Isolation and properties of a constitutive D-xylulokinase from a novel thermophilic Saccharococcus caldoxylosilyticus DSM 12041 (ATCC 700356 (original) (raw)
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Journal of Bacteriology, 1996
JW/SL-YS 489. optimum from Thermoanaerobacterium strain xylose (glucose) isomerase with an acidic pH Purification and cloning of a thermostable http://jb.asm.org/content/178/20/5938 Updated information and services can be found at: These include: CONTENT ALERTS more» cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new articles http://journals.asm.org/site/misc/reprints.xhtml Information about commercial reprint orders: http://journals.asm.org/site/subscriptions/ To subscribe to to another ASM Journal go to: on March 24, 2013 by guest
Microbiology
The nucleotide sequence of the complete xynA gene, encoding a novel multidomain xylanase XynA of 'Caldibacillus cellulovorans', was determined by genomic-walking PCR. The putative XynA comprises an N-terminal domain (D1), recently identified as a xylan-binding domain (XBD), homologous to non-catalytic thermostabilizing domains from other xylanases. D1 is followed by a xylanase catalytic domain (D2) homologous to family 10 glycosyl hydrolases. Downstream of this domain two cellulose-binding domains (CBD), D3 and D4, were found linked via proline-threonine (PT)-rich peptides. Both CBDs showed sequence similarity to family IIIb CBDs. Upstream of xynA an incomplete open reading frame was identified, encoding a putative C-terminal CBD homologous to family IIIb CBDs. Two expression plasmids encoding the N-terminal XBD plus the catalytic domain (XynAd1/2) and the xylanase catalytic domain alone (XynAd2) were constructed and the biochemical properties of the recombinant enzymes comp...
1994
An alkaliphilic, thermophilic BaciUlus sp. (NCIM 59) produced extracellular xylose isomerase at pH 10 and 50°C by using xylose or wheat bran as the carbon source. The distribution of xylose isomerase as a function of growth in comparison with distributions of extraand intracellular marker enzymes such as xylanase and 0-galactosidase revealed that xylose isomerase was truly secreted as an extracellular enzyme and was not released because of sporulation or lysis. The enzyme was purified to homogeneity by ammonium sulfate precipitation followed by gel filtration, preparative polyacrylamide gel electrophoresis, and ion-exchange chromatography. The molecular weight of xylose isomerase was estimated to be 160,000 by gel filtration and 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating the presence of three subunits. The enzyme is most active at pH 8.0 and with incubation at 85°C for 20 min. Divalent metal ions Mg2+, Co2+, and Mn2+ were required for maximum act...
Journal of Bioscience and Bioengineering, 2009
A recombinant β-glucosidase from Caldicellulosiruptor saccharolyticus DSM 8903 with a specific activity of 13 U/mg was purified by heat treatment and His-Trap affinity chromatography and identified as a single 54 kDa band on SDS-PAGE. The molecular mass of the native enzyme was 108 kDa as a dimer by gel filtration. β-Glucosidase showed optimum activity at pH 5.5 and 70°C for p-nitrophenyl (pNP)-β-D-glucopyranoside. The half-lives of the enzyme at 60, 70, and 80°C were 250, 24.3, and 0.4 h, respectively. The enzyme exhibited catalytic efficiency and specific activity for pNP-β-D-fucopyranoside, pNP-β-D-glucopyranoside, and pNP-β-D-galactopyranoside in decreasing order among aryl-β-glycosides, but not for aryl-α-glycosides. Cello-oligosaccharides from n = 2 to 5 as substrates using 4 mM each sugar and 3 U/mg of enzyme were completely hydrolyzed to glucose at 70°C within 16 h. Crown Cellulose, which is an unbranched glucose polymer composed of Dglucose units linked by a 1,4-β-D-glucosidic bond, is the most abundant renewable resource on the earth. Renewable alternative energy has recently attracted much attention because of the shortage of fossil fuels, emission of greenhouse gasses and air pollution caused by incomplete combustion of fossil fuels (1). The hydrolysis of cellulose to glucose is the most important process in the production of bioethanol, an important alternative energy resource.
Microbiology, 1998
A large cellulolytic enzyme (CelA) with the ability to hydrolyse microcrystalline cellulose was isolated from the extremely thermophilic, cellulolytic bacterium 'Anaerocellum thermophilum I . Full-length CelA and a truncated enzyme species designated CelA were purified to homogeneity from culture supernatants. CelA has an apparent molecular mass of 230 kDa. The enzyme exhibited significant activity towards Avicel and was most active towards soluble substrates such as CM-cellulose (CMC) and p-glucan. Maximal activity was observed between pH values of 5 and 6 and temperatures of 95 "C (CM-cellulase) and 85 "C (Avicelase). Cellobiose, glucose and minor amounts of cellotriose were observed as end-products of Avicel degradation. The CelAencoding gene was isolated from genomic DNA of 'A. thermophilum by PCR and the nucleotide sequence was determined. The celA gene encodes a protein of 1711 amino acids (190 kDa) starting with the sequence found at the Nterminus of CelA purified from 'A. thermophilum I . Sequence analysis revealed a multidomain structure consisting of two distinct catalytic domains homologous to glycosyl hydrolase families 9 and 48 and three domains homologous to family 111 cellulose-binding domain linked by Pro-Thr-Ser-rich regions. The enzyme is most closely related to CelA of Caldicellulosiruptor sacchamlyticus (sequence identities of 96 and 97% were found for the N-and C-terminal catalytic domains, respectively). Endoglucanase CelZ of Clostridium stercorarium shows 70.4% sequence identity to the N-terminal family 9 domain and exoglucanase CelY from the same organism has 69.2% amino acid identity with the C-terminal family 48 domain. Consistent with this similarity on the primary structure level, the 90 kDa truncated derivative CelA' containing the N-terminal half of CelA exhibited endoglucanase activity and bound t o microcrystalline cellulose. Due to the significantly enhanced Avicelase activity of full-length CelA, exoglucanase activity may be ascribed to the C-terminal family 48 catalytic domain.
Cloning, purification, and characterization of xylose isomerase from Thermotoga naphthophila RKU-10
Journal of basic microbiology, 2016
A 1.3 kb xyl-A gene encoding xylose isomerase from a hyperthermophilic eubacterium Thermotoga naphthophila RKU-10 (TnapXI) was cloned and over-expressed in Escherichia coli to produce the enzyme in mesophilic conditions that work at high temperature. The enzyme was concentrated by lyophilization and purified by heat treatment, fractional precipitation, and UNOsphere Q anion-exchange column chromatography to homogeneity level. The apparent molecular mass was estimated by SDS-PAGE to be 49.5 kDa. The active enzyme showed a clear zone on Native-PAGE when stained with 2, 3, 5-triphenyltetrazolium chloride. The optimum temperature and pH for D-glucose to D-fructose isomerization were 98 °C and 7.0, respectively. Xylose isomerase retains 85% of its activity at 50 °C (t1/2 1732 min) for 4 h and 32.5% at 90 °C (t1/2 58 min) for 2 h. It retains 90-95% of its activity at pH 6.5-7.5 for 30 min. The enzyme was highly activated (350%) with the addition of 0.5 mM Co(2+) and to a lesser extent abo...
Applied and environmental microbiology, 2014
Thermoanaerobacterium saccharolyticum, a Gram-positive thermophilic anaerobic bacterium, grows robustly on insoluble hemicellulose, which requires a specialized suite of secreted and transmembrane proteins. We report here the characterization of proteins secreted by this organism. Cultures were grown on hemicellulose, glucose, xylose, starch, and xylan in pH-controlled bioreactors, and samples were analyzed via spotted microarrays and liquid chromatography-mass spectrometry. Key hydrolases and transporters employed by T. saccharolyticum for growth on hemicellulose were, for the most part, hitherto uncharacterized and existed in two clusters (Tsac_1445 through Tsac_1464 for xylan/xylose and Tsac_1344 through Tsac_1349 for starch). A phosphotransferase system subunit, Tsac_0032, also appeared to be exclusive to growth on glucose. Previously identified hydrolases that showed strong conditional expression changes included XynA (Tsac_1459), XynC (Tsac_0897), and a pullulanase, Apu (Tsac_...
Glucose (xylose) isomerase production from thermotolerant and thermophilic bacteria
African Journal of Biotechnology, 2012
The latter is used on a wide scale in the production of the high fructose corn syrup (HFCS) from corn starch. The great need of a thermostable GI, which is still active at higher temperatures (up to 90°C), opened the door to screen more microorganisms for the production of a more efficient industrial enzyme. Whole cells of 50 thermotole-rant/thermophilic bacterial isolates were used to evaluate their potential to produce GI when they were grown in broth medium (pH 7.0) containing D-xylose as a sole carbon source. Klebsiella and Pseudomonas showed the highest enzyme activity of 0.7; Bacillus came second with an activity of 0.3; while Acetobacter and Staphylococcus showed moderate activity of 0.3 for both, and Clostridium, Corynebacterium and Enterobacter showed the lowest enzyme activity of 0.2 each. The results reveal the need for optimizing the conditions for enhancing the production of the enzyme from Klebsiella and Pseudomonas isolates.
Biochemistry and Cell Biology, 2001
A thermostable D-xylose-glucose isomerase was isolated from the thermophilic strain Streptomyces thermovulgaris 127, var. 7-86, as a result of mutagenic treatment by gamma-irradiation of the parent strain, by precipitation and sequential chromatographies on DEAE-Sephadex A50, TSK-gel, FPLC-Mono Q/HR, and Superose 12 columns. The N-terminal amino acid sequence and amino acid analysis shows 73-92% homology with xylose-glucose isomerases from other sources. The native molecular mass, determined by gel filtration on a Superose 12 column, is 180 kDa, and 44.6 and 45 kDa were calculated, based on amino acid analysis and 10% SDS-PAGE, respectively. Both, the activity and stability of the enzyme were investigated toward pH, temperature, and denaturation with guanidine hydrochloride. The enzyme activity showed a clear pH optimum between pH 7.2 and 9.0 with D-glucose and 7.4 and 8.3 with D-xylose as substrates, respectively. The enzyme is active up to 60-85 degrees C at pH 7.0, using D-glucose, and up to 50-60 degrees C at pH 7.6, using D-xylose as substrates. The activation energy (Ea = 46 kJ x mol(-1)) and the critical temperature (Tc = 60 degrees C) were determined by fluorescence spectroscopy. Tc is in close coincidence with the melting temperature of denaturation (Tm = 59 degrees C), determined by circular dichroism (CD) spectroscopy. The free energy of stabilization in water after denaturation with Gdn.HCl was calculated to be 12 k x mol(-1). The specific activity (km values) for D-xylose-glucose isomerase at 70 degrees C toward different substrates, D-xylose, D-glucose, and D-ribose, were determined to be 4.4, 55.5, and 13.3 mM, respectively.