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1.
Foley S Stolarczyk E Mouni F Brassart C Vidal O Aïssi E Bouquelet S Krzewinski F 《Archives of microbiology》2008,189(2):157-167
Bifidobacterium bifidum, in contrast to other bifidobacterial species, is auxotrophic for N-acetylglucosamine. Growth experiments revealed assimilation of radiolabelled N-acetylglucosamine in bacterial cell walls and in acetate, an end-product of central metabolism via the bifidobacterial d-fructose-6-phosphate shunt. While supplementation with fructose led to reduced N-acetylglucosamine assimilation via the d-fructose-6-phosphate shunt, no significant difference was observed in levels of radiolabelled N-acetylglucosamine incorporated into cell walls. Considering the central role played by glutamine fructose-6-phosphate transaminase
(GlmS) in linking the biosynthetic pathway for N-acetylglucosamine to hexose metabolism, the GlmS of Bifidobacterium was characterized. The genes encoding the putative GlmS of B. longum DSM20219 and B. bifidum DSM20082 were cloned and sequenced. Bioinformatic analyses of the predicted proteins revealed 43% amino acid identity with
the Escherichia coli GlmS, with conservation of key amino acids in the catalytic domain. The B. longum GlmS was over-produced as a histidine-tagged fusion protein. The purified C-terminal His-tagged GlmS possessed glutamine
fructose-6-phosphate amidotransferase activity as demonstrated by synthesis of glucosamine-6-phosphate from fructose-6-phosphate
and glutamine. It also possesses an independent glutaminase activity, converting glutamine to glutamate in the absence of
fructose-6-phosphate. This is of interest considering the apparently reduced coding potential in bifidobacteria for enzymes
associated with glutamine metabolism.
S. Foley and E. Stolarczyk contributed equally to this work 相似文献
2.
Hansen T Arnfors L Ladenstein R Schönheit P 《Extremophiles : life under extreme conditions》2007,11(1):105-114
Recently, unusual non-regulated ATP-dependent 6-phosphofructokinases (PFK) that belong to the PFK-B family have been described
for the hyperthermophilic archaea Desulfurococcus amylolyticus and Aeropyrum pernix. Putative homologues were found in genomes of several archaea including the hyperthermophilic archaeon Methanocaldococcus jannaschii. In this organism, open reading frame MJ0406 had been annotated as a PFK-B sugar kinase. The gene encoding MJ0406 was cloned
and functionally expressed in Escherichia coli. The purified recombinant enzyme is a homodimer with an apparent molecular mass of 68 kDa composed of 34 kDa subunits. With
a temperature optimum of 85°C and a melting temperature of 90°C, the M. jannaschii nucleotide kinase represents one of the most thermoactive and thermostable members of the PFK-B family described so far.
The recombinant enzyme was characterized as a functional nucleoside kinase rather than a 6-PFK. Inosine, guanosine, and cytidine
were the most effective phosphoryl acceptors. Besides, adenosine, thymidine, uridin and xanthosine were less efficient. Extremely
low activity was found with fructose-6-phosphate. Further, the substrate specificity of closely related PFK-Bs from D. amylolyticus and A. pernix were reanalysed. 相似文献
3.
Anne Taylor Paige Molzahn Tanner Bushnell Clint Cheney Monique LaJeunesse Mohamad Azizian Lewis Semprini 《Journal of industrial microbiology & biotechnology》2018,45(3):201-211
Due to the natural gas boom in North America, there is renewed interest in the production of other chemical products from methane. We investigated the feasibility of immobilizing the obligate methanotrophic bacterium Methylosinus trichosporium OB3b in alginate beads, and selectively inactivating methanol dehydrogenase (MDH) with cyclopropane to produce methanol. In batch cultures and in semi-continuous flow columns, the exposure of alginate-immobilized cells to cyclopropane or cyclopropanol resulted in the loss of the majority of MDH activity (> 80%), allowing methanol to accumulate to significant concentrations while retaining all of M. trichosporium OB3b’s methane monooxygenase capacity. Thereafter, the efficiency of methanol production fell due to recovery of most of the MDH activity; however, subsequent inhibition periods resulted in renewed methanol production efficiency, and immobilized cells retained methane-oxidizing activity for at least 14 days. 相似文献
4.
Fiki AE Metabteb GE Bellebna C Wartmann T Bode R Gellissen G Kunze G 《Applied microbiology and biotechnology》2007,74(6):1292-1299
The yeast Arxula
adeninivorans provides an attractive expression platform and can be exploited as gene source for biotechnologically interesting proteins.
In the following study, a striking example for the combination of both aspects is presented. The transaldolase-encoding A. adeninivorans ATAL gene, including its promoter and terminator elements, was isolated and characterized. The gene includes a coding sequence
of 963 bp encoding a putative 321 amino acid protein of 35.0 kDa. The enzyme characteristics analyzed from isolates of native
strains and recombinant strains overexpressing the ATAL gene revealed a molecular mass of ca. 140 kDa corresponding to a tetrameric structure, a pH optimum of ca. 5.5, and a temperature
optimum of 20°C. The preferred substrates for the enzyme include d-erythrose-4-phosphate and d-fructose-6-phosphate, whereas d-glyceraldehyde is not converted. The ATAL expression level under salt-free conditions was observed to increase in media supplemented with 5% NaCl rendering the ATAL promoter attractive for moderate heterologous gene expression under high-salt conditions. Its suitability was assessed for
the expression of a human serum albumin (HSA) reporter gene. 相似文献
5.
Cellobiohydrolase genes cbhI and cbhII were isolated from Trichoderma viride AS3.3711 and T. viride CICC 13038, respectively, using RT-PCR technique. The cbhI gene from T. viride AS3.3711 contains 1,542 nucleotides and encodes a 514-amino acid protein with a molecular weight of approximately 53.96 kDa.
The cbhII gene from T. viride CICC 13038 was 1,413 bp in length encoding 471 amino acid residues with a molecular weight of approximately 49.55 kDa. The
CBHI protein showed high homology with enzymes belonging to glycoside hydrolase family 7 and CBHII is a member of Glycoside
hydrolase family 6. CBHI and CBHII play a role in the conversion of cellulose to glucose by cutting the disaccharide cellobiose
from the non-reducing end of the cellulose polymer chain. The two cellobiohydrolase (CBHI, CBHII) genes were successfully
expressed in Saccharomyces cerevisiae H158. Maximal activities of transformants Sc-cbhI and Sc-cbhII were 0.03 and 0.089 units ml−1 under galactose induction, respectively. The optimal temperatures of the recombinant enzymes (CBHI, CBHII) were 60 and 70°C,
respectively. The optimal pHs of recombinant enzymes CBHI and CBHII were at pH 5.8 and 5.0, respectively. 相似文献
6.
Nguyen VN Oh IJ Kim YJ Kim KY Kim YC Park RD 《Journal of industrial microbiology & biotechnology》2009,36(2):195-203
Two extracellular chitinases were purified from Paecilomyces variotii DG-3, a chitinase producer and a nematode egg-parasitic fungus, to homogeneity by DEAE Sephadex A-50 and Sephadex G-100 chromatography.
The purified enzymes were a monomer with an apparent molecular mass of 32 kDa (Chi32) and 46 kDa (Chi46), respectively, and
showed chitinase activity bands with 0.01% glycol chitin as a substrate after SDS-PAGE. The first 20 and 15 N-terminal amino
acid sequences of Chi32 and Chi46 were determined to be Asp-Pro-Typ-Gln-Thr-Asn-Val-Val-Tyr-Thr-Gly-Gln-Asp-Phe-Val-Ser-Pro-Asp-Leu-Phe
and Asp-Ala-X-X-Tyr-Arg-Ser-Val-Ala-Tyr-Phe-Val-Asn-Trp-Ala, respectively. Optimal temperature and pH of the Chi32 and Chi46
were found to be both 60°C, and 2.5 and 3.0, respectively. Chi32 was almost inhibited by metal ions Ag+ and Hg2+ while Chi46 by Hg2+ and Pb2+ at a 10 mM concentration but both enzymes were enhanced by 1 mM concentration of Co2+. On analyzing the hydrolyzates of chitin oligomers [(GlcNAc)
n
, n = 2–6)], it was considered that Chi32 degraded chitin oligomers as an exo-type chitinase while Chi46 as an endo-type chitinase. 相似文献
7.
8.
Kaur P Lingner A Singh B Böer E Polajeva J Steinborn G Bode R Gellissen G Satyanarayana T Kunze G 《Antonie van Leeuwenhoek》2007,91(1):45-55
The extracellular acid phosphatase-encoding Arxula adeninivorans APHO1 gene was isolated using degenerated specific oligonucleotide primers in a PCR screening approach. The gene harbours an ORF of 1449 bp encoding a protein of 483 amino acids with a calculated molecular mass of 52.4 kDa. The sequence includes an N-terminal secretion sequence of 17 amino acids. The deduced amino acid sequence exhibits 54% identity to phytases from Aspergillus awamori, Asp. niger and Asp. ficuum and a more distant relationship to phytases of the yeasts Candida albicans and Debaryomyces hansenii (36–39% identity). The sequence contains the phosphohistidine signature and the conserved active site sequence of acid phosphatases. APHO1 expression is induced under conditions of phosphate limitation. Enzyme isolates from wild and recombinant strains with the APHO1 gene expressed under control of the strong A. adeninivorans-derived TEF1 promoter were characterized. For both proteins, a molecular mass of approx. 350 kDa, corresponding to a hexameric structure, a pH optimum of pH 4.8 and a temperature optimum of 60°C were determined. The preferred substrates include p-nitrophenyl-phosphate, pyridoxal-5-phosphate, 3-indoxyl-phosphate, 1-naphthylphosphate, ADP, glucose-6-phosphate, sodium-pyrophosphate, and phytic acid. Thus the enzyme is a secretory acid phosphatase with phytase activity and not a phytase as suggested by strong homology to such enzymes. 相似文献
9.
The class II fructose-1,6-bisphosphatase gene of Corynebacterium glutamicum, fbp, was cloned and expressed with a N-terminal His-tag in Escherichia coli. Purified, His-tagged fructose-1,6-bisphosphatase from C. glutamicum was shown to be tetrameric, with a molecular mass of about 140 kDa for the homotetramer. The enzyme displayed Michaelis-Menten kinetics for the substrate fructose 1,6-bisphosphate with a Km value of about 14 µM and a Vmax of about 5.4 µmol min–1 mg–1 and kcat of about 3.2 s–1. Fructose-1,6-bisphosphatase activity was dependent on the divalent cations Mg2+ or Mn2+ and was inhibited by the monovalent cation Li+ with an inhibition constant of 140 µM. Fructose 6-phosphate, glycerol 3-phosphate, ribulose 1,5-bisphosphate and myo-inositol-monophosphate were not significant substrates of fructose-1,6-bisphosphatase from C. glutamicum. The enzymatic activity was inhibited by AMP and phosphoenolpyruvate and to a lesser extent by phosphate, fructose 6-phosphate, fructose 2,6-bisphosphate, and UDP. Fructose-1,6-bisphosphatase activities and protein levels varied little with respect to the carbon source. Deletion of the chromosomal fbp gene led to the absence of any detectable fructose-1,6-bisphosphatase activity in crude extracts of C. glutamicum WTfbp and to an inability of this strain to grow on the carbon sources acetate, citrate, glutamate, and lactate. Thus, fbp is essential for growth on gluconeogenic carbon sources and likely codes for the only fructose-1,6-bisphosphatase in C. glutamicum. 相似文献
10.
Rosa Garcia Sanchez Bärbel Hahn-Hägerdal Marie F Gorwa-Grauslund 《Microbial cell factories》2010,9(1):40
Background
In Saccharomyces cerevisiae galactose is initially metabolized through the Leloir pathway after which glucose 6-phosphate enters glycolysis. Galactose is controlled both by glucose repression and by galactose induction. The gene PGM2 encodes the last enzyme of the Leloir pathway, phosphoglucomutase 2 (Pgm2p), which catalyses the reversible conversion of glucose 1-phosphate to glucose 6-phosphate. Overexpression of PGM2 has previously been shown to enhance aerobic growth of S. cerevisiae in galactose medium. 相似文献11.
Livingstone JR Yoshida I Tarui Y Hirooka K Yamamoto Y Tsutui N Hirasawa E 《Journal of plant research》2002,115(5):393-400
NAD-dependent aminoaldehyde dehydrogenase (AMADH, EC 1.2.1.-) from Avena shoots was purified by DEAE Sephacel, hydroxyapatite, 5′-AMP Sepharose 4B, Mono Q, and TSK-GEL column chromatographies to
homogeneity by the criterion of native PAGE. SDS–PAGE yielded a single band at a molecular mass of 55 kDa. IEF studies showed
a band with a pI value of 5.3. In contrast to AMADHs from other species, the TSK-GEL chromatography showed that Avena AMADH exists as a monomer in the native state. The purified enzyme catalyzed the oxidations of 3-aminopropionaldehyde (APAL),
4-aminobutyraldehyde (ABAL) N-(3-aminopropyl)-4-aminobutyraldehyde (APBAL), and 4-guanidinobutyraldehyde (GBAL), but not of betaine aldehyde or indoleacetaldehyde.
The K
m values for APAL, ABAL, and GBAL were 1.5×10–6, 2.2×10–6, and 1.3×10–5 M, respectively. Although N-terminal amino acid sequence of Avena AMADH could not be determined due to a modification of the amino residue, the sequence of the fragment of AMADH cleaved by
V8 protease showed greater similarity to the barley BADH than to the pea AMADH.
Electronic Publication 相似文献
12.
A new species of Erysiphe sect. Uncinula is described and illustrated from Patagonia, Argentina. Erysiphe patagoniaca sp. nov., found on leaves of Nothofagus × antarctica, is similar to E. nothofagi and E. kenjiana, but differs in its appendages being twisted throughout their length and the number of appendages, asci, and ascospores.
The two endemic species of Erysiphe sect. Uncinula, E. magellanica and E. nothofagi, coexisted on the same leaves together with Erysiphe patagoniaca.
Received: September 19, 2002 / Accepted: November 28, 2002
Acknowledgments The authors are grateful to Ms. Seiko Niinomi for providing the micrographs of ascomata of Erysiphe spp. on Nothofagus.
Correspondence to:S. Takamatsu 相似文献
13.
ORF PAE1610 from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum was first annotated as the conjectural pgi gene coding for hypothetical phosphoglucose isomerase (PGI). However, we have recently identified this ORF as the putative pgi/pmi gene coding for hypothetical bifunctional phosphoglucose/phosphomannose isomerase (PGI/PMI). To prove its coding function, ORF PAE1610 was overexpressed in Escherichia coli, and the recombinant enzyme was characterized. The 65-kDa homodimeric protein catalyzed the isomerization of both glucose-6-phosphate and mannose-6-phosphate to fructose-6-phosphate at similar catalytic rates, thus characterizing the enzyme as bifunctional PGI/PMI. The enzyme was extremely thermoactive; it had a temperature optimum for catalytic activity of about 100°C and a melting temperature for thermal unfolding above 100°C. 相似文献
14.
Cytochrome c
6
, (cyt c
6) a soluble monoheme electron transport protein, was isolated and characterized from the chlorophyll d-containing cyanobacterium Acaryochoris marina, the type strain MBIC11017. The protein was purified using ammonium sulfate precipitation, ion exchange and gel filtration
column chromatography, and fast performance liquid chromatography. Its molecular mass and pI have been determined to be 8.87 kDa
and less than 4.2, respectively, by mass spectrometry and isoelectrofocusing (IEF). The protein has an alpha helical structure
as indicated by CD (circular dichroism) spectroscopy and a reduction midpoint potential (E
m) of +327 mV versus the normal hydrogen electrode (NHE) as determined by redox potentiometry. Its potential role in electron
transfer processes is discussed. 相似文献
15.
Julie Kaye Ardley Graham W. O’Hara Wayne G. Reeve Ron J. Yates Michael J. Dilworth Ravi P. Tiwari John G. Howieson 《Archives of microbiology》2009,191(4):311-318
The South African legumes Lotononis bainesii, L. listii and L. solitudinis are specifically nodulated by highly effective, pink-pigmented bacteria that are most closely related to Methylobacterium nodulans on the basis of 16S rRNA gene homology. Methylobacterium spp. are characterized by their ability to utilize methanol and other C1 compounds, but 11 Lotononis isolates neither grew on methanol as a sole carbon source nor were able to metabolize it. No product was obtained for PCR
amplification of mxaF, the gene encoding the large subunit of methanol dehydrogenase. Searches for methylotrophy genes in the sequenced genome
of Methylobacterium sp. 4-46, isolated from L. bainesii, indicate that the inability to utilize methanol may be due to the absence of the mxa operon. While methylotrophy appears to contribute to the effectiveness of the Crotalaria/M. nodulans symbiosis, our results indicate that the ability to utilize methanol is not a factor in the Lotononis/Methylobacterium symbiosis. 相似文献
16.
Babu R Jiang CJ Xu X Kottapalli KR Takatsuji H Miyao A Hirochika H Kawasaki S 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2011,122(4):831-854
We evaluated a large collection of Tos17 mutant panel lines for their reaction to three different races of Magnaporthe oryzae and identified a lesion mimic mutant, NF4050-8, that showed lesions similar to naturally occurring spl5 mutant and enhanced
resistance to all the three blast races tested. Nested modified-AFLP using Tos17-specific primers and southern hybridization experiments of segregating individuals indicated that the lesion mimic phenotype
in NF4050-8 is most likely due to a nucleotide change acquired during the culturing process and not due to Tos17 insertion per se. Inheritance and genetic analyses in two japonica × indica populations identified an overlapping genomic
region of 13 cM on short arm of chromosome 7 that was linked with the lesion mimic phenotype. High-resolution genetic mapping
using 950 F3 and 3,821 F4 plants of NF4050-8 × CO39 delimited a 35 kb region flanked by NBARC1 (5.262 Mb) and RM8262 (5.297 Mb), which contained 6
ORFs; 3 of them were ‘resistance gene related’ with typical NBS–LRR signatures. One of them harbored a NB–ARC domain, which
had been previously demonstrated to be associated with cell death in animals. Microarray analysis of NF4050-8 revealed significant
up-regulation of numerous defense/pathogenesis-related genes and down-regulation of heme peroxidase genes. Real-time PCR analysis
of WRKY45 and PR1b genes suggested possible constitutive activation of a defense signaling pathway downstream of salicylic
acid but independent of NH1 in these mutant lines of rice. 相似文献
17.
Moderately thermophilic methanotrophs Methylocaldum szegediense O-12 and Methylococcus
capsulatus Bath exhibit activities of antioxidant protection enzymes: glutathione peroxidase, superoxide dismutase, and cytochrome c peroxidase. The cells of methanotrophs grown at optimal temperatures (57 or 45°C, respectively) produce reactive oxygen species
more actively than those grown at suboptimal temperatures, and exhibit higher activities of the membrane-associated cytochrome
c peroxidase. Glutathione, glutathione peroxidase, and glucose-6-phosphate dehydrogenase levels in Md. szegediense O-12 increased in response to lowering of the cultivation temperature. By contrast, glutathione accumulation in cells of
Mc. capsulatus Bath and the activity of glutathione peroxidase at a suboptimal temperature (29°C) were lower than at the optimal one. The
role of the multilevel system of antioxidant protection in the adaptation of methanotrophs to temperature fluctuations is
discussed. 相似文献
18.
Using 3′-RACE and 5′-RACE, we have cloned and sequenced the genomic gene and complete cDNA encoding l-glutamine d-fructose 6-phosphate amidotransferase (GFAT) from the edible straw mushroom, Volvariella volvacea. Gfat contains five introns, and encodes a predicted protein of 697 amino acids that is homologous to other reported GFAT sequences.
Southern hybridization indicated that a single gfat gene locus exists in the V. volvacea genome. Recombinant native V. volvacea GFAT enzyme, over-expressed using Escherichia coli and partially purified, had an estimated molecular mass of 306 kDa and consisted of four equal-sized subunits of 77 kD. Reciprocal
plots revealed K
m values of 0.55 and 0.75 mM for fructose 6-phosphate and l-glutamine, respectively. V. volvacea GFAT activity was inhibited by the end-product of the hexosamine pathway, UDP-GlcNAc, and by the glutamine analogues N
3-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid and 2-amino-2-deoxy-d-glucitol-6-phosphate. 相似文献
19.
To understand the physiological functions of thermostable fructose-1,6-bisphosphatase (TNA1-Fbp) from Thermococcus onnurineus NA1, its recombinant enzyme was overexpressed in Escherichia coli, purified, and the enzymatic properties were characterized. The enzyme showed maximal activity for fructose-1,6-bisphosphate
at 95°C and pH 8.0 with a half-life (t
1/2) of about 8 h. TNA1-Fbp had broad substrate specificities for fructose-1,6-bisphosphate and its analogues including fructose-1-phosphate,
glucose-1-phosphate, and phosphoenolpyruvate. In addition, its enzyme activity was increased five-fold by addition of 1 mM
Mg2+, while Li+ did not enhance enzymatic activity. TNA1-Fbp activity was inhibited by ATP, ADP, and phosphoenolpyruvate, but AMP up to 100
mM did not have any effect. TNA1-Fbp is currently defined as a class V fructose-1,6-bisphosphatase (FBPase) because it is
very similar to FBPase of Thermococcus kodakaraensis KOD1 based on sequence homology. However, this enzyme shows a different range of substrate specificities. These results suggest
that TNA1-Fbp can establish new criterion for class V FBPases. 相似文献
20.
Bacillus
thuringiensis subsp. kurstaki BUPM255 secretes a chitobiosidase Chi255 having an expected molecular weight of 70.665 kDa. When the corresponding gene,
chi255, was expressed in E. coli, the active form, extracted from the periplasmic fraction of E. coli/pBADchi255, was of about 54 kDa, which suggested that Chi255 was excessively degraded by the action of E. coli proteases. Therefore, in vitro progressive C-terminal Chi255 deleted derivatives were constructed in order to study their
stability and their activity in E. coli. Interestingly, when the chitin binding domain (CBD) was deleted from Chi255, an active form (Chi2555Δ5) of expected size
of about 60 kDa was extracted from the E. coli periplasmic fraction, without the observation of any proteolytic degradation. Compared to Chi255, Chi255Δ5 exhibited a higher
chitinase activity on colloidal chitin. Both of the enzymes exhibit activities at broad pH and temperature ranges with maximal
enzyme activities at pH 5 and pH 6 and at temperatures 50°C and 40°C, respectively for Chi255 and Chi255Δ5. Thus, it was concluded
that the C-terminal deletion of Chi255 CBD might be a nice tool for avoiding the excessive chitinase degradation, observed
in the native chitinase, and for improving its activity. 相似文献