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nicotinoprotein alcohol/aldehyde oxidoreductases. enzymes with bound nad(p) as cofactor. 19979059647
genetic and physiological analysis of the lethal effect of l-(+)-lactate dehydrogenase deficiency in streptococcus mutans: complementation by alcohol dehydrogenase from zymomonas mobilis.ch4ts is a previously isolated recombinant mutant of streptococcus mutans ng8 which produces a thermolabile l-(+)-lactate dehydrogenase (ldh) activity. it does not grow at 42 degrees c under a variety of cultivation conditions. in this study, we show that a batch culture of ch4ts shifted from 30 to 42 degrees c underwent rapid cessation of growth and accelerated cell death. the mutant grew at 42 degrees c in continuous culture under glucose-limiting conditions. under these conditions, lactate pr ...19968926105
6-phosphogluconate dehydratase from zymomonas mobilis: an iron-sulfur-manganese enzyme.the enzyme 6-phosphogluconate dehydratase has been isolated in a stable form by a simple one-step procedure using dye ligand chromatography. the role of metal ions in the activity and stability of the enzyme was investigated. as with aconitase and several other dehydratase enzymes, the active site includes an fe4s4 cluster. in addition, the purified enzyme has been shown to contain one manganese ion per subunit, which is also essential for activity. rapid inactivation by superoxide radical was o ...19968728108
mutagenesis and crystallographic studies of zymomonas mobilis trna-guanine transglycosylase reveal aspartate 102 as the active site nucleophile.procaryotic trna-guanine transglycosylase (tgt) catalyzes the posttranscriptional base exchange of the queuine precursor 7-aminomethyl-7-deazaguanine (preq1) with the genetically encoded guanine at the wobble position of trnas specific for asn, asp, his, and tyr. the x-ray structures of zymomonas mobilis tgt and of its complex with preq1 [romier, c., reuter, k., suck, d., & ficner, r. (1996) embo j. 15, 2850-2857] have revealed a specific preq1 binding pocket and allowed a proposal for trna bind ...19968961936
investigation of the cofactor-binding site of zymomonas mobilis pyruvate decarboxylase by site-directed mutagenesis.several enzymes require thiamin diphosphate (thdp) as an essential cofactor, and we have used one of these, pyruvate decarboxylase (pdc; ec 4.1.1.1) from zymomonas mobilis, as a model for this group of enzymes. it is well suited for this purpose because of its stability, ease of purification and its simple kinetic properties. a sequence motif of approx. 30 residues, beginning with a glycine-aspartate-glycine (-gdg-) triplet and ending with a double asparagine (-nn-) sequence, has been identified ...19948198554
alteration of substrate specificity of zymomonas mobilis alcohol dehydrogenase-2 using in vitro random mutagenesis.random mutagenesis of the gene encoding zymomonas mobilis alcohol dehydrogenase-2 has enabled isolation of variants of the enzyme that have substrate specificities different from that of the wild-type enzyme. after amino acids responsible for the changes were identified, directed mutation at these sites was also carried out. variants that are active on butanol have been investigated in detail. changes at residue 161 and other changes at residues 155 and 165 cause enhanced activity with longer-ch ...19979116506
isoprenoid biosynthesis in bacteria: a novel pathway for the early steps leading to isopentenyl diphosphate.incorporation of 13c-labelled glucose, acetate, pyruvate or erythrose allowed the determination of the origin of the carbon atoms of triterpenoids of the hopane series and/or of the ubiquinones from several bacteria (zymomonas mobilis, methylobacterium fujisawaense, escherichia coli and alicyclobacillus acidoterrestris) confirmed our earlier results obtained by incorporation of 13c-labelled acetate into the hopanoids of other bacteria and led to the identification of a novel biosynthetic route f ...19938240251
development of an arabinose-fermenting zymomonas mobilis strain by metabolic pathway engineering.the substrate fermentation range of the ethanologenic bacterium zymomonas mobilis was expanded to include the pentose sugar, l-arabinose, which is commonly found in agricultural residues and other lignocellulosic biomass. five genes, encoding l-arabinose isomerase (araa), l-ribulokinase (arab), l-ribulose-5-phosphate-4-epimerase (arad), transaldolase (talb), and transketolase (tkta), were isolated from escherichia coli and introduced into z. mobilis under the control of constitutive promoters th ...19968953718
crystal structure of trna-guanine transglycosylase: rna modification by base exchange.trna-guanine transglycosylases (tgt) are enzymes involved in the modification of the anticodon of trnas specific for asn, asp, his and tyr, leading to the replacement of guanine-34 at the wobble position by the hypermodified base queuine. in prokaryotes tgt catalyzes the exchange of guanine-34 with the queuine (.)precursor 7-aminomethyl-7-deazaguanine (preq1). the crystal structure of tgt from zymomonas mobilis was solved by multiple isomorphous replacement and refined to a crystallographic r-fa ...19968654383
the gluemp operon from zymomonas mobilis encodes a high-affinity glutamate carrier with similarity to binding-protein-dependent transport systems.the nucleotide sequence downstream of the grp gene, encoding the glutamate uptake regulatory protein of zymomonas mobilis, was determined. three clustered genes (glue, glum, and glup) close to ghe grp gene, but on the opposite strand, were identified. these genes encode a high-affinity transport system for glutamate and aspartate. the glup gene product is a polypeptide of 25.4 kda and contains segments with significant similarity to the atp-binding proteins of binding-protein-dependent transport ...19968661924
export of the periplasmic nadp-containing glucose-fructose oxidoreductase of zymomonas mobilis.glucose-fructose oxidoreductase (gfor) of the gram-negative bacterium zymomonas mobilis is a periplasmic enzyme with the tightly bound cofactor nadp. the preprotein carries an unusually long n-terminal signal sequence of 52 amino acid residues. a sorbitol-negative mutant strain (acm3963) was found to be deficient in gfor activity and was used for the expression of plasmid-borne copies of the wild-type gfo gene or of alleles encoding alterations in the signal sequence of the pre-gfor protein. z. ...19968661942
stabilization of pet operon plasmids and ethanol production in escherichia coli strains lacking lactate dehydrogenase and pyruvate formate-lyase activities.in the last decade, a major goal of research in biofuels has been to metabolically engineer microorganisms to ferment multiple sugars from biomass or agricultural wastes to fuel ethanol. escherichia coli strains genetically engineered to contain the pet operon (zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase b genes) produce high levels of ethanol. strains carrying the pet operon in plasmid (e.g., e. coli b/ploi297) or in chromosomal (e.g., e. coli ko11) sites require antibiot ...19968953729
identification and characterization of phon-sf, a gene on the large plasmid of shigella flexneri 2a encoding a nonspecific phosphatase.a gene encoding a nonspecific phosphatase, named phon-sf, was identified on the large virulence plasmid (pmysh6000) of shigella flexneri 2a ysh6000. the phosphatase activity in ysh6000 was observed under high-phosphate conditions. however, it was found that low-phosphate conditions induced a slightly higher level of activity. the nucleotide sequence of the phon-sf region cloned from pmysh6000 possessing the phon-sf gene encoded 249 amino acids with a typical signal sequence at the n terminus. th ...19968755883
purification, crystallization, and preliminary x-ray diffraction studies of trna-guanine transglycosylase from zymomonas mobilis.the trna modifying enzyme trna-gnanine transglycosylase (tgt) catalyzes the exchange of guanine in the first position of the anticodon with the quenine precursor 7-aminomethyl-7-deazagnanine. tgt from zymomonas mobilis has been purified by crystallization and further recrystallized to obtain single crystals suitable for x-ray diffraction studies. crystals were grown by vapor diffusion/gel crystallization methods using peg 8,000 as precipitant. macroseeding techniques were employed to produce lar ...19968860000
allosteric control of zymomonas mobilis glucose-6-phosphate dehydrogenase by phosphoenolpyruvate.the second enzyme of the entner-doudoroff glycolytic pathway in zymomonas mobilis, glucose-6-phosphate dehydrogenase, has been found to be inhibited by phosphoenolpyruvate (pep). in the presence of pep levels in the micromolar range, the response of the enzyme to glucose 6-phosphate concentration becomes sigmoidal, with a hill coefficient up to 2. at low ionic strength in the absence of pep, the response to glucose 6-phosphate concentration is michaelis-menten, but at physiological ionic strengt ...19979307022
differential inactivation of alcohol dehydrogenase isoenzymes in zymomonas mobilis by oxygen.zymomonas mobilis is endowed with two isoenzymes of fermentative alcohol dehydrogenase, a zinc-containing enzyme (adh i) and an iron-containing enzyme (adh ii). the activity of adh i remains fully conserved, while adh ii activity decays when anaerobic cultures are shifted to aerobiosis. this differential response depends on the metal present on each isoenzyme, since pure preparations of adh i are resistant to oxidative inactivation and preparations of zinc-containing adh ii, obtained by incubati ...19979023190
the replacement of trp392 by alanine influences the decarboxylase/carboligase activity and stability of pyruvate decarboxylase from zymomonas mobilis.the bulky tryptophan residue 392 located in the deep cleft leading to the active center of pyruvate decarboxylase (pdc) from zymomonas mobilis was changed to alanine which is found in the equivalent position of pdc from yeast. the mutation reduced the decarboxylase activity towards pyruvate by a factor of two (60-70 u/mg), whereas the km (1.1 mm in mes/koh buffer) remains unchanged compared with the wild-type enzyme. the apparent km for thiamine diphosphate (thiamin-p2) in the presence of 5 mm m ...19958536715
an elevation of the molar growth yield of zymomonas mobilis during aerobic exponential growthelevated values of molar growth yield (yx/s = 14-26 g mol-1) were obtained during exponential growth (μ > 0.4 h-1) of zymomonas mobilis atcc 29191 by using reduced concentrations of glucose (6.25-100 mm) and increased oxygen supply (eh > 300 mv) in the growth medium, as compared to the yx/s of anaerobic exponential growth (8-10 g mol-1). aerobically grown cells showed an increased maximum growth rate (μmax), and a reduced specific glucose consumption rate (qs), and specific ethanol formation rat ...19979042757
measurement of platelet aggregation peptide inhibitors by ultrasonic interferometry.several peptide inhibitors of thrombin- or collagen-induced platelet aggregation and of the interaction between glycoprotein ib and von willebrand factor were studied by a new method--ultrasonic interferometry (echo cell). inhibition of aggregate formation in a concentration-dependent manner was observed. the sensitivity of the method was 3 to 40 times higher than that of classical turbidimetry.19989451507
the relationship between growth enhancement and pet expression in escherichia coli.the pet operon consists of genes coding for enzymes responsible for ethanol production and consists of pyruvate dehydrogenase and alcohol dehydrogenase ii from the high-performance ethanologen zymomonas mobilis. this article describes the physiological influence of pet expression in escherichia coli b (atcc 11303) in terms of growth rate and overall concentrations of cell mass and catabolic end products achieved under well-defined cultivation conditions that included constant ph and carbon (ener ...19968669901
factors contributing to the loss of ethanologenicity of escherichia coli b recombinants pl0i297 and ko11.to be economic and to be compatible with modern continuous bioconversion systems, it is imperative that the process organism exhibits an extremely high degree of stability. in the case of ethanol production from lignocellulosic biomass, functional stability of the potential process biocatalyst can be assessed in terms of the capacity to sustain high-performance fermentation during the continuous fermentation of biomass-derived sugars. this investigation employed glucose- or xylose-limited chemos ...19968669902
molecular characterization of the levansucrase gene from the endophytic sugarcane bacterium acetobacter diazotrophicus srt4.the acetobacter diazotrophicus srt4 gene encoding levansucrase (ec 2.4.1.10) (isda) was isolated from a genomic library. the nucleotide sequence of a 2.3 kb dna fragment sufficient for complementation of a levansucrase-deficient mutant (obtained by ems treatment) was determined. the isda gene (1751 bp) coded for a polypeptide of molecular mass 64.9 kda with an isoelectric point of 5.2. the n-terminal amino acid sequence of the extracellular levansucrase indicated the presence of a precursor prot ...19968704949
sampling tube device for monitoring intracellular metabolite dynamics.continuous sampling of microorganisms from a controlled bioreactor with rapid inactivation of metabolism and extraction of metabolites using precooled -40 degrees c perchloric acid solution (35%) was achieved with a sampling tube, thus fixing fast dynamic reactions at a certain position in the tube. after sampling was stopped (200 s) the tube was frozen at -80 degrees c and divided into identical parts and the extracted metabolites were analyzed enzymatically. a high resolution in time was achie ...19979073360
overexpression, purification, and generation of a thermostable variant of zymomonas mobilis fructokinase.the gene encoding fructokinase (ec 2.7.1.4) from zymomonas mobilis has been expressed at high level in escherichia coli by modifying the ribosome binding site using the polymerase chain reaction. a simple two-step purification from extracts of the recombinant cells results in highly purified enzyme suitable for use in fructose determination. using the polymerase chain reaction in mutagenic conditions, a variant of fructokinase was isolated which was more thermostable than the wild type, taking t ...19968776754
classification of rhizomonas suberifaciens, an unnamed rhizomonas species, and sphingomonas spp. in rrna superfamily iv.thermal melting profiles of hybrids between 3h-labeled rrna of rhizomonas suberifaciens, the causal agent of corky root of lettuce, and chromosomal dnas from 27 species of gram-negative bacteria indicated that the genus rhizomonas belongs to superfamily iv of de ley. on the basis of the melting temperatures of dna hybrids with rrnas from the type strains of r. suberifaciens, sphingomonas paucimobilis, and sphingomonas capsulata, rhizomonas strains constitute a separate branch in superfamily iv, ...19938427800
an ultraviolet-sensitive mutant of zymomonas mobilis affecting the stability of its natural plasmid pzmo2.plasmid pzmo2, a 1.9-kb natural plasmid of zymomonas mobilis strain atcc 10988, was found to be absent from the extract of a mutant isolate sensitive to ultraviolet irradiation, methyl methanesulfonic acid, and mitomycin c. this mutant, named uvs51, also exhibited extremely high segregational instability of the recombinant plasmid pds212, whose maintenance in the parental strain is known to be due to pzmo2 sequences. helped conjugations, employing uvs51 as recipient with escherichia coli donors ...19938441765
protein design on pyruvate decarboxylase (pdc) by site-directed mutagenesis. application to mechanistical investigations, and tailoring pdc for the use in organic synthesis.pyruvate decarboxylases (e.c. 4.1.1.1) from various organisms have been studied for many years, mainly with respect to the mechanism of the non-oxidative decarboxylation reaction. although the c-c-bond-forming properties of these enzymes are known and have been applied for many years in biotransformations for the synthesis of chiral alpha-hydroxy ketones, only little is known about the factors influencing the carboligase side-reaction. the present review surveys recent efforts in the study of si ...19979103910
cysteine 265 is in the active site of, but is not essential for catalysis by trna-guanine transglycosylase (tgt) from escherichia coli.site-directed mutagenesis and x-ray absorption spectroscopy studies have previously shown that the trna-guanine transglycosylase (tgt) from escherichia coli is a zinc metalloprotein and identified the enzymic ligands to the zinc [chong et al. (1995), biochemistry 34, 3694-3701; garcia et al. (1966), biochemistry 35, 3133-3139]. during these studies one mutant, tgt (c265a), was found to exhibit a significantly lower specific activity, but was not found to be involved in the zinc site. the present ...19979055203
thermostable variants of zymomonas mobilis alcohol dehydrogenase obtained using pcr-mediated random mutagenesis.using a random mutagenesis technique, the ferrous-ion-activated alcohol dehydrogenase of zymomonas mobilis has been altered to produce more thermally stable variants. after three rounds of mutation, a variant over 10 degrees c more stable at ph 8, with essentially unaltered kinetic characteristics, was produced. however, the ph profile of thermostability of this variant was much altered compared with the wild-type, with a relatively small increase (4 degrees c) at ph 6. sequencing of the variant ...19989473458
surface display of zymomonas mobilis levansucrase by using the ice-nucleation protein of pseudomonas syringae.the ice-nucleation protein (inp) is a glycosyl phosphatidylinositol-anchored outer membrane protein found in some gram-negative bacteria. using pseudomonas syringae inp as an anchoring motif, we investigated the functional display of a foreign protein, zymomonas mobilis levansucrase (levu), on the surface of escherichia coli. the cells expressing inp-levu were found to retain both the ice-nucleation and whole-cell levansucrase enzyme activities, indicating the functional expression of inp-levu h ...19989624691
expression of the zymomonas mobilis gfo gene or nadp-containing glucose:fructose oxidoreductase (gfor) in escherichia coli. formation of enzymatically active pregfor but lack of processing into a stable periplasmic protein.glucose:fructose oxidoreductase (gfor) of the gram-negative bacterium zymomonas mobilis is a periplasmic enzyme with tightly bound cofactor nadp. the preprotein carries an unusually long n-terminal signal peptide of 52 amino acid residues. expression of the gfo gene in cells of escherichia coli k12, under the control of a tac promoter, led to immunologically detectable proteins in western blots, and to the formation of an enzymatically active precursor form (pregfor), located in the cytosol. pro ...19979063452
expression of the escherichia coli pmi gene, encoding phosphomannose-isomerase in zymomonas mobilis, leads to utilization of mannose as a novel growth substrate, which can be used as a selective marker.wild-type zymomonas mobilis can utilize only three substrates (sucrose, glucose, and fructose) as sole carbon sources, which are largely converted into ethanol and carbon dioxide. here, we show that although d-mannose is not used as a growth substrate, it is taken up via the glucose uniport system (glucose facilitator protein) with a vmax similar to that of glucose. moreover, d-mannose was phosphorylated by a side activity of the resident fructokinase to mannose-6-phosphate. fructokinase was pur ...19968900006
extracellular melibiose and fructose are intermediates in raffinose catabolism during fermentation to ethanol by engineered enteric bacteria.contrary to general concepts of bacterial saccharide metabolism, melibiose (25 to 32 g/liter) and fructose (5 to 14 g/liter) accumulated as extracellular intermediates during the catabolism of raffinose (o-alpha-d-galactopyranosyl-1, 6-alpha-d-glucopyranosyl-beta-d-fructofuranoside) (90 g/liter) by ethanologenic recombinants of escherichia coli b, klebsiella oxytoca m5a1, and erwinia chrysanthemi ec16. both hydrolysis products (melibiose and fructose) were subsequently transported and further me ...19979068632
the cis-diol dehydrogenase cbac gene of tn5271 is required for growth on 3-chlorobenzoate but not 3,4-dichlorobenzoate.the nucleotide sequence of cbac, the 1-carboxy-3-chloro-4,5-dihydroxycyclohexa-2,6-diene (cis-diol) dehydrogenase gene from the 3-chlorobenzoate (3-cba) catabolic transposon tn5271 was determined. the functional significance of the deduced open reading frame was evaluated by deletion of an internal bsteii restriction site in cbac and by the creation of nested deletions using exonuclease iii. expression studies were carried out with alcaligenes sp. strain br6024, a chloramphenicol-resistant, tryp ...19979322760
squalene-hopene cyclase from bradyrhizobium japonicum: cloning, expression, sequence analysis and comparison to other triterpenoid cyclases.with the help of a pcr-based screening method, the gene encoding squalenehopene cyclase (shc) of bradyrhizobium japonicum usda 110 was isolated from a cosmid library. the shc catalyses the cyclization of squalene to hopanoids, a class of triterpenoid lipids recently discovered in nitrogen-fixing, root-nodule-forming bradyrhizobium bacteria. hybridization experiments showed that the gene is present in bacteria of all bradyrhizobium strains tested and in photosynthetic bacteria forming stem nodule ...19979141686
the dna-binding protein ii from zymomonas mobilis. complete amino acid sequence and interaction with dna.the primary structure of the dna-binding protein ii from zymomonas mobilis has been determined from data provided by automated edman degradation of the intact protein and of peptides derived from cleavage at aspartic acid and arginine residues. when compared with the homologous protein isolated from other bacteria, the dna-binding protein ii from z mobilis shows many substitutions. several non-conservative substitutions at positions usually highly conserved in this type of protein probably accou ...19989587668
an elevation of the molar growth yield of zymomonas mobilis during aerobic exponential growth.elevated values of molar growth yield (yx/s = 14-26 g mol-1) were obtained during exponential growth (mu > 0.4 h-1) of zymomonas mobilis atcc 29191 by using reduced concentrations of glucose (6. 25-100 mm) and increased oxygen supply (eh > 300 mv) in the growth medium, as compared to the yx/s of anaerobic exponential growth (8-10 g mol-1). aerobically grown cells showed an increased maximum growth rate (mumax), and a reduced specific glucose consumption rate (qs), and specific ethanol formation ...19979133324
modulation of lipoxygenase activity by bacterial hopanoids.tetrahydroxybacteriohopane (1), a bacterial hopanoid, inhibited soybean 15-lipoxygenase with an ic50 of about 10 microm. after per-o-acetylation of 1 no inhibition of the 15-lipoxygenase was observed. two other bacterial hopanoids, tetrahydroxybacteriohopane glucosamine (2) and tetrahydroxybacteriohopane ether (3), stimulated the activity of soybean 15-lipoxygenase. the activities of two other arachidonic acid-metabolizing enzymes, human 5-lipoxygenase and prostaglandin h synthase, were unaffect ...19979134748
a glutamate uptake regulatory protein (grp) in escherichia coli? 19979140981
high resolution crystal structure of pyruvate decarboxylase from zymomonas mobilis. implications for substrate activation in pyruvate decarboxylases.the crystal structure of tetrameric pyruvate decarboxylase from zymomonas mobilis has been determined at 1.9 a resolution and refined to a crystallographic r-factor of 16.2% and rfree of 19.7%. the subunit consists of three domains, all of the alpha/beta type. two of the subunits form a tight dimer with an extensive interface area. the thiamin diphosphate binding site is located at the subunit-subunit interface, and the cofactor, bound in the v conformation, interacts with residues from the n-te ...19989685367
isolation and molecular characterization of high-performance cellobiose-fermenting spontaneous mutants of ethanologenic escherichia coli ko11 containing the klebsiella oxytoca casab operon.escherichia coli ko11 was previously constructed to produce ethanol from acid hydrolysates of hemicellulose (pentoses and hexoses) by the chromosomal integration of zymomonas mobilis genes encoding pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhb). klebsiella oxytoca p2 was constructed in an analogous fashion for the simultaneous saccharification and fermentation of cellulose and contains pts enzymes for cellobiose. in this study, ko11 was further engineered for the fermentation of c ...19979406380
a physical map of the genome of ethanol fermentative bacterium zymomonas mobilis zm4 and localization of genes on the map.a physical map of the zymomonas mobilis zm4 genome has been constructed from the results of reciprocal southern hybridization with pmei, paci, and noti-digested genomic dna fragments and linking cosmid clones. restriction enzyme-digested z. mobilis zm4 genome was electrophoresed with phage lambda dna concatemers as a size standard in a bio-rad chef-drii pulsed-field gel electrophoresis (pfge) system. the restriction enzyme pmei generated 15 fragments (3-625 kb), and paci produced 19 fragments (7 ...19989469936
isolation and characterization of ethanol-tolerant mutants of escherichia coli ko11 for fuel ethanol production.genetically engineered escherichia coli ko11 is capable of efficiently producing ethanol from all sugar constituents of lignocellulose but lacks the high ethanol tolerance of yeasts currently used for commercial starch-based ethanol processes. using an enrichment method which selects alternatively for ethanol tolerance during growth in broth and for ethanol production on solid medium, mutants of ko11 with increased ethanol tolerance were isolated which can produce more than 60 g ethanol l-1 from ...19989611822
no prokaryotic gpi anchoring. 19989743095
continuous ethanol production by zymomonas mobilis and saccharomyces cerevisiae in biofilm reactors.continuous ethanol fermentations were performed in duplicate for 60 days with zymomonas mobilis atcc 331821 or saccharomyces cerevisiae atcc 24859 in packed-bed reactors with polypropylene or plastic composite-supports. the plastic composite-supports used contained polypropylene (75%) with ground soybean-hulls (20%) and zein (5%) for z. mobilis, or with ground soybean-hulls (20%) and soybean flour (5%) for s. cerevisiae. maximum ethanol productivities of 536 g l-1 h-1 (39% yield) and 499 g l-1 h ...19968652117
transition-state theoretical interpretation of the catalytic power of pyruvate decarboxylases: the roles of static and dynamical considerations.the catalytic power of two thiamin diphosphate (thdp)-dependent enzymes, yeast pyruvate decarboxylase (the hysteretically regulated enzyme from saccharomyces cerevisiae, scpdc) and bacterial pyruvate decarboxylase (the unregulated enzyme from zymomonas mobilis, zmpdc), are analyzed by thorough-going application of transition-state theory, i.e. by a static approach that emphasizes the state-function character of the free energy of activation and takes no explicit account of dynamical consideratio ...19989655907
the substitution of a single amino acid residue (ser-116 --> asp) alters nadp-containing glucose-fructose oxidoreductase of zymomonas mobilis into a glucose dehydrogenase with dual coenzyme specificity.glucose-fructose oxidoreductase (gfor, ec 1.1.1.99.-) from the gram-negative bacterium zymomonas mobilis contains the tightly bound cofactor nadp. based on the revision of the gfo dna sequence, the derived gfor sequence was aligned with enzymes catalyzing reactions with similar substrates. a novel consensus motif (agkhvxcekp) for a class of dehydrogenases was detected. from secondary structure analysis the serine-116 residue of gfor was predicted as part of a rossmann-type dinucleotide binding f ...19979148926
antimicrobial activity of flavonoids from leaves of tagetes minuta.the total extract and fractions with different solvents, obtained from leaves of tagetes minuta, showed several degrees of antimicrobial activity against gram positive and gram negative microorganisms. the same fractions were inactive against lactobacillus, zymomonas and saccharomices species. the major component of the extract: quercetagetin-7-arabinosyl-galactoside, showed significant antimicrobial activity on pathogen microorganisms tested. correlation results were carried out using chloramph ...19979201613
membrane d-lactate oxidase in zymomonas mobilis: evidence for a branched respiratory chain.respiratory chain composition of the ethanol-producing bacterium zymomonas mobilis was studied. its membrane d-lactate oxidase was characterised. with nadh, but not d-lactate as substrate, a cytochrome o-like component was seen in co difference spectra. chlorpromazine specifically inhibited reduction of cytochrome d, while myxothiazol eliminated the cytochrome o-like features in co difference spectra. it is suggested that electrons from nadh are distributed between branches terminated by the cyt ...19989812368
improving fermentation performance of recombinant zymomonas in acetic acid-containing media.in the production of ethanol from lignocellulosic biomass, the hydrolysis of the acetylated pentosans in hemicellulose during pretreatment produces acetic acid in the prehydrolysate. the national renewable energy laboratory (nrel) is currently investigating a simultaneous saccharification and cofermentation (sscf) process that uses a proprietary metabolically engineered strain of zymomonas mobilis that can coferment glucose and xylose. acetic acid toxicity represents a major limitation to biocon ...19989627380
changes in the growth and enzyme level of zymomonas mobilis under oxygen-limited conditions at low glucose concentration.zymomonas mobilis growing aerobically with 20 g glucose-1 (carbon-limited) in a chemostat exhibited an increase in both the molar growth yield (yx/s) and the maximum molar growth yield (yx/smax) and a decrease in both the specific substrate consumption rate (qs) and the maintenance energy consumption rate (me). stepwise increase in the input oxygen partial pressure showed that anaerobic-to-aerobic transitional adaptation occurred in four stages: anaerobic (0 mm hgo2), oxygen-limited (7.6- 230 mm ...19979211713
a multistep process is responsible for product-induced inactivation of glucose-fructose oxidoreductase from zymomonas mobilis.glucose-fructose oxidoreductase from the bacterium zymomonas mobilis catalyzes a transhydrogenation reaction in which d-fructose reduction to d-sorbitol is coupled to the oxidation of d-glucose or other aldoses to the corresponding aldonolactones. tightly protein-bound nadp(h) serves as the cofactor. we found that the interaction of glucose-fructose oxidoreductase with its aldonolactone product triggered a sequential process that affects the protein structure conformationally and chemically and, ...19989490072
sequence analysis of a cryptic plasmid from flavobacterium sp. kp1, a psychrophilic bacterium.a cryptic plasmid found at high copy number was isolated from flavobacterium sp. kp1, a psychrophilic gram-negative bacterium, cloned, and sequenced. the sequence will appear in the ddbj/embl/genbank databases under the accession number ab007196. the pfl1 plasmid is 2311 nucleotides in length with 32.7% gc content, and shows a distinctive nucleotide sequence without homology to other plasmids of similar length. the plasmid contains two open reading frames of significant length, orfi and orfii. o ...19999919674
the effects of temperature on the kinetics and stability of mesophilic and thermophilic 3-phosphoglycerate kinases.the effects of temperature on the kinetic parameters kcat and km, for three isolates of the highly conserved monomeric enzyme 3-phosphoglycerate kinase (pgk), were investigated in detail using a rapid automated kinetics apparatus. pgk was purified from the thermophilic bacterium thermoanaerobacter sp. rt8.g4 (optimum growth temperature 68 degrees c), the mesophile zymomonas mobilis (optimum growth temperature 32 degrees c) and a second, unidentified, soil mesophile designated unid a (optimum gro ...19989494072
levansucrase of rahnella aquatilis atcc33071. gene cloning, expression, and levan formation. 19989928133
purification of the pyruvate dehydrogenase multienzyme complex of zymomonas mobilis and identification and sequence analysis of the corresponding genes.the pyruvate dehydrogenase (pdh) complex of the gram-negative bacterium zymomonas mobilis was purified to homogeneity. from 250 g of cells, we isolated 1 mg of pdh complex with a specific activity of 12.6 u/mg of protein. analysis of subunit composition revealed a pdh (e1) consisting of the two subunits e1alpha (38 kda) and e1beta (56 kda), a dihydrolipoamide acetyltransferase (e2) of 48 kda, and a lipoamide dehydrogenase (e3) of 50 kda. the e2 core of the complex is arranged to form a pentagona ...19989515924
aspartate-27 and glutamate-473 are involved in catalysis by zymomonas mobilis pyruvate decarboxylase.zymomonas mobilis pyruvate decarboxylase (ec 4.1.1.1) was subjected to site-directed mutagenesis at two acidic residues near the thiamin diphosphate cofactor in the active site. asp-27 was changed to glu or asn, and glu-473 was mutated to asp (e473d) or gln (e473q). each mutant protein was purified to near-homogeneity, and the kinetic and cofactor-binding properties were compared with those of the wild-type protein. despite the very conservative nature of these alterations, all mutants had a ver ...199910191255
the effect of ethanol and oxygen on the growth of zymomonas mobilis and the levels of hopanoids and other membrane lipids.zymomonas mobilis (atcc 29191) was grown either aerobically or anaerobically in the presence of 2% (wt/vol) glucose and 0, 3, or 6% (vol/vol) ethanol. the rates of growth and the composition of hopanoids, cellular fatty acids, and other lipids in the bacterial membranes were quantitatively analyzed. the bacterium grew in the presence of 3% and 6% ethanol and was more ethanol tolerant when grown anaerobically. in the absence of ethanol, hopanoids comprised about 30% (by mass) of the total cellula ...19979216888
metabolic engineering for the production of carotenoids in non-carotenogenic bacteria and yeasts.the crt gene clusters responsible for the biosynthesis of carotenoids such as lycopene, beta-carotene and astaxanthin have been isolated from carotenogenic bacteria such as erwinia species and the marine bacterium agrobacterium aurantiacum. the functions of the individual genes have been identified. the first substrate of the enzymes encoded by the erwinia crt clusters is farnesyl pyrophosphate which is not only the precursor for carotenoid biosynthesis but also sterols, dolichols and other nume ...19979519479
purification and characterization of a novel levanoctaose-producing levanase from pseudomonas strain k-52.levan-assimilating micro-organisms from soil samples were screened for levanoligosaccharide-generating enzyme production. the isolated strain k-52 produced an extracellular levanoctaose-generating enzyme and was identified as belonging to genus pseudomonas. the levanase was purified to homogeneity by (nh4)2so4 fractionation and successive column chromatography on deae-cellulose, phenyl-toyopearl 650 m, sephadex g-100 and hydroxyapatite. the molecular mass of the enzyme was estimated as approx. 3 ...19989569612
conditions that promote production of lactic acid by zymomonas mobilis in batch and continuous culture.this study documents the similar ph-dependent shift in pyruvate metabolism exhibited by zymomonas mobilis atcc 29191 and atcc 39676 in response to controlled changes in their steady-state growth environments. the usual high degree of ethanol selectivity associated with glucose fermentation by z. mobilis is associated with conditions that promote rapid and robust growth, with about 95% of the substrate (5% w/v glucose) being converted to ethanol and c)2, and the remaining 5% being used for the sy ...19989627381
in-vitro study of interaction between photooxidation and biodegradation of 2-methylphenanthrene by sphingomonas sp. 2mpii.this work reports a study of interactions between reactions of photooxidation and reactions of bacterial degradation of an alkylated polyaromatic hydrocarbon (2-methylphenanthrene). bacterial growth was carried out using artificial sunlight as light source. among the various products detected, the major product was identified as the 2-methylphenanthrene aldehyde. sunlight allows accelerated elimination of the substrate. this enhancement of the biodegradation rate of 2 methylphenanthrene is due t ...199910204235
cloning of a sphingomonas paucimobilis syk-6 gene encoding a novel oxygenase that cleaves lignin-related biphenyl and characterization of the enzyme.sphingomonas paucimobilis syk-6 transforms 2,2'-dihydroxy-3,3'-dimethoxy-5,5'-dicarboxybiphenyl (ddva), a lignin-related biphenyl compound, to 5-carboxyvanillic acid via 2,2',3-trihydroxy-3'-methoxy-5,5'-dicarboxybiphenyl (oh-ddva) as an intermediate (15). the ring fission of oh-ddva is an essential step in the ddva degradative pathway. a 15-kb ecori fragment isolated from the cosmid library complemented the growth deficiency of a mutant on oh-ddva. subcloning and deletion analysis showed that a ...19989647824
increased cellulose production from sucrose with reduced levan accumulation by an acetobacter strain harboring a recombinant plasmid.cellulose production from sucrose by acetobacter strains is accompanied by the accumulation of a water-soluble polysaccharide, called levan. to improve cellulose productivity, a levansucrase-deficient mutant, ld-2, was derived from acetobacter strain 757 and used as a host for the construction of recombinant strains. an ld-2 mutant harboring a plasmid containing the sucrase gene, sucze3, from zymomonas mobilis together with zlis, a gene that encodes a secretion-activating factor under the contro ...19989648211
purification and characterization of a levanbiose-producing levanase from pseudomonas sp. no. 43.a levanbiose-accumulating levanase from pseudomonas sp. no. 43 was purified to a homogeneous state by (nh4)2so4 fractionation and by chromatography on deae-toyopearl 650 m and phenyl-toyopearl 650 m columns. the molecular mass and isoelectric point of the enzyme were estimated to be 36 kda and 5.7 respectively; the optimal ph and temperature for the enzyme reaction were ph 7.0 and 40 degrees c respectively. the purified enzyme was stable in the ph range 6.0-8.0 at 20 degrees c and stable up to 5 ...199910334957
subunit structure, function and organisation of pyruvate decarboxylases from various organisms.the nature of the environment of macromolecules influences and determines the state of their overall structure and the extent of binding of specific (cofactors, substrates) or unspecific ligands. how these interactions between enzyme molecules and ligands influence their quaternary structures and, in this way, the realisation of high catalytic activity will be discussed here for the enzyme pyruvate decarboxylase from various organisms: brewer's yeast, brewer's yeast strain, recombinant wild type ...19989655918
structure and properties of pyruvate decarboxylase and site-directed mutagenesis of the zymomonas mobilis enzyme.pyruvate decarboxylase (ec 4.1.1.1) is a thiamin diphosphate-dependent enzyme that catalyzes the penultimate step in alcohol fermentation. the enzyme is widely distributed in plants and fungi but is rare in prokaryotes and absent in animals. here we review its structure and properties with particular emphasis on how site-directed mutagenesis of the enzyme from zymomonas mobilis has assisted us to understand the function of critical residues.19989655927
gene and subunit organization of bacterial pyruvate dehydrogenase complexes.pyruvate dehydrogenase complexes of bacterial origin are compared with respect to subunit composition, organization of the corresponding genes, and the number and location of lipoyl domains. special attention is given to two unusual examples of pyruvate dehydrogenase complexes, formed by zymomonas mobilis and thiobacillus ferrooxidans.19989655937
control of the association state of tetrameric glucose-fructose oxidoreductase from zymomonas mobilis as the rationale for stabilization of the enzyme in biochemical reactors.tetrameric, nadp-containing glucose-fructose oxidoreductase (gfor) from zymomonas mobilis catalyzes the oxidation of glucose into glucono-delta-lactone coupled to the reduction of fructose to sorbitol. gfor is inactivated during substrate turnover in vitro, the long-term stability of the enzyme during conversions in biochemical reactors thereby being drastically reduced. the process of inactivation is triggered by structural transitions that are induced by the lactone product and involves aggreg ...19989685715
purification and characterization of alkaline phosphatase containing phosphotyrosyl phosphatase activity from the bacterium prevotella intermedia.a novel alkaline phosphatase, designated pialp, has been purified and characterized from prevotella intermedia atcc 25611, an anaerobe implicated in progressive periodontal disease. the enzyme was a homodimer of apparently identical subunits of mr 54 kda. thiol-reducing agents completely inhibited the purified enzyme. the enzyme was highly stable even at 80 degrees c. it exhibited substantial activity against tyrosine-phosphate-containing raytide. the phosphatase activity was sensitive to orthov ...19989654126
ethanol synthesis by genetic engineering in cyanobacteria.cyanobacteria are autotrophic prokaryotes which carry out oxygenic photosynthesis and accumulate glycogen as the major form of stored carbon. in this research, we introduced new genes into a cyanobacterium in order to create a novel pathway for fixed carbon utilization which results in the synthesis of ethanol. the coding sequences of pyruvate decarboxylase (pdc) and alcohol dehydrogenase ii (adh) from the bacterium zymomonas mobilis were cloned into the shuttle vector pcb4 and then used to tran ...19999925577
small-angle x-ray solution-scattering studies on ligand-induced subunit interactions of the thiamine diphosphate dependent enzyme pyruvate decarboxylase from different organisms.the quaternary structures of the thiamine diphosphate dependent enzyme pyruvate decarboxylase (ec 4.1.1.1) from the recombinant wild type of saccharomycescerevisiae and zymomonas mobilis and from germinating pisum sativum seeds were examined by x-ray solution scattering. the dependence of the subunit association equilibrium on the ph and the presence of the cofactors thiamine diphosphate and magnesium ions were compared, and the differences between the catalytic properties of the different enzym ...19989548765
the role of his113 and his114 in pyruvate decarboxylase from zymomonas mobilis.pyruvate decarboxylase (pdc) is one of several enzymes that require thiamin diphosphate (thdp) and a divalent cation as essential cofactors. recently, the three-dimensional structures of the enzyme from two yeasts have been determined. while these structures shed light on the binding of the cofactors and the reaction mechanism, the interactions between the substrate pyruvate and the enzyme remain unclear. we have used pdc from zymomonas mobilis as a model for these enzymes in order to study subs ...19979310361
lactone-ring-cleaving enzyme: genetic analysis, novel rna editing, and evolutionary implications.a lactonohydrolase from fusarium oxysporum aku 3702 is an enzyme catalyzing the hydrolysis of aldonate lactones to the corresponding aldonic acids. the amino acid sequences of the nh2 terminus and internal peptide fragments of the enzyme were determined to prepare synthetic oligonucleotides as primers for the pcr. an approximate 1, 000-base genomic dna fragment thus amplified was used as the probe to clone both genomic dna and cdna for the enzyme. the lactonohydrolase genomic gene consists of si ...19989788992
expression of the extracellular levansucrase and invertase genes from zymomonas mobilis in escherichia coli cells.we investigated the expression and localization in escherichia coli of sucze2 and sucze3, encoding zymomonas mobilis extracellular levansucrase and invertase, respectively, and lacking a typical n-terminal secretion signal. levansucrase and invertase were expressed efficiently under the lac and tac promoters in e. coli cells, and some of the levansucrase produced was localized in the periplasmic space. the sucze2 expression was not lethal to e. coli in the presence of 5% sucrose, and led to the ...19989805385
exceptional characteristics of heterotetrameric (alpha 2 beta 2) e1p of the pyruvate dehydrogenase complex from zymomonas mobilis: expression from an own promoter and a lipoyl domain in e1 beta.in the pyruvate dehydrogenase complex (pdhc) of zymomonas mobilis the beta subunit of the pyruvate dehydrogenase (e1p) as well as the acetyltransferase (e2p) contain an n-terminal lipoyl domain. both lipoyl domains were acetylated in vitro using 2-14c-pyruvate as a substrate, demonstrating that both lipoyl domains can accept acetyl groups from the e1 component. as previously shown the structural genes (pdha alpha beta, pdhb, lpd) encoding the pyruvate dehydrogenase complex of z. mobilis are loca ...199910436929
activation of thiamine diphosphate in pyruvate decarboxylase from zymomonas mobilis.replacement of tryptophan 392 located in the active site cavity of pyruvate decarboxylase (pdc; ec 4.1.1.1) from zymomonas mobilis by methionine or glutamine yields enzymes with smaller catalytic constants of 8.5 s(-1) and 3.6 s(-1) at 4 degrees c, compared to that of the wild-type enzyme (17 s(-1)). the rate constants of the h/d exchange at the c2 of the coenzyme thiamine diphosphate have been determined to be 130 s(-1) for the wild-type enzyme, 56 s(-1) for the methionine and 30 s(-1) for the ...19989891980
active site mutants of pyruvate decarboxylase from zymomonas mobilis--a site-directed mutagenesis study of l112, i472, i476, e473, and n482.the homotetrameric pyruvate decarboxylase (pdc) from zymomonas mobilis requires the cofactors thiamin diphosphate and mg2+ for catalytic activity. we have investigated the role of various amino acid residues in the direct environment of the active site. the role of residue e473 in the catalytic activity and stability of the enzyme was probed by several mutations. all mutant enzymes were either inactive or failed to give any recombinant protein. the close interaction of e473 and n482, which can b ...19989839941
novel selection for isoniazid (inh) resistance genes supports a role for nad+-binding proteins in mycobacterial inh resistance.the genetic basis of isoniazid (inh) resistance remains unknown for a significant proportion of clinical isolates. to identify genes which might confer resistance by detoxifying or sequestering inh, we transformed the escherichia coli oxyr mutant, which is relatively sensitive to inh, with a mycobacterium tuberculosis plasmid library and selected for inh-resistant clones. three genes were identified and called ceo for their ability to complement the escherichia coli oxyr mutant. ceoa was the pre ...19989784509
characterization of the tra2 region of the inchi1 plasmid r27.in this study, the dna sequence of one of the transfer regions of the inchi1 plasmid r27 was determined. this region, which corresponds to coordinates 0-40 on the r27 map has been called the tra2 region, and is believed to be involved in mating pair formation. dna sequence analysis of the transfer region identified 11 open reading frames which showed similarities to the transfer genes from other conjugative systems. the r27 transfer genes appear to most closely resemble the genes from the f plas ...199910366528
metabolic state of zymomonas mobilis in glucose-, fructose-, and xylose-fed continuous cultures as analysed by 13c- and 31p-nmr spectroscopy.the reasons for the well-known significantly different behaviour of the anaerobic, gram-negative, ethanologenic bacterium zymomonas mobilis during growth on fructose (i.e. decreased growth and ethanol yields, increased by-product formation) as compared to that on its second natural substrate, glucose, have remained unexplained. a xylose-fermenting recombinant strain of z. mobilis that was recently constructed in our laboratory also unexpectedly displayed an increased formation of by-products and ...199910369893
bioconversion of glucose and fructose to sorbitol and gluconic acid by untreated cells of zymomonas mobilis.the bioconversion of glucose and fructose to gluconic acid and sorbitol, respectively, by the enzymes glucose-fructose oxidoreductase (gfor) and glucono-delta-lactonase (gl), contained in untreated cells of zymomonas mobilis atcc 29191, was investigated in batch runs with glucose plus fructose concentrations (s0) varying from 100 to 750 g l-1 in equimolar ratio. when s0 was increased to 650 g l-1, the yields were improved, reaching a maximum of 91% for both products, with productivities of 1.6 a ...199910553651
crystal structure of alginate lyase a1-iii from sphingomonas species a1 at 1.78 a resolution.the three-dimensional structure of alginate lyase a1-iii (alyiii) from a sphingomonas species a1 was determined by x-ray crystallography. the enzyme was crystallized by the hanging-drop vapour-diffusion method in the presence of 49% ammonium sulfate at 20 degrees c. the crystals are monoclinic and belong to the space group c2 with unit cell dimensions of a=49.18 a, b=93.08 a, c=82.10 a and beta=104.12 degrees. there was one molecule of alginate lyase in the asymmetric unit of the crystal. the di ...199910390348
probing the location and function of the conserved histidine residue of phosphoglucose isomerase by using an active site directed inhibitor n-bromoacetylethanolamine phosphate.phosphoglucose isomerase (ec 5.3.1.9) catalyzes the interconversion of d-glucopyranose-6-phosphate and d-fructofuranose-6-phosphate by promoting an intrahydrogen transfer between c1 and c2. a conserved histidine exists throughout all phosphoglucose isomerases and was hypothesized to be the base catalyzing the isomerization reaction. in the present study, this conserved histidine, his311, of the enzyme from bacillus stearothermophilus was subjected to mutational analysis, and the mutational effec ...199910595547
development and application of a system for analysis of mixed cultures of microorganisms.development and application of a system for real-time quantitative assessment of individual cell activities in a mixed culture system was investigated. this was based on a concept that the activities of individual cells in a mixed culture can be assessed if the cells are physically separated (in separate compartments) in a vessel while the culture conditions, including the broth components, are maintained the same in all the compartments during the cultivation. on this basis, three different app ...199910394620
kinetics and performance of a co-immobilised system of amyloglucosidase and zymomonas mobilis.high operational stability and productivity of co-immobilised systems are important aspects for their successful application in industrial processes. a dynamic model is required to describe artificially co-immobilised systems because the time needed to reach steady state normally exceeds the operational life span of these systems. time dependent intraparticle concentration profiles and macroscopic conversion were modelled to study the operational stability and productivity of these systems theor ...199910397826
molecular characterization of squalene synthase from the green microalga botryococcus braunii, race b.the green microalga botryococcus braunii produces large amounts of liquid hydrocarbons and is classified into three races, depending on the type of the hydrocarbon produced. the b race produces two types of triterpenoid hydrocarbons, squalene and botryococcene, both of which are putative condensation products of farnesyl diphosphate. in an attempt to better understand the regulation involved in the production of squalene and botryococcene, we have isolated and characterized a squalene synthase ( ...200010620354
construction and characterization of an effector strain of streptococcus mutans for replacement therapy of dental caries.an effector strain has been constructed for use in the replacement therapy of dental caries. recombinant dna methods were used to make the streptococcus mutans supercolonizing strain, jh1140, lactate dehydrogenase deficient by deleting virtually all of the ldh open reading frame (orf). to compensate for the resulting metabolic imbalance, a supplemental alcohol dehydrogenase activity was introduced by substituting the adhb orf from zymomonas mobilis in place of the deleted ldh orf. the resulting ...200010639415
altered regulation of pyruvate kinase or co-overexpression of phosphofructokinase increases glycolytic fluxes in resting escherichia coli.glycolytic fluxes in resting escherichia coli were enhanced by overexpression of heterologous pyruvate kinases (pyk) from bacillus stearothermophilus and zymomonas mobilis, but not homologous pyk. compared to the control, an increase of 10% in specific glucose consumption and of 15% in specific ethanol production rates was found in anaerobic resting cells, expressing the heterologous pyks, that were harvested from exponentially growing aerobic cultures. a further increase in glycolytic flux was ...200010649237
cloning of conserved genes from zymomonas mobilis and bradyrhizobium japonicum that function in the biosynthesis of hopanoid lipids.the squalene-hopene cyclase (shc) is the only enzyme involved in the biosynthesis of hopanoid lipids that has been characterized on the genetic level. to investigate if additional genes involved in hopanoid biosynthesis are clustered with the shc gene, we cloned and analyzed the nucleotide sequences located immediately upstream of the shc genes from zymomonas mobilis and bradyrhizobium japonicum. in z. mobilis, five open reading frames (orfs, designated as hpna-e) were detected in a close arrang ...19989714766
cloning, nucleotide sequence, and expression in escherichia coli of levansucrase genes from the plant pathogens pseudomonas syringae pv. glycinea and p. syringae pv. phaseolicola.plant-pathogenic bacteria produce various extracellular polysaccharides (epss) which may function as virulence factors in diseases caused by these bacteria. the eps levan is synthesized by the extracellular enzyme levansucrase in pseudomonas syringae, erwinia amylovora, and other bacterial species. the lsc genes encoding levansucrase from p. syringae pv. glycinea pg4180 and p. syringae pv. phaseolicola ncppb 1321 were cloned, and their nucleotide sequences were determined. heterologous expressio ...19989726857
fermentations with new recombinant organisms.united states fuel ethanol production in 1998 exceeded the record production of 1.4 billion gallons set in 1995. most of this ethanol was produced from over 550 million bushels of corn. expanding fuel ethanol production will require developing lower-cost feedstocks, and only lignocellulosic feedstocks are available in sufficient quantities to substitute for corn starch. major technical hurdles to converting lignocellulose to ethanol include the lack of low-cost efficient enzymes for saccharifica ...199910514256
cloning and sequencing of the beta-fructofuranosidase gene from bacillus sp. v230.the beta-fructofuranosidase gene (bff) from bacillus sp. v230 has been cloned in escherichia coli and its nucleotide sequence has been analyzed. the product of bff consists of a signal sequence of 32 amino acid (a.a.) residues for secretion and 455 a.a. residues of the extracellular beta-fructofuranosidase. the a.a. sequence of the bff product has similarities with those of the bacillus subtilis levanscrase (63.7% identity), the streptococcus mutans fructosyltransferase (33.7%), and the zymomona ...199910427700
the incidence of oscillatory behavior in the continuous fermentation of zymomonas mobilisthe incidence of oscillatory behavior in the continuous culture of zymomonas mobilis has been examined using a combination of experimental investigations and a predictive model. the tendency to oscillatory behavior was assessed by perturbing the feed substrate concentration and dilution rate in a continuous fermentation starting from a number of distinct initial conditions. the entire range of qualitative dynamic behavior was observed: overdamped, underdamped, and sustained oscillatory responses ...199910441358
enteric bacterial catalysts for fuel ethanol production.the technology is available to produce fuel ethanol from renewable lignocellulosic biomass. the current challenge is to assemble the various process options into a commercial venture and begin the task of incremental improvement. current process designs for lignocellulose are far more complex than grain to ethanol processes. this complexity results in part from the complexity of the substrate and the biological limitations of the catalyst. our work at the university of florida has focused primar ...199910514255
characterization of the mobilization region of the zymomonas mobilis atcc10988 plasmid pzmo3.the 2.7-kb zymomonas mobilis atcc10988 plasmid pzmo3 contains a coding region (orf1) indispensable for mobilization. a cis-acting 409-bp sequence between orf2 (c-terminal) and orf1 (n-terminal) conferred mobilization activity to puc19, when the product of orf1 was provided in trans. in this area, two segments showed homology with previously characterized orit regions.19999887309
engineering endoglucanase-secreting strains of ethanologenic klebsiella oxytoca p2.recombinant klebsiella oxytoca p2 was developed as a biocatalyst for the simultaneous saccharification and fermentation (ssf) of cellulose by chromosomally integrating zymomonas mobilis genes (pdc, adhb) encoding the ethanol pathway. this strain contains the native ability to transport and metabolize cellobiose, eliminating the need to supplement with beta-glucosidase during ssf. to increase the utility of this biocatalyst, we have now chromosomally integrated the celz gene encoding the primary ...199910455486
the efficient export of nadp-containing glucose-fructose oxidoreductase to the periplasm of zymomonas mobilis depends both on an intact twin-arginine motif in the signal peptide and on the generation of a structural export signal induced by cofactor binding.the periplasmic, nadp-containing glucose-fructose oxidoreductase of the gram-negative bacterium zymomonas mobilis belongs to a class of redox cofactor-dependent enzymes which are exported with the aid of a signal peptide containing a so-called twin-arginine motif. in this paper we show that the replacement of one or both arginine residues results in drastically reduced translocation of glucose-fructose oxidoreductase to the periplasm, showing that this motif is essential. mutant proteins which, ...199910406965
continuous fermentation studies with xylos-utilizing recombinant zymomonas mobilis.this study examined the continuous cofermentation performance characteristics of a dilute-acid "prehydrolysate-adapted" recombinant zymomonas 39676:pzb4l and builds on the ph-stat batch fermentations with this recombinant that we reported on last year. substitution of yeast extract by 1% (w/v) corn steep liquor (csl) (50% solids) and mg (2 mm) did not alter the cofermentation performance. using declared assumptions, the cost of using csl and mg was estimated to be 12.5 cents/gal of ethanol with ...200010849797
complement activation by bacterial surface glycolipids: a study with planar bilayer membranes.planar asymmetric glycolipid/phospholipid bilayer membranes were used as a reconstitution model of the lipid matrix of the outer membrane of gram-negative bacteria to study complement (c) activation by various bacterial surface glycolipids with the aim of defining the c activation pathway. as glycolipids the lipopolysaccharides of salmonella enterica serovar minnesota r mutant strains r595 (re lps) and r4 (rd2 lps), pentaacyl lipid a from the lps of the escherichia coli re mutant f515, and glyco ...19999929374
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