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Caldicellulosiruptor lactoaceticus 6A, an anaerobic and extremely thermophilic bacterium, uses natural xylan as carbon source. The encoded genes of C. lactoaceticus 6A for glycoside hydrolase (GH) provide a platform for xylan degradation. The GH family 10 xylanase (Xyn10A) and GH67 α-glucuronidase (Agu67A) from C. lactoaceticus 6A were heterologously expressed, purified and characterized. Both Xyn10A and Agu67A are predicted as intracellular enzymes as no signal peptides identified. Xyn10A and Agu67A had molecular weight of 47.0 kDa and 80.0 kDa respectively as determined by SDS-PAGE, while both appeared as homodimer when analyzed by gel filtration. Xyn10A displayed the highest activity at 80°C and pH 6.5, as 75°C and pH 6.5 for Agu67A. Xyn10A had good stability at 75°C, 80°C, and pH 4.5–8.5, respectively, and was sensitive to various metal ions and reagents. Xyn10A possessed hydrolytic activity towards xylo-oligosaccharides (XOs) and beechwood xylan. At optimum conditions, the specific activity of Xyn10A was 44.6 IU/mg with beechwood xylan as substrate, and liberated branched XOs, xylobiose, and xylose. Agu67A was active on branched XOs with methyl-glucuronic acids (MeGlcA) sub-chains, and primarily generated XOs equivalents and MeGlcA. The specific activity of Agu67A was 1.3 IU/mg with aldobiouronic acid as substrate. The synergistic action of Xyn10A and Agu67A was observed with MeGlcA branched XOs and xylan as substrates, both backbone and branched chain of substrates were degraded, and liberated xylose, xylobiose, and MeGlcA. The synergism of Xyn10A and Agu67A provided not only a thermophilic method for natural xylan degradation, but also insight into the mechanisms for xylan utilization of C. lactoaceticus.
Citation: Jia X, Mi S, Wang J, Qiao W, Peng X, Han Y (2014) Insight into Glycoside Hydrolases for Debranched Xylan Degradation from Extremely Thermophilic Bacterium Caldicellulosiruptor lactoaceticus. PLoS ONE 9(9): e106482. https://doi.org/10.1371/journal.pone.0106482
Editor: Jose M. Sanchez-Ruiz, Universidad de Granada, Spain
Received: March 17, 2014; Accepted: August 5, 2014; Published: September 3, 2014
Copyright: © 2014 Jia et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper.
Funding: This work was funded by the National High Technology Research and Development Program of China (863 Project, No. 2014AA021905), and 100 Talents Program of Institute of Process Engineering, Chinese Academy of Sciences. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Xylan, the main hemicellulose component of plant cell wall, is a heteropolymeric polysaccharide consisted mostly of linear backbone of β-1,4-D-xylopyranoside units which are commonly decorated with 4-O-methyl-glucuronyl, acetyl, and arabinofuranosyl substituents , . In a general sense, the efficient depolymerization of xylan to monosaccharides requires the synergistic function of enzyme system, including endo-β-1,4-xylanase (EC 184.108.40.206), β-xylosidase (EC 220.127.116.11), α-L-arabinofuranosidase (EC 18.104.22.168), α-glucuronidase (EC 22.214.171.124), and acetyl xylan esterase (EC 126.96.36.199) , . Endo-β-1,4-xylanases catalyze the random cleavage of the internal β-1,4-glycosidic linkage between xylose residues in xylan polymer, and have been classified into glycoside hydrolase (GH) families 5, 7, 8, 10, 11 and 43 . The α-glucuronidases (EC 188.8.131.52) cleave the α-1,2-linkage between 4-O-methylglucuronic acid (4-O- MeGlcA) and XOs . Unlike xylanases, α-glucuronidases cluster into either GH family 67 or family 115 based on amino acid sequences , . To date, a large number of xylanolytic enzymes have been identified from a variety of microbial sources (CAZy; http://www.cazy.org/). Thermostable hemicelluloytic enzymes, with a number of advantages over mesophilic enzymes, have thus gained worldwide industrial and biotechnological interest.
Caldicellulosiruptor lactoaceticus 6A, an anaerobic and extremely thermophilic, cellulose and hemicelluloses degrading bacterium, was isolated from an alkaline hot spring in Iceland . It grows efficiently at temperature between 50 and 78°C and pH 5.8–8.2 within optimum near 68°C and 7.0, respectively. Besides xylan, it utilizes cellulose, starch, pectin, cellobiose, xylose, maltose and lactose as carbon sources. Thus its ability to express highly thermostable carbohydrate-active enzymes makes it an ideal candidate for studying extreme temperature biomass conversion. Currently, the complete genome sequence was available for this species , providing new approach to investigate the mechanisms of polysaccharides degradation. The genes encoding thermophilic GHs of C. lactoaceticus 6A provide a platform for degrading natural polysaccharides at higher temperature.
In present study, thermophilic degradation of MeGlcA decorated xylan using C. lactoaceticus 6A GHs was studied. The genes of endo-β-1,4-xylanase Xyn10A and α-glucuronidase Agu67A were identified and cloned in the genome of C. lactoaceticus 6A, and heterologously expressed, purified and biochemically characterized. The synergistically hydrolytic properties of the two enzymes on MeGlcA decorated xylan and XOs were also investigated.
Results and Discussion
Gene cloning and sequence analysis of Xyn10A and Agu67A
Through C. lactoaceticus genome sequence analysis, Calla_1331 and Calla_1259 were annotated as putative GH10 endo-β-1,4-xylanase (Xyn10A) and α-glucuronidase (Agu67A), respectively. Both Xyn10A and Agu67A had no signal peptide, indicating they are intracellular enzymes. Xyn10A only contained a GH10 motif (Figure 1A), the calculated molecular weight (Mw) and deduced pI of Xyn10A were 46,965 Da and 5.65, respectively. The encoding gene xyn10A was amplified using C. lactoaceticus DNA as template. In genes screening analysis in genome DNA of C. lactoaceticus, no other xylan degradation genes except a putative polysaccharide deacetylase upstream of xyn10A was found (Figure 1B). In amino acids sequence blast analysis, Xyn10A showed high identity with other predicted xylanases from Caldicellulosiruptor sp. In addition to Caldicellulosiruptor, Xyn10A exhibited the highest similarity (79.7%) and identity (65.1%) with Thermoanaerobacterium saccharolyticum GH10 xylanase [GenBank: ADQ57411.2], and similarity (71.5%) and identity (57.8%) with GH10 xylanase from Alicyclobacillus sp. A4 [GenBank: ADK91076.1]. However, Xyn10A showed much lower similarity (27.0–41.8%) and identity (14.4–23.5%) with other characterized thermophilic GH10 xylanases, indicating that Xyn10A was a novel thermophilic GH10 xylanase. The amino acid residues Glu161 and Glu266, predicted acting as proton donor and catalytic nucleophile, were also conserved in Xyn10A , . Phylogenetic analysis of defined thermophilic GH10 xylanases revealed the differentiation of the homology of thermophilic GH10 xylanases into three groups (Figure 2A). Xyn10A had the closest relationship with T. saccharolyticum xylanase, and then clustered with Alicyclobacillus sp. xylanase as a single evolutionary clade which was distinct from other thermophilic bacteria and fungi. On the contrary, Xyn10A was far isolated from other reported Caldicellulosiruptor sp. xylanase indicating the distinct interspecific-diversity of xylanases.
A. Modular organization for Xyn10A and Agu67A. NCBI conserved domains database and SignalP 4.1 Server were used for analysis. B. Genomic organization for Xyn10A. Calla_1331 was annotated as a putative endo-β-1,4-xylanase, and upstream of the xylanase is the gene predicted to encode a polysaccharide deacetylase. C. Genomic organization for Agu67A. Calla_1259 was annotated as a putative α-glucuronidase. Gene annotations were performed using the rapid annotations using subsystems technology (RAST) Server.
A. Phylogenetic tree of xylanases in different organisms. B. Phylogenetic tree of α-glucuronidases in different organisms. Trees were constructed using MEGA 5.05 by the Neighbor-Joining method with 1000 bootstrap replicates, and Genbank accession numbers of each protein sequence were given at the end of each species name.
The C. lactoaceticus agu67A gene was predicted to encode a 693-amino-acid GH67 α-glucuronidase with Mw of 80,343 Da and a pI of 8.31. Agu67A had a conserved GH67 N-terminus (14–136 amino acids), a GH67 middle domain (146–467 amino acids), and a GH67 C-terminus (468–692 amino acids) motifs without signal peptide (Figure 1A). Genomic organization analysis did not find any xylanolytic genes around the agu67A gene (Figure 1C). Agu67A shared the highest similarity (79.9%) and identity (64.4%) to that of Thermotoga maritima MSB8 [GenBank: AHD18175.1], and had higher similarity and identity with other α-glucuronidase from Caldanaerobius polysaccharolyticus [GenBank: AFM44650.1] (77.1%, 61.4%), Thermoanaerobacterium xylanolyticum LX-11 [GenBank: AEF17768.1] (73.9%, 58.4%), Geobacillus stearothermophilus [GenBank: ABI49940.1] (73.2%, 58.2%), Bacillus halodurans C-125 [GenBank: BAB04780] (72.0%, 56.0%), and Paenibacillus sp. Strain JDR-2 [GenBank: ABV90485.1] (71.0%, 53.5%), respectively. Analysis of Neighbor-Joining (N-J) tree revealed Agu67A was located in the group of thermophilic bacteria (Figure 2B). Agu67A was closely related to T. maritima MSB8 α-glucuronidase, and then sub-clustered with C. polysaccharolyticus α-glucuronidase with reliable bootstrap values.
Expression and purification of Xyn10A and Agu67A
For functional analysis of the recombinant enzymes, both xyn10A and agu67A were sub-cloned into pET-28b vector and expressed in Escherichia coli BL21 (DE3). Proteins were heat-treated at 65°C for 30 min followed by Ni-affinity chromatography, and further purified through Superdex 200 gel filtration. The recombinant Xyn10A displayed as a single band with Mw of about 47.0 kDa by SDS-PAGE analysis, which was in agreement with the predicted Mw based on the amino acid sequence (Figure 3A). Size exclusion chromatography revealed that Xyn10A eluted as a single peak with Mw of 84.0±1.2 kDa, suggesting that Xyn10A existed as a homodimer in solution (Figure 3B). Other xylanases such as Syncephalastrum racemosum Cohn. (58.0 kDa)  and Trichoderma reesei (90.0 kDa)  are also homodimers, whereas the majorities are identified as monomeric proteins in solution, for instance, Glaciecola mesophila KMM 241 (43.0 kDa) , Cohnella laeviribosi HY-21 (42.0 kDa) , B.halodurans TSEV1 (40.0 kDa) , and Remersonia thermophila CBS 540.69 (42.0 kDa) .
A. SDS-PAGE analysis of purified Xyn10A. B. Quaternary structure analysis of Xyn10A by gel filtration chromatography. C. SDS-PAGE analysis of purified Agu67A. D. Quaternary structure analysis of Agu67A by gel filtration chromatography. Both Xyn10A and Agu67A were purified by Ni-affinity chromatography, followed by Superdex 200 gel filtration.
SDS-PAGE analysis of the purified Agu67A indicated a single protein at approximately 80.0 kDa, which was also consistent with the theoretical Mw (Figure 3C). However, as shown in Figure 3D, Agu67A appeared as a single peak with Mw of 163.7±1.4 kDa, suggesting that Agu67A existed as a homodimer in solution (Figure 3D). It has already been proved that most bacterial α-glucuronidases, including C. polysaccharolyticus (158.0 kDa) , Bacillus stearothermophilus No. 236 (161.0 kDa)  and B.stearothermophilus T-6 (150.0 kDa) , consist of two subunits with Mw of around 75.0 kDa per subunit. In contrast, many fungal α-glucuronidases function as monomeric proteins with a higher Mw of 100.0 kDa per subunit due to the glycosylation .
Substrate specificity of Xyn10A and Agu67A
Both Xyn10A and Agu67A were observed with clearing hydrolytic activity zones on the agar plates containing beechwood xylan or XOs (Figure 4A). In contrast, no clearing zones were found in the case of plates with LBG, soluble starch, Avicel, and CMC as substrates. Reducing sugars were detectable after incubation with beechwood xylan or XOs for 40 min with the Congo red assay (Figure 4B). Both Xyn10A and Agu67A displayed the highest activity with XOs as substrate. These results, therefore, suggested that Xyn10A and Agu67A possessed xylan degrading activity, but not mannanase, amylase, or cellulase activity.
A. Identifications of Xyn10A and Agu67A activity on agar plate. The capacity of enzymes was assessed by incubating each protein on agar plates infused with different substrates at 60°C for 12 h, followed by staining with Congo red. B. The activity of Xyn10A and Agu67A on different substrates with produced reducing sugar assay. Both Xyn10A and Agu67A (0.5 µM each, final concentration) were incubated with different substrates at 80°C and pH 6.5 for 40 min. 1, beechwood xylan; 2, xylo-oligosaccharides; 3, locust bean gum; 4, soluble starch; 5, Avicel; 6, carboxymethyl cellulose. All of the tested substrates were at a fixed concentration of 1.0% (w/v).
It was reported that most of the α-glucuronidases are only active on MeGlcA linked short xylo-oligomers, while few studies have confirmed its high activity against polymeric substrates , . The observable activity of Agu67A detected with beechwood xylan might mainly because of the existing of small amounts of aldobiouronic and aldotriouronic acids in the polysaccharide mixtures . While the composition and structure of XOs depend on the xylan source and production process, enzymatic hydrolysis would produce branched hetero-xylooligosaccharides decorated with MeGlcA . Typically, wood xylan exists as O-acetyl-4-O-methylglucuronoxylan in hardwoods with higher degree of polymerization (DP, 150–200), and as arabino-4-O-methylglucuronoxylan in softwoods .
Biochemical characterization of Xyn10A and Agu67A
Xyn10A displayed the highest activity at 80°C and maintained more than 50% of its activity at 65–85°C (Figure 5A). Moreover, Xyn10A was incubated at 75, 80 and 85°C in the absence of substrate for thermostability determination. Xyn10A was found to be extremely thermostable with over 90% residual activity after incubation at 75°C for 6 h, and still retained approximately 60% activity after incubation at 80°C for 6 h, while lost rapidly of its activity after incubated after incubation at 85°C for 30 min (Figure 5C). Interestingly, Xyn10A was most active at pH 6.5 and retained over 55% activity at pH 6.0–8.5, suggesting it's active in neutral and weak alkaline solution (Figure 5B). Furthermore, for pH stability assay, pure enzyme was pre-incubated in pH 4.0–8.5 buffers for 10 h without substrates at room temperature. Xyn10A exhibited good stability at pH 4.5–8.5, while 45% percent of the activity was reduced after incubation at pH 4.0 for 10 h (Figure 5D). The specific activity of Xyn10A was 44.6 IU/mg with beechwood xylan as substrate at optimum conditions.
A. Temperature profile of Xyn10A. Xylanase activity determination was performed in a temperature range of 40–95°C at pH 6.0 for 3 min. B. pH profile of Xyn10A. Xylanase activity assay was carried out by a 3 min incubation using phosphate-citrate buffers (pH 4.0–8.5) at 80°C. C. Thermostability profile of Xyn10A. The purified Xyn10A was incubated in pH 8.5 buffer at 75, 80 and 85°C, respectively for 0.5, 1, 2, 3, 4 and 6 h, and residual activity was detected under optimal conditions. D. pH stability profile of Xyn10A. The purified Xyn10A was pre-incubated in pH 4.0–8.5 buffers at room temperature for 10 h, and then the residual activity was measured under optimal conditions. E. Temperature profile of Agu67A. The α-glucuronidase activity determination was performed in a temperature range of 40-95°C at pH 6.5 for 5 min. F. pH profile of Agu67A. The α-glucuronidase activity assay was carried out by a 5 min incubation using phosphate-citrate buffers (pH 4.0–8.5) at 75°C. The maximum activity was defined as 100% and values shown were the means of three replicates.
Certain properties of Xyn10A were compared with some other thermophilic xylanases from bacteria and fungi as shown in Table 1. Xyn10A showed good catalytic activity over a broad temperature and pH range. The properties of Xyn10A indicated that enzyme activity remained more stable at temperature below 85°C and pH range 4.5–8.5.
In addition, the influences of various additives including metal ions and reagents on Xyn10A activity were also investigated (Table 2). Most of ions except 1 mM Fe3+ and Zn2+ showed obviously effects on Xyn10A activity. It was strongly activated by 1 mM or 5 mM NH4+, Na+, K+, Mg2+, Ni2+; 1 mM Fe2+; and 5 mM Ca2+. Furthermore, the addition of 1 mM Ca2+ exhibited a moderate elevation to the activity. On the contrary, other concentrations of ions, including 1 mM or 5 mM Co2+, Mn2+, Cu2+, and 5 mM Fe2+, Fe3+, Zn2+, significantly inhibited the xylanase activity. Reductant, detergents, and metal chelator also had influence on the enzyme activity. 1 mM or 5 mM DTT, 0.1% or 0.5% Triton X-100, 5 mM EDTA, along with 0.1% β-ME markedly as well as 1 mM EDTA slightly increased the activity. However, the activity was strongly interrupted by 0.1% or 0.5% SDS and 0.5% β-ME. In addition, both 5% and 10% glycerol showed the highest positive impact on xylanase catalytic activity. But it was noteworthy that the activity was almost completely inhibited by three tested organic reagents (ethanol, isopropanol, and butanol).
In the previous reports, a large number of xylanases were also affected to some extent by these metals which suggested its possible function as a cofactor for maintaining structure stability and aiding enzyme-substrate reaction . Crystal structure of Bacillus sp. NG-27 extracellular GH10 endo-xylanase revealed a metal binding site located at the C-terminal end of the catalytic domain . An Mg2+-binding site was seen and the xylanase activity had a concentration-dependent manner with the presence of Mg2+. Some metals, such as Cu2+ and Zn2+ had strong affinities toward sulphydryl groups hence strongly inhibited the activity . Notably, Xyn10A activity was enhanced by disulfide-reducing agents (0.1% β-ME, 1 mM and 5 mM DTT), indicating that the presence of the thiol group was essential but not absolutely critical for its activity although 0.5% β-ME played an inverse role. Owing to the strong protein denaturation, little xylanases had high SDS resistance even at low concentration of SDS . In the study of Thermotoga thermarum xylanase, the enzyme activity was found to be greatly stimulated by Ca2+, Mn2+ and Co2+ . The T. saccharolyticum NTOU1 XynFCB activity was also enhanced by the metal ions NH4+, K+, Na+, and Ca2+, while strongly inhibited by Cu2+ and Zn2+ . The presence of 1 mM Mn2+, β-ME and EDTA enhanced the Alicyclobacillus sp. A4 XynA4-2 activity, whereas 1 mM Zn2+, Cu2+, and SDS resulted in severe or complete inhibition . Likewise, the activity of Actinomadura sp. strain Cpt20 xylanase was also found to be enhanced by Ca2+ and Co2+, but was inhibited by Fe2+, Zn2+, Cu2+ and Mg2+, yet almost unaffected by K+, Na+ and Mn2+ . In contrast to the results obtained, ethanol, isopropanol and butanol had no effect on the Streptomyces rameus L2001 xylanase activity .
Optimal temperature and pH for Agu67A were 75°C (Figure 5E) and 6.5 (Figure 5F), respectively. The specific activity of Agu67A was 1.3 IU/mg with XOs containing aldobiouronic acid as substrate by analyzing the released xylose under optimal conditions. The C.polysaccharolyticus Agu67A was most active at 60°C and pH 5.5 with specific activity reaching 10.8 IU/mg and 11.6 IU/mg for (4-O-methyl-α-D-glucurono)-D-xylan and birchwood xylan, respectively . Similarly, the temperature and pH profiles of B. stearothermophilus T-6 α-glucuronidase were 65°C and pH 5.5–6.0, respectively .
Hydrolysis of beechwood xylan and XOs by Xyn10A and Agu67A
In order to assay the relationship between Xyn10A capacity and substrate abundance, different concentrations of beechwood xylan were hydrolyzed by constant amount of enzyme. Xylose, xylobiose, xylotriose, xylotetraose, as well as higher polymeric XOs, accumulated with the increasing of substrates (Figure 6A). Meanwhile, reducing ends also elevated quickly with more substrates addition, the results were also found in TLC analysis (Figure 6B).
A. TLC analysis of each hydrolysis products. B. The produced reducing sugar assay in each hydrolysis products. 0.1–2.0% (w/v) beechwood xylan was incubated with Xyn10A (0.5 µM, final concentration) at 80°C and pH 6.5 for 4 hours.
Moreover, to explore their effects on XOs hydrolysis, the two enzymes were incubated separately or combination with XOs, and the products were analyzed. TLC analysis revealed the efficient break down of XOs into xylose and xylobiose in the presence of Xyn10A or two combined enzymes (Figure 7A). As aldobiouronic acid existed in XOs, a small amount of xylose was visible when XOs incubated with Agu67A. As a result, the release of reducing ends by Xyn10A, Agu67A, and Xyn10A&Agu67A were 26.0 mM, 5.1 mM, and 33.5 mM, respectively (Figure 7B). The synergistic activity of Xyn10A&Agu67A was also verified by results of HPLC analysis (data not shown). Xyn10A primarily degraded XOs into xylobiose and xylose, while Agu67A specifically digested aldobiouronic acid to MeGlcA and xylose. As a result, a slightly synergistic effect of xylose and xylobiose equivalents was seen when they coordinately acted together.
A. TLC analysis of the XOs hydrolysis products. The detectable xylose produced by Agu67A was marked with a box. B. Produced reducing sugar assay of the XOs hydrolysis products. C. TLC analysis of the beechwood xylan hydrolysis products after different incubation times. D. Produced reducing sugar assay of the beechwood xylan hydrolysis products. The differences of hydrolysis products after 1 hour were marked with a box. E. HPLC analysis of the beechwood xylan hydrolysis products of 2 hours. F. Details of the HPLC analysis (retention time 13.5–14.5 min). XOs hydrolysis was performed by incubating single or mixed enzyme (2.0 µM each, final concentration) with 2.0% (w/v) XOs at 80°C and pH 6.5 for 4 hours. Beechwood xylan hydrolysis was conducted by incubating Xyn10A (1.75 µM, final concentration), or Agu67A (0.85 µM, final concentration), or Xyn10A (1.75 µM, final concentration) and Agu67A (0.85 µM, final concentration) mixture with 1.0% (w/v) beechwood xylan at 80°C and pH 6.5 for different times (0, 0.25, 1, 2, 6, 12 hours). Xylose (X1), xylobiose (X2), xylotriose (X3), and xylotetraose (X4) were used as standards and labeled.
Furthermore, to evaluate the synergistic activity of Xyn10A and Agu67A, single or mixed enzymes were incubated with beechwood xylan and the products of hydrolysis were analyzed accordingly. As shown by the TLC analysis (Figure 7C), Xyn10A mainly produced xylose, xylobiose, a spot of heterogeneous XOs, and short chain polymers at the beginning. However, the products of Agu67A hydrolysis were undetectable throughout 12 hours incubation. Similarly, the synergistic activity of Xyn10A and Agu67A was detected in the process. When Xyn10A and Agu67A were applied together, the major hydrolysis products were xylose and xylobiose. The reducing sugar increased along the twelve hours reactions, and XOs disappeared after 1 hour reaction. The final reducing sugars were 27.0 mM, 1.8 mM, and 29.1 mM when Xyn10A, Agu67A, and Xyn10A&Agu67A were applied respectively (Figure 7D).
In addition, to further evaluate the synergistic activity of Xyn10A and Agu67A, the products of beechwood xylan hydrolyzed for 2 h were determined by HPLC (Figure 7E). Peaks representing xylose and xylobiose were observed when Xyn10A was applied alone, while the production by Agu67A was negligible (Figure 7F). In the reaction of Xyn10A&Agu67A applied together, the produced xylose and xylobiose was increased compared with that of enzyme added separately, indicating the synergism of the two enzymes. All these results clearly showed that Xyn10A acted on both xylan polymer and XOs, and liberate a large number of xylose and xylobiose, indicating Xyn10A was active on XOs with DP ≥3. While Agu67A was mostly active on branched XOs with methyl-glucuronic acids sub-chains, and primarily generated XOs equivalents. As had been stated, the synergism of Xyn10A and Agu67A would improve the end products of xylobiose and xylose taking MeGlcA branched XOs or xylan as substrates. While due to the complex structure of beechwood xylan, its hydrolysis was even more complex than that of MeGlcA decorated XOs.
Many GH10 xylanases were detected to degrade xylan polymer into a mixture mostly of xylose and XOs with DP lower than five . The action mode of Xyn10A was in accordance with that of xylanase from T. thermarum, in which xylose and xylobiose were the major end products of beechwood xylan after 5 h hydrolysis . Similarly, the hydrolysis products of birchwood xylan by Alicyclobacillus sp. A4 XynA4-2 contained 92.7% xylose and 7.3% xylobiose . However, xylotriose and xylotetraose instead of xylose were formed largely from the hydrolytic reaction of birchwood xylan by C. laeviribosi HY-21 iXylC . Xylobiose, along with higher XOs as intermediates, clearly detected as the predominant end products released by Streptomyces sp. CS428 Xyn428 from beechwood xylan .
Given the possibility that endoxylanases might breakdown xylan into XOs substituted with MeGlcA, microorganisms form α-glucuronidase to remove these side chains hence ensuring the effectiveness of xylan degradation. Nevertheless, a majority of the α-glucuronidases only has the capacity to act on small model xylan or MeGlcA branched XOs , . Thus, the more reducing sugar detected in enzyme-cocktail treatment was related to the synergistic action of Xyn10A and Agu67A on the different parts of xylan polymer or XOs. The backbone-hydrolyzing Xyn10A liberates substituted XOs which are then degraded into monosaccharides by Agu67A and other xylanolytic enzymes. Synergism between xylanase and α-glucuronidase in hydrolysis of xylan has been investigated to some extent. In C. polysaccharolyticus, the release of either xylose or xylobiose tended to be slightly higher from birchwood xylan when incubated with two enzyme mixture than with single xylanase at 65°C . The purified α-glucuronidase of Aspergillus tubingensis observed to liberate minor amounts of MeGlcA from birchwood xylan, and the amounts of MeGlcA and short oligo-saccharides (xylobiose and xylotriose) were improved in the two enzymes combination system . The Schizophyllum commune α-glucuronidase was able to remove the MeGlcA groups from polymeric glucuronoxylan, and 76% of MeGlcA side groups were cleaved from the backbone of glucuronoxylan together with a xylanase .
C. lactoaceticus 6A is an extremely thermophlic plant biomass-degrading bacterium, with capability of utilizing xylan as carbon source . Analysis of the genome of C. lactoaceticus revealed only three genes encoding endo-β-1,4-xylanase (Calla_1331, Calla_1781, and Calla_0206). Both Calla_1781 and Calla_0206 contained one GH10 domain and two to three carbohydrate-binding modules (CBMs). Interestingly, all of the three xylanases were predicted to be intracellular based on the absence of signal peptides. Specifically, no genes encoding putative β-xylosidase were annotated throughout the genome. Hence, other xylan-specific enzymes, as well as their novel synergistic roles need to be further investigated to give a deeper understanding of the mechanisms involved in xylan deconstruction process. In previous study, Calla_1781 was also expressed in E. coli BL21 (DE3) whereas no activity was detected (data not shown). Consequently, Calla_1331 (namely Xyn10A), the first characterized xylanase without CBMs, might played a prominent role in efficient degradation of xylan.
In this study, two novel thermostable xylanolytic enzymes endo-β-1,4-xylanase Xyn10A and α-glucuronidase Agu67A from C.lactoaceticus were obtained and characterized. Xyn10A and Agu67A showed optimum temperature of 80°C and 75°C, respectively. Xyn10A also had good thermostability (75°C and 80°C for 6 h) and broad pH stability (4.5–8.5). Xyn10A could hydrolyze branched xylan and produce xylose, xylobiose, and MeGlcA decorated XOs. Agu67A was active on MeGlcA decorated XOs, and produced MeGlcA and equivalents XOs. The synergistic activity of Xyn10A and Agu67A was detected with both MeGlcA branched xylan and XOs as substrates, and produced xylose, xylobiose, and MeGlcA. The synergistic function of Xyn10A and Agu67A provided a promising way for degrading natural xylan at high temperature. The characterization of the two intracellular GHs also offered an opportunity to systematically evaluate the mechanisms for xylan utilization of C. lactoaceticus.
Materials and Methods
Strains, plasmids and chemicals
C. lactoaceticus DSM 9545 was purchased from DSMZ (Braunschweig, Germany). E. coli Top10 (TianGen, China) and plasmid pET-28b (Novagen, USA) were used for gene cloning, and E. coli BL21 (DE3) was used for protein expression. Beechwood xylan was purchased from Sigma-Aldrich (St. Luis, USA), and XOs (DP, 2–7) was a kind gift from Longlive Bio-Technology Co. (Shandong, China). D-xylose, locust bean gum (LBG), soluble starch, Avicel, carboxymethyl cellulose (CMC), and chemicals for buffer preparations were obtained from Kepujia Reagent Co. (Beijing, China). All other chemicals were of analytical grade unless otherwise stated.
Genomic DNA extraction and amplification
The genomic DNA of C. lactoaceticus 6A was extracted from 5 mL culture using TIANamp Bacteria DNA Kit (TianGen, China). Based on the whole genome of strain 6A , gene xyn10A encoding a hypothetical endo-β-1,4-xylanase [GenBank: YP_004798927.1] and gene agu67A encoding an α-glucuronidase [GenBank: YP_004798856.1] were predicted and primers were designed as follows: xyn10A-F (5′-CTAGCTAGCATGGCTAATTATGAGCATC-3′, Nhe I site underlined), xyn10A-R (5′-CCCAAGCTTTTAAAGAGTAATTTCAATAAACTTG-3′, Hind III site underlined), agu67A-F (5′-GCCGCGCGGCAGCATGATTTTATCAAGTAGCAGTAAC-3′), and agu67A-R (5′-GCGGCCGCAAGCGTTTATGGATATATCACTCTTC-3′). The PCR mixture contained genomic DNA 1 µL, forward primer 1 µL, reverse primer 1 µL, 2×Pfu PCR MasterMix (TianGen) 12.5 µL, and ddH2O 9.5 µL. PCR conditions were as follows: 94°C 5 min, 30 cycles of 94°C 30 s, 55°C 30 s and 72°C 2 min, followed by one cycle of 72°C 5 min. The target PCR products were purified with TIAN gel Midi Purification Kit (TianGen).
Construction and sequencing of the expression vector
The purified PCR products of xyn10A amplification were then digested with Nhe I and Hind III (Takara, Dalian, China) and inserted into pET-28b at the corresponding sites, obtaining the plasmid pET-28b-xyn10A. The purified PCR products of agu67A amplification were then digested with T4 DNA polymerase (Takara) and sub-cloned into pET-28b EK/LIC vector, yielding the plasmid pET-28b-agu67A. Both two plasmid were transferred into E.coli Top10 competent cells by heat shock and grown overnight at 37°C on Lysogeny Broth (LB) agar broth containing kanamycin (50 µg/mL). Positive recombinants were screened by using colony PCR and sequenced with T7 primers from both strands.
Expression and purification of Xyn10A and Agu67A
The recombinant plasmids were then extracted using TIANprep Mini Plasmid Kit (TianGen) and transformed individually into E. coli BL21 (DE3) by heat shock and grown overnight at 37°C on LB agar plates supplemented with kanamycin (50 µg/mL). Seed culture was prepared by growing one single colony separately at 37°C on a rotary shaker (220 rpm) for overnight in 5 mL LB liquid medium containing kanamycin (50 µg/mL). The pre-cultures were then diluted individually 100-fold in fresh LB with antibiotic and cultured at 37°C on a rotary shaker (220 rpm). Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM when the culture reached an optical density of 0.4-0.6 at 600 nm, and incubations were further continued for an additional 16 h at 16°C. The cells were harvested by centrifugation at 4000 rpm for 15 min at 4°C, and re-suspended in binding buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl). The cell suspensions were lysed by sonication and centrifugation at 10,000 g for 15 min at 4°C. The supernatants were heated at 65°C for 30 min and centrifuged at 10,000 g for 15 min at 4°C to remove E. coli heat labile proteins. The supernatants were loaded individually onto a His-Tag Ni-affinity resin (National Engineering Research Centre for Biotechnology, China) pre-equilibrated with binding buffer for five times. Thereafter, the columns were washed with binding buffer for three times to remove the unbound proteins. Finally, the binded target proteins were eluted from the column with elution buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 150 mM Imidazole). The purity of the target proteins were verified by sodium dodecyl sulfate-polycrymide gel electrophoresis (SDS-PAGE) as described by Laemmli . The native Mws of Xyn10A and Agu67A were analyzed by size exclusion chromatography using a 33 mL elution volume Superdex 200 exclusion column. Gel standard protein mixture, or 300 µL of samples was loaded individually onto the column pre-equilibrated with citrate buffer (50 mM sodium citrate, 150 mM NaCl, pH 6.0) at a flow rate of 0.5 mL/min using Huxi chromatographic separation system (Huxi analysis instrument factory, Co. LTD, Shanghai, China). The apparent Mws of two proteins were calculated from the calibration curve of log (Mw) vs. elution volume.
Enzyme assay and protein determination
To get initial reaction velocity for activity assay, preliminary experiments were conducted at different conditions. Appropriate amount of Xyn10A or Agu67A was incubated with excess substrates, and the hydrolysis was terminated at different time to get a linear curve of production versus reaction time. Xylanase activity was assayed by incubating 0.78 µg purified recombinant enzyme with beechwood xylan (0.5%, w/v) in 100 µL citrate buffer (pH 6.5) at 80°C for 2 min. The amount of reducing sugar generated by Xyn10A was determined by using the para-hydroxybenzoic acid hydrazide (PHBAH) method with xylose as a standard . And the activity of Agu67A was assayed by incubating 0.9 µg enzyme with MeGlcA decorated XOs (2.0 mg/mL, final concentration) in 100 µL citrate buffer (pH 6.5) at 75°C for 4 min. The released xylose was measured by high performance liquid chromatography (HPLC) using Hi-Plex Ca column (300×7.7 mm, Agilent Technologies, Tokyo, Japan) with HPLC grade water as mobile phase at 0.6 mL/min, and injection volume of 10 µL. One unit of enzyme activity was defined as the amount of enzyme required to liberate 1 µmol of xylose equivalent per minute under the standard assay conditions. The concentrations of purified proteins were determined by Bradford method using bovine serum albumin (BSA) as a standard. All of the experiments were performed in triplicate.
Substrate specificity assays of Xyn10A and Agu67A
The substrate specificity of Xyn10A and Agu67A were screened respectively with different polysaccharide substrates including beechwood xylan, XOs, LBG, soluble starch, Avicel, and CMC. All of the tested substrates were at a fixed concentration of 1.0% (w/v). Both two enzymes were checked for the ability to hydrolyze various substrates using the Congo red assay . The detection agar plates contained 1.0% (w/v) substrate and 0.8% (w/v) agar with citrate buffer (pH 6.5). Each of 10 µL enzymes was spotted onto the plates and incubated at 60°C for 12 h. After incubation, all the plates were stained with 0.1% (w/v) Congo red for 15 min and washed with 1 M NaCl for 3 times. Meanwhile, substrate specificity of the enzymes was also examined by measuring the produce of reducing sugar from those substrates at 80°C and pH 6.5 for 40 min. All the experiments were performed in triplicate.
Biochemical characterization of Xyn10A and Agu67A
For determination the temperature optimum of Xyn10A, enzyme activity was measured as described above in a temperature range of 40–95°C at pH 6.0. The pH profile of Xyn10A was determined by using phosphate-citrate buffers (pH 4.0–8.5) at 80°C. The optimal temperature and pH of Agu67A was investigated with MeGlcA decorated XOs as substrates as described above. In optimal temperature assay, the assay was conducted at pH 6.5 with temperature of 40-95°C, while the pH profile (4.0–8.5) was conducted at 75°C. To detect the thermostability of Xyn10A, purified Xyn10A was incubated in phosphate buffer (pH 8.5) at 75, 80 and 85°C, respectively, and residual activity under optimal conditions was compared at the moment of 0.5, 1, 2, 3, 4 and 6 h. The pH stability of Xyn10A was measured by assaying the relative activity under optimal conditions after the enzyme was pre-incubated in different pH buffers ranging from 4.0–8.5 at room temperature for 10 h.
In addition, the effects of various additives on the recombinant xylanase activity were assayed by adding 1 mM and 5 mM of various metal ions (NH4+, Na+, K+, Mg2+, Fe2+, Ca2+, Fe3+, Zn2+, Co2+, Mn2+, Cu2+, Ni2+), or 1 mM and 5 mM of chemicals (dithiothreitol, ethylenediamine tetraacetic acid) or 0.1% (v/v) and 0.5% (v/v) of different solvents (β-mercaptoethanol, Triton X-100, sodium dodecyl sulfate) or 5% (v/v) and 10% (v/v) organic reagents (glycerol, ethanol, isopropanol, butanol) respectively in the reaction mixture and incubating at 80°C for 5 min. The residual enzyme activity was determined at optimal conditions and the xylanase activity without addition of metal ions or chemical reagents was defined as 100%. All the experiments were performed in triplicate.
Hydrolysis of beechwood xylan and XOs by Xyn10A and Agu67A
The capacity of Xyn10A and Agu67A to hydrolyze beechwood xylan and XOs was assessed by detecting the hydrolysis products using thin-layer chromatography (TLC), reducing sugar assay, and HPLC analysis. Xyn10A (0.5 µM, final concentration) was incubated with different concentrations (0.1–2.0%, w/v) of beechwood xylan under optimal conditions for 4 hours. Besides, Xyn10A (2.0 µM, final concentration), or Agu67A (2.0 µM, final concentration), or a Xyn10A and Agu67A mixture (2.0 µM each, final concentration) was incubated with XOs (2.0%, w/v) for 4 hours under optimal conditions. To further evaluate the synergistic activity of Xyn10A and Agu67A, Xyn10A (1.75 µM, final concentration), or Agu67A (0.85 µM, final concentration), or a Xyn10A (1.75 µM, final concentration) and Agu67A (0.85 µM, final concentration) mixture was incubated with beechwood xylan (1.0%, w/v) for 0.25, 1, 2, 6 and 12 hours in the same way. Each of the control reaction was performed under the same experimental condition except adding the heat denatured enzyme. After reaction, hydrolysis products were centrifuged at 12,000 g for 10 min. Supernatants were spotted on Silica Gel GF254 TLC plates (Kepujia Reagent Co., Beijing, China) and then developed in 1-butanol/acetic acid/H2O (10∶5∶1, v/v/v) system for 2 hours after drying . Finally, products were visualized by spraying the dried plates with a mixture of methanolic orcinol (0.05%, w/v) and sulfuric acid (5.0%, v/v) followed by heating at 75°C for 10 min . Xylose (X1), xylobiose (X2), xylotriose (X3), and xylotetraose (X4) were used as standards. At the same time, the hydrolysis products of XOs and beechwood xylan were determined by reducing sugar assay using PHBAH method as mentioned above. And the products from the hydrolysis of XOs and 2 h's hydrolysis of beechwood xylan were also detected by HPLC. All the experiments were performed in triplicate.
Conceived and designed the experiments: YH XJ. Performed the experiments: XJ SM WQ. Analyzed the data: XJ YH SM WQ XP. Contributed reagents/materials/analysis tools: XJ SM JW WQ XP. Contributed to the writing of the manuscript: XJ JW YH.
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domain: literature


text:
VIRTUAL Creative Movement and Intro to Ballet classes
Creative Movement and Intro to Ballet classes.
Creative Movement and Imaginative Play at Georgetown Family YMCA
Writers Corner Registration Form for Georgetown Family YMCA
Creative Movement for 3-5 Yrs. Hutto Family YMCA Children that love rhythm and movement and feel safe participating in a class without Mom and Dad are invited to participate in this class.

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Ben Fogle and Kate Humble explore life behind the scenes at Longleat Estate and Safari Park. It's feeding time for the tarantula, and down on the lake the sea lions are expecting an addition to the splash.
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2021 Royal Enfield Classic 350 launch date revealed
Royal Enfield will launch the new generation Classic 350 on September 1
Royal Enfield has released a teaser image revealing that the new generation Classic 350 will launch on September 1. The bike has been spotted testing multiple times before, revealing the same retro-modern design with a round headlamp and taillamp. A new addition to the Classic 350 will be a tripper navigation display. The bike shares its platform and powertrain with the Meteor 350, which means the Classic 350 will be based on the J platform and will get the 349cc single-cylinder engine making 20.2hp and 27Nm of torque.


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There is something so simple and captivating about this little yellow wedding. It may be the stunning bride, or the pop of color, or the throw back style, but it remains one of my favorites. Nicole and Chris tied the knot at the Mountain Winery in the South Bay. It is a rustic venue, but they didn’t let that determine their wedding style. They kept it simple and clean and truly impactful by choosing a bright color and letting it speak.
I think what really makes it work is the accents of black and white. If her bouquet had a white or yellow ribbon on it, it would have looked like just another wedding. But the vision of allowing the black and white to come in through a ribbon really takes her entire wedding ensemble to the next level.
I am mesmerized by the fancy bridesmaid dresses and swept back hair. It looks like they danced out of the 20’s without trying too hard. LOVE! Thank you for letting us be part of your special day, Nicole and Chris. Your love is evident and we are touched to be part of it. Excellent work Tanja (Tanja Lippert Photography) in capturing the day and creating elegance. We love your work!


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Fried Spring Rolls with Vegan Option
Golden, crispy, crunchy, spring rolls that you can make at home. Forget the chinese take-out spring rolls that are a soggy, greasy, and mushy by the time you get them home. These won't disappoint.
Servings: 20 spring rolls
To see the ingredient amounts in metric units, click the blue "METRIC:" button between the ingredients and the instructions for this recipe.
1 deep fryer or wok
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- 1 lb ground pork or Vegan ground pork mix
- 2 cloves garlic
- 8 fresh mushrooms diced
- 2 tsp Umami Powder
- 1½ cups shredded carrots
- 1½ cups shredded cabbage regular or napa
- 1½ tsp Hoisin Sauce
- 2 tsp soy sauce
- 2 tsp cornstarch
- 2 tbsp oil
- 1 package Spring Roll Wrappers 20 - 25 eight-inch square wrappers
- 2 tbsp cornstarch
- 1 tbsp water
- Oil for deep frying
Cook the Filling
Saute the garlic in one tbsp of oil until fragrant.
Add the ground pork, stir to break up the pork, and cook until the pork is white.
If you are making Vegan Spring Rolls, Saute the garlic and add the vegan ground pork mix with the vegetables.
Add the mushrooms, cabbage, and carrots. Cook for about 3 minutes.
Mix the cornstarch, hoisin sauce, soy sauce, and umami powder. Add to the vegetables and pork. Cook for one minute. The mixture should be thickened and slightly sticky. You don't want it watery, or you will have soggy spring rolls.
Spread the filling on a tray and refrigerate for 10 minutes to cool.
Fill the Spring Rolls
Mix the cornstarch and water in a small shallow bowl. This is for sealing the spring rolls
Peel off one spring roll wrapper. Keep the rest of the wrappers under a piece of plastic wrap or damp paper towel so they don't dry out.
Put the wrapper on the work surface in a diamond pattern. Place a heaping tablespoon of the filling at the bottom point and roll up halfway. Fold in the edges and continue rolling to the top point. Seal the roll with the cornstarch/water sludge. (see the video for how to)
Heat the oil in the deep fryer or wok to 375° F, (190° C)
Fry the spring rolls 4-5 at a time until golden brown and crispy.
Reheat leftover Spring Rolls in an air fryer or toaster oven.
Freeze uncooked spring rolls in an airtight container. Deep Fry frozen Spring Rolls and serve.
Serving: 2rolls | Calories: 315kcal | Carbohydrates: 16g | Protein: 6g | Fat: 8g | Saturated Fat: 2g | Polyunsaturated Fat: 1g | Monounsaturated Fat: 4g | Trans Fat: 0.1g | Cholesterol: 18mg | Sodium: 319mg | Potassium: 147mg | Fiber: 1g | Sugar: 1g | Vitamin A: 1642IU | Vitamin C: 3mg | Calcium: 21mg | Iron: 1mg

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text:
Canada opposition leader heads West amid oil scrap
By Jeffrey Jones
CALGARY, Alberta (Reuters) - Canada's federal opposition party leader, who has criticized the oil sands boom as harmful to the country's manufacturing sector, prepared on Wednesday to make his first visit to the massive resource development in Alberta, where his comments have sparked anger.
New Democratic Party leader Thomas Mulcair, slated to tour Suncor Energy Inc's oil sands operation on Thursday, has said the manufacturing sector is being hollowed out by a Canadian dollar that has surged due to the boom in oil exports.
Quebec-based Mulcair's comments, which characterized the situation as "Dutch disease," angered Alberta Premier Alison Redford, who said they threaten to deepen East-West divisions. She will not meet with the leader of the left-wing party, having already committed to an economic forum in the United States.
Mulcair's tar sands tour is seen in Alberta as an important introduction to an industry that has eschewed the left-leaning NDP with its history of support from organized labor. Nationally, recent opinion polls have shown Mulcair and his party approaching and even surpassing Prime Minister Stephen Harper's Conservatives, major supporters of the oil-industry.
"Our simple message is simply that we want to work for sustainable development in all regions of Canada," Mulcair told reporters in Ottawa. "There's nothing specific to the West in our message ... we're going to say that we want legislation enforced and legislation that is there to protect human health and ecosystems."
Joe Oliver, the federal natural resources minister, said the opposition leader's trip to the heart of oil country does nothing to change an anti-industry stance. The NDP has also been highly critical of the environmental impact of development.
"The NDP is trying to dress up an anti-resource agenda in sheep's clothing. It is clear that they want to shut down an industry that employs hundreds of thousands of Canadians and provides billions in revenues to governments across Canada to pay for social programs such as education and health care," Oliver said.
'OIL SANDS FEVER' Continued...

domain: news


Wildfires are annual scourge for the southern country
23 June, 2017
Portugal's leading environmental lobby group, Quercus, blamed the blazes on "forest management errors and bad political decisions" by governments over recent decades. It rebuked authorities for allowing the planting of huge swathes of eucalyptus trees - the country's most common and most profitable species - but one that's often blamed for stoking blazes. Wildfires are an annual scourge in Portugal. Between 1993 and 2013, Portugal recorded the highest annual number of forest fires in southern Europe, according to a report last year by the European Environment Agency.
The government announced a raft of new measures against wildfires in March. They included restrictions on eucalyptus plantations and a simplified and cheaper programme of property registration that seeks to ascertain which land is being neglected. Not all of those reforms have come into force yet.
Fire experts pointed to a series of shortcomings in Portugal's strategy of tackling wildfires, even though the summer blazes have been happening for decades. There is a broad consensus that more work is needed on fire prevention, starting with forest clearing and the creation of fire breaks.
"In Portugal, the main factor in the scale of wildfires is the unbroken stretches of forest," Paulo Fernandes, a forest researcher at Portugal's Tras-os-Montes e Alto Douro University, told the AP.
Statistics show that 35% of Portugal is covered by woodland, slightly above the EU average of 31%. The forest industry, especially the production of paper pulp, accounts for around 3% of the country's GDP.


text:
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domain: economics


text:
Skip to content
- Name: Calicum
- Symbol: Ca
- Atomic Number: 20
- Atomic Weight: 40.078 u
- Period: 4
- Group: 2
22 Calcium Facts for Kids
- Calcium is a chemical element on the periodic table.
- Calcium was isolated and first discovered by Cornish chemist and inventor Humphry Davy in 1808.
- Calcium is a solid at room temperature.
- The symbol for calcium is Ca.
- The atomic number for calcium is 20.
- The standard atomic weight of calcium is 40.078 u.
- Calcium is in the alkaline earth metal group on the periodic table.
- Calcium is a period 4 chemical element, which is the fourth row on the periodic table.
- Calcium is a group 2 chemical element, which is the beryllium group.
- Calcium has five stable isotopes and they are 40Ca, 42Ca, 43Ca, 44Ca and 46Ca.
- The melting point for calcium is 1,548 °F (842 °C).
- The boiling point for calcium is 2,703 °F (1,484 °C).
- Calcium is the 5th most abundant element in the human body.
- Calcium is the 5th most abundant of all the elements found on Earth.
- Calcium is the 3rd most abundant metal found on Earth.
- Calcium is the 5th most abundant element found in the human body, and it’s mostly in the bones and teeth.
- Calcium is important to the human body and considered an essential element.
- Calcium helps the development of bones in children and adolescents.
- Calcium can help treat certain bone diseases, like osteoporosis.
- Dairy products typically are rich in Calcium, like cheese and yogurt.
- Calcium intake that exceeds nutritional recommendations can increase the risk of kidney stones.
- The largest non-nutritional use of Calcium is in the steelmaking process.
Additional Resources on Calcium
- Calcium – RSC – Learn more about the chemical element calcium on the Royal Society of Chemistry website.
- Calcium – JLabs – Find more calcium facts on the Jefferson Science Education website.
- Calcium – NIH – Discover more about calcium on the National Institute of Health website.
- Calcium Facts – Read more calcium facts for kids on the Live Science website.

domain: health


text:
Tennessee outfielders Rubi Silva and Jonathan Mota slammed back-to-back home runs in the fourth, while catcher Charlie Cutler drew a bases-loaded walk-off base on balls in the eleventh inning to give the Smokies a 5-4 victory over the Chattanooga Lookouts on Friday night in front of 5,493 fans at Smokies Stadium.
The win is third consecutive walk-off victory for the Smokies, who improved their second half record to 2-0 and are in position to clinch a series win Saturday evening. The loss drops the Lookouts to 0-2 in the second half, in addition to a 26-36 overall clip.
After both teams were held silent the first extra frame, Tennessee won it in the 11th after loading the bases. CF Jae-Hoon Ha started the final inning with an infield single, and then moved to second on 2B Stephen Bruno’s base knock to center, with both then taking an extra base on an error on the play. The Lookouts would then intentional walk 3B Christian Villanueva to load the bags full for Cutler, who worked a nine-pitch at-bat, taking ball four to end the contest and cue the celebration.
The Smokies had walked off the previous night in regulation thanks to Villanueva’s run-scoring single to center, while SS Elliot Soto recorded a walk-off base knock in the 11th inning in the club’s first half finale on June 15 versus Mississippi. Overall, it was the fifth time Tennessee won a game in their last at bat.
For the second straight evening it was Chattanooga who got out in front early, once again thanks to the big fly. 2B Darnell Sweeney began the frame with a triple off the center field wall, and then came plateward when CF Noel Cuevas exited the yard with a two-run blast to left, putting the Lookouts ahead 2-0.
The Smokies, however, would answer back with their back-to-back jacks with to snag the 3-2 advantage. After Cutler drew a free pass, Silva smacked his second homer in as many nights with an opposite field two-run shot to left, evening the tally at 2-2, while Mota followed with a solo shot off the left field foul pole to put Tennessee ahead. It marked the second time this year the Smokies hit homers in consecutive at bats after doing so on April 13 versus Chattanooga.
Two innings later, the Lookouts would take back the lead with a two-run sixth. After LF Scott Schebler doubled to right, C Chris O’Brien immediately followed with a two-bagger of his own off the right field wall to nod the game even. O’Brien would then come across thanks to a Smokies miscue after SS Angel Sanchez reached on a force out to give Chattanooga their second lead.
That margin did not last long however, as Tennessee came right back to tie the contest at 4-4 in the next half inning. Cutler reached base on his second walk of the evening, and then moved on to third on an error, before coming across on another Chattanooga error to draw the game even and force extra innings.
Bruno (2-6) and Silva (2-5, 1 R, 1 HR, 2 RBI) each recorded two base knocks in the Smokies win, while O’Brien (2-4, 1 R, 1 RBI, 1 BB) and Cuevas (2-6, 1 R, 1 HR, 2 RBI) recorded multi-hit efforts for the Lookouts. In addition, both Villanueva and Cutler each drew three walks, with Villanueva reaching base four times on the evening.
Neither starting pitcher figured in the final decisions, with Tennessee RHP Matt Loosen (5.0 IP, 3 H, 2 R, 2 ER, 3 BB, 5 SO) allowing two earned runs on three hits, adding five strikeouts in five frames, while Chattanooga RHP Matt Shelton (4.0 IP, 4 H, 3 R, 3 ER, 2 BB, 2 SO) yielded three runs on four hits in four frames. Smokies relief man Jeff Lorick (1.0 IP) earned the victory with a perfect 11thh inning to improve to 2-2, while Juan Noriega (0.1 IP, 2 H, 1 R, 1 ER, 2 BB) suffered the defeat.
Tennessee and Chattanooga are set to square off again Saturday night for the third game of their series, with first pitch scheduled for 6:15 p.m. The Smokies are set to give the nod to RHP Corey Black (2-4, 3.25), while LHP Chris Reed is slated to take the hill for the Lookouts.
It’s Margaritaville Night at Smokies Stadium, presented by Margaritaville Pigeon Forge, with a special Jimmy Buffett themed evening planned. There will be food and drink specials throughout the evening, including three dollar cheeseburgers, Coronas and margaritas. In addition, the Jimmy Buffett tribute band Key West will be performing a post-game concert.
Tickets for that contest and the remaining 2014 Smokies home slate are still available and can be purchased online at smokiesbaseball.com, or by calling the Smokies Ticket Office at (865) 286-2300.
Listen to Manager Buddy Bailey’s postgame press conference below:


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Prosecutors said it was not yet clear, and their views could change depending on whether Flynn testifies as a defense witness in next week’s trial in Alexandria of Bijan Rafiekian, who is accused of illegally lobbying for Turkey while running a consulting firm with Flynn.
The government said it is too soon to draw conclusions about Flynn’s cooperation since pleading guilty in Dec. 2017 to lying to FBI agents about his contacts with Russia’s ambassador. In his plea deal in special counsel Robert S. Mueller III’s Russia probe, Flynn agreed to provide information and testify fully and truthfully about all matters requested, or else face the potential for a longer sentence or even additional charges.
In a two-page filing Wednesday, prosecutor Brandon L. Van Grack told U.S. District Judge Emmet G. Sullivan of the District, “At this time the government cannot speculate on how specifically the aforementioned records will impact the government’s sentencing position,” which is that Flynn deserves probation, not prison time.
Van Grack added, “Although the government will no longer call the defendant as a witness in that case, because the defendant could be called to testify by Rafiekian, the government still believes that the sentencing hearing should be scheduled to follow the completion of that trial.”
Flynn’s attorneys have until 5 p.m. Thursday to respond.
U.S. prosecutors in Rafiekian’s case had previously said they expected Flynn to serve as a key witness about the pair’s work with Turkish officials in 2016 to craft a persuasion campaign against exiled cleric Fethullah Gulen.
Prosecutors contend that Dutch-Turkish businessman Ekim Alptekin acted as a go-between so Flynn and Rafiekian could avoid being labeled as foreign agents. Alptekin is charged in the case but remains abroad.
Through attorneys, Flynn maintains that he is still cooperating and has not changed his story about the work of the Flynn Intel Group. In his guilty plea on Dec. 1, 2017, the retired lieutenant general admitted both concealing the nature of his contacts with Russian Ambassador Sergey Kislyak and making false statements regarding his Turkish dealings.
Flynn admitted lying when he retroactively filed under the Foreign Agent Registration Act, including that he wrote an anti-Gulen op-ed on “his own initiative” and that his consulting firm “did not know whether or the extent to which the Republic of Turkey was involved in the Turkey project.”
Flynn is now claiming that it was his former attorneys at Covington & Burling who “under extreme and unprecedented pressure” from the government wrote an inaccurate FARA filing that he did not read before signing.
Flynn fired his Covington defense team last month, replacing them with conservative lawyer and commentator Sidney Powell, a critic of the Mueller investigation.
In a statement Wednesday, Covington & Burling said, “Our ethical obligations, including obligations to former clients, at this time prevent us from commenting publicly on this matter.”
U.S. prosecutors revealed in documents unsealed Tuesday in Rafiekian’s case that they “do not necessarily agree” with Powell’s “characterizations” of Flynn’s new defense position, marking a significant break between Flynn and the government.
Late Tuesday, Judge Anthony J. Trenga, who is presiding over Rafiekian’s case, ruled that at this point, Flynn could not be deemed a co-conspirator in the case. Trump tweeted the news Tuesday night, implying he considered it a victory for his former adviser. But Trenga’s ruling did not rest on any evaluation of Flynn’s cooperation or level of candor; instead he found that the government had failed to provide enough evidence that a conspiracy existed.
Trenga issued an opinion saying evidence of Rafiekian’s guilt is “substantially undercut by his contemporaneous conduct, which included seeking out legal advice concerning his FARA disclosure obligations.”
The judge added that “there is no contention, or evidence proffered” that when writing the FARA filing the attorneys involved were unable to review “e-mails and other documents” that prosecutors say reveal conspiratorial intent.
Trenga also ruled that the government cannot tell jurors that Turkey paid for the project — although they can point to evidence that Rafiekian was told about Turkish funding. But he declined defense attorneys’ motion to dismiss the indictment.
Flynn and Rafiekian hired Covington in late 2016 after officials at the Justice Department reached out, concerned by the op-ed.
The FARA filing for the consulting firm ultimately stated that, “Although the Flynn Intel Group was engaged by a private firm, Inovo BV, and not by a foreign government, because of the subject matter of the engagement, Flynn Intel Group’s work for Inovo could be construed to have principally benefited the Republic of Turkey.”

domain: law


text:
Matt Hopf has joined The Herald-Whig newsroom as a staff writer in 2010. His primary responsibility is covering Illinois region news and features, and also writes the popular "Answers" column. A Palos Heights native, Matt received a master's degree in public affairs from the University of Illinois Springfield and a bachelor's degree in journalism from Eastern Illinois University. Before starting at The Herald-Whig, Matt interned with the GateHouse Media State Capital Bureau. He also interned at newspapers in Palos Heights and Tinley Park. In his free time, Matt roots for the Chicago White Sox and the Green Bay Packers, watches cooking shows religiously and plays trumpet in the Quincy Concert Band. Matt and his wife, Melissa, live in Quincy and have a daughter.
Prosecutors have announced their intention to seek the death penalty in the case of a former Quincy resident accused of drowning her 8-year-old son in 2017.
A mobile substation was parked at the Ameren Illinois substation at North 22nd and Spruce right before the July 13, 2015, windstorm that knocked out power for the majority of the city of Quincy.
Bond was increased to a combined $600,000 for a Quincy man accused of violating an order of protection for a Quincy teenager who drowned Oct. 27.
An expert will be hired for the defense of a Pittsfield man charged in the beating death of another Pittsfield man.
Kaylee Mehochko will be rememberd as a quiet and loving person. She loved animals, music and hoped to become a pediatric physician. "She was very soft spoken, very mild mannered," her uncle Jim May remembered.
Jury selection is expected to take two days in the second trial of the Quincy charged in the shooting death of another Quincy man.


text:
107 High River Ranch DR
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A slice of beautiful Hill Country inside a gated community - perfect setting for this charming country home. Open floorplan with plenty of windows to stream in natural light. Floor-to-ceiling limestone surround fireplace, high ceilings, and the mid-tone wood plank floor make for a very comfortable space central living space. A huge foyer entry is big enough to create a unique space much more than a foyer. The primary ensuite, once again, provides you space to create a very personal owner suite - a space to be imagined. Your living space extends outdoors to a huge stone patio and giant pergola with a built-in grill station and fire pit. 2.3+ acres of green grass and trees waiting to be carved into the lifestyle of your choice!

domain: design


text:
Introducing the G.E.S. Gazette, a new bilingual publication for the Globeville, Elyria, and Swansea neighborhoods and RiNo Art District!
Each month, the G.E.S. Gazette will bring news about local politics, community happenings, arts & culture, and more to residents. The G.E.S. Gazette is a new publication from the creators of The Denver North Star, an award-winning community newspaper focused on the North Denver community West of I-25.
- 8,000 copies each month. 7,200 of those are mailed directly to homes and businesses in the 80216 zip code within Denver. The other 800 will be available at local businesses, libraries and other community locations for visitors to pick up and learn more about businesses and news in the area.
- No corporate ownership means that the news is written by and for local residents and you won’t see the same “local” news story run in the G.E.S. Gazette and a half dozen other community papers.
- The professional, experienced team behind the paper means each issue will be completed and delivered every month on schedule, with nonbiased relevant information.
Full website coming soon!
For General inquires, please email David@DenverNorthStar.com
For advertising opportunities, please email Jill@DenverNorthStar.com
You can also view and download a media kit, including pricing, below.

domain: library


Jay DiBella…a musician that feels that music is an unconditional feeling that translates to all through song. It transcends styles, race, and any barrier known or created by man.
Jay began making drum boxes in 2004 utilizing his knowledge in percussion and timing, and woodworking. Since that time... See Full Bio
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ASHEVILLE, N.C. (AP)Tajion Jones’s 14 points helped UNC Asheville defeat Brevard 94-46 on Saturday.
Jones shot 5 for 8, including 2 for 5 from beyond the arc for the Bulldogs (2-0). Fletcher Abee shot 5 for 7, including 4 for 5 from beyond the arc to add 14 points. Jamon Battle shot 5 of 5 from the field and 3 for 5 from the line to finish with 13 points, while adding seven rebounds.
Charles Reed finished with 11 points and seven rebounds for the Tornados (0-2). Brevard also got 11 points from Jadon Carnes. Carson Evans also had seven points.
Up next for UNC Asheville is a matchup Friday with Texas A&M-Commerce. Brevard visits Western Carolina on Saturday.
The Associated Press created this story using technology provided by Data Skrive and data from Sportradar.


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Tom Youngs v Carlo Festuccia
Playing Leicester inevitably involves a massive confrontation with their powerful front five, and two experienced international hookers will be at the heart of this weekend’s match.
Following Dylan Hartley’s repeated disciplinary problems, 28-year-old Youngs became England’s first-choice hooker at the World Cup.
The converted centre’s speed and handling skills bring another dimension to his game but his relative lack of size makes him a less effective scrummager than some, while his throwing in, although much improved, remains a work-in-progress.
He is working with two new second rows this season in Mike Williams and Mike Fitzgerald and Wasps may scent opportunity to disrupt the hosts’ lineout as a result.
Festuccia is into the veteran stage of his career, but brings the traditional skill-set of an experienced hooker.
Accurate throwing, and the sort of powerful scrummaging rugby fans have come to expect from Italian front-rowers are his stock-in-trade, while his willingness to carry in the loose and tackling also impressed last week.
Festuccia represented Racing Metro with distinction in a league which is renowned for its ferocious scrummaging and is now backed-up by the impressive Ashley Johnson, which gives Wasps a genuine 80-minute threat in the No.2 shirt.
Tom Croft v James Haskell
Two of English rugby’s best-known names will see plenty of each other at Welford Road as Wasps’ skipper Haskell and international team-mate Croft pack down in their clubs’ back rows.
Haskell’s return to Wasps’ line-up was a key factor in last week’s win over Bath. Capped 62 times, he’s an old-school, if-it-moves-smash-it style of blindside flanker whose 18-plus stone frame is very effective at close quarters, both tackling and scrapping for ball at the breakdown.
He is also an experienced captain and useful ball carrier, whose partnership with George Smith and Nathan Hughes provides Dai Young with a perfect blend of back-row skills.
Croft’s 41-cap England career has been regularly interrupted by serious neck and knee injuries in the last four years, but he remains one of the outstanding players of his generation.
He is a totally different style of player to Haskell, with speed in open field that has led him to be described as a three-quarter playing in the pack.
This also allows him to operate effectively as an openside flanker.
Taller and lighter than Haskell, he has consistently been one of Leicester’s main lineout targets, as was shown by his five wins in the Tigers’ narrow win over Harlequins last week.

domain: sports


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Pinto Goncalves Furniture, 2018-01-02 12:31:33. The Organic Crib Mattress. Nowadays most mattress manufacturers claim to have mattress options, and for the most part, they all do. However, they mostly use slightly different outer layer and filling materials. Since there are many safe and healthy options available today, understand that you are not looking for just one option when shopping for an organic mattress. Some items you ought to look for in an organic mattress include: The use of 100% organic cotton and wool - both in the filling and the outer layer of the crib. There are not any herbicides or pesticides used on organically grown cotton crops. The fire retardants that are used in organic mattresses are organic. As we mentioned before, the chemicals that are used in traditional mattresses, to protect against fire can have serious health effects. In-crib fire barriers are required per governmental regulations. Organic fire retardants such as baking soda, hydrated silica, cellulose fiber and wool, which is a natural fire retardant, are some of the products you should look for. These are all natural alternatives that may meet the fire protection requirement of crib mattresses.
Rafaela Barbosa Furniture, 2017-10-17 12:01:18. f you find you are not sleeping well it may be due to an old and uncomfortable bed. How much time we spend in bed can affect its durability. If the mattress is lumpy and the springs are too bouncy it might be time to go shopping. We also change as we age. A person who has once slept best on a soft mattress may be more suited to a harder mattress later in life, or, vice versa. Weight gain and weight loss can all effect how we react to our mattresses. A mattress might be perfect for one person but if two people start using it there could be difficulties. Studies have shown that beds can lose up to 70% of their original hardiness over 10 years. How Do I Select a Mattress?
Rafael Cavalcanti Furniture, 2017-10-18 11:51:32. Hard Vs Soft Mattress. Mattresses come in a wide range of solidity. From the ultra soft to the extremely hard, it is more a matter of preference than necessity. However, there are some basic truths that hold for the majority of us. Very soft cloud-like mattresses can wreak havoc on bad backs. This is because there is very little support in a soft mattress. While hard mattresses might be better for a bad back, very hard mattresses can cause just as much trouble. It all boils down to what works best for each individual.
Rocha Fernandes Furniture, 2017-10-16 12:14:07. Before shopping for a mattress you need to arm yourself with pertinent information. First determine what type of bed is best for you. Do you like soft or hard beds? Would you prefer a standard mattress or a foam mattress? If you are not sure, go try out a few different mattress types. Second, consider your budget. This is not a purchase you want to scrimp on, as a poor nights sleep can affect your whole day. Try to get as much value as you can for your budget. Finally, size is a very important factor especially if there is more than one person sleeping in the bed. Ideally, you should both be able to lie on the bed with your arms behind your heads and not touch. A bed needs to be 10-15 cm longer than the tallest person sleeping in that bed. Also pay attention to the height. Lower beds can be easy to get into but harder to get out of. High beds can be hard to get into, especially for shorter people, but very easy to hop out of in the morning.
Pinto Goncalves Furniture, 2017-10-20 11:40:15. Bedsteads. Bedsteads are what most of us consider a typical bed frame. The mattress support is usually made of wood slats that can be very flexible or tough and rigid. Wire mesh is another material used to make the support. The more rigid and cheap slats are further apart but the gap should not exceed 10cm. In addition, the wider the space between the slats, the more likely your mattress will be to wear out.
Nicole Melo Furniture, 2017-10-23 11:29:38. The mattress exterior is called ticking. Visually it is really not too important to select a "pretty" mattress as it will be covered most of the time. Instead you want to make sure the ticking is sturdy and well made and not prone to ripping. The higher quality ticking is made of material that is knitted or woven using cotton or viscose yarn. Cheaper mattresses are made of polypropylene or polyester. The cheapest mattresses are made of stitchbond or bonded cloth. Manufacturers now make external mattress covers with special features such as anti-allergy, anti-bacterial and anti-static. They may be resistant to staining, water and fire. Amazingly enough, there are even mattresses that give off different scents conducive to a good nights sleep. Spring Mattresses. There are three basic types of spring mattresses. A continuous spring mattress is made from a single piece of wire woven into many springs. The springs are linked in a vertical pattern. The wire is soft and the coils are small. This generates more reaction from the mattress. An open coil mattress is the most common form of mattress. The springs are arranged horizontally and connected at their tops and on the bottom by a twisting wire. The rods that run around the edge of the coils add strength. A pocket spring mattress has coils that are placed in fabric coverings. Instead of working as a unit, these springs function separately. As a result movement between partners on the same bed can not be felt by the other.
Santos Araujo Furniture, 2017-10-12 12:24:05. More and more mattress companies are manufacturing customizable mattresses. This allows consumers to specify such things as mattress firmness and the type of latex used to create their individual sleep system. These are just a few benefits of buying an organic mattress. There is no better time to improve your quality of sleep than today!Manufacturers are permitted to advertise their products as "organic" as long as some of the materials used to make them have been certified organic, under strict U.S. guidelines. Organic wool is one of the most beneficial materials used in a natural mattress. It is hypoallergenic and dust mite resistant. This aids in the reduction of allergens in the air. It is also naturally flame resistant and complies with the 2007 ruling, requiring that all mattresses sold in the United States be fire retardant. This fabric also helps to relieve pressure points, while you sleep, reducing the frequency in which you toss and turn. It is a natural insulator of both heat and cold, which aids in keeping your body temperature at a comfortable level.
Matilde Sousa Furniture, 2017-10-22 11:18:26. They are Organic and Natural. Did you know a quarter of the worlds pesticides are used for cotton crops? To make just one T-shirt, a quarter of a pound of pesticides are used. Thats why choosing organic makes a massive difference for our health and environment. Natural memory foam mattresses are made using organic fabrics. When selecting your mattress, make sure your mattress is organic all the way through from top, fill and back. Choose unbleached organic cotton with organic fill and organic dyes and steer clear of harsh chemically treated fabrics.
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Founded in 1962 and originally released as a quarterly, Film Comment features reviews and analysis of mainstream, art-house, and avant-garde filmmaking from around the world.
Isabelle Huppert in conversation with Joan Dupont, Amy Taubin on White Material, Paul Haggis interviewed by Scott Foundas, Andrew Sarris on Claude Chabrol, Hot Property: Aurora by Chris Chang, Site Specifics: Chris Marker and Second Life by Jesse P. Finnegan, reviews of The King’s Speech, 127 Hours, Black Swan, and The Tempest, plus short takes on All Good Things + Blue Valentine, The Illusionist, Night Catches Us, Rare Exports: A Christmas Tale, and Tiny Furniture
David Fincher’s The Social Network, part two of Paul Brunick’s article on the recent state of film criticism, Tom Mes on Masahiro Shinoda, Andrew Sarris on Sacha Guitry, reviews of Tamara Drewe, Nuremberg, Scott Pilgrim vs. the World, and Last Train Home, plus short takes on Buried, Catfish, Heartbreaker, The Milk of Sorrow, Nowhere Boy, and Vision
Amy Taubin’s interview with Inception director Christopher Nolan, Paul Brunick on the recent state of film criticism, Laura Kern on Animal Kingdom, Kent Jones on Farewell, My Lovely and Cannes, Hot Property: The Hole by Dave Kehr, Site Specifics: UltraSlomo.com by Jesse P. Finnegan, reviews of Cyrus, Spring Fever, I Killed My Mother, Around a Small Mountain, The Law, and Alamar, plus short takes on The Disappearance of Alice Creed, Down Terrace, A Film Unfinished, Lebanon, Restrepo, and Soul Kitchen
Breathless at 50, the newly reconstructed Metropolis, Andrew Sarris on Roman Polanski, the best experimental films of the decade, The Oath, World on a Wire, Trash Humpers, and I Am Love, Alain Resnais interview, Mia Hansen-Løve, Lewis Klahr, Messiah of Evil
Catherine Breillat, Nicole Holofcener, Daniel Schmid, Andrew Sarris on Eric Rohmer, Sundance reports by Amy Taubin and Laura Kern, Greenberg, I Love You Phillip Morris, No One Knows About Persian Cats, and Mother, the 35th annual grosses gloss, your comments about the best of 2009 and the decade
Peter Jackson interview, Andrew Sarris on Vincere, Un lac, African Film Library, A Prophet, the Red Riding trilogy, Wrap-ups of the Films of the Year (including Terra Incognita and Movies That Mattered) and the best of the decade


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Kevin’s picks on DVD this week: Re-Animator Blu-ray, The Office: Season 8, Parks & Recreation: Season 4, Safe, Grey’s Anatomy: Season 8 and The Five-Year Engagement
Stinker of the week: Piranha 3DD
Kevin Carr’s DVD Rundown for 09/04/12
This Week: After a week of estrogen releases, Kevin invites a lady into the Magical Studio in the Sky to talk about the only somewhat estrogen-filled release schedule in the movies houses this weekend. Shannon Hood from Frothygirlz.com joins Kevin to talk about pirates, weddings, Edgar Allan Poe and the merits and downfalls of Jason Statham movies. They also touch on smaller releases like The Sound of My Voice and The Innkeepers before moving onto a Fat Guy Five about awesome pirate movies. Then it’s back to the radio segment to chat about DVDs and whatnot before heading off into the sunset.

domain: entertainment


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Zeus – King of All Seasonings history: Spiro Fokas developed a seasoning recipe in a village called Mavrata on the island of Kefalonia and won many awards over the years. People in Kefalonia called it the King of all Seasonings. Spiro brought his seasoning mix to Charleston SC and opened up Old Towne Grill & Seafood in 1972 to share his flavor with all in Downtown Charleston.
The Fokas family continued their restaurant tradition with opening up North Towne Grill & Seafood in 1990 and Zeus Grill & Seafood in 2001.
Pit Masters all over the country have won awards using our seasoning and 100’s of restaurants use Zeus Seasoning every day.
Today the Fokas Family is committed to continue spreading their special seasoning blend that adds delicious flavor to chicken, steak, salads, pasta, veal, roast, fish, shrimp, octopus, calamari, turkey, ribs, hamburger, omelets, dressings, stews, and vegetable dishes. You’ll love Zeus Seasoning!
King Street, Charleston, SC


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Notice of Effectiveness means a notice upon receipt of which the Seller effectively transfers to the Administrative Agent the exclusive control of the Controlled Account.
Obligor means a Person obligated to make payments pursuant to a Contract (other than any such Person whose indebtedness constitutes Excluded Receivables); provided that in the event that any payments in respect of a Contract are made by any other Person, such other Person shall also be deemed to be an Obligor.
Original Date has the meaning specified in Section 1.02(e)(i) of the Agreement.
Originator means United Rentals (North America), Inc. (f/k/a UR Merger Sub Corporation, as successor in interest to United Rentals (North America), Inc. and United Rentals Northwest, Inc.) and its successors and permitted assigns.
Other Corporations means United Rentals, Inc. and all of its Subsidiaries except the Seller.
Other Costs has the meaning specified in Section 7.04(c).
Other Investors means any Person other than the Seller, the Originator or the Collection Agent.
Other Sellers has the meaning specified in Section 7.04(c).
Outstanding Balance of any Receivable at any time means the then outstanding principal balance thereof.
Parent means United Rentals, Inc. and its successors and permitted assigns.
Performance Undertaking Agreement means the Amended and Restated Performance Undertaking Agreement, dated as of the date hereof, made by United Rentals in favor of the Seller, as the same may, from time to time, be amended, restated, modified or supplemented.
Percentage of any Bank means, (a) with respect to Scotia Capital,
34.838709734.83870968%, (b) with respect to PNC, 12.903225812.90322581%, (c) with respect to BTMU, 12.903225812.90322581%, (d) with respect to ST, 11.612903211.61290323%, (e) with respect to BMO, 11. 612903261290323%, (f) with respect to TD, 16.129032316.12903226%, and (g) with respect to a Bank that has entered into an Assignment and Acceptance, the amount set forth therein as such Banks Percentage, in each case as such amount may be modified by an Assignment and Acceptance entered into between a Bank and an Eligible Assignee.
Periodic Report means the Monthly Report, the Weekly Report or the Daily Report.

domain: finance