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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. PMID:18976825

  2. 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. PMID:19085591

  3. [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

  4. [Stability of whole cell biocatalyst for biodiesel production from renewable oils].

    PubMed

    Sun, Ting; Du, Wei; Liu, Dehua; Li, Wei; Zeng, Jing; Dai, Lingmei

    2009-09-01

    Lipase-mediated biodiesel production becomes increasingly important because of mild reaction conditions, pollution free during the process and easy product separation. Compared with traditional immobilized lipase, whole cell biocatalyst is promising for biodiesel production because it is easy to prepare and has higher enzyme activity recovery. Rhizopus oryzae IFO4697 can be used as the catalyst for biodiesel production. To further study the stability of the whole cell biocatalyst is extremely important for its further application on large scale. This paper focuses on the stability study of Rhizopus oryzae IFO4697 when used for the methanolysis of renewable oils for biodiesel production. The results showed that water content was important for achieving high catalytic activity and good stability of the biocatalyst. The optimum water content was found to be 5%-15%. Both particle size and desiccation methods showed no obvious effect on the stability of the biocatalyst. With GA cross-linking pretreatment, the stability of the biocatalyst could be improved significantly. When Rhizopus oryzae IFO4697 repeatedly used for next batch reaction, direct vacuum filtration was found to be a good way for the maintenance of good stability of the biocatalyst. Under the optimum reaction conditions, the methyl ester yield could keep over 80% during 20 repeated reaction batches. PMID:19938482

  5. 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.

  6. Simple enzymatic procedure for L-carnosine synthesis: whole-cell biocatalysis and efficient biocatalyst recycling.

    PubMed

    Heyland, Jan; Antweiler, Nicolai; Lutz, Jochen; Heck, Tobias; Geueke, Birgit; Kohler, Hans-Peter E; Blank, Lars M; Schmid, Andreas

    2010-01-01

    β-Peptides and their derivates are usually stable to proteolysis and have an increased half-life compared with α-peptides. Recently, β-aminopeptidases were described as a new enzyme class that enabled the enzymatic degradation and formation of β-peptides. As an alternative to the existing chemical synthesis routes, the aim of the present work was to develop a whole-cell biocatalyst for the synthesis and production of β-peptides using this enzymatic activity. For the optimization of the reaction system we chose the commercially relevant β,α-dipeptide L-carnosine (β-alanine-L-histidine) as model product. We were able to show that different recombinant yeast and bacteria strains, which overexpress a β-peptidase, could be used directly as whole-cell biocatalysts for the synthesis of L-carnosine. By optimizing relevant reaction conditions for the best-performing recombinant Escherichia coli strain, such as pH and substrate concentrations, we obtained high l-carnosine yields of up to 71%. Long-time as well as biocatalyst recycling experiments indicated a high stability of the developed biocatalyst for at least five repeated batches. Application of the recombinant E. coli in a fed-batch process enabled the accumulation of l-carnosine to a concentration of 3.7 g l(-1). PMID:21255308

  7. 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

  8. Regioselective synthesis of cytarabine monopropionate by using a fungal whole-cell biocatalyst in nonaqueous medium.

    PubMed

    Yang, Mei-yan; Wu, Hui; Lu, Zhi-hong; Li, Xiao-feng; Lai, Fu-rao; Zhao, Guang-lei

    2014-08-01

    The utilization of a dehydrated fungal biocatalyst of Aspergillus oryzae cells was successfully performed to achieve efficient acylation modification of a polar nucleoside cytarabine (ara-C). Organic solvents showed evident influence on the reaction catalyzed by the A. oryzae whole-cells. Except for hexane-pyridine, the catalytic activity and regioselectivity of the whole-cells clearly increased with increasing the polarity of the hydrophobic organic solvents used. The effects of some crucial factors on the reaction were further examined. The best reaction medium, hydrophobic solvent concentration, vinyl propionate/ara-C ratio, reaction temperature and shaking speed were confirmed as isopropyl ether (IPE)-pyridine, 30% (v/v), 90, 30 °C and 140-180 rpm, respectively. The cell biocatalyst also showed good thermal stabilities in both IPE-pyridine and hexane-pyridine systems. In addition, the desired 3'-O-propional derivative of ara-C was synthesized with the yields of 88.3% and regioselectivity (>70%). The resulting biocatalytic system appears to be an effective alternative, and can thus be employed for application in highly regioselective modification of nucleoside analogues. PMID:24957351

  9. Mucor circinelloides whole-cells as a biocatalyst for the production of ethyl esters based on babassu oil.

    PubMed

    Andrade, Grazielle S S; Carvalho, Ana K F; Romero, Cintia M; Oliveira, Pedro C; de Castro, Heizir F

    2014-12-01

    The intracellular lipase production by Mucor circinelloides URM 4182 was investigated through a step-by-step strategy to attain immobilized whole-cells with high lipase activity. Physicochemical parameters, such as carbon and nitrogen sources, inoculum size and aeration, were studied to determine the optimum conditions for both lipase production and immobilization in polyurethane support. Olive oil and soybean peptone were found to be the best carbon and nitrogen sources, respectively, to enhance the intracellular lipase activity. Low inoculum level and poor aeration rate also provided suitable conditions to attain high lipase activity (64.8 ± 0.8 U g(-1)). The transesterification activity of the immobilized whole- cells was assayed and optimal reaction conditions for the ethanolysis of babassu oil were determined by experimental design. Statistical analysis showed that M. circinelloides whole-cells were able to produce ethyl esters at all tested conditions, with the highest yield attained (98.1 %) at 35 °C using an 1:6 oil-to-ethanol molar ratio. The biocatalyst operational stability was also assayed in a continuous packed bed reactor (PBR) charged with glutaraldehyde (GA) and Aliquat-treated cells revealing half-life of 43.0 ± 0.5 and 20.0 ± 0.8 days, respectively. These results indicate the potential of immobilized M. circinelloides URM 4182 whole-cells as a low-cost alternative to conventional biocatalysts in the production of ethyl esters from babassu oil. PMID:24958521

  10. 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

  11. 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

  12. Synthesis of chiral 2-alkanols from n-alkanes by a P. putida whole-cell biocatalyst.

    PubMed

    Tieves, Florian; Erenburg, Isabelle N; Mahmoud, Osama; Urlacher, Vlada B

    2016-09-01

    The cytochrome P450 monooxygenase CYP154A8 from Nocardia farcinica was previously found to catalyze hydroxylation of linear alkanes (C7 -C9 ) with a high regio- and stereoselectivity. The objective of this study was to integrate CYP154A8 along with suitable redox partners into a whole-cell system for the production of chiral 2-alkanols starting from alkanes. Both recombinant Escherichia coli and Pseudomonas putida whole-cell biocatalysts tested for this purpose showed the ability to produce chiral alkanols, but a solvent tolerant P. putida strain demonstrated several advantages in the applied biphasic reaction system. The optimized P. putida whole-cell system produced ∼16 mM (S)-2-octanol with 87% ee from octane, which is more than sevenfold higher than the previously described system with isolated enzymes. The achieved enantiopurity of the product could further be increased up to 99% ee by adding an alcohol dehydrogenase (ADH) to the alkane-oxidizing P. putida whole-cell systems. By using this setup for the individual conversions of heptane, octane or nonane, 2.6 mM (S)-2-heptanol with 91% ee, 5.4 mM (S)-2-octanol with 97% ee, or 5.5 mM (S)-2-nonanol with 97% ee were produced, respectively. The achieved concentrations of chiral 2-alkanols are the highest reported for a P450-based whole-cell system so far. Biotechnol. Bioeng. 2016;113: 1845-1852. © 2016 Wiley Periodicals, Inc. PMID:26887569

  13. 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

  14. 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

  15. Construction of an efficient Escherichia coli whole-cell biocatalyst for D-mannitol production.

    PubMed

    Reshamwala, Shamlan M S; Pagar, Sandip K; Velhal, Vishal S; Maranholakar, Vijay M; Talangkar, Vishal G; Lali, Arvind M

    2014-12-01

    Mannitol is a six carbon sugar alcohol that finds applications in the pharmaceutical and food industries. A novel Escherichia coli strain capable of converting D-glucose to D-mannitol has been constructed, wherein native mannitol-1-phosphate dehydrogenase (MtlD) and codon-optimized Eimeria tenella mannitol-1-phosphatase (M1Pase) have been overexpressed. Codon-optimized Pseudomonas stutzeri phosphite dehydrogenase (PtxD) was overexpressed for cofactor (NADH) regeneration with the concomitant oxidation of phosphite to phosphate. Whole-cell biotransformation using resting cells in a medium containing D-glucose and equimolar sodium phosphite resulted in d-mannitol yield of 87 mol%. Thus, production of an industrially relevant biochemical without using complex media components and elaborate process control mechanisms has been demonstrated. PMID:24908186

  16. 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

  17. Highly efficient biodiesel production by a whole-cell biocatalyst employing a system with high lipase expression in Aspergillus oryzae.

    PubMed

    Takaya, Tomohiro; Koda, Risa; Adachi, Daisuke; Nakashima, Kazunori; Wada, Junpei; Bogaki, Takayuki; Ogino, Chiaki; Kondo, Akihiko

    2011-05-01

    In the present study, a system with high lipase expression in Aspergillus oryzae was developed using an improved enolase promoter (P-enoA124) and the 5' untranslated region of a heat-shock protein (Hsp-UTR). P-enoA142 enhanced the transcriptional level of a heterologous lipase gene and Hsp-UTR improved its translational efficiency. Fusarium heterosporum lipase (FHL) was inserted into a pSENSU-FHL expression vector harboring P-enoA142 and Hsp-UTR and was transformed into an A. oryzae NS4 strain. Transformants possessing pSENSU-FHL in single (pSENSU-FHL#1) and double copies (pSENSU-FHL#2) were selected to evaluate the lipase activity of the whole-cell biocatalyst. The two strains, pSENSU-FHL#1 and #2, showed excellent lipase activity in hydrolysis compared with the strain transformed with conventional expression vector pNAN8142-FHL. Furthermore, by using pSENSU-FHL#2, methanolysis could proceed much more effectively without deactivation, which allowed a swift addition of methanol to the reaction mixture, thereby reducing reaction time. PMID:21380514

  18. Development of recombinant Escherichia coli whole-cell biocatalyst expressing a novel alkaline lipase-coding gene from Proteus sp. for biodiesel production.

    PubMed

    Gao, Bei; Su, Erzheng; Lin, Jinping; Jiang, Zhengbing; Ma, Yushu; Wei, Dongzhi

    2009-01-15

    A lipase-producing bacterium K107 was isolated from soil samples of China and identified to be a strain of Proteus sp. With genome-walking method, the open reading frame of lipase gene lipK107, encoding 287 amino acids, was cloned and expressed in a heterologous host, Escherichia coli BL21 (DE3). The recombinant lipase was purified and characterized, and the optimum pH of the purified LipK107 was 9, at 35 degrees C. The recombinant E. coli expressing lipK107 was applied in biodiesel production in the form of whole-cell biocatalyst. Activity of the biocatalyst increased significantly when cells were permeabilized with 0.3% (w/v) cetyl-trimethylammoniumbromide (CTAB). This transesterification was carried out efficiently in a mixture containing 5M equivalents of methanol to the oil and 100% water by weight of the substrate. It was the first time to use E. coli whole-cell biocatalyst expressing lipase in biodiesel production, and the biodiesel reached a yield of nearly 100% after 12h reaction at the optimal temperature of 15 degrees C, which was the lowest temperature among all the known catalyst in biodiesel production. PMID:19007827

  19. Construction of a highly efficient Bacillus subtilis 168 whole-cell biocatalyst and its application in the production of L-ornithine.

    PubMed

    Wang, Meizhou; Xu, Meijuan; Rao, Zhiming; Yang, Taowei; Zhang, Xian

    2015-11-01

    L-Ornithine, a non-protein amino acid, is usually extracted from hydrolyzed protein as well as produced by microbial fermentation. Here, we focus on a highly efficient whole-cell biocatalyst for the production of L-ornithine. The gene argI, encoding arginase, which catalyzes the hydrolysis of L-arginine to L-ornithine and urea, was cloned from Bacillus amyloliquefaciens B10-127 and expressed in GRAS strain Bacillus subtilis 168. The recombinant strain exhibited an arginase activity of 21.9 U/mg, which is 26.7 times that of wild B. subtilis 168. The optimal pH and temperature of the purified recombinant arginase were 10.0 and 40 °C, respectively. In addition, the recombinant arginase exhibited a strong Mn(2+) preference. When using whole-cell biocatalyst-based bioconversion, a hyper L-ornithine production of 356.9 g/L was achieved with a fed-batch strategy in a 5-L reactor within 12 h. This whole-cell bioconversion study demonstrates an environmentally friendly strategy for L-ornithine production in industry. PMID:26314414

  20. Combination of phenylpyruvic acid (PPA) pathway engineering and molecular engineering of L-amino acid deaminase improves PPA production with an Escherichia coli whole-cell biocatalyst.

    PubMed

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

    2016-03-01

    In our previous study, we produced phenylpyruvic acid (PPA) in one step from L-phenylalanine by using an Escherichia coli whole-cell biocatalyst expressing an L-amino acid deaminase (L-AAD) from Proteus mirabilis KCTC2566. However, the PPA titer was low due to the degradation of PPA and low substrate specificity of L-AAD. In this study, metabolic engineering of the L-phenylalanine degradation pathway in E. coli and protein engineering of L-AAD from P. mirabilis were performed to improve the PPA titer. First, three aminotransferase genes were knocked out to block PPA degradation, which increased the PPA titer from 3.3 ± 0.2 to 3.9 ± 0.1 g/L and the substrate conversion ratio to 97.5 %. Next, L-AAD was engineered via error-prone polymerase chain reaction, followed by site-saturation mutation to improve its catalytic performance. The triple mutant D165K/F263M/L336M produced the highest PPA titer of 10.0 ± 0.4 g/L, with a substrate conversion ratio of 100 %, which was 3.0 times that of wild-type L-AAD. Comparative kinetics analysis showed that compared with wild-type L-AAD, the triple mutant had higher substrate-binding affinity and catalytic efficiency. Finally, an optimal fed-batch biotransformation process was developed to achieve a maximal PPA titer of 21 ± 1.8 g/L within 8 h. This study developed a robust whole-cell E. coli biocatalyst for PPA production by integrating metabolic and protein engineering, strategies that may be useful for the construction of other biotransformation biocatalysts. PMID:26552798

  1. 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...

  2. 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. PMID:26777245

  3. 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

  4. 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

  5. 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. PMID:22537973

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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

    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

  11. Biodiesel Production: Utilization of Loofah Sponge to Immobilize Rhizopus chinensis CGMCC #3.0232 Cells as a Whole-Cell Biocatalyst.

    PubMed

    He, Qiyang; Xia, Qianjun; Wang, Yuejiao; Li, Xun; Zhang, Yu; Hu, Bo; Wang, Fei

    2016-07-28

    Rhizopus chinensis cells immobilized on loofah (Luffa cylindrica) sponges were used to produce biodiesel via the transesterification of soybean oil. In whole-cell immobilization, loofah sponge is considered to be a superior alternative to conventional biomass carriers because of its biodegradable and renewable properties. During cell cultivation, Rhizopus chinensis mycelia can spontaneously and firmly adhere to the surface of loofah sponge particles. The optimal conditions for processing 9.65 g soybean oil at 40°C and 180 rpm using a 3:1 methanol-to-oil molar ratio were found to be 8% cell addition and 3-10% water content (depending on the oil's weight). Under optimal conditions, an over 90% methyl ester yield was achieved after the first reaction batch. The operational stability of immobilized Rhizopus chinensis cells was assayed utilizing a 1:1 methanol-to-oil molar ratio, thus resulting in a 16.5-fold increase in half-life when compared with immobilized cells of the widely studied Rhizopus oryzae. These results suggest that transesterification of vegetable oil using Rhizopus chinensis whole cells immobilized onto loofah sponge is an effective approach for biodiesel production. PMID:27090185

  12. 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

  13. 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

  14. 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.

  15. 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. PMID:24906465

  16. 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.

  17. 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

  18. Outrunning Nature: Directed Evolution of Superior Biocatalysts

    ERIC Educational Resources Information Center

    Woodyer, Ryan; Chen, Wilfred; Zhao, Huimin

    2004-01-01

    The development of enzymes as biocatalysts for industrial use and the emergence of directed evolution in the invention of advanced biocatalysts are discussed and illustrated. Thus, directed evolution has bridged the functional gap between natural and specially designed biocatalysts.

  19. 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.

  20. 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

  1. 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

  2. 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

  3. Potential of Immobilized Whole-Cell Methylocella tundrae as a Biocatalyst for Methanol Production from Methane.

    PubMed

    Mardina, Primata; Li, Jinglin; Patel, Sanjay K S; Kim, In-Won; Lee, Jung-Kul; Selvaraj, Chandrabose

    2016-07-28

    Methanol is a versatile compound that can be biologically synthesized from methane (CH4) by methanotrophs using a low energy-consuming and environment-friendly process. Methylocella tundrae is a type II methanotroph that can utilize CH4 as a carbon and energy source. Methanol is produced in the first step of the metabolic pathway of methanotrophs and is further oxidized into formaldehyde. Several parameters must be optimized to achieve high methanol production. In this study, we optimized the production conditions and process parameters for methanol production. The optimum incubation time, substrate, pH, agitation rate, temperature, phosphate buffer and sodium formate concentration, and cell concentration were determined to be 24 h, 50% CH4, pH 7, 150 rpm, 30°C, 100 mM and 50 mM, and 18 mg/ml, respectively. The optimization of these parameters significantly improved methanol production from 0.66 to 5.18 mM. The use of alginate-encapsulated cells resulted in enhanced methanol production stability and reusability of cells after five cycles of reuse under batch culture conditions. PMID:27012239

  4. 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

  5. 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

  6. 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

  7. 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

  8. Whole cell biotransformation for reductive amination reactions

    PubMed Central

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

    2014-01-01

    Whole cell biotransformation systems with enzyme cascading increasingly find application in biocatalysis to complement or replace established chemical synthetic routes for production of, e.g., fine chemicals. Recently, we established an Escherichia coli whole cell biotransformation system for reductive amination by coupling a transaminase and an amino acid dehydrogenase with glucose catabolism for cofactor recycling. Transformation of 2-keto-3-methylvalerate to l-isoleucine by E. coli cells was improved by genetic engineering of glucose metabolism for improved cofactor regeneration. Here, we compare this system with different strategies for cofactor regeneration such as cascading with alcohol dehydrogenases, with alternative production hosts such as Pseudomonas species or Corynebacterium glutamicum, and with improving whole cell biotransformation systems by metabolic engineering of NADPH regeneration. PMID:24406456

  9. The future of whole-cell modeling.

    PubMed

    Macklin, Derek N; Ruggero, Nicholas A; Covert, Markus W

    2014-08-01

    Integrated whole-cell modeling is poised to make a dramatic impact on molecular and systems biology, bioengineering, and medicine--once certain obstacles are overcome. From our group's experience building a whole-cell model of Mycoplasma genitalium, we identified several significant challenges to building models of more complex cells. Here we review and discuss these challenges in seven areas: first, experimental interrogation; second, data curation; third, model building and integration; fourth, accelerated computation; fifth, analysis and visualization; sixth, model validation; and seventh, collaboration and community development. Surmounting these challenges will require the cooperation of an interdisciplinary group of researchers to create increasingly sophisticated whole-cell models and make data, models, and simulations more accessible to the wider community. PMID:24556244

  10. Biocatalysts from alkaloid producing plants.

    PubMed

    Kries, Hajo; O'Connor, Sarah E

    2016-04-01

    Metabolic pathways leading to benzylisoquinoline and monoterpene indole alkaloids in plants are revealing remarkable new reactions. Understanding of the enzymes involved in alkaloid biosynthesis provides access to a variety of applications in biocatalysis and bioengineering. In chemo-enzymatic settings, plant biocatalysts can transform medically important scaffolds. Additionally, synthetic biologists are taking alkaloid pathways as templates to assemble pathways in microorganisms that are tailored to the needs of medicinal chemistry. In light of these many recent discoveries, it is expected that plants will continue to be a source of novel biocatalysts for the foreseeable future. PMID:26773811

  11. 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…

  12. Electrochemical reduction of xylose to xylitol by whole cells or crude enzyme of Candida peltata.

    PubMed

    Park, Sun Mi; Sang, Byung In; Park, Dae Won; Park, Doo Hyun

    2005-10-01

    In this study, whole cells and a crude enzyme of Candida peltata were applied to an electrochemical bioreactor, in order to induce an increment of the reduction of xylose to xylitol. Neutral red was utilized as an electron mediator in the whole cell reactor, and a graphite-Mn(IV) electrode was used as a catalyst in the enzyme reactor in order to induce the electrochemical reduction of NAD(+) to NADH. The efficiency with which xylose was converted to xylitol in the electrochemical bioreactor was five times higher than that in the conventional bioreactor, when whole cells were employed as a biocatalyst. Meanwhile, the xylose to xylitol reduction efficiency in the enzyme reactor using the graphite-Mn (IV) electrode and NAD(+) was twice as high as that observed in the conventional bioreactor which utilized NADH as a reducing power. In order to use the graphite-Mn(IV) electrode as a catalyst for the reduction of NAD(+) to NADH, a bioelectrocatalyst was engineered, namely, oxidoreductase (e.g. xylose reductase). NAD(+) can function in this biotransformation procedure without any electron mediator or a second oxidoreductase for NAD(+)/NADH recycling. PMID:16273038

  13. 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

  14. Fast Ion Beam Microscopy of Whole Cells

    NASA Astrophysics Data System (ADS)

    Watt, Frank; Chen, Xiao; Chen, Ce-Belle; Udalagama, Chammika Nb; Ren, Minqin; Pastorin, G.; Bettiol, Andrew

    2013-08-01

    The way in which biological cells function is of prime importance, and the determination of such knowledge is highly dependent on probes that can extract information from within the cell. Probing deep inside the cell at high resolutions however is not easy: optical microscopy is limited by fundamental diffraction limits, electron microscopy is not able to maintain spatial resolutions inside a whole cell without slicing the cell into thin sections, and many other new and novel high resolution techniques such as atomic force microscopy (AFM) and near field scanning optical microscopy (NSOM) are essentially surface probes. In this paper we show that microscopy using fast ions has the potential to extract information from inside whole cells in a unique way. This novel fast ion probe utilises the unique characteristic of MeV ion beams, which is the ability to pass through a whole cell while maintaining high spatial resolutions. This paper first addresses the fundamental difference between several types of charged particle probes, more specifically focused beams of electrons and fast ions, as they penetrate organic material. Simulations show that whereas electrons scatter as they penetrate the sample, ions travel in a straight path and therefore maintain spatial resolutions. Also described is a preliminary experiment in which a whole cell is scanned using a low energy (45 keV) helium ion microscope, and the results compared to images obtained using a focused beam of fast (1.2 MeV) helium ions. The results demonstrate the complementarity between imaging using low energy ions, which essentially produce a high resolution image of the cell surface, and high energy ions, which produce an image of the cell interior. The characteristics of the fast ion probe appear to be ideally suited for imaging gold nanoparticles in whole cells. Using scanning transmission ion microscopy (STIM) to image the cell interior, forward scattering transmission ion microscopy (FSTIM) to improve the

  15. Process limitations of a whole-cell P450 catalyzed reaction using a CYP153A-CPR fusion construct expressed in Escherichia coli.

    PubMed

    Lundemo, M T; Notonier, S; Striedner, G; Hauer, B; Woodley, J M

    2016-02-01

    Cytochrome P450s are interesting biocatalysts due to their ability to hydroxylate non-activated hydrocarbons in a selective manner. However, to date only a few P450-catalyzed processes have been implemented in industry due to the difficulty of developing economically feasible processes. In this study, we have used the CYP153A heme domain from Marinobacter aquaeolei fused to the reductase domain of CYP102A1 from Bacillus megaterium (BM3) expressed in Escherichia coli. This self-sufficient protein chimera CYP153A-CPRBM3 G307A mutant is able to selectively hydroxylate medium and long chain length fatty acids at the terminal position. ω-Hydroxylated fatty acids can be used in the field of high-end polymers and in the cosmetic and fragrance industry. Here, we have identified the limitations for implementation of a whole-cell P450-catalyzed reaction by characterizing the chosen biocatalyst as well as the reaction system. Despite a well-studied whole-cell P450 catalyst, low activity and poor stability of the artificial fusion construct are the main identified limitations to reach sufficient biocatalyst yield (mass of product/mass of biocatalyst) and space-time yield (volumetric productivity) essential for an economically feasible process. Substrate and product inhibition are also challenges that need to be addressed, and the application of solid substrate is shown to be a promising option to improve the process. PMID:26432459

  16. Biocatalytic anti-Prelog reduction of prochiral ketones with whole cells of Acetobacter pasteurianus GIM1.158

    PubMed Central

    2014-01-01

    Background Enantiomerically pure alcohols are important building blocks for production of chiral pharmaceuticals, flavors, agrochemicals and functional materials and appropriate whole-cell biocatalysts offer a highly enantioselective, minimally polluting route to these valuable compounds. At present, most of these biocatalysts follow Prelog’s rule, and thus the (S)-alcohols are usually obtained when the smaller substituent of the ketone has the lower CIP priority. Only a few anti-Prelog (R)-specific whole cell biocatalysts have been reported. In this paper, the biocatalytic anti-Prelog reduction of 2-octanone to (R)-2-octanol was successfully conducted with high enantioselectivity using whole cells of Acetobacter pasteurianus GIM1.158. Results Compared with other microorganisms investigated, Acetobacter pasteurianus GIM1.158 was shown to be more effective for the reduction reaction, affording much higher yield, product enantiomeric excess (e.e.) and initial reaction rate. The optimal temperature, buffer pH, co-substrate and its concentration, substrate concentration, cell concentration and shaking rate were 35°C, 5.0, 500 mmol/L isopropanol, 40 mmol/L, 25 mg/mL and 120 r/min, respectively. Under the optimized conditions, the maximum yield and the product e.e. were 89.5% and >99.9%, respectively, in 70 minutes. Compared with the best available data in aqueous system (yield of 55%), the yield of (R)-2-octanol was greatly increased. Additionally, the efficient whole-cell biocatalytic process was feasible on a 200-mL preparative scale and the chemical yield increased to 95.0% with the product e.e. being >99.9%. Moreover, Acetobacter pasteurianus GIM1.158 cells were proved to be capable of catalyzing the anti-Prelog bioreduction of other prochiral carbonyl compounds with high efficiency. Conclusions Via an effective increase in the maximum yield and the product e.e. with Acetobacter pasteurianus GIM1.158 cells, these results open the way to use of whole cells of

  17. Individual surface-engineered microorganisms as robust Pickering interfacial biocatalysts for resistance-minimized phase-transfer bioconversion.

    PubMed

    Chen, Zhaowei; Ji, Haiwei; Zhao, Chuanqi; Ju, Enguo; Ren, Jinsong; Qu, Xiaogang

    2015-04-13

    A powerful strategy for long-term and diffusional-resistance-minimized whole-cell biocatalysis in biphasic systems is reported where individually encapsulated bacteria are employed as robust and recyclable Pickering interfacial biocatalysts. By individually immobilizing bacterial cells and optimizing the hydrophobic/hydrophilic balance of the encapsulating magnetic mineral shells, the encased bacteria became interfacially active and locate at the Pickering emulsion interfaces, leading to dramatically enhanced bioconversion performances by minimizing internal and external diffusional resistances. Moreover, in situ product separation and biocatalyst recovery was readily achieved using a remote magnetic field. Importantly, the mineral shell effectively protected the entire cell from long-term organic-solvent stress, as shown by the reusability of the biocatalysts for up to 30 cycles, while retaining high stereoselective catalytic activities, cell viabilities, and proliferative abilities. PMID:25706244

  18. Engineering Pichia pastoris for improved NADH regeneration: A novel chassis strain for whole-cell catalysis.

    PubMed

    Geier, Martina; Brandner, Christoph; Strohmeier, Gernot A; Hall, Mélanie; Hartner, Franz S; Glieder, Anton

    2015-01-01

    Many synthetically useful reactions are catalyzed by cofactor-dependent enzymes. As cofactors represent a major cost factor, methods for efficient cofactor regeneration are required especially for large-scale synthetic applications. In order to generate a novel and efficient host chassis for bioreductions, we engineered the methanol utilization pathway of Pichia pastoris for improved NADH regeneration. By deleting the genes coding for dihydroxyacetone synthase isoform 1 and 2 (DAS1 and DAS2), NADH regeneration via methanol oxidation (dissimilation) was increased significantly. The resulting Δdas1 Δdas2 strain performed better in butanediol dehydrogenase (BDH1) based whole-cell conversions. While the BDH1 catalyzed acetoin reduction stopped after 2 h reaching ~50% substrate conversion when performed in the wild type strain, full conversion after 6 h was obtained by employing the knock-out strain. These results suggest that the P. pastoris Δdas1 Δdas2 strain is capable of supplying the actual biocatalyst with the cofactor over a longer reaction period without the over-expression of an additional cofactor regeneration system. Thus, focusing the intrinsic carbon flux of this methylotrophic yeast on methanol oxidation to CO2 represents an efficient and easy-to-use strategy for NADH-dependent whole-cell conversions. At the same time methanol serves as co-solvent, inductor for catalyst and cofactor regeneration pathway expression and source of energy. PMID:26664594

  19. Engineering Pichia pastoris for improved NADH regeneration: A novel chassis strain for whole-cell catalysis

    PubMed Central

    Geier, Martina; Brandner, Christoph; Strohmeier, Gernot A; Hall, Mélanie; Hartner, Franz S

    2015-01-01

    Summary Many synthetically useful reactions are catalyzed by cofactor-dependent enzymes. As cofactors represent a major cost factor, methods for efficient cofactor regeneration are required especially for large-scale synthetic applications. In order to generate a novel and efficient host chassis for bioreductions, we engineered the methanol utilization pathway of Pichia pastoris for improved NADH regeneration. By deleting the genes coding for dihydroxyacetone synthase isoform 1 and 2 (DAS1 and DAS2), NADH regeneration via methanol oxidation (dissimilation) was increased significantly. The resulting Δdas1 Δdas2 strain performed better in butanediol dehydrogenase (BDH1) based whole-cell conversions. While the BDH1 catalyzed acetoin reduction stopped after 2 h reaching ~50% substrate conversion when performed in the wild type strain, full conversion after 6 h was obtained by employing the knock-out strain. These results suggest that the P. pastoris Δdas1 Δdas2 strain is capable of supplying the actual biocatalyst with the cofactor over a longer reaction period without the over-expression of an additional cofactor regeneration system. Thus, focusing the intrinsic carbon flux of this methylotrophic yeast on methanol oxidation to CO2 represents an efficient and easy-to-use strategy for NADH-dependent whole-cell conversions. At the same time methanol serves as co-solvent, inductor for catalyst and cofactor regeneration pathway expression and source of energy. PMID:26664594

  20. Accelerated discovery via a whole-cell model.

    PubMed

    Sanghvi, Jayodita C; Regot, Sergi; Carrasco, Silvia; Karr, Jonathan R; Gutschow, Miriam V; Bolival, Benjamin; Covert, Markus W

    2013-12-01

    To test the promise of whole-cell modeling to facilitate scientific inquiry, we compared growth rates simulated in a whole-cell model with experimental measurements for all viable single-gene disruption Mycoplasma genitalium strains. Discrepancies between simulations and experiments led to predictions about kinetic parameters of specific enzymes that we subsequently validated. These findings represent, to our knowledge, the first application of whole-cell modeling to accelerate biological discovery. PMID:24185838

  1. Directed Evolution of a Filamentous Fungus for Thermotolerance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Filamentous fungi represent the most widely used eukaryotic biocatalysts in industrial and chemical applications. Metarhizium anisopliae is a broad-host-range entomopathogenic fungus currently under intensive investigation as a biologically based alternative to chemical pesticides. One of the most p...

  2. WholeCellSimDB: a hybrid relational/HDF database for whole-cell model predictions

    PubMed Central

    Karr, Jonathan R.; Phillips, Nolan C.; Covert, Markus W.

    2014-01-01

    Mechanistic ‘whole-cell’ models are needed to develop a complete understanding of cell physiology. However, extracting biological insights from whole-cell models requires running and analyzing large numbers of simulations. We developed WholeCellSimDB, a database for organizing whole-cell simulations. WholeCellSimDB was designed to enable researchers to search simulation metadata to identify simulations for further analysis, and quickly slice and aggregate simulation results data. In addition, WholeCellSimDB enables users to share simulations with the broader research community. The database uses a hybrid relational/hierarchical data format architecture to efficiently store and retrieve both simulation setup metadata and results data. WholeCellSimDB provides a graphical Web-based interface to search, browse, plot and export simulations; a JavaScript Object Notation (JSON) Web service to retrieve data for Web-based visualizations; a command-line interface to deposit simulations; and a Python API to retrieve data for advanced analysis. Overall, we believe WholeCellSimDB will help researchers use whole-cell models to advance basic biological science and bioengineering. Database URL: http://www.wholecellsimdb.org Source code repository URL: http://github.com/CovertLab/WholeCellSimDB PMID:25231498

  3. 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

  4. Immobilization of Acetobacter sp. CCTCC M209061 for efficient asymmetric reduction of ketones and biocatalyst recycling

    PubMed Central

    2012-01-01

    Background The bacterium Acetobacter sp. CCTCC M209061 is a promising whole-cell biocatalyst with exclusive anti-Prelog stereoselectivity for the reduction of prochiral ketones that can be used to make valuable chiral alcohols such as (R)-4-(trimethylsilyl)-3-butyn-2-ol. Although it has promising catalytic properties, its stability and reusability are relatively poor compared to other biocatalysts. Hence, we explored various materials for immobilizing the active cells, in order to improve the operational stability of biocatalyst. Results It was found that Ca-alginate give the best immobilized biocatalyst, which was then coated with chitosan to further improve its mechanical strength and swelling-resistance properties. Conditions were optimized for formation of reusable immobilized beads which can be used for repeated batch asymmetric reduction of 4′-chloroacetophenone. The optimized immobilized biocatalyst was very promising, with a specific activity of 85% that of the free-cell biocatalyst (34.66 μmol/min/g dw of cells for immobilized catalyst vs 40.54 μmol/min/g for free cells in the asymmetric reduction of 4′-chloroacetophenone). The immobilized cells showed better thermal stability, pH stability, solvent tolerance and storability compared with free cells. After 25 cycles reaction, the immobilized beads still retained >50% catalytic activity, which was 3.5 times higher than degree of retention of activity by free cells reused in a similar way. The cells could be recultured in the beads to regain full activity and perform a further 25 cycles of the reduction reaction. The external mass transfer resistances were negligible as deduced from Damkohler modulus Da < <1, and internal mass transfer restriction affected the reduction action but was not the principal rate-controlling step according to effectiveness factors η < 1 and Thiele modulus 0.3<∅ <1. Conclusions Ca-alginate coated with chitosan is a highly effective material for immobilization of

  5. A virus-based biocatalyst

    NASA Astrophysics Data System (ADS)

    Carette, Noëlle; Engelkamp, Hans; Akpa, Eric; Pierre, Sebastien J.; Cameron, Neil R.; Christianen, Peter C. M.; Maan, Jan C.; Thies, Jens C.; Weberskirch, Ralf; Rowan, Alan E.; Nolte, Roeland J. M.; Michon, Thierry; van Hest, Jan C. M.

    2007-04-01

    Virus particles are probably the most precisely defined nanometre-sized objects that can be formed by protein self-assembly. Although their natural function is the storage and transport of genetic material, they have more recently been applied as scaffolds for mineralization and as containers for the encapsulation of inorganic compounds. The reproductive power of viruses has been used to develop versatile analytical methods, such as phage display, for the selection and identification of (bio)active compounds. To date, the combined use of self-assembly and reproduction has not been used for the construction of catalytic systems. Here we describe a self-assembled system based on a plant virus that has its coat protein genetically modified to provide it with a lipase enzyme. Using single-object and bulk catalytic studies, we prove that the virus-anchored lipase molecules are catalytically active. This anchored biocatalyst, unlike man-made supported catalysts, has the capability to reproduce itself in vivo, generating many independent catalytically active copies.

  6. Fluidization characteristics of Biobone particles used for biocatalysts

    SciTech Connect

    Ellis, N.; Margaritis, A.; Briens, C.L.; Bergougnou, M.A.

    1996-01-01

    Liquid-solid fluidization characteristics of irregularly shaped Biobone particles were studied in a fluidized bioreactor column 2.35 m high and 0.1 m dia. The wet Biobone particles had a density of 1,890 kg/m{sup 3}. Two size ranges were studied: 1.70--2.36 mm and 1.25--2.36 mm. The Biobone is a natural cheap material composed of collagen, which is a strong protein structure, embedded with microcrystals of hydroxy apatite and calcium phosphate, and it is an excellent matrix of commercial importance used for the immobilization of enzymes, whole cells and other biocatalysts. Fluidization characteristics of Biobone particles, which include measurements of pressure drops, holdups, minimum fluidization velocities, particle entrainment, and residence time distributions at different water superficial velocities, are reported. Because of the highly irregular shape of Biobone particles, new fluidization behavior was observed, which was much different from the fluidization behavior of spherical particles reported in the literature. A new channeling index is proposed to quantify the channeling characteristics of fluidized Biobone particles, and a new parameter was developed to distinguish the transition between fixed and fluidized states of the Biobone particles. Fluidized bioreactor systems are used in bioprocessing for the production of high-value medicinal products, ethanol and beer production, food processing and beverage, wastewater treatment, and other environmental applications.

  7. Improving immobilized biocatalysts by gel phase polymerization

    SciTech Connect

    Kuu, W.Y.; Polack, J.A.

    1983-08-01

    A new method is presented for the treatment of gel-type supports, used for immobilizing microbial cells and enzymes, to obtain high mechanical strength. It is particularly useful for ethanol fermentation over gel beads containing immobilized viable cells, where the beads can be ruptured by gas production and the growth of cells within the gels. This method consists of treating agar or carrageenan gel with polyacrylamide to form a rigid support which retains the high catalytic activity characteristic of the untreated biocatalysts. The size and shape of the biocatalyst is unaffected by this treatment. The method involves the diffusion of acrylamide, N,N'-methylenebisacrylamide and BETA-dimethylaminopropionitrile (or N,N,N',N'-tetramethylethylenediamine) into the preformed biocatalyst beads followed by the addition of an initiator to cause polymerization within the beads. Treated gels have been used for the continuous fermentation of glucose to ethanol in a packed column for over two months.

  8. Efficient whole-cell biotransformation of 5-(hydroxymethyl)furfural into FDCA, 2,5-furandicarboxylic acid.

    PubMed

    Koopman, Frank; Wierckx, Nick; de Winde, Johannes H; Ruijssenaars, Harald J

    2010-08-01

    2,5-furandicarboxylic acid (FDCA) is a promising bio-based platform chemical that may serve as a 'green' substitute for terephthalate in polyesters. Recently, a novel HMF/furfural oxidoreductase from Cupriavidus basilensis HMF14 was identified that converts 5-(hydroxymethyl)furfural (HMF) into FDCA. The hmfH gene encoding this oxidoreductase was introduced into Pseudomonas putida S12 and the resulting whole-cell biocatalyst was employed to produce FDCA from HMF. In fed-batch experiments using glycerol as the carbon source, 30.1 g l(-1) of FDCA was produced from HMF at a yield of 97%. FDCA was recovered from the culture broth as a 99.4% pure dry powder, at 76% recovery using acid precipitation and subsequent tetrahydrofuran extraction. PMID:20363622

  9. 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. PMID:26231123

  10. 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.

  11. The search for the ideal biocatalyst.

    PubMed

    Burton, Stephanie G; Cowan, Don A; Woodley, John M

    2002-01-01

    While the use of enzymes as biocatalysts to assist in the industrial manufacture of fine chemicals and pharmaceuticals has enormous potential, application is frequently limited by evolution-led catalyst traits. The advent of designer biocatalysts, produced by informed selection and mutation through recombinant DNA technology, enables production of process-compatible enzymes. However, to fully realize the potential of designer enzymes in industrial applications, it will be necessary to tailor catalyst properties so that they are optimal not only for a given reaction but also in the context of the industrial process in which the enzyme is applied. PMID:11753360

  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. 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.

  15. 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

  16. 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.

  17. 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

  18. A Whole-Cell Computational Model Predicts Phenotype from Genotype

    PubMed Central

    Karr, Jonathan R.; Sanghvi, Jayodita C.; Macklin, Derek N.; Gutschow, Miriam V.; Jacobs, Jared M.; Bolival, Benjamin; Assad-Garcia, Nacyra; Glass, John I.; Covert, Markus W.

    2012-01-01

    SUMMARY Understanding how complex phenotypes arise from individual molecules and their interactions is a primary challenge in biology that computational approaches are poised to tackle. We report a whole-cell computational model of the life cycle of the human pathogen Mycoplasma genitalium that includes all of its molecular components and their interactions. An integrative approach to modeling that combines diverse mathematics enabled the simultaneous inclusion of fundamentally different cellular processes and experimental measurements. Our whole-cell model accounts for all annotated gene functions and was validated against a broad range of data. The model provides insights into many previously unobserved cellular behaviors, including in vivo rates of protein-DNA association and an inverse relationship between the durations of DNA replication initiation and replication rates. In addition, experimental analysis directed by model predictions identified previously undetected kinetic parameters and biological functions. We conclude that comprehensive whole-cell models can be used to facilitate biological discovery. PMID:22817898

  19. 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. PMID:26109004

  20. 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

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

    PubMed Central

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

    2013-01-01

    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. PMID:23822510

  2. 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.

  3. 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. PMID:16678400

  4. Biocatalyst design for stability and specificity

    SciTech Connect

    Himmel, M.E.; Georgiou, G.

    1991-01-01

    This volume has been developed from a symposium sponsored by the Division of Biochemical Technology of the American Chemical Society at the Fourth Chemical Congress of North America (202nd National Meeting of the American Chemical Society), held in New York, New York, August 25-30, 1991. Papers included here relate to the development of biocatalysts, with an emphasis on the stability and specificity of the catalysts. Major topics of these papers include enzymes, biotechnology, protein engineering, and protein folding.

  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. A Highly Productive, Whole-Cell DERA Chemoenzymatic Process for Production of Key Lactonized Side-Chain Intermediates in Statin Synthesis

    PubMed Central

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

  7. Improving immobilized biocatalysts by gel phase polymerization

    SciTech Connect

    Kuu, W.Y.; Polack, J.A.

    1983-08-01

    A new method is presented for the treatment of gel-type supports, used for immobilizing microbial cells and enzymes, to obtain high mechanical strength. It is particularly useful for ethanol fermentation over gel beads containing immobilized viable cells, where the beads can be ruptured by gas production and the growth of cells within the gels. This method consists of treating agar or carrageenan gel with polyacrylamide to form a rigid support which retains the high catalytic activity characteristic of the untreated biocatalysts. The size and shape of the biocatalyst is unaffected by this treatment. The method involves the diffusion of acrylamide, N,N'-methylenebisacrylamide and ..beta..-dimethylaminopropionitrile (or N,N,N',N'-tetramethylethylenediamine) into the preformed biocatalyst beads followed by the addition of an initiator to cause polymerization within the beads. Treated gels have been used for the continuous fermentation of glucose to ethanol in a packed column for over two months. During this operation, the gel beads maintained their rigidity, and the maximum productivity was as high as 50 gh/sup -1/ L/sup -1/ gel. There was no appreciable decay of cell activity.

  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. 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…

  10. 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

  11. 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

  12. 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

  13. 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

  14. Reaction and catalyst engineering to exploit kinetically controlled whole-cell multistep biocatalysis for terminal FAME oxyfunctionalization.

    PubMed

    Schrewe, Manfred; Julsing, Mattijs K; Lange, Kerstin; Czarnotta, Eik; Schmid, Andreas; Bühler, Bruno

    2014-09-01

    The oxyfunctionalization of unactivated C−H bonds can selectively and efficiently be catalyzed by oxygenase-containing whole-cell biocatalysts. Recombinant Escherichia coli W3110 containing the alkane monooxygenase AlkBGT and the outer membrane protein AlkL from Pseudomonas putida GPo1 have been shown to efficiently catalyze the terminal oxyfunctionalization of renewable fatty acid methyl esters yielding bifunctional products of interest for polymer synthesis. In this study, AlkBGTL-containing E. coli W3110 is shown to catalyze the multistep conversion of dodecanoic acid methyl ester (DAME) via terminal alcohol and aldehyde to the acid, exhibiting Michaelis-Menten-type kinetics for each reaction step. In two-liquid phase biotransformations, the product formation pattern was found to be controlled by DAME availability. Supplying DAME as bulk organic phase led to accumulation of the terminal alcohol as the predominant product. Limiting DAME availability via application of bis(2-ethylhexyl)phthalate (BEHP) as organic carrier solvent enabled almost exclusive acid accumulation. Furthermore, utilization of BEHP enhanced catalyst stability by reducing toxic effects of substrate and products. A further shift towards the overoxidized products was achieved by co-expression of the gene encoding the alcohol dehydrogenase AlkJ, which was shown to catalyze efficient and irreversible alcohol to aldehyde oxidation in vivo. With DAME as organic phase, the aldehyde accumulated as main product using resting cells containing AlkBGT, AlkL, as well as AlkJ. This study highlights the versatility of whole-cell biocatalysis for synthesis of industrially relevant bifunctional building blocks and demonstrates how integrated reaction and catalyst engineering can be implemented to control product formation patterns in biocatalytic multistep reactions. PMID:24852702

  15. 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

  16. 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.

  17. 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

  18. Use of microbial spores as a biocatalyst

    SciTech Connect

    Murata, Kousaku . Research Inst. for Food Science)

    1993-01-01

    Endospores of a bacterium Bacillus subtilis and ascospores of a yeast Saccharomyces cerevisiae contained almost all the activities for the same enzymes as vegetative cells. The biotechnological potential of spores was studied by selecting adenosine 5[prime]-triphosphatase and alkaline phosphatase in bacterial and yeast spores, respectively, as model enzymes. The activity of both enzymes was efficiently expressed when the spores were treated by physical (sonication or electric field pulse) and chemical (organic solvents or detergents) methods. The yeast spores were immobilized in polyacrylamide gel without any appreciable loss of activity. The immobilized spores were packed in a column and used successfully for the continuous reactions of alkaline phosphatase and glyoxalase I. The microbial spores were confirmed to be promising as a biocatalyst for the production of useful chemicals in bioreactor systems.

  19. 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

  20. 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. PMID:26528624

  1. Ultra-High Resolution 3D Imaging of Whole Cells.

    PubMed

    Huang, Fang; Sirinakis, George; Allgeyer, Edward S; Schroeder, Lena K; Duim, Whitney C; Kromann, Emil B; Phan, Thomy; Rivera-Molina, Felix E; Myers, Jordan R; Irnov, Irnov; Lessard, Mark; Zhang, Yongdeng; Handel, Mary Ann; Jacobs-Wagner, Christine; Lusk, C Patrick; Rothman, James E; Toomre, Derek; Booth, Martin J; Bewersdorf, Joerg

    2016-08-11

    Fluorescence nanoscopy, or super-resolution microscopy, has become an important tool in cell biological research. However, because of its usually inferior resolution in the depth direction (50-80 nm) and rapidly deteriorating resolution in thick samples, its practical biological application has been effectively limited to two dimensions and thin samples. Here, we present the development of whole-cell 4Pi single-molecule switching nanoscopy (W-4PiSMSN), an optical nanoscope that allows imaging of three-dimensional (3D) structures at 10- to 20-nm resolution throughout entire mammalian cells. We demonstrate the wide applicability of W-4PiSMSN across diverse research fields by imaging complex molecular architectures ranging from bacteriophages to nuclear pores, cilia, and synaptonemal complexes in large 3D cellular volumes. PMID:27397506

  2. Neisseria meningitidis: serotyping and subtyping by whole cell ELISA.

    PubMed

    Prakash, K; Lakshmy, A; Malhotra, V L

    1993-09-01

    Twenty strains of Neisseria meningitidis isolated from clinically diagnosed cases of meningococcal disease were subjected to serogrouping, employing slide agglutination followed by serotyping and serosubtyping by whole cell ELISA using monoclonal typing antisera. All isolates were from sporadic cases of meningitis during a period of two years from various hospitals in Delhi. All 20 isolates were grouped as serogroup A and typed as serotype 4, except one strain which was untypable. On serosubtyping the isolates were found to belong to P1.9 (7 strains) followed by P1.1 (5), P1.9 (2), P1.16,1 (2), P1.6,10 (2), P1.10,7,1 (1) and non-subtypable (1). PMID:8241833

  3. THE USE OF LIVE BIOCATALYSTS FOR PESTICIDE DETOXIFICATION. (R823663)

    EPA Science Inventory

    During the past decade, numerous microorganisms capable of degrading pesticides have been isolated, and detoxification processes based on these live biocatalysts have been developed. Recently, novel detoxification strategies using genetically engineered microorganisms with extend...

  4. Biocatalysts and their small molecule products from metagenomic studies

    PubMed Central

    Iqbal, Hala A.; Feng, Zhiyang; Brady, Sean F.

    2012-01-01

    The vast majority of bacteria present in environmental samples have never been cultured and therefore they have not been available to exploit their ability to produce useful biocatalysts or collections of biocatalysts that can biosynthesize interesting small molecules. Metagenomic libraries constructed using DNA extracted directly from natural bacterial communities offer access to the genetic information present in the genomes of these as yet uncultured bacteria. This review highlights recent efforts to recover both discrete enzymes and small molecules from metagenomic libraries. PMID:22455793

  5. 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

  6. Understanding biocatalyst inhibition by carboxylic acids.

    PubMed

    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

  7. 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.

  8. 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

  9. 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

  10. 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

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. Reconstitution of the In Vitro Activity of the Cyclosporine-Specific P450 Hydroxylase from Sebekia benihana and Development of a Heterologous Whole-Cell Biotransformation System

    PubMed Central

    Ma, Li; Du, Lei; Chen, Hui; Sun, Yue; Huang, Shan; Zheng, Xianliang

    2015-01-01

    The cytochrome P450 enzyme CYP-sb21 from Sebekia benihana is capable of catalyzing the site-specific hydroxylation of the immunosuppressant cyclosporine (CsA), leading to the single product γ-hydroxy-N-methyl-l-Leu4-CsA (CsA-4-OH). Unlike authentic CsA, this hydroxylated CsA shows significantly reduced immunosuppressive activity while it retains a side effect of CsA, the hair growth stimulation effect. Although CYP-sb21 was previously identified to be responsible for CsA-specific hydroxylation in vivo, the in vitro activity of CYP-sb21 has yet to be established for a deeper understanding of this P450 enzyme and further reaction optimization. In this study, we reconstituted the in vitro activity of CYP-sb21 by using surrogate redox partner proteins of bacterial and cyanobacterial origins. The highest CsA site-specific hydroxylation activity by CYP-sb21 was observed when it was partnered with the cyanobacterial redox system composed of seFdx and seFdR from Synechococcus elongatus PCC 7942. The best bioconversion yields were obtained in the presence of 10% methanol as a cosolvent and an NADPH regeneration system. A heterologous whole-cell biocatalyst using Escherichia coli was also constructed, and the permeability problem was solved by using N-cetyl-N,N,N-trimethylammonium bromide (CTAB). This work provides a useful example for reconstituting a hybrid P450 system and developing it into a promising biocatalyst for industrial application. PMID:26150455

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

    PubMed

    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

    2015-05-01

    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

  17. 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

  18. 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

  19. Asymmetric reduction of ketones with recombinant E. coli whole cells in neat substrates.

    PubMed

    Jakoblinnert, Andre; Mladenov, Radoslav; Paul, Albert; Sibilla, Fabrizio; Schwaneberg, Ulrich; Ansorge-Schumacher, Marion B; de María, Pablo Domínguez

    2011-11-28

    The asymmetric reduction of ketones is performed by using lyophilized whole cells in neat substrates with defined water activity (a(w)). Ketones and alcohols prone to be unstable in aqueous media can now be converted via biocatalysis. PMID:22005469

  20. An improved whole cell pertussis vaccine with reduced content of endotoxin

    PubMed Central

    Dias, Waldely Oliveira; van der Ark, Arno A.J.; Sakauchi, Maria Aparecida; Kubrusly, Flávia Saldanha; Prestes, Ana Fabíola R.O.; Borges, Monamaris Marques; Furuyama, Noemi; Horton, Denise S.P.Q.; Quintilio, Wagner; Antoniazi, Marta; Kuipers, Betsy; van der Zeijst, Bernard A.M.; Raw, Isaias

    2013-01-01

    An improved whole cell pertussis vaccine, designated as Plow, which is low in endotoxicity due to a chemical extraction of lipo-oligosaccharide (LOS) from the outer membrane, was evaluated for safety, immunogenicity and potency, comparatively to a traditional whole cell pertussis vaccine. Current whole cell pertussis vaccines are effective but contain large quantities of endotoxin and consequently display local and systemic adverse reactions after administration. Endotoxin is highly inflammatory and contributes considerably to the reactogenicity as well as the potency of these vaccines. In contrast, acellular pertussis vaccines hardly contain endotoxin and are significantly less reactogenic, but their elevated costs limit their global use, especially in developing countries. In this paper, bulk products of Plow and a traditional whole cell vaccine, formulated as plain monocomponents or combined with diphtheria and tetanus toxoids (DTPlow or DTP, respectively) were compared by in vitro and in vivo assays. Chemical extraction of LOS resulted in a significant decrease in endotoxin content (20%) and a striking decline in endotoxin related toxicity (up to 97%), depending on the used in vitro or in vivo test. The LOS extraction did not affect the integrity of the product and, more importantly, did not affect the potency and/or stability of DTPlow. Moreover, hardly any differences in antibody and T-cell responses were observed. The development of Plow is a significant improvement regarding the endotoxicity of whole cell pertussis vaccines and therefore a promising and affordable alternative to currently available whole cell or acellular pertussis vaccines for developing countries. PMID:23291935

  1. Biotransformation of (-)-α-pinene and geraniol to α-terpineol and p-menthane-3,8-diol by the white rot fungus, Polyporus brumalis.

    PubMed

    Lee, Su-Yeon; Kim, Seon-Hong; Hong, Chang-Young; Park, Se-Yeong; Choi, In-Gyu

    2015-07-01

    In this study, the monoterpenes, α-pinene and geraniol, were biotransformed to synthesize monoterpene alcohol compounds. Polyporus brumalis which is classified as a white rot fungus was used as a biocatalyst. Consequently α-terpineol was synthesized from α-pinene by P. brumalis mycelium, after three days. Moreover, another substrate, the acyclic monoterpenoids geraniol was transformed into the cyclic compound, p-menthane-3, 8-diol (PMD). The main metabolites, i.e., α-terpineol and PMD, are known to be bioactive monoterpene alcohol compounds. This study highlights the potential of fungal biocatalysts for monoterpene transformation. PMID:26115995

  2. 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

  3. 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

  4. [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

  5. 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

  6. 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. PMID:22226194

  7. 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

  8. New techniques for making whole-cell recordings from CNS neurons in vivo.

    PubMed

    Rose, G J; Fortune, E S

    1996-09-01

    Patch-type pipettes increasingly are being used to obtain intracellular 'whole-cell' recordings from neurons. Here we describe our methods for making whole-cell recordings in vivo from midbrain neurons in an electric fish. Novel elements in the procedure are: A device for micropositioning the pipette when near a cell, use of a 'Picospritzer' for cleaning the pipette tip and cell surface, and an electroporetic method for perforating the patch following seal formulation. In addition, we show that extracellular and intracellular recordings can be made from the same neuron. Stable intracellular recordings can be made from neurons at least as small as 10 microns. PMID:8895897

  9. 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

  10. Identification and Characterization of Thymosin B4 in Chicken Macrophages Using Whole Cell MALDI-TOF

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The aim of the study was to determine chicken monocyte and granulocyte associated peptides and proteins using "whole cell" matrix assisted laser desorption/ionization time-of-flight mass spectrometry and to characterize the peptides based on their abundance. The mass spectra showed a prominent peak ...

  11. 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.

  12. 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...

  13. Serum antibodies to whole-cell and recombinant antigens of Borrelia burgdorferi in cottontail rabbits.

    PubMed

    Magnarelli, Louis A; Norris, Steven J; Fikrig, Erol

    2012-01-01

    Archived serum samples, from 95 eastern cottontail rabbits (Sylvilagus floridanus) captured in New York, New York, USA and Millbrook, New York, USA, during 1985-86, were analyzed in solid-phase enzyme-linked immunosorbent assays (ELISA) for total and class-specific immunoglobulin (Ig) M antibodies to whole-cell or recombinant antigens of Borrelia burgdorferi sensu stricto. Using a polyvalent conjugate, rabbit sera contained antibodies to whole-cell and recombinant antigens (protein [p]35, p37, or VlsE) during different seasons, but there was no reactivity to outer surface protein (Osp)A or OspB. Seventy-six of the 102 sera (75%) analyzed were reactive with one or more of the antigens; 61 of the positive samples (80%) reacted to whole-cell antigens, followed by results for the p35 (58%, 44/76), VlsE (43%, 33/76), and p37 (29%, 22/ 76) antigens. Fifty-eight sera (76%) contained antibodies to the VlsE or p35 antigens with or without reactivity to whole-cell antigens. High antibody titers (≥1:2,560) recorded for 52 sera indicate robust antibody production. In analyses for IgM antibodies in an ELISA containing whole-cell antigens, there were 30 positive sera; titers ranged from 1:160 to 1:640. There was minimal cross-reactivity when rabbit antisera to Treponema pallidum or four serovars of Leptospira interrogans were screened against B. burgdorferi antigens. Based on more-specific results, VlsE and p35 antigens appear to be useful markers for detecting possible B. burgdorferi infections. PMID:22247369

  14. SERUM ANTIBODIES TO WHOLE-CELL AND RECOMBINANT ANTIGENS OF BORRELIA BURGDORFERI IN COTTONTAIL RABBITS

    PubMed Central

    Magnarelli, Louis A.; Norris, Steven J.; Fikrig, Erol

    2011-01-01

    Archived serum samples, from 95 eastern cottontail rabbits (Sylvilagus floridanus) captured in New York, New York, USA and Millbrook, New York, USA, during 1985–86, were analyzed in solid-phase enzyme-linked immunosorbent assays (ELISA) for total and class-specific immunoglobulin (Ig) M antibodies to whole-cell or recombinant antigens of Borrelia burgdorferi sensu stricto. Using a polyvalent conjugate, rabbit sera contained antibodies to whole-cell and recombinant antigens (protein [p]35, p37, or VlsE) during different seasons, but there was no reactivity to outer surface protein (Osp)A or OspB. Seventy-six of the 102 sera (75%) analyzed were reactive with one or more of the antigens; 61 of the positive samples (80%) reacted to whole-cell antigens, followed by results for the p35 (58%, 44/76), VlsE (43%, 33/76), and p37 (29%, 22/76) antigens. Fifty-eight sera (76%) contained antibodies to the VlsE or p35 antigens with or without reactivity to whole-cell antigens. High antibody titers (≥1:2,560) recorded for 52 sera indicate robust antibody production. In analyses for IgM antibodies in an ELISA containing whole-cell antigens, there were 30 positive sera; titers ranged from 1:160 to 1:640. There was minimal cross-reactivity when rabbit antisera to Treponema pallidum or four serovars of Leptospira interrogans were screened against B. burgdorferi antigens. Based on more-specific results, VlsE and p35 antigens appear to be useful markers for detecting possible B. burgdorferi infections. PMID:22247369

  15. 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

  16. 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

  17. 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

  18. Development of a mediated whole cell-based electrochemical biosensor for joint toxicity assessment of multi-pollutants using a mixed microbial consortium.

    PubMed

    Gao, Guanyue; Qian, Jun; Fang, Deyu; Yu, Yuan; Zhi, Jinfang

    2016-06-14

    Since most risk assessment for toxicants is based on individual single-species test, the deduction of such results to ecosystem evaluation is afflicted with uncertainties. Herein, we successfully developed a p-benzoquinone mediated whole-cell electrochemical biosensor for multi-pollutants toxicological analysis by co-immobilizing mixed strains of microorganism, including Escherichia coli (gram-negative bacteria), Bacillus subtilis (gram-positive bacteria) and Saccharomyces cerevisiae (fungus). The individual and combined toxicities of heavy metal ions (Cu(2+), Cd(2+)), phenol (3,5-dichlorophenol) and pesticides (Ametryn, Acephate) were examined. The experimental results showed that the order of toxicity for individual toxicant was ranked as Cu(2+) > 3,5-dichlorophenol (DCP) > Ametryn > Cd(2+) > Acephate. Then the toxic unit (TU) model was applied to determine the nature of toxicological interaction of the toxicants which can be classified as concentration additive (IC50mix = 1TU), synergistic (IC50mix < 1TU) and antagonistic (IC50mix > 1TU) responses. The binary combination of Cu(2+) + Cd(2+), Cu(2+) + DCP, Cu(2+) + Acephate, DCP + Acephate, Acephate + Ametryn were analyzed and the three kind of joint toxicity effects (i.e. additive, synergistic and antagonistic) mentioned above were observed according to the dose-response relationship. The results indicate that the whole-cell electrochemical biosensor based on mixed microbial consortium is more reasonable to reflect the joint biotoxicity of multi-pollutants existing in real wastewater, and combined effects of toxicants is extremely necessary to be taken into account in ecological risk assessment. Thus, present study has provided a promising approach to the quality assessment of wastewater and a reliable way for early risk warning of acute biotoxicity. PMID:27181640

  19. 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

  20. Application of genetically engineered microbial whole-cell biosensors for combined chemosensing.

    PubMed

    He, Wei; Yuan, Sheng; Zhong, Wen-Hui; Siddikee, Md Ashaduzzaman; Dai, Chuan-Chao

    2016-02-01

    The progress of genetically engineered microbial whole-cell biosensors for chemosensing and monitoring has been developed in the last 20 years. Those biosensors respond to target chemicals and produce output signals, which offer a simple and alternative way of assessment approaches. As actual pollution caused by human activities usually contains a combination of different chemical substances, how to employ those biosensors to accurately detect real contaminant samples and evaluate biological effects of the combined chemicals has become a realistic object of environmental researches. In this review, we outlined different types of the recent method of genetically engineered microbial whole-cell biosensors for combined chemical evaluation, epitomized their detection performance, threshold, specificity, and application progress that have been achieved up to now. We also discussed the applicability and limitations of this biosensor technology and analyzed the optimum conditions for their environmental assessment in a combined way. PMID:26615397

  1. 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.

  2. Specific initiation by RNA polymerase I in a whole-cell extract from yeast.

    PubMed Central

    Schultz, M C; Choe, S Y; Reeder, R H

    1991-01-01

    A protocol is described for making a soluble whole-cell extract from yeast (Saccharomyces cerevisiae) that supports active and specific transcription initiation by RNA polymerases I, II, and III. Specific initiation by polymerase I decreases in high-density cultures, paralleling the decrease in abundance of the endogenous 35S rRNA precursor. This extract should be useful for studying the molecular mechanisms that regulate rRNA transcription in yeast. Images PMID:1992452

  3. Frequency-selective REDOR and spin-diffusion relays in uniformly labeled whole cells.

    PubMed

    Rice, David M; Romaniuk, Joseph A H; Cegelski, Lynette

    2015-11-01

    Solid-state NMR is a powerful and non-perturbative method to measure and define chemical composition and architecture in bacterial cell walls, even in the context of whole cells. Most NMR studies on whole cells have used selectively labeled samples. Here, we introduce an NMR sequence relay using frequency-selective REDOR (fsREDOR) and spin diffusion elements to probe a unique amine contribution in uniformly (13)C- and (15)N-labeled Staphylococcus aureus whole cells that we attribute to the d-alanine of teichoic acid. In addition to the primary peptidoglycan structural scaffold, cell walls can contain significant amounts of teichoic acid that contribute to cell-wall function. When incorporated into teichoic acid, d-alanine is present as an ester, connected via its carbonyl to a ribitol carbon, and thus has a free amine. Teichoic acid d-Ala is removed during cell-wall isolations and can only be detected in the context of whole cells. The sequence presented here begins with fsREDOR and a chemical shift evolution period for 2D data acquisition, followed by DARR spin diffusion and then an additional fsREDOR period. fsREDOR elements were used for (13)C observation to avoid complications from (13)C-(13)C couplings due to uniform labeling and for (15)N dephasing to achieve selectivity in the nitrogens serving as dephasers. The results show that the selected amine nitrogen of interest is near to teichoic acid ribitol carbons and also the methyl group carbon associated with alanine. In addition, its carbonyl is not significantly dephased by amide nitrogens, consistent with the expected microenvironment around teichoic acid. PMID:26493462

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. Accurate initiation by RNA polymerase II in a whole cell extract from Saccharomyces cerevisiae.

    PubMed

    Woontner, M; Jaehning, J A

    1990-06-01

    We have developed a simple procedure for isolating a transcriptional extract from whole yeast cells which obviates the requirement for nuclear isolation. Detection of accurate mRNA initiation by RNA polymerase II in the extract requires the use of a sensitive assay, recently described by Kornberg and co-workers (Lue, N. F., Flanagan, P. M., Sugimoto, K., and Kornberg, R. D. (1989) Science 246, 661-664) that involves activation by a GAL4-VP16 fusion protein and a template lacking guanosine residues in the coding strand. The extract is prepared from fresh or frozen yeast cells by disruption with glass beads and fractionation of proteins by ammonium sulfate precipitation. The alpha-amanitin-sensitive transcripts synthesized in the assay were identical to those produced in a parallel assay using a yeast nuclear extract. The activity of the whole cell extract is lower per mg of protein than a nuclear extract but proportional to the volume of the nucleus relative to the whole cell. The optimal ranges for several reaction components including template, mono- and divalent cations, and nucleotide substrate concentration were determined. Under optimal conditions the whole cell extract produced a maximum of approximately 1 X 10(-2) transcripts/template molecule in 30 min. PMID:2188968

  10. 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.

  11. A multipurpose immobilized biocatalyst with pectinase, xylanase and cellulase activities

    PubMed Central

    Dalal, Sohel; Sharma, Aparna; Gupta, Munishwar Nath

    2007-01-01

    Background The use of immobilized enzymes for catalyzing various biotransformations is now a widely used approach. In recent years, cross-linked enzyme aggregates (CLEAs) have emerged as a novel and versatile biocatalyst design. The present work deals with the preparation of a CLEA from a commercial preparation, Pectinex™ Ultra SP-L, which contains pectinase, xylanase and cellulase activities. The CLEA obtained could be used for any of the enzyme activities. The CLEA was characterized in terms of kinetic parameters, thermal stability and reusability in the context of all the three enzyme activities. Results Complete precipitation of the three enzyme activities was obtained with n-propanol. When resulting precipitates were subjected to cross-linking with 5 mM glutaraldehyde, the three activities initially present (pectinase, xylanase and cellulase) were completely retained after cross-linking. The Vmax/Km values were increased from 11, 75 and 16 to 14, 80 and 19 in case of pectinase, xylanase and cellulase activities respectively. The thermal stability was studied at 50°C, 60°C and 70°C for pectinase, xylanase and cellulase respectively. Half-lives were improved from 17, 22 and 32 minutes to 180, 82 and 91 minutes for pectinase, xylanase and cellulase respectively. All three of the enzymes in CLEA could be reused three times without any loss of activity. Conclusion A single multipurpose biocatalyst has been designed which can be used for carrying out three different and independent reactions; 1) hydrolysis of pectin, 2) hydrolysis of xylan and 3) hydrolysis of cellulose. The preparation is more stable at higher temperatures as compared to the free enzymes. PMID:17880745

  12. 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

  13. Exploring the mechanism of biocatalyst inhibition in microbial desulfurization.

    PubMed

    Abin-Fuentes, Andres; Mohamed, Magdy El-Said; Wang, Daniel I C; Prather, Kristala L J

    2013-12-01

    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

  14. 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

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

    PubMed

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

    2013-01-01

    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

  16. 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

  17. Hydroxylated polychlorinated biphenyl detection based on a genetically engineered bioluminescent whole-cell sensing system.

    PubMed

    Turner, Kendrick; Xu, Shifen; Pasini, Patrizia; Deo, Sapna; Bachas, Leonidas; Daunert, Sylvia

    2007-08-01

    The metabolites of polychlorinated biphenyls (PCBs), such as hydroxylated PCBs (OH-PCBs), have been identified as environmental contaminants. Various studies have shown that some OH-PCBs can potentially contribute to health problems. Detection of these compounds in environmental and biological samples could provide useful information about their levels and lead to a better understanding of their apparent toxicity. To that end, we have developed a whole-cell sensing system for the detection of OH-PCBs by taking advantage of the recognition of a group of related compounds, i.e., hydroxylated biphenyls, by the product of the hbpR gene in the hbp operon from Pseudomonas azelaica strain HBP1. By fusing the luxAB genes, encoding the reporter protein bacterial luciferase, to the hbp regulator-promoter sequence, a whole-cell sensing system was developed. Here, we describe the optimization and application of this whole-cell sensing system for the detection of a model compound, 2-hydroxy-3',4'-dichlorobiphenyl. A detection limit of 1 x 10(-8) M was achieved using this system. The detection of a broad range of individual OH-PCBs as well as an OH-PCB mixture was investigated. The system can detect OH-PCBs in whole serum samples in a trace amount, which is comparable to the detection of these analytes in medium alone. We envision that the method developed can potentially be employed as a rapid and sensitive way to monitor OH-PCBs for toxicological study in the laboratory, as well as a useful tool to evaluate the presence of bioavailable OH-PCBs in natural environments. PMID:17602671

  18. 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

  19. 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

  20. Reporter Proteins in Whole-Cell Optical Bioreporter Detection Systems, Biosensor Integrations, and Biosensing Applications

    PubMed Central

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

    2009-01-01

    Whole-cell, genetically modified bioreporters are designed to emit detectable signals in response to a target analyte or related group of analytes. When integrated with a transducer capable of measuring those signals, a biosensor results that acts as a self-contained analytical system useful in basic and applied environmental, medical, pharmacological, and agricultural sciences. Historically, these devices have focused on signaling proteins such as green fluorescent protein, aequorin, firefly luciferase, and/or bacterial luciferase. The biochemistry and genetic development of these sensor systems as well as the advantages, challenges, and common applications of each one will be discussed. PMID:22291559

  1. 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

  2. [The type subtyping of meningococci by means of whole-cell immunoenzyme analysis].

    PubMed

    Demina, A A; Koroleva, I S

    1992-01-01

    In this work the method of the whole-cell enzyme immunoassay, used for the serotype-subtyping of meningococci by means of specific monoclonal antibodies, is described. High specificity of the method, the simplicity of the assay procedure and evaluation of its results, as well as the availability of this method for practical use, have been demonstrated. The results of this investigation confirm the importance of the evaluation of type-subtype appurtenance of reference and laboratory strains used in experiments. Study of 72 meningococcal strains obtained from patients has revealed their polyclonal character in respect of their type-subtype signs. PMID:1455966

  3. 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. PMID:26259168

  4. 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

  5. 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

  6. Comparison of Whole-Cell SELEX Methods for the Identification of Staphylococcus Aureus-Specific DNA Aptamers

    PubMed Central

    Moon, Jihea; Kim, Giyoung; Park, Saet Byeol; Lim, Jongguk; Mo, Changyeun

    2015-01-01

    Whole-cell Systemic Evolution of Ligands by Exponential enrichment (SELEX) is the process by which aptamers specific to target cells are developed. Aptamers selected by whole-cell SELEX have high affinity and specificity for bacterial surface molecules and live bacterial targets. To identify DNA aptamers specific to Staphylococcus aureus, we applied our rapid whole-cell SELEX method to a single-stranded ssDNA library. To improve the specificity and selectivity of the aptamers, we designed, selected, and developed two categories of aptamers that were selected by two kinds of whole-cell SELEX, by mixing and combining FACS analysis and a counter-SELEX process. Using this approach, we have developed a biosensor system that employs a high affinity aptamer for detection of target bacteria. FAM-labeled aptamer sequences with high binding to S. aureus, as determined by fluorescence spectroscopic analysis, were identified, and aptamer A14, selected by the basic whole-cell SELEX using a once-off FACS analysis, and which had a high binding affinity and specificity, was chosen. The binding assay was evaluated using FACS analysis. Our study demonstrated the development of a set of whole-cell SELEX derived aptamers specific to S. aureus; this approach can be used in the identification of other bacteria. PMID:25884791

  7. Spectral Snapshots of Bacterial Cell-Wall Composition and the Influence of Antibiotics by Whole-Cell NMR

    PubMed Central

    Nygaard, Rie; Romaniuk, Joseph A.H.; Rice, David M.; Cegelski, Lynette

    2015-01-01

    Gram-positive bacteria surround themselves with a thick cell wall that is essential to cell survival and is a major target of antibiotics. Quantifying alterations in cell-wall composition are crucial to evaluating drug modes of action, particularly important for human pathogens that are now resistant to multiple antibiotics such as Staphylococcus aureus. Macromolecular and whole-cell NMR spectroscopy allowed us to observe the full panel of carbon and nitrogen pools in S. aureus cell walls and intact whole cells. We discovered that one-dimensional 13C and 15N NMR spectra, together with spectroscopic selections based on dipolar couplings as well as two-dimensional spin-diffusion measurements, revealed the dramatic compositional differences between intact cells and cell walls and allowed the identification of cell-wall signatures in whole-cell samples. Furthermore, the whole-cell NMR approach exhibited the sensitivity to detect distinct compositional changes due to treatment with the antibiotics fosfomycin (a cell-wall biosynthesis inhibitor) and chloramphenicol (a protein synthesis inhibitor). Whole cells treated with fosfomycin exhibited decreased peptidoglycan contributions while those treated with chloramphenicol contained a higher percentage of peptidoglycan as cytoplasmic protein content was reduced. Thus, general antibiotic modes of action can be identified by profiling the total carbon pools in intact whole cells. PMID:25809251

  8. [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

  9. 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. PMID:19766668

  10. Efficient biocatalyst by encapsulating lipase into nanoporous gold

    NASA Astrophysics Data System (ADS)

    Du, Xiaoyu; Liu, Xueying; Li, Yufei; Wu, Chao; Wang, Xia; Xu, Ping

    2013-04-01

    Lipases are one of the most important biocatalysts for biotechnological applications. Immobilization is an efficient method to increase the stability and reusability of lipases. In this study, nanoporous gold (NPG), a new kind of nanoporous material with tunable porosity and excellent biocompatibility, was employed as an effective support for lipase immobilization. The pore size of NPG and adsorption time played key roles in the construction of lipase-NPG biocomposites. The morphology and composition of NPG before and after lipase loading are verified using a scanning electron microscope, equipped with an energy-dispersive X-ray spectrometer. The resulting lipase-NPG biocomposites exhibited excellent catalytic activity and remarkable reusability. The catalytic activity of the lipase-NPG biocomposite with a pore size of 35 nm had no decrease after ten recycles. Besides, the lipase-NPG biocomposite exhibited high catalytic activity in a broader pH range and higher temperature than that of free lipase. In addition, the leaching of lipase from NPG could be prevented by matching the protein's diameter and pore size. Thus, the encapsulation of enzymes within NPG is quite useful for establishing new functions and will have wide applications for different chemical processes.

  11. Structural evolution and stability of sol gel biocatalysts

    NASA Astrophysics Data System (ADS)

    Rodgers, L. E.; Knott, R. B.; Holden, P. J.; Pike, K. J.; Hanna, J. V.; Foster, L. J. R.; Bartlett, J. R.

    2006-11-01

    Immobilisation strategies for catalytic enzymes are important as they allow recovery and reuse of the biocatalysts. In this work, sol-gel matrices have been used to immobilise Candida antarctica lipase B (CALB), a commonly used industrial enzyme. The sol-gel bioencapsulate is produced through fluoride-catalysed hydrolysis of mixtures of tetramethylorthosilicate (TMOS) and methyltrimethoxysilane (MTMS) in the presence of CALB, yielding materials with controlled pore sizes and surface chemistries. Sol-gel matrices prolong the catalytic life and enhance the activity of CALB, although the molecular basis for this effect has yet to be elucidated due to the limitations of analytical techniques applied to date. Small angle neutron scattering (SANS) allows such multi-component systems to be characterised through contrast matching. In the sol-gel bioencapsulate system at the contrast match point for silica, residual scattering intensity is due to the CALB and density fluctuations in the matrix. A SANS contrast variation series found the match point for the silica matrix, both with and without enzyme present, to be around 35%. The model presented here proposes a mechanism for the interaction between CALB and the surrounding sol-gel matrix, and the observed improvement in enzyme activity and matrix strength. Essentially, the inclusion of CALB modulates silicate speciation during evolution of the inorganic network, leading to associated variations in SANS contrast. The SANS protocol developed here may be applied more generally to other encapsulated enzyme systems.

  12. Evaluation of enteric-coated tablets as a whole cell inactivated vaccine candidate against Vibrio cholerae.

    PubMed

    Fernández, Sonsire; Año, Gemma; Castaño, Jorge; Pino, Yadira; Uribarri, Evangelina; Riverón, Luis A; Cedré, Bárbara; Valmaseda, Tania; Falero, Gustavo; Pérez, José L; Infante, Juan F; García, Luis G; Solís, Rosa L; Sierra, Gustavo; Talavera, Arturo

    2013-01-01

    A vaccine candidate against cholera was developed in the form of oral tablets to avoid difficulties during application exhibited by current whole cell inactivated cholera vaccines. In this study, enteric-coated tablets were used to improve the protection of the active compound from gastric acidity. Tablets containing heat-killed whole cells of Vibrio cholerae strain C7258 as the active pharmaceutical compound was enteric-coated with the polymer Kollicoat(®) MAE-100P, which protected them efficiently from acidity when a disintegration test was carried out. Enzyme-linked immunosorbent assay (ELISA) anti-lipopolysaccharide (LPS) inhibition test and Western blot assay revealed the presence of V. cholerae antigens as LPS, mannose-sensitive haemagglutinin (MSHA) and outer membrane protein U (Omp U) in enteric-coated tablets. Immunogenicity studies (ELISA and vibriocidal test) carried out by intraduodenal administration in rabbits showed that the coating process of tablets did not affect the immunogenicity of V. cholerae-inactivated cells. In addition, no differences were observed in the immune response elicited by enteric-coated or uncoated tablets, particularly because the animal model and immunization route used did not allow discriminating between acid resistances of both tablets formulations in vivo. Clinical studies with volunteers will be required to elucidate this aspect, but the results suggest the possibility of using enteric-coated tablets as a final pharmaceutical product for a cholera vaccine. PMID:23492079

  13. 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.

  14. 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.

  15. Automated detection of whole-cell mitochondrial motility and its dependence on cytoarchitectural integrity.

    PubMed

    Kandel, Judith; Chou, Philip; Eckmann, David M

    2015-07-01

    Current methodologies used for mitochondrial motility analysis tend to either overlook individual mitochondrial tracks or analyze only peripheral mitochondria instead of mitochondria in all regions of the cell. Furthermore, motility analysis of an individual mitochondrion is usually quantified by establishing an arbitrary threshold for "directed" motion. In this work, we created a custom, publicly available computational algorithm based on a previously published approach (Giedt et al., 2012. Ann Biomed Eng 40:1903-1916) in order to characterize the distribution of mitochondrial movements at the whole-cell level, while still preserving information about single mitochondria. Our technique is easy to use, robust, and computationally inexpensive. Images are first pre-processed for increased resolution, and then individual mitochondria are tracked based on object connectivity in space and time. When our method is applied to microscopy fields encompassing entire cells, we reveal that the mitochondrial net distances in fibroblasts follow a lognormal distribution within a given cell or group of cells. The ability to model whole-cell mitochondrial motility as a lognormal distribution provides a new quantitative paradigm for comparing mitochondrial motility in naïve and treated cells. We further demonstrate that microtubule and microfilament depolymerization shift the lognormal distribution in directions which indicate decreased and increased mitochondrial movement, respectively. These findings advance earlier work on neuronal axons (Morris and Hollenbeck, 1993. J Cell Sci 104:917-927) by relating them to a different cell type, applying them on a global scale, and automating measurement of mitochondrial motility in general. PMID:25678368

  16. 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

  17. 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

  18. Physical and bioengineering properties of polyvinyl alcohol lens-shaped particles versus spherical polyelectrolyte complex microcapsules as immobilisation matrices for a whole-cell Baeyer-Villiger monooxygenase.

    PubMed

    Schenkmayerová, Andrea; Bučko, Marek; Gemeiner, Peter; Treľová, Dušana; Lacík, Igor; Chorvát, Dušan; Ačai, Pavel; Polakovič, Milan; Lipták, Lukáš; Rebroš, Martin; Rosenberg, Michal; Stefuca, Vladimír; Neděla, Vilém; Tihlaříková, Eva

    2014-11-01

    Direct comparison of key physical and chemical-engineering properties of two representative matrices for multipurpose immobilisations was performed for the first time. Polyvinyl alcohol lens-shaped particles LentiKats® and polyelectrolyte complex microcapsules were characterised by advanced techniques with respect to the size distribution of the particles, their inner morphology as revealed by fluorescent probe staining, mechanical resistance, size-exclusion properties, determination of effective diffusion coefficient and environmental scanning electron microscope imaging. While spherical polyelectrolyte complex microcapsules composed of a rigid semipermeable membrane and a liquid core are almost uniform in shape and size (diameter of 0.82 mm; RSD = 5.6 %), lens-shaped LentiKats® are characterised by wider size distribution (diameter of 3.65 mm; RSD = 10.3 % and height of 0.341 mm; RSD = 32.3 %) and showed the same porous structure throughout their whole volume at the mesoscopic (micrometre) level. Despite differences in their inner structure and surface properties, the pore diameter of ∼ 2.75 nm for regular polyelectrolyte complex microcapsules and ∼ 1.89 nm for LentiKats® were similar. These results were used for mathematical modelling, which provided the estimates of the effective diffusion coefficient of sucrose. This value was 1.67 × 10(-10) m(2) s(-1) for polyelectrolyte complex microcapsules and 0.36 × 10(-10) m(2) s(-1) for LentiKats®. Recombinant cells Escherichia coli-overexpressing enzyme cyclopentanone monooxygenase were immobilised in polyelectrolyte complex microcapsules and LentiKats® for comparison of their operational stability using model Baeyer-Villiger oxidation of (±)-cis-bicyclo [3.2.0] hept-2-en-6-one to regioisomeric lactones as important chiral synthons for potential pharmaceuticals. Both immobilisation matrices rendered high operational stability for whole-cell biocatalyst with no reduction in the

  19. 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.

  20. Specific detection of the pesticide chlorpyrifos by a sensitive genetic-based whole cell biosensor.

    PubMed

    Whangsuk, Wirongrong; Thiengmag, Sirinthra; Dubbs, James; Mongkolsuk, Skorn; Loprasert, Suvit

    2016-01-15

    The Sinorhizobium meliloti chpA promoter is highly induced in the presence of the pesticide chlorpyrifos (CPF) through the action of the transcriptional activator, ChpR. A whole-cell biosensor for the detection of CPF was developed and is composed of an Escherichia coli strain carrying a chpR expression vector and a chpA promoter-atsBA transcriptional fusion plasmid encoding sulfatase (atsA) and formylglycine generating enzyme (atsB) from Klebsiella sp. The sulfatase is posttranslationally activated by formylglycine generating enzyme (FGE) and then converts 4-methylumbelliferyl sulfate (4-MUS) to the fluorescent product, 4-methyllumbelliferone (4-MU). This biosensor system exhibited a linear response range from 25 to 500 nM CPF. PMID:26452613

  1. 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

  2. Capillary electrophoresis chips for screening of endotoxin chemotypes from whole-cell lysates.

    PubMed

    Kilár, Anikó; Péterfi, Zoltán; Csorba, Eszter; Kilár, Ferenc; Kocsis, Béla

    2008-10-01

    A fast microchip electrophoresis method was developed to analyze and differentiate bacterial endotoxins directly from whole-cell lysates after removal of the proteinaceous components with proteinase K digestion and a precipitation of the endotoxin components. The partially purified endotoxin components were visualized by the interaction with dodecyl sulphate and then a fluorescent dye. The lipopolysaccharide (LPS) profiles can be directly evaluated from digested bacterial cells, and the electrophoresis patterns very closely resembled to those of pure LPSs, and the R and S chemotypes can be used to assign the strains. The method has been found to be useful in the screening of a large number of bacterial mutants and the structural characterization of endotoxins extracted only from 1 ml cultures. PMID:18692189

  3. 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. PMID:26662854

  4. 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

  5. Whole-cell cancer vaccination: from autologous to allogeneic tumor- and dendritic cell-based vaccines

    PubMed Central

    de Gruijl, Tanja D.; van den Eertwegh, Alfons J. M.; Pinedo, Herbert M.

    2008-01-01

    The field of tumor vaccination is currently undergoing a shift in focus, from individualized tailor-made vaccines to more generally applicable vaccine formulations. Although primarily predicated by financial and logistic considerations, stemming from a growing awareness that clinical development for wide-scale application can only be achieved through backing from major pharmaceutical companies, these new approaches are also supported by a growing knowledge of the intricacies and minutiae of antigen presentation and effector T-cell activation. Here, the development of whole-cell tumor and dendritic cell (DC)-based vaccines from an individualized autologous set-up to a more widely applicable allogeneic approach will be discussed as reflected by translational studies carried out over the past two decades at our laboratories and clinics in the vrije universiteit medical center (VUmc) in Amsterdam, The Netherlands. PMID:18523771

  6. 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

  7. 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.

  8. Whole Cell Patch Clamp for Investigating the Mechanisms of Infrared Neural Stimulation

    PubMed Central

    Brown, William G. A.; Needham, Karina; Nayagam, Bryony A.; Stoddart, Paul R.

    2013-01-01

    It has been demonstrated in recent years that pulsed, infrared laser light can be used to elicit electrical responses in neural tissue, independent of any further modification of the target tissue. Infrared neural stimulation has been reported in a variety of peripheral and sensory neural tissue in vivo, with particular interest shown in stimulation of neurons in the auditory nerve. However, while INS has been shown to work in these settings, the mechanism (or mechanisms) by which infrared light causes neural excitation is currently not well understood. The protocol presented here describes a whole cell patch clamp method designed to facilitate the investigation of infrared neural stimulation in cultured primary auditory neurons. By thoroughly characterizing the response of these cells to infrared laser illumination in vitro under controlled conditions, it may be possible to gain an improved understanding of the fundamental physical and biochemical processes underlying infrared neural stimulation. PMID:23929071

  9. Transcriptional activation in an improved whole-cell extract from Saccharomyces cerevisiae.

    PubMed Central

    Woontner, M; Wade, P A; Bonner, J; Jaehning, J A

    1991-01-01

    We report an improved in vitro transcription system for Saccharomyces cerevisiae. Small changes in assay and whole-cell extraction procedures increase selective initiation by RNA polymerase II up to 60-fold over previous conditions (M. Woontner and J. A. Jaehning, J. Biol. Chem. 265:8979-8982, 1990), to levels comparable to those obtained with nuclear extracts. We have found that the simultaneous use of distinguishable templates with and without an upstream activation sequence is critical to the measurement of apparent activation. Transcription from any template was very sensitive to the concentrations of template and nontemplate DNA, extract, and activator (GAL4/VP16). Alterations in reaction conditions led to proportionately greater changes from a template lacking an upstream activation sequence; thus, the apparent ratio of activation is largely dependent on the level of basal transcription. Using optimal conditions for activation, we have also demonstrated activation by a bona fide yeast activator, heat shock transcription factor. Images PMID:1875938

  10. Transcriptional activation in an improved whole-cell extract from Saccharomyces cerevisiae.

    PubMed

    Woontner, M; Wade, P A; Bonner, J; Jaehning, J A

    1991-09-01

    We report an improved in vitro transcription system for Saccharomyces cerevisiae. Small changes in assay and whole-cell extraction procedures increase selective initiation by RNA polymerase II up to 60-fold over previous conditions (M. Woontner and J. A. Jaehning, J. Biol. Chem. 265:8979-8982, 1990), to levels comparable to those obtained with nuclear extracts. We have found that the simultaneous use of distinguishable templates with and without an upstream activation sequence is critical to the measurement of apparent activation. Transcription from any template was very sensitive to the concentrations of template and nontemplate DNA, extract, and activator (GAL4/VP16). Alterations in reaction conditions led to proportionately greater changes from a template lacking an upstream activation sequence; thus, the apparent ratio of activation is largely dependent on the level of basal transcription. Using optimal conditions for activation, we have also demonstrated activation by a bona fide yeast activator, heat shock transcription factor. PMID:1875938

  11. 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

  12. 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

  13. Microscopic monitoring provides information on structure and properties during biocatalyst immobilization.

    PubMed

    Bidmanova, Sarka; Hrdlickova, Eva; Jaros, Josef; Ilkovics, Ladislav; Hampl, Ales; Damborsky, Jiri; Prokop, Zbynek

    2014-06-01

    Enzymes have a wide range of applications in different industries owing to their high specificity and efficiency. Immobilization is often used to improve biocatalyst properties, operational stability, and reusability. However, changes in the structure of biocatalysts during immobilization and under process conditions are still largely uncertain. Here, three microscopy techniques - bright-field, confocal and electron microscopy - were applied to determine the distribution and structure of an immobilized biocatalyst. Free enzyme (haloalkane dehalogenase), cross-linked enzyme aggregates (CLEAs) and CLEAs entrapped in polyvinyl alcohol lenses (lentikats) were used as model systems. Electron microscopy revealed that sonicated CLEAs underwent morphological changes that strongly correlated with increased catalytic activity compared to less structured, non-treated CLEAs. Confocal microscopy confirmed that loading of the biocatalyst was not the only factor affecting the catalytic activity of the lentikats. Confocal microscopy also showed a significant reduction in the pore size of lentikats exposed to 25% tetrahydrofuran and 50% dioxane. Narrow pores appeared to provide protection to CLEAs from the detrimental action of cosolvents, which significantly correlated with higher activity of CLEAs compared to free enzyme. The results showed that microscopy can provide valuable information about the structure and properties of a biocatalyst during immobilization and under process conditions. PMID:24639415

  14. 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. PMID:26948574

  15. MATLAB-based Simulation of Whole-Cell and Single-Channel Currents

    PubMed Central

    Molitor, Scott C.; Tong, Mingjie; Vora, Deepan

    2006-01-01

    Mathematical models of electrophysiological data are used to investigate biophysical mechanisms that underlie electrical excitability. Although the resources and time required for obtaining experimental data to create these models may not be available to undergraduates enrolled in a biophysics course, computational tools that simulate cellular or single-channel responses to electrophysiological stimuli can be utilized to provide these data. We have developed two MATLAB-based simulation packages that are being used in a cellular electrophysiology course for upper-level undergraduate engineering students to demonstrate the design of electrophysiological stimuli, and the analysis and modeling of ionic currents in excitable tissues. The first package simulates a Hodgkin-Huxley style voltage-gated current elicited during voltage-clamp experiments. Users specify the duration and magnitude of a voltage waveform; the model returns a simulated whole-cell current traces with superimposed noise, and various measurements including peak current, steady state current, and time constants from exponential fits of the current time course. The second package simulates a voltage- or ligand-gated single-channel current as a stochastic process using a state transition matrix. Users specify the membrane voltage, ligand concentration, and number of trials; the model returns simulated single-channel current traces with superimposed noise, and various measurements including amplitude and dwell time histograms. This software has been used during lectures to demonstrate various principles in class, and for class projects in which students derive kinetic models that underlie currents obtained during whole-cell and single-channel recordings. These software packages are freely available and can be downloaded at www.eng.utoledo.edu/∼smolitor/download.htm. PMID:23493427

  16. Whole cell bioconversion of vitamin D3 to calcitriol using Pseudonocardia sp. KCTC 1029BP.

    PubMed

    Kang, Dae-Jung; Im, Jong-Hyuk; Kang, Jae-Hoon; Kim, Kyoung Heon

    2015-07-01

    Calcitriol is an important drug used for treating osteoporosis, which can be produced from vitamin D3. The current method of producing calcitriol from vitamin D3 during cultivation of microbial cells results in low yields of calcitriol and high purification costs. Therefore, in this study, the steps of cell cultivation and bioconversion of vitamin D3 to calcitriol were separated. Cells of Pseudonocardia sp. KCTC 1029BP were utilized as a whole cell catalyst to produce a high level and yield of calcitriol from vitamin D3. In addition, the effects of bioconversion buffers, cyclodextrins, and metal salts on the production of calcitriol were comparatively examined and selected for incorporation in the bioconversion medium, and their compositions were statistically optimized. The optimal bioconversion medium was determined as consisting of 15 mM Trizma base, 25 mM sodium succinate, 2 mM MgSO4, 0.08% β-cyclodextrin, 0.1% NaCl, 0.2% K2HPO4, and 0.03% MnCl2. Using this optimal bioconversion medium, 61.87 mg/L of calcitriol, corresponding to a 30.94% mass yield from vitamin D3, was produced in a 75-L fermentor after 9 days. This calcitriol yield was 3.6 times higher than that obtained using a bioconversion medium lacking β-cyclodextrin, NaCl, K2HPO4, and MnCl2. In conclusion, utilizing whole cells of Pseudonocardia sp. KCTC 1029BP together with the optimal bioconversion medium markedly enhanced the production of calcitriol from vitamin D3. PMID:25666830

  17. [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

  18. Xylanase Immobilized on Novel Multifunctional Hyperbranched Polyglycerol-Grafted Magnetic Nanoparticles: An Efficient and Robust Biocatalyst.

    PubMed

    Landarani-Isfahani, Amir; Taheri-Kafrani, Asghar; Amini, Mina; Mirkhani, Valiollah; Moghadam, Majid; Soozanipour, Asieh; Razmjou, Amir

    2015-08-25

    Although several strategies are now available for immobilization of enzymes to magnetic nanoparticles for bioapplications, little progresses have been reported on the use of dendritic or hyperbranched polymers for the same purpose. Herein, we demonstrated synthesis of magnetic nanoparticles supported hyperbranched polyglycerol (MNP/HPG) and a derivative conjugated with citric acid (MNP/HPG-CA) as unique and convenient nanoplatforms for immobilization of enzymes. Then, an important industrial enzyme, xylanase, was immobilized on the nanocarriers to produce robust biocatalysts. A variety of analytical tools were used to study the morphological, structural, and chemical properties of the biocatalysts. Additionally, the results of biocatalyst systems exhibited the substantial improvement of reactivity, reusability, and stability of xylanase due to this strategy, which might confer them a wider range of applications. PMID:26258956

  19. Reactivity of dog sera to whole-cell or recombinant antigens of Borrelia burgdorferi by ELISA and immunoblot analysis.

    PubMed

    Magnarelli, L A; Levy, S A; Ijdo, J W; Wu, C; Padula, S J; Fikrig, E

    2001-10-01

    Enzyme-linked immunosorbent assays (ELISAs) with separate preparations of 10 purified recombinant antigens of Borrelia burgdorferi sensu stricto were used to test sera from 36 dogs not vaccinated with whole cells of this agent and from five dogs vaccinated with whole-cell B. burgdorferi bacteria. All dogs lived in tick-infested areas of Connecticut and south-eastern New York state, USA. The non-vaccinated dogs had limb or joint disorder, lameness and fever during the period 1984-1991 and had antibodies to B. burgdorferi, as determined by a polyvalent ELISA with whole-cell antigen. In re-analyses of sera for total immunoglobulins in ELISAs with recombinant antigens, reactions were most frequently recorded when outer-surface protein (Osp) F, protein (p)35, p37, p39 and p-41G (a flagellin component) were tested separately. Western immunoblots of a subset of 16 sera, positive by ELISA with whole-cell antigen and representing a range of antibody titres (640-40960), verified immune responses to these or other lysed whole-cell antigens. Sera from vaccinated dogs contained antibodies to OspA, OspB, p22, p37 and p41-G. Therefore, serological reactions to OspF, p35 and p39 were the most important indicators of natural exposure to B. burgdorferi. Serum reactivities to these recombinant antigens in ELISAs can be used to help identify possible natural infections of canine borreliosis in dogs not vaccinated with whole-cell B. burgdorferi and to provide information on the geographic distribution of this bacterium. PMID:11599738

  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. 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.

  3. 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.

  4. Lipases and whole cell biotransformations of 2-hydroxy-2-(ethoxyphenylphosphinyl)acetic acid and its ester.

    PubMed

    Majewska, Paulina; Serafin, Monika; Klimek-Ochab, Magdalena; Brzezińska-Rodak, Małgorzata; Żymańczyk-Duda, Ewa

    2016-06-01

    A wide spectrum of commercially available lipases and microbial whole cells catalysts were tested for biotransformations of 2-hydroxy-2-(ethoxyphenylphosphinyl)acetic acid 1 and its butyryl ester. The best results were achieved for biocatalytic hydrolysis of ester: 2-butyryloxy-2-(ethoxyphenylphosphinyl)acetic acid 2 performed by lipase from Candida cylindracea, what gave optically active products with 85% enantiomeric excess, 50% conversion degree and enantioselectivity 32.9 for one pair of enantiomers. Also enzymatic systems of Penicillium minioluteum and Fusarium oxysporum were able to hydrolyze tested compound with high enantiomeric excess (68-93% ee), enantioselectivity (44 for one pair of enantiomers) and conversion degree about 50-55%. Enzymatic acylation of hydroxyphosphinate was successful in case when porcine pancreas lipase was used. After 4days of biotransformation the conversion reaches 45% but the enantiomeric enrichment of the isomers mixture do not exceed 43%. Obtained chiral compounds are valuable derivatizing agents for spectroscopic (NMR) evaluation of enantiomeric excess for particular compounds (e.g. amino acids). PMID:26989983

  5. Purification and preconcentration of genomic DNA from whole cell lysates using photoactivated polycarbonate (PPC) microfluidic chips

    PubMed Central

    Witek, Małgorzata A.; Llopis, Shawn D.; Wheatley, Abigail; McCarley, Robin L.; Soper, Steven A.

    2006-01-01

    We discuss the use of a photoactivated polycarbonate (PPC) microfluidic chip for the solid-phase, reversible immobilization (SPRI) and purification of genomic DNA (gDNA) from whole cell lysates. The surface of polycarbonate was activated by UV radiation resulting in a photo-oxidation reaction, which produced a channel surface containing carboxylate groups. The gDNA was selectively captured on this photoactivated surface in an immobilization buffer, which consisted of 3% polyethylene glycol, 0.4 M NaCl and 70% ethanol. The methodology reported herein is similar to conventional SPRI in that surface-confined carboxylate groups are used for the selective immobilization of DNA; however, no magnetic beads or a magnetic field are required. As observed by UV spectroscopy, a load of ∼7.6 ± 1.6 µg/ml of gDNA was immobilized onto the PPC bed. The recovery of DNA following purification was estimated to be 85 ± 5%. The immobilization and purification assay using this PPC microchip could be performed within ∼25 min as follows: (i) DNA immobilization ∼6 min, (ii) chip washout with ethanol 10 min, and (iii) drying and gDNA desorption ∼6 min. The PPC microchip could also be used for subsequent assays with no substantial loss in recovery, no observable carryover and no need for ‘reactivation’ of the PC surface with UV light. PMID:16757572

  6. 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. PMID:25558872

  7. 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

  8. 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

  9. 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

  10. Whole Cell Cryo-Electron Tomography Reveals Distinct Disassembly Intermediates of Vaccinia Virus

    PubMed Central

    Cyrklaff, Marek; Linaroudis, Alexandros; Boicu, Marius; Chlanda, Petr; Baumeister, Wolfgang; Griffiths, Gareth; Krijnse-Locker, Jacomine

    2007-01-01

    At each round of infection, viruses fall apart to release their genome for replication, and then reassemble into stable particles within the same host cell. For most viruses, the structural details that underlie these disassembly and assembly reactions are poorly understood. Cryo-electron tomography (cryo-ET), a unique method to investigate large and asymmetric structures at the near molecular resolution, was previously used to study the complex structure of vaccinia virus (VV). Here we study the disassembly of VV by cryo-ET on intact, rapidly frozen, mammalian cells, infected for up to 60 minutes. Binding to the cell surface induced distinct structural rearrangements of the core, such as a shape change, the rearrangement of its surface spikes and de-condensation of the viral DNA. We propose that the cell surface induced changes, in particular the decondensation of the viral genome, are a prerequisite for the subsequent release of the vaccinia DNA into the cytoplasm, which is followed by its cytoplasmic replication. Generally, this is the first study that employs whole cell cryo-ET to address structural details of pathogen-host cell interaction. PMID:17487274

  11. 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.

  12. 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. PMID:26905642

  13. 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

  14. 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

  15. Dose tolerance at helium and nitrogen temperatures for whole cell electron tomography.

    PubMed

    Comolli, Luis R; Downing, Kenneth H

    2005-12-01

    Electron tomography is currently the only method that allows the direct three-dimensional visualization of macromolecules in an unperturbed cellular context. In principle, tomography should enable the identification and localization of the major macromolecular complexes within intact bacteria, embedded in amorphous ice. In an effort to optimize conditions for recording data that would bring us close to the theoretical limits, we present here a comparison of the dose tolerance of Caulobacter crescentus cells embedded in amorphous ice at liquid helium versus liquid nitrogen temperature. The inner and outer cell membranes, and the periodic structure of the S-layer of this Gram-negative bacterium provide ideal features to monitor changes in contrast and order as a function of dose. The loss of order in the S-layer occurs at comparable doses at helium and nitrogen temperatures. Macroscopic bubbling within the cell and the plastic support develops at both temperatures, but more slowly at helium temperature. The texture of the bubbles is finer in initial stages at helium temperature, giving an impression of contrast reversal in some parts of the specimen. Bubbles evolve differently in different organelles, presumably a consequence of their different chemical composition and mechanical properties. Finally, the amorphous ice "flows" at helium temperature, causing changes in the relative positions of markers within the specimen and distorting the cells. We conclude that for cryo-electron tomography of whole cells liquid nitrogen temperature provides better overall data quality. PMID:16198601

  16. Fast detection of extrasynaptic GABA with a whole-cell sniffer

    PubMed Central

    Christensen, Rasmus K.; Petersen, Anders V.; Schmitt, Nicole; Perrier, Jean-François

    2014-01-01

    Gamma-amino-butyric acid (GABA) is the main inhibitory transmitter of the brain. It operates by binding to specific receptors located both inside and outside synapses. The extrasynaptic receptors are activated by spillover from GABAergic synapses and by ambient GABA in the extracellular space. Ambient GABA is essential for adjusting the excitability of neurons. However, due to the lack of suitable methods, little is known about its dynamics. Here we describe a new technique that allows detection of GABA transients and measurement of the steady state GABA concentration with high spatial and temporal resolution. We used a human embryonic kidney (HEK) cell line that stably expresses GABAA receptors composed of α1, β2, and γ2 subunits. We recorded from such a HEK cell with the whole-cell patch-clamp technique. The presence of GABA near the HEK cell generated a measurable electric current whose magnitude increased with concentration. A fraction of the current did not inactivate during prolonged exposition to GABA. This technique, which we refer to as a “sniffer” allows the measurement of ambient GABA concentration inside nervous tissue with a resolution of few tens of nanomolars. In addition, the sniffer detects variations in the extrasynaptic GABA concentration with millisecond time resolution. Pilot experiments demonstrate that the sniffer is able to report spillover of GABA induced by synaptic activation in real time. This is the first report on a GABA sensor that combines the ability to detect fast transients and to measure steady concentrations. PMID:24860433

  17. Whole cell structural imaging at 20 nanometre resolutions using MeV ions

    NASA Astrophysics Data System (ADS)

    Watt, F.; Chen, X.; Chen, C.-B.; Udalagama, CNB; van Kan, J. A.; Bettiol, A. A.

    2013-07-01

    MeV proton and alpha (helium ion) particle beams can now be focused to 20 nm spot sizes, and ion/matter simulations using the DEEP computer code show that these resolutions are maintained through the top micrometre or so of organic material. In addition, the energy deposition profiles of the transmitted ions are laterally constrained to a few nanometers from the initial ion path. This paves the way for high resolution structural imaging of relatively thick biological material, e.g. biological cells. Examples are shown of high resolution structural imaging of whole biological cells (MRC5) using on-axis scanning transmission ion microscopy (STIM). Nanoparticles have the ability to cross the cell membrane, and may therefore prove useful as drug delivery probes. We show that the combination of on-axis STIM for imaging the cell interior, and off-axis STIM for imaging gold nanoparticles with enhanced contrast within the cell, represents a powerful set of ion beam techniques for tracking gold nanoparticles in biological cells. Whole cell imaging at high spatial resolutions represents a new area for nuclear microprobes.

  18. 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

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

    PubMed Central

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

    2013-01-01

    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 set-back. 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 anti-folate. The second and third compounds’ MOA were not clearly identified, likely due to unique MOA not represented within the commercial database. The availability of the database generated in this report for S. 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 distinction of unique or broadly acting MOA at this time is less clear. PMID:22434711

  20. 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...

  1. 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 ...

  2. Culture condition improvement for whole-cell lipase production in submerged fermentation by Rhizopus chinensis using statistical method.

    PubMed

    Teng, Yun; Xu, Yan

    2008-06-01

    Rhizopus chinensis CCTCC M201021 was a versatile strain capable of producing whole-cell lipase with synthetic activity in submerged fermentation. In order to improve the production of whole-cell lipase and study the culture conditions systematically, the combination of taguchi method and response surface methodology was performed. Taguchi method was used for the initial optimization, and eight factors viz., maltose, olive oil, peptone, K2HPO4, agitation, inoculum size, fermentation volume and pH were selected for this study. The whole-cell lipase activity yield was two times higher than the control experiment under initial optimal conditions, and four significant factors (inoculum, olive oil, fermentation volume and peptone) were selected to test the effect on the lipase production using response surface methodology. The optimal fermentation parameters for enhanced whole-cell lipase yield were found to be: inoculum 4.25 x 10(8) spores/L, olive oil 2.367% (w/v), fermentation volume 18 mL/250 mL flask, peptone 4.06% (w/v). Subsequent experimental trails confirmed the validity of the model. These optimal culture conditions in the shake flask led to a lipase yield of 13875 U/L, which 120% increased compare with the non-optimized conditions. PMID:17888652

  3. 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

  4. 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

  5. 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. PMID:22967923

  6. Whole-cell microtiter plate screening assay for terminal hydroxylation of fatty acids by P450s.

    PubMed

    Weissenborn, Martin J; Notonier, Sandra; Lang, Sarah-Luise; Otte, Konrad B; Herter, Susanne; Turner, Nicholas J; Flitsch, Sabine L; Hauer, Bernhard

    2016-05-01

    A readily available galactose oxidase (GOase) variant was used to develop a whole cell screening assay. This endpoint detection system was applied in a proof-of-concept approach by screening a focussed mutant library. This led to the discovery of the thus far most active P450 Marinobacter aquaeolei mutant catalysing the terminal hydroxylation of fatty acids. PMID:27074906

  7. 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

  8. 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

  9. 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. PMID:23170776

  10. 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. PMID:26272428

  11. 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.

  12. 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

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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

  18. 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

  19. Evaluation of a whole cell, p57- vaccine against Renibacterium salmoninarum.

    PubMed

    Piganelli, J D; Wiens, G D; Zhang, J A; Christensen, J M; Kaattari, S L

    1999-04-15

    A whole cell Renibacterium salmoninarum vaccine was developed using 37 degrees C heat treated cells that were subsequently formalin fixed; this treatment reduced bacterial hydrophobicity and cell associated p57. Coho salmon Oncorhynchus kisutch were immunized with the p57- vaccine by either a combination of intraperitoneal (i.p.) and intramuscular (i.m.) injections or per os. In the first experiment, i.p./i.m. vaccination of coho salmon with p57- cells in Freund's Incomplete Adjuvant (FIA) conferred a statistically significant increase in mean time to death after the salmon were i.p. challenged with 4.1 x 10(6) colony forming units (cfu) of R. salmoninarum. There was no significant difference in response between fish immunized with R. salmoninarum cell surface extract in FIA and those immunized with extracellular protein (ECP) concentrated from culture supernatant in FIA. The i.p. challenge dose resulted in complete mortality of all fish by Day 43. In a second experiment, fish were orally vaccinated with p57- R. salmoninarum cells encased in a pH protected, enteric-coated antigen microsphere (ECAM). Fish were bath challenged with 4.2 x 10(6) cfu ml-1 on Day 0 and sampled at time points of 0 (pre-challenge), 50, 90, or 150 d immersion challenge. Vaccine efficacy was determined by monitoring the elaboration of p57 in the kidneys of vaccinated and control fish. Fish vaccinated orally demonstrated a significantly lower concentration of p57 (p < 0.01) at Day 150 post challenge compared to fish receiving ECAMs alone. Fish receiving p57 cells without ECAM coating also showed a significantly lower p57 level (p < 0.03) versus control. In contrast, fish injected intraperitoneally with the p57- cells or fish fed p57+ R. salmoninarum cells in ECAMs demonstrated no significant difference (p > 0.05) versus controls. In summary, these studies suggest the preliminary efficacy of 37 degrees C treatment of R. salmoninarum cells as an oral bacterial kidney disease vaccine. PMID

  20. 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

  1. Modeling and measurement of a whole-cell bioluminescent biosensor based on a single photon avalanche diode.

    PubMed

    Daniel, Ramiz; Almog, Ronen; Ron, Amit; Belkin, Shimshon; Diamand, Yosi Shacahm

    2008-12-01

    Whole-cell biosensors are potential candidates for on-line and in situ environmental monitoring. In this work we present a new design of a whole-cell bioluminescence biosensor for water toxicity detection, based on genetically engineered Escherichia coli bacteria, carrying a recA::luxCDABE promoter-reporter fusion. Sensitive optical detection is achieved using a single photon avalanche photodiode (SPAD) working in the Geiger mode. The present work describes a simple mathematical model for the kinetic process of the bioluminescence based SOS toxin response of E. coli bacteria. We find that initially the bioluminescence signal depends on the time square and we show that the spectral intensity of the bioluminescence signal is inverse proportional to the frequency. We get excellent agreement between the theoretical model and the measured light signal. Furthermore, we present experimental results of the bioluminescent signal measurement using a SPAD and a photomultiplier, and demonstrate improvement of the measurement by applying a matched digital filter. Low intensity bioluminescence signals were measured after the whole-cell sensors were exposed to various toxicant concentrations (5, 15 and 20ppm). PMID:18774705

  2. Target Mechanism-Based Whole-Cell Screening Identifies Bortezomib as an Inhibitor of Caseinolytic Protease in Mycobacteria

    PubMed Central

    Moreira, Wilfried; Ngan, Grace J. Y.; Low, Jian Liang; Poulsen, Anders; Chia, Brian C. S.; Ang, Melgious J. Y.; Yap, Amelia; Fulwood, Justina; Lakshmanan, Umayal; Lim, Jolander; Khoo, Audrey Y. T.; Flotow, Horst; Hill, Jeffrey; Raju, Ravikiran M.; Rubin, Eric J.

    2015-01-01

    ABSTRACT A novel type of antibacterial screening method, a target mechanism-based whole-cell screening method, was developed to combine the advantages of target mechanism- and whole-cell-based approaches. A mycobacterial reporter strain with a synthetic phenotype for caseinolytic protease (ClpP1P2) activity was engineered, allowing the detection of inhibitors of this enzyme inside intact bacilli. A high-throughput screening method identified bortezomib, a human 26S proteasome drug, as a potent inhibitor of ClpP1P2 activity and bacterial growth. A battery of secondary assays was employed to demonstrate that bortezomib indeed exerts its antimicrobial activity via inhibition of ClpP1P2: Down- or upmodulation of the intracellular protease level resulted in hyper- or hyposensitivity of the bacteria, the drug showed specific potentiation of translation error-inducing aminoglycosides, ClpP1P2-specific substrate WhiB1 accumulated upon exposure, and growth inhibition potencies of bortezomib derivatives correlated with ClpP1P2 inhibition potencies. Furthermore, molecular modeling showed that the drug can bind to the catalytic sites of ClpP1P2. This work demonstrates the feasibility of target mechanism-based whole-cell screening, provides chemical validation of ClpP1P2 as a target, and identifies a drug in clinical use as a new lead compound for tuberculosis therapy. PMID:25944857

  3. QSSPN: dynamic simulation of molecular interaction networks describing gene regulation, signalling and whole-cell metabolism in human cells

    PubMed Central

    Fisher, Ciarán P.; Plant, Nicholas J.; Moore, J. Bernadette; Kierzek, Andrzej M.

    2013-01-01

    Motivation: Dynamic simulation of genome-scale molecular interaction networks will enable the mechanistic prediction of genotype–phenotype relationships. Despite advances in quantitative biology, full parameterization of whole-cell models is not yet possible. Simulation methods capable of using available qualitative data are required to develop dynamic whole-cell models through an iterative process of modelling and experimental validation. Results: We formulate quasi-steady state Petri nets (QSSPN), a novel method integrating Petri nets and constraint-based analysis to predict the feasibility of qualitative dynamic behaviours in qualitative models of gene regulation, signalling and whole-cell metabolism. We present the first dynamic simulations including regulatory mechanisms and a genome-scale metabolic network in human cell, using bile acid homeostasis in human hepatocytes as a case study. QSSPN simulations reproduce experimentally determined qualitative dynamic behaviours and permit mechanistic analysis of genotype–phenotype relationships. Availability and implementation: The model and simulation software implemented in C++ are available in supplementary material and at http://sysbio3.fhms.surrey.ac.uk/qsspn/. Contact: a.kierzek@surrey.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online. PMID:24064420

  4. [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

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

    PubMed Central

    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

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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