| pullulanase type i from fervidobacterium pennavorans ven5: cloning, sequencing, and expression of the gene and biochemical characterization of the recombinant enzyme. | the gene encoding the type i pullulanase from the extremely thermophilic anaerobic bacterium fervidobacterium pennavorans ven5 was cloned and sequenced in escherichia coli. the pula gene from f. pennavorans ven5 had 50.1% pairwise amino acid identity with pula from the anaerobic hyperthermophile thermotoga maritima and contained the four regions conserved among all amylolytic enzymes. the pullulanase gene (pula) encodes a protein of 849 amino acids with a 28-residue signal peptide. the pula gene ... | 1999 | 10224005 |
| characterization of a highly thermostable alkaline phosphatase from the euryarchaeon pyrococcus abyssi. | this work reports the first isolation and characterization of an alkaline phosphatase (ap) from a hyperthermophilic archaeon. an ap gene from pyrococcus abyssi, a euryarchaeon isolated from a deep-sea hydrothermal vent, was cloned and the enzyme expressed in escherichia coli. analysis of the sequence showed conservation of the active site and structural elements of the e. coli ap. the recombinant ap was purified and characterized. monomeric and homodimeric active forms were detected, with a mono ... | 2001 | 11571149 |
| pilin-like proteins in the extremely thermophilic bacterium thermus thermophilus hb27: implication in competence for natural transformation and links to type iv pilus biogenesis. | the extreme thermophile thermus thermophilus hb27 exhibits high frequencies of natural transformation. although we recently reported identification of the first competence genes in thermus, the molecular basis of dna uptake is unknown. a pilus-like structure is assumed to be involved. twelve genes encoding prepilin-like proteins were identified in three loci in the genome of t. thermophilus. mutational analyses, described in this paper, revealed that one locus, which contains four genes that enc ... | 2003 | 12839734 |
| cloning, sequencing, and characterization of a heat- and alkali-stable type i pullulanase from anaerobranca gottschalkii. | the gene encoding a type i pullulanase was identified from the genome sequence of the anaerobic thermoalkaliphilic bacterium anaerobranca gottschalkii. in addition, the homologous gene was isolated from a gene library of anaerobranca horikoshii and sequenced. the proteins encoded by these two genes showed 39% amino acid sequence identity to the pullulanases from the thermophilic anaerobic bacteria fervidobacterium pennivorans and thermotoga maritima. the pullulanase gene from a. gottschalkii (en ... | 2004 | 15184138 |
| molecular cloning and expression of a novel trehalose synthase gene from enterobacter hormaechei. | abstract: | 2009 | 19523196 |
| mechanistic analysis of trehalose synthase from mycobacterium smegmatis. | trehalose synthase (tres) catalyzes the reversible interconversion of maltose and trehalose and has been recently shown to function primarily in the mobilisation of trehalose as a glycogen precursor. consequently the mechanism of this intriguing isomerase is of both academic and potential pharmacological interest. tres catalyzes the hydrolytic cleavage of +¦-aryl glucosides, as well as +¦-glucosyl fluoride, thereby allowing facile continuous assays. reaction of tres with 5-fluoroglycosyl fluorid ... | 2011 | 21840994 |
| hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. | enzymes synthesized by hyperthermophiles (bacteria and archaea with optimal growth temperatures of > 80 degrees c), also called hyperthermophilic enzymes, are typically thermostable (i.e., resistant to irreversible inactivation at high temperatures) and are optimally active at high temperatures. these enzymes share the same catalytic mechanisms with their mesophilic counterparts. when cloned and expressed in mesophilic hosts, hyperthermophilic enzymes usually retain their thermal properties, ind ... | 2001 | 11238984 |
| directed evolution of dna polymerases for next-generation sequencing. | | 2010 | 20629059 |
| trehalose metabolism: from osmoprotection to signaling. | trehalose is a non-reducing disaccharide formed by two glucose molecules. it is widely distributed in nature and has been isolated from certain species of bacteria, fungi, invertebrates and plants, which are capable of surviving in a dehydrated state for months or years and subsequently being revived after a few hours of being in contact with water. this disaccharide has many biotechnological applications, as its physicochemical properties allow it to be used to preserve foods, enzymes, vaccines ... | 2009 | 19865519 |
| dna polymerases engineered by directed evolution to incorporate non-standard nucleotides. | dna polymerases have evolved for billions of years to accept natural nucleoside triphosphate substrates with high fidelity and to exclude closely related structures, such as the analogous ribonucleoside triphosphates. however, polymerases that can accept unnatural nucleoside triphosphates are desired for many applications in biotechnology. the focus of this review is on non-standard nucleotides that expand the genetic "alphabet." this review focuses on experiments that, by directed evolution, ha ... | 2014 | 25400626 |
| the rare sugar d-allose acts as a triggering molecule of rice defence via ros generation. | only d-allose, among various rare monosaccharides tested, induced resistance to xanthomonas oryzae pv. oryzae in susceptible rice leaves with defence responses: reactive oxygen species, lesion mimic formation, and pr-protein gene expression. these responses were suppressed by ascorbic acid or diphenylene iodonium. transgenic rice plants overexpressing osrbohc, encoding nadph oxidase, were enhanced in sensitivity to d-allose. d-allose-mediated defence responses were suppressed by the presence of ... | 2013 | 24014866 |
| substrate channel flexibility in pseudomonas aeruginosa murb accommodates two distinct substrates. | biosynthesis of udp-n-acetylmuramic acid in bacteria is a committed step towards peptidoglycan production. in an nadph- and fad-dependent reaction, the udp-n-acetylglucosamine-enolpyruvate reductase (murb) reduces udp-n-acetylglucosamine-enolpyruvate to udp-n-acetylmuramic acid. we determined the three-dimensional structures of the ternary complex of pseudomonas aeruginosa murb with fad and nadp(+) in two crystal forms to resolutions of 2.2 and 2.1 å, respectively, to investigate the structural ... | 2013 | 23805286 |
| expression of a secretory α-glucosidase ii from apis cerana indica in pichia pastoris and its characterization. | α-glucosidase (hbgase) plays a key role in hydrolyzing α-glucosidic linkages. in apis mellifera, three isoforms of hbgase (i, ii and iii) have been reported, which differ in their nucleotide composition, encoding amino acid sequences and enzyme kinetics. recombinant (r)hbgase ii from a. cerana indica (racihbgase ii) was focused upon here due to the fact it is a native and economic honeybee species in thailand. the data is compared to the two other isoforms, acihbgase i and iii from the same bee ... | 2013 | 23419073 |
| diverse allosteric and catalytic functions of tetrameric d-lactate dehydrogenases from three gram-negative bacteria. | nad-dependent d-lactate dehydrogenases (d-ldhs) reduce pyruvate into d-lactate with oxidation of nadh into nad(+). although non-allosteric d-ldhs from lactobacilli have been extensively studied, the catalytic properties of allosteric d-ldhs from gram-negative bacteria except for escherichia coli remain unknown. we characterized the catalytic properties of d-ldhs from three gram-negative bacteria, fusobacterium nucleatum (fnldh), pseudomonas aeruginosa (paldh), and e. coli (ecldh) to gain an insi ... | 2014 | 25401076 |
| the core of allosteric motion in thermus caldophilus l-lactate dehydrogenase. | for thermus caldophilus l-lactate dehydrogenase (tcldh), fructose 1,6-bisphosphate (fbp) reduced the pyruvate s(0.5) value 10(3)-fold and increased the v(max) value 4-fold at 30 °c and ph 7.0, indicating that tcldh has a much more t state-sided allosteric equilibrium than thermus thermophilus l-lactate dehydrogenase, which has only two amino acid replacements, a154g and h179y. the inactive (t) and active (r) state structures of tcldh were determined at 1.8 and 2.0 å resolution, respectively. the ... | 2014 | 25258319 |
| lucy: a versatile new fluorescent reporter protein. | we report on the discovery, isolation, and use of a novel yellow fluorescent protein. lucigen yellow (lucy) binds one fad molecule within its core, thus shielding it from water and maintaining its structure so that fluorescence is 10-fold higher than freely soluble fad. lucy displays excitation and emission spectra characteristic of fad, with 3 excitation peaks at 276 nm, 377 nm, and 460 nm and a single emission peak at 530 nm. these excitation and emission maxima provide the large stokes shift ... | 2015 | 25906065 |
| crystal structure of a dna binding protein from the hyperthermophilic euryarchaeon methanococcus jannaschii. | the sac10b family consists of a group of highly conserved dna binding proteins from both the euryarchaeotal and the crenarchaeotal branches of archaea. the proteins have been suggested to play an architectural role in the chromosomal organization in these organisms. previous studies have mainly focused on the sac10b proteins from the crenarchaeota. here, we report the 2.0 a resolution crystal structure of mja10b from the euryarchaeon methanococcus jannaschii. the model of mja10b has been refined ... | 2003 | 14627741 |
| purification and characterization of a thermostable adp-glucose pyrophosphorylase from thermus caldophilus gk-24. | an extremely thermostable adp-glucose pyrophosphorylase (agpase) has been purified from thermus caldophilus gk-24 to homogeneity by chromatographic methods, including gel filtration and ion-exchange and affinity chromatography. the specific activity of the enzyme was enriched 134.8-fold with a recovery of 10.5%. the purified enzyme was a single band by sds/page with a molecular mass of 52 kda. the homotetrameric structure of the native enzyme was determined by gel filtration analysis, which show ... | 1996 | 8921008 |
| enzymatic biotransformation of ginsenoside rb1 and gypenoside xvii into ginsenosides rd and f2 by recombinant β-glucosidase from flavobacterium johnsoniae. | this study focused on the enzymatic biotransformation of the major ginsenoside rb1 into rd for the mass production of minor ginsenosides using a novel recombinant β-glucosidase from flavobacterium johnsoniae. the gene (bglf3) consisting of 2,235 bp (744 amino acid residues) was cloned and the recombinant enzyme overexpressed in escherichia coli bl21(de3) was characterized. this enzyme could transform ginsenoside rb1 and gypenoside xvii to the ginsenosides rd and f2, respectively. the glutathione ... | 2012 | 23717145 |
| crystallization and preliminary crystallographic analysis of a putative glucokinase/hexokinase from thermus thermophilus. | glucokinase/hexokinase catalyzes the phosphorylation of glucose to glucose 6-phosphate, which is the first step of glycolysis. the open reading frame ttha0299 of the extreme thermophile thermus thermophilus encodes a putative glucokinase/hexokinase which contains the consensus sequence for proteins from the repressors, open reading frames and sugar kinases family. in this study, the glucokinase/hexokinase from t. thermophilus was purified and crystallized using polyethylene glycol 8000 as a prec ... | 2011 | 22139166 |
| heterogeneous nucleation of protein crystals on fluorinated layered silicate. | here, we describe an improved system for protein crystallization based on heterogeneous nucleation using fluorinated layered silicate. in addition, we also investigated the mechanism of nucleation on the silicate surface. crystallization of lysozyme using silicates with different chemical compositions indicated that fluorosilicates promoted nucleation whereas the silicates without fluorine did not. the use of synthesized saponites for lysozyme crystallization confirmed that the substitution of h ... | 2011 | 21818343 |
| structure of the mycobacterium tuberculosis d-alanine:d-alanine ligase, a target of the antituberculosis drug d-cycloserine. | d-alanine:d-alanine ligase (ec 6.3.2.4; ddl) catalyzes the atp-driven ligation of two d-alanine (d-ala) molecules to form the d-alanyl:d-alanine dipeptide. this molecule is a key building block in peptidoglycan biosynthesis, making ddl an attractive target for drug development. d-cycloserine (dcs), an analog of d-ala and a prototype ddl inhibitor, has shown promise for the treatment of tuberculosis. here, we report the crystal structure of mycobacterium tuberculosis ddl at a resolution of 2.1 å. ... | 2010 | 20956591 |
| structure of the mycobacterium tuberculosis d-alanine:d-alanine ligase, a target of the antituberculosis drug d-cycloserine. | d-alanine:d-alanine ligase (ec 6.3.2.4; ddl) catalyzes the atp-driven ligation of two d-alanine (d-ala) molecules to form the d-alanyl:d-alanine dipeptide. this molecule is a key building block in peptidoglycan biosynthesis, making ddl an attractive target for drug development. d-cycloserine (dcs), an analog of d-ala and a prototype ddl inhibitor, has shown promise for the treatment of tuberculosis. here, we report the crystal structure of mycobacterium tuberculosis ddl at a resolution of 2.1 å. ... | 2010 | 20956591 |
| crystallization and preliminary x-ray analysis of a d-alanyl-d-alanine ligase (ecddlb) from escherichia coli. | a recombinant form of escherichia coli ddlb (ecddlb) has been prepared and cocrystallized with adp and d-alanyl-d-alanine to represent the ternary complex of ecddlb. furthermore, ecddlb has been cocrystallized under the same conditions with the ligands atp and d-alanyl-d-alanine, representing the product-inhibited complex. the crystals belonged to space group p2(1)2(1)2(1), with unit-cell parameters a = 53.0, b = 97.6, c = 109.5 a and a = 51.2, b = 97.8, c = 110.1 a, respectively, and both conta ... | 2010 | 20383009 |
| structure of d-alanine-d-alanine ligase from thermus thermophilus hb8: cumulative conformational change and enzyme-ligand interactions. | d-alanine-d-alanine ligase (ddl) is one of the key enzymes in peptidoglycan biosynthesis and is an important target for drug discovery. the enzyme catalyzes the condensation of two d-ala molecules using atp to produce d-ala-d-ala, which is the terminal peptide of a peptidoglycan monomer. the structures of five forms of the enzyme from thermus thermophilus hb8 (ttddl) were determined: unliganded ttddl (2.3 a resolution), ttddl-adenylyl imidodiphosphate (2.6 a), ttddl-adp (2.2 a), ttddl-adp-d-ala ... | 2009 | 19770507 |
| recombinant production and biochemical characterization of a hyperthermostable alpha-glucan/maltodextrin phosphorylase from pyrococcus furiosus. | alpha-glucan phosphorylase catalyzes the reversible cleavage of alpha-1-4-linked glucose polymers into alpha-d-glucose-1-phosphate. we report the recombinant production of an alpha-glucan/maltodextrin phosphorylase (pf1535) from a hyperthermophilic archaeon, pyrococcus furiosus, and the first detailed biochemical characterization of this enzyme from any archaeal source using a mass-spectrometry-based assay. the apparent 98 kda recombinant enzyme was active over a broad range of temperatures and ... | 2008 | 19054743 |
| recombinant production and biochemical characterization of a hyperthermostable alpha-glucan/maltodextrin phosphorylase from pyrococcus furiosus. | alpha-glucan phosphorylase catalyzes the reversible cleavage of alpha-1-4-linked glucose polymers into alpha-d-glucose-1-phosphate. we report the recombinant production of an alpha-glucan/maltodextrin phosphorylase (pf1535) from a hyperthermophilic archaeon, pyrococcus furiosus, and the first detailed biochemical characterization of this enzyme from any archaeal source using a mass-spectrometry-based assay. the apparent 98 kda recombinant enzyme was active over a broad range of temperatures and ... | 2008 | 19054743 |
| chemical diversity of panax ginseng, panax quinquifolium, and panax notoginseng. | the major commercial ginsengs are panax ginseng meyer (korean ginseng), p. quinquifolium l. (american ginseng), and p. notoginseng (burk.) fh chen (notoginseng). p. ginseng is the most commonly used as an adaptogenic agent and has been shown to enhance physical performance, promote vitality, increase resistance to stress and aging, and have immunomodulatory activity. these ginsengs contain saponins, which can be classified as dammarane-type, ocotillol-type and oleanane-type oligoglycosides, and ... | 2012 | 23717099 |
| pullulanase: role in starch hydrolysis and potential industrial applications. | the use of pullulanase (ec 3.2.1.41) has recently been the subject of increased applications in starch-based industries especially those aimed for glucose production. pullulanase, an important debranching enzyme, has been widely utilised to hydrolyse the α-1,6 glucosidic linkages in starch, amylopectin, pullulan, and related oligosaccharides, which enables a complete and efficient conversion of the branched polysaccharides into small fermentable sugars during saccharification process. the indust ... | 2012 | 22991654 |
| biosynthetic origin and mechanism of formation of the aminoribosyl moiety of peptidyl nucleoside antibiotics. | several peptidyl nucleoside antibiotics that inhibit bacterial translocase i involved in peptidoglycan cell wall biosynthesis contain an aminoribosyl moiety, an unusual sugar appendage in natural products. we present here the delineation of the biosynthetic pathway for this moiety upon in vitro characterization of four enzymes (lipm-p) that are functionally assigned as (i) lipo, an l-methionine:uridine-5'-aldehyde aminotransferase; (ii) lipp, a 5'-amino-5'-deoxyuridine phosphorylase; (iii) lipm, ... | 2011 | 21819104 |
| crystallization and preliminary crystallographic analysis of d-alanine-d-alanine ligase from streptococcus mutans. | d-alanine-d-alanine ligase is encoded by the gene ddl (smu_599) in streptococcus mutans. this ligase plays a very important role in cell-wall biosynthesis and may be a potential target for drug design. to study the structure and function of this ligase, the gene ddl was amplified from s. mutans genomic dna and cloned into the expression vector pet28a. the protein was expressed in soluble form in escherichia coli strain bl21 (de3). homogeneous protein was obtained using a two-step procedure consi ... | 2007 | 17768361 |
| evaluation and directed evolution for thermostability improvement of a gh 13 thermostable α-glucosidase from thermus thermophilus tc11. | thermal stable α-glucosidases with transglycosylation activity could be applied to the industrial production of oligosaccharides as well as conjugation of sugars to biologically useful materials. therefore, α-glucosidases isolated from thermophiles have gained attention over the past decade. in this study, the characterization of a highly thermostable α-glucosidase and its thermostability improved mutant from newly isolated strain thermus thermophilus tc11 were investigated. | 2015 | 26490269 |
| characterization of a ginsenoside-transforming β-glucosidase from paenibacillus mucilaginosus and its application for enhanced production of minor ginsenoside f₂. | a novel β-glucosidase (bglpm) was identified from paenibacillus mucilaginosus kctc 3870(t) which has ginsenoside converting activity. the gene, termed bglpm, consists of 1,260 bp and belongs to glycoside hydrolase family 1 (gh1). after being overexpressed and purified from escherichia coli, the enzymatic properties of bglpm were investigated. the enzyme exhibited an optimal activity at 45°c and ph 7.5 and showed high bioconversion ability for major ginsenoside rb1 and rd into ginsenoside f2. thu ... | 2014 | 24475050 |
| production of β-galactosidase from streptococcus thermophilus for galactooligosaccharides synthesis. | efficiency of different methods for disruption of streptococcus thermophilus cells, isolated from different dairy products, to release β-galactosidase and synthesis of gos by extracted enzyme using whey supplemented with different concentrations of lactose as a substrate was studied. unlike most other studies on gos synthesis which used only one method of cell disruption and only few microbial strains, we compared five different cell disruption methods and used 30 strains of s. thermophilus in o ... | 2014 | 26139885 |
| production of β-galactosidase from streptococcus thermophilus for galactooligosaccharides synthesis. | efficiency of different methods for disruption of streptococcus thermophilus cells, isolated from different dairy products, to release β-galactosidase and synthesis of gos by extracted enzyme using whey supplemented with different concentrations of lactose as a substrate was studied. unlike most other studies on gos synthesis which used only one method of cell disruption and only few microbial strains, we compared five different cell disruption methods and used 30 strains of s. thermophilus in o ... | 2014 | 26139885 |