional lipase-catalyzed polymer synthesis in terms of both the concepts and strategies of their synthetic processes.

  3. Efficient immobilization of mushroom tyrosinase utilizing whole cells from Agaricus bisporus and its application for degradation of bisphenol A.

    PubMed

    Kampmann, Markus; Boll, Stefan; Kossuch, Jan; Bielecki, Julia; Uhl, Stefan; Kleiner, Beatrice; Wichmann, Rolf

    2014-06-15

    A simple and efficient procedure for preparation and immobilization of tyrosinase enzyme was developed utilizing whole cells from the edible mushroom Agaricus bisporus, without the need for enzyme purification. Tyrosinase activity in the cell preparation remained constant during storage at 21 °C for at least six months. The cells were entrapped in chitosan and alginate matrix capsules and characterized with respect to their resulting tyrosinase activity. A modification of the alginate with colloidal silica enhanced the activity due to retention of both cells and tyrosinase from fractured cells, which otherwise leached from matrix capsules. The observed activity was similar to the activity that was obtained with immobilized isolated tyrosinase in the same material. Mushroom cells in water were susceptible to rapid inactivation, whereas the immobilized cells maintained 73% of their initial activity after 30 days of storage in water. Application in repeated batch experiments resulted in almost 100% conversion of endocrine disrupting bisphenol A (BPA) for 11 days, under stirring conditions, and 50-60% conversion after 20 days, without stirring under continuous usage. The results represent the longest yet reported application of immobilized tyrosinase for degradation of BPA in environmental water samples.

  4. A comparison of the haemagglutinating and enzymic activities of Bacteroides fragilis whole cells and outer membrane vesicles.

    PubMed

    Patrick, S; McKenna, J P; O'Hagan, S; Dermott, E

    1996-04-01

    The haemagglutinating and enzymic activities of the obligately anaerobic pathogenic bacterium Bacteroides fragilis were examined. Outer membrane vesicles are released from the surface of B. fragilis. They can be detected by electron microscopy in ultrathin sections and bacterial suspensions after negative staining. Electron microscopy and immunogold labelling with a MAb specific for surface polysaccharide of B. fragilis confirmed that the vesicles carried outer membrane associated epitopes. The haemagglutinating activity of whole cells from populations of B. fragilis strains NCTC9343, BE3 and LS66 enriched by Percoll density gradient centrifugation for a large capsule (LC), electron dense layer (EDL); non-capsulate by light microscopy) and outer membrane vesicles (OMV) which had been purified by centrifugation from EDL-enriched populations were compared using human and horse erythrocytes. The enzymic activity of OMV, LC- and EDL-enriched populations, as detected by the API ZYM kit, was compared for strains NCTC 9343 and BE3. Purified OMV from the strains examined exhibited both haemagglutinating and enzymatic activity. Haemagglutination by the EDL-enriched population was sensitive to treatment with sodium periodate. The LC-enriched population haemagglutinated only after ultrasonic removal of the capsule. This indicates that the LC masks a haemagglutinin. The results suggest a potential role for OMV in the virulence of B. fragilis. PMID:8737489

  5. Tetrahydroisoquinolines affect the whole-cell phenotype of Mycobacterium tuberculosis by inhibiting the ATP-dependent MurE ligase

    PubMed Central

    Guzman, Juan D.; Pesnot, Thomas; Barrera, Diana A.; Davies, Heledd M.; McMahon, Eleanor; Evangelopoulos, Dimitrios; Mortazavi, Parisa N.; Munshi, Tulika; Maitra, Arundhati; Lamming, Eleanor D.; Angell, Richard; Gershater, Markus C.; Redmond, Joanna M.; Needham, Deborah; Ward, John M.; Cuca, Luis E.; Hailes, Helen C.; Bhakta, Sanjib

    2015-01-01

    Objectives (S)-Leucoxine, isolated from the Colombian Lauraceae tree Rhodostemonodaphne crenaticupula Madriñan, was found to inhibit the growth of Mycobacterium tuberculosis H37Rv. A biomimetic approach for the chemical synthesis of a wide array of 1-substituted tetrahydroisoquinolines was undertaken with the aim of elucidating a common pharmacophore for these compounds with novel mode(s) of anti-TB action. Methods Biomimetic Pictet–Spengler or Bischler–Napieralski synthetic routes were employed followed by an evaluation of the biological activity of the synthesized compounds. Results In this work, the synthesized tetrahydroisoquinolines were found to inhibit the growth of M. tuberculosis H37Rv and affect its whole-cell phenotype as well as the activity of the ATP-dependent MurE ligase, a key enzyme involved in the early stage of cell wall peptidoglycan biosynthesis. Conclusions As the correlation between the MIC and the half-inhibitory enzymatic concentration was not particularly strong, there is a credible possibility that these compounds have pleiotropic mechanism(s) of action in M. tuberculosis. PMID:25656411

  6. Assaying Bcr-Abl kinase activity and inhibition in whole cell extracts by phosphorylation of substrates immobilized on agarose beads.

    PubMed

    Wu, Ding; Nair-Gill, Evan; Sher, Dorie A; Parker, Laurie L; Campbell, Jennifer M; Siddiqui, Mariah; Stock, Wendy; Kron, Stephen J

    2005-12-01

    There is a current and increasing demand for simple, robust, nonradioactive assays of protein tyrosine kinase activity with applications for clinical diagnosis and high-throughput screening of potential molecularly targeted therapeutic agents. One significant challenge is to detect and measure the activity of specific kinases with key roles in cell signaling as an approach to distinguish normal cells from cancer cells and as a means of evaluating targeted drug efficacy and resistance in cancer cells. Here, we describe a method in which kinase substrates fused to glutathione-S-transferase and immobilized on glutathione agarose beads are phosphorylated, eluted, and then assayed to detect kinase activity. The activity of recombinant, purified c-Abl kinase or Bcr-Abl kinase in whole cell extracts can be detected with equivalent specificity, sensitivity, and reproducibility. Similarly, inhibition of recombinant c-Abl or Bcr-Abl in cells or cell extracts by imatinib mesylate and other Bcr-Abl targeted kinase inhibitors is readily assayed. This simple kinase assay is sufficiently straightforward and robust for use in clinical laboratories and is potentially adaptable to high-throughput assay formats.

  7. Biotechnological production of fucosylated human milk oligosaccharides: Prokaryotic fucosyltransferases and their use in biocatalytic cascades or whole cell conversion systems.

    PubMed

    Petschacher, Barbara; Nidetzky, Bernd

    2016-10-10

    Human milk oligosaccharides (HMOs) constitute a class of complex carbohydrates unique to mother's milk and are strongly correlated to the health benefits of breastfeeding in infants. HMOs are important as functional ingredients of advanced infant formula and have attracted broad interest for use in health-related human nutrition. About 50% of the HMOs structures contain l-fucosyl residues, which are introduced into nascent oligosaccharides by enzymatic transfer from GDP-l-fucose. To overcome limitation in the current availability of fucosylated HMOs, biotechnological approaches for their production have been developed. Functional expression of the fucosyltransferase(s) and effective supply of GDP-l-fucose, respectively, are both bottlenecks of the biocatalytic routes of synthesis. Strategies of in vitro and in vivo production of fucosylated HMOs are reviewed here. Besides metabolic engineering for enhanced HMO production in whole cells, the focus is on the characteristics and the heterologous overexpression of prokaryotic α1,2- and α1,3/4-fucosyltransferases. Up to 20g/L of fucosylated HMOs were obtained in optimized production systems. Optimized expression enabled recovery of purified fucosyltransferases in a yield of up to 45mg/L culture for α1,2-fucosyltransferases and of up to 200mg protein/L culture for α1,3/4-fucosyltransferases.

  8. Chemical Modification in the Design of Immobilized Enzyme Biocatalysts: Drawbacks and Opportunities.

    PubMed

    Rueda, Nazzoly; Dos Santos, Jose C S; Ortiz, Claudia; Torres, Rodrigo; Barbosa, Oveimar; Rodrigues, Rafael C; Berenguer-Murcia, Ángel; Fernandez-Lafuente, Roberto

    2016-06-01

    Chemical modification of enzymes and immobilization used to be considered as separate ways to improve enzyme properties. This review shows how the coupled use of both tools may greatly improve the final biocatalyst performance. Chemical modification of a previously immobilized enzyme is far simpler and easier to control than the modification of the free enzyme. Moreover, if protein modification is performed to improve its immobilization (enriching the enzyme in reactive groups), the final features of the immobilized enzyme may be greatly improved. Chemical modification may be directed to improve enzyme stability, but also to improve selectivity, specificity, activity, and even cell penetrability. Coupling of immobilization and chemical modification with site-directed mutagenesis is a powerful instrument to obtain fully controlled modification. Some new ideas such as photoreceptive enzyme modifiers that change their physical properties under UV exposition are discussed.

  9. Application of biocatalysts to Space Station ECLSS and PMMS water reclamation

    NASA Technical Reports Server (NTRS)

    Jolly, Clifford D.; Bagdigian, Robert M.

    1989-01-01

    Immobilized enzyme reactors have been developed and tested for potential water reclamation applications in the Space Station Freedom Environmental Control and Life Support System (ECLSS) and Process Materials Management System (PMMS). The reactors convert low molecular weight organic contaminants found in ECLSS and PMMS wastewaters to compounds that are more efficiently removed by existing technologies. Demonstration of the technology was successfully achieved with two model reactors. A packed bed reactor containing immobilized urease was found to catalyze the complete decomposition of urea to by-products that were subsequently removed using conventional ion exchange results. A second reactor containing immobilized alcohol oxidase showed promising results relative to its ability to convert methanol and ethanol to the corresponding aldehydes for subsequent removal. Preliminary assessments of the application of biocatalysts to ECLSS and PMMS water reclamation sytems are presented.

  10. Chemical Modification in the Design of Immobilized Enzyme Biocatalysts: Drawbacks and Opportunities.

    PubMed

    Rueda, Nazzoly; Dos Santos, Jose C S; Ortiz, Claudia; Torres, Rodrigo; Barbosa, Oveimar; Rodrigues, Rafael C; Berenguer-Murcia, Ángel; Fernandez-Lafuente, Roberto

    2016-06-01

    Chemical modification of enzymes and immobilization used to be considered as separate ways to improve enzyme properties. This review shows how the coupled use of both tools may greatly improve the final biocatalyst performance. Chemical modification of a previously immobilized enzyme is far simpler and easier to control than the modification of the free enzyme. Moreover, if protein modification is performed to improve its immobilization (enriching the enzyme in reactive groups), the final features of the immobilized enzyme may be greatly improved. Chemical modification may be directed to improve enzyme stability, but also to improve selectivity, specificity, activity, and even cell penetrability. Coupling of immobilization and chemical modification with site-directed mutagenesis is a powerful instrument to obtain fully controlled modification. Some new ideas such as photoreceptive enzyme modifiers that change their physical properties under UV exposition are discussed. PMID:27166751

  11. GEL-STATE NMR OF BALL-MILLED WHOLE CELL WALLS IN DMSO-d6 USING 2D SOLUTION-STATE NMR SPECTROSCOPY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant cell walls were used for obtaining 2D solution-state NMR spectra without actual solubilization or structural modification. Ball-milled whole cell walls were swelled directly in the NMR tube with DMSO-d6 where they formed a gel. There are relatively few gel-state NMR studies. Most have involved...

  12. The relative merits of direct morphometry of reconstructions of whole cells, and statistical morphometry by stereology of random sections of cells.

    PubMed

    Parsons, D F; Marko, M; Leith, A

    1990-12-01

    Stereology, or the derivation of quantitative, three-dimensional (3-D) data about cells by statistical analysis of the structures of random sections, is widely used in cytology and pathology. However, there are situations where this approach is inadequate, and only an analysis of a homogeneous population of whole cells will give the required results. This involved 3-D reconstruction from physical or optical sections, or tomography or photogrammetry of whole-cell mounts. Use of stereo views of individual sections or projections adds considerably to the information available for both contouring and reconstruction. Recent image-processing advances in clinical radiography have shown, for the first time, that rapid, high-resolution digitization and contrast enhancement enable nearly all structural details to be routinely extracted from the micrographs and adequately portrayed. Three-D whole-cell reconstructions provide the digital data for many kinds of morphometric measurements on both whole cells and their individual organelles and membranes. Rapid fixation or freezing allows improved quantitative structure/function correlations of organelles with disturbances in cell metabolism or gene expression. PMID:1714349

  13. Comparison of Chemical Binding to Recombinant Fathead minnow and Human Estrogen Receptor alpha (ERα) in Whole Cell and Cell-Free Assay Systems.

    EPA Science Inventory

    Our objectives were to assess whether binding of chemicals differs significantly between recombinant estrogen receptors from fathead minnow (fhERα) and human (hERα) and to evaluate the performance of these receptors using two different in vitro assay systems: a COS whole cell bin...

  14. Biocatalyst activity in nonaqueous environments correlates with centisecond-range protein motions.

    PubMed

    Eppler, Ross K; Hudson, Elton P; Chase, Shannon D; Dordick, Jonathan S; Reimer, Jeffrey A; Clark, Douglas S

    2008-10-14

    Recent studies exploring the relationship between enzymatic catalysis and protein dynamics in the aqueous phase have yielded evidence that dynamics and enzyme activity are strongly correlated. Given that protein dynamics are significantly attenuated in organic solvents and that proteins exhibit a wide range of motions depending on the specific solvent environment, the nonaqueous milieu provides a unique opportunity to examine the role of protein dynamics in enzyme activity. Variable-temperature kinetic measurements, X-band electron spin resonance spectroscopy, (1)H NMR relaxation, and (19)F NMR spectroscopy experiments were performed on subtilisin Carlsberg colyophilized with several inorganic salts and suspended in organic solvents. The results indicate that salt activation induces a greater degree of transition-state flexibility, reflected by a more positive DeltaDeltaS(dagger), for the more active biocatalyst preparations in organic solvents. In contrast, DeltaDeltaH(dagger) was negligible regardless of salt type or salt content. Electron spin resonance spectroscopy and (1)H NMR relaxation measurements, including spin-lattice relaxation, spin-lattice relaxation in the rotating frame, and longitudinal magnetization exchange, revealed that the enzyme's turnover number (k(cat)) was strongly correlated with protein motions in the centisecond time regime, weakly correlated with protein motions in the millisecond regime, and uncorrelated with protein motions on the piconanosecond timescale. In addition, (19)F chemical shift measurements and hyperfine tensor measurements of biocatalyst formulations inhibited with 4-fluorobenzenesulfonyl fluoride and 4-ethoxyfluorophosphinyl-oxy-TEMPO, respectively, suggest that enzyme activation was only weakly affected by changes in active-site polarity. PMID:18840689

  15. K562-Derived Whole-Cell Vaccine Enhances Antitumor Responses of CAR-Redirected Virus-Specific Cytotoxic-T Lymphocytes in vivo

    PubMed Central

    Caruana, Ignazio; Weber, Gerrit; Ballard, Brandon Corde’; Wood, Michael Scott; Savoldo, Barbara; Dotti, Gianpietro

    2015-01-01

    Purpose Adoptive transfer of Epstein Barr Virus (EBV)- and Cytomegalovirus (CMV)-specific cytotoxic T cells (CTLs) genetically modified to express a Chimeric Antigen Receptor (CAR) induces objective tumor responses in clinical trials. In vivo expansion and persistence of these cells is crucial to achieve sustained clinical responses. We aimed to develop an off-the-shelf whole-cell vaccine to boost CAR-redirected virus-specific CTLs in vivo after adoptive transfer. As proof of principle, we validated our vaccine approach by boosting CMV-specific CTLs (CMV-CTLs) engineered with a CAR that targets the GD2 antigen. Experimental Design We generated the whole-cell vaccine by engineering the K562 cell line to express the CMV-pp65 protein and the immune stimulatory molecules CD40L and OX40L. Single-cell-derived clones were used to stimulate CMV-CTLs in vitro and in vivo in a xenograft model. We also assessed whether the in vivo boosting of CAR-redirected CMV-CTLs with the whole-cell vaccine enhances the antitumor responses. Finally, we addressed potential safety concerns by including the inducible safety switch caspase9 (iC9) gene in the whole-cell vaccine. Results We found that K562 expressing CMV-pp65, CD40L and OX40L effectively stimulates CMV-specific responses in vitro by promoting antigen cross-presentation to professional antigen-presenting cells (APCs). Vaccination also enhances antitumor effects of CAR-redirected CMV-CTLs in xenograft tumor models. Activation of the iC9 gene successfully induces growth arrest of engineered K562 implanted in mice. Conclusions Vaccination with a whole-cell vaccine obtained from K562 engineered to express CMV-pp65, CD40L, OX40L and iC9 can safely enhance the antitumor effects of CAR-redirected CMV-CTLs. PMID:25691731

  16. Development and validation of a whole-cell inhibition assay for bacterial methionine aminopeptidase by surface-enhanced laser desorption ionization-time of flight mass spectrometry.

    PubMed

    Greis, Kenneth D; Zhou, Songtao; Siehnel, Richard; Klanke, Chuck; Curnow, Alan; Howard, Jeremy; Layh-Schmitt, Gerlinde

    2005-08-01

    Bacterial methionine aminopeptidase (MAP) is a protease that removes methionine from the N termini of newly synthesized bacterial proteins after the peptide deformylase enzyme cleaves the formyl group from the initiator formylmethionine. MAP is an essential bacterial gene product and thus represents a potential target for therapeutic intervention. A fundamental challenge in the antibacterial drug discovery field is demonstrating conclusively that compounds with in vitro enzyme inhibition activity produce the desired antibacterial effect by interfering with the same target in whole bacterial cells. One way to address the activity of inhibitor compounds is by profiling cellular biomarkers in whole bacterial cells using compounds that are known inhibitors of a particular target. However, in the case of MAP, no specific inhibitors were available for such studies. Instead, a genetically attenuated MAP strain was generated in which MAP expression was placed under the control of an inducible arabinose promoter. Thus, MAP inhibition in whole cells could be mimicked by growth in the absence of arabinose. This genetically attenuated strain was used as a benchmark for MAP inhibition by profiling whole-cell lysates for unprocessed proteins using surface-enhanced laser desorption ionization-time of flight mass spectrometry (MS). Eight proteins between 4 and 14 kDa were confirmed as being unprocessed and containing the initiator methionine by adding back purified MAP to the preparations prior to MS analysis. Upon establishing these unprocessed proteins as biomarkers for MAP inhibition, the assay was used to screen small-molecule chemical inhibitors of purified MAP for whole-cell activity. Fifteen compound classes yielded three classes of compound with whole-cell activity for further optimization by chemical expansion. This report presents the development, validation, and implementation of a whole-cell inhibition assay for MAP.

  17. Formulation of a killed whole cell pneumococcus vaccine - effect of aluminum adjuvants on the antibody and IL-17 response

    PubMed Central

    2011-01-01

    Background Streptococcus pneumoniae causes widespread morbidity and mortality. Current vaccines contain free polysaccharides or protein-polysaccharide conjugates, and do not induce protection against serotypes that are not included in the vaccines. An affordable and broadly protective vaccine is very desirable. The goal of this study was to determine the optimal formulation of a killed whole cell pneumococcal vaccine with aluminum-containing adjuvants for intramuscular injection. Methods Four aluminium-containing adjuvants were prepared with different levels of surface phosphate groups resulting in different adsorptive capacities and affinities for the vaccine antigens. Mice were immunized three times and the antigen-specific antibody titers and IL-17 responses in blood were analyzed. Results Although all adjuvants induced significantly higher antibody titers than antigen without adjuvant, the vaccine containing aluminum phosphate adjuvant (AP) produced the highest antibody response when low doses of antigen were used. Aluminum hydroxide adjuvant (AH) induced an equal or better antibody response at high doses compared with AP. Vaccines formulated with AH, but not with AP, induced an IL-17 response. The vaccine formulated with AH was stable and retained full immunogenicity when stored at 4°C for 4 months. Conclusions Antibodies are important for protection against systemic streptococcal disease and IL-17 is critical in the prevention of nasopharyngeal colonization by S. pneumoniae in the mouse model. The formulation of the whole killed bacterial cells with AH resulted in a stable vaccine that induced both antibodies and an IL-17 response. These experiments underscore the importance of formulation studies with aluminium containing adjuvants for the development of stable and effective vaccines. PMID:21801401

  18. A Whole-Cell Phenotypic Screening Platform for Identifying Methylerythritol Phosphate Pathway-Selective Inhibitors as Novel Antibacterial Agents

    PubMed Central

    Johnson, L. Jeffrey

    2012-01-01

    Isoprenoid biosynthesis is essential for survival of all living organisms. More than 50,000 unique isoprenoids occur naturally, with each constructed from two simple five-carbon precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Two pathways for the biosynthesis of IPP and DMAPP are found in nature. Humans exclusively use the mevalonate (MVA) pathway, while most bacteria, including all Gram-negative and many Gram-positive species, use the unrelated methylerythritol phosphate (MEP) pathway. Here we report the development of a novel, whole-cell phenotypic screening platform to identify compounds that selectively inhibit the MEP pathway. Strains of Salmonella enterica serovar Typhimurium were engineered to have separately inducible MEP (native) and MVA (nonnative) pathways. These strains, RMC26 and CT31-7d, were then used to differentiate MVA pathway- and MEP pathway-specific perturbation. Compounds that inhibit MEP pathway-dependent bacterial growth but leave MVA-dependent growth unaffected represent MEP pathway-selective antibacterials. This screening platform offers three significant results. First, the compound is antibacterial and is therefore cell permeant, enabling access to the intracellular target. Second, the compound inhibits one or more MEP pathway enzymes. Third, the MVA pathway is unaffected, suggesting selectivity for targeting the bacterial versus host pathway. The cell lines also display increased sensitivity to two reported MEP pathway-specific inhibitors, further biasing the platform toward inhibitors selective for the MEP pathway. We demonstrate development of a robust, high-throughput screening platform that combines phenotypic and target-based screening that can identify MEP pathway-selective antibacterials simply by monitoring optical density as the readout for cell growth/inhibition. PMID:22777049

  19. Effect of Different Adjuvants on Protection and Side-Effects Induced by Helicobacter suis Whole-Cell Lysate Vaccination.

    PubMed

    Bosschem, Iris; Bayry, Jagadeesh; De Bruyne, Ellen; Van Deun, Kim; Smet, Annemieke; Vercauteren, Griet; Ducatelle, Richard; Haesebrouck, Freddy; Flahou, Bram

    2015-01-01

    Helicobacter suis (H. suis) is a widespread porcine gastric pathogen, which is also of zoonotic importance. The first goal of this study was to investigate the efficacy of several vaccine adjuvants (CpG-DNA, Curdlan, Freund's Complete and Incomplete, Cholera toxin), administered either subcutaneously or intranasally along with H. suis whole-cell lysate, to protect against subsequent H. suis challenge in a BALB/c infection model. Subcutaneous immunization with Freund's complete (FC)/lysate and intranasal immunization with Cholera toxin (CT)/lysate were shown to be the best options for vaccination against H. suis, as determined by the amount of colonizing H. suis bacteria in the stomach, although adverse effects such as post-immunization gastritis/pseudo-pyloric metaplasia and increased mortality were observed, respectively. Therefore, we decided to test alternative strategies, including sublingual vaccine administration, to reduce the unwanted side-effects. A CCR4 antagonist that transiently inhibits the migration of regulatory T cells was also included as a new adjuvant in this second study. Results confirmed that immunization with CT (intranasally or sublingually) is among the most effective vaccination protocols, but increased mortality was still observed. In the groups immunized subcutaneously with FC/lysate and CCR4 antagonist/lysate, a significant protection was observed. Compared to the FC/lysate immunized group, gastric pseudo-pyloric metaplasia was less severe or even absent in the CCR4 antagonist/lysate immunized group. In general, an inverse correlation was observed between IFN-γ, IL-4, IL-17, KC, MIP-2 and LIX mRNA expression and H. suis colonization density, whereas lower IL-10 expression levels were observed in partially protected animals.

  20. Arsenic bioavailability in soils before and after soil washing: the use of Escherichia coli whole-cell bioreporters.

    PubMed

    Yoon, Youngdae; Kang, Yerin; Chae, Yooeun; Kim, Sunghoon; Lee, Youngshim; Jeong, Seung-Woo; An, Youn-Joo

    2016-02-01

    We investigated the quantification of bioavailable arsenic in contaminated soils and evaluation of soil-washing processes in the aspect of bioavailability using a novel bacterial bioreporter developed in present study. The whole-cell bioreporter (WCB) was genetically engineered by fusing the promoter of nik operon from Escherichia coli and green fluorescent protein as a sensing domain and reporter domain. Among eight well-known hazardous heavy metals and metalloid, this system responded specifically to arsenic, thereby inferring association of As(III) with NikR inhibits the repression. Moreover, the response was proportional to the concentration of As(III), thereby it was capable to determine the amount of bioavailable arsenic quantitatively in contaminated soils. The bioavailable portion of arsenic was 5.9 (3.46-10.96) and 0.9 (0.27-1.74) % of total from amended and site soils, respectively, suggesting the bioavailability of arsenic in soils was related to the soil properties and duration of aging. On the other hand, only 1.37 (0.21-2.97) % of total arsenic was extracted into soil solutions and 19.88 (11.86-28.27) % of arsenic in soil solution was bioavailable. This result showed that the soluble arsenic is not all bioavailable and most of bioavailable arsenic in soils is water non-extractable. In addition, the bioavailable arsenic was increased after soil-washing while total amount was decreased, thereby suggesting the soil-washing processes release arsenic associated with soil materials to be bioavailable. Therefore, it would be valuable to have a tool to assess bioavailability and the bioavailability should be taken into consideration for soil remediation plans. PMID:26411448

  1. Arsenic bioavailability in soils before and after soil washing: the use of Escherichia coli whole-cell bioreporters.

    PubMed

    Yoon, Youngdae; Kang, Yerin; Chae, Yooeun; Kim, Sunghoon; Lee, Youngshim; Jeong, Seung-Woo; An, Youn-Joo

    2016-02-01

    We investigated the quantification of bioavailable arsenic in contaminated soils and evaluation of soil-washing processes in the aspect of bioavailability using a novel bacterial bioreporter developed in present study. The whole-cell bioreporter (WCB) was genetically engineered by fusing the promoter of nik operon from Escherichia coli and green fluorescent protein as a sensing domain and reporter domain. Among eight well-known hazardous heavy metals and metalloid, this system responded specifically to arsenic, thereby inferring association of As(III) with NikR inhibits the repression. Moreover, the response was proportional to the concentration of As(III), thereby it was capable to determine the amount of bioavailable arsenic quantitatively in contaminated soils. The bioavailable portion of arsenic was 5.9 (3.46-10.96) and 0.9 (0.27-1.74) % of total from amended and site soils, respectively, suggesting the bioavailability of arsenic in soils was related to the soil properties and duration of aging. On the other hand, only 1.37 (0.21-2.97) % of total arsenic was extracted into soil solutions and 19.88 (11.86-28.27) % of arsenic in soil solution was bioavailable. This result showed that the soluble arsenic is not all bioavailable and most of bioavailable arsenic in soils is water non-extractable. In addition, the bioavailable arsenic was increased after soil-washing while total amount was decreased, thereby suggesting the soil-washing processes release arsenic associated with soil materials to be bioavailable. Therefore, it would be valuable to have a tool to assess bioavailability and the bioavailability should be taken into consideration for soil remediation plans.

  2. Enzyme-based glucose delivery as a high content screening tool in yeast-based whole-cell biocatalysis.

    PubMed

    Grimm, T; Grimm, M; Klat, R; Neubauer, A; Palela, M; Neubauer, P

    2012-05-01

    The influence of glucose release on growth and biotransformation of yeasts was examined by using the medium EnBase® Flo in shake flasks. The medium contains a polysaccharide acting as substrate, which is degraded to glucose by the addition of an enzyme. In the present paper, this medium was adapted for the cultivation of yeasts by increasing the complex components (booster) and the enzyme concentrations to guarantee a higher glucose release rate. Important changes were an increase of the complex component booster to 10-15% and an increased glucose release by increasing the enzyme content to 15 U L(-1). The 20 yeasts investigated in the present work showed an improvement of growth and biomass production when cultivated with the EnBase medium in comparison to yeast extract dextrose (YED) medium. Values of optical densities (OD(600)) of approximately 40 AU (corresponding to over 60 g L(-1) wet cell weight) were achieved for all 20 yeast strains tested. During the following screening of the yeasts in whole-cell biotransformation, an improvement of the conversion for 19 out of the 20 yeasts cultivated with the EnBase Flo medium could be observed. The biomass from the EnBase Flo cultivation showed a higher conversion activity in the reduction of 2-butanone to (R/S)-2-butanol. The enantioselectivity (ee) of 15 yeast strains showed an improvement by using the EnBase medium. The number of yeasts with an ee >97% increased from zero with YED to six with EnBase medium. Thus, the use of a glucose release cultivation strategy in the screening process for transformation approaches provides significant benefits compared to standard batch approaches. PMID:22258642

  3. Detection of bioavailable cadmium, lead, and arsenic in polluted soil by tailored multiple Escherichia coli whole-cell sensor set.

    PubMed

    Hou, Qihui; Ma, Anzhou; Wang, Thanh; Lin, Jianqiang; Wang, Hailin; Du, Binghai; Zhuang, Xuliang; Zhuang, Guoqiang

    2015-09-01

    Microbial whole-cell sensor has been widely used to assess bioavailability and risk of toxic elements, but their environmental use is still limited due to the presence of other interfering pollutants and the nonspecific binding in cells, which leads to inaccurate results. Here, we proposed a strategy combining Escherichia coli sensor set with binary regression models for the specific detection of bioavailable cadmium (Cd), lead (Pb), and arsenic (As) in a co-polluted environment. Initial tests suggested that the sensor set respectively termed pcadCluc, pzntRluc, and parsRluc could be classified into two groups according to their specific response to Cd, Pb, and As: group 1 (pcadCluc and pzntRluc) induced by a Cd-Pb mix and group 2 (parsRluc) induced by a Cd-As mix. Based on the variance in responses of each sensor to mixtures of target elements, three binary linear equations for two sensor groups were set up to calculate the individual concentrations in the mixture solutions. This method was then used to quantify the bioavailable Cd, Pb, and As in soils from a co-polluted mining region and to compare the results with other methods. Results showed that the conventional single target sensor method overestimated the bioavailability of each element, while sensor set was credible for accurate bioavailable Cd, Pb, and As quantification and comparable with the results from inductively coupled plasma mass spectrometry (ICP-MS) analysis. Our method can potentially be extended to cover the specific detection of other bioavailable toxic elements in different environmental settings. PMID:26138890

  4. Effect of Different Adjuvants on Protection and Side-Effects Induced by Helicobacter suis Whole-Cell Lysate Vaccination

    PubMed Central

    Bosschem, Iris; Bayry, Jagadeesh; De Bruyne, Ellen; Van Deun, Kim; Smet, Annemieke; Vercauteren, Griet; Ducatelle, Richard; Haesebrouck, Freddy; Flahou, Bram

    2015-01-01

    Helicobacter suis (H. suis) is a widespread porcine gastric pathogen, which is also of zoonotic importance. The first goal of this study was to investigate the efficacy of several vaccine adjuvants (CpG-DNA, Curdlan, Freund’s Complete and Incomplete, Cholera toxin), administered either subcutaneously or intranasally along with H. suis whole-cell lysate, to protect against subsequent H. suis challenge in a BALB/c infection model. Subcutaneous immunization with Freund’s complete (FC)/lysate and intranasal immunization with Cholera toxin (CT)/lysate were shown to be the best options for vaccination against H. suis, as determined by the amount of colonizing H. suis bacteria in the stomach, although adverse effects such as post-immunization gastritis/pseudo-pyloric metaplasia and increased mortality were observed, respectively. Therefore, we decided to test alternative strategies, including sublingual vaccine administration, to reduce the unwanted side-effects. A CCR4 antagonist that transiently inhibits the migration of regulatory T cells was also included as a new adjuvant in this second study. Results confirmed that immunization with CT (intranasally or sublingually) is among the most effective vaccination protocols, but increased mortality was still observed. In the groups immunized subcutaneously with FC/lysate and CCR4 antagonist/lysate, a significant protection was observed. Compared to the FC/lysate immunized group, gastric pseudo-pyloric metaplasia was less severe or even absent in the CCR4 antagonist/lysate immunized group. In general, an inverse correlation was observed between IFN-γ, IL-4, IL-17, KC, MIP-2 and LIX mRNA expression and H. suis colonization density, whereas lower IL-10 expression levels were observed in partially protected animals. PMID:26115373

  5. Reconstitution of TCA cycle with DAOCS to engineer Escherichia coli into an efficient whole cell catalyst of penicillin G.

    PubMed

    Lin, Baixue; Fan, Keqiang; Zhao, Jian; Ji, Junjie; Wu, Linjun; Yang, Keqian; Tao, Yong

    2015-08-11

    Many medically useful semisynthetic cephalosporins are derived from 7-aminodeacetoxycephalosporanic acid (7-ADCA), which has been traditionally made by the polluting chemical method. Here, a whole-cell biocatalytic process based on an engineered Escherichia coli strain expressing 2-oxoglutarate-dependent deacetoxycephalosporin C synthase (DAOCS) for converting penicillin G to G-7-ADCA is developed. The major engineering strategy is to reconstitute the tricarboxylic acid (TCA) cycle of E. coli to force the metabolic flux to go through DAOCS catalyzed reaction for 2-oxoglutarate to succinate conversion. Then the glyoxylate bypass was disrupted to eliminate metabolic flux that may circumvent the reconstituted TCA cycle. Additional engineering steps were taken to reduce the degradation of penicillin G and G-7-ADCA in the bioconversion process. These steps include engineering strategies to reduce acetate accumulation in the biocatalytic process and to knock out a host β-lactamase involved in the degradation of penicillin G and G-7-ADCA. By combining these manipulations in an engineered strain, the yield of G-7-ADCA was increased from 2.50 ± 0.79 mM (0.89 ± 0.28 g/L, 0.07 ± 0.02 g/gDCW) to 29.01 ± 1.27 mM (10.31 ± 0.46 g/L, 0.77 ± 0.03 g/gDCW) with a conversion rate of 29.01 mol%, representing an 11-fold increase compared with the starting strain (2.50 mol%).

  6. Biosynthesis of 2-deoxysugars using whole-cell catalyst expressing 2-deoxy-D-ribose 5-phosphate aldolase.

    PubMed

    Li, Jitao; Yang, Jiangang; Men, Yan; Zeng, Yan; Zhu, Yueming; Dong, Caixia; Sun, Yuanxia; Ma, Yanhe

    2015-10-01

    2-Deoxy-D-ribose 5-phosphate aldolase (DERA) accepts a wide variety of aldehydes and is used in de novo synthesis of 2-deoxysugars, which have important applications in drug manufacturing. However, DERA has low preference for non-phosphorylated substrates. In this study, DERA from Klebsiella pneumoniae (KDERA) was mutated to increase its enzyme activity and substrate tolerance towards non-phosphorylated polyhydroxy aldehyde. Mutant KDERA(K12) (S238D/F200I/ΔY259) showed a 3.15-fold improvement in enzyme activity and a 1.54-fold increase in substrate tolerance towards D-glyceraldehyde compared with the wild type. Furthermore, a whole-cell transformation strategy using resting cells of the BL21(pKDERA12) strain, containing the expressed plasmid pKDERA12, resulted in increase in 2-deoxy-D-ribose yield from 0.41 mol/mol D-glyceraldehyde to 0.81 mol/mol D-glyceraldehyde and higher substrate tolerance from 0.5 to 3 M compared to in vitro assays. With further optimization of the transformation process, the BL21(pKDERA12) strain produced 2.14 M (287.06 g/L) 2-deoxy-D-robose (DR), with a yield of 0.71 mol/mol D-glyceraldehyde and average productivity of 0.13 mol/L·h (17.94 g/L·h). These results demonstrate the potential for large-scale production of 2-deoxy-D-ribose using the BL21(pKDERA12) strain. Furthermore, the BL21(pKDERA12) strain also exhibited the ability to efficiently produce 2-deoxy-D-altrose from D-erythrose, as well as 2-deoxy-L-xylose and 2-deoxy-L-ribose from L-glyceraldehyde.

  7. Detection of bioavailable cadmium, lead, and arsenic in polluted soil by tailored multiple Escherichia coli whole-cell sensor set.

    PubMed

    Hou, Qihui; Ma, Anzhou; Wang, Thanh; Lin, Jianqiang; Wang, Hailin; Du, Binghai; Zhuang, Xuliang; Zhuang, Guoqiang

    2015-09-01

    Microbial whole-cell sensor has been widely used to assess bioavailability and risk of toxic elements, but their environmental use is still limited due to the presence of other interfering pollutants and the nonspecific binding in cells, which leads to inaccurate results. Here, we proposed a strategy combining Escherichia coli sensor set with binary regression models for the specific detection of bioavailable cadmium (Cd), lead (Pb), and arsenic (As) in a co-polluted environment. Initial tests suggested that the sensor set respectively termed pcadCluc, pzntRluc, and parsRluc could be classified into two groups according to their specific response to Cd, Pb, and As: group 1 (pcadCluc and pzntRluc) induced by a Cd-Pb mix and group 2 (parsRluc) induced by a Cd-As mix. Based on the variance in responses of each sensor to mixtures of target elements, three binary linear equations for two sensor groups were set up to calculate the individual concentrations in the mixture solutions. This method was then used to quantify the bioavailable Cd, Pb, and As in soils from a co-polluted mining region and to compare the results with other methods. Results showed that the conventional single target sensor method overestimated the bioavailability of each element, while sensor set was credible for accurate bioavailable Cd, Pb, and As quantification and comparable with the results from inductively coupled plasma mass spectrometry (ICP-MS) analysis. Our method can potentially be extended to cover the specific detection of other bioavailable toxic elements in different environmental settings.

  8. Fungus-insect gall of Phlebopus portentosus.

    PubMed

    Zhang, Chun-Xia; He, Ming-Xia; Cao, Yang; Liu, Jing; Gao, Feng; Wang, Wen-Bing; Ji, Kai-Ping; Shao, Shi-Cheng; Wang, Yun

    2015-01-01

    Phlebopus portentosus is a popular edible wild mushroom found in the tropical Yunnan, China, and northern Thailand. In its natural habitats, a gall often has been found on some plant roots, around which fungal fruiting bodies are produced. The galls are different from common insect galls in that their cavity walls are not made from plant tissue but rather from the hyphae of P. portentosus. Therefore we have termed this phenomenon "fungus-insect gall". Thus far six root mealy bug species in the family Pseudococcidae that form fungus-insect galls with P. portentosus have been identified: Formicococcus polysperes, Geococcus satellitum, Planococcus minor, Pseudococcus cryptus, Paraputo banzigeri and Rastrococcus invadens. Fungus-insect galls were found on the roots of more than 21 plant species, including Delonix regia, Citrus maxima, Coffea arabica and Artocarpus heterophyllus. Greenhouse inoculation trials showed that fungus-insect galls were found on the roots of A. heterophyllus 1 mo after inoculation. The galls were subglobose to globose, fulvous when young and became dark brown at maturation. Each gall harbored one or more mealy bugs and had a chimney-like vent for ventilation and access to the gall. The cavity wall had three layers. Various shaped mealy bug wax deposits were found inside the wall. Fungal hyphae invaded the epidermis of plant roots and sometimes even the cortical cells during the late stage of gall development. The identity of the fungus inside the cavity was confirmed by molecular methods. PMID:25344264

  9. Fungus-insect gall of Phlebopus portentosus.

    PubMed

    Zhang, Chun-Xia; He, Ming-Xia; Cao, Yang; Liu, Jing; Gao, Feng; Wang, Wen-Bing; Ji, Kai-Ping; Shao, Shi-Cheng; Wang, Yun

    2015-01-01

    Phlebopus portentosus is a popular edible wild mushroom found in the tropical Yunnan, China, and northern Thailand. In its natural habitats, a gall often has been found on some plant roots, around which fungal fruiting bodies are produced. The galls are different from common insect galls in that their cavity walls are not made from plant tissue but rather from the hyphae of P. portentosus. Therefore we have termed this phenomenon "fungus-insect gall". Thus far six root mealy bug species in the family Pseudococcidae that form fungus-insect galls with P. portentosus have been identified: Formicococcus polysperes, Geococcus satellitum, Planococcus minor, Pseudococcus cryptus, Paraputo banzigeri and Rastrococcus invadens. Fungus-insect galls were found on the roots of more than 21 plant species, including Delonix regia, Citrus maxima, Coffea arabica and Artocarpus heterophyllus. Greenhouse inoculation trials showed that fungus-insect galls were found on the roots of A. heterophyllus 1 mo after inoculation. The galls were subglobose to globose, fulvous when young and became dark brown at maturation. Each gall harbored one or more mealy bugs and had a chimney-like vent for ventilation and access to the gall. The cavity wall had three layers. Various shaped mealy bug wax deposits were found inside the wall. Fungal hyphae invaded the epidermis of plant roots and sometimes even the cortical cells during the late stage of gall development. The identity of the fungus inside the cavity was confirmed by molecular methods.

  10. A New Biocatalyst for Production of Optically Pure Aryl Epoxides by Styrene Monooxygenase from Pseudomonas fluorescens ST

    PubMed Central

    Di Gennaro, Patrizia; Colmegna, Andrea; Galli, Enrica; Sello, Guido; Pelizzoni, Francesca; Bestetti, Giuseppina

    1999-01-01

    We developed a biocatalyst by cloning the styrene monooxygenase genes (styA and styB) from Pseudomonas fluorescens ST responsible for the oxidation of styrene to its corresponding epoxide. Recombinant Escherichia coli was able to oxidize different aryl vinyl and aryl ethenyl compounds to their corresponding optically pure epoxides. The results of bioconversions indicate the broad substrate preference of styrene monooxygenase and its potential for the production of several fine chemicals. PMID:10347083

  11. Resolving the mechanism of bacterial inhibition by plant secondary metabolites employing a combination of whole-cell biosensors.

    PubMed

    Chan, Andrea C; Ager, Duane; Thompson, Ian P

    2013-06-01

    Tightening regulations regarding the use of biocides have stimulated interest in investigating alternatives to current antimicrobial strategies. Plant essential oils and their constituent compounds are promising candidates as novel antimicrobial agents because of their excellent ability in killing microbes while being non-toxic to humans at antimicrobially-active concentrations. Allyl isothiocyanate (AIT), carvacrol, cinnamaldehyde (CNAD), citral, and thymol were investigated for their antibacterial activity against Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. The five compounds were screened via disc diffusion assay and broth microdilution method, by which inhibition zone diameters, minimum inhibitory concentrations (MICs), and minimum bactericidal concentrations (MBCs) were determined. AIT and CNAD displayed the greatest inhibitory effects against all species tested, with AIT yielding MICs of 156.25mg/L and MBCs of 156.25 to 312.5mg/L, and CNAD yielding MICs of 78.125 to 156.25mg/L and MBCs of 78.125 to 312.5mg/L. Based on these results, AIT and CNAD were selected for closer examination of their toxic effects. Two complementary bioluminescence-based bacterial biosensors, E. coli HB101_pUCD607_lux and Acinetobacter baylyi ADP1_recA_lux, were employed to examine the dose-response relationships and mechanism of action of AIT and CNAD. This is the first reported study to employ a lux-based biosensor assay coupled with parallel plate count experiments to demonstrate that AIT and CNAD not only damaged cell membranes, but also disrupted cellular metabolism and energy production in bacteria. It is also the first to use genotoxicity-sensing whole-cell bioreporters to demonstrate that neither AIT nor CNAD induced expression of the universal DNA repair gene, recA. This suggests that AIT and CNAD were not genotoxic. As an antimicrobial agent, it is advantageous that the compound be genetically non-damaging so that toxicity towards

  12. Open-Ended Experimentation with the Fungus Pilobolus.

    ERIC Educational Resources Information Center

    Coble, Charles R.; Bland, Charles E.

    This paper describes open-ended experimentation with the fungus Pilobolus for laboratory work by high school students. The fungus structure and reproduction is described and sources of the fungus are suggested. Four areas for investigation are suggested: the effect of a diffuse light source, the effect of a point light source, the effect of light…

  13. White-nose syndrome fungus (Geomyces destructans) in bat, France.

    PubMed

    Puechmaille, Sebastien J; Verdeyroux, Pascal; Fuller, Hubert; Gouilh, Meriadeg Ar; Bekaert, Michael; Teeling, Emma C

    2010-02-01

    White-nose syndrome is caused by the fungus Geomyces destructans and is responsible for the deaths of >1,000,000 bats since 2006. This disease and fungus had been restricted to the northeastern United States. We detected this fungus in a bat in France and assessed the implications of this finding. PMID:20113562

  14. The Touch and Zap Method for In Vivo Whole-Cell Patch Recording of Intrinsic and Visual Responses of Cortical Neurons and Glial Cells

    PubMed Central

    Schramm, Adrien E.; Marinazzo, Daniele; Gener, Thomas; Graham, Lyle J.

    2014-01-01

    Whole-cell patch recording is an essential tool for quantitatively establishing the biophysics of brain function, particularly in vivo. This method is of particular interest for studying the functional roles of cortical glial cells in the intact brain, which cannot be assessed with extracellular recordings. Nevertheless, a reasonable success rate remains a challenge because of stability, recording duration and electrical quality constraints, particularly for voltage clamp, dynamic clamp or conductance measurements. To address this, we describe “Touch and Zap”, an alternative method for whole-cell patch clamp recordings, with the goal of being simpler, quicker and more gentle to brain tissue than previous approaches. Under current clamp mode with a continuous train of hyperpolarizing current pulses, seal formation is initiated immediately upon cell contact, thus the “Touch”. By maintaining the current injection, whole-cell access is spontaneously achieved within seconds from the cell-attached configuration by a self-limited membrane electroporation, or “Zap”, as seal resistance increases. We present examples of intrinsic and visual responses of neurons and putative glial cells obtained with the revised method from cat and rat cortices in vivo. Recording parameters and biophysical properties obtained with the Touch and Zap method compare favourably with those obtained with the traditional blind patch approach, demonstrating that the revised approach does not compromise the recorded cell. We find that the method is particularly well-suited for whole-cell patch recordings of cortical glial cells in vivo, targeting a wider population of this cell type than the standard method, with better access resistance. Overall, the gentler Touch and Zap method is promising for studying quantitative functional properties in the intact brain with minimal perturbation of the cell's intrinsic properties and local network. Because the Touch and Zap method is performed semi

  15. Proline porter II is activated by a hyperosmotic shift in both whole cells and membrane vesicles of Escherichia coli K12.

    PubMed

    Milner, J L; Grothe, S; Wood, J M

    1988-10-15

    Proline porter II is rapidly activated when nongrowing bacteria are subjected to a hyperosmotic shift (Grothe, S., Krogsrud, R. L., McClellan, D. J., Milner, J. L., and Wood, J. M. (1986) J. Bacteriol. 166, 253-259). Proline porter II was active in membrane vesicles prepared from bacteria grown under optimal conditions, nutritional stress, or osmotic stress. That activity was: (i) dependent on the presence of the energy sources phenazine methosulphate plus ascorbate or D-lactate; (ii) observed only when a hyperosmotic shift accompanied the transport measurement; (iii) inhibited by glycine betaine in a manner analogous to that observed in whole cells; and (iv) eliminated by lesions in proP. Membrane vesicles were able to transport serine but not glutamine and serine transport was reduced by the hyperosmotic shift. In whole cells, proline porter II activity was supported by glucose and by D-lactate in a strain defective for proline porters I and III and the F1F0-ATPase. Glucose energized proline uptake was eliminated by carbonyl cyanide m-chlorophenylhydrazone and KCN as was serine uptake. These results suggested that proline porter II was respiration-dependent and probably ion-linked. Activation of proline porter II in whole cells by sucrose or NaCl was sustained over 30 min, whereas activation by glycerol was transient. Proline porter II was activated by NaCl and sucrose with a half-time of approximately 1 min in both whole cells and membrane vesicles. Thus, activation of proline porter II was reversible. It occurred at a rate comparable to that of K+ influx and much more rapid than the genetic regulatory responses that follow a hyperosmotic shift. PMID:3049595

  16. Cell Surface Display Fungal Laccase as a Renewable Biocatalyst for Degradation of Persistent Micropollutants Bisphenol A and Sulfamethoxazole.

    PubMed

    Chen, Yingying; Stemple, Brooke; Kumar, Manish; Wei, Na

    2016-08-16

    Fungal laccases have high activity in degrading various persistent organic pollutants. However, using enzymes in solution for water treatment has limitations of nonreusability, short enzyme lifetimes, and high cost of single use. In this study, we developed a new type of biocatalyst by immobilizing fungal laccase on the surface of yeast cells using synthetic biology techniques. The biocatalyst, referred to as surface display laccase (SDL), had an enzyme activity of 104 ± 3 mU/g dry cell (with 2,2-azinobis-3-ethylbenzothiazoline-6-sulfonate (ABTS)). The SDL retained over 90% of the initial enzyme activity after 25 days storage at room temperature, while, in contrast, activity of free laccase declined to 60% of its initial activity. The SDL could be reused with high stability as it retained 74% of initial activity after eight repeated batch reactions. Proof-of-concept evaluations of the effectiveness of SDL in treating contaminants of emerging concern were performed with bisphenol A and sulfamethoxazole. Results from contaminant degradation kinetics and the effects of redox mediator amendment provided insights into the factors affecting the efficacy of the SDL system. This study reports, for the first time, the development of a surface display enzyme biocatalyst as an effective and renewable alternative for treating recalcitrant organic micropollutants. PMID:27414990

  17. Distinguishing nuclei-specific benzo[a]pyrene-induced effects from whole-cell alterations in MCF-7 cells using Fourier-transform infrared spectroscopy.

    PubMed

    Obinaju, Blessing E; Fullwood, Nigel J; Martin, Francis L

    2015-09-01

    Exposure to chemicals such as benzo[a]pyrene (B[a]P) can generate intracellular toxic mechanisms. Fourier-transform infrared (FTIR) spectroscopy is a novel approach that allows the non-destructive analysis of underlying chemical bond alterations in patho-physiological processes. This study set out to examine whether B[a]P-induced whole cell alterations could be distinguished from effects on nuclei of exposed cells. Using attenuated total reflection FTIR (ATR-FTIR) spectroscopy, alterations in nuclei isolated from B[a]P-treated MCF-7 cells concentrated either in G0/G1- or S-phase were observed. B[a]P-induced effects in whole-cells included alterations to lipids, DNA and protein spectral regions. Absorbance areas for protein and DNA/RNA regions in B[a]P-treated whole cells differed significantly (P<0.0001) from vehicle controls and these observations correlated with alterations noted in isolated nuclei. Our findings provide evidence that FTIR spectroscopy has the ability to identify specific chemical-induced alterations.

  18. Seroprevalence of Antibodies to Pertussis Toxin among Different Age Groups in Thailand after 37 Years of Universal Whole-Cell Pertussis Vaccination

    PubMed Central

    Wanlapakorn, Nasamon; Ngaovithunvong, Varisara; Thongmee, Thanunrat; Vichaiwattana, Preeyaporn; Vongpunsawad, Sompong; Poovorawan, Yong

    2016-01-01

    Despite the high coverage of prophylactic vaccine against Bordetella pertussis infection in many countries for more than three decades, pertussis remains a common vaccine-preventable disease. Infections have been detected more commonly in countries using acellular pertussis vaccine in their Expanded Program of Immunization. Thailand implemented a routine infant immunization program with whole-cell pertussis vaccine in 1977, and since 1992, the national vaccine policy has offered a five-dose whole-cell pertussis vaccine for children given at the ages of 2, 4, 6, 18, and 48 months. This study aimed to investigate the seroprevalence of antibodies to pertussis toxin among healthy people across all ages to determine the level of whole-cell vaccine-induced immunity in the population, and to identify which age group should be targeted for a booster dose. The lowest seronegative rate and highest geometric mean concentrations were found in the 0–10 years age group, corresponding to their recent pertussis vaccination. The proportion of people with undetectable IgG level was prominent, starting after 11 years of age onwards. Now that a reduced-dose pertussis vaccine with fewer adverse effects is available, a booster dose during adolescence should be considered in order to reduce the incidence of pertussis disease. Further studies exploring how long the reduced-dose pertussis vaccine can provide protective immunity against pertussis disease when administered to adults and adolescents should also be performed. PMID:26837004

  19. Seroprevalence of Antibodies to Pertussis Toxin among Different Age Groups in Thailand after 37 Years of Universal Whole-Cell Pertussis Vaccination.

    PubMed

    Wanlapakorn, Nasamon; Ngaovithunvong, Varisara; Thongmee, Thanunrat; Vichaiwattana, Preeyaporn; Vongpunsawad, Sompong; Poovorawan, Yong

    2016-01-01

    Despite the high coverage of prophylactic vaccine against Bordetella pertussis infection in many countries for more than three decades, pertussis remains a common vaccine-preventable disease. Infections have been detected more commonly in countries using acellular pertussis vaccine in their Expanded Program of Immunization. Thailand implemented a routine infant immunization program with whole-cell pertussis vaccine in 1977, and since 1992, the national vaccine policy has offered a five-dose whole-cell pertussis vaccine for children given at the ages of 2, 4, 6, 18, and 48 months. This study aimed to investigate the seroprevalence of antibodies to pertussis toxin among healthy people across all ages to determine the level of whole-cell vaccine-induced immunity in the population, and to identify which age group should be targeted for a booster dose. The lowest seronegative rate and highest geometric mean concentrations were found in the 0-10 years age group, corresponding to their recent pertussis vaccination. The proportion of people with undetectable IgG level was prominent, starting after 11 years of age onwards. Now that a reduced-dose pertussis vaccine with fewer adverse effects is available, a booster dose during adolescence should be considered in order to reduce the incidence of pertussis disease. Further studies exploring how long the reduced-dose pertussis vaccine can provide protective immunity against pertussis disease when administered to adults and adolescents should also be performed. PMID:26837004

  20. Acidophilic bacteria and archaea: acid stable biocatalysts and their potential applications.

    PubMed

    Sharma, Archana; Kawarabayasi, Yutaka; Satyanarayana, T

    2012-01-01

    Acidophiles are ecologically and economically important group of microorganisms, which thrive in acidic natural (solfataric fields, sulfuric pools) as well as artificial man-made (areas associated with human activities such as mining of coal and metal ores) environments. They possess networked cellular adaptations to regulate pH inside the cell. Several extracellular enzymes from acidophiles are known to be functional at much lower pH than the cytoplasmic pH. Enzymes like amylases, proteases, ligases, cellulases, xylanases, α-glucosidases, endoglucanases, and esterases stable at low pH are known from various acidophilic microbes. The possibility of improving them by genetic engineering and directed evolution will further boost their industrial applications. Besides biocatalysts, other biomolecules such as plasmids, rusticynin, and maltose-binding protein have also been reported from acidophiles. Some strategies for circumventing the problems encountered in expressing genes encoding proteins from extreme acidophiles have been suggested. The investigations on the analysis of crystal structures of some acidophilic proteins have thrown light on their acid stability. Attempts are being made to use thermoacidophilic microbes for biofuel production from lignocellulosic biomass. The enzymes from acidophiles are mainly used in polymer degradation.

  1. Mevalonate production by engineered acetogen biocatalyst during continuous fermentation of syngas or CO₂/H₂ blend.

    PubMed

    Kiriukhin, Michael; Tyurin, Michael

    2014-02-01

    Naturally mevalonate-resistant acetogen Clostridium sp. MT1243 produced only 425 mM acetate during syngas fermentation. Using Clostridium sp. MT1243 we engineered biocatalyst selectively producing mevalonate from synthesis gas or CO₂/H₂ blend. Acetate production and spore formation were eliminated from Clostridium sp. MT1243 using Cre-lox66/lox71-system. Cell energy released via elimination of phosphotransacetylase, acetate kinase and early stage sporulation genes powered mevalonate accumulation in fermentation broth due to expression of synthetic thiolase, HMG-synthase, and HMG-reductase, three copies of each, integrated using Tn7-approach. Recombinants produced 145 mM mevalonate in five independent single-step fermentation runs 25 days each in five repeats using syngas blend 60% CO and 40% H₂ (v/v) (p < 0.005). Mevalonate production was 97 mM if only CO₂/H₂ blend was fed instead of syngas (p < 0.005). Mevalonate from CO₂/H₂ blend might serve as a commercial route to mitigate global warming in proportion to CO₂ fermentation scale worldwide.

  2. Recombinant Lipases and Phospholipases and Their Use as Biocatalysts for Industrial Applications.

    PubMed

    Borrelli, Grazia M; Trono, Daniela

    2015-01-01

    Lipases and phospholipases are interfacial enzymes that hydrolyze hydrophobic ester linkages of triacylglycerols and phospholipids, respectively. In addition to their role as esterases, these enzymes catalyze a plethora of other reactions; indeed, lipases also catalyze esterification, transesterification and interesterification reactions, and phospholipases also show acyltransferase, transacylase and transphosphatidylation activities. Thus, lipases and phospholipases represent versatile biocatalysts that are widely used in various industrial applications, such as for biodiesels, food, nutraceuticals, oil degumming and detergents; minor applications also include bioremediation, agriculture, cosmetics, leather and paper industries. These enzymes are ubiquitous in most living organisms, across animals, plants, yeasts, fungi and bacteria. For their greater availability and their ease of production, microbial lipases and phospholipases are preferred to those derived from animals and plants. Nevertheless, traditional purification strategies from microbe cultures have a number of disadvantages, which include non-reproducibility and low yields. Moreover, native microbial enzymes are not always suitable for biocatalytic processes. The development of molecular techniques for the production of recombinant heterologous proteins in a host system has overcome these constraints, as this allows high-level protein expression and production of new redesigned enzymes with improved catalytic properties. These can meet the requirements of specific industrial process better than the native enzymes. The purpose of this review is to give an overview of the structural and functional features of lipases and phospholipases, to describe the recent advances in optimization of the production of recombinant lipases and phospholipases, and to summarize the information available relating to their major applications in industrial processes.

  3. Synthetic biology approaches to improve biocatalyst identification in metagenomic library screening

    PubMed Central

    Guazzaroni, María-Eugenia; Silva-Rocha, Rafael; Ward, Richard John

    2015-01-01

    There is a growing demand for enzymes with improved catalytic performance or tolerance to process-specific parameters, and biotechnology plays a crucial role in the development of biocatalysts for use in industry, agriculture, medicine and energy generation. Metagenomics takes advantage of the wealth of genetic and biochemical diversity present in the genomes of microorganisms found in environmental samples, and provides a set of new technologies directed towards screening for new catalytic activities from environmental samples with potential biotechnology applications. However, biased and low level of expression of heterologous proteins in Escherichia coli together with the use of non-optimal cloning vectors for the construction of metagenomic libraries generally results in an extremely low success rate for enzyme identification. The bottleneck arising from inefficient screening of enzymatic activities has been addressed from several perspectives; however, the limitations related to biased expression in heterologous hosts cannot be overcome by using a single approach, but rather requires the synergetic implementation of multiple methodologies. Here, we review some of the principal constraints regarding the discovery of new enzymes in metagenomic libraries and discuss how these might be resolved by using synthetic biology methods. PMID:25123225

  4. Enzymes from solvent-tolerant microbes: useful biocatalysts for non-aqueous enzymology.

    PubMed

    Gupta, Anshu; Khare, S K

    2009-01-01

    Solvent-tolerant microbes are a newly emerging class that possesses the unique ability to thrive in the presence of organic solvents. Their enzymes adapted to mediate cellular and metabolic processes in a solvent-rich environment and are logically stable in the presence of organic solvents. Enzyme catalysis in non-aqueous/low-water media is finding increasing applications for the synthesis of industrially important products, namely peptides, esters, and other trans-esterification products. Solvent stability, however, remains a prerequisite for employing enzymes in non-aqueous systems. Enzymes, in general, get inactivated or give very low rates of reaction in non-aqueous media. Thus, early efforts, and even some recent ones, have aimed at stabilization of enzymes in organic media by immobilization, surface modifications, mutagenesis, and protein engineering. Enzymes from solvent-tolerant microbes appear to be the choicest source for studying solvent-stable enzymes because of their unique ability to survive in the presence of a range of organic solvents. These bacteria circumvent the solvent's toxic effects by virtue of various adaptations, e.g. at the level of the cytoplasmic membrane, by degradation and transformation of solvents, and by active excretion of solvents. The recent screening of these exotic microbes has generated some naturally solvent-stable proteases, lipases, cholesterol oxidase, cholesterol esterase, cyclodextrin glucanotransferase, and other important enzymes. The unique properties of these novel biocatalysts have great potential for applications in non-aqueous enzymology for a range of industrial processes.

  5. [Biocatalyst of redox mediators on the denitrification by Paracoccus versutus strain GW1].

    PubMed

    Li, Hai-Bo; Lian, Jing; Guo, Yan-Kai; Zhao, Li-Jun; Du, Hai-Feng; Yang, Jing-Liang; Guo, Jian-Bo

    2012-07-01

    The quinone respiration process of Paracoccus versutus strain GW1 was characterized and the effects of the four redox mediators on the denitrification process were studied. The experiment results suggested that quinones were utilized by Paracoccus versutus strain GW1 as electron acceptors in the respiratory chain and reduced to hydroquinone. Batch experiments were carried out to investigate the biocatalyst effect of redox mediators as catalyst on the denitrification process at 35 degrees C. All four redox mediators tested were able to enhance the nitrate removal efficiency and the denitrification efficiency by 1.14-1.63 fold and 1.12-2.02 fold, respectively. The accelerating effect from high to low was AQDS > 1,5-AQDS > AQS > alpha-AQS. In the presence of redox mediators, the stabilized ORP values in the nitrate decomposition process were reduced by 33-75 mV. The pH variations in denitrification with redox mediators showed similar tendency to that of the conventional nitrate removal process. In the concentration range of 0-0.32 mmol x L(-1), AQDS had the best accelerating effect and a linear correlation was found for the denitrification rate K and the AQDS concentration cAQDS. This study indicated that the application of redox mediators significantly improved the denitrification process by enhancing the decomposition rate. PMID:23002627

  6. Efficient aspartic acid production by a psychrophile-based simple biocatalyst.

    PubMed

    Tajima, Takahisa; Hamada, Mai; Nakashimada, Yutaka; Kato, Junichi

    2015-10-01

    We previously constructed a Psychrophile-based Simple bioCatalyst (PSCat) reaction system, in which psychrophilic metabolic enzymes are inactivated by heat treatment, and used it here to study the conversion of aspartic acid from fumaric acid mediated by the activity of aspartate ammonia-lyase (aspartase). In Escherichia coli, the biosynthesis of aspartic acid competes with that of L-malic acid produced from fumaric acid by fumarase. In this study, E. coli aspartase was expressed in psychrophilic Shewanella livingstonensis Ac10 heat treated at 50 °C for 15 min. The resultant PSCat could convert fumaric acid to aspartic acid without the formation of L-malic acid because of heat inactivation of psychrophilic fumarase activity. Furthermore, alginate-immobilized PSCat produced high yields of aspartic acid and could be re-used nine times. The results of our study suggest that PSCat can be applied in biotechnological production as a new approach to increase the yield of target compounds. PMID:26254042

  7. Recombinant Lipases and Phospholipases and Their Use as Biocatalysts for Industrial Applications.

    PubMed

    Borrelli, Grazia M; Trono, Daniela

    2015-01-01

    Lipases and phospholipases are interfacial enzymes that hydrolyze hydrophobic ester linkages of triacylglycerols and phospholipids, respectively. In addition to their role as esterases, these enzymes catalyze a plethora of other reactions; indeed, lipases also catalyze esterification, transesterification and interesterification reactions, and phospholipases also show acyltransferase, transacylase and transphosphatidylation activities. Thus, lipases and phospholipases represent versatile biocatalysts that are widely used in various industrial applications, such as for biodiesels, food, nutraceuticals, oil degumming and detergents; minor applications also include bioremediation, agriculture, cosmetics, leather and paper industries. These enzymes are ubiquitous in most living organisms, across animals, plants, yeasts, fungi and bacteria. For their greater availability and their ease of production, microbial lipases and phospholipases are preferred to those derived from animals and plants. Nevertheless, traditional purification strategies from microbe cultures have a number of disadvantages, which include non-reproducibility and low yields. Moreover, native microbial enzymes are not always suitable for biocatalytic processes. The development of molecular techniques for the production of recombinant heterologous proteins in a host system has overcome these constraints, as this allows high-level protein expression and production of new redesigned enzymes with improved catalytic properties. These can meet the requirements of specific industrial process better than the native enzymes. The purpose of this review is to give an overview of the structural and functional features of lipases and phospholipases, to describe the recent advances in optimization of the production of recombinant lipases and phospholipases, and to summarize the information available relating to their major applications in industrial processes. PMID:26340621

  8. Reduction of Technetium by Desulfovibrio desulfuricans: Biocatalyst Characterization and Use in a Flowthrough Bioreactor

    PubMed Central

    Lloyd, J. R.; Ridley, J.; Khizniak, T.; Lyalikova, N. N.; Macaskie, L. E.

    1999-01-01

    Resting cells of Desulfovibrio desulfuricans coupled the oxidation of a range of electron donors to Tc(VII) reduction. The reduced technetium was precipitated as an insoluble low-valence oxide. The optimum electron donor for the biotransformation was hydrogen, although rapid rates of reduction were also supported when formate or pyruvate was supplied to the cells. Technetium reduction was less efficient when the growth substrates lactate and ethanol were supplied as electron donors, while glycerol, succinate, acetate, and methanol supported negligible reduction. Enzyme activity was stable for several weeks and was insensitive to oxygen. Transmission electron microscopy showed that the radionuclide was precipitated at the periphery of the cell. Cells poisoned with Cu(II), which is selective for periplasmic but not cytoplasmic hydrogenases, were unable to reduce Tc(VII), a result consistent with the involvement of a periplasmic hydrogenase in Tc(VII) reduction. Resting cells, immobilized in a flowthrough membrane bioreactor and supplied with Tc(VII)-supplemented solution, accumulated substantial quantities of the radionuclide when formate was supplied as the electron donor, indicating the potential of this organism as a biocatalyst to treat Tc-contaminated wastewaters. PMID:10347062

  9. Multilayer enzyme-coupled magnetic nanoparticles as efficient, reusable biocatalysts and biosensors.

    PubMed

    Garcia, Josep; Zhang, Yue; Taylor, Hannah; Cespedes, Oscar; Webb, Michael E; Zhou, Dejian

    2011-09-01

    Herein we report the development of a highly active, magnetically retrievable and reusable biocatalyst using multilayer enzyme coupled-magnetic nanoparticles (MNPs) prepared by layer-by-layer assembly using two well-studied enzymes, horseradish peroxidase (HRP) and glucose oxidase (GOX), as a model enzyme system. We show that by combining the use of a biocompatible linker as well as biospecific immobilisation, the first layer enzyme in our HRP(1)-MNP system retains the native activity of the enzyme in solution, and the overall catalytic activity of the multilayer enzyme system, HRP(x)-MNP, increases linearly with the increasing number of enzyme layers. Furthermore, the HRP(x)-MNP system can be conveniently retrieved by using an external magnetic field and reused for 10 consecutive cycles without apparent reduction of catalytic activity. We also report the development of a novel coupled bienzyme, GOX/HRP(x)-MNP, system that can perform bi-enzymatic reactions to couple the colourless GOX-catalyzed reaction to the chromophoric HRP-catalyzed reaction via H(2)O(2) production. This model bienzyme-MNP system can be used for simple, rapid colorimetric quantification of micromolar glucose. PMID:21792451

  10. Preparation of a stable biocatalyst of bovine liver catalase using immobilization and postimmobilization techniques.

    PubMed

    Betancor, Lorena; Hidalgo, Aurelio; Fernández-Lorente, Gloria; Mateo, Cesar; Fernández-Lafuente, Roberto; Guisan, José M

    2003-01-01

    Bovine liver catalase was immobilized on different supports. The tetrameric nature of this enzyme was found to cause its rapid inactivation in diluted conditions due to subunit dissociation, a fact that may rule out its industrial use. Multi-subunit immobilization using highly activated glyoxyl agarose was not enough to involve all enzyme subunits. In fact, washing the derivative produced a strong decrease in the enzyme activity. Further cross-linking of previously immobilized enzyme with tailor-made dextran-aldehyde permitted the multimeric structure to be fully stabilized using either multisubunit preparations immobilized onto highly activated glyoxyl-agarose support or one subunit enzymes immobilized onto poorly activated glyoxyl-agarose. The highest stability of the final biocatalyst was observed using the multisubunit immobilized derivative cross-linked with dextran-aldehyde. The optimal derivative retained around 60% of the immobilized activity, did not release any enzyme subunits after boiling in the presence of SDS, and did not lose activity during washing, and its stability did not depend on the dilution. This derivative was used for 10 cycles in the destruction of 10 mM hydrogen peroxide without any decrease in the enzyme activity. PMID:12790636

  11. Synthetic biology approaches to improve biocatalyst identification in metagenomic library screening.

    PubMed

    Guazzaroni, María-Eugenia; Silva-Rocha, Rafael; Ward, Richard John

    2015-01-01

    There is a growing demand for enzymes with improved catalytic performance or tolerance to process-specific parameters, and biotechnology plays a crucial role in the development of biocatalysts for use in industry, agriculture, medicine and energy generation. Metagenomics takes advantage of the wealth of genetic and biochemical diversity present in the genomes of microorganisms found in environmental samples, and provides a set of new technologies directed towards screening for new catalytic activities from environmental samples with potential biotechnology applications. However, biased and low level of expression of heterologous proteins in Escherichia coli together with the use of non-optimal cloning vectors for the construction of metagenomic libraries generally results in an extremely low success rate for enzyme identification. The bottleneck arising from inefficient screening of enzymatic activities has been addressed from several perspectives; however, the limitations related to biased expression in heterologous hosts cannot be overcome by using a single approach, but rather requires the synergetic implementation of multiple methodologies. Here, we review some of the principal constraints regarding the discovery of new enzymes in metagenomic libraries and discuss how these might be resolved by using synthetic biology methods.

  12. Incorporating unnatural amino acids to engineer biocatalysts for industrial bioprocess applications.

    PubMed

    Ravikumar, Yuvaraj; Nadarajan, Saravanan Prabhu; Hyeon Yoo, Tae; Lee, Chong-Soon; Yun, Hyungdon

    2015-12-01

    The bioprocess engineering with biocatalysts broadly spans its development and actual application of enzymes in an industrial context. Recently, both the use of bioprocess engineering and the development and employment of enzyme engineering techniques have been increasing rapidly. Importantly, engineering techniques that incorporate unnatural amino acids (UAAs) in vivo has begun to produce enzymes with greater stability and altered catalytic properties. Despite the growth of this technique, its potential value in bioprocess applications remains to be fully exploited. In this review, we explore the methodologies involved in UAA incorporation as well as ways to synthesize these UAAs. In addition, we summarize recent efforts to increase the yield of UAA engineered proteins in Escherichia coli and also the application of this tool in enzyme engineering. Furthermore, this protein engineering tool based on the incorporation of UAA can be used to develop immobilized enzymes that are ideal for bioprocess applications. Considering the potential of this tool and by exploiting these engineered enzymes, we expect the field of bioprocess engineering to open up new opportunities for biocatalysis in the near future.

  13. Production and optimization of biodiesel using mixed immobilized biocatalysts in packed bed reactor.

    PubMed

    Bakkiyaraj, S; Syed, Mahin Basha; Devanesan, M G; Thangavelu, Viruthagiri

    2016-05-01

    Vegetable oils are used as raw materials for biodiesel production using transesterification reaction. Several methods for the production of biodiesel were developed using chemical (alkali and acidic compounds) and biological catalysts (lipases). Biodiesel production catalyzed by lipases is energy and cost-saving processes and is carried out at normal temperature and pressure. The need for an efficient method for screening larger number of variables has led to the adoption of statistical experimental design. In the present study, packed bed reactor was designed to study with mixed immobilized biocatalysts to have higher productivity under optimum conditions. Contrary to the single-step acyl migration mechanism, a two-step stepwise reaction mechanism involving immobilized Candida rugosa lipase and immobilized Rhizopus oryzae cells was employed for the present work. This method was chosen because enzymatic hydrolysis followed by esterification can tolerate high free fatty acid containing oils. The effects of flow rate and bed height on biodiesel yield were studied using two factors five-level central composite design (CCD) and response surface methodology (RSM). Maximum biodiesel yield of 85 and 81 % was obtained for jatropha oil and karanja oil with the optimum bed height and optimum flow rate of 32.6 cm and 1.35 L/h, and 32.6 cm and 1.36 L/h, respectively.

  14. Process optimization of enzyme catalyzed production of dietary diacylglycerol (DAG) using TLIM as biocatalyst.

    PubMed

    Dhara, Rupali; Singhal, Rekha S

    2014-01-01

    Diacylglycerol (DAG)-rich sunflower oil was prepared and the optimal conditions for synthesis of DAG-rich oil by glycerolysis using biocatalyst TLIM was determined. A maximum production of 59.8% DAG was obtained after 5 h of constant reaction under vacuum (756 mm of Hg). The optimum temperature for glycerolysis was found to be 50°C, while stoichiometric molar ratio of sunflower oil:glycerol was 2:1 for this reaction. A minimum acid value of 0.48 mg of KOH.g(-1) of oil was observed under these conditions. The fatty acid composition of DAG-rich oil was found to be similar to the original TAG-rich sunflower oil used in the work. The lipase catalysed glycerolysis using 1,3 specific lipase was used to promote the formation of 1,3 isoform of DAG as this isoform is known to possess anti-obesity effect. DAG content was determined by HPTLC and GCMS. The DAG-rich oil contained 59.75% DAG of which 63.34% was found as 1,3-DAG and 36.65% was 1,2-DAG/2,3-DAG.

  15. Recombinant Lipases and Phospholipases and Their Use as Biocatalysts for Industrial Applications

    PubMed Central

    Borrelli, Grazia M.; Trono, Daniela

    2015-01-01

    Lipases and phospholipases are interfacial enzymes that hydrolyze hydrophobic ester linkages of triacylglycerols and phospholipids, respectively. In addition to their role as esterases, these enzymes catalyze a plethora of other reactions; indeed, lipases also catalyze esterification, transesterification and interesterification reactions, and phospholipases also show acyltransferase, transacylase and transphosphatidylation activities. Thus, lipases and phospholipases represent versatile biocatalysts that are widely used in various industrial applications, such as for biodiesels, food, nutraceuticals, oil degumming and detergents; minor applications also include bioremediation, agriculture, cosmetics, leather and paper industries. These enzymes are ubiquitous in most living organisms, across animals, plants, yeasts, fungi and bacteria. For their greater availability and their ease of production, microbial lipases and phospholipases are preferred to those derived from animals and plants. Nevertheless, traditional purification strategies from microbe cultures have a number of disadvantages, which include non-reproducibility and low yields. Moreover, native microbial enzymes are not always suitable for biocatalytic processes. The development of molecular techniques for the production of recombinant heterologous proteins in a host system has overcome these constraints, as this allows high-level protein expression and production of new redesigned enzymes with improved catalytic properties. These can meet the requirements of specific industrial process better than the native enzymes. The purpose of this review is to give an overview of the structural and functional features of lipases and phospholipases, to describe the recent advances in optimization of the production of recombinant lipases and phospholipases, and to summarize the information available relating to their major applications in industrial processes. PMID:26340621

  16. Identification and Characterization of Lipopeptides from Bacillus subtilis B1 Against Sapstain Fungus of Rubberwood Through MALDI-TOF-MS and RT-PCR.

    PubMed

    Sajitha, K L; Dev, Suma Arun; Maria Florence, E J

    2016-07-01

    Bacillus subtilis is a potent biocontrol agent producing a wide array of antifungal lipopeptides for the inhibition of fungal growth. B. subtilis B1 isolated from market-available compost provided an efficient control of rubberwood sapstain fungus, Lasiodiplodia theobromae. The current study is aimed to identify and characterize the lipopeptides responsible for the biocontrol of rubberwood sapstain fungus by Bacillus subtilis B1. The bacterial whole-cell surface extract from the dual culture of B. subtilis B1 and sapstain fungus (L. theobromae) was analysed using MALDI-TOF-MS. The protonated as well as sodium, potassium adducts of homologues of iturin C, surfactin, bacillomycin D and fengycin A and B were identified and expression of the lipopeptide biosynthetic genes could be confirmed through RT-PCR. This is the first report of mycobacillin and trimethylsilyl derivative of bacilysin during antagonism through MALDI-TOF-MS. MALDI-TOF-MS with RT-PCR offered easy platforms to characterize the antifungal lipopeptides. The identification of antifungal lipopeptides can lead to the formulation of prospective biocontrol by-products which have wide-scale utility. PMID:27004481

  17. Ant-fungus species combinations engineer physiological activity of fungus gardens.

    PubMed

    Seal, J N; Schiøtt, M; Mueller, U G

    2014-07-15

    Fungus-gardening insects are among the most complex organisms because of their extensive co-evolutionary histories with obligate fungal symbionts and other microbes. Some fungus-gardening insect lineages share fungal symbionts with other members of their lineage and thus exhibit diffuse co-evolutionary relationships, while others exhibit little or no symbiont sharing, resulting in host-fungus fidelity. The mechanisms that maintain this symbiont fidelity are currently unknown. Prior work suggested that derived leaf-cutting ants in the genus Atta interact synergistically with leaf-cutter fungi (Attamyces) by exhibiting higher fungal growth rates and enzymatic activities than when growing a fungus from the sister-clade to Attamyces (so-called 'Trachymyces'), grown primarily by the non-leaf cutting Trachymyrmex ants that form, correspondingly, the sister-clade to leaf-cutting ants. To elucidate the enzymatic bases of host-fungus specialization in leaf-cutting ants, we conducted a reciprocal fungus-switch experiment between the ant Atta texana and the ant Trachymyrmex arizonensis and report measured enzymatic activities of switched and sham-switched fungus gardens to digest starch, pectin, xylan, cellulose and casein. Gardens exhibited higher amylase and pectinase activities when A. texana ants cultivated Attamyces compared with Trachymyces fungi, consistent with enzymatic specialization. In contrast, gardens showed comparable amylase and pectinase activities when T. arizonensis cultivated either fungal species. Although gardens of leaf-cutting ants are not known to be significant metabolizers of cellulose, T. arizonensis were able to maintain gardens with significant cellulase activity when growing either fungal species. In contrast to carbohydrate metabolism, protease activity was significantly higher in Attamyces than in Trachymyces, regardless of the ant host. Activity of some enzymes employed by this symbiosis therefore arises from complex interactions between the

  18. Production of microparticles of molinate degrading biocatalysts using the spray drying technique.

    PubMed

    Lopes, Ana R; Sousa, Vera M; Estevinho, Berta N; Leite, José P; Moreira, Nuno F F; Gales, Luís; Rocha, Fernando; Nunes, Olga C

    2016-10-01

    Previous studies demonstrated the capability of mixed culture DC1 to mineralize the thiocarbamate herbicide molinate through the activity of molinate hydrolase (MolA). Because liquid suspensions are not compatible with long-term storage and are not easy to handle when bioremediation strategies are envisaged, in this study spray drying was evaluated as a cost-effective method to store and transport these molinate biocatalysts. Microparticles of mixed culture DC1 (DC1) and of cell free crude extracts containing MolA (MA) were obtained without any carrier polymer, and with calcium alginate (CA) or modified chitosan (MCt) as immobilizing agents. All the DC1 microparticles showed high molinate degrading activity upon storage for 6 months, or after 9 additions of ∼0.4 mM molinate over 1 month. The DC1-MCt microparticles were those with the highest survival rate and lowest heterogeneity. For MA microparticles, only MA-MCt degraded molinate. However, its Vmax was only 1.4% of that of the fresh cell free extract (non spray dried). The feasibility of using the DC1-MCt and MA-MCt microparticles in bioaugmentation processes was assessed in river water microcosms, using mass (g):volume (L) ratios of 1:13 and 1:0.25, respectively. Both type of microparticles removed ∼65-75% of the initial 1.5 mg L(-1) molinate, after 7 days of incubation. However, only DC1-MCt microparticles were able to degrade this environmental concentration of molinate without disturbing the native bacterial community. These results suggest that spray drying can be successfully used to produce DC1-MCt microparticles to remediate molinate polluted sites through a bioaugmentation strategy.

  19. [Report on a fungus parasitizing Entamoeba histolytica].

    PubMed

    Cao, C Q; Feng, Y S

    1989-01-01

    Infection of Entamoeba histolytica with chytridiaceous fungus Sphaerita was observed in some specimens obtained from a farmer and stained with iron-haematoxylin. The fungi were found in 78% of the cysts, mostly immature ones. Within the amoebae this parasite occurred singly, in groups, or in the form of a sporangium. It was located in the cytoplasm, the glycogen mass or the chromatoidal bars. In the same specimen, the parasitic fungus was also found in 18% of E. coli cysts; in 11% of E. nana cysts; while only one of 16 E. hartmanni cysts was parasitized. It is an interesting case of superimposed parasitism so far reported in China as well as a rare case of several species of amoebae being heavily involved with the same in the scientific literature. PMID:2548767

  20. Characterization of CHO-K1 cells stably expressing PDE-IV enzymes. Whole-cell cAMP determinations vs broken-cell enzymatic assays.

    PubMed

    Pon, D J; Plant, M; Tkach, J; Boulet, L; Muise, E; Allen, R A; Rodger, I W

    1998-01-01

    A CHO-K1 cell line stably expressing a recombinant full-length human PDE-IVa (HSPDE4A4B) enzyme was established under hygromycin B selection. Full-length expression of the protein was determined by Western blot analysis, which revealed the presence of a 125-kDa immunoreactive band using rabbit anti-PDE-IVa antibodies. The potency of inhibitor compounds was examined by their ability to increase cAMP in the whole-cell, and by their ability to inhibit cAMP hydrolysis in a 100,000 g supernatant (soluble enzyme preparation) obtained from the same cell line. Inhibition of the expressed PDE-IVa activity by selective PDE-IV inhibitors--(R) and (S)-rolipram, RS 14203, and CDP 840--at 100 nM substrate demonstrated that RS 14203 and CDP 840 were the most potent with IC50 = 9 nM, followed by (R)-rolipram (IC50 = 110 nM) and (S)-rolipram (IC50 = 420 nM). The rank order of potencies of the inhibitors in elevating cAMP in the whole-cell assay was quite different from that on the soluble enzyme. RS 14203 was still the most potent compound in elevating cAMP. Moreover, the relative rank order of potencies between CDP 840 and (R)-rolipram changed dramatically, such that (R)-rolipram was more potent than CDP 840 = (S)-rolipram. An apparent 30-fold stereoselectivity between (R)- and (S)-rolipram was also noted. The whole-cell rank order of potencies was also maintained when PKA activity ratios were measured in place of cAMP levels. The ability of the compounds to elevate cAMP in the stable CHO-K1 cells appeared to track better with the potency of the compounds against the high-affinity (Sr) conformer of the enzyme rather than the low-affinity catalytic state.

  1. Identification and use of an alkane transporter plug-in for applications in biocatalysis and whole-cell biosensing of alkanes

    NASA Astrophysics Data System (ADS)

    Grant, Chris; Deszcz, Dawid; Wei, Yu-Chia; Martínez-Torres, Rubéns Julio; Morris, Phattaraporn; Folliard, Thomas; Sreenivasan, Rakesh; Ward, John; Dalby, Paul; Woodley, John M.; Baganz, Frank

    2014-07-01

    Effective application of whole-cell devices in synthetic biology and biocatalysis will always require consideration of the uptake of molecules of interest into the cell. Here we demonstrate that the AlkL protein from Pseudomonas putida GPo1 is an alkane import protein capable of industrially relevant rates of uptake of C7-C16 n-alkanes. Without alkL expression, native E.coli n-alkane uptake was the rate-limiting step in both the whole-cell bioconversion of C7-C16 n-alkanes and in the activation of a whole-cell alkane biosensor by C10 and C11 alkanes. By coexpression of alkL as a transporter plug-in, specific yields improved by up to 100-fold for bioxidation of >C12 alkanes to fatty alcohols and acids. The alkL protein was shown to be toxic to the host when overexpressed but when expressed from a vector capable of controlled induction, yields of alkane oxidation were improved a further 10-fold (8 g/L and 1.7 g/g of total oxidized products). Further testing of activity on n-octane with the controlled expression vector revealed the highest reported rates of 120 μmol/min/g and 1 g/L/h total oxidized products. This is the first time AlkL has been shown to directly facilitate enhanced uptake of C10-C16 alkanes and represents the highest reported gain in product yields resulting from its use.

  2. Novel biocatalysts based on S-layer self-assembly of Geobacillus stearothermophilus NRS 2004/3a: a nanobiotechnological approach.

    PubMed

    Schäffer, Christina; Novotny, René; Küpcü, Seta; Zayni, Sonja; Scheberl, Andrea; Friedmann, Jacqueline; Sleytr, Uwe B; Messner, Paul

    2007-09-01

    The crystalline cell-surface (S) layer sgsE of Geobacillus stearothermophilus NRS 2004/3a represents a natural protein self-assembly system with nanometer-scale periodicity that is evaluated as a combined carrier/patterning element for the conception of novel types of biocatalyst aiming at the controllable display of biocatalytic epitopes, storage stability, and reuse. The glucose-1-phosphate thymidylyltransferase RmlA is used as a model enzyme and chimeric proteins are constructed by translational fusion of rmlA to the C-terminus of truncated forms of sgsE (rSgsE (131-903), rSgsE(331-903)) and used for the construction of three principal types of biocatalysts: soluble (monomeric), self-assembled in aqueous solution, and recrystallized on negatively charged liposomes. Enzyme activity of the biocatalysts reaches up to 100 % compared to sole RmlA cloned from the same bacterium. The S-layer portion of the biocatalysts confers significantly improved shelf life to the fused enzyme without loss of activity over more than three months, and also enables biocatalyst recycling. These nanopatterned composites may open up new functional concepts for biocatalytic applications in nanobiotechnology. PMID:17786898

  3. New tricycloalternarenes from fungus Alternaria sp.

    PubMed

    Shi, Xiu; Wei, Wei; Zhang, Wen-Jing; Hua, Cheng-Pin; Chen, Chao-Jun; Ge, Hui-Ming; Tan, Ren-Xiang; Jiao, Rui-Hua

    2015-01-01

    Two new tricycloalternarenes I (1) and J (2), together with five known derivatives (3-7), were isolated from the culture of marine fungus Alternaria sp. The structures were elucidated by a combination of spectroscopic approach ((1)H, (13)C NMR, HMBC, COSY, and NOESY) and the low-temperature (100 K) single-crystal X-ray crystallography analysis. The antimicrobial assays of tricycloalternarenes I (1) and J (2) were tested.

  4. Reduced risk of pertussis in whole-cell compared to acellular vaccine recipients is not confounded by age or receipt of booster-doses.

    PubMed

    Sheridan, Sarah L; Ware, Robert S; Grimwood, Keith; Lambert, Stephen B

    2015-09-22

    Several observational studies provide evidence that acellular pertussis vaccines (aP) are less protective against pertussis disease than highly effective whole-cell pertussis vaccines (wP), however, concerns have been raised that some of these findings may be confounded by age. By undertaking age-stratified and restricted analyses on a cohort of Australian children primed with either aP-only, wP-only or mixed pertussis vaccine schedules, we demonstrate that compared to aP the association of wP with increased protection from pertussis is not confounded by age, nor by aP booster-dose receipt.

  5. Correlation between the physicochemical properties of organic solvents and their biocompatibility toward epoxide hydrolase activity in whole-cells of a yeast, Rhodotorula sp.

    PubMed

    Lotter, Jeanette; Botes, Adriana L; Van Dyk, Martha S; Breytenbach, Jaco C

    2004-08-01

    Epoxides are often highly hydrophobic substrates and the presence of an organic co-solvent within an aqueous bioreactor is in such cases indicated. The effect of 40 water-miscible and -immiscible organic solvents on epoxide hydrolase activity in whole-cells of the yeast Rhodotorula sp. UOFS Y-0448 was investigated. No formal correlation between solvent biocompatibility and physicochemical properties was deductible, although the introduction of hydroxyl groups increased biocompatibility. 1-Pentanol, 2-methylcyclohexanol and 1-octanol were the most biocompatible resulting in relatively low activity losses when used at up to 20% (v/v).

  6. Genome tailoring powered production of isobutanol in continuous CO2/H2 blend fermentation using engineered acetogen biocatalyst.

    PubMed

    Gak, Eugene; Tyurin, Michael; Kiriukhin, Michael

    2014-05-01

    The cell energy fraction that powered maintenance and expression of genes encoding pro-phage elements, pta-ack cluster, early sporulation, sugar ABC transporter periplasmic proteins, 6-phosphofructokinase, pyruvate kinase, and fructose-1,6-disphosphatase in acetogen Clostridium sp. MT871 was re-directed to power synthetic operon encoding isobutanol biosynthesis at the expense of these genes achieved via their elimination. Genome tailoring decreased cell duplication time by 7.0 ± 0.1 min (p < 0.05) compared to the parental strain, with intact genome and cell duplication time of 68 ± 1 min (p < 0.05). Clostridium sp. MT871 with tailored genome was UVC-mutated to withstand 6.1 % isobutanol in fermentation broth to prevent product inhibition in an engineered commercial biocatalyst producing 5 % (674.5 mM) isobutanol during two-step continuous fermentation of CO2/H2 gas blend. Biocatalyst Clostridium sp. MT871RG- 11IBR6 was engineered to express six copies of synthetic operon comprising optimized synthetic format dehydrogenase, pyruvate formate lyase, acetolactate synthase, acetohydroxyacid reductoisomerase, 2,3-dihydroxy-isovalerate dehydratase, branched-chain alpha-ketoacid decarboxylase gene, aldehyde dehydrogenase, and alcohol dehydrogenase, regaining cell duplication time of 68 ± 1 min (p < 0.05) for the parental strain. This is the first report on isobutanol production by an engineered acetogen biocatalyst suitable for commercial manufacturing of this chemical/fuel using continuous fermentation of CO2/H2 blend thus contributing to the reversal of global warming.

  7. Confluence of structural and chemical biology: plant polyketide synthases as biocatalysts for a bio-based future.

    PubMed

    Stewart, Charles; Vickery, Christopher R; Burkart, Michael D; Noel, Joseph P

    2013-06-01

    Type III plant polyketide synthases (PKSs) biosynthesize a dazzling array of polyphenolic products that serve important roles in both plant and human health. Recent advances in structural characterization of these enzymes and new tools from the field of chemical biology have facilitated exquisite probing of plant PKS iterative catalysis. These tools have also been used to exploit type III PKSs as biocatalysts to generate new chemicals. Going forward, chemical, structural and biochemical analyses will provide an atomic resolution understanding of plant PKSs and will serve as a springboard for bioengineering and scalable production of valuable molecules in vitro, by fermentation and in planta.

  8. Development of A Flexible System for the Simultaneous Conversion of Biomass to Industrial Chemicals and the Production of Industrial Biocatalysts

    SciTech Connect

    Gao, Johnway; Hooker, Brian S.; Skeen, R S.; Anderson, D B.; Lankey, R. L.; Anastas, P. T.

    2002-01-01

    A flexible system was developed for the simultaneous conversion of biomass to industrial chemicals and the production of industrial biocatalysts. In particular, the expression of a bacterial enzyme, beta-glucuronidase (GUS), was investigated using a genetically modified starch-degrading Saccharomyces strain in suspension cultures in starch media. Different sources of starch including corn and waste potato starch were used for yeast biomass accumulation and GUS expression studies under controls of inducible and constitutive promoters. A thermostable bacterial cellulase, Acidothermus cellulolyticus E1 endoglucanase gene was also cloned into an episomal plasmid expression vector and expressed in the starch-degrading Saccharomyces strain.

  9. Mining for hemicellulases in the fungus-growing termite Pseudacanthotermes militaris using functional metagenomics

    PubMed Central

    2013-01-01

    degradation of biomass within the termite mound, although further investigation will be needed to clarify the complex synergies that might exist between the different microbiomes that constitute the termitosphere of fungus-growing termites. This study exemplifies the power of functional metagenomics for the discovery of biomass-active enzymes and has provided a collection of potentially interesting biocatalysts for further study. PMID:23672637

  10. Differentiation and numerical analysis of oral yeasts based on SDS-Page profiles. Influence of the culture media on the whole-cell protein extracts.

    PubMed

    Höfling, J F; Rosa, E A; Pereira, C V; Boriollo, M F; Rodrigues, J A

    2001-08-01

    The application of gel electrophoresis and numerical analysis of yeast soluble proteins analysis to the investigation of 12 oral yeast strains belonging to five species is described. It involves one-dimensional electrophoresis of SDS-solubilized whole-cell proteins using different culture media for the cultivation of the cells, integration densitometries in the areas of the gels and percentages of the proteins extraction. These extracts were prepared from four isolates of Candida albicans, two of C. tropicalis, C. guilliermondii, C. parapsilosis and C. krusei. The extracts from whole-cells proteins using different culture media for the cultivation of the cells were fractionated by slab electrophoresis using a discontinuous buffer system. The corresponding patterns showed at least 36 polypeptides in the range of 14.4-200 kDa. Different isolates of each species were clearly different in each of the five species. The data obtained suggest that different nutritional compositions led to the expression of different proteins derived from alternatives metabolic pathways expressed by the electrophoretic profiles. The construction of a database of protein fingerprints and numerical analysis based on such data, may have some implications in the classification and identification of such species with epidemiological, ecological and taxonomic purposes. A well defined or synthetic culture media seems to be much properly.

  11. Serological diagnosis of pertussis: evaluation of IgA against whole cell and specific Bordetella pertussis antigens as markers of recent infection.

    PubMed Central

    Poynten, M.; Hanlon, M.; Irwig, L.; Gilbert, G. L.

    2002-01-01

    In Australia, notification of pertussis cases in older children or adults has increased significantly in recent years. In most cases, laboratory diagnosis is based only on a positive serological test for IgA antibody against whole cell Bordetella pertussis. During a 3-month period, 318 consecutive sera submitted for diagnosis of pertussis were tested for IgA antibody against whole cell (WC) sonicated B. pertussis, pertussis toxin (PT), filamentous haemagglutinin (FHA) and pertactin (PRN). Results of one or more of these tests were positive in sera from 175 subjects and clinical information was obtained by telephone interview from 90 subjects. Using a clinical case definition as the reference standard, the sensitivities of the four IgA assays were variable but quite low (24-64%), but the specificities were high (93-98%). For diagnosis of pertussis in subjects with a compatible clinical illness, these and other findings support the use of serological testing for IgA antibody. PMID:12002533

  12. Mapping N-linked Glycosylation Sites in the Secretome and Whole Cells of Aspergillus niger Using Hydrazide Chemistry and Mass Spectrometry

    SciTech Connect

    Wang, Lu; Aryal, Uma K.; Dai, Ziyu; Mason, Alisa C.; Monroe, Matthew E.; Tian, Zhixin; Zhou, Jianying; Su, Dian; Weitz, Karl K.; Liu, Tao; Camp, David G.; Smith, Richard D.; Baker, Scott E.; Qian, Weijun

    2012-01-01

    Protein glycosylation is known to play an essential role in both cellular functions and the secretory pathways; however, little information is available on the dynamics of glycosylated N-linked glycosites of fungi. Herein we present the first extensive mapping of glycosylated N-linked glycosites in industrial strain Aspergillus niger by applying an optimized solid phase enrichment of glycopeptide protocol using hydrazide modified magnetic beads. The enrichment protocol was initially optimized using mouse plasma and A. niger secretome samples, which was then applied to profile N-linked glycosites from both the secretome and whole cell lysates of A. niger. A total of 847 unique N-linked glycosites and 330 N-linked glycoproteins were confidently identified by LC-MS/MS. Based on gene ontology analysis, the identified N-linked glycoproteins in the whole cell lysate were primarily localized in the plasma membrane, endoplasmic reticulum, golgi apparatus, lysosome, and storage vacuoles. The identified N-linked glycoproteins are involved in a wide range of biological processes including gene regulation and signal transduction, protein folding and assembly, protein modification and carbohydrate metabolism. The extensive coverage of glycosylated N-linked glycosites along with identification of partial N-linked glycosylation in those enzymes involving in different biochemical pathways provide useful information for functional studies of N-linked glycosylation and their biotechnological applications in A. niger.

  13. A multiscale computational model of spatially resolved calcium cycling in cardiac myocytes: from detailed cleft dynamics to the whole cell concentration profiles

    PubMed Central

    Vierheller, Janine; Neubert, Wilhelm; Falcke, Martin; Gilbert, Stephen H.; Chamakuri, Nagaiah

    2015-01-01

    Mathematical modeling of excitation-contraction coupling (ECC) in ventricular cardiac myocytes is a multiscale problem, and it is therefore difficult to develop spatially detailed simulation tools. ECC involves gradients on the length scale of 100 nm in dyadic spaces and concentration profiles along the 100 μm of the whole cell, as well as the sub-millisecond time scale of local concentration changes and the change of lumenal Ca2+ content within tens of seconds. Our concept for a multiscale mathematical model of Ca2+ -induced Ca2+ release (CICR) and whole cardiomyocyte electrophysiology incorporates stochastic simulation of individual LC- and RyR-channels, spatially detailed concentration dynamics in dyadic clefts, rabbit membrane potential dynamics, and a system of partial differential equations for myoplasmic and lumenal free Ca2+ and Ca2+-binding molecules in the bulk of the cell. We developed a novel computational approach to resolve the concentration gradients from dyadic space to cell level by using a quasistatic approximation within the dyad and finite element methods for integrating the partial differential equations. We show whole cell Ca2+-concentration profiles using three previously published RyR-channel Markov schemes. PMID:26441674

  14. Simultaneous recording of the action potential and its whole-cell associated ion current on NG108-15 cells cultured over a MWCNT electrode

    NASA Astrophysics Data System (ADS)

    Morales-Reyes, I.; Seseña-Rubfiaro, A.; Acosta-García, M. C.; Batina, N.; Godínez-Fernández, R.

    2016-08-01

    It is well known that, in excitable cells, the dynamics of the ion currents (I i) is extremely important to determine both the magnitude and time course of an action potential (A p). To observe these two processes simultaneously, we cultured NG108-15 cells over a multi-walled carbon nanotubes electrode (MWCNTe) surface and arranged a two independent Patch Clamp system configuration (Bi-Patch Clamp). The first system was used in the voltage or current clamp mode, using a glass micropipette as an electrode. The second system was modified to connect the MWCNTe to virtual ground. While the A p was recorded through the micropipette electrode, the MWCNTe was used to measure the underlying whole-cell current. This configuration allowed us to record both the membrane voltage (V m) and the current changes simultaneously. Images acquired by atomic force microscopy (AFM) and scanning electron microscopy (SEM) indicate that cultured cells developed a complex network of neurites, which served to establish the necessary close contact and strong adhesion to the MWCNTe surface. These features were a key factor to obtain the recording of the whole-cell I i with a high signal to noise ratio (SNR). The experimental results were satisfactorily reproduced by a theoretical model developed to simulate the proposed system. Besides the contribution to a better understanding of the fundamental mechanisms involved in cell communication, the developed method could be useful in cell physiology studies, pharmacology and diseases diagnosis.

  15. Electron transfer from Proteus vulgaris to a covalently assembled, single walled carbon nanotube electrode functionalised with osmium bipyridine complex: application to a whole cell biosensor.

    PubMed

    Rawson, Frankie J; Garrett, David J; Leech, Donal; Downard, Alison J; Baronian, Keith H R

    2011-01-15

    We report the fabrication and use of electrodes constructed from single walled carbon nanotubes (SWCNTs) chemically assembled on a carbon surface and functionalised with an osmium(II) bipyridine complex (Osbpy). The ability of the electrodes to transduce biologically generated currents from Proteus vulgaris has been established. Our investigations show that there are two contributions to the current: one from electroactive species secreted into solution and another from cell redox sites. The modified electrode can be used to monitor cell metabolism, thereby acting as a whole cell biosensor. The biosensor was used in a 1-h assay to investigate the toxicity of ethanol, sodium azide and the antibiotic ampicillin and gave quantitative data that were closely correlated with standard cell plate viability assays. The results provide proof of principle that the whole cell biosensor could be used for high throughput screening of antimicrobial activity. One of the modified electrodes was used for approximately 1000 measurements over four months demonstrating the robustness of the system.

  16. Anesthetized- and awake-patched whole-cell recordings in freely moving rats using UV-cured collar-based electrode stabilization.

    PubMed

    Lee, Doyun; Shtengel, Gleb; Osborne, Jason E; Lee, Albert K

    2014-12-01

    Intracellular recording allows precise measurement and manipulation of individual neurons, but it requires stable mechanical contact between the electrode and the cell membrane, and thus it has remained challenging to perform in behaving animals. Whole-cell recordings in freely moving animals can be obtained by rigidly fixing ('anchoring') the pipette electrode to the head; however, previous anchoring procedures were slow and often caused substantial pipette movement, resulting in loss of the recording or of recording quality. We describe a UV-transparent collar and UV-cured adhesive technique that rapidly (within 15 s) anchors pipettes in place with virtually no movement, thus substantially improving the reliability, yield and quality of freely moving whole-cell recordings. Recordings are first obtained from anesthetized or awake head-fixed rats. UV light cures the thin adhesive layers linking pipette to collar to head. Then, the animals are rapidly and smoothly released for recording during unrestrained behavior. The anesthetized-patched version can be completed in ∼4-7 h (excluding histology) and the awake-patched version requires ∼1-4 h per day for ∼2 weeks. These advances should greatly facilitate studies of neuronal integration and plasticity in identified cells during natural behaviors. PMID:25375992

  17. Differentiation of Shewanella putrefaciens and Shewanella alga on the basis of whole-cell protein profiles, ribotyping, phenotypic characterization, and 16S rRNA gene sequence analysis.

    PubMed Central

    Vogel, B F; Jørgensen, K; Christensen, H; Olsen, J E; Gram, L

    1997-01-01

    Seventy-six presumed Shewanella putrefaciens isolates from fish, oil drillings, and clinical specimens, the type strain of Shewanella putrefaciens (ATCC 8071), the type strain of Shewanella alga (IAM 14159), and the type strain of Shewanella hanedai (ATCC 33224) were compared by several typing methods. Numerical analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell protein and ribotyping patterns showed that the strains were separated into two distinct clusters with 56% +/- 10% and 40% +/- 14% similarity for whole-cell protein profiling and ribotyping, respectively. One cluster consisted of 26 isolates with 52 to 55 mol% G + C and included 15 human isolates, mostly clinical specimens, 8 isolates from marine waters, and the type strain of S. alga. This homogeneous cluster of mesophilic, halotolerant strains was by all analyses identical to the recently defined species S. alga (U. Simidu et al., Int. J. Syst. Bacteriol, 40:331-336, 1990). Fifty-two typically psychrotolerant strains formed the other, more heterogeneous major cluster, with 43 to 47 mol% G + C. The type strain of S. putrefaciens was included in this group. The two groups were confirmed by 16S rRNA gene sequence analysis. It is concluded that the isolates must be considered two different species, S. alga and S. putrefaciens, and that most mesophilic isolates formerly identified as S. putrefaciens belong to S. alga. The ecological role and potential pathogenicity of S. alga can be evaluated only if the organism is correctly identified. PMID:9172338

  18. Displaying Candida antarctica lipase B on the cell surface of Aspergillus niger as a potential food-grade whole-cell catalyst.

    PubMed

    Pan, Zhi-You; Yang, Zhi-Ming; Pan, Li; Zheng, Sui-Ping; Han, Shuang-Yan; Lin, Ying

    2014-04-01

    Aspergillus niger is a recognized workhorse used to produce food processing enzymes because of its extraordinarily high protein-producing capacity. We have developed a new cell surface display system de novo in A. niger using expression elements from generally recognized as safe certified microorganisms. Candida antarctica lipase B (CALB), a widely used hydrolase, was fused to an endogenous cell wall mannoprotein, CwpA, and functionally displayed on the cell surface. Localization of CALB was confirmed by enzymatic assay and immunofluorescence analysis using laser scanning confocal microscopy. After induction by maltose for 45 h, the hydrolytic activity and synthesis activity of A. niger mycelium-surface displayed CALB (AN-CALB) reached 400 and 240 U/g dry cell, respectively. AN-CALB was successfully used as a whole-cell catalyst for the enzymatic production of ethyl esters from a series of fatty acids of different chain lengths and ethanol. In a solvent-free system, AN-CALB showed great synthetic activity and afforded high substrate mole conversions, which amounted to 87 % for ethyl hexanoate after 2 h, 89 % for ethyl laurate after 2 h, and 84 % for ethyl stearate after 3 h. These results suggested that CwpA can act as an efficient anchoring motif for displaying enzyme on A. niger, and AN-CALB is a robust, green, and cost-effective alternative food-grade whole-cell catalyst to commercial lipase.

  19. Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently

    PubMed Central

    Currin, Andrew; Swainston, Neil; Day, Philip J.

    2015-01-01

    , simultaneously, this offers opportunities for protein improvement not readily available to natural evolution on rapid timescales. Intelligent landscape navigation, informed by sequence-activity relationships and coupled to the emerging methods of synthetic biology, offers scope for the development of novel biocatalysts that are both highly active and robust. PMID:25503938

  20. Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently.

    PubMed

    Currin, Andrew; Swainston, Neil; Day, Philip J; Kell, Douglas B

    2015-03-01

    offers opportunities for protein improvement not readily available to natural evolution on rapid timescales. Intelligent landscape navigation, informed by sequence-activity relationships and coupled to the emerging methods of synthetic biology, offers scope for the development of novel biocatalysts that are both highly active and robust.

  1. Identification of Yersinia pestis and Escherichia coli strains by whole cell and outer membrane protein extracts with mass spectrometry-based proteomics.

    PubMed

    Jabbour, Rabih E; Wade, Mary Margaret; Deshpande, Samir V; Stanford, Michael F; Wick, Charles H; Zulich, Alan W; Snyder, A Peter

    2010-07-01

    Whole cell protein and outer membrane protein (OMP) extracts were compared for their ability to differentiate and delineate the correct database organism to an experimental sample and for the degree of dissimilarity to the nearest neighbor database organism strains. These extracts were isolated from pathogenic and nonpathogenic strains of Yersinia pestis and Escherichia coli using ultracentrifugation and a sarkosyl extraction method followed by protein digestion and analysis using liquid chromatography tandem mass spectrometry (MS). Whole cell protein extracts contain many different types of proteins resident in an organism at a given phase in its growth cycle. OMPs, however, are often associated with virulence in Gram-negative pathogens and could prove to be model biomarkers for strain differentiation among bacteria. The mass spectra of bacterial peptides were searched, using the SEQUEST algorithm, against a constructed proteome database of microorganisms in order to determine the identity and number of unique peptides for each bacterial sample. Data analysis was performed with the in-house BACid software. It calculated the probabilities that a peptide sequence assignment to a product ion mass spectrum was correct and used accepted spectrum-to-sequence matches to generate a sequence-to-bacterium (STB) binary matrix of assignments. Validated peptide sequences, either present or absent in various strains (STB matrices), were visualized as assignment bitmaps and analyzed by the BACid module that used phylogenetic relationships among bacterial species as part of a decision tree process. The bacterial classification and identification algorithm used assignments of organisms to taxonomic groups (phylogenetic classification) based on an organized scheme that begins at the phylum level and follows through the class, order, family, genus, and species to the strain level. For both Gram-negative organisms, the number of unique distinguishing proteins arrived at by the whole

  2. Symbiotic Fungi Produce Laccases Potentially Involved in Phenol Degradation in Fungus Combs of Fungus-Growing Termites in Thailand†

    PubMed Central

    Taprab, Yaovapa; Johjima, Toru; Maeda, Yoshimasa; Moriya, Shigeharu; Trakulnaleamsai, Savitr; Noparatnaraporn, Napavarn; Ohkuma, Moriya; Kudo, Toshiaki

    2005-01-01

    Fungus-growing termites efficiently decompose plant litter through their symbiotic relationship with basidiomycete fungi of the genus Termitomyces. Here, we investigated phenol-oxidizing enzymes in symbiotic fungi and fungus combs (a substrate used to cultivate symbiotic fungi) from termites belonging to the genera Macrotermes, Odontotermes, and Microtermes in Thailand, because these enzymes are potentially involved in the degradation of phenolic compounds during fungus comb aging. Laccase activity was detected in all the fungus combs examined as well as in the culture supernatants of isolated symbiotic fungi. Conversely, no peroxidase activity was detected in any of the fungus combs or the symbiotic fungal cultures. The laccase cDNA fragments were amplified directly from RNA extracted from fungus combs of five termite species and a fungal isolate using degenerate primers targeting conserved copper binding domains of basidiomycete laccases, resulting in a total of 13 putative laccase cDNA sequences being identified. The full-length sequences of the laccase cDNA and the corresponding gene, lcc1-2, were identified from the fungus comb of Macrotermes gilvus and a Termitomyces strain isolated from the same fungus comb, respectively. Partial purification of laccase from the fungus comb showed that the lcc1-2 gene product was a dominant laccase in the fungus comb. These findings indicate that the symbiotic fungus secretes laccase to the fungus comb. In addition to laccase, we report novel genes that showed a significant similarity with fungal laccases, but the gene product lacked laccase activity. Interestingly, these genes were highly expressed in symbiotic fungi of all the termite hosts examined. PMID:16332742

  3. A new biocatalyst: Penicillin G acylase immobilized in sol-gel micro-particles with magnetic properties.

    PubMed

    Bernardino, Susana M S A; Fernandes, Pedro; Fonseca, Luís P

    2009-05-01

    The present work focuses on the development and basic characterization of a new magnetic biocatalyst, namely penicillin G acylase (PGA), immobilized in sol-gel matrices with magnetic properties, ultimately aimed for application in cephalexin (CEX) synthesis. A mechanically stable carrier, based on porous xerogels silica matrixes starting from tetramethoxysilane (TMOS), was prepared leading to micro-carriers with medium sized particles of 30 microm, as determined by scanning electron microscopy. An immobilization yield of 95-100% and a recovered activity of 50-65% at 37 degrees C, as determined by penicillin G (PG) hydrolysis (pH STAT method), were observed. These results clearly exceed those reported in a previous work on PGA immobilization in sol-gel, where only 10% of activity was recovered. The values of activity were kept constant for 6 months. Immobilized PGA (682 U/g(dry weight)) retained high specific activity throughout ten consecutive runs for PG hydrolysis, suggesting adequate biocatalyst stability. The CEX synthesis was performed at 14 degrees C, using the free and immobilized PGA in aqueous medium. Phenylglycine methyl ester was used as acyl donor at 90 mM and 7-aminodeacetoxycephalosporanic acid was the limiting substrate at 30 mM. The CEX stoichiometric yield after 1-h reaction was close to 68% (23 mM CEX/h) and 65% (19 mM CEX/h), respectively. PMID:19418472

  4. Corynebacterium glutamicum as a potent biocatalyst for the bioconversion of pentose sugars to value-added products.

    PubMed

    Gopinath, Vipin; Murali, Anusree; Dhar, Kiran S; Nampoothiri, K Madhavan

    2012-01-01

    Corynebacterium glutamicum, the industrial microbe traditionally used for the production of amino acids, proved its value for the fermentative production of diverse products through genetic/metabolic engineering. A successful demonstration of the heterologous expression of arabinose and xylose utilization genes made them interesting biocatalysts for pentose fermentation, which are the main components in lignocellulosic hydrolysates. Its ability to withstand substantial amount of general growth inhibitors like furfurals, hydroxyl methyl furfurals and organic acids generated from the acid/alkali hydrolysis of lignocellulosics in growth arrested conditions and its ability to produce amino acids like glutamate and lysine in acid hydrolysates of rice straw and wheat bran, indicate the future prospective of this bacterium as a potent biocatalyst in fermentation biotechnology. However, the efforts so far on these lines have not yet been reviewed, and hence an attempt is made to look into the efficacy and prospects of C. glutamicum to utilize the normally non-fermentable pentose sugars from lignocellulosic biomass for the production of commodity chemicals. PMID:22094976

  5. A new biocatalyst: Penicillin G acylase immobilized in sol-gel micro-particles with magnetic properties.

    PubMed

    Bernardino, Susana M S A; Fernandes, Pedro; Fonseca, Luís P

    2009-05-01

    The present work focuses on the development and basic characterization of a new magnetic biocatalyst, namely penicillin G acylase (PGA), immobilized in sol-gel matrices with magnetic properties, ultimately aimed for application in cephalexin (CEX) synthesis. A mechanically stable carrier, based on porous xerogels silica matrixes starting from tetramethoxysilane (TMOS), was prepared leading to micro-carriers with medium sized particles of 30 microm, as determined by scanning electron microscopy. An immobilization yield of 95-100% and a recovered activity of 50-65% at 37 degrees C, as determined by penicillin G (PG) hydrolysis (pH STAT method), were observed. These results clearly exceed those reported in a previous work on PGA immobilization in sol-gel, where only 10% of activity was recovered. The values of activity were kept constant for 6 months. Immobilized PGA (682 U/g(dry weight)) retained high specific activity throughout ten consecutive runs for PG hydrolysis, suggesting adequate biocatalyst stability. The CEX synthesis was performed at 14 degrees C, using the free and immobilized PGA in aqueous medium. Phenylglycine methyl ester was used as acyl donor at 90 mM and 7-aminodeacetoxycephalosporanic acid was the limiting substrate at 30 mM. The CEX stoichiometric yield after 1-h reaction was close to 68% (23 mM CEX/h) and 65% (19 mM CEX/h), respectively.

  6. Corynebacterium glutamicum as a potent biocatalyst for the bioconversion of pentose sugars to value-added products.

    PubMed

    Gopinath, Vipin; Murali, Anusree; Dhar, Kiran S; Nampoothiri, K Madhavan

    2012-01-01

    Corynebacterium glutamicum, the industrial microbe traditionally used for the production of amino acids, proved its value for the fermentative production of diverse products through genetic/metabolic engineering. A successful demonstration of the heterologous expression of arabinose and xylose utilization genes made them interesting biocatalysts for pentose fermentation, which are the main components in lignocellulosic hydrolysates. Its ability to withstand substantial amount of general growth inhibitors like furfurals, hydroxyl methyl furfurals and organic acids generated from the acid/alkali hydrolysis of lignocellulosics in growth arrested conditions and its ability to produce amino acids like glutamate and lysine in acid hydrolysates of rice straw and wheat bran, indicate the future prospective of this bacterium as a potent biocatalyst in fermentation biotechnology. However, the efforts so far on these lines have not yet been reviewed, and hence an attempt is made to look into the efficacy and prospects of C. glutamicum to utilize the normally non-fermentable pentose sugars from lignocellulosic biomass for the production of commodity chemicals.

  7. Expanding Distribution of Lethal Amphibian Fungus Batrachochytrium salamandrivorans in Europe.

    PubMed

    Spitzen-van der Sluijs, Annemarieke; Martel, An; Asselberghs, Johan; Bales, Emma K; Beukema, Wouter; Bletz, Molly C; Dalbeck, Lutz; Goverse, Edo; Kerres, Alexander; Kinet, Thierry; Kirst, Kai; Laudelout, Arnaud; Marin da Fonte, Luis F; Nöllert, Andreas; Ohlhoff, Dagmar; Sabino-Pinto, Joana; Schmidt, Benedikt R; Speybroeck, Jeroen; Spikmans, Frank; Steinfartz, Sebastian; Veith, Michael; Vences, Miguel; Wagner, Norman; Pasmans, Frank; Lötters, Stefan

    2016-07-01

    Emerging fungal diseases can drive amphibian species to local extinction. During 2010-2016, we examined 1,921 urodeles in 3 European countries. Presence of the chytrid fungus Batrachochytrium salamandrivorans at new locations and in urodeles of different species expands the known geographic and host range of the fungus and underpins its imminent threat to biodiversity.

  8. Phomalactone from a phytopathogenic fungus infecting Zinnia elegans (Asteraceae) leaves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Zinnia elegans plants are infected by a fungus that causes necrosis with dark red spots particularly in late spring to the middle of summer in the Mid-South part of the United States. This fungal disease when untreated causes the leaves to wilt and eventually kills the plant. The fungus was isolated...

  9. Genome Sequence of the Pathogenic Fungus Sporothrix schenckii (ATCC 58251).

    PubMed

    Cuomo, Christina A; Rodriguez-Del Valle, Nuri; Perez-Sanchez, Lizaida; Abouelleil, Amr; Goldberg, Jonathan; Young, Sarah; Zeng, Qiandong; Birren, Bruce W

    2014-05-22

    Sporothrix schenckii is a pathogenic dimorphic fungus that grows as a yeast and as mycelia. This species is the causative agent of sporotrichosis, typically a skin infection. We report the genome sequence of S. schenckii, which will facilitate the study of this fungus and of the Sporothrix schenckii group.

  10. Expanding Distribution of Lethal Amphibian Fungus Batrachochytrium salamandrivorans in Europe

    PubMed Central

    Spitzen-van der Sluijs, Annemarieke; Martel, An; Asselberghs, Johan; Bales, Emma K.; Beukema, Wouter; Bletz, Molly C.; Dalbeck, Lutz; Goverse, Edo; Kerres, Alexander; Kinet, Thierry; Kirst, Kai; Laudelout, Arnaud; Marin da Fonte, Luis F.; Nöllert, Andreas; Ohlhoff, Dagmar; Sabino-Pinto, Joana; Schmidt, Benedikt R.; Speybroeck, Jeroen; Spikmans, Frank; Steinfartz, Sebastian; Veith, Michael; Vences, Miguel; Wagner, Norman; Pasmans, Frank

    2016-01-01

    Emerging fungal diseases can drive amphibian species to local extinction. During 2010–2016, we examined 1,921 urodeles in 3 European countries. Presence of the chytrid fungus Batrachochytrium salamandrivorans at new locations and in urodeles of different species expands the known geographic and host range of the fungus and underpins its imminent threat to biodiversity. PMID:27070102

  11. Expanding Distribution of Lethal Amphibian Fungus Batrachochytrium salamandrivorans in Europe.

    PubMed

    Spitzen-van der Sluijs, Annemarieke; Martel, An; Asselberghs, Johan; Bales, Emma K; Beukema, Wouter; Bletz, Molly C; Dalbeck, Lutz; Goverse, Edo; Kerres, Alexander; Kinet, Thierry; Kirst, Kai; Laudelout, Arnaud; Marin da Fonte, Luis F; Nöllert, Andreas; Ohlhoff, Dagmar; Sabino-Pinto, Joana; Schmidt, Benedikt R; Speybroeck, Jeroen; Spikmans, Frank; Steinfartz, Sebastian; Veith, Michael; Vences, Miguel; Wagner, Norman; Pasmans, Frank; Lötters, Stefan

    2016-07-01

    Emerging fungal diseases can drive amphibian species to local extinction. During 2010-2016, we examined 1,921 urodeles in 3 European countries. Presence of the chytrid fungus Batrachochytrium salamandrivorans at new locations and in urodeles of different species expands the known geographic and host range of the fungus and underpins its imminent threat to biodiversity. PMID:27070102

  12. Assembly of complex plant–fungus networks

    PubMed Central

    Toju, Hirokazu; Guimarães, Paulo R.; Olesen, Jens M.; Thompson, John N.

    2014-01-01

    Species in ecological communities build complex webs of interaction. Although revealing the architecture of these networks is fundamental to understanding ecological and evolutionary dynamics in nature, it has been difficult to characterize the structure of most species-rich ecological systems. By overcoming this limitation through next-generation sequencing technology, we herein uncover the network architecture of below-ground plant–fungus symbioses, which are ubiquitous to terrestrial ecosystems. The examined symbiotic network of a temperate forest in Japan includes 33 plant species and 387 functionally and phylogenetically diverse fungal taxa, and the overall network architecture differs fundamentally from that of other ecological networks. In contrast to results for other ecological networks and theoretical predictions for symbiotic networks, the plant–fungus network shows moderate or relatively low levels of interaction specialization and modularity and an unusual pattern of ‘nested’ network architecture. These results suggest that species-rich ecological networks are more architecturally diverse than previously recognized. PMID:25327887

  13. Bacterial farming by the fungus Morchella crassipes

    PubMed Central

    Pion, Martin; Spangenberg, Jorge E.; Simon, Anaele; Bindschedler, Saskia; Flury, Coralie; Chatelain, Auriel; Bshary, Redouan; Job, Daniel; Junier, Pilar

    2013-01-01

    The interactions between bacteria and fungi, the main actors of the soil microbiome, remain poorly studied. Here, we show that the saprotrophic and ectomycorrhizal soil fungus Morchella crassipes acts as a bacterial farmer of Pseudomonas putida, which serves as a model soil bacterium. Farming by M. crassipes consists of bacterial dispersal, bacterial rearing with fungal exudates, as well as harvesting and translocation of bacterial carbon. The different phases were confirmed experimentally using cell counting and 13C probing. Common criteria met by other non-human farming systems are also valid for M. crassipes farming, including habitual planting, cultivation and harvesting. Specific traits include delocalization of food production and consumption and separation of roles in the colony (source versus sink areas), which are also found in human agriculture. Our study evidences a hitherto unknown mutualistic association in which bacteria gain through dispersal and rearing, while the fungus gains through the harvesting of an additional carbon source and increased stress resistance of the mycelium. This type of interaction between fungi and bacteria may play a key role in soils. PMID:24174111

  14. Assessing fungus prevalence in domestic interiors.

    PubMed

    Solomon, W R

    1975-09-01

    Single-plate, Andersen sampler collections of mesonphilic imperfect fungi were made at three points in and immediately outside a series of midwestern homes. During frost-free periods, emanations of dark-spored form genera predominated at both points with indoor levels averaging 25% of those in outside air. At these times, volumetric recoveries and those by 30-min exposure of open culture plates have correlated tenuously (r = 0.29) in bedroom air of 20 homes. During winter, form species of Penicillium, Aspergillus, Oospora, Sporothrix, yeasts, etc. predominated indoors, with levels exceeding 1,000 particles/M3 noted in over 18% of homes; outdoor concentrations never exceeded 230 particles/M3. Comparisons of volumetric and open-plate recoveries from 50 homes during winter have revealed an almost random relationship (r = 0.06). These findings reflect the case with which outdoor spore clouds may penetrate structures and obscure evidence of internal fungus cources. The data also imply that, because of size-related undersampling, open plates often seriously misrepresent prevalence levels and occasionally can exclude abundant types from recovery. The fungus flora of enclosed spaces merits further critical study by volumetric techniques of calculable efficiency in a setting that minimizes contamination from without.

  15. Hazardous waste treatment using fungus enters marketplace

    SciTech Connect

    Illman, D.L.

    1993-07-01

    When the announcement was made eight years ago that a common fungus had been found that could degrade a variety of environmental pollutants, the news stirred interest in the scientific community, the private sector, and the general public. Here was the promise of a new technology that might be effective and economical in treating hazardous waste, especially the most recalcitrant of toxic pollutants. Today, commercialization is beginning amid a mixture of optimism and skepticism. The organism in question is white rot fungus, or Phanerochaete chrysosporium, and it belongs to a family of woodrotting fungi common all over North America. The fungi secrete enzymes that break down lignin in wood to carbon dioxide and water--a process called mineralization. These lignin-degrading enzymes are not very discriminating, however. The white rot fungi have been shown to degrade such materials as DDT, the herbicide (2,4,5-trichlorophenoxy)acetic acid (2,4,5-T), 2,4,6-trinitrotoluene (TNT), pentachlorophenol (PCP), creosote, coal tars, and heavy fuels, in many cases mineralizing these pollutants to a significant extent.

  16. Bacterial farming by the fungus Morchella crassipes.

    PubMed

    Pion, Martin; Spangenberg, Jorge E; Simon, Anaele; Bindschedler, Saskia; Flury, Coralie; Chatelain, Auriel; Bshary, Redouan; Job, Daniel; Junier, Pilar

    2013-12-22

    The interactions between bacteria and fungi, the main actors of the soil microbiome, remain poorly studied. Here, we show that the saprotrophic and ectomycorrhizal soil fungus Morchella crassipes acts as a bacterial farmer of Pseudomonas putida, which serves as a model soil bacterium. Farming by M. crassipes consists of bacterial dispersal, bacterial rearing with fungal exudates, as well as harvesting and translocation of bacterial carbon. The different phases were confirmed experimentally using cell counting and (13)C probing. Common criteria met by other non-human farming systems are also valid for M. crassipes farming, including habitual planting, cultivation and harvesting. Specific traits include delocalization of food production and consumption and separation of roles in the colony (source versus sink areas), which are also found in human agriculture. Our study evidences a hitherto unknown mutualistic association in which bacteria gain through dispersal and rearing, while the fungus gains through the harvesting of an additional carbon source and increased stress resistance of the mycelium. This type of interaction between fungi and bacteria may play a key role in soils. PMID:24174111

  17. A Randomized, Placebo-Controlled Trial Evaluating Safety and Immunogenicity of the Killed, Bivalent, Whole-Cell Oral Cholera Vaccine in Ethiopia.

    PubMed

    Desai, Sachin N; Akalu, Zenebe; Teshome, Samuel; Teferi, Mekonnen; Yamuah, Lawrence; Kim, Deok Ryun; Yang, Jae Seung; Hussein, Jemal; Park, Ju Yeong; Jang, Mi Seon; Mesganaw, Chalachew; Taye, Hawult; Beyene, Demissew; Bedru, Ahmed; Singh, Ajit Pal; Wierzba, Thomas F; Aseffa, Abraham

    2015-09-01

    Killed whole-cell oral cholera vaccine (OCV) has been a key component of a comprehensive package including water and sanitation measures for recent cholera epidemics. The vaccine, given in a two-dose regimen, has been evaluated in a large number of human volunteers in India, Vietnam, and Bangladesh, where it has demonstrated safety, immunogenicity, and clinical efficacy. We conducted a double-blind randomized placebo-controlled trial in Ethiopia, where we evaluated the safety and immunogenicity of the vaccine in 216 healthy adults and children. OCV was found to be safe and elicited a robust immunological response against Vibrio cholerae O1, with 81% adults and 77% children demonstrating seroconversion 14 days after the second dose of vaccine. This is the first study to evaluate safety and immunogenicity of the vaccine in a population outside Asia using a placebo-controlled, double-blind, randomized study design.

  18. A simple method of fast extracellular solution exchange for the study of whole-cell or single channel currents using patch-clamp technique.

    PubMed

    Hering, S; Beech, D J; Bolton, T B

    1987-10-01

    A new concentration-jump technique was devised for the rapid application of drugs to single, isolated cells attached to the base of the experimental chamber while recording from them with patch-clamp technique. Cells were placed in a micro-drop (less than 0.1 microliter) in a small inner bath which was separated from an outer bath by a ring of "Sylgard" polymer. Stable whole-cell recordings were made in the micro-drop and rapid solution exchange took place when a much larger volume of test solution from the outer bath was flooded over the Sylgard ring and mixed with the micro-drop. Complete equilibration occurred within less than 10 ms.

  19. General metabolism of the dimorphic and pathogenic fungus Paracoccidioides brasiliensis.

    PubMed

    Arraes, Fabrício B M; Benoliel, Bruno; Burtet, Rafael T; Costa, Patrícia L N; Galdino, Alexandro S; Lima, Luanne H A; Marinho-Silva, Camila; Oliveira-Pereira, Luciana; Pfrimer, Pollyanna; Procópio-Silva, Luciano; Reis, Viviane Castelo-Branco; Felipe, Maria Sueli S

    2005-06-30

    Annotation of the transcriptome of the dimorphic fungus Paracoccidioides brasiliensis has set the grounds for a global understanding of its metabolism in both mycelium and yeast forms. This fungus is able to use the main carbohydrate sources, including starch, and it can store reduced carbons in the form of glycogen and trehalose; these provide energy reserves that are relevant for metabolic adaptation, protection against stress and infectivity mechanisms. The glyoxylate cycle, which is also involved in pathogenicity, is present in this fungus. Classical pathways of lipid biosynthesis and degradation, including those of ketone body and sterol production, are well represented in the database of P. brasiliensis. It is able to synthesize de novo all nucleotides and amino acids, with the sole exception of asparagine, which was confirmed by the fungus growth in minimal medium. Sulfur metabolism, as well as the accessory synthetic pathways of vitamins and co-factors, are likely to exist in this fungus.

  20. A genetically engineered whole-cell pigment-based bacterial biosensing system for quantification of N-butyryl homoserine lactone quorum sensing signal.

    PubMed

    Yong, Yang-Chun; Zhong, Jian-Jiang

    2009-09-15

    N-acyl homoserine lactone (AHL) is a widely conserved quorum sensing (QS) signal of gram-negative bacteria and has received attention in fighting against human diseases and environmental pollution. However, a method for quantifying AHL is lacking although it is urgently required for diagnosis and bioprocess manipulation. This work screened out an aromatics degrader Pseudomonas aeruginosa for biosensing system development, which produced a blue-green pigment regulated by the RhlI-RhlR QS system. By taking advantage of the recognition of N-butyryl homoserine lactone (BHL, the signal molecule of RhlI-RhlR QS system and an AHL) by the product of rhlR, a new whole-cell biosensor P. aeruginosa Delta rhlIR/pYC-rhlR (rhlI(-)rhlR(++)) was developed. It was constructed through abolishing its BHL production by in-frame deletion of rhlIR and over-expressing rhlR by introducing a multi-copy plasmid pYC-rhlR into Delta rhlIR. By using the pigment production which responded to exogenous BHL as biosensor output, BHL quantification in samples was simply done spectrophotometrically. Under optimum conditions, the calibration curve had the limit of detection (LOD), the 50% activation/effect concentration, the limit of quantification (LOQ), and the quantitative detection range of 1.3 nM, 2.77+/-0.45 microM, 5.7 nM and 0.11-49.7 microM, respectively. The biosensor output was stable, culture samples could be stored 10 days under -20 degrees C, and this sensing system was resistant to interferences by toxic aromatic pollutants. It was successfully applied to environmental samples even without extraction. The new whole-cell biosensing system provided a simple, stable, toxic pollutants-tolerant, and cost-effective tool for quantitative investigation of the QS signals' role in environmental processes.

  1. Study on Toxicity Reduction and Potency Induction in Whole-cell Pertussis Vaccine by Developing a New Optimal Inactivation Condition Processed on Bordetella pertussis

    PubMed Central

    Mohammadpour Dounighi, Naser; Razzaghi-Abyane, Mehdi; Nofeli, Mojtaba; Zolfagharian, Hossein; Shahcheraghi, Fereshteh

    2016-01-01

    Background Whooping cough is caused by Bordetella pertussis, and it remains a public health concern. Whole-cell pertussis vaccines have been commonly employed for expanded immunization. There is no doubt of the efficacy of whole cell pertussis vaccine, but it is necessary to improve the vaccine to decrease its toxicity. Objectives In this study, an inactivation process of dealing with pertussis bacteria is optimized in order to decrease the bacteria content in human doses of vaccines and reduce the vaccine’s toxicity. Materials and Methods The bacterial suspensions of pertussis strains 509 and 134 were divided into 21 sample parts from F1 to F21 and inactivated under different conditions. The inactivated suspensions of both strains were tested for opacity, non-viability, agglutination, purity, and sterility; the same formulation samples that passed quality tests were then pooled together. The pool of inactivated suspensions were analyzed for sterility, agglutination, opacity, specific toxicity, and potency. Results The harvest of both bacterial strains showed purity. The opacity of various samples were lost under different treatment conditions by heat from 8% to 12%, formaldehyde 6% to 8%, glutaraldehyde 6% to 8%, and thimerosal 5% to 8%. Tests on suspensions after inactivation and on pooled suspensions showed inactivation conditions not degraded agglutinins of both strains. The samples of F2, F4, F8, F12, F15, and F17 passed the toxicity test. The potency (ED50) of these samples showed following order F17 > F12 > F8 > F15, F4 > F2, and F17 revealed higher potency compared to other formulations. Conclusions It can be concluded that F17 showed desirable outcomes in the toxicity test and good immunogenicity with a low bacterial number content. Consequently, lower adverse effects and good immunogenicity are foreseeable for vaccine preparation with this method.

  2. Vaccination of ducks with a whole-cell vaccine expressing duck hepatitis B virus core antigen elicits antiviral immune responses that enable rapid resolution of de novo infection.

    PubMed

    Miller, Darren S; Halpern, Michael; Kotlarski, Ieva; Jilbert, Allison R

    2006-05-10

    As a first step in developing immuno-therapeutic vaccines for patients with chronic hepatitis B virus infection, we examined the ability of a whole-cell vaccine, expressing the duck hepatitis B virus (DHBV) core antigen (DHBcAg), to target infected cells leading to the resolution of de novo DHBV infections. Three separate experiments were performed. In each experiment, ducks were vaccinated at 7 and 14 days of age with primary duck embryonic fibroblasts (PDEF) that had been transfected 48 h earlier with plasmid DNA expressing DHBcAg with and without the addition of anti-DHBcAg (anti-DHBc) antibodies. Control ducks were injected with either 0.7% NaCl or non-transfected PDEF. The ducks were then challenged at 18 days of age by intravenous inoculation with DHBV (5 x 10(8) viral genome equivalents). Liver biopsies obtained on day 4 post-challenge demonstrated that vaccination did not prevent infection of the liver as similar numbers of infected hepatocytes were detected in all vaccinated and control ducks. However, analysis of liver tissue obtained 9 or more days post-challenge revealed that 9 out of 11 of the PDEF-DHBcAg vaccinated ducks and 8 out of 11 ducks vaccinated with PDEF-DHBcAg plus anti-DHBc antibodies had rapidly resolved the DHBV infection with clearance of infected cells. In contrast, 10 out of 11 of the control unvaccinated ducks developed chronic DHBV infection. In conclusion, vaccination of ducks with a whole-cell PDEF vaccine expressing DHBcAg elicited immune responses that induced a rapid resolution of DHBV infection. The results establish that chronic infection can be prevented via the vaccine-mediated induction of a core-antigen-specific immune response.

  3. Strain variation among Bordetella pertussis isolates in finland, where the whole-cell pertussis vaccine has been used for 50 years.

    PubMed

    Elomaa, Annika; Advani, Abdolreza; Donnelly, Declan; Antila, Mia; Mertsola, Jussi; Hallander, Hans; He, Qiushui

    2005-08-01

    Pertussis is an infectious disease of the respiratory tract caused by Bordetella pertussis. Despite the introduction of mass vaccination against pertussis in Finland in 1952, pertussis has remained an endemic disease with regular epidemics. To monitor changes in the Finnish B. pertussis population, 101 isolates selected from 1991 to 2003 and 21 isolates selected from 1953 to 1982 were studied together with two Finnish vaccine strains. The analyses included serotyping of fimbriae (Fim), genotyping of the pertussis toxin S1 subunit (ptxA) and pertactin (prn), and pulsed-field gel electrophoresis (PFGE) after digestion of B. pertussis genomic DNA with XbaI restriction enzyme. Strains isolated before 1977 were found to harbor the same ptxA as the strains used in the Finnish whole-cell pertussis vaccine, and strains isolated before 1982 harbored the same prn as the strains used in the Finnish whole-cell pertussis vaccine. All recent isolates, however, represented genotypes distinct from those of the two vaccine strains. A marked shift of predominant serotype from Fim serotype 2 (Fim2) to Fim3 has been observed since the late 1990s. Temporal changes were seen in the genome of B. pertussis by PFGE analysis. Three PFGE profiles (BpSR1, BpSR11, and BpSR147) were distinguished by their prevalence between 1991 and 2003. The yearly emergence of the three profiles was distributed periodically. Our study stresses the importance of the continuous monitoring of emerging strains of B. pertussis and the need to obtain a better understanding of the relationship of the evolution of B. pertussis in vaccinated populations.

  4. Strain Variation among Bordetella pertussis Isolates in Finland, Where the Whole-Cell Pertussis Vaccine Has Been Used for 50 Years

    PubMed Central

    Elomaa, Annika; Advani, Abdolreza; Donnelly, Declan; Antila, Mia; Mertsola, Jussi; Hallander, Hans; He, Qiushui

    2005-01-01

    Pertussis is an infectious disease of the respiratory tract caused by Bordetella pertussis. Despite the introduction of mass vaccination against pertussis in Finland in 1952, pertussis has remained an endemic disease with regular epidemics. To monitor changes in the Finnish B. pertussis population, 101 isolates selected from 1991 to 2003 and 21 isolates selected from 1953 to 1982 were studied together with two Finnish vaccine strains. The analyses included serotyping of fimbriae (Fim), genotyping of the pertussis toxin S1 subunit (ptxA) and pertactin (prn), and pulsed-field gel electrophoresis (PFGE) after digestion of B. pertussis genomic DNA with XbaI restriction enzyme. Strains isolated before 1977 were found to harbor the same ptxA as the strains used in the Finnish whole-cell pertussis vaccine, and strains isolated before 1982 harbored the same prn as the strains used in the Finnish whole-cell pertussis vaccine. All recent isolates, however, represented genotypes distinct from those of the two vaccine strains. A marked shift of predominant serotype from Fim serotype 2 (Fim2) to Fim3 has been observed since the late 1990s. Temporal changes were seen in the genome of B. pertussis by PFGE analysis. Three PFGE profiles (BpSR1, BpSR11, and BpSR147) were distinguished by their prevalence between 1991 and 2003. The yearly emergence of the three profiles was distributed periodically. Our study stresses the importance of the continuous monitoring of emerging strains of B. pertussis and the need to obtain a better understanding of the relationship of the evolution of B. pertussis in vaccinated populations. PMID:16081896

  5. Detection of bioavailable heavy metals in EILATox-Oregon samples using whole-cell luminescent bacterial sensors in suspension or immobilized onto fibre-optic tips.

    PubMed

    Hakkila, Kaisa; Green, Tal; Leskinen, Piia; Ivask, Angela; Marks, Robert; Virta, Marko

    2004-01-01

    At the EILATox-Oregon Workshop, nine luminescent whole-cell bacterial sensors were used for the determination of bioavailable metals in blind samples (17 synthetic and 3 environmental). A non-inducible luminescent control strain was used to determine sample matrix effects and bacterial toxicity. Whole-cell bacterial sensors capable of determining arsenic, inorganic mercury and its organic derivatives, cadmium, lead or copper were used in suspensions and a bacterial sensor for the detection of inorganic mercury was immobilized onto fibre-optic tips using calcium alginate. Bioavailable amounts of metals were estimated using calibration plots, that were constructed to determine the range of metals giving rise to a linear relationship between luminescence and the amount of metals present in the standard solutions. EILATox-Oregon sample 5, which contained 74 mg l(-1) of Hg, gave a significant response with both formats of the mercury sensor. The bioavailable amounts of mercury according to the measurement of bacterial sensor in suspension and immobilized onto a fibre-optic tip were 76 and 93 mg l(-1), respectively. The bacterial sensor for arsenic and copper showed a response with sample 6 (58 mg l(-1) of As) and sample 8 (400 mg l(-1) of metham sodium), respectively. This study showed that the bacterial sensors in suspension or immobilized onto optical fibres are capable of quantifying bioavailable metals from unknown samples. The measurement protocol of bacterial sensors is simple and possible to perform in the field. Moreover, the samples do not need any pretreatment before analysis. Construction and characterization of the strain for the detection of bioavailable copper are described.

  6. Study on Toxicity Reduction and Potency Induction in Whole-cell Pertussis Vaccine by Developing a New Optimal Inactivation Condition Processed on Bordetella pertussis

    PubMed Central

    Mohammadpour Dounighi, Naser; Razzaghi-Abyane, Mehdi; Nofeli, Mojtaba; Zolfagharian, Hossein; Shahcheraghi, Fereshteh

    2016-01-01

    Background Whooping cough is caused by Bordetella pertussis, and it remains a public health concern. Whole-cell pertussis vaccines have been commonly employed for expanded immunization. There is no doubt of the efficacy of whole cell pertussis vaccine, but it is necessary to improve the vaccine to decrease its toxicity. Objectives In this study, an inactivation process of dealing with pertussis bacteria is optimized in order to decrease the bacteria content in human doses of vaccines and reduce the vaccine’s toxicity. Materials and Methods The bacterial suspensions of pertussis strains 509 and 134 were divided into 21 sample parts from F1 to F21 and inactivated under different conditions. The inactivated suspensions of both strains were tested for opacity, non-viability, agglutination, purity, and sterility; the same formulation samples that passed quality tests were then pooled together. The pool of inactivated suspensions were analyzed for sterility, agglutination, opacity, specific toxicity, and potency. Results The harvest of both bacterial strains showed purity. The opacity of various samples were lost under different treatment conditions by heat from 8% to 12%, formaldehyde 6% to 8%, glutaraldehyde 6% to 8%, and thimerosal 5% to 8%. Tests on suspensions after inactivation and on pooled suspensions showed inactivation conditions not degraded agglutinins of both strains. The samples of F2, F4, F8, F12, F15, and F17 passed the toxicity test. The potency (ED50) of these samples showed following order F17 > F12 > F8 > F15, F4 > F2, and F17 revealed higher potency compared to other formulations. Conclusions It can be concluded that F17 showed desirable outcomes in the toxicity test and good immunogenicity with a low bacterial number content. Consequently, lower adverse effects and good immunogenicity are foreseeable for vaccine preparation with this method. PMID:27679704

  7. Microbial Biotransformation of Gentiopicroside by the Endophytic Fungus Penicillium crustosum 2T01Y01

    PubMed Central

    Zeng, Wen-Liang; Li, Wan-Kui; Han, Han; Tao, Yan-Yan; Yang, Li; Chen, Kai-Xian

    2014-01-01

    Endophytic fungi are symbiotic with plants and possess multienzyme systems showing promising metabolite potency with region selectivity and stereoselectivity. The aim of this study was to use these special microorganisms as an in vitro model to mimic the potential mammalian metabolites of a natural iridoid gentiopicroside (GPS, compound 1). The fungi isolated from a medicinal plant, Dendrobium candidum Wall. ex Lindl., were screened for their biotransformation abilities with GPS as the substrate, and one strain with high converting potency was identified as Penicillium crustosum 2T01Y01 on the basis of the sequence of the internal transcribed spacer of the ribosomal DNA region. Upon the optimized incubation of P. crustosum 2T01Y01 with the substrate, seven deglycosylated metabolites were detected by ultraperformance liquid chromatography/quadrupole time of flight mass spectrometry (UPLC/Q-TOF MS). Preparative-scale biotransformation with whole cells of the endophytic fungus resulted in the production of five metabolites, including three novel ones, 5α-(hydroxymethyl)-6β-methyl-3,4,5,6-tetrahydropyrano[3,4-c]pyran-1(8H)-one (compound 2), (Z)-4-(1-hydroxybut-3-en-2-yl)-5,6-dihydropyran-2-one (compound 3), and (E)-4-(1-hydroxybut-3-en-2-yl)-5,6-dihydropyran-2-one (compound 4), along with two known ones, 5α-(hydroxymethyl)-6β-methyl-1H,3H-5,6-dihydropyrano[3,4-c]pyran-1(3H)-one (compound 5) and 5α-(hydroxymethyl)-6α-methyl-5,6-dihydropyrano[3,4-c]pyran-1(3H)-one (compound 6), aided by nuclear magnetic resonance and high-resolution mass spectral analyses. The other two metabolites were tentatively identified by online UPLC/Q-TOF MS as 5-hydroxymethyl-5,6-dihydroisochromen-1-one (compound 7) and 5-hydroxymethyl-3,4,5,6-tetrahydroisochromen-1-one (compound 8), and compound 8 is a new metabolite. To test the metabolic mechanism, the β-glucosidase activity of the fungus P. crustosum 2T01Y01 was assayed with ρ-nitrophenyl-β-d-glucopyranoside as a probe substrate

  8. Magnetic Cross-Linked Enzyme Aggregates (mCLEAs) of Candida antarctica Lipase: An Efficient and Stable Biocatalyst for Biodiesel Synthesis

    PubMed Central

    Cruz-Izquierdo, Álvaro; Picó, Enrique A.; López, Carmen; Serra, Juan L.; Llama, María J.

    2014-01-01

    Enzyme-catalyzed production of biodiesel is the object of extensive research due to the global shortage of fossil fuels and increased environmental concerns. Herein we report the preparation and main characteristics of a novel biocatalyst consisting of Cross-Linked Enzyme Aggregates (CLEAs) of Candida antarctica lipase B (CALB) which are covalently bound to magnetic nanoparticles, and tackle its use for the synthesis of biodiesel from non-edible vegetable and waste frying oils. For this purpose, insolubilized CALB was covalently cross-linked to magnetic nanoparticles of magnetite which the surface was functionalized with –NH2 groups. The resulting biocatalyst combines the relevant catalytic properties of CLEAs (as great stability and feasibility for their reutilization) and the magnetic character, and thus the final product (mCLEAs) are superparamagnetic particles of a robust catalyst which is more stable than the free enzyme, easily recoverable from the reaction medium and reusable for new catalytic cycles. We have studied the main properties of this biocatalyst and we have assessed its utility to catalyze transesterification reactions to obtain biodiesel from non-edible vegetable oils including unrefined soybean, jatropha and cameline, as well as waste frying oil. Using 1% mCLEAs (w/w of oil) conversions near 80% were routinely obtained at 30°C after 24 h of reaction, this value rising to 92% after 72 h. Moreover, the magnetic biocatalyst can be easily recovered from the reaction mixture and reused for at least ten consecutive cycles of 24 h without apparent loss of activity. The obtained results suggest that mCLEAs prepared from CALB can become a powerful biocatalyst for application at industrial scale with better performance than those currently available. PMID:25551445

  9. Magnetic Cross-Linked Enzyme Aggregates (mCLEAs) of Candida antarctica lipase: an efficient and stable biocatalyst for biodiesel synthesis.

    PubMed

    Cruz-Izquierdo, Álvaro; Picó, Enrique A; López, Carmen; Serra, Juan L; Llama, María J

    2014-01-01

    Enzyme-catalyzed production of biodiesel is the object of extensive research due to the global shortage of fossil fuels and increased environmental concerns. Herein we report the preparation and main characteristics of a novel biocatalyst consisting of Cross-Linked Enzyme Aggregates (CLEAs) of Candida antarctica lipase B (CALB) which are covalently bound to magnetic nanoparticles, and tackle its use for the synthesis of biodiesel from non-edible vegetable and waste frying oils. For this purpose, insolubilized CALB was covalently cross-linked to magnetic nanoparticles of magnetite which the surface was functionalized with -NH2 groups. The resulting biocatalyst combines the relevant catalytic properties of CLEAs (as great stability and feasibility for their reutilization) and the magnetic character, and thus the final product (mCLEAs) are superparamagnetic particles of a robust catalyst which is more stable than the free enzyme, easily recoverable from the reaction medium and reusable for new catalytic cycles. We have studied the main properties of this biocatalyst and we have assessed its utility to catalyze transesterification reactions to obtain biodiesel from non-edible vegetable oils including unrefined soybean, jatropha and cameline, as well as waste frying oil. Using 1% mCLEAs (w/w of oil) conversions near 80% were routinely obtained at 30°C after 24 h of reaction, this value rising to 92% after 72 h. Moreover, the magnetic biocatalyst can be easily recovered from the reaction mixture and reused for at least ten consecutive cycles of 24 h without apparent loss of activity. The obtained results suggest that mCLEAs prepared from CALB can become a powerful biocatalyst for application at industrial scale with better performance than those currently available. PMID:25551445

  10. Magnetic Cross-Linked Enzyme Aggregates (mCLEAs) of Candida antarctica lipase: an efficient and stable biocatalyst for biodiesel synthesis.

    PubMed

    Cruz-Izquierdo, Álvaro; Picó, Enrique A; López, Carmen; Serra, Juan L; Llama, María J

    2014-01-01

    Enzyme-catalyzed production of biodiesel is the object of extensive research due to the global shortage of fossil fuels and increased environmental concerns. Herein we report the preparation and main characteristics of a novel biocatalyst consisting of Cross-Linked Enzyme Aggregates (CLEAs) of Candida antarctica lipase B (CALB) which are covalently bound to magnetic nanoparticles, and tackle its use for the synthesis of biodiesel from non-edible vegetable and waste frying oils. For this purpose, insolubilized CALB was covalently cross-linked to magnetic nanoparticles of magnetite which the surface was functionalized with -NH2 groups. The resulting biocatalyst combines the relevant catalytic properties of CLEAs (as great stability and feasibility for their reutilization) and the magnetic character, and thus the final product (mCLEAs) are superparamagnetic particles of a robust catalyst which is more stable than the free enzyme, easily recoverable from the reaction medium and reusable for new catalytic cycles. We have studied the main properties of this biocatalyst and we have assessed its utility to catalyze transesterification reactions to obtain biodiesel from non-edible vegetable oils including unrefined soybean, jatropha and cameline, as well as waste frying oil. Using 1% mCLEAs (w/w of oil) conversions near 80% were routinely obtained at 30°C after 24 h of reaction, this value rising to 92% after 72 h. Moreover, the magnetic biocatalyst can be easily recovered from the reaction mixture and reused for at least ten consecutive cycles of 24 h without apparent loss of activity. The obtained results suggest that mCLEAs prepared from CALB can become a powerful biocatalyst for application at industrial scale with better performance than those currently available.

  11. Scale-up and intensification of (S)-1-(2-chlorophenyl)ethanol bioproduction: economic evaluation of whole cell-catalyzed reduction of o-chloroacetophenone.

    PubMed

    Eixelsberger, Thomas; Woodley, John M; Nidetzky, Bernd; Kratzer, Regina

    2013-08-01

    Escherichia coli cells co-expressing genes coding for Candida tenuis xylose reductase and Candida boidinii formate dehydrogenase were used for the bioreduction of o-chloroacetophenone with in situ coenzyme recycling. The product, (S)-1-(2-chlorophenyl)ethanol, is a key chiral intermediate in the synthesis of polo-like kinase 1 inhibitors, a new class of chemotherapeutic drugs. Production of the alcohol in multi-gram scale requires intensification and scale-up of the biocatalyst production, biotransformation, and downstream processing. Cell cultivation in a 6.9-L bioreactor led to a more than tenfold increase in cell concentration compared to shaken flask cultivation. The resultant cells were used in conversions of 300 mM substrate to (S)-1-(2-chlorophenyl)ethanol (e.e. >99.9%) in high yield (96%). Results obtained in a reaction volume of 500 mL were identical to biotransformations carried out in 1 mL (analytical) and 15 mL (preparative) scale. Optimization of product isolation based on hexane extraction yielded 86% isolated product. Biotransformation and extraction were accomplished in a stirred tank reactor equipped with pH and temperature control. The developed process lowered production costs by 80% and enabled (S)-1-(2-chlorophenyl)ethanol production within previously defined economic boundaries. A simple and efficient way to synthesize (S)-1-(2-chlorophenyl)ethanol in an isolated amount of 20 g product per reaction batch was demonstrated.

  12. Simultaneous biocatalyst production and Baeyer-Villiger oxidation for bioconversion of cyclohexanone by recombinant Escherichia coli expressing cyclohexanone monooxygenase.

    PubMed

    Lee, Won-Heong; Park, Yong-Cheol; Lee, Dae-Hee; Park, Kyungmoon; Seo, Jin-Ho

    2005-01-01

    Cyclohexanone monooxygenase (CHMO) catalyzing Baeyer-Villiger oxidation converts cyclic ketones into optically pure lactones, which have been used as building blocks in organic synthesis. A recombinant Escherichia coli BL21(DE3)/pMM4 expressing CHMO originated from Acinetobacter sp. NCIB 9871 was used to produce epsilon-caprolactone through a simultaneous biocatalyst production and Baeyer-Villiger oxidation (SPO) process. A fed-batch process was designed to obtain high cell density for improving production of epsilon-caprolactone. The fed-batch SPO process gave the best results, 10.2 g/L of epsilon-caprolactone and 0.34 g/(L.h) of productivity, corresponding to a 10.5- and 3.4-fold enhancement compared with those of the batch SPO, respectively.

  13. Novel Flow Sheet for Low Energy CO2 Capture Enabled by Biocatalyst Delivery System

    SciTech Connect

    Reardon, John; Shaffer, Alex; Vaysman, Vladimir

    2015-02-01

    This report documents a preliminary Techno-Economic Assessment (TEA) for processes utilizing Akermin’s second generation biocatalyst delivery system to enhance AKM24, a non- volatile salt solution for CO2 capture. Biocatalyst enhanced AKM24 offers the potential to reduce the cost of CO2 capture in flue gas applications due to its improved equilibrium and stoichiometric properties that result in double the absorption capacity relative to previously demonstrated biocatalyst enhanced solvents. The study assumes a new supercritical pulverized coal fired power plant with a net output of 550 MWe after 90% CO2 capture and uses the June 2011 cost basis (August 2012 update of Bituminous Baseline Study, or BBS). Power plant modeling, capital cost review, and economic calculations were provided by WorleyParsons. Rate-based CO2 capture process modeling and equipment sizing was performed by Akermin using AspenPlus® V8.4, customized to accurately predict thermodynamics, kinetics, and physical properties of the AKM-24 solvent based on available laboratory data. Equipment capital costs were estimated using Aspen Process Economic Analyzer™ which compared well with published baseline cost estimates. Quotes of equipment costs and power consumption for vacuum blower and CO2 compression equipment were also provided by Man Diesel & Turbo. Three process scenarios were examined for Akermin biocatalyst enhanced solvent systems including: Case-1A: an absorption-desorption system operated with a reboiler pressure of 0.16 bara (60°C); Case-2A: an absorption-desorption system with moderate vacuum assisted regeneration at 0.40 bara (80°C); and finally, Case-2B: a conventional absorption-desorption system with near atmospheric pressure regeneration at 1.07 bara (105°C). The estimated increases in cost of electricity (ICOE) for these cases were $58.1/MWh, $47.3/MWh and $46.4/MWh, respectively. Case 2B had the best results for this analysis

  14. Recombinant sterol esterase from Ophiostoma piceae: an improved biocatalyst expressed in Pichia pastoris

    PubMed Central

    2012-01-01

    heterologous production of recombinant sterol esterase from O. piceae, yielding higher activity levels than those obtained with the saprophytic fungus. The enzyme showed improved kinetic parameters because of its modified N-terminus, which allowed changes in its aggregation behaviour, suggesting that its hydrophobicity has been modified. PMID:22676486

  15. Adaptive alterations in the fatty acids composition under induced oxidative stress in heavy metal-tolerant filamentous fungus Paecilomyces marquandii cultured in ascorbic acid presence.

    PubMed

    Słaba, Mirosława; Gajewska, Ewa; Bernat, Przemysław; Fornalska, Magdalena; Długoński, Jerzy

    2013-05-01

    The ability of the heavy metal-tolerant fungus Paecilomyces marquandii to modulate whole cells fatty acid composition and saturation in response to IC50 of Cd, Pb, Zn, Ni, and Cu was studied. Cadmium and nickel caused the most significant growth reduction. In the mycelia cultured with all tested metals, with the exception of nickel, a rise in the fatty acid unsaturation was noted. The fungus exposure to Pb, Cu, and Ni led to significantly higher lipid peroxidation. P. marquandii incubated in the presence of the tested metals responded with an increase in the level of linoleic acid and escalation of electrolyte leakage. The highest efflux of electrolytes was caused by lead. In these conditions, the fungus was able to bind up to 100 mg g(-1) of lead, whereas the content of the other metals in the mycelium was significantly lower and reached from 3.18 mg g(-1) (Cu) to 15.21 mg g(-1) (Zn). Additionally, it was shown that ascorbic acid at the concentration of 1 mM protected fungal growth and prevented the changes in the fatty acid composition and saturation but did not alleviate lipid peroxidation or affect the increased permeability of membranes after lead exposure. Pro-oxidant properties of ascorbic acid in the copper-stressed cells manifested strong growth inhibition and enhanced metal accumulation as a result of membrane damage. Toxic metals action caused cellular modulations, which might contributed to P. marquandii tolerance to the studied metals. Moreover, these changes can enhance metal removal from contaminated environment. PMID:23132407

  16. Adaptive alterations in the fatty acids composition under induced oxidative stress in heavy metal-tolerant filamentous fungus Paecilomyces marquandii cultured in ascorbic acid presence.

    PubMed

    Słaba, Mirosława; Gajewska, Ewa; Bernat, Przemysław; Fornalska, Magdalena; Długoński, Jerzy

    2013-05-01

    The ability of the heavy metal-tolerant fungus Paecilomyces marquandii to modulate whole cells fatty acid composition and saturation in response to IC50 of Cd, Pb, Zn, Ni, and Cu was studied. Cadmium and nickel caused the most significant growth reduction. In the mycelia cultured with all tested metals, with the exception of nickel, a rise in the fatty acid unsaturation was noted. The fungus exposure to Pb, Cu, and Ni led to significantly higher lipid peroxidation. P. marquandii incubated in the presence of the tested metals responded with an increase in the level of linoleic acid and escalation of electrolyte leakage. The highest efflux of electrolytes was caused by lead. In these conditions, the fungus was able to bind up to 100 mg g(-1) of lead, whereas the content of the other metals in the mycelium was significantly lower and reached from 3.18 mg g(-1) (Cu) to 15.21 mg g(-1) (Zn). Additionally, it was shown that ascorbic acid at the concentration of 1 mM protected fungal growth and prevented the changes in the fatty acid composition and saturation but did not alleviate lipid peroxidation or affect the increased permeability of membranes after lead exposure. Pro-oxidant properties of ascorbic acid in the copper-stressed cells manifested strong growth inhibition and enhanced metal accumulation as a result of membrane damage. Toxic metals action caused cellular modulations, which might contributed to P. marquandii tolerance to the studied metals. Moreover, these changes can enhance metal removal from contaminated environment.

  17. Vanadium Oxide Electrochemical Capacitors: An Investigation into Aqueous Capacitive Degradation, Alternate Electrolyte-Solvent Systems, Whole Cell Performance and Graphene Oxide Composite Electrodes

    NASA Astrophysics Data System (ADS)

    Engstrom, Allison Michelle

    Vanadium oxide has emerged as a potential electrochemical capacitor material due to its attractive pseudocapacitive performance; however, it is known to suffer from capacitive degradation upon sustained cycling. In this work, the electrochemical cycling behavior of anodically electrodeposited vanadium oxide films with various surface treatments in aqueous solutions is investigated at different pH. Quantitative compositional analysis and morphological studies provide additional insight into the mechanism responsible for capacitive degradation. Furthermore, the capacitance and impedance behavior of vanadium oxide electrochemical capacitor electrodes is compared for both aqueous and nonaqueous electrolyte-solvent systems. Alkali metal chloride and bromide electrolytes were studied in aqueous systems, and nonaqueous systems containing alkali metal bromides were studied in polar aprotic propylene carbonate (PC) or dimethyl sulfoxide (DMSO) solvents. The preferred aqueous and nonaqueous systems identified in the half-cell studies were utilized in symmetric vanadium oxide whole-cells. An aqueous system utilizing a 3.0 M NaCl electrolyte at pH 3.0 exhibited an excellent 96% capacitance retention over 3000 cycles at 10 mV s-1. An equivalent system tested at 500 mV s-1 displayed an increase in capacitance over the first several thousands of cycles, and eventually stabilized over 50,000 cycles. Electrodes cycled in nonaqueous 1.0 M LiBr in PC exhibited mostly non-capacitive charge-storage, and electrodes cycled in LiBr-DMSO exhibited a gradual capacitive decay over 10,000 cycles at 500 mV s-1. Morphological and compositional analyses, as well as electrochemical impedance modeling, provide additional insight into the cause of the cycing behavior. Lastly, reduced graphene oxide and vanadium oxide nanowire composites have been successfully synthesized using electrophoretic deposition for electrochemical capacitor electrodes. The composite material was found to perform with a

  18. The role of mites in insect-fungus associations.

    PubMed

    Hofstetter, R W; Moser, J C

    2014-01-01

    The interactions among insects, mites, and fungi are diverse and complex but poorly understood in most cases. Associations among insects, mites, and fungi span an almost incomprehensible array of ecological interactions and evolutionary histories. Insects and mites often share habitats and resources and thus interact within communities. Many mites and insects rely on fungi for nutrients, and fungi benefit from them with regard to spore dispersal, habitat provision, or nutrient resources. Mites have important impacts on community dynamics, ecosystem processes, and biodiversity within many insect-fungus systems. Given that mites are understudied but highly abundant, they likely have bigger, more important, and more widespread impacts on communities than previously recognized. We describe mutualistic and antagonistic effects of mites on insect-fungus associations, explore the processes that underpin ecological and evolutionary patterns of these multipartite communities, review well-researched examples of the effects of mites on insect-fungus associations, and discuss approaches for studying mites within insect-fungus communities.

  19. An insect parasitoid carrying an ochratoxin producing fungus

    NASA Astrophysics Data System (ADS)

    Vega, Fernando E.; Posada, Francisco; Gianfagna, Thomas J.; Chaves, Fabio C.; Peterson, Stephen W.

    2006-06-01

    The insect parasitoid Prorops nasuta has been introduced from Africa to many coffee-producing countries in an attempt to control the coffee berry borer. In this paper, we report on the sequencing of the ITS LSU-rDNA and beta-tubulin loci used to identify a fungus isolated from the cuticle of a P. nasuta that emerged from coffee berries infected with the coffee berry borer. The sequences were compared with deposits in GenBank and the fungus was identified as Aspergillus westerdijkiae. The fungus tested positive for ochratoxin A production, with varying levels depending on the media in which it was grown. These results raise the possibility that an insect parasitoid might be disseminating an ochratoxin-producing fungus in coffee plantations.

  20. High-Throughput Screening Using a Whole-Cell Virus Replication Reporter Gene Assay to Identify Inhibitory Compounds against Rift Valley Fever Virus Infection.

    PubMed

    Islam, Md Koushikul; Baudin, Maria; Eriksson, Jonas; Öberg, Christopher; Habjan, Matthias; Weber, Friedemann; Överby, Anna K; Ahlm, Clas; Evander, Magnus

    2016-04-01

    Rift Valley fever virus (RVFV) is an emerging virus that causes serious illness in humans and livestock. There are no approved vaccines or treatments for humans. The purpose of the study was to identify inhibitory compounds of RVFV infection without any preconceived idea of the mechanism of action. A whole-cell-based high-throughput drug screening assay was developed to screen 28,437 small chemical compounds targeting RVFV infection. To accomplish both speed and robustness, a replication-competent NSs-deleted RVFV expressing a fluorescent reporter gene was developed. Inhibition of fluorescence intensity was quantified by spectrophotometry and related to virus infection in human lung epithelial cells (A549). Cell toxicity was assessed by the Resazurin cell viability assay. After primary screening, 641 compounds were identified that inhibited RVFV infection by ≥80%, with ≥50% cell viability at 50 µM concentration. These compounds were subjected to a second screening regarding dose-response profiles, and 63 compounds with ≥60% inhibition of RVFV infection at 3.12 µM compound concentration and ≥50% cell viability at 25 µM were considered hits. Of these, six compounds with high inhibitory activity were identified. In conclusion, the high-throughput assay could efficiently and safely identify several promising compounds that inhibited RVFV infection.

  1. Establishment and Validation of Whole-Cell Based Fluorescence Assays to Identify Anti-Mycobacterial Compounds Using the Acanthamoeba castellanii - Mycobacterium marinum Host-Pathogen System

    PubMed Central

    Kicka, Sébastien; Trofimov, Valentin; Harrison, Christopher; Ouertatani-Sakouhi, Hajer; McKinney, John; Scapozza, Leonardo; Hilbi, Hubert; Cosson, Pierre; Soldati, Thierry

    2014-01-01

    Tuberculosis is considered to be one of the world’s deadliest disease with 2 million deaths each year. The need for new antitubercular drugs is further exacerbated by the emergence of drug-resistance strains. Despite multiple recent efforts, the majority of the hits discovered by traditional target-based screening showed low efficiency in vivo. Therefore, there is heightened demand for whole-cell based approaches directly using host-pathogen systems. The phenotypic host-pathogen assay described here is based on the monitoring of GFP-expressing Mycobacterium marinum during infection of the amoeba Acanthamoeba castellanii. The assay showed straight-forward medium-throughput scalability, robustness and ease of manipulation, demonstrating its qualities as an efficient compound screening system. Validation with a series of known antitubercular compounds highlighted the advantages of the assay in comparison to previously published macrophage-Mycobacterium tuberculosis-based screening systems. Combination with secondary growth assays based on either GFP-expressing D. discoideum or M. marinum allowed us to further fine-tune compound characterization by distinguishing and quantifying growth inhibition, cytotoxic properties and antibiotic activities of the compounds. The simple and relatively low cost system described here is most suitable to detect anti-infective compounds, whether they present antibiotic activities or not, in which case they might exert anti-virulence or host defense boosting activities, both of which are largely overlooked by classical screening approaches. PMID:24498207

  2. Whole-cell method for phenol detection based on the color reaction of phenol with 4-aminoantipyrine catalyzed by CotA laccase on endospore surfaces.

    PubMed

    Zeng, Zhiming; Tian, Longjian; Li, Zheng; Jia, Lina; Zhang, Xinya; Xia, Miaomiao; Hu, Yonggang

    2015-07-15

    A green method for phenol spectrophotometric determination was developed based on the color reaction of phenol with 4-aminoantipyrine catalyzed by addition of Bacillus amyloliquefaciens endospores in the presence of O2. The catalytic activity of the endospores may be attributed to the presence of coat protein A on the cell surfaces. This deduction was confirmed by cotA gene knock-out from B. amyloliquefaciens using the homologous double-exchange method. Under optimal conditions, linear responses were obtained over phenol concentrations ranging from 5.0×10(-5)gL(-1) to 1.0×10(-2)gL(-1) (r=0.9984) with a detection limit of 2.1×10(-5)gL(-1) (3σ). Repeatability measurements of 1.0mgL(-1) phenol provided reproducible results with a relative standard deviation of 5.3% (n=11). Standard addition tests indicated recoveries ranging from 92.78% to 107.60%. The proposed whole-cell method was successfully used to detect total phenol in synthetic samples. Results confirmed the potential use of the developed method in practical applications.

  3. Caspase-1-independent interleukin-1β is required for clearance of Bordetella pertussis infections and whole-cell vaccine-mediated immunity.

    PubMed

    Place, David E; Muse, Sarah J; Kirimanjeswara, Girish S; Harvill, Eric T

    2014-01-01

    Whooping cough remains a significant disease worldwide and its re-emergence in highly vaccinated populations has been attributed to a combination of imperfect vaccines and evolution of the pathogen. The focus of this study was to examine the role of IL-1α/β and the inflammasome in generation of the interleukin-1 (IL-1) response, which is required for the clearance of Bordetella pertussis. We show that IL-1β but not IL-1α is required for mediating the clearance of B. pertussis from the lungs of mice. We further found that IL-1β and IL-1R deficient mice, compared to wild-type, have similar but more persistent levels of inflammation, characterized by immune cell infiltration, with significantly increased IFNγ and a normal IL-17A response during B. pertussis infection. Contrary to expectations, the cleavage of precursor IL-1β to its mature form did not require caspase-1 during primary infections within the lung despite being required by bone marrow-derived macrophages exposed to live bacteria. We also found that the caspase-1 inflammasome was not required for protective immunity against a B. pertussis challenge following vaccination with heat-killed whole cell B. pertussis, despite IL-1R signaling being required. These findings demonstrate that caspase-1-independent host factors are involved in the processing of protective IL-1β responses that are critical for bacterial clearance and vaccine-mediated immunity.

  4. Evaluation of parallel milliliter-scale stirred-tank bioreactors for the study of biphasic whole-cell biocatalysis with ionic liquids.

    PubMed

    Dennewald, Danielle; Hortsch, Ralf; Weuster-Botz, Dirk

    2012-01-01

    As clear structure-activity relationships are still rare for ionic liquids, preliminary experiments are necessary for the process development of biphasic whole-cell processes involving these solvents. To reduce the time investment and the material costs, the process development of such biphasic reaction systems would profit from a small-scale high-throughput platform. Exemplarily, the reduction of 2-octanone to (R)-2-octanol by a recombinant Escherichia coli in a biphasic ionic liquid/water system was studied in a miniaturized stirred-tank bioreactor system allowing the parallel operation of up to 48 reactors at the mL-scale. The results were compared to those obtained in a 20-fold larger stirred-tank reactor. The maximum local energy dissipation was evaluated at the larger scale and compared to the data available for the small-scale reactors, to verify if similar mass transfer could be obtained at both scales. Thereafter, the reaction kinetics and final conversions reached in different reactions setups were analysed. The results were in good agreement between both scales for varying ionic liquids and for ionic liquid volume fractions up to 40%. The parallel bioreactor system can thus be used for the process development of the majority of biphasic reaction systems involving ionic liquids, reducing the time and resource investment during the process development of this type of applications.

  5. Whole-Cell Scan using Automatic Variable-Angle and Variable-Illumination-Depth Pseudo—Total Internal Reflection Fluorescence Microscopy

    SciTech Connect

    Sun, Wei; Xu, Aoshuang; Marchuk, Kyle; Wang, Gufeng; Fang, Ning

    2011-08-01

    An automatic calibration and angle-scanning prism-type total internal reflection fluorescence microscope (TIRFM) was modified to function in both TIRFM and pseudo-TIRFM modes. When the incident angle of the excitation laser beam was controlled to be larger than the critical angle, the instrument served as a variable-angle TIRFM. A homemade computer program automatically calibrates the laser illumination spot in the sample to overlap with the center of the microscope's field of view. Then, by measuring the fluorescence intensities at different incident angles, the z-positions of fluorescent nanospheres close to the cell basolateral membrane can be extracted. When the incident angle is reduced to be in the subcritical range, the instrument works as a pseudo-TIRFM. The whole cell body from bottom to top can be imaged in a vertical scan process. Furthermore, the illumination field depth in the pseudo-TIRFM can be controlled by changing the incident angle or the horizontal position of the laser spot.

  6. The Cardiomyopathy Lamin A/C D192G Mutation Disrupts Whole-Cell Biomechanics in Cardiomyocytes as Measured by Atomic Force Microscopy Loading-Unloading Curve Analysis

    PubMed Central

    Lanzicher, Thomas; Martinelli, Valentina; Puzzi, Luca; Del Favero, Giorgia; Codan, Barbara; Long, Carlin S.; Mestroni, Luisa; Taylor, Matthew R. G.; Sbaizero, Orfeo

    2015-01-01

    Atomic force microscopy (AFM) cell loading/unloading curves were used to provide comprehensive insights into biomechanical behavior of cardiomyocytes carrying the lamin A/C (LMNA) D192G mutation known to cause defective nuclear wall, myopathy and severe cardiomyopathy. Our results suggested that the LMNA D192G mutation increased maximum nuclear deformation load, nuclear stiffness and fragility as compared to controls. Furthermore, there seems to be a connection between this lamin nuclear mutation and cell adhesion behavior since LMNA D192G cardiomyocytes displayed loss of AFM probe-to-cell membrane adhesion. We believe that this loss of adhesion involves the cytoskeletal architecture since our microscopic analyses highlighted that mutant LMNA may also lead to a morphological alteration in the cytoskeleton. Furthermore, chemical disruption of the actin cytoskeleton by cytochalasin D in control cardiomyocytes mirrored the alterations in the mechanical properties seen in mutant cells, suggesting a defect in the connection between the nucleoskeleton, cytoskeleton and cell adhesion molecules in cells expressing the mutant protein. These data add to our understanding of potential mechanisms responsible for this fatal cardiomyopathy, and show that the biomechanical effects of mutant lamin extend beyond nuclear mechanics to include interference of whole-cell biomechanical properties. PMID:26323789

  7. Two-dimensional blue native/SDS-PAGE analysis of whole cell lysate protein complexes of rice in response to salt stress.

    PubMed

    Hashemi, Amenehsadat; Gharechahi, Javad; Nematzadeh, Ghorbanali; Shekari, Faezeh; Hosseini, Seyed Abdollah; Salekdeh, Ghasem Hosseini

    2016-08-01

    To understand the biology of a plant in response to stress, insight into protein-protein interactions, which almost define cell behavior, is thought to be crucial. Here, we provide a comparative complexomics analysis of leaf whole cell lysate of two rice genotypes with contrasting responses to salt using two-dimensional blue native/SDS-PAGE (2D-BN/SDS-PAGE). We aimed to identify changes in subunit composition and stoichiometry of protein complexes elicited by salt. Using mild detergent for protein complex solubilization, we were able to identify 9 protein assemblies as hetero-oligomeric and 30 as homo-oligomeric complexes. A total of 20 proteins were identified as monomers in the 2D-BN/SDS-PAGE gels. In addition to identifying known protein complexes that confirm the technical validity of our analysis, we were also able to discover novel protein-protein interactions. Interestingly, an interaction was detected for glycolytic enzymes enolase (ENO1) and triosephosphate isomerase (TPI) and also for a chlorophyll a-b binding protein and RuBisCo small subunit. To show changes in subunit composition and stoichiometry of protein assemblies during salt stress, the differential abundance of interacting proteins was compared between salt-treated and control plants. A detailed exploration of some of the protein complexes provided novel insight into the function, composition, stoichiometry and dynamics of known and previously uncharacterized protein complexes in response to salt stress. PMID:27362847

  8. Harnessing Candida tenuis and Pichia stipitis in whole-cell bioreductions of o-chloroacetophenone: stereoselectivity, cell activity, in situ substrate supply and product removal.

    PubMed

    Gruber, Christoph; Krahulec, Stefan; Nidetzky, Bernd; Kratzer, Regina

    2013-06-01

    Generally, recombinant and native microorganisms can be employed as whole-cell catalysts. The application of native hosts, however, shortens the process development time by avoiding multiple steps of strain construction. Herein, we studied the NAD(P)H-dependent reduction of o-chloroacetophenone by isolated xylose reductases and their native hosts Candida tenuis and Pichia stipitis. The natural hosts were benchmarked against Escherichia coli strains co-expressing xylose reductase and a dehydrogenase for co-enzyme recycling. Xylose-grown cells of C. tenuis and P. stipitis displayed specific o-chloroacetophenone reductase activities of 366 and 90 U gCDW (-1) , respectively, in the cell-free extracts. Fresh biomass was employed in batch reductions of 100 mM o-chloroacetophenone using glucose as co-substrate. Reaction stops at a product concentration of about 15 mM, which suggests sensitivity of the catalyst towards the formed product. In situ substrate supply and product removal by the addition of 40% hexane increased catalyst stability. Optimisation of the aqueous phase led to a (S)-1-(2-chlorophenyl)ethanol concentration of 71 mM (ee > 99.9%) obtained with 44 gCDW L(-1) of C. tenuis. The final difference in productivities between native C. tenuis and recombinant E. coli was < 1.7-fold. The optically pure product is a required key intermediate in the synthesis of a new class of chemotherapeutic substances (polo-like kinase 1 inhibitors). PMID:23589466

  9. The effect of pertussis toxin and whole-cell pertussis vaccine on haemodynamics and autonomic responsiveness in the rat depends on route of administration and age.

    PubMed

    van Amsterdam, J G; te Biesebeek, J D; van de Kuil, T; van der Laan, J W; Wemer, J; de Wildt, D J; Vleeming, W

    1998-04-01

    Vaccination of children with Diphtheria, Tetanus, Poliomyelitis and pertussis vaccine (DTPoP-vaccine) containing the whole-cell pertussis component is known to be associated with manifestation of side-effects such as acute encephalopathy, convulsions and hypotensive-hyporesponsive episodes. In young and adult rats the effects of pertussis toxin and DTPoP-vaccine on haemodynamics and autonomic responsiveness are evaluated following treatment with high dose via different routes of administration (s.c., i.p. and i.v.). The effect of pertussis toxin is dose-dependent (between 1 and 20 micrograms kg-1) and largest responses are observed after i.v. administration. At 20 micrograms kg-1, i.v. pertussis toxin decreases baseline diastolic blood pressure and increases baseline heart rate by 31% and inhibits autonomic responsiveness (salbutamol-induced increase in diastolic blood pressure and arecoline-induced decrease in heart rate). In adult rats DTPoP-vaccine induces generally more prominent effects than in young rats. In adult rats DTPoP-vaccine reduces baseline diastolic blood pressure by 25% while no response is observed in young rats. In adult rats DTPoP inhibits the adrenergic response though less compared to treatment of pertussis toxin. After treatment with DTPoP-vaccine (single or twice) only minor differences are observed between young and adult rats. Present results show that adult rats are more sensitive to pertussis toxin and pertussis vaccine than young rats and that the responses depend on the route of administration.

  10. Progress against Escherichia coli with the Oxazolidinone Class of Antibacterials: Test Case for a General Approach To Improving Whole-Cell Gram-Negative Activity.

    PubMed

    Takrouri, Khuloud; Cooper, Harold D; Spaulding, Andrew; Zucchi, Paola; Koleva, Bilyana; Cleary, Dillon C; Tear, Westley; Beuning, Penny J; Hirsch, Elizabeth B; Aggen, James B

    2016-06-10

    Novel antibacterials with activity against the Gram-negative bacteria associated with nosocomial infections, including Escherichia coli and other Enterobacteriaceae, are urgently needed due to the increasing prevalence of multidrug-resistant strains. A major obstacle that has stalled progress on nearly all small-molecule classes with potential for activity against these species has been achieving sufficient whole-cell activity, a difficult challenge due to the formidable outer membrane and efflux barriers intrinsic to these species. Using a set of compound design principles derived from available information relating physicochemical properties to Gram-negative entry or activity, we synthesized and evaluated a focused library of oxazolidinone analogues, a currently narrow spectrum class of antibacterials active only against Gram-positive bacteria. In this series, we have explored the effectiveness for improving Gram-negative activity by identifying and combining beneficial structural modifications in the C-ring region. We have found polar and/or charge-carrying modifications that, when combined in hybrid C-ring analogues, appear to largely overcome the efflux and/or permeability barriers, resulting in improved Gram-negative activity. In particular, those analogues least effected by efflux and the permeation barrier had significant zwitterionic character. PMID:27627629

  11. Phylogenetic diversity and whole-cell hybridization of oxymonad flagellates from the hindgut of the wood-feeding lower termite Reticulitermes flavipes.

    PubMed

    Stingl, Ulrich; Brune, Andreas

    2003-04-01

    SSU rRNA genes of oxymonad protists from the hindgut of the wood-feeding termite Reticulitermes flavipes were PCR-amplified using a newly designed oxymonad-specific forward primer and a newly designed reverse primer specific for termite gut flagellates. After cloning, the clone library was sorted into four groups by RFLP analysis and nearly full-length SSU rRNA gene sequences were obtained for representative clones from each group. Phylogenetic analysis revealed that sequences of all four groups formed a monophyletic cluster with the only other existing SSU rRNA gene sequence of oxymonads. Using whole-cell hybridization with clone-specific fluorescently labeled probes, each of the four clone groups could be assigned to a specific morphotype, which were identified as Dinenympha gracilis, Dinenympha fimbriata, and so-far undescribed species of Pyrsonympha and Dinenympha. Our results demonstrate that the morphological variety of oxymonads is not caused by the presence of different developmental stages of the same organism, but that the various morphotypes represent different species.

  12. Use of DNA and peptide nucleic acid molecular beacons for detection and quantification of rRNA in solution and in whole cells.

    PubMed

    Xi, Chuanwu; Balberg, Michal; Boppart, Stephen A; Raskin, Lutgarde

    2003-09-01

    DNA and peptide nucleic acid (PNA) molecular beacons were successfully used to detect rRNA in solution. In addition, PNA molecular beacon hybridizations were found to be useful for the quantification of rRNA: hybridization signals increased in a linear fashion with the 16S rRNA concentrations used in this experiment (between 0.39 and 25 nM) in the presence of 50 nM PNA MB. DNA and PNA molecular beacons were successfully used to detect whole cells in fluorescence in situ hybridization (FISH) experiments without a wash step. The FISH results with the PNA molecular beacons were superior to those with the DNA molecular beacons: the hybridization kinetics were much faster, the signal-to-noise ratio was much higher, and the specificity was much better for the PNA molecular beacons. Finally, it was demonstrated that the combination of the use of PNA molecular beacons in FISH and flow cytometry makes it possible to rapidly collect quantitative FISH data. Thus, PNA molecular beacons might provide a solution for limitations of traditional FISH methods, such as variable target site accessibility, poor sensitivity for target cells with low rRNA content, background fluorescence, and applications of FISH in microfluidic devices.

  13. Whole-Cell Hybridization of Frankia Strains with Fluorescence- or Digoxigenin-Labeled, 16S rRNA-Targeted Oligonucleotide Probes

    PubMed Central

    Hahn, Dittmar; Amann, Rudolf I.; Zeyer, Josef

    1993-01-01

    Whole-cell hybridization with non-radioactively labeled oligonucleotide probes was used to detect and identify Frankia strains in pure cultures and in nodules. Digoxigenin-labeled probes, which were detected with antibody-alkaline phosphatase conjugates, were more suitable for in situ detection of Frankia strains than fluorescent probes since the sensitivity of the former was higher and problems arising from the autofluorescence of cells and plant material were avoided. Successful detection of Frankia strains in paraformaldehyde-fixed cell material with digoxigenin-labeled oligonucleotide probes depended on pretreatments to permeabilize the cells. Specific hybridization signals on vesicles were obtained after lysozyme pretreatment (1 mg ml-1 for 30 min at 20°C). Reliable penetration of the antibody-enzyme conjugate into hyphae required additional washing with the detergent Nonidet P-40 (0.1%) and toluene (1% in ethanol) after lysozyme treatment. Identification of Frankia vesicles in nodule homogenates was possible only after the removal of the polysaccharide capsule surrounding the vesicles. Incubation with H2O2 (15% in water for 1 h at room temperature) before lysozyme and detergent treatments was found to facilitate specific hybridization. No filaments or spores could be detected in nodule homogenates. This technique should be a powerful tool in the identification of Frankia isolates, in the characterization of as-yet-uncultured nodule populations, and in the confirmation of the origin of unusual Frankia isolates. Images PMID:16348948

  14. Effectiveness and economic analysis of the whole cell/recombinant B subunit (WC/rbs) inactivated oral cholera vaccine in the prevention of traveller's diarrhoea

    PubMed Central

    2009-01-01

    Background Nowadays there is a debate about the indication of the oral whole-cell/recombinant B-subunit cholera vaccine (WC/rBS) in traveller's diarrhoea. However, a cost-benefit analysis based on real data has not been published. Methods A cost-effectiveness and cost-benefit study of the oral cholera vaccine (WC/rBS), Dukoral® for the prevention of traveller's diarrhoea (TD) was performed in subjects travelling to cholera risk areas. The effectiveness of WC/rBS vaccine in the prevention of TD was analyzed in 362 travellers attending two International Vaccination Centres in Spain between May and September 2005. Results The overall vaccine efficacy against TD was 42,6%. Direct healthcare-related costs as well as indirect costs (lost vacation days) subsequent to the disease were considered. Preventive vaccination against TD resulted in a mean saving of 79.26 € per traveller. Conclusion According to the cost-benefit analysis performed, the recommendation for WC/rBS vaccination in subjects travelling to zones at risk of TD is beneficial for the traveller, regardless of trip duration and visited continent. PMID:19445712

  15. High-Throughput Screening Using a Whole-Cell Virus Replication Reporter Gene Assay to Identify Inhibitory Compounds against Rift Valley Fever Virus Infection.

    PubMed

    Islam, Md Koushikul; Baudin, Maria; Eriksson, Jonas; Öberg, Christopher; Habjan, Matthias; Weber, Friedemann; Överby, Anna K; Ahlm, Clas; Evander, Magnus

    2016-04-01

    Rift Valley fever virus (RVFV) is an emerging virus that causes serious illness in humans and livestock. There are no approved vaccines or treatments for humans. The purpose of the study was to identify inhibitory compounds of RVFV infection without any preconceived idea of the mechanism of action. A whole-cell-based high-throughput drug screening assay was developed to screen 28,437 small chemical compounds targeting RVFV infection. To accomplish both speed and robustness, a replication-competent NSs-deleted RVFV expressing a fluorescent reporter gene was developed. Inhibition of fluorescence intensity was quantified by spectrophotometry and related to virus infection in human lung epithelial cells (A549). Cell toxicity was assessed by the Resazurin cell viability assay. After primary screening, 641 compounds were identified that inhibited RVFV infection by ≥80%, with ≥50% cell viability at 50 µM concentration. These compounds were subjected to a second screening regarding dose-response profiles, and 63 compounds with ≥60% inhibition of RVFV infection at 3.12 µM compound concentration and ≥50% cell viability at 25 µM were considered hits. Of these, six compounds with high inhibitory activity were identified. In conclusion, the high-throughput assay could efficiently and safely identify several promising compounds that inhibited RVFV infection. PMID:26762502

  16. Biological control of Ascaris suum eggs by Pochonia chlamydosporia fungus.

    PubMed

    Ferreira, Sebastião Rodrigo; de Araújo, Jackson Victor; Braga, Fábio Ribeiro; Araujo, Juliana Milani; Frassy, Luiza Neme; Ferreira, Aloízio Soares

    2011-12-01

    Ascaris suum is a gastrointestinal nematode parasite of swines. The aim of this study was to observe Pochonia chlamydosporia fungus on biological control of A. suum eggs after fungus passage through swines gastrointestinal tract. Eighteen pigs, previously dewormed, were randomly divided into three groups: group 1, treated with the fungus isolate VC4; group 2, treated with the fungus isolate VC1 and group 3 did not receive fungus (control). In the treated groups, each animal received a 9 g single dose of mycelium mass containing P. chlamydosporia (VC1 or VC4). Thereafter, animal fecal samples were collected at the following intervals: 8, 12, 24, 36, 48, 72 and 96 h after treatment beginning and these were poured in Petri dishes containing 2% water-agar culture medium. Then, 1,000 A. suum eggs were poured into each dish and kept in an incubator at 26 °C and in the dark for 30 days. After this period, approximately 100 eggs were removed from each Petri dish and morphologically analyzed under light microscopy following the ovicidal activity parameters. The higher percentage observed for isolated VC4 eggs destruction was 57.5% (36 h) after fungus administration and for isolate VC1 this percentage was 45.8% (24 h and 72 h) (p > 0.01). P. chlamydosporia remained viable after passing through the gastrointestinal tract of swines, maintaining its ability of destroying A. suum eggs.

  17. Biological control of Ascaris suum eggs by Pochonia chlamydosporia fungus.

    PubMed

    Ferreira, Sebastião Rodrigo; de Araújo, Jackson Victor; Braga, Fábio Ribeiro; Araujo, Juliana Milani; Frassy, Luiza Neme; Ferreira, Aloízio Soares

    2011-12-01

    Ascaris suum is a gastrointestinal nematode parasite of swines. The aim of this study was to observe Pochonia chlamydosporia fungus on biological control of A. suum eggs after fungus passage through swines gastrointestinal tract. Eighteen pigs, previously dewormed, were randomly divided into three groups: group 1, treated with the fungus isolate VC4; group 2, treated with the fungus isolate VC1 and group 3 did not receive fungus (control). In the treated groups, each animal received a 9 g single dose of mycelium mass containing P. chlamydosporia (VC1 or VC4). Thereafter, animal fecal samples were collected at the following intervals: 8, 12, 24, 36, 48, 72 and 96 h after treatment beginning and these were poured in Petri dishes containing 2% water-agar culture medium. Then, 1,000 A. suum eggs were poured into each dish and kept in an incubator at 26 °C and in the dark for 30 days. After this period, approximately 100 eggs were removed from each Petri dish and morphologically analyzed under light microscopy following the ovicidal activity parameters. The higher percentage observed for isolated VC4 eggs destruction was 57.5% (36 h) after fungus administration and for isolate VC1 this percentage was 45.8% (24 h and 72 h) (p > 0.01). P. chlamydosporia remained viable after passing through the gastrointestinal tract of swines, maintaining its ability of destroying A. suum eggs. PMID:21796329

  18. Development of a carbonate absorption-based process for post-combustion CO2 capture: The role of biocatalyst to promote CO2 absorption rate

    USGS Publications Warehouse

    Lu, Y.; Ye, X.; Zhang, Z.; Khodayari, A.; Djukadi, T.

    2011-01-01

    An Integrated Vacuum Carbonate Absorption Process (IVCAP) for post-combustion carbon dioxide (CO2) capture is described. IVCAP employs potassium carbonate (PC) as a solvent, uses waste or low quality steam from the power plant for CO2 stripping, and employs a biocatalyst, carbonic anhydrase (CA) enzyme, for promoting the CO2 absorption into PC solution. A series of experiments were performed to evaluate the activity of CA enzyme mixed in PC solutions in a stirred tank reactor system under various temperatures, CA dosages, CO2 loadings, CO2 partial pressures, and the presence of major flue gas contaminants. It was demonstrated that CA enzyme is an effective biocatalyst for CO2 absorption under IVCAP conditions. ?? 2011 Published by Elsevier Ltd.

  19. L-glutamic acid production in a continuous stirred tank bioreactor using coimmobilized bio-catalyst using a fluorosensor.

    PubMed

    Prabhu, N; Babu, J Sarat Chandra; Sundaram, S

    2002-01-01

    The production of L-Glutamic acid has been studied using coimmobilized whole cells of pseudomonas reptilivora and micrococcus glutamicus in a two litre Tokyo Rikakikai fermentor using glucose as selected production medium. The process was carried out at an optimum temperature of 32 degree Celsius and a pH of 7.2. The progress of the reaction was recorded using Dr. Ingold fluorosensor. The effect of initial substrate concentration, speed of agitation, volume ofcalcium alginate beads and aeration rate on the yield of glutamic acid has been investigated. It has been found that the acid production increases exponentially with substrate concentration, and mass transfer co-efficient varied linearly with aeration rate. The kinetic parameters also had been estimated.

  20. Iron competition in fungus-plant interactions

    PubMed Central

    López-Berges, Manuel S.; Turrà, David; Capilla, Javier; Schafferer, Lukas; Matthijs, Sandra; Jöchl, Christoph; Cornelis, Pierre; Guarro, Josep; Haas, Hubertus; Di Pietro, Antonio

    2013-01-01

    Soilborne fungal pathogens are highly persistent and provoke important crop losses. During saprophytic and infectious stages in the soil, these organisms face situations of nutrient limitation and lack of essential elements, such as iron. We investigated the role of the bZIP transcription factor HapX as a central regulator of iron homeostasis and virulence in the vascular wilt fungus Fusarium oxysporum. This root-infecting plant pathogen attacks more than hundred different crops and is an emerging human opportunistic invader. Although iron uptake remains unaffected in a strain lacking HapX, de-repression of genes implicated in iron-consuming processes such as respiration, amino acid metabolism, TCA cycle and heme biosynthesis lead to severely impaired growth under iron-limiting conditions. HapX is required for full virulence of F. oxysporum in tomato plants and essential for infection in immunodepressed mice. Virulence attenuation of the ΔhapX strain on tomato plants is more pronounced by co-inoculation of roots with the biocontrol strain Pseudomonas putida KT2440, but not with a mutant deficient in siderophores production. These results demonstrate that HapX is required for iron competition of F. oxysporum in the tomato rhizosphere and establish a conserved role for HapX-mediated iron homeostasis in fungal infection of plants and mammals. PMID:23299422