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Biotechnology Applications (biotechnology + application)
Selected AbstractsFunctional expression and stabilization of horseradish peroxidase by directed evolution in Saccharomyces cerevisiaeBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2001Birgit Morawski Abstract Biotechnology applications of horseradish peroxidase (HRP) would benefit from access to tailor-made variants with greater specific activity, lower Km for peroxide, and higher thermostability. Starting with a mutant that is functionally expressed in Saccharomyces cerevisiae, we used random mutagenesis, recombination, and screening to identify HRP-C mutants that are more active and stable to incubation in hydrogen peroxide at 50°C. A single mutation (N175S) in the HRP active site was found to improve thermal stability. Introducing this mutation into an HRP variant evolved for higher activity yielded HRP 13A7-N175S, whose half-life at 60°C and pH 7.0 is three times that of wild-type (recombinant) HRP and a commercially available HRP preparation from Sigma (St. Louis, MO). The variant is also more stable in the presence of H2O2, SDS, salts (NaCl and urea), and at different pH values. Furthermore, this variant is more active towards a variety of small organic substrates frequently used in diagnostic applications. Site-directed mutagenesis to replace each of the four methionine residues in HRP (M83, M181, M281, M284) with isoleucine revealed no mutation that significantly increased the enzyme's stability to hydrogen peroxide. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 76: 99,107, 2001. [source] Characteristics of Immobilized Lipase on Hydrophobic Superparamagnetic Microspheres To Catalyze EsterificationBIOTECHNOLOGY PROGRESS, Issue 2 2004Zheng Guo A novel immobilized lipase (from Candida rugosa) on hydrophobic and superparamagnetic microspheres was prepared and used as a biocatalyst to catalyze esterification reactions in diverse solvents and reaction systems. The results showed that the immobilized lipase had over 2-fold higher activities in higher log P value solvents. An exponential increase of lipase activity against log P of two miscible solvent mixtures was observed for the first time. Both free and immobilized lipase achieved its maximum activity at the range of water activity ( aw) 0.5,0.8 or higher. At aw 0.6, the immobilized lipase exhibited markedly higher activities in heptane and a solvent-free system than did the native lipase. In multicompetitive reactions, the alcohol specificity of the lipase showed a strong chain-length dependency, and the immobilized enzyme exhibited more preference for a longer-chain alcohol, which is different from previous reports. The immobilized lipase showed higher specificities for butyric acid and the medium-chain-length fatty acids (C8,C12). Then, the immobilized lipase was extended to solvent-free synthesis of glycerides from glycerol and fatty acids. Recovered by magnetic separation, the immobilized lipase exhibited good reusability in repeated batch reaction, indicating its promising feature for biotechnology application. [source] In Pursuit of Zero: Polymer Brushes that Resist the Adsorption of ProteinsADVANCED MATERIALS, Issue 23 2009Angus Hucknall Abstract Protein resistant or "non-fouling" surfaces are of great interest for a variety of biomedical and biotechnology applications. This article briefly reviews the development of protein resistant surfaces, followed by recent research on a new methodology to fabricate non-fouling surfaces by surface-initiated polymerization. We show that polymer brushes synthesized by surface-initiated polymerization that present short oligo(ethylene glycol) side chains are exceptionally resistant to protein adsorption and cell adhesion. The importance of the protein and cell resistance conferred by these polymer brushes is illustrated by their use as substrates for the fabrication of antibody microarrays that exhibit femtomolar limits of detection in complex fluids such as serum and blood with relaxed requirements for intermediate wash steps. This example highlights the important point that the reduction in background noise afforded by protein-resistant surfaces can greatly simplify the development of ultrasensitive heterogeneous, surface-based clinical and proteomic assays with increased sensitivity and utility. [source] Bovine fetal microchimerism in normal and embryo transfer pregnancies and its implications for biotechnology applications in cattleBIOTECHNOLOGY JOURNAL, Issue 4 2007Lauretta Turin Dr. Abstract Fetal cells and DNA have been detected in the maternal circulation during and after pregnancy in a few mammalian species. The incidence of similar microchimerism in cattle could have repercussion for the application of modern biotechnologies such as the transfer of transgenic embryos. To determine if feto-maternal leakage can occur in pregnant cows, we have analyzed maternal blood samples for the presence of fetal DNA during gestation and post-partum periods. Y chromosome-specific DNA was detected in up to 73% of blood samples from naturally mated heifers carrying conventional bull calves and a transgene-specific sequence in up to 50% of recipient cows carrying transgenic fetuses. These findings document for the first time that transplacental leakage of fetal DNA into the maternal circulation can occur in cattle despite the epitheliochorial placenta of ruminants, with potential implications for the utilization of recipient cows in the food chain. [source] Optimization of pH and nitrogen for enhanced hydrogen production by Synechocystis sp.BIOTECHNOLOGY PROGRESS, Issue 4 2009PCC 6803 via statistical, machine learning methods Abstract The nitrogen (N) concentration and pH of culture media were optimized for increased fermentative hydrogen (H2) production from the cyanobacterium, Synechocystis sp. PCC 6803. The optimization was conducted using two procedures, response surface methodology (RSM), which is commonly used, and a memory-based machine learning algorithm, Q2, which has not been used previously in biotechnology applications. Both RSM and Q2 were successful in predicting optimum conditions that yielded higher H2 than the media reported by Burrows et al., Int J Hydrogen Energy. 2008;33:6092,6099 optimized for N, S, and C (called EHB-1 media hereafter), which itself yielded almost 150 times more H2 than Synechocystis sp. PCC 6803 grown on sulfer-free BG-11 media. RSM predicted an optimum N concentration of 0.63 mM and pH of 7.77, which yielded 1.70 times more H2 than EHB-1 media when normalized to chlorophyll concentration (0.68 ± 0.43 ,mol H2 mg Chl,1 h,1) and 1.35 times more when normalized to optical density (1.62 ± 0.09 nmol H2 OD730,1 h,1). Q2 predicted an optimum of 0.36 mM N and pH of 7.88, which yielded 1.94 and 1.27 times more H2 than EHB-1 media when normalized to chlorophyll concentration (0.77 ± 0.44 ,mol H2 mg Chl,1 h,1) and optical density (1.53 ± 0.07 nmol H2 OD730,1 h,1), respectively. Both optimization methods have unique benefits and drawbacks that are identified and discussed in this study. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Global Gene Expression Differences Associated with Changes in Glycolytic Flux and Growth Rate in Escherichia coli during the Fermentation of Glucose and XyloseBIOTECHNOLOGY PROGRESS, Issue 1 2002Ramon Gonzalez The simplicity of the fermentation process (anaerobic with pH, temperature, and agitation control) in ethanologenic Escherichia coli KO11 and LY01 makes this an attractive system to investigate the utility of gene arrays for biotechnology applications. By using this system, gene expression, glycolytic flux, and growth rate have been compared in glucose-grown and xylose-grown cells. Although the initial metabolic steps differ, ethanol yields from both sugars were essentially identical on a weight basis, and little carbon was diverted to biosynthesis. Expression of only 27 genes changed by more than 2-fold in both strains. These included induction of xylose-specific operons ( xylE, xylFGHR, and xylAB) regulated by XylR and the cyclic AMP,CRP system and repression of Mlc-regulated genes encoding glucose uptake ( ptsHIcrr, ptsG) and mannose uptake ( manXYZ) during growth on xylose. However, expression of genes encoding central carbon metabolism and biosynthesis differed by less than 2-fold. Simple statistical methods were used to investigate these more subtle changes. The reproducibility (coefficient of variation of 12%) of expression measurements (mRNA as cDNA) was found to be similar to that typically observed for in vitro measurements of enzyme activities. Using Studentapos;s t test, many smaller but significant sugar-dependent changes were identified ( p < 0.05 in both strains). A total of 276 genes were more highly expressed during growth on xylose; 307 genes were more highly expressed with glucose. Slower growth (lower ATP yield) on xylose was accompanied by decreased expression of 62 genes concerned with the biosynthesis of small molecules (amino acids, nucleotides, cofactors, and lipids), transcription, and translation; 5 such genes were expressed at a higher level. In xylose-grown cells, 90 genes associated with the transport, catabolism, and regulation of pathways for alternative carbon sources were expressed at higher levels than in glucose-grown cells, consistent with a relaxation of control by the cyclic AMP,CRP regulatory system. Changes in expression of genes encoding the Embden,Meyerhof,Parnas (EMP) pathway were in excellent agreement with calculated changes in flux for individual metabolites. Flux through all but one step, pyruvate kinase, was predicted to be higher during glucose fermentation. Expression levels (glucose/xylose) were higher in glucose-grown cells for all EMP genes except the isoenzymes encoding pyruvate kinase ( pykA and pykF). Expression of both isoenzymes was generally higher during xylose fermentation but statistically higher in both strains only for pykF encoding the isoenzyme activated by fructose-6-phosphate, a key metabolite connecting pentose metabolism to the EMP pathway. The coordinated changes in expression of genes encoding the EMP pathway suggest the presence of a common regulatory system and that flux control within the EMP pathway may be broadly distributed. In contrast, expression levels for genes encoding the Pentose,Phosphate pathway did not differ significantly between glucose-grown and xylose-grown cells. [source] | ||||||||||