Science.gov

Sample records for epimerase enzymatic function

  1. Discovery of a Dipeptide Epimerase Enzymatic Function Guided by Homology Modeling and Virtual Screening

    SciTech Connect

    Kalyanaraman, C.; Imker, H; Fedorov, A; Fedorov, E; Glasner, M; Babbitt, P; Almo, S; Gerlt, J; Jacobson, M

    2008-01-01

    We have developed a computational approach to aid the assignment of enzymatic function for uncharacterized proteins that uses homology modeling to predict the structure of the binding site and in silico docking to identify potential substrates. We apply this method to proteins in the functionally diverse enolase superfamily that are homologous to the characterized L-Ala-D/L-Glu epimerase from Bacillus subtilis. In particular, a protein from Thermotoga martima was predicted to have different substrate specificity, which suggests that it has a different, but as yet unknown, biological function. This prediction was experimentally confirmed, resulting in the assignment of epimerase activity for L-Ala-D/L-Phe, L-Ala-D/L-Tyr, and L-Ala-D/L-His, whereas the enzyme is annotated incorrectly in GenBank as muconate cycloisomerase. Subsequently, crystal structures of the enzyme were determined in complex with three substrates, showing close agreement with the computational models and revealing the structural basis for the observed substrate selectivity.

  2. Chemical Genetic Analysis and Functional Characterization of Staphylococcal Wall Teichoic Acid 2-Epimerases Reveals Unconventional Antibiotic Drug Targets.

    PubMed

    Mann, Paul A; Müller, Anna; Wolff, Kerstin A; Fischmann, Thierry; Wang, Hao; Reed, Patricia; Hou, Yan; Li, Wenjin; Müller, Christa E; Xiao, Jianying; Murgolo, Nicholas; Sher, Xinwei; Mayhood, Todd; Sheth, Payal R; Mirza, Asra; Labroli, Marc; Xiao, Li; McCoy, Mark; Gill, Charles J; Pinho, Mariana G; Schneider, Tanja; Roemer, Terry

    2016-05-01

    Here we describe a chemical biology strategy performed in Staphylococcus aureus and Staphylococcus epidermidis to identify MnaA, a 2-epimerase that we demonstrate interconverts UDP-GlcNAc and UDP-ManNAc to modulate substrate levels of TarO and TarA wall teichoic acid (WTA) biosynthesis enzymes. Genetic inactivation of mnaA results in complete loss of WTA and dramatic in vitro β-lactam hypersensitivity in methicillin-resistant S. aureus (MRSA) and S. epidermidis (MRSE). Likewise, the β-lactam antibiotic imipenem exhibits restored bactericidal activity against mnaA mutants in vitro and concomitant efficacy against 2-epimerase defective strains in a mouse thigh model of MRSA and MRSE infection. Interestingly, whereas MnaA serves as the sole 2-epimerase required for WTA biosynthesis in S. epidermidis, MnaA and Cap5P provide compensatory WTA functional roles in S. aureus. We also demonstrate that MnaA and other enzymes of WTA biosynthesis are required for biofilm formation in MRSA and MRSE. We further determine the 1.9Å crystal structure of S. aureus MnaA and identify critical residues for enzymatic dimerization, stability, and substrate binding. Finally, the natural product antibiotic tunicamycin is shown to physically bind MnaA and Cap5P and inhibit 2-epimerase activity, demonstrating that it inhibits a previously unanticipated step in WTA biosynthesis. In summary, MnaA serves as a new Staphylococcal antibiotic target with cognate inhibitors predicted to possess dual therapeutic benefit: as combination agents to restore β-lactam efficacy against MRSA and MRSE and as non-bioactive prophylactic agents to prevent Staphylococcal biofilm formation. PMID:27144276

  3. Chemical Genetic Analysis and Functional Characterization of Staphylococcal Wall Teichoic Acid 2-Epimerases Reveals Unconventional Antibiotic Drug Targets

    PubMed Central

    Mann, Paul A.; Müller, Anna; Wolff, Kerstin A.; Fischmann, Thierry; Wang, Hao; Reed, Patricia; Hou, Yan; Li, Wenjin; Müller, Christa E.; Xiao, Jianying; Murgolo, Nicholas; Sher, Xinwei; Mayhood, Todd; Sheth, Payal R.; Mirza, Asra; Labroli, Marc; Xiao, Li; McCoy, Mark; Gill, Charles J.; Pinho, Mariana G.; Schneider, Tanja; Roemer, Terry

    2016-01-01

    Here we describe a chemical biology strategy performed in Staphylococcus aureus and Staphylococcus epidermidis to identify MnaA, a 2-epimerase that we demonstrate interconverts UDP-GlcNAc and UDP-ManNAc to modulate substrate levels of TarO and TarA wall teichoic acid (WTA) biosynthesis enzymes. Genetic inactivation of mnaA results in complete loss of WTA and dramatic in vitro β-lactam hypersensitivity in methicillin-resistant S. aureus (MRSA) and S. epidermidis (MRSE). Likewise, the β-lactam antibiotic imipenem exhibits restored bactericidal activity against mnaA mutants in vitro and concomitant efficacy against 2-epimerase defective strains in a mouse thigh model of MRSA and MRSE infection. Interestingly, whereas MnaA serves as the sole 2-epimerase required for WTA biosynthesis in S. epidermidis, MnaA and Cap5P provide compensatory WTA functional roles in S. aureus. We also demonstrate that MnaA and other enzymes of WTA biosynthesis are required for biofilm formation in MRSA and MRSE. We further determine the 1.9Å crystal structure of S. aureus MnaA and identify critical residues for enzymatic dimerization, stability, and substrate binding. Finally, the natural product antibiotic tunicamycin is shown to physically bind MnaA and Cap5P and inhibit 2-epimerase activity, demonstrating that it inhibits a previously unanticipated step in WTA biosynthesis. In summary, MnaA serves as a new Staphylococcal antibiotic target with cognate inhibitors predicted to possess dual therapeutic benefit: as combination agents to restore β-lactam efficacy against MRSA and MRSE and as non-bioactive prophylactic agents to prevent Staphylococcal biofilm formation. PMID:27144276

  4. Functions, structures, and applications of cellobiose 2-epimerase and glycoside hydrolase family 130 mannoside phosphorylases.

    PubMed

    Saburi, Wataru

    2016-07-01

    Carbohydrate isomerases/epimerases are essential in carbohydrate metabolism, and have great potential in industrial carbohydrate conversion. Cellobiose 2-epimerase (CE) reversibly epimerizes the reducing end d-glucose residue of β-(1→4)-linked disaccharides to d-mannose residue. CE shares catalytic machinery with monosaccharide isomerases and epimerases having an (α/α)6-barrel catalytic domain. Two histidine residues act as general acid and base catalysts in the proton abstraction and addition mechanism. β-Mannoside hydrolase and 4-O-β-d-mannosyl-d-glucose phosphorylase (MGP) were found as neighboring genes of CE, meaning that CE is involved in β-mannan metabolism, where it epimerizes β-d-mannopyranosyl-(1→4)-d-mannose to β-d-mannopyranosyl-(1→4)-d-glucose for further phosphorolysis. MGPs form glycoside hydrolase family 130 (GH130) together with other β-mannoside phosphorylases and hydrolases. Structural analysis of GH130 enzymes revealed an unusual catalytic mechanism involving a proton relay and the molecular basis for substrate and reaction specificities. Epilactose, efficiently produced from lactose using CE, has superior physiological functions as a prebiotic oligosaccharide. PMID:27031293

  5. Structural and functional studies on a 3'-epimerase involved in the biosynthesis of dTDP-6-deoxy-D-allose.

    PubMed

    Kubiak, Rachel L; Phillips, Rebecca K; Zmudka, Matthew W; Ahn, Melissa R; Maka, E Malaika; Pyeatt, Gwen L; Roggensack, Sarah J; Holden, Hazel M

    2012-11-20

    Unusual deoxy sugars are often attached to natural products such as antibiotics, antifungals, and chemotherapeutic agents. One such sugar is mycinose, which has been found on the antibiotics chalcomycin and tylosin. An intermediate in the biosynthesis of mycinose is dTDP-6-deoxy-D-allose. Four enzymes are required for the production of dTDP-6-deoxy-D-allose in Streptomyces bikiniensis, a soil-dwelling microbe first isolated from the Bikini and Rongelap atolls. Here we describe a combined structural and functional study of the enzyme ChmJ, which reportedly catalyzes the third step in the pathway leading to dTDP-6-deoxy-D-allose formation. Specifically, it has been proposed that ChmJ is a 3'-epimerase that converts dTDP-4-keto-6-deoxyglucose to dTDP-4-keto-6-deoxyallose. This activity, however, has never been verified in vitro. As reported here, we demonstrate using (1)H nuclear magnetic resonance that ChmJ, indeed, functions as a 3'-epimerase. In addition, we determined the structure of ChmJ complexed with dTDP-quinovose to 2.0 Å resolution. The structure of ChmJ shows that it belongs to the well-characterized "cupin" superfamily. Two active site residues, His 60 and Tyr 130, were subsequently targeted for study via site-directed mutagenesis and kinetic analyses, and the three-dimensional architecture of the H60N/Y130F mutant protein was determined to 1.6 Å resolution. Finally, the structure of the apoenzyme was determined to 2.2 Å resolution. It has been previously suggested that the position of a conserved tyrosine, Tyr 130 in the case of ChmJ, determines whether an enzyme in this superfamily functions as a mono- or diepimerase. Our results indicate that the orientation of the tyrosine residue in ChmJ is a function of the ligand occupying the active site cleft. PMID:23116432

  6. Functional reassignment of Cellvibrio vulgaris EpiA to cellobiose 2-epimerase and an evaluation of the biochemical functions of the 4-O-β-D-mannosyl-D-glucose phosphorylase-like protein, UnkA.

    PubMed

    Saburi, Wataru; Tanaka, Yuka; Muto, Hirohiko; Inoue, Sota; Odaka, Rei; Nishimoto, Mamoru; Kitaoka, Motomitsu; Mori, Haruhide

    2015-01-01

    The aerobic soil bacterium Cellvibrio vulgaris has a β-mannan-degradation gene cluster, including unkA, epiA, man5A, and aga27A. Among these genes, epiA has been assigned to encode an epimerase for converting D-mannose to D-glucose, even though the amino acid sequence of EpiA is similar to that of cellobiose 2-epimerases (CEs). UnkA, whose function currently remains unknown, shows a high sequence identity to 4-O-β-D-mannosyl-D-glucose phosphorylase. In this study, we have investigated CE activity of EpiA and the general characteristics of UnkA using recombinant proteins from Escherichia coli. Recombinant EpiA catalyzed the epimerization of the 2-OH group of sugar residue at the reducing end of cellobiose, lactose, and β-(1→4)-mannobiose in a similar manner to other CEs. Furthermore, the reaction efficiency of EpiA for β-(1→4)-mannobiose was 5.5 × 10(4)-fold higher than it was for D-mannose. Recombinant UnkA phosphorolyzed β-D-mannosyl-(1→4)-D-glucose and specifically utilized D-glucose as an acceptor in the reverse reaction, which indicated that UnkA is a typical 4-O-β-D-mannosyl-D-glucose phosphorylase. PMID:25704402

  7. Structural and biochemical insights into nucleotide-rhamnose synthase/epimerase-reductase from Arabidopsis thaliana.

    PubMed

    Han, Xiaodong; Qian, Lei; Zhang, Lianwen; Liu, Xinqi

    2015-10-01

    L-Rhamnose (Rha) is synthesized via a similar enzymatic pathway in bacteria, plants and fungi. In plants, nucleotide-rhamnose synthase/epimerase-reductase (NRS/ER) catalyzes the final step in the conversion of dTDP/UDP-α-D-Glc to dTDP/UDP-β-L-Rha in an NAD(P)H dependent manner. Currently, only biochemical evidence for the function of NRS/ER has been described. In this study, a crystal structure for Arabidopsis thaliana NRS/ER was determined, which is the first report of a eukaryotic rhamnose synthase with both epimerase and reductase activities. NRS/ER functions as a metal ion independent homodimer that forms through hydrophobic interactions via a four-helix bundle. Each monomer exhibits α/β folding that can be divided into two regions, nucleotide cofactor binding domain and sugar substrate binding domain. The affinities of ligands with NRS/ER were measured using isothermal titration calorimetry, which showed that NRS/ER has a preference for dTDP over UDP, while the cofactor binding site has a similar affinity for NADH and NADPH. Structural analysis coupled to site-directed mutagenesis suggested C115 and K183 as the acid/base pair responsible for epimerization, while T113, Y144 and K148 are the conserved residues in reduction. These findings shed light on the molecular mechanism of NRS/ER and were helpful to explore other eukaryotic enzymes involved in L-Rha synthesis. PMID:26116145

  8. Structural and Functional Characterization of the R-modules in Alginate C-5 Epimerases AlgE4 and AlgE6 from Azotobacter vinelandii

    PubMed Central

    Buchinger, Edith; Knudsen, Daniel H.; Behrens, Manja A.; Pedersen, Jan Skov; Aarstad, Olav A.; Tøndervik, Anne; Valla, Svein; Skjåk-Bræk, Gudmund; Wimmer, Reinhard; Aachmann, Finn L.

    2014-01-01

    The bacterium Azotobacter vinelandii produces a family of seven secreted and calcium-dependent mannuronan C-5 epimerases (AlgE1–7). These epimerases are responsible for the epimerization of β-d-mannuronic acid (M) to α-l-guluronic acid (G) in alginate polymers. The epimerases display a modular structure composed of one or two catalytic A-modules and from one to seven R-modules having an activating effect on the A-module. In this study, we have determined the NMR structure of the three individual R-modules from AlgE6 (AR1R2R3) and the overall structure of both AlgE4 (AR) and AlgE6 using small angle x-ray scattering. Furthermore, the alginate binding ability of the R-modules of AlgE4 and AlgE6 has been studied with NMR and isothermal titration calorimetry. The AlgE6 R-modules fold into an elongated parallel β-roll with a shallow, positively charged groove across the module. Small angle x-ray scattering analyses of AlgE4 and AlgE6 show an overall elongated shape with some degree of flexibility between the modules for both enzymes. Titration of the R-modules with defined alginate oligomers shows strong interaction between AlgE4R and both oligo-M and MG, whereas no interaction was detected between these oligomers and the individual R-modules from AlgE6. A combination of all three R-modules from AlgE6 shows weak interaction with long M-oligomers. Exchanging the R-modules between AlgE4 and AlgE6 resulted in a novel epimerase called AlgE64 with increased G-block forming ability compared with AlgE6. PMID:25266718

  9. D-Ribulose 5-Phosphate 3-Epimerase: Functional and Structural Relationships to Members of the Ribulose-Phosphate Binding (beta/alpha)8-Barrel Superfamily

    SciTech Connect

    Akana,J.; Federov, A.; Federov, E.; Novak, W.; Babbitt, P.; Almo, S.; Gerlt, J.

    2006-01-01

    The 'ribulose phosphate binding' superfamily defined by the Structural Classification of Proteins (SCOP) database is considered the result of divergent evolution from a common ({beta}/{alpha}){sub 8}-barrel ancestor. The superfamily includes D-ribulose 5-phosphate 3-epimerase (RPE), orotidine 5'-monophosphate decarboxylase (OMPDC), and 3-keto-L-gulonate 6-phosphate decarboxylase (KGPDC), members of the OMPDC suprafamily, as well as enzymes involved in histidine and tryptophan biosynthesis that utilize phosphorylated metabolites as substrates. We now report studies of the functional and structural relationships of RPE to the members of the superfamily. As suggested by the results of crystallographic studies of the RPEs from rice and Plasmodium falciparum, the RPE from Streptococcus pyogenes is activated by Zn{sup 2+} which binds with a stoichiometry of one ion per polypeptide. Although wild type RPE has a high affinity for Zn{sup 2+} and inactive apoenzyme cannot be prepared, the affinity for Zn{sup 2+} is decreased by alanine substitutions for the two histidine residues that coordinate the Zn{sup 2+} ion (H34A and H67A); these mutant proteins can be prepared in an inactive, metal-free form and activated by exogenous Zn{sup 2+}. The crystal structure of the RPE was solved at 1.8 Angstroms resolution in the presence of D-xylitol 5-phosphate, an inert analogue of the D-xylulose 5-phosphate substrate. This structure suggests that the 2,3-enediolate intermediate in the 1,1-proton transfer reaction is stabilized by bidentate coordination to the Zn{sup 2+} that also is liganded to His 34, Asp 36, His 67, and Asp 176; the carboxylate groups of the Asp residues are positioned also to function as the acid/base catalysts. Although the conformation of the bound analogue resembles those of ligands bound in the active sites of OMPDC and KGPDC, the identities of the active site residues that coordinate the essential Zn{sup 2+} and participate as acid/base catalysts are not

  10. Improved operational stability of d-psicose 3-epimerase by a novel protein engineering strategy, and d-psicose production from fruit and vegetable residues.

    PubMed

    Patel, Satya Narayan; Sharma, Manisha; Lata, Kusum; Singh, Umesh; Kumar, Vinod; Sangwan, Rajender S; Singh, Sudhir P

    2016-09-01

    The aim of the present work was to improve stability of d-psicose 3-epimerase and biotransformation of fruit and vegetable residues for d-psicose production. The study established that N-terminal fusion of a yeast homolog of SUMO protein - Smt3 - can confer elevated optimal temperature and improved operational stability to d-psicose 3-epimerase. The Smt3-d-psicose 3-epimerase conjugate system exhibited relatively better catalytic efficiency, and improved productivity in terms of space-time yields of about 8.5kgL(-1)day(-1). It could serve as a promising catalytic tool for the pilot scale production of the functional sugar, d-psicose. Furthermore, a novel approach for economical production of d-psicose was developed by enzymatic and microbial bioprocessing of fruit and vegetable residues, aimed at epimerization of in situd-fructose to d-psicose. The bioprocessing led to achievement of d-psicose production to the extent of 25-35% conversion (w/w) of d-fructose contained in the sample. PMID:27235974

  11. Structural and Functional Characterization of the Clostridium perfringens N-Acetylmannosamine-6-phosphate 2-Epimerase Essential for the Sialic Acid Salvage Pathway*

    PubMed Central

    Pélissier, Marie-Cécile; Sebban-Kreuzer, Corinne; Guerlesquin, Françoise; Brannigan, James A.; Bourne, Yves; Vincent, Florence

    2014-01-01

    Pathogenic bacteria are endowed with an arsenal of specialized enzymes to convert nutrient compounds from their cell hosts. The essential N-acetylmannosamine-6-phosphate 2-epimerase (NanE) belongs to a convergent glycolytic pathway for utilization of the three amino sugars, GlcNAc, ManNAc, and sialic acid. The crystal structure of ligand-free NanE from Clostridium perfringens reveals a modified triose-phosphate isomerase (β/α)8 barrel in which a stable dimer is formed by exchanging the C-terminal helix. By retaining catalytic activity in the crystalline state, the structure of the enzyme bound to the GlcNAc-6P product identifies the topology of the active site pocket and points to invariant residues Lys66 as a putative single catalyst, supported by the structure of the catalytically inactive K66A mutant in complex with substrate ManNAc-6P. 1H NMR-based time course assays of native NanE and mutated variants demonstrate the essential role of Lys66 for the epimerization reaction with participation of neighboring Arg43, Asp126, and Glu180 residues. These findings unveil a one-base catalytic mechanism of C2 deprotonation/reprotonation via an enolate intermediate and provide the structural basis for the development of new antimicrobial agents against this family of bacterial 2-epimerases. PMID:25320079

  12. Rational design of functional and tunable oscillating enzymatic networks

    NASA Astrophysics Data System (ADS)

    Semenov, Sergey N.; Wong, Albert S. Y.; van der Made, R. Martijn; Postma, Sjoerd G. J.; Groen, Joost; van Roekel, Hendrik W. H.; de Greef, Tom F. A.; Huck, Wilhelm T. S.

    2015-02-01

    Life is sustained by complex systems operating far from equilibrium and consisting of a multitude of enzymatic reaction networks. The operating principles of biology's regulatory networks are known, but the in vitro assembly of out-of-equilibrium enzymatic reaction networks has proved challenging, limiting the development of synthetic systems showing autonomous behaviour. Here, we present a strategy for the rational design of programmable functional reaction networks that exhibit dynamic behaviour. We demonstrate that a network built around autoactivation and delayed negative feedback of the enzyme trypsin is capable of producing sustained oscillating concentrations of active trypsin for over 65 h. Other functions, such as amplification, analog-to-digital conversion and periodic control over equilibrium systems, are obtained by linking multiple network modules in microfluidic flow reactors. The methodology developed here provides a general framework to construct dissipative, tunable and robust (bio)chemical reaction networks.

  13. Rational design of functional and tunable oscillating enzymatic networks.

    PubMed

    Semenov, Sergey N; Wong, Albert S Y; van der Made, R Martijn; Postma, Sjoerd G J; Groen, Joost; van Roekel, Hendrik W H; de Greef, Tom F A; Huck, Wilhelm T S

    2015-02-01

    Life is sustained by complex systems operating far from equilibrium and consisting of a multitude of enzymatic reaction networks. The operating principles of biology's regulatory networks are known, but the in vitro assembly of out-of-equilibrium enzymatic reaction networks has proved challenging, limiting the development of synthetic systems showing autonomous behaviour. Here, we present a strategy for the rational design of programmable functional reaction networks that exhibit dynamic behaviour. We demonstrate that a network built around autoactivation and delayed negative feedback of the enzyme trypsin is capable of producing sustained oscillating concentrations of active trypsin for over 65 h. Other functions, such as amplification, analog-to-digital conversion and periodic control over equilibrium systems, are obtained by linking multiple network modules in microfluidic flow reactors. The methodology developed here provides a general framework to construct dissipative, tunable and robust (bio)chemical reaction networks. PMID:25615670

  14. Studies of the V94M-substituted human UDPgalactose-4-epimerase enzyme associated with generalized epimerase-deficiency galactosaemia.

    PubMed

    Wohlers, T M; Fridovich-Keil, J L

    2000-11-01

    Impairment of the human enzyme UDPgalactose 4-epimerase (hGALE) results in epimerase-deficiency galactosaemia, an inborn error of metabolism with variable biochemical presentation and clinical outcomes reported to range from benign to severe. Molecular studies of the hGALE loci from patients with epimerase deficiency reveal significant allelic heterogeneity, raising the possibility that variable genotypes may constitute at least one factor contributing to the biochemical and clinical heterogeneity observed. Previously we have identified a single substitution mutation, V94M, present in the homozygous state in all patients genotyped with the severe, generalized form of epimerase-deficiency galactosaemia. We report here further studies of the V94M-hGALE enzyme, overexpressed and purified from a null-background yeast expression system. Our results demonstrate that the mutant protein is impaired relative to the wild-type enzyme predominantly at the level of Vmax rather than of Km. Studies using UDP-N-acetylgalactosamine as a competitor of UDPgalactose further demonstrate that the Km values for these two substrates vary by less than a factor of 3 for both the wild-type and mutant proteins. Finally, we have explored the impact of the V94M substitution on susceptibility of yeast expressing human GALE to galactose toxicity, including changes in the levels of galactose 1-phosphate (gal-1-P) accumulated in these cells at different times following exposure to galactose. We have observed an inverse correlation between the level of GALE activity expressed in a given culture and the degree of galactose toxicity observed. We have further observed an inverse correlation between the level of GALE activity expressed in a culture and the concentration of gal-1-P accumulated in the cells. These data support the hypothesis that elevated levels of gal-1-P may underlie the observed toxicity. They further raise the intriguing possibility that yeast may provide a valuable model not only for

  15. A new robust kinetic assay for DAP epimerase activity.

    PubMed

    Hor, Lilian; Peverelli, Martin G; Perugini, Matthew A; Hutton, Craig A

    2013-10-01

    DAP epimerase is the penultimate enzyme in the lysine biosynthesis pathway. The most versatile assay for DAP epimerase catalytic activity employs a coupled DAP epimerase-DAP dehydrogenase enzyme system with a commercial mixture of DAP isomers as substrate. DAP dehydrogenase converts meso-DAP to THDP with concomitant reduction of NADP(+) to NADPH. We show that at high concentrations, accumulation of NADPH results in inhibition of DAPDH, resulting in spurious kinetic data. A new assay has been developed employing DAP decarboxylase that allows the reliable characterisation of DAP epimerase enzyme kinetics. PMID:23838343

  16. Enzymatic regulation of functional vascular networks using gelatin hydrogels

    PubMed Central

    Chuang, Chia-Hui; Lin, Ruei-Zeng; Tien, Han-Wen; Chu, Ya-Chun; Li, Yen-Cheng; Melero-Martin, Juan M.; Chen, Ying-Chieh

    2015-01-01

    To manufacture tissue engineering-based functional tissues, scaffold materials that can be sufficiently vascularized to mimic the functionality and complexity of native tissues are needed. Currently, vascular network bioengineering is largely carried out using natural hydrogels as embedding scaffolds, but most natural hydrogels have poor mechanical stability and durability, factors that critically limit their widespread use. In this study, we examined the suitability of gelatin-phenolic hydroxyl (gelatin-Ph) hydrogels that can be enzymatically crosslinked, allowing tuning of the storage modulus and the proteolytic degradation rate, for use as injectable hydrogels to support the human progenitor cell-based formation of a stable and mature vascular network. Porcine gelatin-Ph hydrogels were found to be cytocompatible with human blood-derived endothelial colony-forming cells and white adipose tissue-derived mesenchymal stem cells, resulting in >87% viability, and cell proliferation and spreading could be modulated by using hydrogels with different proteolytic degradability and stiffness. In addition, gelatin was extracted from mouse dermis and murine gelatin-Ph hydrogels were prepared. Importantly, implantation of human cell-laden porcine or murine gelatin-Ph hydrogels into immunodeficient mice resulted in the rapid formation of functional anastomoses between the bioengineered human vascular network and the mouse vasculature. Furthermore, the degree of enzymatic crosslinking of the gelatin-Ph hydrogels could be used to modulate cell behavior and the extent of vascular network formation in vivo. Our report details a technique for the synthesis of gelatin-Ph hydrogels from allogeneic or xenogeneic dermal skin and suggests that these hydrogels can be used for biomedical applications that require the formation of microvascular networks, including the development of complex engineered tissues. PMID:25749296

  17. Intramolecular Epistasis and the Evolution of a New Enzymatic Function

    PubMed Central

    Noor, Sajid; Taylor, Matthew C.; Russell, Robyn J.; Jermiin, Lars S.; Jackson, Colin J.; Oakeshott, John G.; Scott, Colin

    2012-01-01

    Atrazine chlorohydrolase (AtzA) and its close relative melamine deaminase (TriA) differ by just nine amino acid substitutions but have distinct catalytic activities. Together, they offer an informative model system to study the molecular processes that underpin the emergence of new enzymatic function. Here we have constructed the potential evolutionary trajectories between AtzA and TriA, and characterized the catalytic activities and biophysical properties of the intermediates along those trajectories. The order in which the nine amino acid substitutions that separate the enzymes could be introduced to either enzyme, while maintaining significant catalytic activity, was dictated by epistatic interactions, principally between three amino acids within the active site: namely, S331C, N328D and F84L. The mechanistic basis for the epistatic relationships is consistent with a model for the catalytic mechanisms in which protonation is required for hydrolysis of melamine, but not atrazine. PMID:22768133

  18. 5-methylcytosine in RNA: detection, enzymatic formation and biological functions

    PubMed Central

    Motorin, Yuri; Lyko, Frank; Helm, Mark

    2010-01-01

    The nucleobase modification 5-methylcytosine (m5C) is widespread both in DNA and different cellular RNAs. The functions and enzymatic mechanisms of DNA m5C-methylation were extensively studied during the last decades. However, the location, the mechanism of formation and the cellular function(s) of the same modified nucleobase in RNA still remain to be elucidated. The recent development of a bisulfite sequencing approach for efficient m5C localization in various RNA molecules puts ribo-m5C in a highly privileged position as one of the few RNA modifications whose detection is amenable to PCR-based amplification and sequencing methods. Additional progress in the field also includes the characterization of several specific RNA methyltransferase enzymes in various organisms, and the discovery of a new and unexpected link between DNA and RNA m5C-methylation. Numerous putative RNA:m5C-MTases have now been identified and are awaiting characterization, including the identification of their RNA substrates and their related cellular functions. In order to bring these recent exciting developments into perspective, this review provides an ordered overview of the detection methods for RNA methylation, of the biochemistry, enzymology and molecular biology of the corresponding modification enzymes, and discusses perspectives for the emerging biological functions of these enzymes. PMID:20007150

  19. Redox control of enzymatic functions: The electronics of life's circuitry.

    PubMed

    Bonini, Marcelo G; Consolaro, Marcia E L; Hart, Peter C; Mao, Mao; de Abreu, Andre Luelsdorf Pimenta; Master, Alyssa M

    2014-03-26

    The field of redox biology has changed tremendously over the past 20 years. Formerly regarded as bi-products of the aerobic metabolism exclusively involved in tissue damage, reactive oxygen species (ROS) are now recognized as active participants of cell signaling events in health and in disease. In this sense, ROS and the more recently defined reactive nitrogen species (RNS) are, just like hormones and second messengers, acting as fundamental orchestrators of cell signaling pathways. The chemical modification of enzymes by ROS and RNS (that result in functional enzymatic alterations) accounts for a considerable fraction of the transient and persistent perturbations imposed by variations in oxidant levels. Upregulation of ROS and RNS in response to stress is a common cellular response that foments adaptation to a variety of physiologic alterations (hypoxia, hyperoxia, starvation, and cytokine production). Frequently, these are beneficial and increase the organisms' resistance against subsequent acute stress (preconditioning). Differently, the sustained ROS/RNS-dependent rerouting of signaling produces irreversible alterations in cellular functioning, often leading to pathogenic events. Thus, the duration and reversibility of protein oxidations define whether complex organisms remain "electronically" healthy. Among the 20 essential amino acids, four are particularly susceptible to oxidation: cysteine, methionine, tyrosine, and tryptophan. Here, we will critically review the mechanisms, implications, and repair systems involved in the redox modifications of these residues in proteins while analyzing well-characterized prototypic examples. Occasionally, we will discuss potential consequences of amino acid oxidation and speculate on the biologic necessity for such events in the context of adaptative redox signaling. © 2014 IUBMB Life, 2014. PMID:24668617

  20. UDP-hexose 4-epimerases: a view on structure, mechanism and substrate specificity.

    PubMed

    Beerens, Koen; Soetaert, Wim; Desmet, Tom

    2015-09-23

    UDP-sugar 4-epimerase (GalE) belongs to the short-chain dehydrogenase/reductase (SDR) superfamily of proteins and is one of enzymes in the Leloir pathway. They have been shown to be important virulence factors in a number of Gram-negative pathogens and to be involved in the biosynthesis of different polysaccharide structures. The metabolic disease type III galactosemia is caused by detrimental mutations in the human GalE. GalE and related enzymes display unusual enzymologic, chemical, and stereochemical properties; including irreversible binding of the cofactor NAD and uridine nucleotide-induced activation of this cofactor. These epimerases have been found active on UDP-hexoses, the N-acetylated and uronic acid forms thereof as well as UDP-pentoses. As they are involved in different pathways and functions, a deeper understanding of the enzymes, and their substrate promiscuity and/or selectivity, could lead to drug and vaccine design as well as antibiotic and probiotic development. This review summarizes the research performed on UDP-sugar 4-epimerases' structure, mechanism and substrate promiscuity. PMID:26162744

  1. A Structural Basis for the Allosteric Regulatin of Non-Hydrolysing UDP-G1cNAc 2-Epimerases

    SciTech Connect

    Velloso,L.; Bhaskaran, S.; Schuch, R.; Fischetti, V.; Stebbins, C.

    2008-01-01

    The non-hydrolysing bacterial UDP-N-acetylglucosamine 2-epimerase (UDP-GlcNAc 2-epimerase) catalyses the conversion of UDP-GlcNAc into UDP-N-acetylmannosamine, an intermediate in the biosynthesis of several cell-surface polysaccharides. This enzyme is allosterically regulated by its substrate UDP-GlcNAc. The structure of the ternary complex between the Bacillus anthracis UDP-GlcNAc 2-epimerase, its substrate UDP-GlcNAc and the reaction intermediate UDP, showed direct interactions between UDP and its substrate, and between the complex and highly conserved enzyme residues, identifying the allosteric site of the enzyme. The binding of UDP-GlcNAc is associated with conformational changes in the active site of the enzyme. Kinetic data and mutagenesis of the highly conserved UDP-GlcNAc-interacting residues confirm their importance in the substrate binding and catalysis of the enzyme. This constitutes the first example to our knowledge, of an enzymatic allosteric activation by direct interaction between the substrate and the allosteric activator.

  2. Epimerase-Deficiency Galactosemia Is Not a Binary Condition

    PubMed Central

    Openo, Kimberly K.; Schulz, Jenny M.; Vargas, Claudia A.; Orton, Corey S.; Epstein, Michael P.; Schnur, Rhonda E.; Scaglia, Fernando; Berry, Gerard T.; Gottesman, Gary S.; Ficicioglu, Can; Slonim, Alfred E.; Schroer, Richard J.; Yu, Chunli; Rangel, Vanessa E.; Keenan, Jennifer; Lamance, Kerri; Fridovich-Keil, Judith L.

    2006-01-01

    Epimerase-deficiency galactosemia results from the impairment of UDP-galactose 4′-epimerase (GALE), the third enzyme in the Leloir pathway of galactose metabolism. Originally identified as a clinically benign “peripheral” condition with enzyme impairment restricted to circulating blood cells, GALE deficiency was later demonstrated also to exist in a rare but clinically severe “generalized” form, with enzyme impairment affecting a range of tissues. Isolated cases of clinically and/or biochemically intermediate cases of epimerase deficiency have also been reported. We report here studies of 10 patients who, in the neonatal period, received the diagnosis of hemolysate epimerase deficiency. We have characterized these patients with regard to three parameters: (1) GALE activity in transformed lymphoblasts, representing a “nonperipheral” tissue, (2) metabolic sensitivity of those lymphoblasts to galactose challenge in culture, and (3) evidence of normal versus abnormal galactose metabolism in the patients themselves. Our results demonstrate two important points. First, whereas some of the patients studied exhibited near-normal levels of GALE activity in lymphoblasts, consistent with a diagnosis of peripheral epimerase deficiency, many did not. We detected a spectrum of GALE activity levels ranging from 15%–64% of control levels, demonstrating that epimerase deficiency is not a binary condition; it is a continuum disorder. Second, lymphoblasts demonstrating the most severe reduction in GALE activity also demonstrated abnormal metabolite levels in the presence of external galactose and, in some cases, also in the absence of galactose. These abnormalities included elevated galactose-1P, elevated UDP-galactose, and deficient UDP-glucose. Moreover, some of the patients themselves also demonstrated metabolic abnormalities, both on and off galactose-restricted diet. Long-term follow-up studies of these and other patients will be required to elucidate the

  3. Recent advances in the elucidation of enzymatic function in natural product biosynthesis

    PubMed Central

    Tan, Gao-Yi; Deng, Zixin; Liu, Tiangang

    2016-01-01

    With the successful production of artemisinic acid in yeast, the promising potential of synthetic biology for natural product biosynthesis is now being realized. The recent total biosynthesis of opioids in microbes is considered to be another landmark in this field. The importance and significance of enzymes in natural product biosynthetic pathways have been re-emphasized by these advancements. Therefore, the characterization and elucidation of enzymatic function in natural product biosynthesis are undoubtedly fundamental for the development of new drugs and the heterologous biosynthesis of active natural products. Here, discoveries regarding enzymatic function in natural product biosynthesis over the past year are briefly reviewed. PMID:26989472

  4. Catalytic Mechanism and Mode of Action of the Periplasmic Alginate Epimerase AlgG*

    PubMed Central

    Wolfram, Francis; Kitova, Elena N.; Robinson, Howard; Walvoort, Marthe T. C.; Codée, Jeroen D. C.; Klassen, John S.; Howell, P. Lynne

    2014-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen that forms chronic biofilm infections in the lungs of cystic fibrosis patients. A major component of the biofilm during these infections is the exopolysaccharide alginate, which is synthesized at the inner membrane as a homopolymer of 1–4-linked β-d-mannuronate. As the polymer passages through the periplasm, 22–44% of the mannuronate residues are converted to α-l-guluronate by the C5-epimerase AlgG to produce a polymer of alternating β-d-mannuronate and α-l-guluronate blocks and stretches of polymannuronate. To understand the molecular basis of alginate epimerization, the structure of Pseudomonas syringae AlgG has been determined at 2.1-Å resolution, and the protein was functionally characterized. The structure reveals that AlgG is a long right-handed parallel β-helix with an elaborate lid structure. Functional analysis of AlgG mutants suggests that His319 acts as the catalytic base and that Arg345 neutralizes the acidic group during the epimerase reaction. Water is the likely catalytic acid. Electrostatic surface potential and residue conservation analyses in conjunction with activity and substrate docking studies suggest that a conserved electropositive groove facilitates polymannuronate binding and contains at least nine substrate binding subsites. These subsites likely align the polymer in the correct register for catalysis to occur. The presence of multiple subsites, the electropositive groove, and the non-random distribution of guluronate in the alginate polymer suggest that AlgG is a processive enzyme. Moreover, comparison of AlgG and the extracellular alginate epimerase AlgE4 of Azotobacter vinelandii provides a structural rationale for the differences in their Ca2+ dependence. PMID:24398681

  5. Catalytic mechanism and mode of action of the periplasmic alginate epimerase AlgG.

    PubMed

    Wolfram, Francis; Kitova, Elena N; Robinson, Howard; Walvoort, Marthe T C; Codée, Jeroen D C; Klassen, John S; Howell, P Lynne

    2014-02-28

    Pseudomonas aeruginosa is an opportunistic pathogen that forms chronic biofilm infections in the lungs of cystic fibrosis patients. A major component of the biofilm during these infections is the exopolysaccharide alginate, which is synthesized at the inner membrane as a homopolymer of 1-4-linked β-D-mannuronate. As the polymer passages through the periplasm, 22-44% of the mannuronate residues are converted to α-L-guluronate by the C5-epimerase AlgG to produce a polymer of alternating β-D-mannuronate and α-L-guluronate blocks and stretches of polymannuronate. To understand the molecular basis of alginate epimerization, the structure of Pseudomonas syringae AlgG has been determined at 2.1-Å resolution, and the protein was functionally characterized. The structure reveals that AlgG is a long right-handed parallel β-helix with an elaborate lid structure. Functional analysis of AlgG mutants suggests that His(319) acts as the catalytic base and that Arg(345) neutralizes the acidic group during the epimerase reaction. Water is the likely catalytic acid. Electrostatic surface potential and residue conservation analyses in conjunction with activity and substrate docking studies suggest that a conserved electropositive groove facilitates polymannuronate binding and contains at least nine substrate binding subsites. These subsites likely align the polymer in the correct register for catalysis to occur. The presence of multiple subsites, the electropositive groove, and the non-random distribution of guluronate in the alginate polymer suggest that AlgG is a processive enzyme. Moreover, comparison of AlgG and the extracellular alginate epimerase AlgE4 of Azotobacter vinelandii provides a structural rationale for the differences in their Ca(2+) dependence. PMID:24398681

  6. Enzymatic Functionalization of Carbon-Hydrogen Bonds1

    PubMed Central

    Lewis, Jared C.; Coelho, Pedro S.

    2010-01-01

    The development of new catalytic methods to functionalize carbon-hydrogen (C-H) bonds continues to progress at a rapid pace due to the significant economic and environmental benefits of these transformations over traditional synthetic methods. In nature, enzymes catalyze regio- and stereoselective C-H bond functionalization using transformations ranging from hydroxylation to hydroalkylation under ambient reaction conditions. The efficiency of these enzymes relative to analogous chemical processes has led to their increased use as biocatalysts in preparative and industrial applications. Furthermore, unlike small molecule catalysts, enzymes can be systematically optimized via directed evolution for a particular application and can be expressed in vivo to augment the biosynthetic capability of living organisms. While a variety of technical challenges must still be overcome for practical application of many enzymes for C-H bond functionalization, continued research on natural enzymes and on novel artificial metalloenzymes will lead to improved synthetic processes for efficient synthesis of complex molecules. In this critical review, we discuss the most prevalent mechanistic strategies used by enzymes to functionalize non-acidic C-H bonds, the application and evolution of these enzymes for chemical synthesis, and a number of potential biosynthetic capabilities uniquely enabled by these powerful catalysts. PMID:21079862

  7. Energy Landscape of Alginate-Epimerase Interactions Assessed by Optical Tweezers and Atomic Force Microscopy

    PubMed Central

    Håti, Armend Gazmeno; Aachmann, Finn Lillelund; Stokke, Bjørn Torger; Skjåk-Bræk, Gudmund; Sletmoen, Marit

    2015-01-01

    Mannuronan C-5 epimerases are a family of enzymes that catalyze epimerization of alginates at the polymer level. This group of enzymes thus enables the tailor-making of various alginate residue sequences to attain various functional properties, e.g. viscosity, gelation and ion binding. Here, the interactions between epimerases AlgE4 and AlgE6 and alginate substrates as well as epimerization products were determined. The interactions of the various epimerase–polysaccharide pairs were determined over an extended range of force loading rates by the combined use of optical tweezers and atomic force microscopy. When studying systems that in nature are not subjected to external forces the access to observations obtained at low loading rates, as provided by optical tweezers, is a great advantage since the low loading rate region for these systems reflect the properties of the rate limiting energy barrier. The AlgE epimerases have a modular structure comprising both A and R modules, and the role of each of these modules in the epimerization process were examined through studies of the A- module of AlgE6, AlgE6A. Dynamic strength spectra obtained through combination of atomic force microscopy and the optical tweezers revealed the existence of two energy barriers in the alginate-epimerase complexes, of which one was not revealed in previous AFM based studies of these complexes. Furthermore, based on these spectra estimates of the locations of energy transition states (xβ), lifetimes in the absence of external perturbation (τ0) and free energies (ΔG#) were determined for the different epimerase–alginate complexes. This is the first determination of ΔG# for these complexes. The values determined were up to 8 kBT for the outer barrier, and smaller values for the inner barriers. The size of the free energies determined are consistent with the interpretation that the enzyme and substrate are thus not tightly locked at all times but are able to relocate. Together with the

  8. Enzymatic Halogenases and Haloperoxidases: Computational Studies on Mechanism and Function.

    PubMed

    Timmins, Amy; de Visser, Sam P

    2015-01-01

    Despite the fact that halogenated compounds are rare in biology, a number of organisms have developed processes to utilize halogens and in recent years, a string of enzymes have been identified that selectively insert halogen atoms into, for instance, a CH aliphatic bond. Thus, a number of natural products, including antibiotics, contain halogenated functional groups. This unusual process has great relevance to the chemical industry for stereoselective and regiospecific synthesis of haloalkanes. Currently, however, industry utilizes few applications of biological haloperoxidases and halogenases, but efforts are being worked on to understand their catalytic mechanism, so that their catalytic function can be upscaled. In this review, we summarize experimental and computational studies on the catalytic mechanism of a range of haloperoxidases and halogenases with structurally very different catalytic features and cofactors. This chapter gives an overview of heme-dependent haloperoxidases, nonheme vanadium-dependent haloperoxidases, and flavin adenine dinucleotide-dependent haloperoxidases. In addition, we discuss the S-adenosyl-l-methionine fluoridase and nonheme iron/α-ketoglutarate-dependent halogenases. In particular, computational efforts have been applied extensively for several of these haloperoxidases and halogenases and have given insight into the essential structural features that enable these enzymes to perform the unusual halogen atom transfer to substrates. PMID:26415843

  9. Enzymatic synthesis of lignin-siloxane hybrid functional polymers.

    PubMed

    Prasetyo, Endry Nugroho; Kudanga, Tukayi; Fischer, Roman; Eichinger, Reinhard; Nyanhongo, Gibson S; Guebitz, Georg M

    2012-02-01

    This study combines the properties of siloxanes and lignin polymers to produce hybrid functional polymers that can be used as adhesives, coating materials, and/or multifunctionalized thin-coating films. Lignin-silica hybrid copolymers were synthesized by using a sol-gel process. Laccases from Trametes hirsuta were used to oxidize lignosulphonates to enhance their reactivity towards siloxanes and then were incorporated into siloxane precursors undergoing a sol-gel process. In vitro copolymerization studies using pure lignin monomers with aminosilanes or ethoxytrimethylsilane and analysis by ²⁹Si NMR spectroscopy revealed hybrid products. Except for kraft lignin, an increase in lignin concentration positively affected the tensile strength in all samples. Similarly, the viscosity generally increased in all samples with increasing lignin concentration and also affected the curing time. PMID:21751391

  10. Glucose level determination with a multi-enzymatic cascade reaction in a functionalized glass chip.

    PubMed

    Costantini, Francesca; Tiggelaar, Roald; Sennato, Simona; Mura, Francesco; Schlautmann, Stefan; Bordi, Federico; Gardeniers, Han; Manetti, Cesare

    2013-09-01

    In this work we show the functionalization of the interior of microfluidic glass chips with poly(2-hydroxyethyl methacrylate) polymer brushes as anchors for co-immobilization of the enzymes glucose-oxidase and horseradish peroxidase. The formation of the brush layer and subsequent immobilization of these enzymes have been characterized on flat surfaces by atomic force microscopy and Fourier transform infrared spectroscopy, and studied inside glass chips by field emission scanning microscopy. Enzyme-functionalized glass chips have been applied for performing a multi-enzymatic cascade reaction for the fast (20 s) determination of glucose in human blood samples and the result is in excellent agreement with values obtained from the conventional hospital laboratory. The limit of detection of this bi-enzymatic method is 60 μM. With the advantages of high selectivity and reproducibility, this functionalization method can be used for improving the efficiency of glucose sensors. PMID:23831561

  11. Overlapping and Distinct Roles of Aspergillus fumigatus UDP-glucose 4-Epimerases in Galactose Metabolism and the Synthesis of Galactose-containing Cell Wall Polysaccharides*

    PubMed Central

    Lee, Mark J.; Gravelat, Fabrice N.; Cerone, Robert P.; Baptista, Stefanie D.; Campoli, Paolo V.; Choe, Se-In; Kravtsov, Ilia; Vinogradov, Evgeny; Creuzenet, Carole; Liu, Hong; Berghuis, Albert M.; Latgé, Jean-Paul; Filler, Scott G.; Fontaine, Thierry; Sheppard, Donald C.

    2014-01-01

    The cell wall of Aspergillus fumigatus contains two galactose-containing polysaccharides, galactomannan and galactosaminogalactan, whose biosynthetic pathways are not well understood. The A. fumigatus genome contains three genes encoding putative UDP-glucose 4-epimerases, uge3, uge4, and uge5. We undertook this study to elucidate the function of these epimerases. We found that uge4 is minimally expressed and is not required for the synthesis of galactose-containing exopolysaccharides or galactose metabolism. Uge5 is the dominant UDP-glucose 4-epimerase in A. fumigatus and is essential for normal growth in galactose-based medium. Uge5 is required for synthesis of the galactofuranose (Galf) component of galactomannan and contributes galactose to the synthesis of galactosaminogalactan. Uge3 can mediate production of both UDP-galactose and UDP-N-acetylgalactosamine (GalNAc) and is required for the production of galactosaminogalactan but not galactomannan. In the absence of Uge5, Uge3 activity is sufficient for growth on galactose and the synthesis of galactosaminogalactan containing lower levels of galactose but not the synthesis of Galf. A double deletion of uge5 and uge3 blocked growth on galactose and synthesis of both Galf and galactosaminogalactan. This study is the first survey of glucose epimerases in A. fumigatus and contributes to our understanding of the role of these enzymes in metabolism and cell wall synthesis. PMID:24257745

  12. Physico-chemical characterization and enzymatic functionalization of Enteromorpha sp. cellulose.

    PubMed

    Jmel, Mohamed Amine; Ben Messaoud, Ghazi; Marzouki, M Nejib; Mathlouthi, Mohamed; Smaali, Issam

    2016-01-01

    Although green macro-algae represent a renewable and highly abundant biomass, they remain poorly exploited in terms of carbohydrate polymers compared to red and brown ones and other lignocellulosic materials. In this study, cellulose from the green macro-algae Enteromorpha sp. was isolated, physico-chemically characterized and enzymatically functionalized. The cellulose content was about 21.4% (w/w). FTIR analyses indicated an absence of acetyl or uronic esters confirming the absence of hemicellulose contamination. The 36% crystallinity index of the extracted cellulose revealed a high amorphous character as determined by X-ray diffraction. The moisture adsorption isotherms and specific surface measurements were respectively 42.87g/100g and 8.34m(2)/g. The Enteromorpha sp. cellulose was first enzymatically saccharified by a commercial cellulase preparation from Aspergillus niger with a hydrolysis yield of 70.4%. Besides, it was successfully functionalized based on the transglycosylation mechanism of the same enzymatic preparation, to produce highly added-value biosurfactants (butyl-glucoside) with a concentration of 8mM. PMID:26453878

  13. Hydrophobic surface functionalization of lignocellulosic jute fabrics by enzymatic grafting of octadecylamine.

    PubMed

    Dong, Aixue; Fan, Xuerong; Wang, Qiang; Yu, Yuanyuan; Cavaco-Paulo, Artur

    2015-08-01

    Enzymatic grafting of synthetic molecules onto lignins provides a mild and eco-friendly alternative for the functionalization of lignocellulosic materials. In this study, laccase-mediated grafting of octadecylamine (OA) onto lignin-rich jute fabrics was investigated for enhancing the surface hydrophobicity. First, the lignins in jute fabrics were isolated and analyzed in the macromolecular level by MALDI-TOF MS, (1)H NMR, (13)C NMR, and HSQC-NMR. Then, the surface of jute fabrics was characterized by FT-IR, XPS, and SEM. Subsequently, the nitrogen content of jute fabrics was determined by the micro-Kjeldahl method, and the grafting percentage (Gp) and grafting efficiency (GE) of the enzymatic reaction were calculated. Finally, the surface hydrophobicity of the jute fabrics was estimated by contact angle and wetting time measurements. The results indicate that the OA monomers were successfully grafted onto the lignin moieties on the jute fiber surface by laccase with Gp and GE values of 0.712% and 10.571%, respectively. Moreover, the modified jute fabrics via OA-grafting showed an increased wetting time of 18.5 min and a contact angle of 116.72°, indicating that the surface hydrophobicity of the jute fabrics increased after the enzymatic grafting modification with hydrophobic OA molecules. PMID:25987460

  14. Selection and validation of enzymatic activities as functional markers in wood biotechnology and fungal ecology.

    PubMed

    Mathieu, Yann; Gelhaye, Eric; Dumarçay, Stéphane; Gérardin, Philippe; Harvengt, Luc; Buée, Marc

    2013-02-15

    The dead wood and forest soils are sources of diversity and under-explored fungal strains with biotechnological potential, which require to be studied. Numerous enzymatic tests have been proposed to investigate the functional potential of the soil microbial communities or to test the functional abilities of fungal strains. Nevertheless, the diversity of these functional markers and their relevance in environmental studies or biotechnological screening does still have not been demonstrated. In this work, we assessed ten different extracellular enzymatic activities involved in the wood decaying process including β-etherase that specifically cleaves the β-aryl ether linkages in the lignin polymer. For this purpose, a collection of 26 fungal strains, distributed within three ecological groups (white, brown and soft rot fungi), has been used. Among the ten potential functional markers, the combinatorial use of only six of them allowed separation between the group of white and soft rot fungi from the brown rot fungi. Moreover, our results suggest that extracellular β-etherase is a rare and dispensable activity among the wood decay fungi. Finally, we propose that this set of markers could be useful for the analysis of fungal communities in functional and environmental studies, and for the selection of strains with biotechnological interests. PMID:23206919

  15. Identification and characterization of a mutation, in the human UDP-galactose-4-epimerase gene, associated with generalized epimerase-deficiency galactosemia.

    PubMed

    Wohlers, T M; Christacos, N C; Harreman, M T; Fridovich-Keil, J L

    1999-02-01

    Epimerase-deficiency galactosemia results from impairment of the human enzyme UDP-galactose-4-epimerase (hGALE). We and others have identified substitution mutations in the hGALE alleles of patients with the clinically mild, peripheral form of epimerase deficiency. We report here the first identification of an hGALE mutation in a patient with the clinically severe, generalized form of epimerase deficiency. The mutation, V94M, was found on both GALE alleles of this patient. This same mutation also was found in the homozygous state in two additional patients with generalized epimerase deficiency. The specific activity of the V94M-hGALE protein expressed in yeast was severely reduced with regard to UDP-galactose and partially reduced with regard to UDP-N-acetylgalactosamine. In contrast, two GALE-variant proteins associated with peripheral epimerase deficiency, L313M-hGALE and D103G-hGALE, demonstrated near-normal levels of activity with regard to both substrates, but a third allele, G90E-hGALE, demonstrated little, if any, detectable activity, despite near-normal abundance. G90E originally was identified in a heterozygous patient whose other allele remains uncharacterized. Thermal lability and protease-sensitivity studies demonstrated compromised stability in all of the partially active mutant enzymes. PMID:9973283

  16. Identification and characterization of a mutation, in the human UDP-galactose-4-epimerase gene, associated with generalized epimerase-deficiency galactosemia.

    PubMed Central

    Wohlers, T M; Christacos, N C; Harreman, M T; Fridovich-Keil, J L

    1999-01-01

    Epimerase-deficiency galactosemia results from impairment of the human enzyme UDP-galactose-4-epimerase (hGALE). We and others have identified substitution mutations in the hGALE alleles of patients with the clinically mild, peripheral form of epimerase deficiency. We report here the first identification of an hGALE mutation in a patient with the clinically severe, generalized form of epimerase deficiency. The mutation, V94M, was found on both GALE alleles of this patient. This same mutation also was found in the homozygous state in two additional patients with generalized epimerase deficiency. The specific activity of the V94M-hGALE protein expressed in yeast was severely reduced with regard to UDP-galactose and partially reduced with regard to UDP-N-acetylgalactosamine. In contrast, two GALE-variant proteins associated with peripheral epimerase deficiency, L313M-hGALE and D103G-hGALE, demonstrated near-normal levels of activity with regard to both substrates, but a third allele, G90E-hGALE, demonstrated little, if any, detectable activity, despite near-normal abundance. G90E originally was identified in a heterozygous patient whose other allele remains uncharacterized. Thermal lability and protease-sensitivity studies demonstrated compromised stability in all of the partially active mutant enzymes. PMID:9973283

  17. Enzymatic hydrolysis of recovered protein from frozen small croaker and functional properties of its hydrolysates.

    PubMed

    Choi, Yeung Joon; Hur, Sungik; Choi, Byeong-Dae; Konno, Kunihiko; Park, Jae W

    2009-01-01

    Fish protein isolate were recovered from frozen small croaker using pH shift. The partial enzymatic hydrolysates were fractionated as soluble and insoluble parts. They were dried using the drum dryer and their functional properties were examined. The total nitrogen content of the enzymatic hydrolysates ranged from 12.9% to 13.7%. The degree of hydrolysis of precipitates was 18.2% and 12.2% for croaker hydrolysates treated with Protamex 1.5 MG (Bacilllus protease complex) and Flavourzyme 500 MG (endoproteases and exoproteases, Aspergillus oryzae), respectively. The TCA supernatant, after centrifugation of hydrolysates, contained numerous peptides ranging from 100 to 4000 daltons. The solubility of the supernatants was higher than that of the precipitates at 0% to 3% NaCl and pH 2 to 10. The precipitate of Flavourzyme- and Protamex-treated hydrolysates showed a high emulsion activity index value compared to egg white and bovine plasma protein. In addition, the highest emulsion stability was observed for Protamex-treated precipitate hydrolysates. Emulsion stability of Protamex-treated precipitate hydrolysates was comparable to those of protein additives (egg white, bovine plasma protein, and soy protein concentrate). Water and fat binding capacity of precipitates were higher than those of supernatant. The results indicate that precipitate hydrolysate from undersized croaker can be used in processed muscle foods as a functional and nutritional ingredient. PMID:19200081

  18. Molecular and structural discrimination of proline racemase and hydroxyproline-2-epimerase from nosocomial and bacterial pathogens.

    PubMed

    Goytia, Maira; Chamond, Nathalie; Cosson, Alain; Coatnoan, Nicolas; Hermant, Daniel; Berneman, Armand; Minoprio, Paola

    2007-01-01

    The first eukaryotic proline racemase (PRAC), isolated from the human Trypanosoma cruzi pathogen, is a validated therapeutic target against Chagas' disease. This essential enzyme is implicated in parasite life cycle and infectivity and its ability to trigger host B-cell nonspecific hypergammaglobulinemia contributes to parasite evasion and persistence. Using previously identified PRAC signatures and data mining we present the identification and characterization of a novel PRAC and five hydroxyproline epimerases (HyPRE) from pathogenic bacteria. Single-mutation of key HyPRE catalytic cysteine abrogates enzymatic activity supporting the presence of two reaction centers per homodimer. Furthermore, evidences are provided that Brucella abortus PrpA [for 'proline racemase' virulence factor A] and homologous proteins from two Brucella spp are bona fide HyPREs and not 'one way' directional PRACs as described elsewhere. Although the mechanisms of aminoacid racemization and epimerization are conserved between PRAC and HyPRE, our studies demonstrate that substrate accessibility and specificity partly rely on constraints imposed by aromatic or aliphatic residues distinctively belonging to the catalytic pockets. Analysis of PRAC and HyPRE sequences along with reaction center structural data disclose additional valuable elements for in silico discrimination of the enzymes. Furthermore, similarly to PRAC, the lymphocyte mitogenicity displayed by HyPREs is discussed in the context of bacterial metabolism and pathogenesis. Considering tissue specificity and tropism of infectious pathogens, it would not be surprising if upon infection PRAC and HyPRE play important roles in the regulation of the intracellular and extracellular amino acid pool profiting the microrganism with precursors and enzymatic pathways of the host. PMID:17849014

  19. Molecular and Structural Discrimination of Proline Racemase and Hydroxyproline-2-Epimerase from Nosocomial and Bacterial Pathogens

    PubMed Central

    Goytia, Maira; Chamond, Nathalie; Cosson, Alain; Coatnoan, Nicolas; Hermant, Daniel; Berneman, Armand; Minoprio, Paola

    2007-01-01

    The first eukaryotic proline racemase (PRAC), isolated from the human Trypanosoma cruzi pathogen, is a validated therapeutic target against Chagas' disease. This essential enzyme is implicated in parasite life cycle and infectivity and its ability to trigger host B-cell nonspecific hypergammaglobulinemia contributes to parasite evasion and persistence. Using previously identified PRAC signatures and data mining we present the identification and characterization of a novel PRAC and five hydroxyproline epimerases (HyPRE) from pathogenic bacteria. Single-mutation of key HyPRE catalytic cysteine abrogates enzymatic activity supporting the presence of two reaction centers per homodimer. Furthermore, evidences are provided that Brucella abortus PrpA [for ‘proline racemase’ virulence factor A] and homologous proteins from two Brucella spp are bona fide HyPREs and not ‘one way’ directional PRACs as described elsewhere. Although the mechanisms of aminoacid racemization and epimerization are conserved between PRAC and HyPRE, our studies demonstrate that substrate accessibility and specificity partly rely on contraints imposed by aromatic or aliphatic residues distinctively belonging to the catalytic pockets. Analysis of PRAC and HyPRE sequences along with reaction center structural data disclose additional valuable elements for in silico discrimination of the enzymes. Furthermore, similarly to PRAC, the lymphocyte mitogenicity displayed by HyPREs is discussed in the context of bacterial metabolism and pathogenesis. Considering tissue specificity and tropism of infectious pathogens, it would not be surprising if upon infection PRAC and HyPRE play important roles in the regulation of the intracellular and extracellular amino acid pool profiting the microrganism with precursors and enzymatic pathways of the host. PMID:17849014

  20. Antioxidant activity and functional properties of enzymatic protein hydrolysates from common carp (Cyprinus carpio) roe (egg).

    PubMed

    Chalamaiah, M; Jyothirmayi, T; Diwan, Prakash V; Dinesh Kumar, B

    2015-09-01

    Previously, we have reported the composition, molecular mass distribution and in vivo immunomodulatory effects of common carp roe protein hydrolysates. In the current study, antioxidative activity and functional properties of common carp (Cyprinus carpio) roe (egg) protein hydrolysates, prepared by pepsin, trypsin and Alcalase, were evaluated. The three hydrolysates showed excellent antioxidant activities in a dose dependent manner in various in vitro models such as 2,2 diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, 2,2'-azino-bis(3-ethylbenzthiazoline-6)-sulfonic acid (ABTS(+)) radical scavenging activity, ferric reducing antioxidant power (FRAP) and ferrous ion (Fe(2+)) chelating ability. Enzymatic hydrolysis significantly increased protein solubility of the hydrolysates to above 62 % over a wide pH range (2-12). Carp roe hydrolysates exhibited good foaming and emulsification properties. The results suggest that bioactive carp roe protein hydrolysates (CRPHs) with good functional properties could be useful in health food/nutraceutical/pharmaceutical industry for various applications. PMID:26344996

  1. Effects of enzymatic hydrolysis on conformational and functional properties of chickpea protein isolate.

    PubMed

    Mokni Ghribi, Abir; Maklouf Gafsi, Ines; Sila, Assaâd; Blecker, Christophe; Danthine, Sabine; Attia, Hamadi; Bougatef, Ali; Besbes, Souhail

    2015-11-15

    The impact of enzymatic hydrolysis by Alcalase on the conformational and functional properties of chickpea protein isolate (CPI) was investigated. The physicochemical, interfacial tension and surface characteristics of CPI and their hydrolysates (CPH) according to the degree of hydrolysis (DH) were also determined. These parameters were then related to the changes in the emulsification activity (EAI) and stability (ESI). The enzymatic hydrolysis was found to improve protein recovery and solubility, leading to a reduction in the molecular weight bands with a concomitant increase in the intensity and appearance of protein bands having apparent molecular mass below 20 kDa. The interfacial tension decreased from ∼ 66.5 mN m(-1) for CPI to ∼ 59.1 m Nm(-1) for CPH. A similar trend was observed for the surface charge which declined from -27.55 mV to -16.4 mV for the CPI and CPH, respectively. These changes were found to have a detrimental effect on the EAI and ESI values. PMID:25977033

  2. Enzymatic Conversion of CO2 to Bicarbonate in Functionalized Mesoporous Silica

    SciTech Connect

    Yu, Yuehua; Chen, Baowei; Qi, Wen N.; Li, Xiaolin; Shin, Yongsoon; Lei, Chenghong; Liu, Jun

    2012-05-01

    We report here that carbonic anhydrase (CA), the fastest enzyme that can covert carbon dioxide to bicarbonate, can be spontaneously entrapped in functionalized mesoporous silica (FMS) with super-high loading density (up to 0.5 mg of protein/mg of FMS) due to the dominant electrostatic interaction. The binding of CA to HOOC-FMS can result in the protein’s conformational change comparing to the enzyme free in solution, but can be overcome with increased protein loading density. The higher the protein loading density, the less conformational change, hence the higher enzymatic activity and the higher enzyme immobilization efficiency. The electrostatically bound CA can be released by changing pH. The released enzyme still displayed the native conformational structure and the same high enzymatic activity as that prior to the enzyme entrapment. This work opens up a new approach converting carbon dioxide to biocarbonate in a biomimetic nanoconfiguration that can be integrated with the other part of biosynthesis process for the assimilation of carbon dioxide.

  3. Linking Microbial Enzymatic Activities and Functional Diversity of Soil around Earthworm Burrows and Casts

    PubMed Central

    Lipiec, Jerzy; Frąc, Magdalena; Brzezińska, Małgorzata; Turski, Marcin; Oszust, Karolina

    2016-01-01

    The aim of this work was to evaluate the effect of earthworms (Lumbricidae) on the enzymatic activity and microbial functional diversity in the burrow system [burrow wall (BW) 0–3 mm, transitional zone (TZ) 3–7 mm, bulk soil (BS) > 20 mm from the BW] and cast aggregates of a loess soil under a pear orchard. The dehydrogenase, β-glucosidase, protease, alkaline phosphomonoesterase, and acid phosphomonoesterase enzymes were assessed using standard methods. The functional diversity (catabolic potential) was assessed using the Average Well Color Development and Richness Index following the community level physiological profiling from Biolog Eco Plates. All measurements were done using soil from each compartment immediately after in situ sampling in spring. The enzymatic activites including dehydrogenase, protease, β-glucosidase and alkaline phosphomonoesterase were appreciably greater in the BW or casts than in BS and TZ. Conversely, acid phosphomonoesterase had the largest value in the BS. Average Well Color Development in both the TZ and the BS (0.98–0.94 A590 nm) were more than eight times higher than in the BWs and casts. The lowest richness index in the BS (15 utilized substrates) increased by 86–113% in all the other compartments. The PC1 in principal component analysis mainly differentiated the BWs and the TZ. Utilization of all substrate categories was the lowest in the BS. The PC2 differentiated the casts from the other compartments. The enhanced activity of a majority of the enzymes and increased microbial functional diversity in most earthworm-influenced compartments make the soils less vulnerable to degradation and thus increases the stability of ecologically relevant processes in the orchard ecosystem. PMID:27625645

  4. Linking Microbial Enzymatic Activities and Functional Diversity of Soil around Earthworm Burrows and Casts.

    PubMed

    Lipiec, Jerzy; Frąc, Magdalena; Brzezińska, Małgorzata; Turski, Marcin; Oszust, Karolina

    2016-01-01

    The aim of this work was to evaluate the effect of earthworms (Lumbricidae) on the enzymatic activity and microbial functional diversity in the burrow system [burrow wall (BW) 0-3 mm, transitional zone (TZ) 3-7 mm, bulk soil (BS) > 20 mm from the BW] and cast aggregates of a loess soil under a pear orchard. The dehydrogenase, β-glucosidase, protease, alkaline phosphomonoesterase, and acid phosphomonoesterase enzymes were assessed using standard methods. The functional diversity (catabolic potential) was assessed using the Average Well Color Development and Richness Index following the community level physiological profiling from Biolog Eco Plates. All measurements were done using soil from each compartment immediately after in situ sampling in spring. The enzymatic activites including dehydrogenase, protease, β-glucosidase and alkaline phosphomonoesterase were appreciably greater in the BW or casts than in BS and TZ. Conversely, acid phosphomonoesterase had the largest value in the BS. Average Well Color Development in both the TZ and the BS (0.98-0.94 A590 nm) were more than eight times higher than in the BWs and casts. The lowest richness index in the BS (15 utilized substrates) increased by 86-113% in all the other compartments. The PC1 in principal component analysis mainly differentiated the BWs and the TZ. Utilization of all substrate categories was the lowest in the BS. The PC2 differentiated the casts from the other compartments. The enhanced activity of a majority of the enzymes and increased microbial functional diversity in most earthworm-influenced compartments make the soils less vulnerable to degradation and thus increases the stability of ecologically relevant processes in the orchard ecosystem. PMID:27625645

  5. How the rice weevil breaks down the pectin network: Enzymatic synergism and sub-functionalization.

    PubMed

    Kirsch, Roy; Heckel, David G; Pauchet, Yannick

    2016-04-01

    Pectin is the most complex polysaccharide in nature and highly abundant in plant cell walls and middle lamellae, where it functions in plant growth and development. Phytopathogens utilize plant pectin as an energy source through enzyme-mediated degradation. These pectolytic enzymes include polygalacturonases (PGs) of the GH28 family and pectin methylesterases (PMEs) of the CE8 family. Recently, PGs were also identified in herbivorous insects of the distantly related plant bug, stick insect and Phytophaga beetle lineages. Unlike all other insects, weevils possess PMEs in addition to PGs. To investigate pectin digestion in insects and the role of PMEs in weevils, all PME and PG family members of the rice weevil Sitophilus oryzae were heterologously expressed and functionally characterized. Enzymatically active and inactive PG and PME family members were identified. The loss of activity can be explained by a lack of substrate binding correlating with substitutions of functionally important amino acid residues. We found subfunctionalization in both enzyme families, supported by expression pattern and substrate specificities as well as evidence for synergistic pectin breakdown. Our data suggest that the rice weevil might be able to use pectin as an energy source, and illustrates the potential of both PG and PME enzyme families to functionally diversify after horizontal gene transfer. PMID:26899322

  6. Generation of Functional RNAs from Inactive Oligonucleotide Complexes by Non-enzymatic Primer Extension

    PubMed Central

    2014-01-01

    The earliest genomic RNAs had to be short enough for efficient replication, while simultaneously serving as unfolded templates and effective ribozymes. A partial solution to this paradox may lie in the fact that many functional RNAs can self-assemble from multiple fragments. Therefore, in early evolution, genomic RNA fragments could have been significantly shorter than unimolecular functional RNAs. Here, we show that unstable, nonfunctional complexes assembled from even shorter 3′-truncated oligonucleotides can be stabilized and gain function via non-enzymatic primer extension. Such short RNAs could act as good templates due to their minimal length and complex-forming capacity, while their minimal length would facilitate replication by relatively inefficient polymerization reactions. These RNAs could also assemble into nascent functional RNAs and undergo conversion to catalytically active forms, by the same polymerization chemistry used for replication that generated the original short RNAs. Such phenomena could have substantially relaxed requirements for copying efficiency in early nonenzymatic replication systems. PMID:25521912

  7. Association of fecal microbial diversity and taxonomy with selected enzymatic functions.

    PubMed

    Flores, Roberto; Shi, Jianxin; Gail, Mitchell H; Gajer, Pawel; Ravel, Jacques; Goedert, James J

    2012-01-01

    Few microbial functions have been compared to a comprehensive survey of the human fecal microbiome. We evaluated determinants of fecal microbial β-glucuronidase and β-glucosidase activities, focusing especially on associations with microbial alpha and beta diversity and taxonomy. We enrolled 51 healthy volunteers (26 female, mean age 39) who provided questionnaire data and multiple aliquots of a stool, from which proteins were extracted to quantify β-glucuronidase and β-glucosidase activities, and DNA was extracted to amplify and pyrosequence 16S rRNA gene sequences to classify and quantify microbiome diversity and taxonomy. Fecal β-glucuronidase was elevated with weight loss of at least 5 lb. (P = 0.03), whereas β-glucosidase was marginally reduced in the four vegetarians (P = 0.06). Both enzymes were correlated directly with microbiome richness and alpha diversity measures, directly with the abundance of four Firmicutes Clostridia genera, and inversely with the abundance of two other genera (Firmicutes Lactobacillales Streptococcus and Bacteroidetes Rikenellaceae Alistipes) (all P = 0.05-0.0001). Beta diversity reflected the taxonomic associations. These observations suggest that these enzymatic functions are performed by particular taxa and that diversity indices may serve as surrogates of bacterial functions. Independent validation and deeper understanding of these associations are needed, particularly to characterize functions and pathways that may be amenable to manipulation. PMID:22761886

  8. Enzymatic Characterization and In Vivo Function of Five Terminal Oxidases in Pseudomonas aeruginosa

    PubMed Central

    Kawakami, Takuro; Osamura, Tatsuya; Hirai, Takehiro; Sakai, Yoshiaki; Ishii, Masaharu

    2014-01-01

    The ubiquitous opportunistic pathogen Pseudomonas aeruginosa has five aerobic terminal oxidases: bo3-type quinol oxidase (Cyo), cyanide-insensitive oxidase (CIO), aa3-type cytochrome c oxidase (aa3), and two cbb3-type cytochrome c oxidases (cbb3-1 and cbb3-2). These terminal oxidases are differentially regulated under various growth conditions and are thought to contribute to the survival of this microorganism in a wide variety of environmental niches. Here, we constructed multiple mutant strains of P. aeruginosa that express only one aerobic terminal oxidase to investigate the enzymatic characteristics and in vivo function of each enzyme. The Km values of Cyo, CIO, and aa3 for oxygen were similar and were 1 order of magnitude higher than those of cbb3-1 and cbb3-2, indicating that Cyo, CIO, and aa3 are low-affinity enzymes and that cbb3-1 and cbb3-2 are high-affinity enzymes. Although cbb3-1 and cbb3-2 exhibited different expression patterns in response to oxygen concentration, they had similar Km values for oxygen. Both cbb3-1 and cbb3-2 utilized cytochrome c4 as the main electron donor under normal growth conditions. The electron transport chains terminated by cbb3-1 and cbb3-2 generate a proton gradient across the cell membrane with similar efficiencies. The electron transport chain of aa3 had the highest proton translocation efficiency, whereas that of CIO had the lowest efficiency. The enzymatic properties of the terminal oxidases reported here are partially in agreement with their regulatory patterns and may explain the environmental adaptability and versatility of P. aeruginosa. PMID:25182500

  9. Protein engineering of a bacterial N-acyl-d-glucosamine 2-epimerase for improved stability under process conditions.

    PubMed

    Klermund, Ludwig; Riederer, Amelie; Hunger, Annique; Castiglione, Kathrin

    2016-06-01

    Enzymatic cascade reactions, i.e. the combination of several enzyme reactions in one pot without isolation of intermediates, have great potential for the establishment of sustainable chemical processes. However, many cascade reactions suffer from cross-inhibitions and enzyme inactivation by components of the reaction system. This study focuses on the two-step enzymatic synthesis of N-acetylneuraminic acid (Neu5Ac) using an N-acyl-d-glucosamine 2-epimerase from Anabaena variabilis ATCC 29413 (AvaAGE) in combination with an N-acetylneuraminate lyase (NAL) from Escherichia coli. AvaAGE epimerizes N-acetyl-d-glucosamine (GlcNAc) to N-acetyl-d-mannosamine (ManNAc), which then reacts with pyruvate in a NAL-catalyzed aldol condensation to form Neu5Ac. However, AvaAGE is inactivated by high pyruvate concentrations, which are used to push the NAL reaction toward the product side. A biphasic inactivation was observed in the presence of 50-800mM pyruvate resulting in activity losses of the AvaAGE of up to 60% within the first hour. Site-directed mutagenesis revealed that pyruvate modifies one of the four lysine residues in the ATP-binding site of AvaAGE. Because ATP is an allosteric activator of the epimerase and the binding of the nucleotide is crucial for its catalytic properties, saturation mutagenesis at position K160 was performed to identify the most compatible amino acid exchanges. The best variants, K160I, K160N and K160L, showed no inactivation by pyruvate, but significantly impaired kinetic parameters. For example, depending on the mutant, the turnover number kcat was reduced by 51-68% compared with the wild-type enzyme. A mechanistic model of the Neu5Ac synthesis was established, which can be used to select the AvaAGE variant that is most favorable for a given process condition. The results show that mechanistic models can greatly facilitate the choice of the right enzyme for an enzymatic cascade reaction with multiple cross-inhibitions and inactivation phenomena

  10. Characterization of functionalized multiwalled carbon nanotubes for use in an enzymatic sensor.

    PubMed

    Guadarrama-Fernández, Leonor; Chanona-Pérez, Jorge; Manzo-Robledo, Arturo; Calderón-Domínguez, Georgina; Martínez-Rivas, Adrián; Ortiz-López, Jaime; Vargas-García, Jorge Roberto

    2014-10-01

    Carbon nanotubes (CNT) have proven to be materials with great potential for the construction of biosensors. Development of fast, simple, and low cost biosensors to follow reactions in bioprocesses, or to detect food contaminants such as toxins, chemical compounds, and microorganisms, is presently an important research topic. This report includes microscopy and spectroscopy to characterize raw and chemically modified multiwall carbon nanotubes (MWCNTs) synthesized by chemical vapor deposition with the intention of using them as the active transducer in bioprocessing sensors. MWCNT were simultaneously purified and functionalized by an acid mixture involving HNO3-H2SO4 and amyloglucosidase attached onto the chemically modified MWCNT surface. A 49.0% decrease in its enzymatic activity was observed. Raw, purified, and enzyme-modified MWCNTs were analyzed by scanning and transmission electron microscopy and Raman and X-ray photoelectron spectroscopy. These studies confirmed purification and functionalization of the CNTs. Finally, cyclic voltammetry electrochemistry was used for electrical characterization of CNTs, which showed promising results that can be useful for construction of electrochemical biosensors applied to biological areas. PMID:25156941

  11. Optical Detection of Enzymatic Activity and Inhibitors on Non-Covalently Functionalized Fluorescent Graphene Oxide.

    PubMed

    Kang, Tae Woog; Jeon, Su-Ji; Kim, Hye-In; Park, Jung Hyun; Yim, DaBin; Lee, Hye-Rim; Ju, Jong-Min; Kim, Man-Jin; Kim, Jong-Ho

    2016-05-24

    It has been of great interest to measure the activity of acetylcholinesterase (AChE) and its inhibitor, as AChE is known to accelerate the aggregation of the amyloid beta peptides that underlie Alzheimer's disease. Herein, we report the development of graphene oxide (GO) fluorescence-based biosensors for the detection of AChE activity and AChE inhibitors. To this end, GO was non-covalently functionalized with phenoxy-modified dextran (PhO-dex-GO) through hydrophobic interaction; the resulting GO showed excellent colloidal stability and intense fluorescence in various aqueous solutions as compared to pristine GO and the GO covalently functionalized with dextran. The fluorescence of PhO-dex-GO remarkably increased as AChE catalyzed the hydrolysis of acetylthiocholine (ATCh) to give thiocholine and acetic acid. It was found that the turn-on fluorescence response of PhO-dex-GO to AChE activity was induced by protonation of carboxyl groups on it from the product of the enzymatic hydrolysis reaction, acetic acid. On the basis of its turn-on fluorescence response, PhO-dex-GO was able to report kinetic and thermodynamic parameters involving a maximum velocity, a Michaelis constant, and an inhibition dissociation constant for AChE activity and inhibition. These parameters enable us to determine the activity of AChE and the efficiency of the inhibitor. PMID:27136042

  12. Degradation kinetics and structural characteristics of pectin under simultaneous sonochemical-enzymatic functions.

    PubMed

    Ma, Xiaobin; Wang, Wenjun; Wang, Danli; Ding, Tian; Ye, Xingqian; Liu, Donghong

    2016-12-10

    This study investigated the degradation kinetics and structural properties of pectin with combining ultrasound and pectinase treatment. Ultrasound at an intensity of 4.5WmL(-1) and a time of 10min significantly enhanced the enzymatic degradation of pectin weight-average molecular weight (Mw). The degradation kinetics model of pectin followed 1/Mwt-1/Mw0=kt, suggesting the randomness of the degradation process. Synergistic effects of ultrasound and pectinase were observed at 20-60°C and were more effective at lower temperatures. Furthermore, the degree of methoxylation (DM) of sonoenzymolysis pectin significantly decreased whereas the degree of acetylation (DAc) remained unchanged compared to the original and enzymolysis pectin. Simultaneous functions of ultrasound and pectinase caused severe decomposition in pectin homogalacturonan (HG) regions without altering the monosaccharides types, configurations and glycoside linkages of the pectin samples. The complex polymeric structures of pectin transformed into smaller units with simpler branches and shorter chains after sonoenzymolysis reactions. PMID:27577908

  13. Enzymatic and nonenzymatic functions of viral RNA-dependent RNA polymerases within oligomeric arrays

    PubMed Central

    Spagnolo, Jeannie F.; Rossignol, Evan; Bullitt, Esther; Kirkegaard, Karla

    2010-01-01

    Few antivirals are effective against positive-strand RNA viruses, primarily because the high error rate during replication of these viruses leads to the rapid development of drug resistance. One of the favored current targets for the development of antiviral compounds is the active site of viral RNA-dependent RNA polymerases. However, like many subcellular processes, replication of the genomes of all positive-strand RNA viruses occurs in highly oligomeric complexes on the cytosolic surfaces of the intracellular membranes of infected host cells. In this study, catalytically inactive polymerases were shown to participate productively in functional oligomer formation and catalysis, as assayed by RNA template elongation. Direct protein transduction to introduce either active or inactive polymerases into cells infected with mutant virus confirmed the structural role for polymerase molecules during infection. Therefore, we suggest that targeting the active sites of polymerase molecules is not likely to be the best antiviral strategy, as inactivated polymerases do not inhibit replication of other viruses in the same cell and can, in fact, be useful in RNA replication complexes. On the other hand, polymerases that could not participate in functional RNA replication complexes were those that contained mutations in the amino terminus, leading to altered contacts in the folded polymerase and mutations in a known polymerase–polymerase interaction in the two-dimensional protein lattice. Thus, the functional nature of multimeric arrays of RNA-dependent RNA polymerase supplies a novel target for antiviral compounds and provides a new appreciation for enzymatic catalysis on membranous surfaces within cells. PMID:20051491

  14. Evolution of New Enzymatic Function by Structural Modulation of Cysteine Reactivity in Pseudomonas fluorescens Isocyanide Hydratase

    SciTech Connect

    Lakshminarasimhan, Mahadevan; Madzelan, Peter; Nan, Ruth; Milkovic, Nicole M.; Wilson, Mark A.

    2010-09-13

    Isocyanide (formerly isonitrile) hydratase (EC 4.2.1.103) is an enzyme of the DJ-1 superfamily that hydrates isocyanides to yield the corresponding N-formamide. In order to understand the structural basis for isocyanide hydratase (ICH) catalysis, we determined the crystal structures of wild-type and several site-directed mutants of Pseudomonas fluorescens ICH at resolutions ranging from 1.0 to 1.9 {angstrom}. We also developed a simple UV-visible spectrophotometric assay for ICH activity using 2-naphthyl isocyanide as a substrate. ICH contains a highly conserved cysteine residue (Cys101) that is required for catalysis and interacts with Asp17, Thr102, and an ordered water molecule in the active site. Asp17 has carboxylic acid bond lengths that are consistent with protonation, and we propose that it activates the ordered water molecule to hydrate organic isocyanides. In contrast to Cys101 and Asp17, Thr102 is tolerant of mutagenesis, and the T102V mutation results in a substrate-inhibited enzyme. Although ICH is similar to human DJ-1 (1.6 {angstrom} C-{alpha} root mean square deviation), structural differences in the vicinity of Cys101 disfavor the facile oxidation of this residue that is functionally important in human DJ-1 but would be detrimental to ICH activity. The ICH active site region also exhibits surprising conformational plasticity and samples two distinct conformations in the crystal. ICH represents a previously uncharacterized clade of the DJ-1 superfamily that possesses a novel enzymatic activity, demonstrating that the DJ-1 core fold can evolve diverse functions by subtle modulation of the environment of a conserved, reactive cysteine residue.

  15. Crystal Structure of Human Senescence Marker Protein 30: Insights Linking Structural, Enzymatic, and Physiological Functions

    SciTech Connect

    Chakraborti, Subhendu; Bahnson, Brian J.

    2010-05-25

    Human senescence marker protein 30 (SMP30), which functions enzymatically as a lactonase, hydrolyzes various carbohydrate lactones. The penultimate step in vitamin-C biosynthesis is catalyzed by this enzyme in nonprimate mammals. It has also been implicated as an organophosphate hydrolase, with the ability to hydrolyze diisopropyl phosphofluoridate and other nerve agents. SMP30 was originally identified as an aging marker protein, whose expression decreased androgen independently in aging cells. SMP30 is also referred to as regucalcin and has been suggested to have functions in calcium homeostasis. The crystal structure of the human enzyme has been solved from X-ray diffraction data collected to a resolution of 1.4 {angstrom}. The protein has a 6-bladed {beta}-propeller fold, and it contains a single metal ion. Crystal structures have been solved with the metal site bound with either a Ca{sup 2+} or a Zn{sup 2+} atom. The catalytic role of the metal ion has been confirmed by mutagenesis of the metal coordinating residues. Kinetic studies using the substrate gluconolactone showed a k{sub cat} preference of divalent cations in the order Zn{sup 2+} > Mn{sup 2+} > Ca{sup 2+} > Mg{sup 2+}. Notably, the Ca{sup 2+} had a significantly higher value of K{sub d} compared to those of the other metal ions tested (566, 82, 7, and 0.6 {micro}m for Ca{sup 2+}, Mg{sup 2+}, Zn{sup 2+}, and Mn{sup 2+}, respectively), suggesting that the Ca{sup 2+}-bound form may be physiologically relevant for stressed cells with an elevated free calcium level.

  16. Structure of d-tagatose 3-epimerase-like protein from Methanocaldococcus jannaschii

    PubMed Central

    Uechi, Keiko; Takata, Goro; Yoneda, Kazunari; Ohshima, Toshihisa; Sakuraba, Haruhiko

    2014-01-01

    The crystal structure of a d-tagatose 3-epimerase-like protein (MJ1311p) encoded by a hypothetical open reading frame, MJ1311, in the genome of the hyperthermophilic archaeon Methanocaldococcus jannaschii was determined at a resolution of 2.64 Å. The asymmetric unit contained two homologous subunits, and the dimer was generated by twofold symmetry. The overall fold of the subunit proved to be similar to those of the d-tagatose 3-epimerase from Pseudomonas cichorii and the d-psicose 3-epimerases from Agrobacterium tumefaciens and Clostridium cellulolyticum. However, the situation at the subunit–subunit interface differed substantially from that in d-tagatose 3-epimerase family enzymes. In MJ1311p, Glu125, Leu126 and Trp127 from one subunit were found to be located over the metal-ion-binding site of the other subunit and appeared to contribute to the active site, narrowing the substrate-binding cleft. Moreover, the nine residues comprising a trinuclear zinc centre in endonuclease IV were found to be strictly conserved in MJ1311p, although a distinct groove involved in DNA binding was not present. These findings indicate that the active-site architecture of MJ1311p is quite unique and is substantially different from those of d-tagatose 3-epimerase family enzymes and endonuclease IV. PMID:25005083

  17. Enzymatic and Functional Analysis of a Protein Phosphatase, Pph3, from Myxococcus xanthus ▿

    PubMed Central

    Kimura, Yoshio; Mori, Yumi; Ina, Youhei; Takegawa, Kaoru

    2011-01-01

    A protein phosphatase, designated Pph3, from Myxococcus xanthus showed the enzymatic characteristics of PP2C-type serine/threonine protein phosphatases, which are metal ion-dependent, okadaic acid-insensitive protein phosphatases. The pph3 mutant under starvation conditions formed immature fruiting bodies and reduced sporulation. PMID:21398555

  18. Heavy metal concentrations and enzymatic activities in the functional zone sediments of Haizhou Bay, Lianyungang, Jiangsu, China.

    PubMed

    Li, Yu; Liu, Fu-cheng

    2015-11-01

    Surface sediments were collected at 31 sites covering five functional zones of Haizhou Bay, Lianyungang, Jiangsu, China. Heavy metal concentrations and enzymatic activity of phosphatase and urease were determined on a dry-weight basis of sediments. Metal concentrations in sediments were comparable to the Chinese National Standard of Marine Sediment Quality and were as follows: Cu, 8.60-55.8 mg kg(-1); Zn, 107-384 mg kg(-1); Pb, 33.6-200 mg kg(-1); Cd, 0.24-2.57 mg kg(-1); Cr, 30.3-92.1 mg kg(-1); As, 12.9-110 mg kg(-1); Ni, 15.8-49.6 mg kg(-1); Mn, 379-1272 mg kg(-1); and Fe, 13,790-38,240 mg kg(-1). A geoaccumulation index (I geo) was calculated to help researchers understand the status of pollutants in the sediments. I geo showed that Cd and As contamination existed in the study area. The mobility of the metals and the relationship between heavy metal concentrations of chemical fractions and enzymatic activities were also investigated. Results showed that Cd and Mn had higher mobility than other metals, and enzymatic activities may play an important role in controlling the bioavailability and transformation trend of heavy metals from one fraction to another in sediments. PMID:26431704

  19. Highly efficient production of rare sugars D-psicose and L-tagatose by two engineered D-tagatose epimerases.

    PubMed

    Bosshart, Andreas; Wagner, Nina; Lei, Lei; Panke, Sven; Bechtold, Matthias

    2016-02-01

    Rare sugars are monosaccharides that do not occur in nature in large amounts. However, many of them demonstrate high potential as low-calorie sweetener, chiral building blocks or active pharmaceutical ingredients. Their production by enzymatic means from broadly abundant epimers is an attractive alternative to synthesis by traditional organic chemical means, but often suffers from low space-time yields and high enzyme costs due to rapid enzyme degradation. Here we describe the detailed characterization of two variants of d-tagatose epimerase under operational conditions that were engineered for high stability and high catalytic activity towards the epimerization of d-fructose to d-psicose and l-sorbose to l-tagatose, respectively. A variant optimized for the production of d-psicose showed a very high total turnover number (TTN) of up to 10(8) catalytic events over a catalyst's lifetime, determined under operational conditions at high temperatures in an enzyme-membrane reactor (EMR). Maximum space-time yields as high as 10.6 kg L(-1) d(-1) were obtained with a small laboratory-scale EMR, indicating excellent performance. A variant optimized for the production of l-tagatose performed less stable in the same setting, but still showed a very good TTN of 5.8 × 10(5) and space-time yields of up to 478 g L(-1) d(-1) . Together, these results confirm that large-scale enzymatic access to rare sugars is feasible. PMID:25615556

  20. The structural basis of substrate promiscuity in UDP-hexose 4-epimerase from the hyperthermophilic Eubacterium Thermotoga maritima.

    PubMed

    Shin, Sun-Mi; Choi, Jin Myung; di Luccio, Eric; Lee, Yong-Jik; Lee, Sang-Jae; Lee, Sang Jun; Lee, Sung Haeng; Lee, Dong-Woo

    2015-11-01

    UDP-galactose 4-epimerase (GalE) catalyzes the interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal), which is a pivotal step in the Leloir pathway for d-galactose metabolism. Although GalE is widely distributed in prokaryotes and eukaryotes, little information is available regarding hyperthermophilic GalE. We overexpressed the TM0509 gene, encoding a putative GalE from Thermotoga maritima (TMGalE), in Escherichia coli and characterized the encoded protein. To further investigate the molecular basis of this enzyme's catalytic function, we determined the crystal structures of TMGalE and TMGalE bound to UDP-Glc at resolutions of 1.9 Å and 2.0 Å, respectively. The enzyme was determined to be a homodimer with a molecular mass of 70 kDa. The enzyme could reversibly catalyze the epimerization of UDP-GalNAc/UDP-GlcNAc as well as UDP-Gal/UDP-Glc at elevated temperatures, with an apparent optimal temperature and pH of 80 °C and 7.0, respectively. Our data showed that TM0509 is a UDP-galactosugar 4-epimerase involved in d-galactose metabolism; consequently, this study provides the first detailed characterization of a hyperthermophilic GalE. Moreover, the promiscuous substrate specificity of TMGalE, which is more similar to human GalE than E. coli GalE, supports the notion that TMGalE might exhibit the earliest form of sugar-epimerizing enzymes in the evolution of galactose metabolism. PMID:26344854

  1. Towards a better understanding of the substrate specificity of the UDP-N-acetylglucosamine C4 epimerase WbpP

    PubMed Central

    2005-01-01

    WbpP is the only genuine UDP-GlcNAc (UDP-N-acetylglucosamine) C4 epimerase for which both biochemical and structural data are available. This represents a golden opportunity to elucidate the molecular basis for its specificity for N-acetylated substrates. Based on the comparison of the substrate binding site of WbpP with that of other C4 epimerases that convert preferentially non-acetylated substrates, or that are able to convert both acetylated and non-acetylated substrates equally well, specific residues of WbpP were mutated, and the substrate specificity of the mutants was determined by direct biochemical assays and kinetic analyses. Most of the mutations tested were anticipated to trigger a significant switch in substrate specificity, mostly towards a preference for non-acetylated substrates. However, only one of the mutations (A209H) had the expected effect, and most others resulted in enhanced specificity of WbpP for N-acetylated substrates (Q201E, G102K, Q201E/G102K, A209N and S143A). One mutation (S144K) totally abolished enzyme activity. These data indicate that, although all residues targeted in the present study turned out to be important for catalysis, determinants of substrate specificity are not confined to the substrate-binding pocket and that longer range interactions are essential in allowing proper positioning of various ligands in the binding pocket. Hence prediction or engineering of substrate specificity solely based on sequence analysis, or even on modelling of the binding pocket, might lead to incorrect functional assignments. PMID:15752069

  2. Discovery of Novel Putative Inhibitors of UDP-GlcNAc 2-Epimerase as Potent Antibacterial Agents.

    PubMed

    Xu, Yong; Brenning, Benjamin; Clifford, Adrianne; Vollmer, David; Bearss, Jared; Jones, Carissa; McCarthy, Virgil; Shi, Chongtie; Wolfe, Bradley; Aavula, Bhasker; Warner, Steve; Bearss, David J; McCullar, Michael V; Schuch, Raymond; Pelzek, Adam; Bhaskaran, Shyam S; Stebbins, C Erec; Goldberg, Allan R; Fischetti, Vincent A; Vankayalapati, Hariprasad

    2013-12-12

    We present the discovery and optimization of a novel series of inhibitors of bacterial UDP-N-acetylglucosamine 2-epimerase (called 2-epimerase in this paper). Starting from virtual screening hits, the activity of various inhibitory molecules was optimized using a combination of structure-based and rational design approaches. We successfully designed and identified a 2-epimerase inhibitor (compound 12-ES-Na, that we named Epimerox) which blocked the growth of methicillin-resistant Staphylococcus aureus (MRSA) at 3.9 μM MIC (minimum inhibitory concentration) and showed potent broad-range activity against all Gram-positive bacteria that were tested. Additionally a microplate coupled assay was performed to further confirm that the 2-epimerase inhibition of Epimerox was through a target-specific mechanism. Furthermore, Epimerox demonstrated in vivo efficacy and had a pharmacokinetic profile that is consonant with it being developed into a promising new antibiotic agent for treatment of infections caused by Gram-positive bacteria. PMID:24443700

  3. Thermostability enhancement of cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus by site-directed mutagenesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cellobiose 2-epimerase from the thermophile Caldicellulosiruptor saccharolyticus (CsCE) catalyzes the isomerization of lactose into lactulose, a non-digestible disaccharide widely used in food and pharmaceutical industries. Semi-rational approaches were applied to enhance the thermostability of CsCE...

  4. GneZ, a UDP-GlcNAc 2-Epimerase, Is Required for S-Layer Assembly and Vegetative Growth of Bacillus anthracis

    PubMed Central

    Wang, Ya-Ting; Missiakas, Dominique

    2014-01-01

    Bacillus anthracis, the causative agent of anthrax, forms an S-layer atop its peptidoglycan envelope and displays S-layer proteins and Bacillus S-layer-associated (BSL) proteins with specific functions to support cell separation of vegetative bacilli and growth in infected mammalian hosts. S-layer and BSL proteins bind via the S-layer homology (SLH) domain to the pyruvylated secondary cell wall polysaccharide (SCWP) with the repeat structure [→4)-β-ManNAc-(1→4)-β-GlcNAc-(1→6)-α-GlcNAc-(1→]n, where α-GlcNAc and β-GlcNAc are substituted with two and one galactosyl residues, respectively. B. anthracis gneY (BAS5048) and gneZ (BAS5117) encode nearly identical UDP-GlcNAc 2-epimerase enzymes that catalyze the reversible conversion of UDP-GlcNAc and UDP-ManNAc. UDP-GlcNAc 2-epimerase enzymes have been shown to be required for the attachment of the phage lysin PlyG with the bacterial envelope and for bacterial growth. Here, we asked whether gneY and gneZ are required for the synthesis of the pyruvylated SCWP and for S-layer assembly. We show that gneZ, but not gneY, is required for B. anthracis vegetative growth, rod cell shape, S-layer assembly, and synthesis of pyruvylated SCWP. Nevertheless, inducible expression of gneY alleviated all the defects associated with the gneZ mutant. In contrast to vegetative growth, neither germination of B. anthracis spores nor the formation of spores in mother cells required UDP-GlcNAc 2-epimerase activity. PMID:24914184

  5. Three-dimensional Structure and Enzymatic Function of Proapoptotic Human p53-inducible Quinone Oxidoreductase PIG3*

    PubMed Central

    Porté, Sergio; Valencia, Eva; Yakovtseva, Evgenia A.; Borràs, Emma; Shafqat, Naeem; Debreczeny, Judit É.; Pike, Ashley C. W.; Oppermann, Udo; Farrés, Jaume; Fita, Ignacio; Parés, Xavier

    2009-01-01

    Tumor suppressor p53 regulates the expression of p53-induced genes (PIG) that trigger apoptosis. PIG3 or TP53I3 is the only known member of the medium chain dehydrogenase/reductase superfamily induced by p53 and is used as a proapoptotic marker. Although the participation of PIG3 in the apoptotic pathway is proven, the protein and its mechanism of action were never characterized. We analyzed human PIG3 enzymatic function and found NADPH-dependent reductase activity with ortho-quinones, which is consistent with the classification of PIG3 in the quinone oxidoreductase family. However, the activity is much lower than that of ζ-crystallin, a better known quinone oxidoreductase. In addition, we report the crystallographic structure of PIG3, which allowed the identification of substrate- and cofactor-binding sites, with residues fully conserved from bacteria to human. Tyr-59 in ζ-crystallin (Tyr-51 in PIG3) was suggested to participate in the catalysis of quinone reduction. However, kinetics of Tyr/Phe and Tyr/Ala mutants of both enzymes demonstrated that the active site Tyr is not catalytic but may participate in substrate binding, consistent with a mechanism based on propinquity effects. It has been proposed that PIG3 contribution to apoptosis would be through oxidative stress generation. We found that in vitro activity and in vivo overexpression of PIG3 accumulate reactive oxygen species. Accordingly, an inactive PIG3 mutant (S151V) did not produce reactive oxygen species in cells, indicating that enzymatically active protein is necessary for this function. This supports that PIG3 action is through oxidative stress produced by its enzymatic activity and provides essential knowledge for eventual control of apoptosis. PMID:19349281

  6. Restoration of Gibberellin Production in Fusarium proliferatum by Functional Complementation of Enzymatic Blocks

    PubMed Central

    Malonek, S.; Rojas, M. C.; Hedden, P.; Hopkins, P.; Tudzynski, B.

    2005-01-01

    Nine biological species, or mating populations (MPs), denoted by letters A to I, and at least 29 anamorphic Fusarium species have been identified within the Gibberella fujikuroi species complex. Members of this species complex are the only species of the genus Fusarium that contain the gibberellin (GA) biosynthetic gene cluster or at least parts of it. However, the ability of fusaria to produce GAs is so far restricted to Fusarium fujikuroi, although at least six other MPs contain all the genes of the GA biosynthetic gene cluster. Members of Fusarium proliferatum, the closest related species, have lost the ability to produce GAs as a result of the accumulation of several mutations in the coding and 5′ noncoding regions of genes P450-4 and P450-1, both encoding cytochrome P450 monooxygenases, resulting in metabolic blocks at the early stages of GA biosynthesis. In this study, we have determined additional enzymatic blocks at the first specific steps in the GA biosynthesis pathway of F. proliferatum: the synthesis of geranylgeranyl diphosphate and the synthesis of ent-kaurene. Complementation of these enzymatic blocks by transferring the corresponding genes from GA-producing F. fujikuroi to F. proliferatum resulted in the restoration of GA production. We discuss the reasons for Fusarium species outside the G. fujikuroi species complex having no GA biosynthetic genes, whereas species distantly related to Fusarium, e.g., Sphaceloma spp. and Phaeosphaeria spp., produce GAs. PMID:16204516

  7. Antioxidant activities and functional properties of enzymatic protein hydrolysates from defatted Camellia oleifera seed cake.

    PubMed

    Li, Xu; Deng, Junlin; Shen, Shian; Li, Tian; Yuan, Ming; Yang, Ruiwu; Ding, Chunbang

    2015-09-01

    Seed cake protein (SCP) from Camellia oleifera was hydrolyzed by five commercial proteases (Flavorzyme, Trypsin, Neutrase, Papain, Alcalase). Amino acid composition, molecular weight distribution, antioxidant activity and functional property of the seed cake protein hydrolysates (SCPH) were investigated. Enzymatic hydrolysis improved protein solubility significantly but impaired the foaming and emulsifying property. Hydrolysate generated by alcalase had the highest hydrolysis degree (DH) and antioxidant activity, and displayed excellent protein solubility over wide range of pH, while hydrolysate prepared by flavorzyme showed better copper chelating capacity and emulsifying stability with low molecular weight distribution. Trypsin-treated SCPH showed better foaming property than original protein. The results indicated that enzyme type greatly influenced the molecular weight, functional property and antioxidant activity of SCPH. It was also found that electing appropriate protease and controlling the DH could be enhanced or reduced functional property according to actual applications. PMID:26344981

  8. Effects of different bulking agents on the maturity, enzymatic activity, and microbial community functional diversity of kitchen waste compost.

    PubMed

    Wang, Xiaojuan; Zhang, Wenwei; Gu, Jie; Gao, Hua; Qin, Qingjun

    2016-10-01

    Aerobic composting is an effective method for the disposal and utilization of kitchen waste. However, the addition of a bulking agent is necessary during kitchen waste composting because of its high moisture content and low C/N ratio. In order to select a suitable bulking agent, we investigated the influence of leaf litter (LL), sawdust (SD), and wheat straw (WS) on the enzymatic activity, microbial community functional diversity, and maturity indices during the kitchen waste composting process. The results showed that the addition of WS yielded the highest maturity (the C/N ratio decreased from 25 to 13, T value = 0.5, and germination index (GI) = 114.7%), whereas the compost containing SD as a bulking agent had the lowest maturity (GI = 32.4%). The maximum cellulase and urease activities were observed with the WS treatment on day 8, whereas the SD treatment had the lowest cellulase activity and the LL treatment had the lowest urease activity. The compost temperature and microbial activity (as the average well color development) showed that bulking the composts with SD prolonged the composting process. The diversity index based on the community-level physiological profile showed that the composts bulked with LL and WS had greater microbial community functional diversity compared with those bulked with SD. Thus, the maturity indexes and enzymatic activities suggest that WS is a suitable bulking agent for use in kitchen waste composting systems. PMID:26895274

  9. Purification, crystallization and preliminary X-ray diffraction studies of d-tagatose 3-epimerase from Pseudomonas cichorii

    SciTech Connect

    Yoshida, Hiromi; Yamada, Mitsugu; Nishitani, Takeyori; Takada, Goro; Izumori, Ken; Kamitori, Shigehiro

    2007-02-01

    Recombinant d-tagatose 3-epimerase from P. cichorii was purified and crystallized. Diffraction data were collected to 2.5 Å resolution. d-Tagatose 3-epimerase (D-TE) from Pseudomonas cichorii catalyzes the epimerization of various ketohexoses at the C3 position. The epimerization of d-psicose has not been reported with epimerases other than P. cichorii D-TE and d-psicose 3-epimerase from Agrobacterium tumefaciens. Recombinant P. cichorii D-TE has been purified and crystallized. Crystals of P. cichorii D-TE were obtained by the sitting-drop method at room temperature. The crystal belongs to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 76.80, b = 94.92, c = 91.73 Å, β = 102.82°. Diffraction data were collected to 2.5 Å resolution. The asymmetric unit is expected to contain four molecules.

  10. Functional dependency of structures of ionic liquids: do substituents govern the selectivity of enzymatic glycerolysis?

    PubMed

    Guo, Zheng; Chen, Biqiang; López Murillo, Rafael; Tan, Tianwei; Xu, Xuebing

    2006-07-21

    The concept of regulating the preference of a reversible multi-step reaction by adjusting the substituents of ionic liquids (ILs) has been successfully exemplified with a group of tetraammonium-based ionic liquids as medium for the enzymatic glycerolysis. Simultaneous existence of long chain hydrophobic substituents and hydrophilic ethoxyl or hydroxyl moieties is found, respectively, to be essential for triglycerides (TG) dissolving and equilibrium shifting. The reactions in the ILs with cations consisting of long chain and free hydroxyl groups gave markedly higher conversion of TG and better preference to monoglyceride formation. Interestingly the predicted results from COSMO-RS (a quantum chemical model programme) achieved a good agreement with the experimental data, mapping out the specific solvation from the ILs as well as demonstrating the interaction between ILs, substrates and products being the intrinsic causes that govern reaction evolution and direct equilibrium shifting. PMID:16826302

  11. Production and physicochemical properties of functional-butterfat through enzymatic interesterification in a continuous reactor.

    PubMed

    Shin, Jung-Ah; Akoh, Casimir C; Lee, Ki-Teak

    2009-02-11

    Modified-butterfat (MBF) was synthesized with four blends (8:6:6, 6:6:8, 6:6:9, and 4:6:10, by weight) of anhydrous butterfat (ABF), palm stearin (PS) and flaxseed oil (FSO) through enzymatic interesterification in a continuous packed-bed reactor. Flow rate effect of 3, 5, 8 and 10 mL/min on enzymatic interesterification was investigated. By increasing the enzyme contact time with substrates (decreased flow rates), not only did melting and crystallization points shift to lower temperature but also the equivalent carbon number, ECN 36-38 from FSO decreased. Further all reactions were performed at flow rate of 5 mL/min (contact time 140 min) in a continuous reactor packed with 150 g of Lipozyme RM IM. After short path distillation, alpha-linolenic acid composition (%) of 8:6:6, 6:6:8, 6:6:9, and 4:6:10 MBFs were 16, 21, 23 and 25%, respectively. The contents of ECN 36-38, and ECN 48-50 decreased in the blends and MBFs for each substrate ratio. ECN 42-46 in the newly produced TAG increased. Melting points of MBFs were 38 degrees C (8:6:6), 35.5 degrees C (6:6:8), 34 degrees C (6:6:9), and 32 degrees C (4:6:10). MBFs interesterified with FSO contained phytosterols (17-36 mg/100 g) and tocopherols (116-173 microg/g). The products of 8:6:6, 6:6:8, 6:6:9 and 4:6:10 MBFs were softer (69, 88, 80, and 92%, respectively) than pure butterfat at refrigeration temperature. The polymorphic form changed from beta form (blends) to desirable crystalline structure of beta' form (MBFs). Crystal morphology of MBFs also changed and was composed of small spherulites of varying density. PMID:19138078

  12. Enzymatic process of rice bran: a stabilized functional food with nutraceuticals and nutrients.

    PubMed

    S Vallabha, Vishwanath; Indira, T N; Jyothi Lakshmi, A; Radha, C; Tiku, Purnima Kaul

    2015-12-01

    Rice bran (RB), a byproduct of rice milling industry, is a rich source of nutraceuticals and nutrients. However its utility is limited due to the presence of lipase and lipoxygenase which initiates rancidity on milling. The aim of this investigation is to prevent oxidation of free fatty acids by enzymatic approach for its effective utilization. The enzymatic treatment comprised of alcalase treatment for complete inactivation of lipase along with reduction in lipoxygenase (LOX) activity and endoglucanase for improving the soluble fiber content. The enzyme treated rice bran was drum dried for further use. The nutraceutical molecules like γ-oryzanol, α-tocopherol and polyphenols were retained in the range of 68 to 110 % and the total antioxidant activity was improved. By the action of endoglucanase the complex carbohydrate was converted into glucose (72.28 %), cellobiose (18.36 %) and cellotriose (9.36 %). The prebiotic effect of enzyme treated rice bran was evaluated by the action of lactobacillus which was measured through the release of the short chain free fatty acids (SCFAs) analyzed by HPLC. The SCFAs; acetic acid and propionic acid increased by 1.72 folds and 2.12 folds respectively. B-complex vitamins showed maximum retention with vitamins like B1 (66.3 %), B2 (68.3 %) and B3 (55.0 %) after enzyme treatment. At different humidity levels, storage studies showed no change in LOX activity and also retained ubiquinol-10 in reduced state in enzyme treated RB for a period of 3 months. A stabilized RB has been developed enriched with short chain prebiotics and antioxidant molecules. PMID:26604401

  13. Co-expression of D-glucose isomerase and D-psicose 3-epimerase: development of an efficient one-step production of D-psicose.

    PubMed

    Men, Yan; Zhu, Yueming; Zeng, Yan; Izumori, Ken; Sun, Yuanxia; Ma, Yanhe

    2014-10-01

    D-Psicose has been attracting attention in recent years because of its alimentary activities and is used as an ingredient in a range of foods and dietary supplements. To develop a one-step enzymatic process of D-psicose production, thermoactive D-glucose isomerase and the D-psicose 3-epimerase obtained from Bacillus sp. and Ruminococcus sp., respectively, were successfully co-expressed in Escherichia coli BL21 strain. The substrate of one-step enzymatic process was D-glucose. The co-expression system exhibited maximum activity at 65 °C and pH 7.0. Mg(2+) could enhance the output of D-psicose by 2.32 fold to 1.6 g/L from 10 g/L of D-glucose. When using high-fructose corn syrup (HFCS) as substrate, 135 g/L D-psicose was produced under optimum conditions. The mass ratio of D-glucose, D-fructose, and D-psicose was almost 3.0:2.7:1.0, when the reaction reached equilibrium after an 8h incubation time. This co-expression system approaching to produce D-psicose has potential application in food and beverage products, especially softdrinks. PMID:25152409

  14. Inelastic X-ray scattering studies of the short-time collective vibrational motions in hydrated lysozyme powders and their possible relation to enzymatic function.

    PubMed

    Wang, Zhe; Bertrand, Christopher E; Chiang, Wei-Shan; Fratini, Emiliano; Baglioni, Piero; Alatas, Ahmet; Alp, E Ercan; Chen, Sow-Hsin

    2013-01-31

    High-resolution inelastic X-ray scattering was used to investigate the collective vibrational excitations in hydrated lysozyme powders as a function of hydration level and temperature. It is found that the samples with strong enzymatic function are "soft", in the sense that they exhibit low frequency and large amplitude intraprotein collective vibrational motions on certain length scales. This is not the case for samples with weak or no enzymatic activity. Thus, we identify a possible correlation between the short-time intraprotein collective vibrational motions and the establishment of enzymatic function in hydrated lysozyme powders, and bring new insight to notions of protein "conformational flexibility" and "softness" in terms of these motions. PMID:23301848

  15. New N-acyl-D-glucosamine 2-epimerases from cyanobacteria with high activity in the absence of ATP and low inhibition by pyruvate.

    PubMed

    Klermund, Ludwig; Groher, Anna; Castiglione, Kathrin

    2013-11-01

    N-Acetylneuraminic acid, an important component of glycoconjugates with various biological functions, can be produced from N-acetyl-D-glucosamine (GlcNAc) and pyruvate using a one-pot, two-enzyme system consisting of N-acyl-D-glucosamine 2-epimerase (AGE) and N-acetylneuraminate lyase (NAL). In this system, the epimerase catalyzes the conversion of GlcNAc into N-acetyl-D-mannosamine (ManNAc). However, all currently known AGEs have one or more disadvantages, such as a low specific activity, substantial inhibition by pyruvate and strong dependence on allosteric activation by ATP. Therefore, four novel AGEs from the cyanobacteria Acaryochloris marina MBIC 11017, Anabaena variabilis ATCC 29413, Nostoc sp. PCC 7120, and Nostoc punctiforme PCC 73102 were characterized. Among these enzymes, the AGE from the Anabaena strain showed the most beneficial characteristics. It had a high specific activity of 117±2 U mg(-1) at 37 °C (pH 7.5) and an up to 10-fold higher inhibition constant for pyruvate as compared to other AGEs indicating a much weaker inhibitory effect. The investigation of the influence of ATP revealed that the nucleotide has a more pronounced effect on the Km for the substrate than on the enzyme activity. At high substrate concentrations (≥200 mM) and without ATP, the enzyme reached up to 32% of the activity measured with ATP in excess. PMID:23850800

  16. Efficient chemo-enzymatic gluten detoxification: reducing toxic epitopes for celiac patients improving functional properties

    PubMed Central

    Ribeiro, Miguel; Nunes, Fernando M.; Guedes, Sofia; Domingues, Pedro; Silva, Amélia M.; Carrillo, Jose Maria; Rodriguez-Quijano, Marta; Branlard, Gérard; Igrejas, Gilberto

    2015-01-01

    Protein engineering of gluten, the exogenous effector in celiac disease, seeking its detoxification by selective chemical modification of toxic epitopes is a very attractive strategy and promising technology when compared to pharmacological treatment or genetic engineering of wheat. Here we present a simple and efficient chemo-enzymatic methodology that decreases celiac disease toxic epitopes of gluten proteins improving its technological value through microbial transglutaminase-mediated transamidation of glutamine with n-butylamine under reducing conditions. First, we found that using low concentrations of amine-nucleophile under non-reducing conditions, the decrease in toxic epitopes is mainly due to transglutaminase-mediated cross-linking. Second, using high amine nucleophile concentrations protein cross-linking is substantially reduced. Third, reducing conditions increase 7-fold the transamidation reaction further decreasing toxic epitopes amount. Fourth, using n-butylamine improves gluten hydrophobicity that strengthens the gluten network. These results open the possibility of tailoring gluten for producing hypoallergenic flours while still taking advantage of the unique viscoelastic properties of gluten. PMID:26691232

  17. Efficient chemo-enzymatic gluten detoxification: reducing toxic epitopes for celiac patients improving functional properties.

    PubMed

    Ribeiro, Miguel; Nunes, Fernando M; Guedes, Sofia; Domingues, Pedro; Silva, Amélia M; Carrillo, Jose Maria; Rodriguez-Quijano, Marta; Branlard, Gérard; Igrejas, Gilberto

    2015-01-01

    Protein engineering of gluten, the exogenous effector in celiac disease, seeking its detoxification by selective chemical modification of toxic epitopes is a very attractive strategy and promising technology when compared to pharmacological treatment or genetic engineering of wheat. Here we present a simple and efficient chemo-enzymatic methodology that decreases celiac disease toxic epitopes of gluten proteins improving its technological value through microbial transglutaminase-mediated transamidation of glutamine with n-butylamine under reducing conditions. First, we found that using low concentrations of amine-nucleophile under non-reducing conditions, the decrease in toxic epitopes is mainly due to transglutaminase-mediated cross-linking. Second, using high amine nucleophile concentrations protein cross-linking is substantially reduced. Third, reducing conditions increase 7-fold the transamidation reaction further decreasing toxic epitopes amount. Fourth, using n-butylamine improves gluten hydrophobicity that strengthens the gluten network. These results open the possibility of tailoring gluten for producing hypoallergenic flours while still taking advantage of the unique viscoelastic properties of gluten. PMID:26691232

  18. Reaction of uridine diphosphate galactose 4-epimerase with a suicide inactivator

    SciTech Connect

    Flentke, G.R.; Frey, P.A. )

    1990-03-06

    UDPgalactose 4-epimerase from Escherichia coli is rapidly inactivated by the compounds uridine 5{prime}-diphosphate chloroacetol (UDC) and uridine 5{prime}-diphosphate bromoacetol (UCB). Both UDC and UDB inactivate the enzyme in neutral solution concomitant with the appearance of chromophores absorbing maximally at 325 and 328 nm, respectively. The reaction of UDC with the enzyme follows saturation kinetics characterized by a K{sub D} of 0.110 mM and k{sub inact} of 0.84 min{sup {minus}1} at pH 8.5 and ionic strength 0.2 M. The inactivation by UDC is competitively inhibited by competitive inhibitors of UDPgalactose 4-epimerase, and it is accompanied by the tight but noncovalent binding of UDC to the enzyme in a stoichiometry of 1 mol of UDC/mol of enzyme dimer, corresponding to 1 mol of UDC/mol of enzyme-bound NAD{sup +}. The inactivation of epimerase by uridine 5{prime}-diphosphate ({sup 2}H{sub 2})chloroacetol proceeds with a primary kinetic isotope effect (k{sub H}/k{sub D}) of 1.4. The inactivation mechanism is proposed to involve a minimum of three steps: (a) reversible binding of UDC to the active site of UDPgalactose 4-epimerase; (b) enolization of the chloroacetol moiety of enzyme-bound UDC, catalyzed by an enzymic general base at the active site; (c) alkylation of the nicotinamide ring of NAD{sup +} at the active site by the chloroacetol enolate. The resulting adduct between UDC and NAD{sup +} is proposed to be the chromophore with {lambda}{sub max} at 325 nm. The enzymic general base required to facilitate proton transfer in redox catalysis by this enzyme may be the general base that facilitates enolization of the chloroacetol moiety of UDC in the inactivation reaction.

  19. Synthetic substrates specific to activated plasmin can monitor the enzymatic functional status in situ in breast cancer cells.

    PubMed

    Gohda, Keigo; Fujimori, Ko; Teno, Naoki; Wanaka, Keiko; Tsuda, Yuko

    2014-01-01

    We here strove to overcome the limitations of expression analyses such as PCR and IHC, based on molecular recognition between target and probe molecules, by designing synthetic substrates specific to the target molecules to directly estimate the enzymatic functionality in situ. The specific substrate contains a probing unit, which is an organic fragment for specific enzyme binding, and a reactive unit, which is a natural peptide subject to catalysis. In this study, the activation of plasminogen to plasmin was examined in MDA-MB231 breast cancer cells using the plasmin-specific synthetic substrates designed from their inhibitors. The localization and function of the activated plasmin were successfully visualized by fluorophore combined with the specific substrate concurrently. This would be the first time for activated plasmin at work in situ by direct observation. Our concept to directly monitor the functionality of target enzymes can be used straightforwardly for other proteases such as cathepsins or caspases. Also, this substrate concept as a 'tailor-made substrate' would be utilized as a novel functional molecular probe in vivo with appropriate detectable probes. PMID:24112688

  20. Solvent environments significantly affect the enzymatic function of Escherichia coli dihydrofolate reductase: comparison of wild-type protein and active-site mutant D27E.

    PubMed

    Ohmae, Eiji; Miyashita, Yurina; Tate, Shin-Ichi; Gekko, Kunihiko; Kitazawa, Soichiro; Kitahara, Ryo; Kuwajima, Kunihiro

    2013-12-01

    To investigate the contribution of solvent environments to the enzymatic function of Escherichia coli dihydrofolate reductase (DHFR), the salt-, pH-, and pressure-dependence of the enzymatic function of the wild-type protein were compared with those of the active-site mutant D27E in relation to their structure and stability. The salt concentration-dependence of enzymatic activity indicated that inorganic cations bound to and inhibited the activity of wild-type DHFR at neutral pH. The BaCl2 concentration-dependence of the (1)H-(15)N HSQC spectra of the wild-type DHFR-folate binary complex showed that the cation-binding site was located adjacent to the Met20 loop. The insensitivity of the D27E mutant to univalent cations, the decreased optimal pH for its enzymatic activity, and the increased Km and Kd values for its substrate dihydrofolate suggested that the substrate-binding cleft of the mutant was slightly opened to expose the active-site side chain to the solvent. The marginally increased fluorescence intensity and decreased volume change due to unfolding of the mutant also supported this structural change or the modified cavity and hydration. Surprisingly, the enzymatic activity of the mutant increased with pressurization up to 250MPa together with negative activation volumes of -4.0 or -4.8mL/mol, depending on the solvent system, while that of the wild-type was decreased and had positive activation volumes of 6.1 or 7.7mL/mol. These results clearly indicate that the insertion of a single methylene at the active site could substantially change the enzymatic reaction mechanism of DHFR, and solvent environments play important roles in the function of this enzyme. PMID:24140567

  1. Structure of a d-tagatose 3-epimerase-related protein from the hyperthermophilic bacterium Thermotoga maritima

    PubMed Central

    Sakuraba, Haruhiko; Yoneda, Kazunari; Satomura, Takenori; Kawakami, Ryushi; Ohshima, Toshihisa

    2009-01-01

    The crystal structure of a d-tagatose 3-epimerase-related protein (TM0416p) encoded by the hypothetical open reading frame TM0416 in the genome of the hyperthermophilic bacterium Thermotoga maritima was determined at a resolution of 2.2 Å. The asymmetric unit contained two homologous subunits and a dimer was generated by twofold symmetry. The main-chain coordinates of the enzyme monomer proved to be similar to those of d-tagatose 3-­epimerase from Pseudomonas cichorii and d-psicose 3-epimerase from Agrobacterium tumefaciens; however, TM0416p exhibited a unique solvent-accessible substrate-binding pocket that reflected the absence of an α-helix that covers the active-site cleft in the two aforementioned ketohexose 3-epimerases. In addition, the residues responsible for creating a hydrophobic environment around the substrate in TM0416p differ entirely from those in the other two enzymes. Collectively, these findings suggest that the substrate specificity of TM0416p is likely to differ substantially from those of other d-tagatose 3-­epimerase family enzymes. PMID:19255464

  2. Composition and enzymatic function of particle-associated and free-living bacteria: a coastal/offshore comparison

    PubMed Central

    D'Ambrosio, Lindsay; Ziervogel, Kai; MacGregor, Barbara; Teske, Andreas; Arnosti, Carol

    2014-01-01

    We compared the function and composition of free-living and particle-associated microbial communities at an inshore site in coastal North Carolina and across a depth profile on the Blake Ridge (offshore). Hydrolysis rates of six different polysaccharide substrates were compared for particle-associated (>3 μm) and free-living (<3 to 0.2 μm) microbial communities. The 16S rRNA- and rDNA-based clone libraries were produced from the same filters used to measure hydrolysis rates. Particle-associated and free-living communities resembled one another; they also showed similar enzymatic hydrolysis rates and substrate preferences. All six polysaccharides were hydrolyzed inshore. Offshore, only a subset was hydrolyzed in surface water and at depths of 146 and 505 m; just three polysaccharides were hydrolyzed at 505 m. The spectrum of bacterial taxa changed more subtly between inshore and offshore surface waters, but changed greatly with depth offshore. None of the OTUs occurred at all sites: 27 out of the 28 major OTUs defined in this study were found either exclusively in a surface or in a mid-depth/bottom water sample. This distinction was evident with both 16S rRNA and rDNA analyses. At the offshore site, despite the low community overlap, bacterial communities maintained a degree of functional redundancy on the whole bacterial community level with respect to hydrolysis of high-molecular-weight substrates. PMID:24763371

  3. Enzymatic hydrolysis of ovomucin and the functional and structural characteristics of peptides in the hydrolysates.

    PubMed

    Abeyrathne, E D N S; Lee, H Y; Jo, C; Suh, J W; Ahn, D U

    2016-02-01

    Ovomucin was hydrolyzed using enzymes or by heating under alkaline conditions (pH 12.0), and the functional, structural and compositional characteristics of the peptides in the hydrolysates were determined. Among the treatments, heating at 100 °C for 15 min under alkaline conditions (OM) produced peptides with the highest iron-binding and antioxidant capacities. Ovomucin hydrolyzed with papain (OMPa) or alcalase (OMAl) produced peptides with high ACE-inhibitory activity. The mass spectrometry analysis indicated that most of the peptides from OMPa were <2 kDa, but peptides from OMTr and OM were >2 kDa. OMAl hydrolyzed ovomucin almost completely and no peptides within 700-5000 Da were found in the hydrolasate. The results indicated that the number and size of peptides were closely related to the functionality of the hydrolysates. Considering the time, cost and activities of the hydrolysates, OM was the best treatment for hydrolyzing ovomucin to produce functional peptides. PMID:26304326

  4. The Closure of the Cycle: Enzymatic Synthesis and Functionalization of Bio-Based Polyesters.

    PubMed

    Pellis, Alessandro; Herrero Acero, Enrique; Ferrario, Valerio; Ribitsch, Doris; Guebitz, Georg M; Gardossi, Lucia

    2016-04-01

    The polymer industry is under pressure to mitigate the environmental cost of petrol-based plastics. Biotechnologies contribute to the gradual replacement of petrol-based chemistry and the development of new renewable products, leading to the closure of carbon circle. An array of bio-based building blocks is already available on an industrial scale and is boosting the development of new generations of sustainable and functionally competitive polymers, such as polylactic acid (PLA). Biocatalysts add higher value to bio-based polymers by catalyzing not only their selective modification, but also their synthesis under mild and controlled conditions. The ultimate aim is the introduction of chemical functionalities on the surface of the polymer while retaining its bulk properties, thus enlarging the spectrum of advanced applications. PMID:26806112

  5. The Molecular Dynamics of Trypanosoma brucei UDP-Galactose 4′-Epimerase: A Drug Target for African Sleeping Sickness

    PubMed Central

    Friedman, Aaron J; Durrant, Jacob D; Pierce, Levi C T; McCorvie, Thomas J; Timson, David J; McCammon, J Andrew

    2012-01-01

    During the past century, several epidemics of human African trypanosomiasis, a deadly disease caused by the protist Trypanosoma brucei, have afflicted sub-Saharan Africa. Over 10 000 new victims are reported each year, with hundreds of thousands more at risk. As current drug treatments are either highly toxic or ineffective, novel trypanocides are urgently needed. The T. brucei galactose synthesis pathway is one potential therapeutic target. Although galactose is essential for T. brucei survival, the parasite lacks the transporters required to intake galactose from the environment. UDP-galactose 4′-epimerase (TbGalE) is responsible for the epimerization of UDP-glucose to UDP-galactose and is therefore of great interest to medicinal chemists. Using molecular dynamics simulations, we investigate the atomistic motions of TbGalE in both the apo and holo states. The sampled conformations and protein dynamics depend not only on the presence of a UDP-sugar ligand, but also on the chirality of the UDP-sugar C4 atom. This dependence provides important insights into TbGalE function and may help guide future computer-aided drug discovery efforts targeting this protein. PMID:22487100

  6. Enzymatic Oxidation of Cholesterol: Properties and Functional Effects of Cholestenone in Cell Membranes

    PubMed Central

    Neuvonen, Maarit; Manna, Moutusi; Mokkila, Sini; Javanainen, Matti; Rog, Tomasz; Liu, Zheng; Bittman, Robert; Vattulainen, Ilpo; Ikonen, Elina

    2014-01-01

    Bacterial cholesterol oxidase is commonly used as an experimental tool to reduce cellular cholesterol content. That the treatment also generates the poorly degradable metabolite 4-cholesten-3-one (cholestenone) has received less attention. Here, we investigated the membrane partitioning of cholestenone using simulations and cell biological experiments and assessed the functional effects of cholestenone in human cells. Atomistic simulations predicted that cholestenone reduces membrane order, undergoes faster flip-flop and desorbs more readily from membranes than cholesterol. In primary human fibroblasts, cholestenone was released from membranes to physiological extracellular acceptors more avidly than cholesterol, but without acceptors it remained in cells over a day. To address the functional effects of cholestenone, we studied fibroblast migration during wound healing. When cells were either cholesterol oxidase treated or part of cellular cholesterol was exchanged for cholestenone with cyclodextrin, cell migration during 22 h was markedly inhibited. Instead, when a similar fraction of cholesterol was removed using cyclodextrin, cells replenished their cholesterol content in 3 h and migrated similarly to control cells. Thus, cholesterol oxidation produces long-term functional effects in cells and these are in part due to the generated membrane active cholestenone. PMID:25157633

  7. Mapping the Mutual Information Network of Enzymatic Families in the Protein Structure to Unveil Functional Features

    PubMed Central

    Aguilar, Daniel; Oliva, Baldo; Marino Buslje, Cristina

    2012-01-01

    Amino acids committed to a particular function correlate tightly along evolution and tend to form clusters in the 3D structure of the protein. Consequently, a protein can be seen as a network of co-evolving clusters of residues. The goal of this work is two-fold: first, we have combined mutual information and structural data to describe the amino acid networks within a protein and their interactions. Second, we have investigated how this information can be used to improve methods of prediction of functional residues by reducing the search space. As a main result, we found that clusters of co-evolving residues related to the catalytic site of an enzyme have distinguishable topological properties in the network. We also observed that these clusters usually evolve independently, which could be related to a fail-safe mechanism. Finally, we discovered a significant enrichment of functional residues (e.g. metal binding, susceptibility to detrimental mutations) in the clusters, which could be the foundation of new prediction tools. PMID:22848494

  8. Mapping the mutual information network of enzymatic families in the protein structure to unveil functional features.

    PubMed

    Aguilar, Daniel; Oliva, Baldo; Marino Buslje, Cristina

    2012-01-01

    Amino acids committed to a particular function correlate tightly along evolution and tend to form clusters in the 3D structure of the protein. Consequently, a protein can be seen as a network of co-evolving clusters of residues. The goal of this work is two-fold: first, we have combined mutual information and structural data to describe the amino acid networks within a protein and their interactions. Second, we have investigated how this information can be used to improve methods of prediction of functional residues by reducing the search space. As a main result, we found that clusters of co-evolving residues related to the catalytic site of an enzyme have distinguishable topological properties in the network. We also observed that these clusters usually evolve independently, which could be related to a fail-safe mechanism. Finally, we discovered a significant enrichment of functional residues (e.g. metal binding, susceptibility to detrimental mutations) in the clusters, which could be the foundation of new prediction tools. PMID:22848494

  9. Fold conservation and proteolysis in zebrafish IRBP structure: Clues to possible enzymatic function?

    PubMed

    Ghosh, Debashis; Haswell, Karen M; Sprada, Molly; Gonzalez-Fernandez, Federico

    2016-06-01

    Multiple functions for Interphotoreceptor Retinoid-Binding Protein (IRBP) may explain its localization in the retina, vitreous and pineal gland and association with retinitis pigmentosa and myopia. We have been engaged in uncovering the structure-function relationships of this interesting protein long thought to bind visual-cycle retinoids and fatty acids in the subretinal space. Although hydrophobic domains capable of binding such ligands have now been found, we ask what other structural domains might be present that could predict new functions? Interestingly, IRBP possesses a fold similar to C-terminal processing proteases (CTPases) but is missing the PDZ domain. Here we present structural evidence that this fold may have a role in a recently observed autoproteolytic activity of the two-module zebrafish (z) IRBP (Ghosh et al. Exp. Eye Res., 2015). When the structure of Scenedesmus obliquus D1 CTPase (D1P) is superimposed with the first module of zIRBP (z1), the PDZ domain of D1P occupies roughly the same position in the amino acid sequence as the inter-domain tether in z1, between residues P71 and P85. The catalytic triad K397, S372 and E375 of D1P is located at the inter-domain interfacial cleft, similarly as the tetrad K241, S243, D177 and T179 of z1 residues, presumed to have proteolytic function. Packing of two adjacent symmetry-related molecules within the z1 crystal show that the helix α8 penetrates the interfacial cleft underneath the inter-domain tether, forming a simple intermolecular "knot". The full-length zIRBP is cleaved at or immediately after T309, which is located at the end of α8 and is the ninth residue of the second module z2. We propose that the helix α8 within intact zIRBP bends at P301, away from the improbable knotted fold, and positions the cleavage site T309 near the putative catalytic tetrad of the neighboring zIRBP to be proteolytically cleaved. The conservation of this functional catalytic domain suggests that possible

  10. Enzymatic hydrolysis of ovomucoid and the functional properties of its hydrolysates.

    PubMed

    Abeyrathne, E D N S; Lee, H Y; Jo, C; Suh, J W; Ahn, D U

    2015-09-01

    Ovomucoid is well known as a "trypsin inhibitor" and is considered to be the main food allergen in egg. However, the negative functions of ovomucoid can be eliminated if the protein is cut into small peptides. The objectives of this study were to hydrolyze ovomucoid using various enzyme combinations, and compare the functional properties of the hydrolysates. Purified ovomucoid was dissolved in distilled water (20 mg/mL) and treated with 1% of pepsin, α-chymotrypsin, papain, and alcalase, singly or in combinations. Sodium sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) results of the hydrolysates indicated that pepsin (OMP), alcalase (OMAl), alcalase+trypsin (OMAlTr), and alcalase+papain (OMAlPa) treatments best hydrolyzed the ovomucoid, and the 4 treatments were selected to determine their functional characteristics. Among the 4 enzyme treatments, hydrolysate from OMAlTr showed the highest iron-chelating and antioxidant activities, while OMP showed higher ACE-inhibitory activity, but lower Fe-chelating activity than the other treatments. However, no difference in the copper-chelating activity among the treatments was found. MS/MS analysis identified numerous peptides from the hydrolysates of OMAlPa and OMAlTr, and majority of the peptides produced were <2 kDa. Pepsin treatment (OMP), however, hydrolyzed ovomucoid almost completely and produced only amino acid monomers, di- and tri-peptides. The ACE-inhibitory, antioxidant and iron-chelating activities of the enzyme hydrolysates were not consistent with the number and size of peptides in the hydrolysates, but we do not have information about the quantity of each peptide present in the hydrolysates at this point. PMID:26195809

  11. Phosphomolybdic acid functionalized graphene loading copper nanoparticles modified electrodes for non-enzymatic electrochemical sensing of glucose.

    PubMed

    Xu, Jiaoyan; Cao, Xiyue; Xia, Jianfei; Gong, Shida; Wang, Zonghua; Lu, Lin

    2016-08-31

    A sensitive non-enzymatic glucose electrochemical biosensor (Cu/PMo12-GR/GCE) was developed based on the combination of copper nanoparticles (CuNPs) and phosphomolybdic acid functionalized graphene (PMo12-GR). PMo12-GR films were modified on the surface of glassy carbon electrode (GCE) through electrostatic self-assembly with the aid of poly diallyl dimethyl ammonium chloride (PDDA). Then CuNPs were successfully decorated onto the PMo12-GR modified GCE through electrodeposition. The morphology of Cu/PMo12-GR/GCE was characterized by scanning electron microscope (SEM). Cyclic voltammetry (CV) and chronoamperometry were used to investigate the electrochemical performances of the biosensor. The results indicated that the modified electrode displayed a synergistic effect of PMo12-GR sheets and CuNPs towards the electro-oxidation of glucose in the alkaline solution. At the optimal detection potential of 0.50 V, the response towards glucose presented a linear response ranging from 0.10 μM to 1.0 mM with a detection limit of 3.0 × 10(-2) μM (S/N = 3). In addition, Cu/PMo12-GR/GCE possessed a high selectivity, good reproducibility, excellent stability and acceptable recovery, which indicating the potential application in clinical field. PMID:27506342

  12. Characterization of the Methylthioadenosine Phosphorylase Polymorphism rs7023954 - Incidence and Effects on Enzymatic Function in Malignant Melanoma

    PubMed Central

    Limm, Katharina; Dettmer, Katja; Reinders, Jörg; Oefner, Peter J.; Bosserhoff, Anja-Katrin

    2016-01-01

    Deficiency of methylthioadenosine phosphorylase (MTAP) supports melanoma development and progression through accumulation of its substrate 5’-methylthioadenosine (MTA), which leads amongst others to a constitutive inhibition of protein arginine methyltransferases (PRMTs) and activation of the transcription factor AP-1 via the receptor ADORA2B. Genetic association studies have also suggested that genetic polymorphism in MTAP may modulate the risk of melanoma. Here, we investigated the only globally common non-synonymous single nucleotide polymorphism (SNP) reported to date for MTAP. The SNP rs7023954 is located in exon 3 (c.166G>A), and leads to the conservative substitution of one branched-chain amino acid residue (valine) for another (isoleucine) at position 56 (p.Val56Ile). Whereas genotype frequencies in normal and primary melanoma tissues or cell lines were in Hardy-Weinberg equilibrium based on cDNA amplicon sequencing, a marked (P = 0.00019) deviation was observed in metastatic melanoma tissues and cell lines due to a deficit of heterozygotes. Enzyme assays conducted on the co-dominantly expressed alleles revealed no difference in the conversion rate of MTA to adenine and 5-methylthioribose-1-phosphate, indicating that this known enzymatic activity does not modulate the tumor suppressive function of MTAP. PMID:27479139

  13. Preparation of Yeast Hydrolysate Enriched in Cyclo-His-Pro (CHP) by Enzymatic Hydrolysis and Evaluation of Its Functionality

    PubMed Central

    Lee, Hyun Jung; Son, Heung Soo; Park, Chung; Suh, Hyung Joo

    2015-01-01

    In this study, we attempted to enrich cyclo-His-Pro (CHP) using enzymatic hydrolysis of yeast and to evaluate the functionality of yeast hydrolysate (YH)-enriched CHP. Flavourzyme offered a better performance in enhancing CHP content than other proteases. The CHP enrichment conditions were optimized as follows: addition of 1% Flavourzyme, 48-h incubation at 60°C, and pH 6.0. The CHP content significantly increased by 20-fold after ultra-filtration (UF). Maximal CHP translation was obtained after heating for 8 h at 50°C and pH 7.0. YH showed poor foaming capacity between pH 3.0 to 9.0. The emulsifying activities of YHs were slightly higher at near acidic pH. Increase in heating temperature and time resulted in decreased CHP content. The results indicate that YH is more heat stable after UF. Therefore, the CHP in YH after UF can be used as a food additive with physiological CHP activity and high heat stability. PMID:26770916

  14. Enzymatic hydrolysis of blue whiting (Micromesistius poutassou); functional and bioactive properties.

    PubMed

    Geirsdottir, Margret; Sigurgisladottir, Sjofn; Hamaguchi, Patricia Y; Thorkelsson, Gudjon; Johannsson, Ragnar; Kristinsson, Hordur G; Kristjansson, Magnus M

    2011-01-01

    Functional and biochemical properties of fish protein hydrolysates (FPH) from blue whiting (BW) were studied. FPH (2.5%, 5%, 10%, and 15% degree of hydrolysis [DH]) were made from isolated proteins from headed and gutted BW with Alcalase 2.4 L. The properties of dried BW mince and protein isolate compared to 4 reference proteins (soy and milk protein) were studied: color, solubility, water-holding capacity (WHC), oil-binding capacity (OBC), emulsion capacity (EC), and emulsion stability (ES). The angiotensin I-converting enzyme (ACE) inhibitory activities of the soluble fraction of BW powders were also investigated. Furthermore, the products were characterized by analyzing their chemical composition. Chemical composition, solubility, OBC, and EC of the BW powders was significantly (P < 0.05) different with different DH, while color, ES, and WHC were not significantly (P > 0.05) different. Salt content of the FPH was high (4% to 19%) and increased with increased DH. Protein solubility varied from 10% to 70% and increased with increased DH. WHC of the FPH was around 97% and was higher than that of all the reference proteins tested. OBC decreased with increased DH (from 3.5 to 2.1 g oil/g protein) and was higher than OBC of the soy and milk proteins (1.6 to 1.9 g oil/g protein). EC of FPH was similar or lower than the reference proteins. ES of FPH (60% to 90%) was similar to or lower than soy and whey proteins (60% to 98%) but higher than casein (20%). ACE inhibition activity increased as DH was increased. Practical Application: The results from this study demonstrate that a functional bioactive hydrolysate can be produced from BW, which is an underutilized fish species, and may aid the industry in better utilizing this raw material. The novelty of this research was the use of BW as a raw material where the protein has been isolated with the pH shift method. Furthermore, it was novel that bioactivity and functionality was measured in the same samples. PMID:21535642

  15. Probing Mechanisms for Enzymatic Activity Enhancement of Organophosphorus Hydrolase in Functionalized Mesoporous Silica

    SciTech Connect

    Chen, Baowei; Lei, Chenghong; Shin, Yongsoon; Liu, Jun

    2009-12-25

    We have previously reported that organophosphorus hydrolase (OPH) can be spontaneously entrapped in functionalized mesoporous silica (FMS) with HOOC - as the functional groups and the entrapped OPH in HOOC-FMS showed enhanced enzyme specific activity. This work is to study the mechanisms that why OPH entrapped in FMS displayed the enhanced activity in views of OPH-FMS interactions using spectroscopic methods. The circular dichroism (CD) spectra show that, comparing to the secondary structure of OPH free in solution, OPH in HOOC-FMS displayed increased a-helix/b-strand transition of OPH with increased OPH loading density. The fluorescence emission spectra of Trp residues were used to assess the tertiary structural changes of the enzyme. There was a 42% increase in fluorescence. This is in agreement with the fact that the fluorescence intensity of OPH was increased accompanying with the increased OPH activity when decreasing urea concentrations in solution. The steady-state anisotropy was increased after OPH entrapping in HOOC-FMS comparing to the free OPH in solution, indicating that protein mobility was reduced upon entrapment. The solvent accessibility of Trp residues of OPH was probed by using acrylamide as a collisional quencher. Trp residues of OPH-FMS had less solvent exposure comparing with free OPH in solution due to its electrostatical binding to HOOC-FMS thereby displaying the increased fluorescence intensity. These results suggest the interactions of OPH with HOOC-FMS resulted in the protein immobilization and a favorable conformational change for OPH in the crowded confinement space and accordingly the enhanced activity.

  16. Bioconversion of D-glucose to D-psicose with immobilized D-xylose isomerase and D-psicose 3-epimerase on Saccharomyces cerevisiae spores.

    PubMed

    Li, Zijie; Li, Yi; Duan, Shenglin; Liu, Jia; Yuan, Peng; Nakanishi, Hideki; Gao, Xiao-Dong

    2015-08-01

    Saccharomyces cerevisiae spores are dormant cells, which can tolerate various types of environmental stress. In our previous work, we successfully developed biological and chemical methods for enzyme immobilization based on the structures of S. cerevisiae spore wall. In this study, we employed biological and chemical approaches for the immobilization of D-xylose isomerase (XI) from Thermus thermophilus and D-psicose 3-epimerase (DPEase) from Agrobacterium tumefaciens with yeast spores, respectively. The enzymatic properties of both immobilized XI and DPEase were characterized and the immobilized enzymes exhibit higher thermostability, broader pH tolerance, and good repeatability compared with free enzymes. Furthermore, we established a two-step approach for the bioconversion of D-glucose to D-psicose using immobilized enzymes. To improve the conversion yield, a multi-pot strategy was adopted for D-psicose production by repeating the two-step process continually. As a result, the yield of D-psicose was obviously improved and the highest yield reached about 12.0 %. PMID:26065389

  17. A non-enzymatic function of 17beta-hydroxysteroid dehydrogenase type 10 is required for mitochondrial integrity and cell survival.

    PubMed

    Rauschenberger, Katharina; Schöler, Katja; Sass, Jörn Oliver; Sauer, Sven; Djuric, Zdenka; Rumig, Cordula; Wolf, Nicole I; Okun, Jürgen G; Kölker, Stefan; Schwarz, Heinz; Fischer, Christine; Grziwa, Beate; Runz, Heiko; Nümann, Astrid; Shafqat, Naeem; Kavanagh, Kathryn L; Hämmerling, Günter; Wanders, Ronald J A; Shield, Julian P H; Wendel, Udo; Stern, David; Nawroth, Peter; Hoffmann, Georg F; Bartram, Claus R; Arnold, Bernd; Bierhaus, Angelika; Oppermann, Udo; Steinbeisser, Herbert; Zschocke, Johannes

    2010-02-01

    Deficiency of the mitochondrial enzyme 2-methyl-3-hydroxybutyryl-CoA dehydrogenase involved in isoleucine metabolism causes an organic aciduria with atypical neurodegenerative course. The disease-causing gene is HSD17B10 and encodes 17beta-hydroxysteroid dehydrogenase type 10 (HSD10), a protein also implicated in the pathogenesis of Alzheimer's disease. Here we show that clinical symptoms in patients are not correlated with residual enzymatic activity of mutated HSD10. Loss-of-function and rescue experiments in Xenopus embryos and cells derived from conditional Hsd17b10(-/-) mice demonstrate that a property of HSD10 independent of its enzymatic activity is essential for structural and functional integrity of mitochondria. Impairment of this function in neural cells causes apoptotic cell death whilst the enzymatic activity of HSD10 is not required for cell survival. This finding indicates that the symptoms in patients with mutations in the HSD17B10 gene are unrelated to accumulation of toxic metabolites in the isoleucine pathway and, rather, related to defects in general mitochondrial function. Therefore alternative therapeutic approaches to an isoleucine-restricted diet are required. PMID:20077426

  18. Rescue of Enzymatic Function for Disease-associated RPE65 Proteins Containing Various Missense Mutations in Non-active Sites*

    PubMed Central

    Li, Songhua; Izumi, Tadahide; Hu, Jane; Jin, Heather H.; Siddiqui, Ahmed-Abdul A.; Jacobson, Samuel G.; Bok, Dean; Jin, Minghao

    2014-01-01

    Over 70 different missense mutations, including a dominant mutation, in RPE65 retinoid isomerase are associated with distinct forms of retinal degeneration; however, the disease mechanisms for most of these mutations have not been studied. Although some mutations have been shown to abolish enzyme activity, the molecular mechanisms leading to the loss of enzymatic function and retinal degeneration remain poorly understood. Here we show that the 26 S proteasome non-ATPase regulatory subunit 13 (PSMD13), a newly identified negative regulator of RPE65, plays a critical role in regulating pathogenicity of three mutations (L22P, T101I, and L408P) by mediating rapid degradation of mutated RPE65s via a ubiquitination- and proteasome-dependent non-lysosomal pathway. These mutant RPE65s were misfolded and formed aggregates or high molecular complexes via disulfide bonds. Interaction of PSMD13 with mutant RPE65s promoted degradation of misfolded but not properly folded mutant RPE65s. Many mutations, including L22P, T101I, and L408P, were mapped on non-active sites. Although their activities were very low, these mutant RPE65s were catalytically active and could be significantly rescued at low temperature, whereas mutant RPE65s with a distinct active site mutation could not be rescued under the same conditions. Sodium 4-phenylbutyrate and glycerol displayed a significant synergistic effect on the low temperature rescue of the mutant RPE65s by promoting proper folding, reducing aggregation, and increasing membrane association. Our results suggest that a low temperature eye mask and sodium 4-phenylbutyrate, a United States Food and Drug Administration-approved oral medicine, may provide a promising “protein repair therapy” that can enhance the efficacy of gene therapy by reducing the cytotoxic effect of misfolded mutant RPE65s. PMID:24849605

  19. Advances in the enzymatic production of L-hexoses.

    PubMed

    Chen, Ziwei; Zhang, Wenli; Zhang, Tao; Jiang, Bo; Mu, Wanmeng

    2016-08-01

    Rare sugars have recently drawn attention because of their potential applications and huge market demands in the food and pharmaceutical industries. All L-hexoses are considered rare sugars, as they rarely occur in nature and are thus very expensive. L-Hexoses are important components of biologically relevant compounds as well as being used as precursors for certain pharmaceutical drugs and thus play an important role in the pharmaceutical industry. Many general strategies have been established for the synthesis of L-hexoses; however, the only one used in the biotechnology industry is the Izumoring strategy. In hexose Izumoring, four entrances link the D- to L-enantiomers, ketose 3-epimerases catalyze the C-3 epimerization of L-ketohexoses, and aldose isomerases catalyze the specific bioconversion of L-ketohexoses and the corresponding L-aldohexoses. In this article, recent studies on the enzymatic production of various L-hexoses are reviewed based on the Izumoring strategy. PMID:27344591

  20. Physico-chemical and functional properties of Resistant starch prepared from red kidney beans (Phaseolus vulgaris.L) starch by enzymatic method.

    PubMed

    Reddy, Chagam Koteswara; Suriya, M; Haripriya, Sundaramoorthy

    2013-06-01

    The objective of this study was to evaluate the production, physico-chemical and functional properties of Resistant starch (RS) from red kidney bean starch by enzymatic method. Native and gelatinized starch were subjected to enzymatic hydrolysis (pullulanase, 40 U/g/10 h), autoclaved (121 °C/30 min), stored under refrigeration (4 °C/24 h), and lyophilized. The enzymatic hydrolysis and thermal treatment of starch increased the formation of RS which showed an increase in water absorption and water solubility indexes and a decrease in swelling power due to hydrolytic and thermal process. The process for obtaining RS changed the crystallinity pattern from C to B and increased the crystallinity due to the retrogradation process. RS obtained from hydrolysis showed a reduction in viscosity, indicating the rupture of starch molecules. The viscosity was found to be inversely proportional to the RS content in the sample. The thermal properties of RS increased due to the retrogradation and recrystallization (P<0.05). PMID:23618263

  1. Enzymatically catalytic deposition of gold nanoparticles by glucose oxidase-functionalized gold nanoprobe for ultrasensitive electrochemical immunoassay.

    PubMed

    Cheng, Hui; Lai, Guosong; Fu, Li; Zhang, Haili; Yu, Aimin

    2015-09-15

    A novel ultrasensitive immunoassay method was developed by combination of the enzymatically catalytic gold deposition with the prepared gold nanoprobe and the gold stripping analysis at an electrochemical chip based immunosensor. The immunosensor was constructed through covalently immobilizing capture antibody at a carbon nanotube (CNT) modified screen-printed carbon electrode. The gold nanoprobe was prepared by loading signal antibody and high-content glucose oxidase (GOD) on the nanocarrier of gold nanorod (Au NR). After sandwich immunoreaction, the GOD-Au NR nanoprobe could be quantitatively captured onto the immunosensor surface and then induce the deposition of gold nanoparticles (Au NPs) via the enzymatically catalytic reaction. Based on the electrochemical stripping analysis of the Au NR nanocarriers and the enzymatically produced Au NPs, sensitive electrochemical signal was obtained for the immunoassay. Both the GOD-induced deposition of Au NPs by the nanoprobe and the sensitive electrochemical stripping analysis on the CNTs based sensing surface greatly amplified the signal response, leading to the ultrahigh sensitivity of this method. Using carcinoembryonic antigen as a model analyte, excellent analytical performance including a wide linear range from 0.01 to 100 ng/mL and a detection limit down to 4.2 pg/mL was obtained. In addition, this immunosensor showed high specificity and satisfactory reproducibility, stability and reliability. The relatively positive detection potential excluded the conventional interference from dissolved oxygen. Thus this electrochemical chip based immunosensing method provided great potentials for practical applications. PMID:25932794

  2. Unraveling the Enzymatic Basis of Wine "Flavorome": A Phylo-Functional Study of Wine Related Yeast Species.

    PubMed

    Belda, Ignacio; Ruiz, Javier; Alastruey-Izquierdo, Ana; Navascués, Eva; Marquina, Domingo; Santos, Antonio

    2016-01-01

    Non-Saccharomyces yeasts are a heterogeneous microbial group involved in the early stages of wine fermentation. The high enzymatic potential of these yeasts makes them a useful tool for increasing the final organoleptic characteristics of wines in spite of their low fermentative power. Their physiology and contribution to wine quality are still poorly understood, with most current knowledge being acquired empirically and in most cases based in single species and strains. This work analyzed the metabolic potential of 770 yeast isolates from different enological origins and representing 15 different species, by studying their production of enzymes of enological interest and linking phylogenetic and enzymatic data. The isolates were screened for glycosidase enzymes related to terpene aroma release, the β-lyase activity responsible for the release of volatile thiols, and sulfite reductase. Apart from these aroma-related activities, protease, polygalacturonase and cellulase activities were also studied in the entire yeast collection, being related to the improvement of different technological and sensorial features of wines. In this context, and in terms of abundance, two different groups were established, with α-L-arabinofuranosidase, polygalacturonase and cellulase being the less abundant activities. By contrast, β-glucosidase and protease activities were widespread in the yeast collection studied. A classical phylogenetic study involving the partial sequencing of 26S rDNA was conducted in conjunction with the enzymatic profiles of the 770 yeast isolates for further typing, complementing the phylogenetic relationships established by using 26S rDNA. This has rendered it possible to foresee the contribution different yeast species make to wine quality and their potential applicability as pure inocula, establishing species-specific behavior. These consistent results allowed us to design future targeted studies on the impact different non-Saccharomyces yeast species

  3. Unraveling the Enzymatic Basis of Wine “Flavorome”: A Phylo-Functional Study of Wine Related Yeast Species

    PubMed Central

    Belda, Ignacio; Ruiz, Javier; Alastruey-Izquierdo, Ana; Navascués, Eva; Marquina, Domingo; Santos, Antonio

    2016-01-01

    Non-Saccharomyces yeasts are a heterogeneous microbial group involved in the early stages of wine fermentation. The high enzymatic potential of these yeasts makes them a useful tool for increasing the final organoleptic characteristics of wines in spite of their low fermentative power. Their physiology and contribution to wine quality are still poorly understood, with most current knowledge being acquired empirically and in most cases based in single species and strains. This work analyzed the metabolic potential of 770 yeast isolates from different enological origins and representing 15 different species, by studying their production of enzymes of enological interest and linking phylogenetic and enzymatic data. The isolates were screened for glycosidase enzymes related to terpene aroma release, the β-lyase activity responsible for the release of volatile thiols, and sulfite reductase. Apart from these aroma-related activities, protease, polygalacturonase and cellulase activities were also studied in the entire yeast collection, being related to the improvement of different technological and sensorial features of wines. In this context, and in terms of abundance, two different groups were established, with α-L-arabinofuranosidase, polygalacturonase and cellulase being the less abundant activities. By contrast, β-glucosidase and protease activities were widespread in the yeast collection studied. A classical phylogenetic study involving the partial sequencing of 26S rDNA was conducted in conjunction with the enzymatic profiles of the 770 yeast isolates for further typing, complementing the phylogenetic relationships established by using 26S rDNA. This has rendered it possible to foresee the contribution different yeast species make to wine quality and their potential applicability as pure inocula, establishing species-specific behavior. These consistent results allowed us to design future targeted studies on the impact different non-Saccharomyces yeast species

  4. Suppression subtractive hybridization identifies genes induced in response to UV-B irradiation in apple skin: isolation of a putative UDP-glucose 4-epimerase.

    PubMed

    Ban, Yusuke; Honda, Chikako; Bessho, Hideo; Pang, Xiao-Ming; Moriguchi, Takaya

    2007-01-01

    Suppression subtractive hybridization (SSH) successfully identified 11 cDNAs in apple skin with highly induced expression as a result of ultraviolet (UV)-B irradiation. Apart from three putative flavonoid biosynthetic genes, chalcone synthase (CHS; A5C), flavanone-3-hydroxylase (F3H; B5F), and flavonol synthase (FLS; D1F), five clones (A1H, A10E, B11G, D5F, and D11H) were induced by low temperature (17 degrees C) as well, which is also known to induce anthocyanin accumulation in apple skin. Moreover, four clones (A1H, A10E, B11G, and D11H), showing higher expression levels in the skin, accumulated higher anthocyanin concentrations than their counterparts. Of the four clones, only A10E, a putative UDP-glucose 4-epimerase (UGE), was deemed to play an important role in anthocyanin accumulation in apple skin based on the facts that: (i) its transcription level was higher in the deep red cultivar, 'Jonathan', than in the pale red cultivar, 'Tsugaru'; and (ii) it could reversibly catalyse UDP-glucose to UDP-galactose, and the latter molecule is a major sugar donor for cyanidin-glycoside in apple. Therefore, the full-length cDNA of A10E was isolated by rapid amplification of cDNA ends (RACE) and designated as MdUGE1. Further analysis demonstrated that UGE enzymatic activity was positively correlated with anthocyanin accumulation in apple skin. Thus, MdUGE1 isolated by SSH could play an important role in anthocyanin biosynthesis in apple skin in concert with other flavonoid biosynthetic genes. PMID:17404384

  5. The cloning and sequencing of the UDP-galactose 4-epimerase gene (galE) from Avibacterium paragallinarum.

    PubMed

    Roodt, Yolande; Bragg, Robert; Albertyn, Jacobus

    2007-08-01

    The putative uridine diphosphate (UDP)-galactose 4-epimerase encoding gene, galE, was isolated from Avibacterium paragallinarum with the use of degenerate primers, colony hybridization and inverse PCR. The data revealed an open reading frame of 1017 bp encoding a protein of 338 amino acids with a molecular weight of 37 kDa and an isoelectric point of 5.5. High sequence homology was obtained with an 87, 91 and 89% sequence identity on protein level towards the galE genes from Actinobacillus pleuropneumoniae, Haemophilus influenza and Pasteurella multocida, respectively. To verify that the cloned galE gene encodes for a UDP-galactose 4-epimeras, this gene was cloned into the pYES-2 expression vector, followed by transformation in a Saccharomyces cerevisiae gal10 deletion strain. Complementation of the gal10 deletion mutant with the galE gene confirmed that this gene encodes a UDP-galactose 4-epimerase. PMID:17541831

  6. Functionalization of monolithic and porous three-dimensional graphene by one-step chitosan electrodeposition for enzymatic biosensor.

    PubMed

    Liu, Jiyang; Wang, Xiaohui; Wang, Tianshu; Li, Dan; Xi, Fengna; Wang, Jin; Wang, Erkang

    2014-11-26

    Biological modification of monolithic and porous 3D graphene is of great significance for extending its application in fabricating highly sensitive biosensors. The present work reports on the first biofunctionalization of monolithic and freestanding 3D graphene foam for one-step preparation of reagentless enzymatic biosensors by controllable chitosan (CS) electrodeposition technology. Using a homogeneous three-component electrodeposition solution containing a ferrocene (Fc) grafted CS hybrid (Fc-CS), glucose oxidase (GOD), and single-walled carbon nanotubes (SWNTs), a homogeneous biocomposite film of Fc-CS/SWNTs/GOD was immobilized on the surface of 3D graphene foam by one-step electrodeposition. The Fc groups grafted on chitosan can be stably immobilized on the 3D graphene surface and keep their original electrochemical activity. The SWNTs doped into the Fc-CS matrix act as a nanowire to facilitate electron transfer and improve the conductivity of the biocomposite film. Combined with the extraordinary properties of 3D graphene foam including large active surface area, high conductivity, and fast mass transport dynamics, the 3D graphene based enzymatic biosensor achieved a large linear range (5.0 μM to 19.8 mM), a low detection limit (1.2 μM), and rapid response (reaching the 95% steady-state response within 8 s) for reagentless detection of glucose in the phosphate buffer solution. PMID:25384251

  7. Mechanism and inhibition of human UDP-GlcNAc 2-epimerase, the key enzyme in sialic acid biosynthesis

    PubMed Central

    Chen, Sheng-Chia; Huang, Chi-Hung; Lai, Shu-Jung; Yang, Chia Shin; Hsiao, Tzu-Hung; Lin, Ching-Heng; Fu, Pin-Kuei; Ko, Tzu-Ping; Chen, Yeh

    2016-01-01

    The bifunctional enzyme UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) plays a key role in sialic acid production. It is different from the non-hydrolyzing enzymes for bacterial cell wall biosynthesis, and it is feed-back inhibited by the downstream product CMP-Neu5Ac. Here the complex crystal structure of the N-terminal epimerase part of human GNE shows a tetramer in which UDP binds to the active site and CMP-Neu5Ac binds to the dimer-dimer interface. The enzyme is locked in a tightly closed conformation. By comparing the UDP-binding modes of the non-hydrolyzing and hydrolyzing UDP-GlcNAc epimerases, we propose a possible explanation for the mechanistic difference. While the epimerization reactions of both enzymes are similar, Arg113 and Ser302 of GNE are likely involved in product hydrolysis. On the other hand, the CMP-Neu5Ac binding mode clearly elucidates why mutations in Arg263 and Arg266 can cause sialuria. Moreover, full-length modelling suggests a channel for ManNAc trafficking within the bifunctional enzyme. PMID:26980148

  8. Structural Basis for Substrate Specificity in Phosphate Binding (β/α)8-Barrels: D-Allulose 6-Phosphate 3-Epimerase from Escherichia coli K-12†

    PubMed Central

    Chan, Kui K.; Fedorov, Alexander A.; Fedorov, Elena V.; Almo, Steven C.; Gerlt, John A.

    2008-01-01

    Enzymes that share the (β/α)8-barrel fold catalyze a diverse range of reactions. Many utilize phosphorylated substrates and share a conserved C-terminal (β/α)2-quarter barrel subdomain that provides a binding motif for the dianionic phosphate group. We recently reported functional and structural studies of D-ribulose 5-phosphate 3-epimerase (RPE) from Streptococcus pyogenes that catalyzes the equilibration of the pentulose 5-phosphates D-ribulose 5-phosphate and D-xylulose 5-phosphate in the pentose phosphate pathway [J. Akana, A. A. Fedorov, E. Fedorov, W. R. P. Novack, P. C. Babbitt, S. C. Almo, and J. A. Gerlt (2006) Biochemistry 45, 2493–2503]. We now report functional and structural studies of D-allulose 6-phosphate 3-epimerase (ALSE) from Escherichia coli K-12 that catalyzes the equilibration of the hexulose 6-phosphates D-allulose 6-phosphate and D-fructose 6-phosphate in a catabolic pathway for D-allose. ALSE and RPE prefer their physiological substrates but are promiscuous for each other’s substrate. The active sites (RPE complexed with D-xylitol 5-phosphate and ALSE complexed with D-glucitol 6-phosphate) are superimposable (as expected from their 39% sequence identity), with the exception of the phosphate binding motif. The loop following the eighth β-strand in ALSE is one residue longer than the homologous loop in RPE, so the binding site for the hexulose 6-phosphate substrate/product in ALSE is elongated relative to that for the pentulose 5-phosphate substrate/product in RPE. We constructed three single-residue deletion mutants of the loop in ALSE, ΔT196, ΔS197 and ΔG198, to investigate the structural bases for the differing substrate specificities; for each, the promiscuity is altered so that D-ribulose 5-phosphate is the preferred substrate. The changes in kcat/Km are dominated by changes in kcat, suggesting that substrate discrimination results from differential transition state stabilization. In both ALSE and RPE, the phosphate group

  9. Structural Basis for Substrate Specificity in Phosphate Binding (beta/alpha)8-Barrels: D-Allulose 6-Phosphate 3-Epimerase from Escherichia coli K-12

    SciTech Connect

    Chan,K.; Fedorov, A.; Almo, S.; Gerlt, J.

    2008-01-01

    Enzymes that share the ({beta}/{alpha})8-barrel fold catalyze a diverse range of reactions. Many utilize phosphorylated substrates and share a conserved C-terminal ({beta}/a)2-quarter barrel subdomain that provides a binding motif for the dianionic phosphate group. We recently reported functional and structural studies of d-ribulose 5-phosphate 3-epimerase (RPE) from Streptococcus pyogenes that catalyzes the equilibration of the pentulose 5-phosphates d-ribulose 5-phosphate and d-xylulose 5-phosphate in the pentose phosphate pathway [J. Akana, A. A. Fedorov, E. Fedorov, W. R. P. Novack, P. C. Babbitt, S. C. Almo, and J. A. Gerlt (2006) Biochemistry 45, 2493-2503]. We now report functional and structural studies of d-allulose 6-phosphate 3-epimerase (ALSE) from Escherichia coli K-12 that catalyzes the equilibration of the hexulose 6-phosphates d-allulose 6-phosphate and d-fructose 6-phosphate in a catabolic pathway for d-allose. ALSE and RPE prefer their physiological substrates but are promiscuous for each other's substrate. The active sites (RPE complexed with d-xylitol 5-phosphate and ALSE complexed with d-glucitol 6-phosphate) are superimposable (as expected from their 39% sequence identity), with the exception of the phosphate binding motif. The loop following the eighth {beta}-strand in ALSE is one residue longer than the homologous loop in RPE, so the binding site for the hexulose 6-phosphate substrate/product in ALSE is elongated relative to that for the pentulose 5-phosphate substrate/product in RPE. We constructed three single-residue deletion mutants of the loop in ALSE, ?T196, ?S197 and ?G198, to investigate the structural bases for the differing substrate specificities; for each, the promiscuity is altered so that d-ribulose 5-phosphate is the preferred substrate. The changes in kcat/Km are dominated by changes in kcat, suggesting that substrate discrimination results from differential transition state stabilization. In both ALSE and RPE, the phosphate

  10. D-ribulose-5-phosphate 3-epimerase: Cloning and heterologous expression of the spinach gene, and purification and characterization of the recombinant enzyme

    SciTech Connect

    Chen, Y.R.; Hartman, F.C.; Lu, T.Y.S.; Larimer, F.W.

    1998-09-01

    The authors have achieved, to their knowledge, the first high-level heterologous expression of the gene encoding D-ribulose-5-phosphate 3-epimerase from any source, thereby permitting isolation and characterization of the epimerase as found in photosynthetic organisms. The extremely labile recombinant spinach (Spinacia oleracea L.) enzyme was stabilized by DL-{alpha}-glycerophosphate or ethanol and destabilized by D-ribulose-5-phosphate or 2-mercaptoethanol. Despite this lability, the unprecedentedly high specific activity of the purified material indicates that the structural integrity of the enzyme is maintained throughout isolation. Ethylenediaminetetraacetate and divalent metal cations did not affect epimerase activity, thereby excluding a requirement for the latter in catalysis. As deduced from the sequence of the cloned spinach gene and the electrophoretic mobility under denaturing conditions of the purified recombinant enzyme, its 25-kD subunit size was about the same as that of the corresponding epimerases of yeast and mammals. However, in contrast to these other species, the recombinant spinach enzyme was octameric rather than dimeric, as assessed by gel filtration and polyacrylamide gel electrophoresis under nondenaturing conditions. Western-blot analyses with antibodies to the purified recombinant enzyme confirmed that the epimerase extracted from spinach leaves is also octameric.

  11. Enhancement of isomerization activity and lactulose production of cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus.

    PubMed

    Shen, Qiuyun; Zhang, Yuzhu; Yang, Ruijin; Pan, Siyi; Dong, Juan; Fan, Yuting; Han, Liang

    2016-09-15

    Industrial application of Caldicellulosiruptor saccharolyticus cellobiose 2-epimerase (CsCE) for lactulose synthesis is limited by low enzyme activity and formation of epilactose as by-product. After four sequential rounds of random mutagenesis and screening, an optimal mutant G4-C5 was obtained. Compared with wild type (WT) enzyme, mutant G4-C5 demonstrated 2.8- and 3.0-fold increases in specific activity and kcat/Km for lactulose production, respectively, without compromising thermostability. DNA sequencing of mutant G4-C5 revealed five amino acid substitutions, namely, R5M, I52V, A12S, K328I and F231L, which were located on the protein surface, except for the mutation I52V. The yield of lactulose catalyzed by mutant G4-C5 increased to approximately 76% with no obvious epilactose detected, indicating that mutant G4-C5 was more suitable for lactulose production than the WT enzyme. PMID:27080880

  12. Rare case of homozygous epimerase deficiency and heterozygous of duarte 2 variant.

    PubMed

    Koliofoti, Eleana Georgia; Gkentzi, Despoina; Varvarigou, Anastasia; Trigka, Maria; Schulpis, Kleopatra

    2014-09-01

    We present a rare case of galactosemia identified by a positive screening test. A 20-day-old female infant was admitted with jaundice and bloody stained diarrhea. There was no history of fever, convulsions, abdominal distention, or bleeding from other sites. Laboratory findings indicated elevated total billirubin, alanine transaminase, aspartate aminotransferase, alkaline phosphatase, and gamma-glutamyl transferase. International normalized ratio (INR), prothrombin time (PT) and activated partial thromboplastin time (aPTT) were prolonged. Total vitamin D was low. Quantitative assay for GALT in hemolysates of RBC: 17 μmol/min/mg protein (normal values: 20-35) (compound heterozygous for D2/N: 16-19). GALE level in RBC hemolysate: 11.5 μmol/h/g Hb (normal values 19-35). Our patient was homozygous for the peripheral form of epimerase deficiency galactosemia, as well as heterozygous for GALT/(D2) deficiency. She was started on galactose restricted diet and vitamin supplementation. At the age of 10 months, the patient appeared normal with no signs of developmental delay or eye-cataract. PMID:24859500

  13. Chemical synthesis and enzymatic, stereoselective hydrolysis of a functionalized dihydropyrimidine for the synthesis of β-amino acids.

    PubMed

    Slomka, Christin; Zhong, Sabilla; Fellinger, Anna; Engel, Ulrike; Syldatk, Christoph; Bräse, Stefan; Rudat, Jens

    2015-12-01

    A novel substrate, 6-(4-nitrophenyl)dihydropyrimidine-2,4(1H,3H)-dione (pNO2PheDU), was chemically synthesized and analytically verified for the potential biocatalytic synthesis of enantiopure β-amino acids. The hydantoinase (EC 3.5.2.2) from Arthrobacter crystallopoietes DSM20117 was chosen to prove the enzymatic hydrolysis of this substrate, since previous investigations showed activities of this enzyme toward 6-monosubstituted dihydrouracils. Whole cell biotransformations with recombinant Escherichia coli expressing the hydantoinase showed degradation of pNO2PheDU. Additionally, the corresponding N-carbamoyl-β-amino acid (NCarbpNO2 βPhe) was chemically synthesized, an HPLC-method with chiral stationary phases for detection of this product was established and thus (S)-enantioselectivity toward pNO2PheDU has been shown. Consequently this novel substrate is a potential precursor for the enantiopure β-amino acid para-nitro-β-phenylalanine (pNO2 βPhe). PMID:26705241

  14. Marek's disease virus (MDV) ubiquitin-specific protease (USP) performs critical functions beyond its enzymatic activity during virus replication.

    PubMed

    Veiga, Inês B; Jarosinski, Keith W; Kaufer, Benedikt B; Osterrieder, Nikolaus

    2013-03-15

    Marek's disease virus (MDV) encodes an ubiquitin-specific protease (USP) within its UL36 gene. USP is highly conserved among herpesviruses and was shown to be important for MDV replication and pathogenesis in MDV's natural host, the chicken. To further investigate the role of MDV USP, several recombinant (r) MDVs were generated and their in vitro phenotypes were evaluated using plaque size and growth kinetics assays. We discovered that the N-terminus of pUL36 is essential for MDV replication and could not be complemented by ectopic expression of MDV USP. In addition, we demonstrated that the region located between the conserved glutamine (Q85) and leucine (L106) residues comprising the active site cysteine (C98) is also essential for MDV replication. Based on the analyses of the rMDVs generated here, we concluded that MDV USP likely contributes to the structure and/or stability of pUL36 and affects replication and oncogenesis of MDV beyond its enzymatic activity. PMID:23399034

  15. Study of oxidative, enzymatic mitochondrial respiratory chain function and apoptosis in perinatally HIV-infected pediatric patients.

    PubMed

    Morén, Constanza; Garrabou, Glòria; Noguera-Julian, Antoni; Rovira, Núria; Catalán, Marc; Hernández, Sandra; Tobías, Ester; Cardellach, Francesc; Fortuny, Clàudia; Miró, Òscar

    2013-10-01

    Mitochondrial toxicity in perinatally human immunodeficiency virus (HIV)-infected pediatric patients has been scarcely investigated. Limited data are available about HIV or antiretroviral (ARV)-mediated mitochondrial damage in this population group, specifically, regarding oxygen consumption and apoptosis approach. We aimed to elucidate whether a given mitochondrial DNA depletion is reflected at downstream levels, to gain insight on the pathology of HIV and highly active antiretroviral therapy (HAART) in perinatally HIV-infected pediatric patients. We studied 10 healthy control participants and 20 perinatally HIV-infected pediatric patients (10 under ARV treatment and 10 off treatment). We determined mitochondrial mass, subunits II and IV of complex IV, global and specific mitochondrial enzymatic and oxidative activities, and apoptosis from peripheral blood mononuclear cells. Global oxygen consumption was significantly compromised in HIV-infected untreated patients, compared to the control group (0.76 ± 0.01 versus 1.59 ± 0.15; P = 0.014). Apoptosis showed a trend to increase in untreated patients as well. The overall complex (C) CI-III-IV activity of the mitochondrial respiratory chain (MRC) was significantly decreased in HIV-infected treated patients with respect to the control group (1.52 ± 0.38 versus 6.38 ± 1.53; P = 0.02). No statistically significant differences were found between untreated and HAART-treated patients. These findings suggest the pathogenic role of both HIV and HAART in mitochondrial dysfunction in vertical infection. The abnormalities in mitochondrial genome may be downstream reflected through a global alteration of the MRC. Mitochondrial impairment associated with HIV and HAART was generalized, rather than localized, in this series of perinatally HIV-infected patients. PMID:23534415

  16. Enzymatic Synthesis and Functional Characterization of Bioactive Microcin C-Like Compounds with Altered Peptide Sequence and Length

    PubMed Central

    Bantysh, Olga; Serebryakova, Marina; Zukher, Inna; Kulikovsky, Alexey; Tsibulskaya, Darya; Dubiley, Svetlana

    2015-01-01

    ABSTRACT Escherichia coli microcin C (McC) consists of a ribosomally synthesized heptapeptide attached to a modified adenosine. McC is actively taken up by sensitive Escherichia coli strains through the YejABEF transporter. Inside the cell, McC is processed by aminopeptidases, which release nonhydrolyzable aminoacyl adenylate, an inhibitor of aspartyl-tRNA synthetase. McC is synthesized by the MccB enzyme, which terminally adenylates the MccA heptapeptide precursor MRTGNAN. Earlier, McC analogs with shortened peptide lengths were prepared by total chemical synthesis and were shown to have strongly reduced biological activity due to decreased uptake. Variants with longer peptides were difficult to synthesize, however. Here, we used recombinant MccB to prepare and characterize McC-like molecules with altered peptide moieties, including extended peptide lengths. We find that N-terminal extensions of E. coli MccA heptapeptide do not affect MccB-catalyzed adenylation and that some extended-peptide-length McC analogs show improved biological activity. When the peptide length reaches 20 amino acids, both YejABEF and SbmA can perform facilitated transport of toxic peptide adenylates inside the cell. A C-terminal fusion of the carrier maltose-binding protein (MBP) with the MccA peptide is also recognized by MccB in vivo and in vitro, allowing highly specific adenylation and/or radioactive labeling of cellular proteins. IMPORTANCE Enzymatic adenylation of chemically synthesized peptides allowed us to generate biologically active derivatives of the peptide-nucleotide antibiotic microcin C with improved bioactivity and altered entry routes into target cells, opening the way for development of various McC-based antibacterial compounds not found in nature. PMID:26195597

  17. UDP-GlcNAc2-epimerase regulates cell surface sialylation and ceramide-induced cell death in human malignant lymphoma.

    PubMed

    Suzuki, Osamu; Tasaki, Kazuhiro; Kusakabe, Takashi; Abe, Masafumi

    2008-09-01

    Stress signals induce ceramide (cer) through sphingomyelinase activation, and metabolites of cer such as sphingosine (Sph) and sphingosine-1-phoshate (S-1-P) play a significant role in many biological processes. This study aimed to elucidate the association between the alteration in cell surface sialylation and ceramide-induced cell death in the human Burkitt's lymphoma cell line, HBL-8. The highly sialylated 3G3 clone was less sensitive to C6-ceramide-induced cell death. On the other hand, the hyposialylated 3D2 clone was more sensitive to C6-ceramide-induced cell death. Neuraminidase treatment or knockdown by siRNA of uridine diphosphate-N-acetylglucosamine 2-epimerase (UDP-GlcNAc2-epimerase), which is a key enzyme of sialic acid biosynthesis, enhanced the amount of cell death induced by C6-ceramide in the highly sialylated 3G3 clone. Sialic acid metabolic complementation assays using several precursors of sialic acid showed that cell surface resialylation by N-acetyl-D-mannosamine (ManNAc) inhibited C6-ceramide-induced cell death. The amount of cell death by C6-ceramide was enhanced after pretreatment with phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002 in both clones. In addition, clone 3G3 was less sensitive to Sph than the 3D2 clone. In conclusion, in human malignant lymphoma, ceramide and its metabolite-induced cell death is regulated by the amount of sialic acid on the cell surface which in turn is regulated by mRNA expression of UDP-GlcNAc2-epimerase. PMID:18698493

  18. Carbon felt-based biocatalytic enzymatic flow-through detectors: chemical modification of tyrosinase onto amino-functionalized carbon felt using various coupling reagents.

    PubMed

    Wang, Yue; Hasebe, Yasushi

    2009-09-15

    Tyrosinase (TYR) was covalently immobilized onto amino-functionalized carbon felt (CF) surface via eight different coupling reagents. Prior to the TYR-immobilization, primary amino group was introduced to the CF surface by the treatment with 3-aminopropyltriethoxysilane (APTES). The APTES modification of the CF surface was confirmed by XPS and SEM measurements. The terminal amino groups on the CF surface were cross-linked with protein lysine group (or cysteine group) using various coupling reagents. The resulting TYR-immobilized CF (TYR-CF) was utilized as a working electrode unit of a biocatalytic enzymatic flow-through detector. Catechol and 4-chlorophenol (4-CP) were used as model analytes for the evaluation of catecholase activity and phenolase activity, respectively, and flow injection peaks based on the electro-reduction of the enzymatically produced o-quinone species were monitored at -0.05 V vs. Ag/AgCl. Among eight coupling reagents, glutaraldehyde (GA) exhibited the best results on the sensitivity, the operational stability and the storage stability. The detection limits of catechol and 4-CP obtained by the GA-coupling method were found to be 6.0 x 10(-9)M and 1.5 x 10(-8)M, respectively with the sample through-put of 36 samples/h. No serious degradation of the peak current was observed over 30 consecutive samples injections on the GA-coupling method, while gradual decrease in the peak currents was observed on other seven coupling reagents. The GA-coupling method showed the best results on the storage stability, and 85% of original activity for catechol oxidation remained after 25 days storage. PMID:19615522

  19. Vi Antigen Biosynthesis in Salmonella typhi: Characterization of UDP-N-acetylglucosamine C-6 Dehydrogenase (TviB) and UDP-N-acetylglucosaminuronic Acid C-4 Epimerase (TviC)†

    PubMed Central

    Zhang, Hua; Zhou, Ying; Bao, Hongbo; Liu, Hung-wen

    2008-01-01

    Vi antigen, the virulence factor of Salmonella typhi, has been used clinically as a molecular vaccine. TviB and TviC are two enzymes involved in the formation of Vi antigen, a linear polymer consisting of α-1,4-linked N-acetylgalactosaminuronate. Protein sequence analysis suggests that TviB is a dehydrogenase and TviC is an epimerase. Both enzymes are expected to be NAD+ dependent. In order to verify their functions, TviB and TviC were cloned, expressed in Escherichia coli, and characterized. The C-terminal His6-tagged TviB protein, purified from soluble cell fractions in the presence of 10 mM DTT, shows UDP-N-acetylglucosamine 6-dehydrogenase activity, and is capable of catalyzing the conversion of UDP-N-acetylglucosamine (UDP-GlcNAc) to UDP-N-acetylglucosaminuronic acid (UDP-GlcNAcA) with a kcat value of 15.5 ± 1.0 min−1. The Km values of TviB for UDP-GlcNAc and NAD+ are 77 ± 9 μM and 276 ± 52 μM, respectively. TviC, purified as C-terminal hexahistidine-tagged protein, shows UDP-GlcNAcA 4-epimerase and UDP-N-acetylgalactosamine (UDP-GalNAc) 4-epimerase activities. The Km values of TviC for UDP-GlcNAcA and UDP-N-acetylgalactosaminuronic acid (UDP-GalNAcA) are 20 ± 1 μM and 42 ± 2 μM, respectively. The kcat value for the conversion of UDP-GlcNAcA to UDP-GalNAcA is 56.8 ± 0.5 min−1, while that for the reverse reaction is 39.1 ± 0.6 min−1. These results show that the biosynthesis of Vi antigen is initiated by the TviB-catalyzed oxidation of UDP-GlcNAc to UDP-GalNAc, followed by the TviC-catalyzed epimerization at C-4 to form UDP-GalNAcA, which serves as the building block for the formation of Vi polymer. These results set the stage for future in vitro biosynthesis of Vi antigen. These enzymes may also be drug targets to inhibit Vi antigen production. PMID:16800641

  20. Enzymatic Hydrolysis of Cellulosic Biomass

    SciTech Connect

    Yang, Bin; Dai, Ziyu; Ding, Shi-You; Wyman, Charles E.

    2011-08-22

    Biological conversion of cellulosic biomass to fuels and chemicals offers the high yields to products vital to economic success and the potential for very low costs. Enzymatic hydrolysis that converts lignocellulosic biomass to fermentable sugars may be the most complex step in this process due to substrate-related and enzyme-related effects and their interactions. Although enzymatic hydrolysis offers the potential for higher yields, higher selectivity, lower energy costs, and milder operating conditions than chemical processes, the mechanism of enzymatic hydrolysis and the relationship between the substrate structure and function of various glycosyl hydrolase components are not well understood. Consequently, limited success has been realized in maximizing sugar yields at very low cost. This review highlights literature on the impact of key substrate and enzyme features that influence performance to better understand fundamental strategies to advance enzymatic hydrolysis of cellulosic biomass for biological conversion to fuels and chemicals. Topics are summarized from a practical point of view including characteristics of cellulose (e.g., crystallinity, degree of polymerization, and accessible surface area) and soluble and insoluble biomass components (e.g., oligomeric xylan, lignin, etc.) released in pretreatment, and their effects on the effectiveness of enzymatic hydrolysis. We further discuss the diversity, stability, and activity of individual enzymes and their synergistic effects in deconstructing complex lignocellulosic biomass. Advanced technologies to discover and characterize novel enzymes and to improve enzyme characteristics by mutagenesis, post-translational modification, and over-expression of selected enzymes and modifications in lignocellulosic biomass are also discussed.

  1. Optimization of extraction efficiency by shear emulsifying assisted enzymatic hydrolysis and functional properties of dietary fiber from deoiled cumin (Cuminum cyminum L.).

    PubMed

    Ma, Mengmei; Mu, Taihua; Sun, Hongnan; Zhang, Miao; Chen, Jingwang; Yan, Zhibin

    2015-07-15

    This study evaluated the optimal conditions for extracting dietary fiber (DF) from deoiled cumin by shear emulsifying assisted enzymatic hydrolysis (SEAEH) using the response surface methodology. Fat adsorption capacity (FAC), glucose adsorption capacity (GAC), and bile acid retardation index (BRI) were measured to evaluate the functional properties of the extracted DF. The results revealed that the optimal extraction conditions included an enzyme to substrate ratio of 4.5%, a reaction temperature of 57 °C, a pH value of 7.7, and a reaction time of 155 min. Under these conditions, DF extraction efficiency and total dietary fiber content were 95.12% and 84.18%, respectively. The major components of deoiled cumin DF were hemicellulose (37.25%) and cellulose (33.40%). FAC and GAC increased with decreasing DF particle size (51-100 μm), but decreased with DF particle sizes <26 μm; BRI increased with decreasing DF particle size. The results revealed that SEAEH is an effective method for extracting DF. DF with particle size 26-51 μm had improved functional properties. PMID:25722165

  2. High-level soluble expression of a bacterial N-acyl-d-glucosamine 2-epimerase in recombinant Escherichia coli.

    PubMed

    Klermund, Ludwig; Riederer, Amelie; Groher, Anna; Castiglione, Kathrin

    2015-07-01

    N-Acyl-d-glucosamine 2-epimerase (AGE) is an important enzyme for the biocatalytic synthesis of N-acetylneuraminic acid (Neu5Ac). Due to the wide range of biological applications of Neu5Ac and its derivatives, there has been great interest in its large-scale synthesis. Thus, suitable strategies for achieving high-level production of soluble AGE are needed. Several AGEs from various organisms have been recombinantly expressed in Escherichia coli. However, the soluble expression level was consistently low with an excessive formation of inclusion bodies. In this study, the effects of different solubility-enhancement tags, expression temperatures, chaperones and host strains on the soluble expression of the AGE from the freshwater cyanobacterium Anabaena variabilis ATCC 29413 (AvaAGE) were examined. The optimum combination of tag, expression temperature, co-expression of chaperones and host strain (His6-tag, 37°C, GroEL/GroES, E. coli BL21(DE3)) led to a 264-fold improvement of the volumetric epimerase activity, a measure of the soluble expression, compared to the starting conditions (His6-maltose-binding protein-tag, 20°C, without chaperones, E. coli BL21(DE3)). A maximum yield of 22.5mg isolated AvaAGE per liter shake flask culture was obtained. PMID:25804337

  3. Value Added Processing of Peanut Meal: Enzymatic Hydrolysis to Improve Functional and Nutritional Properties of Water Soluble Extracts

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Value added applications are needed for peanut meal, which is the high protein byproduct of commercial peanut oil production. Peanut meal dispersions were hydrolyzed with alcalase, flavourzyme and pepsin in an effort to improve functional and nutritional properties of the resulting water soluble ex...

  4. Ex vivo Enzymatic Treatment of Aged CD4 T Cells Restores Cognate T-cell Helper Function and Enhances Antibody Production in Mice

    PubMed Central

    Perkey, Eric; Miller, Richard A.; Garcia, Gonzalo G.

    2012-01-01

    Previous in vitro studies have shown that CD4 T cells from old mice have defects in T cell receptor (TCR) signaling, immune synapse formation, activation, and proliferation. We have reported that removing a specific set of surface glycoproteins by ex vivo treatment with O-sialoglycoprotein endopeptidase (OSGE) can reverse many aspects of the age-related decline in CD4 T cell function. However, the specific mechanism by which this process occurs remains unclear, and it is unknown whether this enzymatic treatment can also restore important aspects of adaptive immunity in vivo. By using an in vivo model of the immune response based on adoptive transfer of CD4 T cells from pigeon cytochrome C (PCC)-specific transgenic H-2(k/k) TCR-Vα11Vβ3 CD4+ mice to syngeneic hosts, we now demonstrat that aging diminishes CD28 costimulatory signals in CD4 T cells. These age-associated defects include changes in phosphorylation of AKT and expression of glucose transporter type I, inducible T-cell costimulatory molecule, and CD40 ligand, suggesting that the lack of CD28 costimulation contributes to age-dependent loss of CD4 function. All of these deficits can be reversed by ex vivo OSGE treatment. Blocking B7-CD28 interactions on T cells prevents OSGE-mediated restoration of T cell function, suggesting that changes in surface glycosylation, including CD28, may be responsible for age-related costimulation decline. Finally, we showed that the age-related decline in CD4 cognate helper function for immunoglobin G production and long-term humoral immunity can also be restored by OSGE treatments of CD4 T cells prior to adoptive transfer. PMID:23136198

  5. Use of protein trans-splicing to produce active and segmentally 2H, 15N labeled mannuronan C5-epimerase AlgE4

    PubMed Central

    Buchinger, Edith; Aachmann, Finn L; Aranko, A Sesilja; Valla, Svein; Skjåk-BræK, Gudmund; Iwaï, Hideo; Wimmer, Reinhard

    2010-01-01

    Alginate epimerases are large multidomain proteins capable of epimerising C5 on β-d-mannuronic acid (M) turning it into α-l-guluronic acid (G) in a polymeric alginate. Azotobacter vinelandii secretes a family of seven epimerases, each of which is capable of producing alginates with characteristic G distribution patterns. All seven epimerases consist of two types of modules, denoted A and R, in varying numbers. Attempts to study these enzymes with solution-state NMR are hampered by their size—the smallest epimerase, AlgE4, consisting of one A- and one R-module, is 58 kDa, resulting in heavy signal overlap impairing the interpretation of NMR spectra. Thus we obtained segmentally 2H, 15N labeled AlgE4 isotopomeres (A-[2H, 15N]-R and [2H, 15N]-A-R) by protein trans-splicing using the naturally split intein of Nostoc punctiforme. The NMR spectra of native AlgE4 and the ligated versions coincide well proving the conservation of protein structure. The activity of the ligated AlgE4 was verified by two different enzyme activity assays, demonstrating that ligated AlgE4 displays the same catalytic activity as wild-type AlgE4. PMID:20552686

  6. Microbial conversion of L-arabinose to xylitol by coexpression of L-arabinose isomerase, D-tagatose 3-epimerase, and L-xylulose reductase in Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A microbial strain has been developed that can produce xylitol from L-arabinose at a high yield by transforming Escherichia coli with a new xylitol biosynthetic pathway consisting of L-arabinose isomerase, D-tagatose 3-epimerase, and L-xylulose reductase. An E. coli strain that heterologously expre...

  7. IMPORTANCE OF ENZYMATIC BIOTRANSFORMATION IN IMMUNOTOXICOLOGY

    EPA Science Inventory

    Many immunotoxic compounds, such as benzene and other organic solvents, pesticides, mycotoxins and polycyclic aromatic hydrocarbons, can alter immune function only after undergoing enzyme-mediated reactions within various tissues. In the review that follows, the role of enzymatic...

  8. L-amino acid ligase from Pseudomonas syringae producing tabtoxin can be used for enzymatic synthesis of various functional peptides.

    PubMed

    Arai, Toshinobu; Arimura, Yasuhiro; Ishikura, Shun; Kino, Kuniki

    2013-08-01

    Functional peptides are expected to be beneficial compounds that improve our quality of life. To address the growing need for functional peptides, we have examined peptide synthesis by using microbial enzymes. l-Amino acid ligase (Lal) catalyzes the condensation of unprotected amino acids in an ATP-dependent manner and is applicable to fermentative production. Hence, Lal is a promising enzyme to achieve cost-effective synthesis. To obtain a Lal with novel substrate specificity, we focused on the putative Lal involved in the biosynthesis of the dipeptidic phytotoxin designated tabtoxin. The tabS gene was cloned from Pseudomonas syringae NBRC14081 and overexpressed in Escherichia coli cells. The recombinant TabS protein produced showed the broadest substrate specificity of any known Lal; it detected 136 of 231 combinations of amino acid substrates when dipeptide synthesis was examined. In addition, some new substrate specificities were identified and unusual amino acids, e.g., l-pipecolic acid, hydroxy-l-proline, and β-alanine, were found to be acceptable substrates. Furthermore, kinetic analysis and monitoring of the reactions over a short time revealed that TabS showed distinct substrate selectivity at the N and C termini, which made it possible to specifically synthesize a peptide without by-products such as homopeptides and heteropeptides with the reverse sequence. TabS specifically synthesized the following functional peptides, including their precursors: l-arginyl-l-phenylalanine (antihypertensive effect; yield, 62%), l-leucyl-l-isoleucine (antidepressive effect; yield, 77%), l-glutaminyl-l-tryptophan (precursor of l-glutamyl-l-tryptophan, which has antiangiogenic activity; yield, 54%), l-leucyl-l-serine (enhances saltiness; yield, 83%), and l-glutaminyl-l-threonine (precursor of l-glutamyl-l-threonine, which enhances saltiness; yield, 96%). Furthermore, our results also provide new insights into tabtoxin biosynthesis. PMID:23770908

  9. Preparation of interstitial lung cells by enzymatic digestion of tissue slices: preliminary characterization by morphology and performance in functional assays

    PubMed Central

    Holt, P. G.; Degebrodt, A.; Venaille, T.; O'Leary, C.; Krska, K.; Flexman, J.; Farrell, H.; Shellam, G.; Young, P.; Penhale, J.; Robertson, T.; Papadimitriou, J. M.

    1985-01-01

    A technique is reported here for the quantitative extraction of live cells from the lung interstitium; it involves the incubation of slices of perfused lung in a mixture containing optimal concentrations of collagenase, DNAse, and fetal calf serum, followed by the simultaneous recovery and fractionation of cells released from the tissue matrix on a six-step discontinuous percoll gradient. Yields in the order of 108 viable cells per gram of lung were routinely achieved with tissues from rat, mouse and guinea-pig. Preliminary characterization of these cells has been performed in the rat by histological techniques (Giemsa staining, transmission electron microscopy), cytochemistry (acid phosphatase, esterase, peroxidase), by the capacity to bind monoclonal antibodies directed at lymphocyte surface markers, and by a range of functional tests. The cells comprised, on average, 32% macrophages, 44% lymphocytes (T and B cells and large granular lymphocytes), with small numbers of eosinophils, mast cells and epithelial cells. Transmission electron microscopy revealed minimal ultrastructural damage to extracted cells, with such functions as phagocytosis, FcR activity, mitogen responsiveness, antigen presentation, and NK-cell activity, being readily demonstrable. In addition, these activities segregated into defined areas of the six-step density gradient. PMID:2982730

  10. Two tomato GDP-D-mannose epimerase isoforms involved in ascorbate biosynthesis play specific roles in cell wall biosynthesis and development.

    PubMed

    Mounet-Gilbert, Louise; Dumont, Marie; Ferrand, Carine; Bournonville, Céline; Monier, Antoine; Jorly, Joana; Lemaire-Chamley, Martine; Mori, Kentaro; Atienza, Isabelle; Hernould, Michel; Stevens, Rebecca; Lehner, Arnaud; Mollet, Jean Claude; Rothan, Christophe; Lerouge, Patrice; Baldet, Pierre

    2016-08-01

    GDP-D-mannose epimerase (GME, EC 5.1.3.18) converts GDP-D-mannose to GDP-L-galactose, and is considered to be a central enzyme connecting the major ascorbate biosynthesis pathway to primary cell wall metabolism in higher plants. Our previous work demonstrated that GME is crucial for both ascorbate and cell wall biosynthesis in tomato. The aim of the present study was to investigate the respective role in ascorbate and cell wall biosynthesis of the two SlGME genes present in tomato by targeting each of them through an RNAi-silencing approach. Taken individually SlGME1 and SlGME2 allowed normal ascorbate accumulation in the leaf and fruits, thus suggesting the same function regarding ascorbate. However, SlGME1 and SlGME2 were shown to play distinct roles in cell wall biosynthesis, depending on the tissue considered. The RNAi-SlGME1 plants harbored small and poorly seeded fruits resulting from alterations of pollen development and of pollination process. In contrast, the RNAi-SlGME2 plants exhibited vegetative growth delay while fruits remained unaffected. Analysis of SlGME1- and SlGME2-silenced seeds and seedlings further showed that the dimerization state of pectin rhamnogalacturonan-II (RG-II) was altered only in the RNAi-SlGME2 lines. Taken together with the preferential expression of each SlGME gene in different tomato tissues, these results suggest sub-functionalization of SlGME1 and SlGME2 and their specialization for cell wall biosynthesis in specific tomato tissues. PMID:27382114

  11. Two tomato GDP-D-mannose epimerase isoforms involved in ascorbate biosynthesis play specific roles in cell wall biosynthesis and development

    PubMed Central

    Mounet-Gilbert, Louise; Dumont, Marie; Ferrand, Carine; Bournonville, Céline; Monier, Antoine; Jorly, Joana; Lemaire-Chamley, Martine; Mori, Kentaro; Atienza, Isabelle; Hernould, Michel; Stevens, Rebecca; Lehner, Arnaud; Mollet, Jean Claude; Rothan, Christophe; Lerouge, Patrice; Baldet, Pierre

    2016-01-01

    GDP-D-mannose epimerase (GME, EC 5.1.3.18) converts GDP-D-mannose to GDP-L-galactose, and is considered to be a central enzyme connecting the major ascorbate biosynthesis pathway to primary cell wall metabolism in higher plants. Our previous work demonstrated that GME is crucial for both ascorbate and cell wall biosynthesis in tomato. The aim of the present study was to investigate the respective role in ascorbate and cell wall biosynthesis of the two SlGME genes present in tomato by targeting each of them through an RNAi-silencing approach. Taken individually SlGME1 and SlGME2 allowed normal ascorbate accumulation in the leaf and fruits, thus suggesting the same function regarding ascorbate. However, SlGME1 and SlGME2 were shown to play distinct roles in cell wall biosynthesis, depending on the tissue considered. The RNAi-SlGME1 plants harbored small and poorly seeded fruits resulting from alterations of pollen development and of pollination process. In contrast, the RNAi-SlGME2 plants exhibited vegetative growth delay while fruits remained unaffected. Analysis of SlGME1- and SlGME2-silenced seeds and seedlings further showed that the dimerization state of pectin rhamnogalacturonan-II (RG-II) was altered only in the RNAi-SlGME2 lines. Taken together with the preferential expression of each SlGME gene in different tomato tissues, these results suggest sub-functionalization of SlGME1 and SlGME2 and their specialization for cell wall biosynthesis in specific tomato tissues. PMID:27382114

  12. Enzymatically catalytic signal tracing by a glucose oxidase and ferrocene dually functionalized nanoporous gold nanoprobe for ultrasensitive electrochemical measurement of a tumor biomarker.

    PubMed

    Cheng, Hui; Xu, Lingling; Zhang, Haili; Yu, Aimin; Lai, Guosong

    2016-07-21

    A nanoporous gold nanosphere (pAu NS) was synthesized to load high-content glucose oxidase (GOx) and ferrocene (Fc) for the successful preparation of a new gold nanoprobe. After the specific recognition of the tumor biomarker of carcinoembryonic antigen (CEA) at a gold electrode based aptasensor, this GOx and Fc dually functionalized pAu NS nanoprobe was further used for sandwich immunoreaction and signal tracing. Based on the Fc-mediated GOx-catalytic reaction, the gold nanoprobes quantitatively captured onto the electrode surface produced a sensitive electrochemical signal corresponding to the protein recognition events, which led to the development of a new biosensing method for CEA measurement. Both the high loading of GOx and Fc on the pAu NS nanocarrier and the enzymatically catalytic reaction of the nanoprobe greatly amplify the electrochemical signal; meanwhile, the immobilization of the Fc mediator on this enzyme nanoprobe and the highly specific aptamer recognition drastically decrease the background current, resulting in the achievement of ultrahigh sensitivity of the method. Under optimum conditions, this method shows an excellent analytical performance including a wide linear relationship of five-order of magnitude and a low detection limit down to 0.45 pg mL(-1). Thus this pAu NS based gold nanoprobe and the proposed immunoassay method provide great potential for practical applications. PMID:27186605

  13. A non enzymatic glucose biosensor based on an ultrasensitive calix[4]arene functionalized boronic acid gold nanoprobe for sensing in human blood serum.

    PubMed

    Pandya, Alok; Sutariya, Pinkesh G; Menon, Shobhana K

    2013-04-21

    We developed a new, advanced, simple and non enzymatic approach for the colorimetric detection of glucose based on calix[4]arene/phenyl boronic acid (CX-PBA)functionalized gold nanoparticles (AuNPs). This molecular receptor proficiently and selectively recognizes glucose due to its ability to reversibly bind diol-containing compounds. The assembly was characterized by transmission electron micrograph (TEM), dynamic light scattering (DLS), UV-Vis, FT-IR, ESI-MS and (1)H NMR spectrometry, which demonstrates the binding affinity for glucose via a boronic acid-diol interaction. The linear range for glucose was found to be 5-100 nM with phosphate buffer pH 10, with a lower detection limit of 4.3 nM. Interference by other saccharides was negligible. The biosensor has been successfully applied to estimate the glucose in human blood serum samples and the results compared well to an automatic analyzer. With the advantages of high sensitivity, selectivity and low sample volume, this method is potentially suitable for the on-site monitoring of glucose. PMID:23476922

  14. Functional significance of Asn-linked glycosylation of proteinase 3 for enzymatic activity, processing, targeting, and recognition by anti-neutrophil cytoplasmic antibodies.

    PubMed

    Specks, Ulrich; Fass, David N; Finkielman, Javier D; Hummel, Amber M; Viss, Margaret A; Litwiller, Robert D; McDonald, Cari J

    2007-01-01

    Proteinase 3 (PR3) is a neutral serine protease stored in neutrophil granules. It has substantial sequence homology with elastase, cathepsin G and azurocidin. PR3 is the target antigen for autoantibodies (ANCA) in Wegener's granulomatosis, a necrotizing vasculitis syndrome. ANCA have been implicated in the pathogenesis of this disease. PR3 has two potential Asn-linked glycosylation sites. This study was designed to determine the occupancy of these glycosylation sites, and to evaluate their effect on enzymatic function, intracellular processing, targeting to granules and recognition by ANCA. We found that glycosylation occurs at both sites in native neutrophil PR3 and in wild type recombinant PR3 (rPR3) expressed in HMC-1 cells. Using glycosylation deficient rPR3 mutants we found that glycosylation at Asn-147, but not at Asn-102, is critical for thermal stability, and for optimal hydrolytic activity of PR3. Efficient amino-terminal proteolytic processing of rPR3 is dependent on glycosylation at Asn-102. Targeting to granules is not dependent on glycosylation, but unglycosylated rPR3 gets secreted preferentially into media supernatants. Finally, a capture ELISA for ANCA detection, using rPR3 glycosylation variants as target antigens, reveals that in about 20% of patients, epitope recognition by ANCA is affected by the glycosylation status of PR3. PMID:17158864

  15. Green polymer chemistry VIII: synthesis of halo-ester-functionalized poly(ethylene glycol)s via enzymatic catalysis.

    PubMed

    Castano, Marcela; Seo, Kwang Su; Kim, Eun Hye; Becker, Matthew L; Puskas, Judit E

    2013-09-01

    Halo-ester-functionalized poly(ethylene glycol)s (PEGs) are successfully prepared by the transesterification of alkyl halo-esters with PEGs using Candida antarctica lipase B (CALB) as a biocatalyst under the solventless conditions. Transesterifications of chlorine, bromine, and iodine esters with tetraethylene glycol monobenzyl ether (BzTEG) are quantitative in less than 2.5 h. The transesterification of halo-esters with PEGs are complete in 4 h. (1) H and (13) C NMR spectroscopy with MALDI-ToF and ESI mass spectrometry confirm the structure and purity of the products. This method provides a convenient and "green" process to effectively produce halo-ester PEGs. PMID:23877930

  16. Macrophage migration inhibitory factor (MIF) is rendered enzymatically inactive by myeloperoxidase-derived oxidants but retains its immunomodulatory function.

    PubMed

    Dickerhof, Nina; Schindler, Lisa; Bernhagen, Jürgen; Kettle, Anthony J; Hampton, Mark B

    2015-12-01

    Macrophage migration inhibitory factor (MIF) is an important player in the regulation of the inflammatory response. Elevated plasma MIF is found in sepsis, arthritis, cystic fibrosis and atherosclerosis. Immunomodulatory activities of MIF include the ability to promote survival and recruitment of inflammatory cells and to amplify pro-inflammatory cytokine production. MIF has an unusual nucleophilic N-terminal proline with catalytic tautomerase activity. It remains unclear whether tautomerase activity is required for MIF function, but small molecules that inhibit tautomerase activity also inhibit the pro-inflammatory activities of MIF. A prominent feature of the acute inflammatory response is neutrophil activation and production of reactive oxygen species, including myeloperoxidase (MPO)-derived hypochlorous acid and hypothiocyanous acid. We hypothesized that MPO-derived oxidants would oxidize the N-terminal proline of MIF and alter its biological activity. MIF was exposed to hypochlorous acid and hypothiocyanous acid and the oxidative modifications on MIF were examined by LC-MS/MS. Imine formation and carbamylation was observed on the N-terminal proline in response to MPO-dependent generation of hypochlorous and hypothiocyanous acid, respectively. These modifications led to a complete loss of tautomerase activity. However, modified MIF still increased CXCL-8/IL-8 production by peripheral blood mononuclear cells (PBMCs) and blocked neutrophil apoptosis, indicating that tautomerase activity is not essential for these biological functions. Pre-treatment of MIF with hypochlorous acid protected the protein from covalent modification by the MIF inhibitor 4-iodo-6-phenylpyrimidine (4-IPP). Therefore, oxidant generation at inflammatory sites may protect MIF from inactivation by more disruptive electrophiles, including drugs designed to target the tautomerase activity of MIF. PMID:26453918

  17. Enzymatically Regulated Peptide Pairing and Catalysis for the Bioanalysis of Extracellular Prometastatic Activities of Functionally Linked Enzymes

    PubMed Central

    Li, Hao; Huang, Yue; Yu, Yue; Li, Tianqi; Li, Genxi; Anzai, Jun-ichi

    2016-01-01

    Diseases such as cancer arise from systematical reconfiguration of interactions of exceedingly large numbers of proteins in cell signaling. The study of such complicated molecular mechanisms requires multiplexed detection of the inter-connected activities of several proteins in a disease-associated context. However, the existing methods are generally not well-equipped for this kind of application. Here a method for analyzing functionally linked protein activities is developed based on enzyme controlled pairing between complementary peptide helix strands, which simultaneously enables elaborate regulation of catalytic activity of the paired peptides. This method has been used to detect three different types of protein modification enzymes that participate in the modification of extracellular matrix and the formation of invasion front in tumour. In detecting breast cancer tissue samples using this method, up-regulated activity can be observed for two of the assessed enzymes, while the third enzyme is found to have a subtle fluctuation of activity. These results may point to the application of this method in evaluating prometastatic activities of proteins in tumour. PMID:27140831

  18. Degradation-by-design: Surface modification with functional substrates that enhance the enzymatic degradation of carbon nanotubes.

    PubMed

    Sureshbabu, Adukamparai Rajukrishnan; Kurapati, Rajendra; Russier, Julie; Ménard-Moyon, Cécilia; Bartolini, Isacco; Meneghetti, Moreno; Kostarelos, Kostas; Bianco, Alberto

    2015-12-01

    Biodegradation of carbon-based nanomaterials has been pursued intensively in the last few years, as one of the most crucial issues for the design of safe, clinically relevant conjugates for biomedical applications. In this paper it is demonstrated that specific functional molecules can enhance the catalytic activity of horseradish peroxidase (HRP) and xanthine oxidase (XO) for the degradation of carbon nanotubes. Two different azido coumarins and one cathecol derivative are linked to multi-walled carbon nanotubes (MWCNTs). These molecules are good reducing substrates and strong redox mediators to enhance the catalytic activity of HRP. XO, known to metabolize various molecules mainly in the mammalian liver, including human, was instead used to test the biodegradability of MWCNTs modified with an azido purine. The products of the biodegradation process are characterized by transmission electron microscopy and Raman spectroscopy. The results indicate that coumarin and catechol moieties have enhanced the biodegradation of MWCNTs compared to oxidized nanotubes, likely due to the capacity of these substrates to better interact with and activate HRP. Although azido purine-MWCNTs are degraded less effectively by XO than oxidized nanotubes, the data uncover the importance of XO in the biodegradation of carbon-nanomaterials leading to their better surface engineering for biomedical applications. PMID:26342557

  19. Functional characterization of three Coffea arabica L. monoterpene synthases: insights into the enzymatic machinery of coffee aroma.

    PubMed

    Del Terra, Lorenzo; Lonzarich, Valentina; Asquini, Elisa; Navarini, Luciano; Graziosi, Giorgio; Suggi Liverani, Furio; Pallavicini, Alberto

    2013-05-01

    The chemical composition of the coffee beverage is extremely complex, being made up of hundreds of volatile and non-volatile compounds, many of which are generated in the thermal reactions that occur during the roasting process. However, in the raw coffee bean there are also compounds that survive roasting and are therefore extracted into the beverage. Monoterpenes are an example of this category, as their presence has been reported in the coffee flower, fruit, seed, roasted bean and in the beverage aroma. The present work describes the isolation, heterologous expression and functional characterization of three Coffea arabica cDNAs coding for monoterpene synthases. RNA was purified from C. arabica (cv. Catuai Red) flowers, seeds and fruits at 4 successive ripening stages. Degenerate primers were designed on the most conserved regions of the monoterpene synthase gene family, and then used to isolate monoterpene synthase-like sequences from the cDNA libraries. After 5'- and 3'-RACE, the complete transcripts of 4 putative C. arabica monoterpene synthases (CofarTPS) were obtained. Gene expression in different tissues and developmental stages was analysed. After heterologous expression in Escherichia coli, enzyme activity and substrate specificity were evaluated in vitro by incubation of the recombinant proteins with geranyl pyrophosphate (GPP), geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP), precursors respectively of mono-, di- and sesquiterpenes. The reaction products were characterized by HS-SPME GC-MS. CofarTPS1 was classified as a limonene synthase gene, while CofarTPS2 and 3 showed lower activity with the production of linalool and β-myrcene. PMID:23398891

  20. What’s New in Enzymatic Halogenations

    PubMed Central

    Fujimori, Danica Galoniæ; Walsh, Christopher T.

    2007-01-01

    Summary The halogenation of thousands of natural products occurs during biosynthesis and often confers important functional properties. While haloperoxidases had been the default paradigm for enzymatic incorporation of halogens, via X+ equivalents into organic scaffolds, a combination of microbial genome sequencing, enzymatic studies and structural biology have provided deep new insights into enzymatic transfer of halide equivalents in three oxidation states. These are: (1) the halide ions (X−) abundant in nature, (2) halogen atoms (X•), and (3) the X+ equivalents. The mechanism of halogen incorporation is tailored to the electronic demands of specific substrates and involves enzymes with distinct redox coenzyme requirements. PMID:17881282

  1. Modulation of Enzymatic Activity and Biological Function of Listeria monocytogenes Broad-Range Phospholipase C by Amino Acid Substitutions and by Replacement with the Bacillus cereus Ortholog

    PubMed Central

    Zückert, Wolfram R.; Marquis, Hélène; Goldfine, Howard

    1998-01-01

    The secreted broad-range phosphatidylcholine (PC)-preferring phospholipase C (PC-PLC) of Listeria monocytogenes plays a role in the bacterium’s ability to escape from phagosomes and spread from cell to cell. Based on comparisons with two orthologs, Clostridium perfringens α-toxin and Bacillus cereus PLC (PLCBc), we generated PC-PLC mutants with altered enzymatic activities and substrate specificities and analyzed them for biological function in tissue culture and mouse models of infection. Two of the conserved active-site zinc-coordinating histidines were confirmed by single amino acid substitutions H69G and H118G, which resulted in proteins inactive in broth culture and unstable intracellularly. Substitutions D4E and H56Y remodeled the PC-PLC active site to more closely resemble the PLCBc active site, while a gene replacement resulted in L. monocytogenes secreting PLCBc. All of these mutants yielded similar amounts of active enzyme as wild-type PC-PLC both in broth culture and intracellularly. D4E increased activity on and specificity for PC, while H56Y and D4E H56Y showed higher activity on both PC and sphingomyelin, with reduced specificity for PC. As expected, PLCBc expressed by L. monocytogenes was highly specific for PC. During early intracellular growth in human epithelial cells, the D4E mutant and the PLCBc-expressing strain performed significantly better than the wild type, while the H56Y and D4E H56Y mutants showed a significant defect. In assays for cell-to-cell spread, the H56Y and D4E mutants had close to wild-type characteristics, while the spreading efficiency of PLCBc was significantly lower. These studies emphasize the species-specific features of PC-PLC important for growth in mammalian cells. PMID:9746585

  2. Identification and Characterization of Bifunctional Proline Racemase/Hydroxyproline Epimerase from Archaea: Discrimination of Substrates and Molecular Evolution

    PubMed Central

    Watanabe, Seiya; Tanimoto, Yoshiaki; Nishiwaki, Hisashi; Watanabe, Yasuo

    2015-01-01

    Proline racemase (ProR) is a member of the pyridoxal 5’-phosphate-independent racemase family, and is involved in the Stickland reaction (fermentation) in certain clostridia as well as the mechanisms underlying the escape of parasites from host immunity in eukaryotic Trypanosoma. Hydroxyproline epimerase (HypE), which is in the same protein family as ProR, catalyzes the first step of the trans-4-hydroxy-L-proline metabolism of bacteria. Their substrate specificities were previously considered to be very strict, in spite of similarities in their structures and catalytic mechanisms, and no racemase/epimerase from the ProR superfamily has been found in archaea. We here characterized the ProR-like protein (OCC_00372) from the hyperthermophilic archaeon, Thermococcus litoralis (TlProR). This protein could reversibly catalyze not only the racemization of proline, but also the epimerization of 4-hydroxyproline and 3-hydroxyproline with similar kinetic constants. Among the four (putative) ligand binding sites, one amino acid substitution was detected between TlProR (tryptophan at the position of 241) and natural ProR (phenylalanine). The W241F mutant showed a significant preference for proline over hydroxyproline, suggesting that this (hydrophobic and bulky) tryptophan residue played an importance role in the recognition of hydroxyproline (more hydrophilic and bulky than proline), and substrate specificity for hydroxyproline was evolutionarily acquired separately between natural HypE and ProR. A phylogenetic analysis indicated that such unique broad substrate specificity was derived from an ancestral enzyme of this superfamily. PMID:25786142

  3. Chemo-Enzymatic Synthesis of Oligoglycerol Derivatives.

    PubMed

    Singh, Abhishek K; Nguyen, Remi; Galy, Nicolas; Haag, Rainer; Sharma, Sunil K; Len, Christophe

    2016-01-01

    A cleaner and greener method has been developed and used to synthesize 14 different functionalized oligomer derivatives of glycerol in moderate 29%-39% yields over three steps. After successive regioselective enzymatic acylation of the primary hydroxyl groups, etherification or esterification of the secondary hydroxyl groups and chemoselective enzymatic saponification, the target compounds can efficiently be used as versatile building blocks in organic and supramolecular chemistry. PMID:27517886

  4. Pretreatment and Enzymatic Hydrolysis

    SciTech Connect

    2006-06-01

    Activities in this project are aimed at overcoming barriers associated with high capital and operating costs and sub-optimal sugar yields resulting from pretreatment and subsequent enzymatic hydrolysis of biomass.

  5. Enzymatic modification of schizophyllan

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An enzymatic method was developed for the progressive modification of the polysaccharide schizophyllan. Fungal strains Hypocrea nigricans NRRL 62555, Penicillium crustosum NRRL 62558, and Penicillium simplicissimum NRRL 62550 were previously identified as novel sources of ß-endoglucanase with specif...

  6. Enzymatic desulfurization of coal

    SciTech Connect

    Marquis, J.K. . School of Medicine); Kitchell, J.P. )

    1988-10-07

    Our current efforts to develop clean coal technology, emphasize the advantages of enzymatic desulfurization techniques and have specifically addressed the potential of using partially-purified extracellular microbial enzymes or commercially available enzymes. Our work is focused on the treatment of model'' organic sulfur compounds such as dibenzothiophene (DBT) and ethylphenylsulfide (EPS). Furthermore, we are designing experiments to facilitate the enzymatic process by means of a hydrated organic solvent matrix.

  7. Enzymatic desulfurization of coal

    SciTech Connect

    Marquis, J.K. . School of Medicine); Kitchell, J.P. )

    1988-12-15

    Our current efforts to develop clean coal technology emphasize the advantages of enzymatic desulfurization techniques and have specifically addressed the potential of using partially-purified extracellular microbial enzymes or commercially available enzymes. Our work is focused on the treatment of model'' organic sulfur compounds such as dibenzothiophene (DBT) and ethylphenylsulfide (EPS). Furthermore, we are designing experiments to facilitate the enzymatic process by means of a hydrated organic solvent matrix.

  8. Enzymatic desulfurization of coal

    SciTech Connect

    Boyer, Y.N.; Crooker, S.C.; Kitchell, J.P.; Nochur, S.V. ); Marquis, J.K. . School of Medicine)

    1989-06-16

    Our current efforts to develop clean coal technology emphasize the advantages of enzymatic desulfurization techniques and have specifically addressed the potential of using partially-purified extracellular microbial enzymes as well as commercially available enzymes. Our work is focused on the treatment of model'' organic sulfur compounds such as dibenzothiophene (DBT) and ethylphenylsulfide (EPS). Furthermore, we are designing experiments to facilitate the enzymatic process by means of a hydrated organic solvent matrix.

  9. A Drug Delivery Strategy: Binding Enkephalin to Asialoglycoprotein Receptor by Enzymatic Galactosylation

    PubMed Central

    Christie, Michelle P.; Simerská, Pavla; Jen, Freda E.-C.; Hussein, Waleed M.; Rawi, Mohamad F. M.; Hartley-Tassell, Lauren E.; Day, Christopher J.; Jennings, Michael P.; Toth, Istvan

    2014-01-01

    Glycosylation of biopharmaceuticals can mediate cell specific delivery by targeting carbohydrate receptors. Additionally, glycosylation can improve the physico-chemical (drug-like) properties of peptide based drug candidates. The main purpose of this study was to examine if glycosylation of the peptide enkephalin could facilitate its binding to the carbohydrate receptor, asialoglycoprotein. Firstly, we described the one-pot enzymatic galactosylation of lactose modified enkephalin in the presence of uridine-5′-diphosphogalactose 4-epimerase and lipopolysaccharyl α-1,4-galactosyltransferase. Stability experiments using human plasma and Caco-2 cell homogenates showed that glycosylation considerably improved the stability of enkephalin (at least 60% remained stable after a 2 hr incubation at 37°C). In vitro permeability experiments using Caco-2 cells revealed that the permeability of mono- and trisaccharide conjugated enkephalins was 14 and 28 times higher, respectively, than that of enkephalin alone (Papp 3.1×10−8 cm/s). By the methods of surface plasmon resonance and molecular modeling, we demonstrated that the enzymatic glycosylation of enkephalin enabled binding the asialoglycoprotein receptor. The addition of a trisaccharide moiety to enkephalin improved the binding of enkephalin to the asialoglycoprotein receptor two fold (KD = 91 µM). The docking scores from molecular modeling showed that the binding modes and affinities of the glycosylated enkephalin derivatives to the asialoglycoprotein receptor complemented the results from the surface plasmon resonance experiments. PMID:24736570

  10. Food-Grade Expression of d-Psicose 3-Epimerase with Tandem Repeat Genes in Bacillus subtilis.

    PubMed

    He, Weiwei; Mu, Wanmeng; Jiang, Bo; Yan, Xin; Zhang, Tao

    2016-07-20

    An integrative food-grade expression system with tandem repeat target genes was constructed for the expression of d-psicose 3-epimerase (DPEase; EC 5.1.3.30). The DPEase gene fused with the P43 promoter constituted an independent monomeric expression cassette. Multimers of the expression cassette were constructed in vitro using the isocaudamer strategy. The recombinant integration plasmids pDG-nDPE (n = 1, 2, 3), which contained one, two, or three consecutive P43-DPEase tandem repeats, were integrated into the genome of B. subtilis. Then, the antibiotic resistance gene was deleted by the Cre/lox system, and the food-grade recombinant B. subtilis 1A751-nDPE (n = 1, 2, 3) with integrated tandem repeats of the P43-DPEase expression cassette were generated. The specific activity of the B. subtilis 1A751-3DPE was the highest among the recombinant strains and was ∼2.2-fold that of the 1A751-1DPE strain. Under the optimal conditions, 8 g/L of freeze-dried enzyme powder could convert 20% d-fructose (300 g/L) into d-allulose after 1 h. PMID:27358033

  11. Alteration of cell wall polysaccharides through transgenic expression of UDP-Glc 4-epimerase-encoding genes in potato tubers.

    PubMed

    Huang, Jie-Hong; Kortstee, Anne; Dees, Dianka C T; Trindade, Luisa M; Schols, Henk A; Gruppen, Harry

    2016-08-01

    Uridine diphosphate (UDP)-glucose 4-epimerase (UGE) catalyzes the conversion of UDP-glucose to UDP-galactose. Cell wall materials from the cv. Kardal (wild-type, background) and two UGE transgenic lines (UGE 45-1 and UGE 51-16) were isolated and fractionated. The galactose (Gal) content (mg/100g tuber) from UGE 45-1 transgenic line was 38% higher than that of wild-type, and resulted in longer pectin side chains. The Gal content present in UGE 51-16 was 17% lower than that of wild-type, although most pectin populations maintained the same level of Gal. Both UGE transgenic lines showed unexpectedly a decrease in acetylation and an increase in methyl-esterification of pectin. Both UGE transgenic lines showed similar proportions of homogalacturonan and rhamnogalacturonan I within pectin backbone as the wild-type, except for the calcium-bound pectin fraction exhibiting relatively less rhamnogalacturonan I. Next to pectin modification, xyloglucan populations from both transgenic lines were altered resulting in different XSGG and XXGG proportion in comparison to wild-type. PMID:27112882

  12. Enzymatic DNA molecules

    NASA Technical Reports Server (NTRS)

    Joyce, Gerald F. (Inventor); Breaker, Ronald R. (Inventor)

    1998-01-01

    The present invention discloses deoxyribonucleic acid enzymes--catalytic or enzymatic DNA molecules--capable of cleaving nucleic acid sequences or molecules, particularly RNA, in a site-specific manner, as well as compositions including same. Methods of making and using the disclosed enzymes and compositions are also disclosed.

  13. Enzymatic Modifications of Polysaccharides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polysaccharides are often modified chemically in order to improve its properties or to impart specific characteristics. Indeed quite a few commercial products are based on modified polysaccharides. In this talk, I shall describe a new set of modified polysaccharides based on enzymatic reactions. ...

  14. Comparison of enzymatic and non-enzymatic nitroethane anion formation: thermodynamics and contribution of tunneling.

    PubMed

    Valley, Michael P; Fitzpatrick, Paul F

    2004-05-26

    In the reaction of nitroalkane oxidase (NAO), the oxidation of nitroalkanes to the corresponding aldehydes or ketones is initiated by the deprotonation of the neutral nitroalkane. The energetics of nitroethane ionization for both the enzymatic and non-enzymatic reactions have been determined by measuring rate constants as a function of temperature. At 25 degrees C, the rate constant for the acetate-catalyzed reaction is a billionfold smaller than the kcat/Km value for NAO. This corresponds to a difference of 12.3 kcal/mol in the free energy of activation that is largely due to a difference in the activation enthalpy. Analysis of the temperature dependence of the deuterium kinetic isotope effects on the reactions yields similar DeltaEa and AH/AD values for the acetate, phosphate, and NAO-catalyzed reactions that fall within the semiclassical limits, consistent with similar contributions of tunneling to the enzymatic and non-enzymatic reactions. PMID:15149217

  15. Review: Enzymatic Hydrolysis of Cellulosic Biomass

    SciTech Connect

    Yang, Bin; Dai, Ziyu; Ding, Shi-You; Wyman, Charles E.

    2011-07-16

    Biological conversion of cellulosic biomass to fuels and chemicals offers the high yields to products vital to economic success and the potential for very low costs. Enzymatic hydrolysis that converts lignocellulosic biomass to fermentable sugars may be the most complex step in this process due to substrate-related and enzyme-related effects and their interactions. Although enzymatic hydrolysis offers the potential for higher yields, higher selectivity, lower energy costs, and milder operating conditions than chemical processes, the mechanism of enzymatic hydrolysis and the relationship between the substrate structure and function of various glycosyl hydrolase components are not well understood. Consequently, limited success has been realized in maximizing sugar yields at very low cost. This review highlights literature on the impact of key substrate and enzyme features that influence performance to better understand fundamental strategies to advance enzymatic hydrolysis of cellulosic biomass for biological conversion to fuels and chemicals. Topics are summarized from a practical point of view including characteristics of cellulose (e.g., crystallinity, degree of polymerization, and accessible surface area) and soluble and insoluble biomass components (e.g., oligomeric xylan, lignin, etc.) released in pretreatment, and their effects on the effectiveness of enzymatic hydrolysis. We further discuss the diversity, stability, and activity of individual enzymes and their synergistic effects in deconstructing complex lignocellulosic biomass. Advanced technologies to discover and characterize novel enzymes and to improve enzyme characteristics by mutagenesis, post-translational modification, and over-expression of selected enzymes and modifications in lignocellulosic biomass are also discussed.

  16. Graphene based enzymatic bioelectrodes and biofuel cells

    NASA Astrophysics Data System (ADS)

    Karimi, Anahita; Othman, Ali; Uzunoglu, Aytekin; Stanciu, Lia; Andreescu, Silvana

    2015-04-01

    The excellent electrical conductivity and ease of functionalization make graphene a promising material for use in enzymatic bioelectrodes and biofuel cells. Enzyme based biofuel cells have attracted substantial interest due to their potential to harvest energy from organic materials. This review provides an overview of the functional properties and applications of graphene in the construction of biofuel cells as alternative power sources. The review covers the current state-of-the-art research in graphene based nanomaterials (physicochemical properties and surface functionalities), the role of these parameters in enhancing electron transfer, the stability and activity of immobilized enzymes, and how enhanced power density can be achieved. Specific examples of enzyme immobilization methods, enzyme loading, stability and function on graphene, functionalized graphene and graphene based nanocomposite materials are discussed along with their advantages and limitations. Finally, a critical evaluation of the performance of graphene based enzymatic biofuel cells, the current status, challenges and future research needs are provided.

  17. Enzymatic production of cyclodextrins.

    PubMed

    Biwer, A; Antranikian, G; Heinzle, E

    2002-09-01

    Cyclodextrins (CD) are enzymatically modified starches with a wide range of applications in food, pharmaceutical and chemical industries, agriculture and environmental engineering. They are produced from starch via enzymatic conversion using cyclodextrin glycosyl transferases (CGTases) and partly alpha-amylases. Due to its low solubility in water, separation and purification of beta-CD is relatively easy compared to alpha- and gamma-CD. In recent years more economic processes for gamma-CD and especially alpha-CD production have been developed using improved CGTases and downstream processing. New purification steps, e.g. affinity adsorption, may reduce the use of complexing agents. The implementation of thermostable CGTases can simplify the production process and increase the selectivity of the reaction. A tabular overview of alpha-CD production processes is presented. PMID:12226716

  18. Enzymatic desulfurization of coal

    SciTech Connect

    Boyer, Y.N.; Crooker, S.C.; Kitchell, J.P.; Nochur, S.V.

    1991-05-16

    The overall objective of this program was to investigate the feasibility of an enzymatic desulfurization process specifically intended for organic sulfur removal from coal. Toward that end, a series of specific objectives were defined: (1) establish the feasibility of (bio)oxidative pretreatment followed by biochemical sulfate cleavage for representative sulfur-containing model compounds and coals using commercially-available enzymes; (2) investigate the potential for the isolation and selective use of enzyme preparations from coal-utilizing microbial systems for desulfurization of sulfur-containing model compounds and coals; and (3) develop a conceptual design and economic analysis of a process for enzymatic removal of organic sulfur from coal. Within the scope of this program, it was proposed to carry out a portion of each of these efforts concurrently. (VC)

  19. DOTA-Functionalized Polylysine: A High Number of DOTA Chelates Positively Influences the Biodistribution of Enzymatic Conjugated Anti-Tumor Antibody chCE7agl

    PubMed Central

    Sarko, Dikran; Dennler, Patrick; Zimmermann, Kurt; Mier, Walter; Schibli, Roger

    2013-01-01

    Site-specific enzymatic reactions with microbial transglutaminase (mTGase) lead to a homogenous species of immunoconjugates with a defined ligand/antibody ratio. In the present study, we have investigated the influence of different numbers of 1,4,7,10-tetraazacyclododecane-N-N′-N′′-N′′′-tetraacetic acid (DOTA) chelats coupled to a decalysine backbone on the in vivo behavior of the chimeric monoclonal anti-L1CAM antibody chCE7agl. The enzymatic conjugation of (DOTA)1-decalysine, (DOTA)3-decalysine or (DOTA)5-decalysine to the antibody heavy chain (via Gln295/297) gave rise to immunoconjugates containing two, six or ten DOTA moieties respectively. Radiolabeling of the immunoconjugates with 177Lu yielded specific activities of approximately 70 MBq/mg, 400 MBq/mg and 700 MBq/mg with increasing numbers of DOTA chelates. Biodistribution experiments in SKOV3ip human ovarian cancer cell xenografts demonstrated a high and specific accumulation of radioactivity at the tumor site for all antibody derivatives with a maximal tumor accumulation of 43.6±4.3% ID/g at 24 h for chCE7agl-[(DOTA)-decalysine]2, 30.6±12.0% ID/g at 24 h for chCE7agl-[(DOTA)3-decalysine]2 and 49.9±3.1% ID/g at 48 h for chCE7agl-[(DOTA)5-decalysine)]2. The rapid elimination from the blood of chCE7agl-[(DOTA)-decalysine]2 (1.0±0.1% ID/g at 24 h) is associated with a high liver accumulation (23.2±4.6% ID/g at 24 h). This behavior changed depending on the numbers of DOTA moieties coupled to the decalysine peptide with a slower blood clearance (5.1±1.0 (DOTA)3 versus 11.7±1.4% ID/g (DOTA)5, p<0.005 at 24 h) and lower radioactivity levels in the liver (21.4±3.4 (DOTA)3 versus 5.8±0.7 (DOTA)5, p<0.005 at 24 h). We conclude that the site-specific and stoichiometric uniform conjugation of the highly DOTA-substituted decalysine ((DOTA)5-decalysine) to an anti-tumor antibody leads to the formation of immunoconjugates with high specific activity and excellent in vivo behavior and is a valuable

  20. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of N-acetylmannosamine-6-phosphate 2-epimerase from methicillin-resistant Staphylococcus aureus.

    PubMed

    North, Rachel A; Kessans, Sarah A; Griffin, Michael D W; Watson, Andrew J A; Fairbanks, Antony J; Dobson, Renwick C J

    2014-05-01

    Sialic acids are one of the most important carbohydrate classes in biology. Some bacterial pathogens can scavenge sialic acids from their surrounding environment and degrade them as a source of carbon, nitrogen and energy. This sequestration and subsequent catabolism of sialic acid require a cluster of genes known as the `Nan-Nag' cluster. The enzymes coded by these genes are important for pathogen colonization and persistence. Importantly, the Nan-Nag genes have proven to be essential for Staphylococcus aureus growth on sialic acids, suggesting that the pathway is a viable antibiotic drug target. The enzyme N-acetylmannosamine-6-phosphate 2-epimerase is involved in the catabolism of sialic acid; specifically, the enzyme converts N-acetylmannosamine-6-phosphate into N-acetylglucosamine-6-phosphate. The gene was cloned into an appropriate expression vector, and recombinant protein was expressed in Escherichia coli BL21 (DE3) cells and purified via a three-step procedure. Purified N-acetylmannosamine-6-phosphate 2-epimerase was screened for crystallization. The best crystal diffracted to a resolution of beyond 1.84 Å in space group P21212. Understanding the structural nature of this enzyme from methicillin-resistant S. aureus will provide us with the insights necessary for the development of future antibiotics. PMID:24817730

  1. Sizing up single-molecule enzymatic conformational dynamics.

    PubMed

    Lu, H Peter

    2014-02-21

    Enzymatic reactions and related protein conformational dynamics are complex and inhomogeneous, playing crucial roles in biological functions. The relationship between protein conformational dynamics and enzymatic reactions has been a fundamental focus in modern enzymology. It is extremely difficult to characterize and analyze such complex dynamics in an ensemble-averaged measurement, especially when the enzymes are associated with multiple-step, multiple-conformation complex chemical interactions and transformations. Beyond the conventional ensemble-averaged studies, real-time single-molecule approaches have been demonstrated to be powerful in dissecting the complex enzymatic reaction dynamics and related conformational dynamics. Single-molecule enzymology has come a long way since the early demonstrations of the single-molecule spectroscopy studies of enzymatic dynamics about two decades ago. The rapid development of this fundamental protein dynamics field is hand-in-hand with the new development of single-molecule imaging and spectroscopic technology and methodology, theoretical model analyses, and correlations with biological preparation and characterization of the enzyme protein systems. The complex enzymatic reactions can now be studied one molecule at a time under physiological conditions. Most exciting developments include active manipulation of enzymatic conformational changes and energy landscape to regulate and manipulate the enzymatic reactivity and associated conformational dynamics, and the new advancements have established a new stage for studying complex protein dynamics beyond by simply observing but by actively manipulating and observing the enzymatic dynamics at the single-molecule sensitivity temporally and spatially. PMID:24306450

  2. Enzymatic temperature change indicator

    DOEpatents

    Klibanov, Alexander M.; Dordick, Jonathan S.

    1989-01-21

    A temperature change indicator is described which is composed of an enzyme and a substrate for that enzyme suspended in a solid organic solvent or mixture of solvents as a support medium. The organic solvent or solvents are chosen so as to melt at a specific temperature or in a specific temperature range. When the temperature of the indicator is elevated above the chosen, or critical temperature, the solid organic solvent support will melt, and the enzymatic reaction will occur, producing a visually detectable product which is stable to further temperature variation.

  3. Enzymatic cascade bioreactor

    DOEpatents

    Simmons, Blake A.; Volponi, Joanne V.; Ingersoll, David; Walker, Andrew

    2007-09-04

    Disclosed is an apparatus and method for continuously converting sucrose to .beta.-D-glucose. The method comprises a three stage enzymatic reactor in which an aqueous solution of sucrose is first converted into a solution of fructose and .alpha.-D-glucose by passing it through a porous, packed column containing an inert media on which invertase is immobilized. This solution is then sent through a second packed column containing glucose isomerase and finally a third packed column containing mutarotase. Solution temperature and pH are adjusted to maximize glucose output.

  4. Investigation of enzymatic hydrolysis conditions on the properties of protein hydrolysate from fish muscle (Collichthys niveatus) and evaluation of its functional properties.

    PubMed

    Shen, Qing; Guo, Rui; Dai, Zhiyuan; Zhang, Yanping

    2012-05-23

    This study was carried out to investigate the enzymatic hydrolysis conditions on the properties of protein hydrolysate from fish muscle of the marine fish species Collichthys niveatus. About 160 fish samples were tested, and the analyzed fish species was found to be a lean fish with low fat (1.77 ± 0.01%) and high protein (16.76 ± 1.21%). Fish muscle of C. niveatus was carefully collected and hydrolyzed with four commercial enzymes: Alcalase, Neutrase, Protamex, and Flavourzyme under the conditions recommended by the manufacturers. Among the tested proteases, Neutrase catalyzed the hydrolysis process most effectively since the hydrolysate generated by Neutrase has the highest content of sweet and umami taste amino acids (SUA). The effect of hydrolysis conditions was further optimized using response surface methodology (RSM), and the optimum values for temperature, pH, and enzyme/substrate ratio (E/S ratio) were found to be 40.7 °C, 7.68, and 0.84%, respectively. Finally, the amino acid composition of the hydrolysate was analyzed by AccQ·Tag derivatization and HPLC-PDA determination. Major amino acids of the muscle of C. niveatus were threonine, glutamic acid, phenyalanine, tryptophan, and lysine, accounting for respectively 10.92%, 10.85%, 10.79%, 9.86%, and 9.76% of total amino acid content. The total content of essential amino acids was 970.7 ng·mL(-1), while that of nonessential amino acids was 709.1 ng·mL(-1). The results suggest that the fish muscle and its protein hydrolysate from C. niveatus provide a versatile supply of the benefits and can be incorporated as supplements in health-care foods. PMID:22530872

  5. Influence of Feeding Enzymatically Hydrolyzed Yeast Cell Wall on Growth Performance and Digestive Function of Feedlot Cattle during Periods of Elevated Ambient Temperature

    PubMed Central

    Salinas-Chavira, J.; Arzola, C.; González-Vizcarra, V.; Manríquez-Núñez, O. M.; Montaño-Gómez, M. F.; Navarrete-Reyes, J. D.; Raymundo, C.; Zinn, R. A.

    2015-01-01

    In experiment 1, eighty crossbred steers (239±15 kg) were used in a 229-d experiment to evaluate the effects of increasing levels of enzymatically hydrolyzed yeast (EHY) cell wall in diets on growth performance feedlot cattle during periods of elevated ambient temperature. Treatments consisted of steam-flaked corn-based diets supplemented to provide 0, 1, 2, or 3 g EHY/hd/d. There were no effects on growth performance during the initial 139-d period. However, from d 139 to harvest, when 24-h temperature humidity index averaged 80, EHY increased dry matter intake (DMI) (linear effect, p<0.01) and average daily gain (ADG) (linear effect, p = 0.01). There were no treatment effects (p>0.10) on carcass characteristics. In experiment 2, four Holstein steers (292±5 kg) with cannulas in the rumen and proximal duodenum were used in a 4×4 Latin Square design experiment to evaluate treatments effects on characteristics of ruminal and total tract digestion in steers. There were no treatment effects (p>0.10) on ruminal pH, total volatile fatty acid, molar proportions of acetate, butyrate, or estimated methane production. Supplemental EHY decreased ruminal molar proportion of acetate (p = 0.08), increased molar proportion of propionate (p = 0.09), and decreased acetate:propionate molar ratio (p = 0.07) and estimated ruminal methane production (p = 0.09). It is concluded that supplemental EHY may enhance DMI and ADG of feedlot steers during periods of high ambient temperature. Supplemental EHY may also enhance ruminal fiber digestion and decrease ruminal acetate:propionate molar ratios in feedlot steers fed steam-flaked corn-based finishing diets. PMID:26194225

  6. Glutathione S-transferase can be used as a C-terminal, enzymatically active dimerization module for a recombinant protease inhibitor, and functionally secreted into the periplasm of Escherichia coli.

    PubMed Central

    Tudyka, T.; Skerra, A.

    1997-01-01

    Glutathione S-transferase (GST) from Schistosoma japonicum, which is widely used for the production of fusion proteins in the cytoplasm of Escherichia coli, was employed as a functional fusion module that effects dimer formation of a recombinant protein and confers enzymatic reporter activity at the same time. For this purpose GST was linked via a flexible spacer to the C-terminus of the thiol-protease inhibitor cystatin, whose binding properties for papain were to be studied. The fusion protein was secreted into the bacterial periplasm by means of the OmpA signal peptide to ensure formation of the two disulfide bonds in cystatin. The formation of wrong crosslinks in the oxidizing milieu was prevented by replacing three of the four exposed cysteine residues in GST. Using the tetracycline promoter for tightly controlled gene expression the soluble fusion protein could be isolated from the periplasmic protein fraction. Purification to homogeneity was achieved in one step by means of an affinity column with glutathione agarose. Alternatively, the protein was isolated via streptavidin affinity chromatography after the Strep-tag had been appended to its C terminus. The GST moiety of the fusion protein was enzymatically active and the kinetic parameters were determined using glutathione and 1-chloro-2,4-dinitrobenzene as substrates. Furthermore, strong binding activity for papain was detected in an ELISA. The signal with the cystatin-GST fusion protein was much higher than with cystatin itself, demonstrating an avidity effect due to the dimer formation of GST. The quaternary structure was further confirmed by chemical crosslinking, which resulted in a specific reaction product with twice the molecular size. Thus, engineered GST is suitable as a moderately sized, secretion-competent fusion partner that can confer bivalency to a protein of interest and promote detection of binding interactions even in cases of low affinity. PMID:9336840

  7. Structural Basis for the Aldolase and Epimerase Activities of Staphylococcus aureus Dihydroneopterin Aldolase

    SciTech Connect

    Blaszczyk,J.; Li, Y.; Gan, J.; Yan, H.; Ji, X.

    2007-01-01

    Dihydroneopterin aldolase (DHNA) catalyzes the conversion of 7,8-dihydroneopterin (DHNP) to 6-hydroxymethyl-7,8-dihydropterin (HP) and also the epimerization of DHNP to 7,8-dihydromonopterin (DHMP). Although crystal structures of the enzyme from several microorganisms have been reported, no structural information is available about the critical interactions between DHNA and the trihydroxypropyl moiety of the substrate, which undergoes bond cleavage and formation. Here, we present the structures of Staphylococcus aureus DHNA (SaDHNA) in complex with neopterin (NP, an analog of DHNP) and with monapterin (MP, an analog of DHMP), filling the gap in the structural analysis of the enzyme. In combination with previously reported SaDHNA structures in its ligand-free form (PDB entry 1DHN) and in complex with HP (PDB entry 2DHN), four snapshots for the catalytic center assembly along the reaction pathway can be derived, advancing our knowledge about the molecular mechanism of SaDHNA-catalyzed reactions. An additional step appears to be necessary for the epimerization of DHMP to DHNP. Three active site residues (E22, K100, and Y54) function coordinately during catalysis: together, they organize the catalytic center assembly, and individually, each plays a central role at different stages of the catalytic cycle.

  8. Enzymatic modification of schizophyllan.

    PubMed

    Leathers, Timothy D; Sutivisedsak, Nongnuch; Nunnally, Melinda S; Price, Neil P J; Stanley, April M

    2015-03-01

    An enzymatic method was developed for the progressive modification of the polysaccharide schizophyllan. Fungal strains Hypocrea nigricans NRRL 62555, Penicillium crustosum NRRL 62558, and Penicillium simplicissimum NRRL 62550 were previously identified as novel sources of β-endoglucanase with specificity towards schizophyllan. Concentrated enzyme preparations from these strains showed specific activities of 1.7-4.3 U β-glucanase/mg protein. Using dilutions of these enzymes in time course digestions, schizophyllan was progressively modified to reduced molecular weight species. Glucose and oligosaccharides were found only in the more complete digestions, and thus modified schizophyllan can be produced quantitatively, without loss, to small molecules. Permethylation analysis confirmed that modified schizophyllan retains the fundamental linkage structure of native schizophyllan. Modified schizophyllan species showed progressively reduced viscosity profiles, and all exhibited pseudoplasticity in response to shear thinning. PMID:25335747

  9. Recent insights in enzymatic synthesis of fructooligosaccharides from inulin.

    PubMed

    Singh, Ram Sarup; Singh, Rupinder Pal; Kennedy, John F

    2016-04-01

    In the past few years, people are paying more attention to their dietary habits, and functional foods are playing a key role in maintaining the health of man. Prebiotics are considered as a main component of the functional foods which are usually composed of short chains of carbohydrates. Fructooligosaccharides (FOSs) are considered as one of the main group of prebiotics which have recognisable bifidogenic properties. FOSs are obtained either by extraction from various plant materials or by enzymatic synthesis from different substrates. Enzymatically, these can be obtained either from sucrose using fructosyltransferase or from inulin by endoinulinase. Inulin is a potent substrate for the enzymatic production of FOSs. This review article will provide an overview on the inulin as potent substrate, microbial sources of endoinulinases, enzymatic synthesis of FOSs from inulin, commercial status of FOSs, and their future perspectives. PMID:26791586

  10. Enzymatic Targets in Trypanosoma brucei.

    PubMed

    Scotti, Luciana; Mendonça, Francisco J B; da Silva, Marcelo S; Scotti, Marcus T

    2016-01-01

    One of the most neglected disease is the Sleeping sickness or Human African Trypanosomiasis (HAT), which is mostly restricted to poor regions of Africa. The disease is caused by parasitic infection with Trypanosoma brucei (T. brucei), and is acquired through the bite of the tsetse fly. In the first stage of the disease, the parasite is in the blood, but in stage 2, the infective form reaches the brain, causing great weakness and death. The few existing drugs against this infection, are highly toxic, and can cause the emergence of resistant forms of the parasite. Also, these drugs are not readily available. New drugs are needed. Many researchers are investigating new enzyme targets for the parasite, searching for more efficient and selective inhibitors that are capable to cause the parasite death with less toxicity to the host. Trypanothione reductase, farnesyl diphosphate synthase, 6-phospho-gluconate dehydrogenase, and UDP 4'-galactose epimerase are some of the enzymes involved in the studies reported on this review. In addition, we have applied ligandbased- virtual screening, using Random Forest associated with structure-based-virtual screening (docking), to a small dataset of 225 alkaloids from the Menispermaceae family (in-house data bank). The aim of this study is to select structures with potential inhibitory activity against trypanothione reductase from Trypanosoma brucei. The computer-aided drug design study selected certain alkaloids that might be worth further investigation. PMID:26983886

  11. miRNA-218 contributes to the regulation of D-glucuronyl C5-epimerase expression in normal and tumor breast tissues

    PubMed Central

    Prudnikova, Tatiana Y.; Mostovich, Luydmila A.; Kashuba, Vladimir I.; Ernberg, Ingemar; Zabarovsky, Eugene R.; Grigorieva, Elvira V.

    2012-01-01

    microRNAs (miRNAs) are key posttranscriptional regulators of gene expression. In the present study, regulation of tumor-suppressor gene D-glucuronyl C5-epimerase (GLCE) by miRNA-218 was investigated. Significant downregulation of miRNA-218 expression was shown in primary breast tumors. Exogenous miRNA-218/anti-miRNA-218 did not affect GLCE mRNA but regulated GLCE protein level in MCF7 breast carcinoma cells in vitro. Comparative analysis showed a positive correlation between miRNA-218 and GLCE mRNA, and negative correlation between miRNA-218 and GLCE protein levels in breast tissues and primary tumors in vivo, supporting a direct involvement of miRNA-218 in posttranscriptional regulation of GLCE in human breast tissue. A common scheme for the regulation of GLCE expression in normal and tumor breast tissues is suggested. PMID:22968430

  12. Computer-Aided Identification of Trypanosoma brucei Uridine Diphosphate Galactose 4′-Epimerase Inhibitors: Toward the Development of Novel Therapies for African Sleeping Sickness

    PubMed Central

    2010-01-01

    Trypanosoma brucei, the causative agent of human African trypanosomiasis, affects tens of thousands of sub-Saharan Africans. As current therapeutics are inadequate due to toxic side effects, drug resistance, and limited effectiveness, novel therapies are urgently needed. UDP-galactose 4′-epimerase (TbGalE), an enzyme of the Leloir pathway of galactose metabolism, is one promising T. brucei drug target. We here use the relaxed complex scheme, an advanced computer-docking methodology that accounts for full protein flexibility, to identify inhibitors of TbGalE. An initial hit rate of 62% was obtained at 100 μM, ultimately leading to the identification of 14 low-micromolar inhibitors. Thirteen of these inhibitors belong to a distinct series with a conserved binding motif that may prove useful in future drug design and optimization. PMID:20527952

  13. A D-psicose 3-epimerase with neutral pH optimum from Clostridium bolteae for D-psicose production: cloning, expression, purification, and characterization.

    PubMed

    Jia, Min; Mu, Wanmeng; Chu, Feifei; Zhang, Xiaoming; Jiang, Bo; Zhou, Liuming Leon; Zhang, Tao

    2014-01-01

    D-Tagatose 3-epimerase family enzymes can efficiently catalyze the epimerization of free keto-sugars, which could be used for D-psicose production from D-fructose. In previous studies, all optimum pH values of these enzymes were found to be alkaline. In this study, a D-psicose 3-epimerase (DPEase) with neutral pH optimum from Clostridium bolteae (ATCC BAA-613) was identified and characterized. The gene encoding the recombinant DPEase was cloned and expressed in Escherichia coli. In order to characterize the catalytic properties, the recombinant DPEase was purified to electrophoretic homogeneity using nickel-affinity chromatography. Ethylenediaminetetraacetic acid was shown to inhibit the enzyme activity completely; therefore, the enzyme was identified as a metalloprotein that exhibited the highest activity in the presence of Co²⁺. Although the DPEase demonstrated the most activity at a pH ranging from 6.5 to 7.5, it exhibited optimal activity at pH 7.0. The optimal temperature for the recombinant DPEase was 55 °C, and the half-life was 156 min at 55 °C. Using D-psicose as the substrate, the apparent K(m), k(cat), and catalytic efficiency (k(cat)/K(m)) were 27.4 mM, 49 s⁻¹, and 1.78 s⁻¹ mM⁻¹, respectively. Under the optimal conditions, the equilibrium ratio of D-fructose to D-psicose was 69:31. For high production of D-psicose, 216 g/L D-psicose could be produced with 28.8 % turnover yield at pH 6.5 and 55 °C. The recombinant DPEase exhibited weak-acid stability and thermostability and had a high affinity and turnover for the substrate D-fructose, indicating that the enzyme was a potential D-psicose producer for industrial production. PMID:23644747

  14. Localization of UDP-GlcNAc 2-epimerase/ManAc kinase (GNE) in the Golgi complex and the nucleus of mammalian cells

    SciTech Connect

    Krause, Sabine; Hinderlich, Stephan; Amsili, Shira; Horstkorte, Ruediger; Wiendl, Heinz; Argov, Zohar; Mitrani-Rosenbaum, Stella; Lochmueller, Hanns . E-mail: hanns@lmb.uni-muenchen.de

    2005-04-01

    The bifunctional enzyme UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE) is essential for early embryonic development and catalyzes the rate limiting step in sialic acid biosynthesis. Although epimerase and kinase activities have been attributed to GNE, little is known about the regulation, differential expression, and subcellular localization of GNE in vivo. Mutations in GNE cause a rare inherited muscle disorder in humans called hereditary inclusion body myopathy (HIBM). However, the role of GNE in HIBM pathogenesis has not been defined yet. Here, we show that the GNE protein is expressed in various mammalian cells and tissues with highest levels found in cancer cells and liver. In human skeletal muscle, GNE protein is developmentally regulated: high levels are found in immature myoblasts but low levels in mature skeletal muscle. The GNE protein colocalizes with resident proteins of the Golgi compartment in a variety of human cells including muscle. Drug-induced disruption of the Golgi and subsequent recovery reveals co-distribution of GNE along with Golgi-targeted proteins. This subcellular localization of GNE is in good agreement with its established role as the key enzyme of sialic acid biosynthesis, since the sialylation of glycoconjugates takes place in the Golgi complex. Surprisingly, GNE is also detected in the nucleus. Upon nocodazole treatment, GNE redistributes to the cytoplasm suggesting that GNE may act as a nucleocytoplasmic shuttling protein. A regulatory role for GNE shifting between the nuclear and the Golgi compartment is proposed. Further insight into GNE regulation may promote the understanding of HIBM pathogenesis.

  15. Enzymatic intracrine regulation of white adipose tissue.

    PubMed

    DiSilvestro, David; Petrosino, Jennifer; Aldoori, Ayat; Melgar-Bermudez, Emiliano; Wells, Alexandra; Ziouzenkova, Ouliana

    2014-07-01

    Abdominal fat formation has become a permanent risk factor for metabolic syndrome and various cancers in one-third of the world's population of obese and even lean patients. Formation of abdominal fat involves additional mechanisms beyond an imbalance in energy intake and expenditure, which explains systemic obesity. In this review, we briefly summarized autonomous regulatory circuits that locally produce hormones from inactive precursors or nutrients for intra-/auto-/paracrine signaling in white adipose depots. Enzymatic pathways activating steroid and thyroid hormones in adipose depots were compared with enzymatic production of retinoic acid from vitamin A. We discussed the role of intracrine circuits in fat-depot functions and strategies to reduce abdominal adiposity through thermogenic adipocytes with interrupted generation of retinoic acid. PMID:25390015

  16. Enzymatic intracrine regulation of white adipose tissue

    PubMed Central

    DiSilvestro, David; Petrosino, Jennifer; Aldoori, Ayat; Melgar-Bermudez, Emiliano; Wells, Alexandra; Ziouzenkova, Ouliana

    2015-01-01

    Abdominal fat formation has become a permanent risk factor for metabolic syndrome and various cancers in one-third of the world's population of obese and even lean patients. Formation of abdominal fat involves additional mechanisms beyond an imbalance in energy intake and expenditure, which explains systemic obesity. In this review, we briefly summarized autonomous regulatory circuits that locally produce hormones from inactive precursors or nutrients for intra-/auto-/paracrine signaling in white adipose depots. Enzymatic pathways activating steroid and thyroid hormones in adipose depots were compared with enzymatic production of retinoic acid from vitamin A. We discussed the role of intracrine circuits in fat-depot functions and strategies to reduce abdominal adiposity through thermogenic adipocytes with interrupted generation of retinoic acid. PMID:25390015

  17. Enzymatically active ultrathin pepsin membranes.

    PubMed

    Raaijmakers, Michiel J T; Schmidt, Thomas; Barth, Monika; Tutus, Murat; Benes, Nieck E; Wessling, Matthias

    2015-05-11

    Enzymatically active proteins enable efficient and specific cleavage reactions of peptide bonds. Covalent coupling of the enzymes permits immobilization, which in turn reduces autolysis-induced deactivation. Ultrathin pepsin membranes were prepared by facile interfacial polycondensation of pepsin and trimesoyl chloride. The pepsin membrane allows for simultaneous enzymatic conversion and selective removal of digestion products. The large water fluxes through the membrane expedite the transport of large molecules through the pepsin layers. The presented method enables the large-scale production of ultrathin, cross-linked, enzymatically active membranes. PMID:25779668

  18. Enzymatic conjugation of a bioactive peptide into an injectable hyaluronic acid-tyramine hydrogel system to promote the formation of functional vasculature.

    PubMed

    Wang, Li-Shan; Lee, Fan; Lim, Jaehong; Du, Chan; Wan, Andrew C A; Lee, Su Seong; Kurisawa, Motoichi

    2014-06-01

    In this study, one-step enzyme-mediated preparation of a multi-functional injectable hyaluronic-acid-based hydrogel system is reported. Hydrogel was formed through the in situ coupling of phenol moieties by horseradish peroxidase (HRP) and hydrogen peroxide (H2O2), and bioactive peptides were simultaneously conjugated into the hydrogel during the gel formation process. The preparation of this multi-functional hydrogel was made possible by synthesizing peptides containing phenols which could couple with the phenol moieties of hyaluronic-acid-tyramine (HA-Tyr) during the HRP-mediated crosslinking reaction. Preliminary studies demonstrated that two phenol moieties per molecule resulted in a consistently high degree of conjugation into the HA-Tyr hydrogel network, unlike the one modified with one phenol moiety per molecule. Therefore, an Arg-Gly-Asp (RGD) peptide bearing two phenol moieties (phenol2-poly(ethylene glycol)-RGD) was designed for conjugation to endow the HA-Tyr hydrogel with adhesion signals and enhance its bioactivities. Human umbilical vein endothelial cells (HUVECs) cultured on or within the RGD-modified hydrogels showed significantly different adhesion behavior, from non-adherence on the HA-Tyr hydrogel to strong adhesion on hydrogels modified with phenol2-poly(ethylene glycol)-RGD. This altered cell adhesion behavior led to improved cell proliferation, migration and formation of capillary-like network in the hydrogel in vitro. More importantly, when HUVECs and human fibroblasts (HFF1) were encapsulated together in the RGD-modified HA-Tyr hydrogel, functional vasculature was observed inside the cell-laden gel after 2weeks in the subcutaneous tissue. Taken together, the in situ conjugation of phenol2-poly(ethylene glycol)-RGD into HA-Tyr hydrogel system, coupled with the ease of incorporating cells, offers a simple and effective means to introduce biological signals for preparation of multi-functional injectable hydrogels for tissue engineering

  19. Homogeneous, Heterogeneous, and Enzymatic Catalysis.

    ERIC Educational Resources Information Center

    Oyama, S. Ted; Somorjai, Gabor A.

    1988-01-01

    Discusses three areas of catalysis: homegeneous, heterogeneous, and enzymatic. Explains fundamentals and economic impact of catalysis. Lists and discusses common industrial catalysts. Provides a list of 107 references. (MVL)

  20. Fluorous enzymatic synthesis of phosphatidylinositides.

    PubMed

    Huang, Weigang; Proctor, Angela; Sims, Christopher E; Allbritton, Nancy L; Zhang, Qisheng

    2014-03-18

    A fluorous tagging strategy coupled with enzymatic synthesis is introduced to efficiently synthesize multiple phosphatidylinositides, which are then directly immobilized on a fluorous polytetrafluoroethylene (PTFE) membrane to probe protein-lipid interactions. PMID:24496473

  1. Enzymatic reactions in confined environments

    NASA Astrophysics Data System (ADS)

    Küchler, Andreas; Yoshimoto, Makoto; Luginbühl, Sandra; Mavelli, Fabio; Walde, Peter

    2016-05-01

    Within each biological cell, surface- and volume-confined enzymes control a highly complex network of chemical reactions. These reactions are efficient, timely, and spatially defined. Efforts to transfer such appealing features to in vitro systems have led to several successful examples of chemical reactions catalysed by isolated and immobilized enzymes. In most cases, these enzymes are either bound or adsorbed to an insoluble support, physically trapped in a macromolecular network, or encapsulated within compartments. Advanced applications of enzymatic cascade reactions with immobilized enzymes include enzymatic fuel cells and enzymatic nanoreactors, both for in vitro and possible in vivo applications. In this Review, we discuss some of the general principles of enzymatic reactions confined on surfaces, at interfaces, and inside small volumes. We also highlight the similarities and differences between the in vivo and in vitro cases and attempt to critically evaluate some of the necessary future steps to improve our fundamental understanding of these systems.

  2. Enzymatic reactions in confined environments.

    PubMed

    Küchler, Andreas; Yoshimoto, Makoto; Luginbühl, Sandra; Mavelli, Fabio; Walde, Peter

    2016-05-01

    Within each biological cell, surface- and volume-confined enzymes control a highly complex network of chemical reactions. These reactions are efficient, timely, and spatially defined. Efforts to transfer such appealing features to in vitro systems have led to several successful examples of chemical reactions catalysed by isolated and immobilized enzymes. In most cases, these enzymes are either bound or adsorbed to an insoluble support, physically trapped in a macromolecular network, or encapsulated within compartments. Advanced applications of enzymatic cascade reactions with immobilized enzymes include enzymatic fuel cells and enzymatic nanoreactors, both for in vitro and possible in vivo applications. In this Review, we discuss some of the general principles of enzymatic reactions confined on surfaces, at interfaces, and inside small volumes. We also highlight the similarities and differences between the in vivo and in vitro cases and attempt to critically evaluate some of the necessary future steps to improve our fundamental understanding of these systems. PMID:27146955

  3. Molecular crowding and protein enzymatic dynamics.

    PubMed

    Echeverria, Carlos; Kapral, Raymond

    2012-05-21

    The effects of molecular crowding on the enzymatic conformational dynamics and transport properties of adenylate kinase are investigated. This tridomain protein undergoes large scale hinge motions in the course of its enzymatic cycle and serves as prototype for the study of crowding effects on the cyclic conformational dynamics of proteins. The study is carried out at a mesoscopic level where both the protein and the solvent in which it is dissolved are treated in a coarse grained fashion. The amino acid residues in the protein are represented by a network of beads and the solvent dynamics is described by multiparticle collision dynamics that includes effects due to hydrodynamic interactions. The system is crowded by a stationary random array of hard spherical objects. Protein enzymatic dynamics is investigated as a function of the obstacle volume fraction and size. In addition, for comparison, results are presented for a modification of the dynamics that suppresses hydrodynamic interactions. Consistent with expectations, simulations of the dynamics show that the protein prefers a closed conformation for high volume fractions. This effect becomes more pronounced as the obstacle radius decreases for a given volume fraction since the average void size in the obstacle array is smaller for smaller radii. At high volume fractions for small obstacle radii, the average enzymatic cycle time and characteristic times of internal conformational motions of the protein deviate substantially from their values in solution or in systems with small density of obstacles. The transport properties of the protein are strongly affected by molecular crowding. Diffusive motion adopts a subdiffusive character and the effective diffusion coefficients can change by more than an order of magnitude. The orientational relaxation time of the protein is also significantly altered by crowding. PMID:22476233

  4. The high-resolution crystal structure of periplasmic Haemophilus influenzae NAD nucleotidase reveals a novel enzymatic function of human CD73 related to NAD metabolism.

    PubMed

    Garavaglia, Silvia; Bruzzone, Santina; Cassani, Camilla; Canella, Laura; Allegrone, Gianna; Sturla, Laura; Mannino, Elena; Millo, Enrico; De Flora, Antonio; Rizzi, Menico

    2012-01-01

    Haemophilus influenzae is a major pathogen of the respiratory tract in humans that has developed the capability to exploit host NAD(P) for its nicotinamide dinucleotide requirement. This strategy is organized around a periplasmic enzyme termed NadN (NAD nucleotidase), which plays a central role by degrading NAD into adenosine and NR (nicotinamide riboside), the latter being subsequently internalized by a specific permease. We performed a biochemical and structural investigation on H. influenzae NadN which determined that the enzyme is a Zn2+-dependent 5'-nucleotidase also endowed with NAD(P) pyrophosphatase activity. A 1.3 Å resolution structural analysis revealed a remarkable conformational change that occurs during catalysis between the open and closed forms of the enzyme. NadN showed a broad substrate specificity, recognizing either mono- or di-nucleotide nicotinamides and different adenosine phosphates with a maximal activity on 5'-adenosine monophosphate. Sequence and structural analysis of H. influenzae NadN led us to discover that human CD73 is capable of processing both NAD and NMN, therefore disclosing a possible novel function of human CD73 in systemic NAD metabolism. Our data may prove to be useful for inhibitor design and disclosed unanticipated fascinating evolutionary relationships. PMID:21933152

  5. Structure of l-Xylulose-5-Phosphate 3-Epimerase (UlaE) from the Anaerobic l-Ascorbate Utilization Pathway of Escherichia coli: Identification of a Novel Phosphate Binding Motif within a TIM Barrel Fold▿

    PubMed Central

    Shi, Rong; Pineda, Marco; Ajamian, Eunice; Cui, Qizhi; Matte, Allan; Cygler, Miroslaw

    2008-01-01

    Three catabolic enzymes, UlaD, UlaE, and UlaF, are involved in a pathway leading to fermentation of l-ascorbate under anaerobic conditions. UlaD catalyzes a β-keto acid decarboxylation reaction to produce l-xylulose-5-phosphate, which undergoes successive epimerization reactions with UlaE (l-xylulose-5-phosphate 3-epimerase) and UlaF (l-ribulose-5-phosphate 4-epimerase), yielding d-xylulose-5-phosphate, an intermediate in the pentose phosphate pathway. We describe here crystallographic studies of UlaE from Escherichia coli O157:H7 that complete the structural characterization of this pathway. UlaE has a triosephosphate isomerase (TIM) barrel fold and forms dimers. The active site is located at the C-terminal ends of the parallel β-strands. The enzyme binds Zn2+, which is coordinated by Glu155, Asp185, His211, and Glu251. We identified a phosphate-binding site formed by residues from the β1/α1 loop and α3′ helix in the N-terminal region. This site differs from the well-characterized phosphate-binding motif found in several TIM barrel superfamilies that is located at strands β7 and β8. The intrinsic flexibility of the active site region is reflected by two different conformations of loops forming part of the substrate-binding site. Based on computational docking of the l-xylulose 5-phosphate substrate to UlaE and structural similarities of the active site of this enzyme to the active sites of other epimerases, a metal-dependent epimerization mechanism for UlaE is proposed, and Glu155 and Glu251 are implicated as catalytic residues. Mutation and activity measurements for structurally equivalent residues in related epimerases supported this mechanistic proposal. PMID:18849419

  6. Structure of L-Xylulose-5-Phosphate 3-Epimerase (UlaE) from the Anaerobic L-Ascorbate Utilization Pathway of Escherichia coli: Identification of a Novel Phosphate Binding Motif within a TIM Barrel Fold

    SciTech Connect

    Shi, Rong; Pineda, Marco; Ajamian, Eunice; Cui, Qizhi; Matte, Allan; Cygler, Miroslaw

    2009-01-15

    Three catabolic enzymes, UlaD, UlaE, and UlaF, are involved in a pathway leading to fermentation of L-ascorbate under anaerobic conditions. UlaD catalyzes a {beta}-keto acid decarboxylation reaction to produce L-xylulose-5-phosphate, which undergoes successive epimerization reactions with UlaE (L-xylulose-5-phosphate 3-epimerase) and UlaF (L-ribulose-5-phosphate 4-epimerase), yielding D-xylulose-5-phosphate, an intermediate in the pentose phosphate pathway. We describe here crystallographic studies of UlaE from Escherichia coli O157:H7 that complete the structural characterization of this pathway. UlaE has a triosephosphate isomerase (TIM) barrel fold and forms dimers. The active site is located at the C-terminal ends of the parallel {beta}-strands. The enzyme binds Zn{sup 2+}, which is coordinated by Glu155, Asp185, His211, and Glu251. We identified a phosphate-binding site formed by residues from the {beta}1/{alpha}1 loop and {alpha}3' helix in the N-terminal region. This site differs from the well-characterized phosphate-binding motif found in several TIM barrel superfamilies that is located at strands {beta}7 and {beta}8. The intrinsic flexibility of the active site region is reflected by two different conformations of loops forming part of the substrate-binding site. Based on computational docking of the L-xylulose 5-phosphate substrate to UlaE and structural similarities of the active site of this enzyme to the active sites of other epimerases, a metal-dependent epimerization mechanism for UlaE is proposed, and Glu155 and Glu251 are implicated as catalytic residues. Mutation and activity measurements for structurally equivalent residues in related epimerases supported this mechanistic proposal.

  7. A rare variant in human fibroblast activation protein associated with ER stress, loss of enzymatic function and loss of cell surface localisation.

    PubMed

    Osborne, Brenna; Yao, Tsun-Wen; Wang, Xin Maggie; Chen, Yiqian; Kotan, L Damla; Nadvi, Naveed A; Herdem, Mustafa; McCaughan, Geoffrey W; Allen, John D; Yu, Denise M T; Topaloglu, A Kemal; Gorrell, Mark D

    2014-07-01

    Fibroblast activation protein (FAP) is a focus of interest as a potential cancer therapy target. This membrane bound protease possesses the unique catalytic activity of hydrolysis of the post-proline bond two or more residues from the N-terminus of substrates. FAP is highly expressed in activated fibroblastic cells in tumours, arthritis and fibrosis. A rare, novel, human polymorphism, C1088T, encoding Ser363 to Leu, occurring in the sixth blade of the β propeller domain, was identified in a family. Both in primary human fibroblasts and in Ser363LeuFAP transfected cells, we showed that this single substitution ablates FAP dimerisation and causes loss of enzyme activity. Ser363LeuFAP was detectable only in endoplasmic reticulum (ER), in contrast to the distribution of wild-type FAP on the cell surface. The variant FAP showed decreased conformational antibody binding, consistent with an altered tertiary structure. Ser363LeuFAP expression was associated with upregulation of the ER chaperone BiP/GRP78, ER stress sensor ATF6, and the ER stress response target phospho-eIF2α, all indicators of ER stress. Proteasomal inhibition resulted in accumulation of Ser363LeuFAP, indicating the involvement of ER associated degradation (ERAD). Neither CHOP expression nor apoptosis was elevated, so ERAD is probably important for protecting Ser363LeuFAP expressing cells. These data on the first loss of function human FAP gene variant indicates that although the protein is vulnerable to an amino acid substitution in the β-propeller domain, inactive, unfolded FAP can be tolerated by cells. PMID:24717288

  8. Using Non-Enzymatic Chemistry to Influence Microbial Metabolism

    PubMed Central

    Wallace, Stephen; Schultz, Erica E.; Balskus, Emily P.

    2015-01-01

    The structural manipulation of small molecule metabolites occurs in all organisms and plays a fundamental role in essentially all biological processes. Despite an increasing interest in developing new, non-enzymatic chemical reactions capable of functioning in the presence of living organisms, the ability of such transformations to interface with cellular metabolism and influence biological function is a comparatively underexplored area of research. This review will discuss efforts to combine non-enzymatic chemistry with microbial metabolism. We will highlight recent and historical uses of non-biological reactions to study microbial growth and function, the use of non-enzymatic transformations to rescue auxotrophic microorganisms, and the combination of engineered microbial metabolism and biocompatible chemical reactions for organic synthesis. PMID:25579453

  9. Enzymatic processing of protein-based fibers.

    PubMed

    Fu, Jiajia; Su, Jing; Wang, Ping; Yu, Yuanyuan; Wang, Qiang; Cavaco-Paulo, Artur

    2015-12-01

    Wool and silk are major protein fiber materials used by the textile industry. Fiber protein structure-function relationships are briefly described here, and the major enzymatic processing routes for textiles and other novel applications are deeply reviewed. Fiber biomodification is described here with various classes of enzymes such as protease, transglutaminase, tyrosinase, and laccase. It is expected that the reader will get a perspective on the research done as a basis for new applications in other areas such as cosmetics and pharma. PMID:26428240

  10. Engineering aspects of enzymatic signal transduction: photoreceptors in the retina.

    PubMed Central

    Detwiler, P B; Ramanathan, S; Sengupta, A; Shraiman, B I

    2000-01-01

    Identifying the basic module of enzymatic amplification as an irreversible cycle of messenger activation/deactivation by a "push-pull" pair of opposing enzymes, we analyze it in terms of gain, bandwidth, noise, and power consumption. The enzymatic signal transduction cascade is viewed as an information channel, the design of which is governed by the statistical properties of the input and the noise and dynamic range constraints of the output. With the example of vertebrate phototransduction cascade we demonstrate that all of the relevant engineering parameters are controlled by enzyme concentrations and, from functional considerations, derive bounds on the required protein numbers. Conversely, the ability of enzymatic networks to change their response characteristics by varying only the abundance of different enzymes illustrates how functional diversity may be built from nearly conserved molecular components. PMID:11106590

  11. Bioluminescence methods for enzymatic determinations

    SciTech Connect

    Bostick, W.D.; Denton, M.S.; Dinsmore, S.R.

    1982-11-02

    An enzymatic method for continuous, on-line and rapid detection of diagnostically useful biomarkers, which are symptomatic of disease or trauma-related tissue damage, is disclosed. The method is characterized by operability on authentic samples of complex biological fluids which contain the biomarkers.

  12. Bioluminescence methods for enzymatic determinations

    SciTech Connect

    Bostick, William D.; Denton, Mark S.; Dinsmore, Stanley R.

    1982-01-01

    An enzymatic method for continuous, on-line and rapid detection of diagnostically useful biomarkers, which are symptomatic of disease or trauma-related tissue damage, is disclosed. The method is characterized by operability on authentic samples of complex biological fluids which contain the biomarkers.

  13. Enzymatic hydrolysis of organic phosphorus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Orthophosphate-releasing enzymatic hydrolysis is an alternative means for characterizing organic phosphorus (Po) in animal manure. The approach is not only simple and fast, but can also provide information difficult to obtain by other methods. Currently, commercially available phosphatases are mainl...

  14. Enzymatic conversion of carbon dioxide.

    PubMed

    Shi, Jiafu; Jiang, Yanjun; Jiang, Zhongyi; Wang, Xueyan; Wang, Xiaoli; Zhang, Shaohua; Han, Pingping; Yang, Chen

    2015-10-01

    With the continuous increase in fossil fuels consumption and the rapid growth of atmospheric CO2 concentration, the harmonious state between human and nature faces severe challenges. Exploring green and sustainable energy resources and devising efficient methods for CO2 capture, sequestration and utilization are urgently required. Converting CO2 into fuels/chemicals/materials as an indispensable element for CO2 capture, sequestration and utilization may offer a win-win strategy to both decrease the CO2 concentration and achieve the efficient exploitation of carbon resources. Among the current major methods (including chemical, photochemical, electrochemical and enzymatic methods), the enzymatic method, which is inspired by the CO2 metabolic process in cells, offers a green and potent alternative for efficient CO2 conversion due to its superior stereo-specificity and region/chemo-selectivity. Thus, in this tutorial review, we firstly provide a brief background about enzymatic conversion for CO2 capture, sequestration and utilization. Next, we depict six major routes of the CO2 metabolic process in cells, which are taken as the inspiration source for the construction of enzymatic systems in vitro. Next, we focus on the state-of-the-art routes for the catalytic conversion of CO2 by a single enzyme system and by a multienzyme system. Some emerging approaches and materials utilized for constructing single-enzyme/multienzyme systems to enhance the catalytic activity/stability will be highlighted. Finally, a summary about the current advances and the future perspectives of the enzymatic conversion of CO2 will be presented. PMID:26055659

  15. Correlation of Activities of the Enzymes α-Phosphoglucomutase, UDP-Galactose 4-Epimerase, and UDP-Glucose Pyrophosphorylase with Exopolysaccharide Biosynthesis by Streptococcus thermophilus LY03

    PubMed Central

    Degeest, Bart; De Vuyst, Luc

    2000-01-01

    The effects of different carbohydrates or mixtures of carbohydrates as substrates on bacterial growth and exopolysaccharide (EPS) production were studied for the yoghurt starter culture Streptococcus thermophilus LY03. This strain produces two heteropolysaccharides with the same monomeric composition (galactose and glucose in the ratio 4:1) but with different molecular masses. Lactose and glucose were fermented by S. thermophilus LY03 only when they were used as sole energy and carbohydrate sources. Fructose was also fermented when it was applied in combination with lactose or glucose. Both the amount of EPS produced and the carbohydrate source consumption rates were clearly influenced by the type of energy and carbohydrate source used, while the EPS monomeric composition remained constant (galactose-glucose, 4:1) under all circumstances. A combination of lactose and glucose resulted in the largest amounts of EPS. Measurements of the activities of enzymes involved in EPS biosynthesis, and of those involved in sugar nucleotide biosynthesis and the Embden-Meyerhof-Parnas pathway, demonstrated that the levels of activity of α-phosphoglucomutase, UDP-galactose 4-epimerase, and UDP-glucose pyrophosphorylase are highly correlated with the amount of EPS produced. Furthermore, a weaker relationship or no relationship between the amounts of EPS and the enzymes involved in either the rhamnose nucleotide synthetic branch of the EPS biosynthesis or the pathway leading to glycolysis was observed for S. thermophilus LY03. PMID:10919816

  16. Characterization of a radical S-adenosyl-L-methionine epimerase, NeoN, in the last step of neomycin B biosynthesis.

    PubMed

    Kudo, Fumitaka; Hoshi, Shota; Kawashima, Taiki; Kamachi, Toshiaki; Eguchi, Tadashi

    2014-10-01

    The last step of neomycin biosynthesis is the epimerization at C-5‴ of neomycin C to give neomycin B. A candidate enzyme responsible for the epimerization was a putative radical S-adenosyl-L-methionine (SAM) enzyme, NeoN, which is uniquely encoded in the neomycin biosynthetic gene cluster and remained an unassigned protein in the neomycin biosynthesis. The reconstituted and reduced NeoN showed the expected epimerization activity in the presence of SAM. In the epimerization, 1 equiv of SAM was consumed to convert neomycin C into neomycin B. The site of neomycin C reactive toward epimerization was clearly confirmed to be C-5‴ by detecting the incorporation of a deuterium atom from the deuterium oxide-based buffer solution. Further, alanine scanning of the NeoN cysteine residues revealed that C249 is a critical amino acid residue that provides a hydrogen atom to complete the epimerization. Furthermore, electron paramagnetic resonance analysis of the C249A variant in the presence of SAM and neomycin C revealed that a radical intermediate is generated at the C-5‴ of neomycin C. Therefore, the present study clearly illustrates that the epimerization of neomycin C to neomycin B is catalyzed by a unique radical SAM epimerase NeoN with a radical reaction mechanism. PMID:25230155

  17. D-Allulose Production from D-Fructose by Permeabilized Recombinant Cells of Corynebacterium glutamicum Cells Expressing D-Allulose 3-Epimerase Flavonifractor plautii

    PubMed Central

    Park, Chul-Soon; Kim, Taeyong; Hong, Seung-Hye; Shin, Kyung-Chul; Kim, Kyoung-Rok; Oh, Deok-Kun

    2016-01-01

    A d-allulose 3-epimerase from Flavonifractor plautii was cloned and expressed in Escherichia coli and Corynebacterium glutamicum. The maximum activity of the enzyme purified from recombinant E. coli cells was observed at pH 7.0, 65°C, and 1 mM Co2+ with a half-life of 40 min at 65°C, Km of 162 mM, and kcat of 25280 1/s. For increased d-allulose production, recombinant C. glutamicum cells were permeabilized via combined treatments with 20 mg/L penicillin and 10% (v/v) toluene. Under optimized conditions, 10 g/L permeabilized cells produced 235 g/L d-allulose from 750 g/L d-fructose after 40 min, with a conversion rate of 31% (w/w) and volumetric productivity of 353 g/L/h, which were 1.4- and 2.1-fold higher than those obtained for nonpermeabilized cells, respectively. PMID:27467527

  18. The metastability of human UDP-galactose 4'-epimerase (GALE) is increased by variants associated with type III galactosemia but decreased by substrate and cofactor binding.

    PubMed

    Pey, Angel L; Padín-Gonzalez, Esperanza; Mesa-Torres, Noel; Timson, David J

    2014-11-15

    Type III galactosemia is an inherited disease caused by mutations which affect the activity of UDP-galactose 4'-epimerase (GALE). We evaluated the impact of four disease-associated variants (p.N34S, p.G90E, p.V94M and p.K161N) on the conformational stability and dynamics of GALE. Thermal denaturation studies showed that wild-type GALE denatures at temperatures close to physiological, and disease-associated mutations often reduce GALE's thermal stability. This denaturation is under kinetic control and results partly from dimer dissociation. The natural ligands, NAD(+) and UDP-glucose, stabilize GALE. Proteolysis studies showed that the natural ligands and disease-associated variations affect local dynamics in the N-terminal region of GALE. Proteolysis kinetics followed a two-step irreversible model in which the intact protein is cleaved at Ala38 forming a long-lived intermediate in the first step. NAD(+) reduces the rate of the first step, increasing the amount of undigested protein whereas UDP-glucose reduces the rate of the second step, increasing accumulation of the intermediate. Disease-associated variants affect these rates and the amounts of protein in each state. Our results also suggest communication between domains in GALE. We hypothesize that, in vivo, concentrations of natural ligands modulate GALE stability and that it should be possible to discover compounds which mimic the stabilising effects of the natural ligands overcoming mutation-induced destabilization. PMID:25150110

  19. High cell density cultivation of recombinant Escherichia coli strains expressing 2-O-sulfotransferase and C5-epimerase for the production of bioengineered heparin.

    PubMed

    Zhang, Jianhua; Suflita, Matt; Li, Guoyun; Zhong, Weihong; Li, Lingyun; Dordick, Jonathan S; Linhardt, Robert J; Zhang, Fuming

    2015-03-01

    Bioengineered heparin is being investigated as a potential substitute for the animal-sourced anticoagulant drug. One step in the current process to prepare bioengineered heparin involves the conversion of N-sulfo heparosan, rich in → 4)GlcNS(1 → 4) GlcA(1 → sequences (where S is sulfo, GlcN is α-D-glucosamine, and GlcA is β-D-glucuronic acid), to a critical intermediate, rich in → 4)GlcNS(1 → 4) IdoA2S(1 → sequences (where S is sulfo and IdoA is α-L-iduronic acid), using 2-O-sulfotransferase (2-OST) and C5 epimerase (C5-epi). Until now, these heparan sulfate biosynthetic enzymes have been expressed in Escherichia coli grown in shake flask culture as fusion proteins. The current study is focused on the high cell density fed-batch cultivation of recombinant E. coli strains expressing both enzymes. We report the high productivity expression of active 2-OST and C5-epi enzymes of 6.0 and 2.2 mg/g dry cell weight, respectively. PMID:25586487

  20. NMR Binding and Functional Assays for Detecting Inhibitors of S. aureus MnaA.

    PubMed

    Hou, Yan; Mayhood, Todd; Sheth, Payal; Tan, Christopher M; Labroli, Marc; Su, Jing; Wyss, Daniel F; Roemer, Terry; McCoy, Mark A

    2016-07-01

    Nonessential enzymes in the staphylococcal wall teichoic acid (WTA) pathway serve as highly validated β-lactam potentiation targets. MnaA (UDP-GlcNAc 2-epimerase) plays an important role in an early step of WTA biosynthesis by providing an activated form of ManNAc. Identification of a selective MnaA inhibitor would provide a tool to interrogate the contribution of the MnaA enzyme in the WTA pathway as well as serve as an adjuvant to restore β-lactam activity against methicillin-resistant Staphylococcus aureus (MRSA). However, development of an epimerase functional assay can be challenging since both MnaA substrate and product (UDP-GlcNAc/UDP-ManNAc) share an identical molecular weight. Herein, we developed a nuclear magnetic resonance (NMR) functional assay that can be combined with other NMR approaches to triage putative MnaA inhibitors from phenotypic cell-based screening campaigns. In addition, we determined that tunicamycin, a potent WTA pathway inhibitor, inhibits both S. aureus MnaA and a functionally redundant epimerase, Cap5P. PMID:27028606

  1. Enzymatic desulfurization of coal

    SciTech Connect

    Boyer, Y.N.; Crooker, S.C.; Kitchell, J.P.; Nochur, S.V.

    1990-03-23

    Our experimental approach focuses on the use of enzymes which catalyze the addition of oxygen to organic compounds. In tailoring the application of these enzymes to coal processing, we are particularly interested in ensuring that oxidation occurs at sulfur and not at carbon-carbon bonds. Previous studies with DBT have shown that the reaction most frequently observed in microbial oxidative pathways is one in which DBT is oxidized at ring carbons. These reactions, as we have said, are accompanied by a considerable decrease in the energy content of the compound. In addition, microbial pathways have been identified in which the sulfur atom is sequentially oxidized to sulfoxide, to sulfone, to sulfonate, and finally to sulfuric acid. In this case, the fuel value of the desulfurized compounds is largely retained. We are evaluating the potential of commercially available enzymes to perform this function.

  2. Enzymatic desulfurization of coal

    SciTech Connect

    Boyer, Y.N.; Crooker, S.C.; Kitchell, J.P.; Nochur, S.V.

    1989-12-14

    Our experimental approach focuses on the use of enzymes which catalyze the addition of oxygen to organic compounds., In tailoring the application of these enzymes to coal processing, we are particularly interested in ensuring that oxidation occurs at sulfur and not at carbon-carbon bonds. Previous studies with DBT have shown that the reaction most frequently observed in microbial oxidative pathways is one in which DBT is oxidized at ring carbons. These reactions, as we have said, are accompanied by a considerable decrease in the energy content of the compound. In addition, microbial pathways have been identified in which the sulfur atom is sequentially oxidized to sulfoxide, to sulfone, to sulfonate, and finally to sulfuric acid. In this case, the fuel value of the desulfurized compounds is largely retained. We are evaluating the potential of commercially available enzymes to perform this function.

  3. Biosynthesis of dermatan sulphate. Defructosylated Escherichia coli K4 capsular polysaccharide as a substrate for the D-glucuronyl C-5 epimerase, and an indication of a two-base reaction mechanism.

    PubMed Central

    Hannesson, H H; Hagner-McWhirter, A; Tiedemann, K; Lindahl, U; Malmström, A

    1996-01-01

    The capsular polysaccharide from Escherichia coli K4 consists of a chondroitin ([GlcA(beta 1-->3)GalNAc(beta 1-->4)]n) backbone, to which beta-fructofuranose units are linked to C-3 of D-glucuronic acid (GlcA) residues. Removal of the fructose units by mild acid hydrolysis provided a substrate for the GlcA C-5 epimerase, which is involved in the generation of L-iduronic acid (IdoA) units during dermatan sulphate biosynthesis. Incubation of this substrate with solubilized fibroblast microsomal enzyme in the presence of 3H2O resulted in the incorporation of tritium at C-5 of hexuronyl units. A Km of 67 x 10(-6) M hexuronic acid (equivalent to disaccharide units) was determined, which is similar to that (80 x 10(-6) M) obtained for dermatan (desulphated dermatan sulphate). Vmax was about 4 times higher with dermatan than with the K4 substrate. A defructosylated K4 polysaccharide isolated after incubation of bacteria with D-[5-3H]glucose released 3H2O on reaction with the epimerase, and thus could be used to assay the enzyme. Incubation of a K4 substrate with solubilized microsomal epimerase for 6 h in the presence of 3H2O resulted in the formation of about 5% IdoA and approximately equal amounts of 3H in GlcA and IdoA. A corresponding incubation of dermatan yielded approx. 22% GlcA, which contained virtually all the 3H label. These results are tentatively explained in terms of a two-base reaction mechanism, involving a monoprotic L-ido-specific base and a polyprotic D-gluco-specific base. Most of the IdoA residues generated by the enzyme occurred singly, although some formation of two or three consecutive IdoA-containing disaccharide units was observed. PMID:8573097

  4. Photon-Regulated DNA-Enzymatic Nanostructures by Molecular Assembly

    PubMed Central

    You, Mingxu; Wang, Ruo-Wen; Zhang, Xiaobing; Chen, Yan; Wang, Kelong; Peng, Lu; Tan, Weihong

    2011-01-01

    Future smart nanostructures will have to rely on molecular assembly for unique or advanced desired functions. For example, the evolved ribosome in nature is one example of functional self-assembly of nucleic acids and proteins employed in nature to perform specific tasks. Artificial self-assembled nanodevices have also been developed to mimic key biofunctions, and various nucleic acid- and protein-based functional nanoassemblies have been reported. However, functionally regulating these nanostructures is still a major challenge. Here we report a general approach to fine-tune the catalytic function of DNA-enzymatic nanosized assemblies by taking advantage of the trans-cis isomerization of azobenzene molecules. To the best of our knowledge, this is the first study to precisely modulate the structures and functions of an enzymatic assembly based on light-induced DNA scaffold switching. Via photocontrolled DNA conformational switching, the proximity of multiple enzyme catalytic centers can be adjusted, as well as the catalytic efficiency of cofactor-mediated DNAzymes. We expect that this approach will lead to the advancement of DNA-enzymatic functional nanostructures in future biomedical and analytical applications. PMID:22098552

  5. Abrogation of fibroblast activation protein enzymatic activity attenuates tumor growth.

    PubMed

    Cheng, Jonathan D; Valianou, Matthildi; Canutescu, Adrian A; Jaffe, Eileen K; Lee, Hyung-Ok; Wang, Hao; Lai, Jack H; Bachovchin, William W; Weiner, Louis M

    2005-03-01

    Tumor-associated fibroblasts are functionally and phenotypically distinct from normal fibroblasts that are not in the tumor microenvironment. Fibroblast activation protein is a 95 kDa cell surface glycoprotein expressed by tumor stromal fibroblasts, and has been shown to have dipeptidyl peptidase and collagenase activity. Site-directed mutagenesis at the catalytic site of fibroblast activation protein, Ser624 --> Ala624, resulted in an approximately 100,000-fold loss of fibroblast activation protein dipeptidyl peptidase (DPP) activity. HEK293 cells transfected with wild-type fibroblast activation protein, enzymatic mutant (S624A) fibroblast activation protein, or vector alone, were inoculated subcutaneously into immunodeficient mouse to assess the contribution of fibroblast activation protein enzymatic activity to tumor growth. Overexpression of wild-type fibroblast activation protein showed growth potentiation and enhanced tumorigenicity compared with both fibroblast activation protein S624A and vector-transfected HEK293 xenografts. HEK293 cells transfected with fibroblast activation protein S624A showed tumor growth rates and tumorigenicity potential similar only to vector-transfected HEK293. In vivo assessment of fibroblast activation protein DPP activity of these tumors showed enhanced enzymatic activity of wild-type fibroblast activation protein, with only baseline levels of fibroblast activation protein DPP activity in either fibroblast activation protein S624A or vector-only xenografts. These results indicate that the enzymatic activity of fibroblast activation protein is necessary for fibroblast activation protein-driven tumor growth in the HEK293 xenograft model system. This establishes the proof-of-principle that the enzymatic activity of fibroblast activation protein plays an important role in the promotion of tumor growth, and provides an attractive target for therapeutics designed to alter fibroblast activation protein-induced tumor growth by targeting

  6. Neocartilage integration in temporomandibular joint discs: physical and enzymatic methods

    PubMed Central

    Murphy, Meghan K.; Arzi, Boaz; Prouty, Shannon M.; Hu, Jerry C.; Athanasiou, Kyriacos A.

    2015-01-01

    Integration of engineered musculoskeletal tissues with adjacent native tissues presents a significant challenge to the field. Specifically, the avascularity and low cellularity of cartilage elicit the need for additional efforts in improving integration of neocartilage within native cartilage. Self-assembled neocartilage holds significant potential in replacing degenerated cartilage, though its stabilization and integration in native cartilage require further efforts. Physical and enzymatic stabilization methods were investigated in an in vitro model for temporomandibular joint (TMJ) disc degeneration. First, in phase 1, suture, glue and press-fit constructs were compared in TMJ disc intermediate zone defects. In phase 1, suturing enhanced interfacial shear stiffness and strength immediately; after four weeks, a 15-fold increase in stiffness and a ninefold increase in strength persisted over press-fit. Neither suture nor glue significantly altered neocartilage properties. In phase 2, the effects of the enzymatic stabilization regimen composed of lysyl oxidase, CuSO4 and hydroxylysine were investigated. A full factorial design was employed, carrying forward the best physical method from phase 1, suturing. Enzymatic stabilization significantly increased interfacial shear stiffness after eight weeks. Combined enzymatic stabilization and suturing led to a fourfold increase in shear stiffness and threefold increase in strength over press-fit. Histological analysis confirmed the presence of a collagen-rich interface. Enzymatic treatment additionally enhanced neocartilage mechanical properties, yielding a tensile modulus over 6 MPa and compressive instantaneous modulus over 1200 kPa at eight weeks. Suturing enhances stabilization of neocartilage, and enzymatic treatment enhances functional properties and integration of neocartilage in the TMJ disc. Methods developed here are applicable to other orthopaedic soft tissues, including knee meniscus and hyaline articular

  7. The TCF4/β-catenin pathway and chromatin structure cooperate to regulate D-glucuronyl C5-epimerase expression in breast cancer

    PubMed Central

    Mostovich, Luydmila A.; Prudnikova, Tatiana Y.; Kondratov, Aleksandr G.; Gubanova, Natalya V.; Kharchenko, Olga A.; Kutsenko, Olesya S.; Vavilov, Pavel V.; Haraldson, Klas; Kashuba, Vladimir I.; Ernberg, Ingemar; Zabarovsky, Eugene R.; Grigorieva, Elvira V.

    2012-01-01

    D-glucuronyl C5-epimerase (GLCE) is a potential tumor-suppressor gene involved in heparan sulfate biosynthesis. GLCE expression is significantly decreased in breast tumors; however, the underlying molecular mechanisms remain unclear. This study examined the possible epigenetic mechanisms for GLCE inactivation in breast cancer. Very little methylation of the GLCE promoter region was detected in breast tumors in vivo and in breast cancer cells (MCF7 and T47D) in vitro and GLCE expression in breast cancer cells was not altered by 5-deoxyazacytidine (5-aza-dC) treatment, suggesting that promoter methylation is not involved in regulating GLCE expression. Chromatin activation by Trichostatin A (TSA) or 5-aza-dC/TSA treatment increased GLCE expression by two to 3-fold due to an increased interaction between the GLCE promoter and the TCF4/β-catenin transactivation complex, or H3K9ac and H3K4Me3 histone modifications. However, ectopic expression of TCF4/β-catenin was not sufficient to activate GLCE expression in MCF7 cells, suggesting that chromatin structure plays a key role in GLCE regulation. Although TSA treatment significantly repressed canonical WNT signaling in MCF7 cells, it did not influence endogenous TCF4/β-catenin mRNA levels and activated TCF4/β-catenin-driven transcription from the GLCE promoter, indicating GLCE as a novel target for TCF4/β-catenin complex in breast cancer cells. A correlation was observed between GLCE, TCF4 and β-catenin expression in breast cancer cells and primary tumors, suggesting an important role for TCF4/β-catenin in regulating GLCE expression both in vitro and in vivo. Taken together, the results indicate that GLCE expression in breast cancer is regulated by a combination of chromatin structure and TCF4/β-catenin complex activity. PMID:22805760

  8. Crystal Structure of Binary and Ternary Complexes of Archaeal UDP-galactose 4-Epimerase-like l-Threonine Dehydrogenase from Thermoplasma volcanium*

    PubMed Central

    Yoneda, Kazunari; Sakuraba, Haruhiko; Araki, Tomohiro; Ohshima, Toshihisa

    2012-01-01

    A gene from the thermophilic archaeon Thermoplasma volcanium encoding an l-threonine dehydrogenase (l-ThrDH) with a predicted amino acid sequence that was remarkably similar to the sequence of UDP-galactose 4-epimerase (GalE) was overexpressed in Escherichia coli, and its product was purified and characterized. The expressed enzyme was moderately thermostable, retaining more than 90% of its activity after incubation for 10 min at up to 70 °C. The catalytic residue was assessed using site-directed mutagenesis, and Tyr137 was found to be essential for catalysis. To clarify the structural basis of the catalytic mechanism, four different crystal structures were determined using the molecular replacement method: l-ThrDH-NAD+, l-ThrDH in complex with NAD+ and pyruvate, Y137F mutant in complex with NAD+ and l-threonine, and Y137F in complex with NAD+ and l-3-hydroxynorvaline. Each monomer consisted of a Rossmann-fold domain and a C-terminal catalytic domain, and the fold of the catalytic domain showed notable similarity to that of the GalE-like l-ThrDH from the psychrophilic bacterium Flavobacterium frigidimaris KUC-1. The substrate binding model suggests that the reaction proceeds through abstraction of the β-hydroxyl hydrogen of l-threonine via direct proton transfer driven by Tyr137. The factors contributing to the thermostability of T. volcanium l-ThrDH were analyzed by comparing its structure to that of F. frigidimaris l-ThrDH. This comparison showed that the presence of extensive inter- and intrasubunit ion pair networks are likely responsible for the thermostability of T. volcanium l-ThrDH. This is the first description of the molecular basis for the substrate recognition and thermostability of a GalE-like l-ThrDH. PMID:22374996

  9. Decaprenylphosphoryl-β-D-Ribose 2′-Epimerase, the Target of Benzothiazinones and Dinitrobenzamides, Is an Essential Enzyme in Mycobacterium smegmatis

    PubMed Central

    Crellin, Paul K.; Brammananth, Rajini; Coppel, Ross L.

    2011-01-01

    Background The unique cell wall of bacteria of the suborder Corynebacterineae is essential for the growth and survival of significant human pathogens including Mycobacterium tuberculosis and Mycobacterium leprae. Drug resistance in mycobacteria is an increasingly common development, making identification of new antimicrobials a priority. Recent studies have revealed potent anti-mycobacterial compounds, the benzothiazinones and dinitrobenzamides, active against DprE1, a subunit of decaprenylphosphoribose 2′ epimerase which forms decaprenylphosphoryl arabinose, the arabinose donor for mycobacterial cell wall biosynthesis. Despite the exploitation of Mycobacterium smegmatis in the identification of DprE1 as the target of these new antimicrobials and its use in the exploration of mechanisms of resistance, the essentiality of DprE1 in this species has never been examined. Indeed, direct experimental evidence of the essentiality of DprE1 has not been obtained in any species of mycobacterium. Methodology/Principal Findings In this study we constructed a conditional gene knockout strain targeting the ortholog of dprE1 in M. smegmatis, MSMEG_6382. Disruption of the chromosomal copy of MSMEG_6382 was only possible in the presence of a plasmid-encoded copy of MSMEG_6382. Curing of this “rescue” plasmid from the bacterial population resulted in a cessation of growth, demonstrating gene essentiality. Conclusions/Significance This study provides the first direct experimental evidence for the essentiality of DprE1 in mycobacteria. The essentiality of DprE1 in M. smegmatis, combined with its conservation in all sequenced mycobacterial genomes, suggests that decaprenylphosphoryl arabinose synthesis is essential in all mycobacteria. Our findings indicate a lack of redundancy in decaprenylphosphoryl arabinose synthesis in M. smegmatis, despite the relatively large coding capacity of this species, and suggest that no alternative arabinose donors for cell wall biosynthesis exist

  10. The widespread role of non-enzymatic reactions in cellular metabolism

    PubMed Central

    Keller, Markus A; Piedrafita, Gabriel; Ralser, Markus

    2015-01-01

    Enzymes shape cellular metabolism, are regulated, fast, and for most cases specific. Enzymes do not however prevent the parallel occurrence of non-enzymatic reactions. Non-enzymatic reactions were important for the evolution of metabolic pathways, but are retained as part of the modern metabolic network. They divide into unspecific chemical reactivity and specific reactions that occur either exclusively non-enzymatically as part of the metabolic network, or in parallel to existing enzyme functions. Non-enzymatic reactions resemble catalytic mechanisms as found in all major enzyme classes and occur spontaneously, small molecule (e.g. metal-) catalyzed or light-induced. The frequent occurrence of non-enzymatic reactions impacts on stability and metabolic network structure, and has thus to be considered in the context of metabolic disease, network modeling, biotechnology and drug design. PMID:25617827

  11. Structural Perspective on Enzymatic Halogenation

    PubMed Central

    2008-01-01

    Simple halogen substituents frequently afford key structural features that account for the potency and selectivity of natural products, including antibiotics and hormones. For example, when a single chlorine atom on the antibiotic vancomycin is replaced by hydrogen, the resulting antibacterial activity decreases by up to 70% (HarrisC. M.; KannanR.; KopeckaH.; HarrisT. M.J. Am. Chem. Soc.1985, 107, 6652−6658). This Account analyzes how structure underlies mechanism in halogenases, the molecular machines designed by nature to incorporate halogens into diverse substrates. Traditional synthetic methods of integrating halogens into complex molecules are often complicated by a lack of specificity and regioselectivity. Nature, however, has developed a variety of elegant mechanisms for halogenating specific substrates with both regio- and stereoselectivity. An improved understanding of the biological routes toward halogenation could lead to the development of novel synthetic methods for the creation of new compounds with enhanced functions. Already, researchers have co-opted a fluorinase from the microorganism Streptomyces cattleya to produce 18F-labeled molecules for use in positron emission tomography (PET) (DengH.; CobbS. L.; GeeA. D.; LockhartA.; MartarelloL.; McGlincheyR. P.; O’HaganD.; OnegaM.Chem. Commun.2006, 652−654). Therefore, the discovery and characterization of naturally occurring enzymatic halogenation mechanisms has become an active area of research. The catalogue of known halogenating enzymes has expanded from the familiar haloperoxidases to include oxygen-dependent enzymes and fluorinases. Recently, the discovery of a nucleophilic halogenase that catalyzes chlorinations has expanded the repertoire of biological halogenation chemistry (DongC.; HuangF.; DengH.; SchaffrathC.; SpencerJ. B.; O’HaganD.; NaismithJ. H.Nature2004, 427, 561−56514765200). Structural characterization has provided a basis toward a mechanistic understanding of the specificity

  12. The unique enzymatic and mechanistic properties of plant myosins.

    PubMed

    Henn, Arnon; Sadot, Einat

    2014-12-01

    Myosins are molecular motors that move along actin-filament tracks. Plants express two main classes of myosins, myosin VIII and myosin XI. Along with their relatively conserved sequence and functions, plant myosins have acquired some unique features. Myosin VIII has the enzymatic characteristics of a tension sensor and/or a tension generator, similar to functions found in other eukaryotes. Interestingly, class XI plant myosins have gained a novel function that consists of propelling the exceptionally rapid cytoplasmic streaming. This specific class includes the fastest known translocating molecular motors, which can reach an extremely high velocity of about 60μms(-1). However, the enzymatic properties and mechanistic basis for these remarkable manifestations are not yet fully understood. Here we review recent progress in understanding the uniqueness of plant myosins, while emphasizing the unanswered questions. PMID:25435181

  13. Thermal-induced conformational changes in the product release area drive the enzymatic activity of xylanases 10B: Crystal structure, conformational stability and functional characterization of the xylanase 10B from Thermotoga petrophila RKU-1

    SciTech Connect

    Santos, Camila Ramos; Meza, Andreia Navarro; Hoffmam, Zaira Bruna; Silva, Junio Cota; Alvarez, Thabata Maria; Ruller, Roberto; Giesel, Guilherme Menegon; Verli, Hugo; Squina, Fabio Marcio; Prade, Rolf Alexander; Murakami, Mario Tyago

    2010-12-10

    Research highlights: {yields} The hyperthermostable xylanase 10B from Thermotoga petrophila RKU-1 produces exclusively xylobiose at the optimum temperature. {yields} Circular dichroism spectroscopy suggests a coupling effect of temperature-induced structural changes with its enzymatic behavior. {yields} Crystallographic and molecular dynamics studies indicate that conformational changes in the product release area modulate the enzyme action mode. -- Abstract: Endo-xylanases play a key role in the depolymerization of xylan and recently, they have attracted much attention owing to their potential applications on biofuels and paper industries. In this work, we have investigated the molecular basis for the action mode of xylanases 10B at high temperatures using biochemical, biophysical and crystallographic methods. The crystal structure of xylanase 10B from hyperthermophilic bacterium Thermotoga petrophila RKU-1 (TpXyl10B) has been solved in the native state and in complex with xylobiose. The complex crystal structure showed a classical binding mode shared among other xylanases, which encompasses the -1 and -2 subsites. Interestingly, TpXyl10B displayed a temperature-dependent action mode producing xylobiose and xylotriose at 20 {sup o}C, and exclusively xylobiose at 90 {sup o}C as assessed by capillary zone electrophoresis. Moreover, circular dichroism spectroscopy suggested a coupling effect of temperature-induced structural changes with this particular enzymatic behavior. Molecular dynamics simulations supported the CD analysis suggesting that an open conformational state adopted by the catalytic loop (Trp297-Lys326) provokes significant modifications in the product release area (+1,+2 and +3 subsites), which drives the enzymatic activity to the specific release of xylobiose at high temperatures.

  14. Macrophage Migration Inhibitory Factor (MIF) Enzymatic Activity and Lung Cancer

    PubMed Central

    Mawhinney, Leona; Armstrong, Michelle E; O’ Reilly, Ciaran; Bucala, Richard; Leng, Lin; Fingerle-Rowson, Gunter; Fayne, Darren; Keane, Michael P; Tynan, Aisling; Maher, Lewena; Cooke, Gordon; Lloyd, David; Conroy, Helen; Donnelly, Seamas C

    2014-01-01

    The cytokine macrophage migration inhibitory factor (MIF) possesses unique tautomerase enzymatic activity, which contributes to the biological functional activity of MIF. In this study, we investigated the effects of blocking the hydrophobic active site of the tautomerase activity of MIF in the pathogenesis of lung cancer. To address this, we initially established a Lewis lung carcinoma (LLC) murine model in Mif-KO and wild-type (WT) mice and compared tumor growth in a knock-in mouse model expressing a mutant MIF lacking enzymatic activity (Mif P1G). Primary tumor growth was significantly attenuated in both Mif-KO and Mif P1G mice compared with WT mice. We subsequently undertook a structure-based, virtual screen to identify putative small molecular weight inhibitors specific for the tautomerase enzymatic active site of MIF. From primary and secondary screens, the inhibitor SCD-19 was identified, which significantly attenuated the tautomerase enzymatic activity of MIF in vitro and in biological functional screens. In the LLC murine model, SCD-19, given intraperitoneally at the time of tumor inoculation, was found to significantly reduce primary tumor volume by 90% (p < 0.001) compared with the control treatment. To better replicate the human disease scenario, SCD-19 was given when the tumor was palpable (at d 7 after tumor inoculation) and, again, treatment was found to significantly reduce tumor volume by 81% (p < 0.001) compared with the control treatment. In this report, we identify a novel inhibitor that blocks the hydrophobic pocket of MIF, which houses its specific tautomerase enzymatic activity, and demonstrate that targeting this unique active site significantly attenuates lung cancer growth in in vitro and in vivo systems. PMID:25826675

  15. Enzymatic approach to biodiesel production.

    PubMed

    Akoh, Casimir C; Chang, Shu-Wei; Lee, Guan-Chiun; Shaw, Jei-Fu

    2007-10-31

    The need for alternative energy sources that combine environmental friendliness with biodegradability, low toxicity, renewability, and less dependence on petroleum products has never been greater. One such energy source is referred to as biodiesel. This can be produced from vegetable oils, animal fats, microalgal oils, waste products of vegetable oil refinery or animal rendering, and used frying oils. Chemically, they are known as monoalkyl esters of fatty acids. The conventional method for producing biodiesel involves acid and base catalysts to form fatty acid alkyl esters. Downstream processing costs and environmental problems associated with biodiesel production and byproducts recovery have led to the search for alternative production methods and alternative substrates. Enzymatic reactions involving lipases can be an excellent alternative to produce biodiesel through a process commonly referred to alcoholysis, a form of transesterification reaction, or through an interesterification (ester interchange) reaction. Protein engineering can be useful in improving the catalytic efficiency of lipases as biocatalysts for biodiesel production. The use of recombinant DNA technology to produce large quantities of lipases, and the use of immobilized lipases and immobilized whole cells, may lower the overall cost, while presenting less downstream processing problems, to biodiesel production. In addition, the enzymatic approach is environmentally friendly, considered a "green reaction", and needs to be explored for industrial production of biodiesel. PMID:17902621

  16. Enzymatic synthesis of magnetic nanoparticles.

    PubMed

    Kolhatkar, Arati G; Dannongoda, Chamath; Kourentzi, Katerina; Jamison, Andrew C; Nekrashevich, Ivan; Kar, Archana; Cacao, Eliedonna; Strych, Ulrich; Rusakova, Irene; Martirosyan, Karen S; Litvinov, Dmitri; Lee, T Randall; Willson, Richard C

    2015-01-01

    We report the first in vitro enzymatic synthesis of paramagnetic and antiferromagnetic nanoparticles toward magnetic ELISA reporting. With our procedure, alkaline phosphatase catalyzes the dephosphorylation of l-ascorbic-2-phosphate, which then serves as a reducing agent for salts of iron, gadolinium, and holmium, forming magnetic precipitates of Fe45±14Gd5±2O50±15 and Fe42±4Ho6±4O52±5. The nanoparticles were found to be paramagnetic at 300 K and antiferromagnetic under 25 K. Although weakly magnetic at 300 K, the room-temperature magnetization of the nanoparticles found here is considerably greater than that of analogous chemically-synthesized LnxFeyOz (Ln = Gd, Ho) samples reported previously. At 5 K, the nanoparticles showed a significantly higher saturation magnetization of 45 and 30 emu/g for Fe45±14Gd5±2O50±15 and Fe42±4Ho6±4O52±5, respectively. Our approach of enzymatically synthesizing magnetic labels reduces the cost and avoids diffusional mass-transfer limitations associated with pre-synthesized magnetic reporter particles, while retaining the advantages of magnetic sensing. PMID:25854425

  17. Enzymatic Synthesis of Magnetic Nanoparticles

    PubMed Central

    Kolhatkar, Arati G.; Dannongoda, Chamath; Kourentzi, Katerina; Jamison, Andrew C.; Nekrashevich, Ivan; Kar, Archana; Cacao, Eliedonna; Strych, Ulrich; Rusakova, Irene; Martirosyan, Karen S.; Litvinov, Dmitri; Lee, T. Randall; Willson, Richard C.

    2015-01-01

    We report the first in vitro enzymatic synthesis of paramagnetic and antiferromagnetic nanoparticles toward magnetic ELISA reporting. With our procedure, alkaline phosphatase catalyzes the dephosphorylation of l-ascorbic-2-phosphate, which then serves as a reducing agent for salts of iron, gadolinium, and holmium, forming magnetic precipitates of Fe45±14Gd5±2O50±15 and Fe42±4Ho6±4O52±5. The nanoparticles were found to be paramagnetic at 300 K and antiferromagnetic under 25 K. Although weakly magnetic at 300 K, the room-temperature magnetization of the nanoparticles found here is considerably greater than that of analogous chemically-synthesized LnxFeyOz (Ln = Gd, Ho) samples reported previously. At 5 K, the nanoparticles showed a significantly higher saturation magnetization of 45 and 30 emu/g for Fe45±14Gd5±2O50±15 and Fe42±4Ho6±4O52±5, respectively. Our approach of enzymatically synthesizing magnetic labels reduces the cost and avoids diffusional mass-transfer limitations associated with pre-synthesized magnetic reporter particles, while retaining the advantages of magnetic sensing. PMID:25854425

  18. Multiple Peroxisomal Enzymatic Deficiency Disorders

    PubMed Central

    Vamecq, Joseph; Draye, Jean-Pierre; Van Hoof, François; Misson, Jean-Paul; Evrard, Philippe; Verellen, Gaston; Eyssen, Hendrik J.; Van Eldere, Johan; Schutgens, Ruud B. H.; Wanders, Ronald J. A.; Roels, Frank; Goldfischer, Sidney L.

    1986-01-01

    Biologic, morphologic, and biochemical investigations performed in 2 patients demonstrate multiple peroxisomal deficiencies in the cerebrohepatorenal syndrome of Zellweger (CHRS) and neonatal adrenoleukodystrophy (NALD). Very long chain fatty acids, abnormal bile acids, including bile acid precursors (di- and trihydroxycoprostanoic acids), and C29-dicarboxylic acid accumulated in plasma in both patients. Generalized hyperaminoaciduria was also present. Peroxisomes could not be detected in CHRS liver and kidney; however, in the NALD patient, small and sparse cytoplasmic bodies resembling altered peroxisomes were found in hepatocytes. Hepatocellular and Kupffer cell lysosomes were engorged with ferritin and contained clefts and trilaminar structures believed to represent very long chain fatty acids. Enzymatic deficiencies reflected the peroxisomal defects. Hepatic glycolate oxidase and palmitoyl-CoA oxidase activities were deficient. No particle-bound catalase was found in cultured fibroblasts, and ether glycerolipid (plasmalogen) biosynthesis was markedly reduced. Administration of phenobarbital and clofibrate, an agent that induces peroxisomal proliferation and enzymatic activities, to the NALD patient did not bring about any changes in plasma metabolites, liver peroxisome population, or oxidizing activities. ImagesFigure 1Figure 2Figure 3Figure 4Figure 5 PMID:2879480

  19. Economics of enzymatic hydrolysis processes

    SciTech Connect

    Wright, J.D.

    1988-02-01

    Enzymatic hydrolysis processes have the ability to produce high yields of sugars for fermentation to fuel ethanol from lignocellulosic biomass. However, these systems have been plagued with yields, product concentrations, and reactions rates far below those that are theoretically possible. Engineering and economic analyses are presented on several fungal enzyme hydrolysis processes to illustrate the effects of the important process parameters, to quantify the progress that has been made to date, and to estimate the cost reductions that can be made through research improvements. All enzymatic hydrolysis processes require pretreatment, hydrolysis, fermentation, and enzyme production. The key effect of pretreatment is to allow access of the enzymes to the substrate. Pretreatments have been devised that make the biomass completely digestible that increase the xylose yield and concentration, and that integrate pretreatment with lignin utilization. Major improvements in enzyme activity and use of simultaneous saccharification and fermentation (SSF) have greatly reduced the inhibition of the enzymes. It now appears that ethanol inhibition of the yeast is the limiting factor. Enzyme production costs have been dramatically reduced because use of SSF has reduced enzyme loading. However, further improvements may be possible by using soluble carbon sources for production. Over the past decade, the predicted cost of ethanol from such processes has dropped from more than $4.00/gallon to approximately $1.60. Research is currently under way in the United States and has the potential to reduce the projected cost to less than $1.00/gallon. 65 refs., 16 figs., 1 tab.

  20. New developments of polysaccharide synthesis via enzymatic polymerization

    PubMed Central

    Kobayashi, Shiro

    2007-01-01

    This review focuses on the in vitro synthesis of polysaccharides, the method of which is “enzymatic polymerization” mainly developed by our group. Polysaccharides are formed by repeated glycosylation reactions between a glycosyl donor and a glycosyl acceptor. A hydrolysis enzyme was found very efficient as catalyst, where the monomer is designed based on the new concept of a “transition-state analogue substrate” (TSAS); sugar fluoride monomers for polycondensation and sugar oxazoline monomers for ring-opening polyaddition. Enzymatic polymerization enabled the first in vitro synthesis of natural polysaccharides such as cellulose, xylan, chitin, hyaluronan and chondroitin, and also of unnatural polysaccharides such as a cellulose–chitin hybrid, a hyaluronan–chondroitin hybrid, and others. Supercatalysis of hyaluronidase was disclosed as unusual enzymatic multi-catalyst functions. Mutant enzymes were very useful for synthetic and mechanistic studies. In situ observations of enzymatic polymerization by SEM, TEM, and combined SAS methods revealed mechanisms of the polymerization and of the self-assembling of high-order molecular structure formed by elongating polysaccharide molecules. PMID:24367148

  1. Enzymatic reactivity of glucose oxidase confined in nanochannels.

    PubMed

    Yu, Jiachao; Zhang, Yuanjian; Liu, Songqin

    2014-05-15

    The construction of nanodevices coupled with an integrated real-time detection system for evaluation of the function of biomolecules in biological processes, and enzymatic reaction kinetics occurring at the confined space or interface is a significant challenge. In this work, a nanochannel-enzyme system in which the enzymatic reaction could be investigated with an electrochemical method was constructed. The model system was established by covalently linking glucose oxidase (GOD) onto the inner wall of the nanochannels of the porous anodic alumina (PAA) membrane. An Au disc was attached at the end of the nanochannels of the PAA membrane as the working electrode for detection of H2O2 product of enzymatic reaction. The effects of ionic strength, amount of immobilized enzyme and pore diameter of the nanochannels on the enzymatic reaction kinetics were illustrated. The GOD confined in nanochannels showed high stability and reactivity. Upon addition of glucose to the nanochannel-enzyme system, the current response had a calibration range span from 0.005 to 2 mM of glucose concentration. The apparent Michaelis-Menten constant (K(m)(app)) of GOD confined in nanochannel was 0.4 mM. The presented work provided a platform for real-time monitoring of the enzyme reaction kinetics confined in nanospaces. Such a nanochannel-enzyme system could also help design future biosensors and enzyme reactors with high sensitivity and efficiency. PMID:24412427

  2. beta. -Sulfopyruvate: chemical and enzymatic syntheses and enzymatic assay

    SciTech Connect

    Weinstein, C.L.; Griffith, O.W.

    1986-01-01

    BETA-Sulfopyruvic acid (2-carboxy-2-oxoethanesulfonic acid) is prepared in greater than 90% yield by reaction of bromopyruvic acid with sodium sulfite. ..beta..-(/sup 35/S)Sulfopyruvate is prepared by transamination between (/sup 35/)cysteinesulfonate (cysteate) and ..cap alpha..-ketoglutarate using mitochondrial aspartate aminotransferase isolated from rat liver. Following either chemical or enzymatic synthesis the crude reaction product is conveniently purified by chromatography on Dowex 1; ..beta..-sulfopyruvate is isolated as the stable, water-soluble dilithium salt. ..beta..-Sulfopyruvate is shown to be an alternative substrate of mitochondrial malate dehydrogenase; in the presence of 0.25 mM NADH, ..beta..-sulfopyruvate is reduced with an apparent K/sub m/ of 6.3 mM and a V/sub max/ equal to about 40% of that observed with oxaloacetate. This finding forms the basis of a convenient spectrophotometric assay of ..beta..-sulfopyruvate.

  3. Autoantibodies with Enzymatic Properties in Human Autoimmune Diseases

    PubMed Central

    Wootla, Bharath; Lacroix-Desmazes, Sébastien; Warrington, Arthur E.; Bieber, Allan J.; Kaveri, Srini V.; Rodriguez, Moses

    2011-01-01

    Immunoglobulins (Ig) or antibodies are heavy plasma proteins, with sugar chains added to amino acid residues by N-linked glycosylation and occasionally by O-linked glycosylation. The versatility of antibodies is demonstrated by the various functions that they mediate such as neutralization, agglutination, fixation with activation of complement and activation of effector cells. In addition to this plethora of functions, some antibodies express enzymatic activity. Antibodies endowed with enzymatic properties have been described in human autoimmune manifestations for more than a decade in a variety of disorders such as autoimmune thyroiditis, systemic erythematosus (SLE), scleroderma, rheumatoid arthritis (RA), multiple sclerosis (MS) and acquired hemophilia (AH). Antibodies isolated from these conditions were able to specifically hydrolyze thyroglobulin, DNA, RNA, myelin basic protein (MBP), and factor VIII (FVIII) or factor IX (FIX), respectively. The therapeutic relevance of these findings is discussed. PMID:21624820

  4. Enzymatic regeneration of adenosine triphosphate cofactor

    NASA Technical Reports Server (NTRS)

    Marshall, D. L.

    1974-01-01

    Regenerating adenosine triphosphate (ATP) from adenosine diphosphate (ADP) by enzymatic process which utilizes carbamyl phosphate as phosphoryl donor is technique used to regenerate expensive cofactors. Process allows complex enzymatic reactions to be considered as candidates for large-scale continuous processes.

  5. Force-Manipulation Single-Molecule Spectroscopy Studies of Enzymatic Dynamics

    NASA Astrophysics Data System (ADS)

    Lu, H. Peter; He, Yufan; Lu, Maolin; Cao, Jin; Guo, Qing

    2014-03-01

    Subtle conformational changes play a crucial role in protein functions, especially in enzymatic reactions involving complex substrate-enzyme interactions and chemical reactions. We applied AFM-enhanced and magnetic tweezers-correlated single-molecule spectroscopy to study the mechanisms and dynamics of enzymatic reactions involved with kinase and lysozyme proteins. Enzymatic reaction turnovers and the associated structure changes of individual protein molecules were observed simultaneously in real-time by single-molecule FRET detections. Our single-molecule spectroscopy measurements of enzymatic conformational dynamics have revealed time bunching effect and intermittent coherence in conformational state change dynamics involving in enzymatic reaction cycles. The coherent conformational state dynamics suggests that the enzymatic catalysis involves a multi-step conformational motion along the coordinates of substrate-enzyme complex formation and product releasing. Our results support a multiple-conformational state model, being consistent with a complementary conformation selection and induced-fit enzymatic loop-gated conformational change mechanism in substrate-enzyme active complex formation.

  6. Computational Investigations on Enzymatic Catalysis and Inhibition

    NASA Astrophysics Data System (ADS)

    Simard, Daniel

    Enzymes are the bimolecular "workhorses" of the cell due to their range of functions and their requirement for cellular success. The atomistic details of how they function can provide key insights into the fundamentals of catalysis and in turn, provide a blueprint for biotechnological advances. A wide range of contemporary computational techniques has been applied with the aim to characterize recently discovered intermediates or to provide insights into enzymatic mechanisms and inhibition. More specifically, an assessment of methods was conducted to evaluate the presence of the growing number 3-- and 4--coordinated sulfur intermediates in proteins/enzymes. Furthermore, two mechanisms have been investigated, the mu-OH mechanism of the hydrolysis of dimethylphosphate in Glycerophosphodiesterase (GpdQ) using five different homonuclear metal combinations Zn(II)/Zn(II), Co(II)/Co(II), Mn(II)/Mn(II), Cd(II)/Cd(II) and Ca(II)/Ca(II) as well as a preliminary study into the effectivness of boron as an inhibitor in the serine protease reaction of class A TEM-1 beta-lactamases.

  7. Both myo-inositol to chiro-inositol epimerase activities and chiro-inositol to myo-inositol ratios are decreased in tissues of GK type 2 diabetic rats compared to Wistar controls.

    PubMed

    Sun, Tie-hua; Heimark, Douglas B; Nguygen, Thang; Nadler, Jerry L; Larner, Joseph

    2002-05-10

    Previous data from our and other labs demonstrated a decreased chiro-inositol content in urine and tissues of human subjects and animals with type 2 diabetes. In urine this decrease in chiro-inositol was accompanied by an increase in myo-inositol content. Decreased urine levels of chiro-inositol in monkeys were next correlated with the severity of underlying insulin resistance determined by five separate assays. To investigate the decreased chiro-inositol and the accompanying increased myo-inositol excretions in urine in humans and monkeys, we postulated a defect in the epimerization of myo-inositol to chiro-inositol. [(3)H]Myo-inositol was then shown to be converted to [(3)H]chiro-inositol in rats in vivo and in fibroblasts in vitro in a process stimulated by insulin. We next demonstrated that the conversion of [(3)H]myo-inositol to [(3)H]chiro-inositol in vivo was markedly decreased in GK type 2 diabetic rats compared to Wistar controls in liver, muscle, and fat, insulin sensitive tissues. Decreases of 20-25% conversion to baseline levels of under 5% conversion were observed. In the present work, we initially compared the total contents of myo-inositol and chiro-inositol in GK type 2 diabetic rat kidney, liver, and muscle compared to Wistar controls. We demonstrated a consistent decreased total chiro-inositol to myo-inositol ratio in kidney, liver, and muscle compared to controls. We next established the presence of a myo-inositol to chiro-inositol epimerase activity in rat liver cytosol. Enzyme activity was shown to be time and enzyme concentration dependent with a broad pH optimum. It required NADH and NADPH for full activity, which is compatible with its action via an oxido-reductive mechanism. Lastly, we demonstrated that the epimerase enzyme bioactivity was significantly decreased in muscle, liver, and fat cytosolic extracts of GK type 2 diabetic rats versus Wistar controls. Decreased myo-inositol to chiro-inositol epimerase activity may therefore play a

  8. Enzymatic synthesis of oligo- and polysaccharide fatty acid esters.

    PubMed

    van den Broek, Lambertus A M; Boeriu, Carmen G

    2013-03-01

    Amphiphilic oligo- and polysaccharides (e.g. polysaccharide alkyl or alkyl-aryl esters) form a new class of polymers with exceptional properties. They function as polymeric surfactants, whilst maintaining most of the properties of the starting polymeric material such as emulsifying, gelling, and film forming properties combined with partial water solubility or permeability. At present carbohydrate fatty acid esters are generally obtained by chemical methods using toxic solvents and organic and inorganic catalysts that leave residual traces in the final products. Enzymatic reactions offer an attractive alternative route for the synthesis of polysaccharide esters. In this review the state of the art of enzymatic synthesis of oligo- and polysaccharides fatty esters has been described. PMID:23465902

  9. Enzymatic Vitrectomy and Pharmacologic Vitreodynamics.

    PubMed

    Shah, Ankoor R; Trese, Michael T

    2016-01-01

    The field of vitreoretinal surgery has evolved substantially over the last several decades. Scientific advances have improved our understanding of disease pathophysiology, and new surgical adjuncts and techniques have decreased surgical time and improved patient outcomes. Pharmacologic agents have recently been developed for intraocular use in order to enhance vitreous removal and even as a nonsurgical treatment for pathology due to an abnormal vitreoretinal interface. Plasmin can successfully cause vitreous liquefaction and induce a posterior vitreous detachment. Additionally, ocriplasmin has been approved for symptomatic vitreomacular adhesion and others appear to be promising for pharmacologic manipulation of the vitreous. The ability to induce vitreous liquefaction and a complete posterior vitreous detachment (PVD) with a single intravitreal injection has potential implications for the management of multiple vitreoretinopathies. Enzymatic vitrectomy may help to reduce vitreous viscosity, thereby facilitating removal during vitrectomy and reducing surgical time, especially when using smaller-gauge vitrectomy instruments. The induction of a PVD also has the potential to reduce intraoperative complications. As we improve our understanding of the molecular flux in the vitreous cavity, pharmacologic vitreodynamics will likely become more important as it may allow for improved manipulation of intravitreal molecules. PMID:26501959

  10. Light-driven Enzymatic Decarboxylation

    PubMed Central

    Köninger, Katharina; Grote, Marius; Zachos, Ioannis; Hollmann, Frank; Kourist, Robert

    2016-01-01

    Oxidoreductases belong to the most-applied industrial enzymes. Nevertheless, they need external electrons whose supply is often costly and challenging. Recycling of the electron donors NADH or NADPH requires the use of additional enzymes and sacrificial substrates. Interestingly, several oxidoreductases accept hydrogen peroxide as electron donor. While being inexpensive, this reagent often reduces the stability of enzymes. A solution to this problem is the in situ generation of the cofactor. The continuous supply of the cofactor at low concentration drives the reaction without impairing enzyme stability. This paper demonstrates a method for the light-catalyzed in situ generation of hydrogen peroxide with the example of the heme-dependent fatty acid decarboxylase OleTJE. The fatty acid decarboxylase OleTJE was discovered due to its unique ability to produce long-chain 1-alkenes from fatty acids, a hitherto unknown enzymatic reaction. 1-alkenes are widely used additives for plasticizers and lubricants. OleTJE has been shown to accept electrons from hydrogen peroxide for the oxidative decarboxylation. While addition of hydrogen peroxide damages the enzyme and results in low yields, in situ generation of the cofactor circumvents this problem. The photobiocatalytic system shows clear advantages regarding enzyme activity and yield, resulting in a simple and efficient system for fatty acid decarboxylation. PMID:27286035

  11. Light-driven Enzymatic Decarboxylation.

    PubMed

    Köninger, Katharina; Grote, Marius; Zachos, Ioannis; Hollmann, Frank; Kourist, Robert

    2016-01-01

    Oxidoreductases belong to the most-applied industrial enzymes. Nevertheless, they need external electrons whose supply is often costly and challenging. Recycling of the electron donors NADH or NADPH requires the use of additional enzymes and sacrificial substrates. Interestingly, several oxidoreductases accept hydrogen peroxide as electron donor. While being inexpensive, this reagent often reduces the stability of enzymes. A solution to this problem is the in situ generation of the cofactor. The continuous supply of the cofactor at low concentration drives the reaction without impairing enzyme stability. This paper demonstrates a method for the light-catalyzed in situ generation of hydrogen peroxide with the example of the heme-dependent fatty acid decarboxylase OleTJE. The fatty acid decarboxylase OleTJE was discovered due to its unique ability to produce long-chain 1-alkenes from fatty acids, a hitherto unknown enzymatic reaction. 1-alkenes are widely used additives for plasticizers and lubricants. OleTJE has been shown to accept electrons from hydrogen peroxide for the oxidative decarboxylation. While addition of hydrogen peroxide damages the enzyme and results in low yields, in situ generation of the cofactor circumvents this problem. The photobiocatalytic system shows clear advantages regarding enzyme activity and yield, resulting in a simple and efficient system for fatty acid decarboxylation. PMID:27286035

  12. Two-Photon Small Molecule Enzymatic Probes.

    PubMed

    Qian, Linghui; Li, Lin; Yao, Shao Q

    2016-04-19

    Enzymes are essential for life, especially in the development of disease and on drug effects, but as we cannot yet directly observe the inside interactions and only partially observe biochemical outcomes, tools "translating" these processes into readable information are essential for better understanding of enzymes as well as for developing effective tools to fight against diseases. Therefore, sensitive small molecule probes suitable for direct in vivo monitoring of enzyme activities are ultimately desirable. For fulfilling this desire, two-photon small molecule enzymatic probes (TSMEPs) producing amplified fluorescent signals based on enzymatic conversion with better photophysical properties and deeper penetration in intact tissues and whole animals have been developed and demonstrated to be powerful in addressing the issues described above. Nonetheless, currently available TSMEPs only cover a small portion of enzymes despite the distinct advantages of two-photon fluorescence microscopy. In this Account, we would like to share design principles for TSMEPs as potential indicators of certain pathology-related biomarkers together with their applications in disease models to inspire more elegant work to be done in this area. Highlights will be addressed on how to equip two-photon fluorescent probes with features amenable for direct assessment of enzyme activities in complex pathological environments. We give three recent examples from our laboratory and collaborations in which TSMEPs are applied to visualize the distribution and activity of enzymes at cellular and organism levels. The first example shows that we could distinguish endogenous phosphatase activity in different organelles; the second illustrates that TSMEP is suitable for specific and sensitive detection of a potential Parkinson's disease marker (monoamine oxidase B) in a variety of biological systems from cells to patient samples, and the third identifies that TSMEPs can be applied to other enzyme

  13. Enzymatic Activity of Xyloglucan Xylosyltransferase 51[OPEN

    PubMed Central

    Culbertson, Alan T.; Chou, Yi-Hsiang; Smith, Adrienne L.; Young, Zachary T.; Tietze, Alesia A.; Cottaz, Sylvain

    2016-01-01

    Xyloglucan, the most abundant hemicellulosic component of the primary cell wall of flowering plants, is composed of a β-(1,4)-glucan backbone decorated with d-xylosyl residues. Three xyloglucan xylosyltransferases (XXTs) participate in xyloglucan biosynthesis in Arabidopsis (Arabidopsis thaliana). Two of these, XXT1 and XXT2, have been shown to be active in vitro, whereas the catalytic activity of XXT5 has yet to be demonstrated. By optimizing XXT2 expression in a prokaryotic system and in vitro activity assay conditions, we demonstrate that nonglycosylated XXT2 lacking its cytosolic amino-terminal and transmembrane domain displays high catalytic activity. Using this optimized procedure for the expression of XXT5, we report, to our knowledge for the first time, that recombinant XXT5 shows enzymatic activity in vitro, although at a significantly slower rate than XXT1 and XXT2. Kinetic analysis showed that XXT5 has a 7-fold higher Km and 9-fold lower kcat compared with XXT1 and XXT2. Activity assays using XXT5 in combination with XXT1 or XXT2 indicate that XXT5 is not specific for their products. In addition, mutagenesis experiments showed that the in vivo function and in vitro catalytic activity of XXT5 require the aspartate-serine-aspartate motif. These results demonstrate that XXT5 is a catalytically active xylosyltransferase involved in xylosylation of the xyloglucan backbone. PMID:27208276

  14. Enzymatic Activity of Xyloglucan Xylosyltransferase 5.

    PubMed

    Culbertson, Alan T; Chou, Yi-Hsiang; Smith, Adrienne L; Young, Zachary T; Tietze, Alesia A; Cottaz, Sylvain; Fauré, Régis; Zabotina, Olga A

    2016-07-01

    Xyloglucan, the most abundant hemicellulosic component of the primary cell wall of flowering plants, is composed of a β-(1,4)-glucan backbone decorated with d-xylosyl residues. Three xyloglucan xylosyltransferases (XXTs) participate in xyloglucan biosynthesis in Arabidopsis (Arabidopsis thaliana). Two of these, XXT1 and XXT2, have been shown to be active in vitro, whereas the catalytic activity of XXT5 has yet to be demonstrated. By optimizing XXT2 expression in a prokaryotic system and in vitro activity assay conditions, we demonstrate that nonglycosylated XXT2 lacking its cytosolic amino-terminal and transmembrane domain displays high catalytic activity. Using this optimized procedure for the expression of XXT5, we report, to our knowledge for the first time, that recombinant XXT5 shows enzymatic activity in vitro, although at a significantly slower rate than XXT1 and XXT2. Kinetic analysis showed that XXT5 has a 7-fold higher Km and 9-fold lower kcat compared with XXT1 and XXT2. Activity assays using XXT5 in combination with XXT1 or XXT2 indicate that XXT5 is not specific for their products. In addition, mutagenesis experiments showed that the in vivo function and in vitro catalytic activity of XXT5 require the aspartate-serine-aspartate motif. These results demonstrate that XXT5 is a catalytically active xylosyltransferase involved in xylosylation of the xyloglucan backbone. PMID:27208276

  15. Nanocrystal Bioassembly: Asymmetry, Proximity, and Enzymatic Manipulation

    SciTech Connect

    Claridge, Shelley A.

    2008-05-01

    Research at the interface between biomolecules and inorganic nanocrystals has resulted in a great number of new discoveries. In part this arises from the synergistic duality of the system: biomolecules may act as self-assembly agents for organizing inorganic nanocrystals into functional materials; alternatively, nanocrystals may act as microscopic or spectroscopic labels for elucidating the behavior of complex biomolecular systems. However, success in either of these functions relies heavily uponthe ability to control the conjugation and assembly processes.In the work presented here, we first design a branched DNA scaffold which allows hybridization of DNA-nanocrystal monoconjugates to form discrete assemblies. Importantly, the asymmetry of the branched scaffold allows the formation of asymmetric2assemblies of nanocrystals. In the context of a self-assembled device, this can be considered a step toward the ability to engineer functionally distinct inputs and outputs.Next we develop an anion-exchange high performance liquid chromatography purification method which allows large gold nanocrystals attached to single strands of very short DNA to be purified. When two such complementary conjugates are hybridized, the large nanocrystals are brought into close proximity, allowing their plasmon resonances to couple. Such plasmon-coupled constructs are of interest both as optical interconnects for nanoscale devices and as `plasmon ruler? biomolecular probes.We then present an enzymatic ligation strategy for creating multi-nanoparticle building blocks for self-assembly. In constructing a nanoscale device, such a strategy would allow pre-assembly and purification of components; these constructs can also act as multi-label probes of single-stranded DNA conformational dynamics. Finally we demonstrate a simple proof-of-concept of a nanoparticle analog of the polymerase chain reaction.

  16. Self-evolving microstructured systems upon enzymatic catalysis.

    PubMed

    Chopineau, J; Lesieur, S; Carion-Taravella, B; Ollivon, M

    1998-01-01

    The consequences of cell microstructuration on enzyme functions is discussed in the framework of self-evolving microstructured systems. Molecular assemblies of amphiphiles or lipids are spontaneously formed by self-organisation. Among these different structures, reversed micelles, liquid crystalline mesophases and vesicles are hosts for enzymatic reaction studies. Inside a living cell, phospholipid metabolism is responsible for membrane structural modifications; the catalytic behaviour of lipolytic enzymes, mainly phospholipase (PL) A2, is described in relation with structural aspects of biological membranes. The implication in cellular regulation events of PLC and PLD is discussed in relation with the role of their reaction products as second messengers in membrane fusion processes. The in vitro synthesis of dialkyl phosphatidylcholines, via the enzymatic 'salvage pathway' which leads to the formation of vesicles upon phospholipid formation, is considered in relation with autopoiesis. More recent studies on self-evolving systems based on enzyme-surfactants reactions are detailed. The interactions between amphiphilic aggregates and enzymes allow to explore the OG/octanol/water phase diagram. Enzymatic formation of dipalmitoylphosphatidylcholine (DPPC) liposomes and non-ionic surfactant vesicles (NSV), starting from mixed micelles or open structures, finally sets an example of a biomimetic self-evolving system. PMID:9782383

  17. Elucidation of Factors Effecting Enzymatic Saccharification using Transgenic Hardwoods

    NASA Astrophysics Data System (ADS)

    Min, Douyong

    Three groups of transgenic wood samples were used as starting materials to elucidate the recalcitrance of enzymatic saccharification with/without pretreatments. The first group of transgenic wood samples is low lignin P. trichocarpa. The second group is low xylan P. trichocarpa. The third one is 12 hybrid poplars which have different levels of S/V ratio and lignin content. Four pretreatments were carried out in this research including dilute sulfuric acid, green liquor, auto hydrolysis and ozone delignification. The behavior among pretreatments as a function of removal of lignin appears to be different. Lignin is the major factor of recalcitrance of the lignocellulosic material to ethanol conversion process. Xylan also plays key role in this process. In addition, the crude milled wood lignin was isolated from these three groups of transgenic samples. Lignin carbohydrate complexes was characterized by 1H-13C HMQC and 13C NMR. Thus the effect of LCCs on enzymatic saccharification was elucidated. High S/V ratio propels the lignin removal during pretreatments however; high S/V ratio retards the enzymatic saccharification on the lignocellulosic material without pretreatments. The level of LCCs linkages accounts for additional recalcitrance of the lignocellulosic material to ethanol conversion process. The amount of LCCs linkages is affected by xylan content, lignin content and S/V ratio.

  18. Controlling enzymatic activity and kinetics in swollen mesophases by physical nano-confinement

    NASA Astrophysics Data System (ADS)

    Sun, Wenjie; Vallooran, Jijo J.; Zabara, Alexandru; Mezzenga, Raffaele

    2014-05-01

    Bicontinuous lipid cubic mesophases are widely investigated as hosting matrices for functional enzymes to build biosensors and bio-devices due to their unique structural characteristics. However, the enzymatic activity within standard mesophases (in-meso) is severely hindered by the relatively small diameter of the mesophase aqueous channels, which provide only limited space for enzymes, and restrict them into a highly confined environment. We show that the enzymatic activity of a model enzyme, horseradish peroxidase (HRP), can be accurately controlled by relaxing its confinement within the cubic phases' water channels, when the aqueous channel diameters are systematically swollen with varying amount of hydration-enhancing sugar ester. The in-meso activity and kinetics of HRP are then systematically investigated by UV-vis spectroscopy, as a function of the size of the aqueous mesophase channels. The enzymatic activity of HRP increases with the swelling of the water channels. In swollen mesophases with water channel diameter larger than the HRP size, the enzymatic activity is more than double that measured in standard mesophases, approaching again the enzymatic activity of free HRP in bulk water. We also show that the physically-entrapped enzymes in the mesophases exhibit a restricted-diffusion-induced initial lag period and report the first observation of in-meso enzymatic kinetics significantly deviating from the normal Michaelis-Menten behaviour observed in free solutions, with deviations vanishing when enzyme confinement is released by swelling the mesophase.Bicontinuous lipid cubic mesophases are widely investigated as hosting matrices for functional enzymes to build biosensors and bio-devices due to their unique structural characteristics. However, the enzymatic activity within standard mesophases (in-meso) is severely hindered by the relatively small diameter of the mesophase aqueous channels, which provide only limited space for enzymes, and restrict them

  19. Enzymatic Enantioselective Decarboxylative Protonation of Heteroaryl Malonates

    PubMed Central

    Lewin, Ross; Goodall, Mark; Thompson, Mark L; Leigh, James; Breuer, Michael; Baldenius, Kai; Micklefield, Jason

    2015-01-01

    The enzyme aryl/alkenyl malonate decarboxylase (AMDase) catalyses the enantioselective decarboxylative protonation (EDP) of a range of disubstituted malonic acids to give homochiral carboxylic acids that are valuable synthetic intermediates. AMDase exhibits a number of advantages over the non-enzymatic EDP methods developed to date including higher enantioselectivity and more environmentally benign reaction conditions. In this report, AMDase and engineered variants have been used to produce a range of enantioenriched heteroaromatic α-hydroxycarboxylic acids, including pharmaceutical precursors, from readily accessible α-hydroxymalonates. The enzymatic method described here represents an improvement upon existing synthetic chemistry methods that have been used to produce similar compounds. The relationship between the structural features of these new substrates and the kinetics associated with their enzymatic decarboxylation is explored, which offers further insight into the mechanism of AMDase. PMID:25766433

  20. Enzymatic desulfurization of coal: Third quarterly report

    SciTech Connect

    Marquis, J.K.; Kitchell, J.P.

    1989-03-14

    Our current efforts to develop clean coal technology emphasize the advantages of enzymatic desulfurization techniques and have specifically addressed the potential of using partially-purified extracellular microbial enzymes or commercially available enzymes. Our work is focused on the treatment of ''model'' organic sulfur compounds such as dibenzothiophene (DBT) and ethylphenylsulfide (EPS). Furthermore, we are designing experiments to facilitate the enzymatic process by means of a hydrated organic solvent matrix. In this quarter we obtained important results both with the development of our understanding of the enzyme reaction systems and also with the microbial work at Woods Hole. 12 figs., 11 tabs.

  1. Reaction graph kernels predict EC numbers of unknown enzymatic reactions in plant secondary metabolism

    PubMed Central

    2010-01-01

    Background Understanding of secondary metabolic pathway in plant is essential for finding druggable candidate enzymes. However, there are many enzymes whose functions are not yet discovered in organism-specific metabolic pathways. Towards identifying the functions of those enzymes, assignment of EC numbers to the enzymatic reactions they catalyze plays a key role, since EC numbers represent the categorization of enzymes on one hand, and the categorization of enzymatic reactions on the other hand. Results We propose reaction graph kernels for automatically assigning EC numbers to unknown enzymatic reactions in a metabolic network. Reaction graph kernels compute similarity between two chemical reactions considering the similarity of chemical compounds in reaction and their relationships. In computational experiments based on the KEGG/REACTION database, our method successfully predicted the first three digits of the EC number with 83% accuracy. We also exhaustively predicted missing EC numbers in plant's secondary metabolism pathway. The prediction results of reaction graph kernels on 36 unknown enzymatic reactions are compared with an expert's knowledge. Using the same data for evaluation, we compared our method with E-zyme, and showed its ability to assign more number of accurate EC numbers. Conclusion Reaction graph kernels are a new metric for comparing enzymatic reactions. PMID:20122204

  2. Inhibition of enzymatic cellulolysis by phenolic compounds.

    PubMed

    Tejirian, Ani; Xu, Feng

    2011-03-01

    Phenolics derived from lignin and other plant components can pose significant inhibition on enzymatic conversion of cellulosic biomass materials to useful chemicals. Understanding the mechanism of such inhibition is of importance for the development of viable biomass conversion technologies. In native plant cell wall, most of the phenolics and derivatives are found in polymeric lignin. When biomass feedstocks are pretreated (prior to enzymatic hydrolysis), simple or oligomeric phenolics and derivatives are often generated from lignin modification/degradation, which can inhibit biomass-converting enzymes. To further understand how such phenolic substances may affect cellulase reaction, we carried out a comparative study on a series of simple and oligomeric phenolics representing or mimicking the composition of lignin or its degradation products. Consistent to previous studies, we observed that oligomeric phenolics could exert more inhibition on enzymatic cellulolysis than simple phenolics. Oligomeric phenolics could inactivate cellulases by reversibly complexing them. Simple and oligomeric phenolics could also inhibit enzymatic cellulolysis by adsorbing onto cellulose. Individual cellulases showed different susceptibility toward these inhibitions. Polyethylene glycol and tannase could respectively bind and degrade the studied oligomeric phenolics, and by doing so mitigate the oligomeric phenolic's inhibition on cellulolysis. PMID:22112906

  3. Frank Westheimer's Early Demonstration of Enzymatic Specificity

    ERIC Educational Resources Information Center

    Ault, Addison

    2008-01-01

    In this article I review one of the most significant accomplishments of Frank H. Westheimer, one of the most respected chemists of the 20th century. This accomplishment was a series of stereospecific enzymatic oxidation and reduction experiments that led chemists to recognize what we now call the enantiotopic and diastereotopic relationships of…

  4. Ultrasonic acceleration of enzymatic processing of cotton

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enzymatic bio-processing of cotton generates significantly less hazardous wastewater effluents, which are readily biodegradable, but it also has several critical shortcomings that impede its acceptance by industries: expensive processing costs and slow reaction rates. It has been found that the intr...

  5. pH & Rate of Enzymatic Reactions.

    ERIC Educational Resources Information Center

    Clariana, Roy B.

    1991-01-01

    A quantitative and inexpensive way to measure the rate of enzymatic reaction is provided. The effects of different pH levels on the reaction rate of an enzyme from yeast are investigated and the results graphed. Background information, a list of needed materials, directions for preparing solutions, procedure, and results and discussion are…

  6. Starch: chemistry, microstructure, processing and enzymatic degradation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Starch is recognized as one of the most abundant and important commodities containing value added attributes for a vast number of industrial applications. Its chemistry, structure, property and susceptibility to various chemical, physical and enzymatic modifications offer a high technological value ...

  7. Multi-parametric MRI characterization of enzymatically degraded articular cartilage.

    PubMed

    Nissi, Mikko J; Salo, Elli-Noora; Tiitu, Virpi; Liimatainen, Timo; Michaeli, Shalom; Mangia, Silvia; Ellermann, Jutta; Nieminen, Miika T

    2016-07-01

    Several laboratory and rotating frame quantitative MRI parameters were evaluated and compared for detection of changes in articular cartilage following selective enzymatic digestion. Bovine osteochondral specimens were subjected to 44 h incubation in control medium or in collagenase or chondroitinase ABC to induce superficial collagen or proteoglycan (glycosaminoglycan) alterations. The samples were scanned at 9.4 T for T1 , T1 Gd (dGEMRIC), T2 , adiabatic T1 ρ , adiabatic T2 ρ , continuous-wave T1 ρ , TRAFF2 , and T1 sat relaxation times and for magnetization transfer ratio (MTR). For reference, glycosaminoglycan content, collagen fibril orientation and biomechanical properties were determined. Changes primarily in the superficial cartilage were noted after enzymatic degradation. Most of the studied parameters were sensitive to the destruction of collagen network, whereas glycosaminoglycan depletion was detected only by native T1 and T1 Gd relaxation time constants throughout the tissue and by MTR superficially. T1 , adiabatic T1 ρ , adiabatic T2 ρ , continuous-wave T1 ρ , and T1 sat correlated significantly with the biomechanical properties while T1 Gd correlated with glycosaminoglycan staining. The findings indicated that most of the studied MRI parameters were sensitive to both glycosaminoglycan content and collagen network integrity, with changes due to enzymatic treatment detected primarily in the superficial tissue. Strong correlation of T1 , adiabatic T1ρ , adiabatic T2 ρ , continuous-wave T1 ρ , and T1 sat with the altered biomechanical properties, reflects that these parameters were sensitive to critical functional properties of cartilage. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1111-1120, 2016. PMID:26662555

  8. Aromatic–Aromatic Interactions Enhance Interfiber Contacts for Enzymatic Formation of a Spontaneously Aligned Supramolecular Hydrogel

    PubMed Central

    2015-01-01

    Anisotropy or alignment is a critical feature of functional soft materials in living organisms, but it remains a challenge for spontaneously generating anisotropic gel materials. Here we report a molecular design that increases intermolecular aromatic–aromatic interactions of hydrogelators during enzymatic hydrogelation for spontaneously forming an anisotropic hydrogel. This process, relying on both aromatic–aromatic interactions and enzyme catalysis, results in spontaneously aligned supramolecular nanofibers as the matrices of a monodomain hydrogel that exhibits significant birefringence. This work, as the first example of monodomain hydrogels formed via an enzymatic reaction, illustrates a new biomimetic approach for generating aligned anisotropic soft materials. PMID:24512553

  9. Modeling Enzymatic Reactions in Proteins.

    NASA Astrophysics Data System (ADS)

    Friesner, Richard

    2007-03-01

    We will discuss application of our density functional (DFT)-based QM/MM methodology to modeling a variety of protein active sites, including methane monooxygenase, myoglobin, and cytochrome P450. In addition to the calculation of intermediates, transition states, and rate constants, we will discuss modeling of reactions requiring protein conformational changes. Our methodology reliably achieves small errors as a result of imposition of the QM/MM boundary. However, the accuracy of DFT methods can vary significantly with the type of system under study. We will discuss a novel approach to the reduction of errors in gradient corrected and hybrid DFT functionals, using empirical localized orbital corrections (DFT-LOC), which addresses this problem effectively. For example, the mean unsigned error in atomization energies for the G3 data set using the B3LYP-LOC model is 0.8 kcal/mole, as compared with 4.8 kcal/mole for B3LYP and 1.0 kcal/mole for G3 theory.

  10. Enzymatic passaging of human embryonic stem cells alters central carbon metabolism and glycan abundance

    PubMed Central

    Badur, Mehmet G.; Zhang, Hui; Metallo, Christian M.

    2016-01-01

    To realize the potential of human embryonic stem cells (hESCs) in regenerative medicine and drug discovery applications, large numbers of cells that accurately recapitulate cell and tissue function must be robustly produced. Previous studies have suggested that genetic instability and epigenetic changes occur as a consequence of enzymatic passaging. However, the potential impacts of such passaging methods on the metabolism of hESCs have not been described. Using stable isotope tracing and mass spectrometry-based metabolomics, we have explored how different passaging reagents impact hESC metabolism. Enzymatic passaging caused significant decreases in glucose utilization throughout central carbon metabolism along with attenuated de novo lipogenesis. In addition, we developed and validated a method for rapidly quantifying glycan abundance and isotopic labeling in hydrolyzed biomass. Enzymatic passaging reagents significantly altered levels of glycans immediately after digestion but surprisingly glucose contribution to glycans was not affected. These results demonstrate that there is an immediate effect on hESC metabolism after enzymatic passaging in both central carbon metabolism and biosynthesis. HESCs subjected to enzymatic passaging are routinely placed in a state requiring re-synthesis of biomass components, subtly influencing their metabolic needs in a manner that may impact cell performance in regenerative medicine applications. PMID:26289220