Science.gov

Sample records for alginate lyase activity

  1. A rapid, sensitive, simple plate assay for detection of microbial alginate lyase activity.

    PubMed

    Sawant, Shailesh S; Salunke, Bipinchandra K; Kim, Beom Soo

    2015-09-01

    Screening of microorganisms capable of producing alginate lyase enzyme is commonly carried out by investigating their abilities to grow on alginate-containing solid media plates and occurrence of a clearance zone after flooding the plates with agents such as 10% (w/v) cetyl pyridinium chloride (CPC), which can form complexes with alginate. Although the CPC method is good, advantageous, and routinely used, the agar in the media interferes with the action of CPC, which makes judgment about clearance zones very difficult. In addition, this method takes a minimum of 30 min to obtain the zone of hydrolysis after flooding and the hydrolyzed area is not sharply discernible. An improved plate assay is reported herein for the detection of extracellular alginate lyase production by microorganisms. In this method, alginate-containing agar plates are flooded with Gram's iodine instead of CPC. Gram's iodine forms a bluish black complex with alginate but not with hydrolyzed alginate, giving sharp, distinct zones around the alginate lyase producing microbial colonies within 2-3 min. Gram's iodine method was found to be more effective than the CPC method in terms of visualization and measurement of zone size. The alginate-lyase-activity area indicated using the Gram's iodine method was found to be larger than that indicated by the CPC method. Both methods (CPC and Gram's iodine) showed the largest alginate lyase activity area for Saccharophagus degradans (ATCC 43961) followed by Microbulbifer mangrovi (KCTC 23483), Bacillus cereus (KF801505) and Paracoccus sp. LL1 (KP288668) grown on minimal sea salt medium. The rate of growth and metabolite production in alginate-containing minimal sea salt liquid medium, followed trends similar to that of the zone activity areas for the four bacteria under study. These results suggested that the assay developed in this study of Gram's iodine could be useful to predict the potential of microorganisms to produce alginate lyase. The method also

  2. Characterization of an extracellular biofunctional alginate lyase from marine Microbulbifer sp. ALW1 and antioxidant activity of enzymatic hydrolysates.

    PubMed

    Zhu, Yanbing; Wu, Liyun; Chen, Yanhong; Ni, Hui; Xiao, Anfeng; Cai, Huinong

    2016-01-01

    A novel alginate-degrading marine bacterium Microbulbifer sp. ALW1 was isolated from rotten brown alga. An extracellular alginate lyase was purified to electrophoretic homogeneity and had a molecular mass of about 26.0 kDa determined by SDS-PAGE and size exclusion chromatography. This enzyme showed activities towards both polyguluronate and polymannuronate indicating its bifunctionality while with preference for the former substrate. Using sodium alginate as a substrate, strain ALW1 alginate lyase was optimally active at 45 °C and pH 7.0. It was stable at 25 °C, 30 °C, 35 °C and 40 °C, but not stable at 50 °C. This alginate lyase showed good stability over a broad pH range (5.0-9.0). The enzyme activity was increased to 5.1 times by adding NaCl to a final concentration of 0.5M. Strain ALW1 alginate lyase produced disaccharide (majority) and trisaccharide from alginate indicating that this enzyme could be a good tool for preparation of alginate oligosaccharides with low degree of polymerization (DP). The alginate oligosaccharides displayed the scavenging abilities towards radicals (DPPH, ABTS(+) and hydroxyl) and the reducing power. Therefore, the hydrolysates exhibited the antioxidant activity and had potential as a natural antioxidant.

  3. Characterization of a New Cold-Adapted and Salt-Activated Polysaccharide Lyase Family 7 Alginate Lyase from Pseudoalteromonas sp. SM0524

    PubMed Central

    Chen, Xiu-Lan; Dong, Sheng; Xu, Fei; Dong, Fang; Li, Ping-Yi; Zhang, Xi-Ying; Zhou, Bai-Cheng; Zhang, Yu-Zhong; Xie, Bin-Bin

    2016-01-01

    Marine bacterial alginate lyases play a role in marine alginate degradation and carbon cycling. Although a large number of alginate lyases have been characterized, reports on alginate lyases with special characteristics are still rather less. Here, a gene alyPM encoding an alginate lyase of polysaccharide lyase family 7 (PL7) was cloned from marine Pseudoalteromonas sp. SM0524 and expressed in Escherichia coli. AlyPM shows 41% sequence identity to characterized alginate lyases, indicating that AlyPM is a new PL7 enzyme. The optimal pH for AlyPM activity was 8.5. AlyPM showed the highest activity at 30°C and remained 19% of the highest activity at 5°C. AlyPM was unstable at temperatures above 30°C and had a low Tm of 37°C. These data indicate that AlyPM is a cold-adapted enzyme. Moreover, AlyPM is a salt-activated enzyme. AlyPM activity in 0.5–1.2 M NaCl was sixfolds higher than that in 0 M NaCl, probably caused by a significant increase in substrate affinity, because the Km of AlyPM in 0.5 M NaCl decreased more than 20-folds than that in 0 M NaCl. AlyPM preferably degraded polymannuronate and mainly released dimers and trimers. These data indicate that AlyPM is a new PL7 endo-alginate lyase with special characteristics. PMID:27486451

  4. Alginate lyases from alginate-degrading Vibrio splendidus 12B01 are endolytic.

    PubMed

    Badur, Ahmet H; Jagtap, Sujit Sadashiv; Yalamanchili, Geethika; Lee, Jung-Kul; Zhao, Huimin; Rao, Christopher V

    2015-03-01

    Alginate lyases are enzymes that degrade alginate through β-elimination of the glycosidic bond into smaller oligomers. We investigated the alginate lyases from Vibrio splendidus 12B01, a marine bacterioplankton species that can grow on alginate as its sole carbon source. We identified, purified, and characterized four polysaccharide lyase family 7 alginates lyases, AlyA, AlyB, AlyD, and AlyE, from V. splendidus 12B01. The four lyases were found to have optimal activity between pH 7.5 and 8.5 and at 20 to 25°C, consistent with their use in a marine environment. AlyA, AlyB, AlyD, and AlyE were found to exhibit a turnover number (kcat) for alginate of 0.60 ± 0.02 s(-1), 3.7 ± 0.3 s(-1), 4.5 ± 0.5 s(-1), and 7.1 ± 0.2 s(-1), respectively. The Km values of AlyA, AlyB, AlyD, and AlyE toward alginate were 36 ± 7 μM, 22 ± 5 μM, 60 ± 2 μM, and 123 ± 6 μM, respectively. AlyA and AlyB were found principally to cleave the β-1,4 bonds between β-d-mannuronate and α-l-guluronate and subunits; AlyD and AlyE were found to principally cleave the α-1,4 bonds involving α-l-guluronate subunits. The four alginate lyases degrade alginate into longer chains of oligomers.

  5. Alginate Lyases from Alginate-Degrading Vibrio splendidus 12B01 Are Endolytic

    PubMed Central

    Badur, Ahmet H.; Jagtap, Sujit Sadashiv; Yalamanchili, Geethika; Lee, Jung-Kul; Zhao, Huimin

    2015-01-01

    Alginate lyases are enzymes that degrade alginate through β-elimination of the glycosidic bond into smaller oligomers. We investigated the alginate lyases from Vibrio splendidus 12B01, a marine bacterioplankton species that can grow on alginate as its sole carbon source. We identified, purified, and characterized four polysaccharide lyase family 7 alginates lyases, AlyA, AlyB, AlyD, and AlyE, from V. splendidus 12B01. The four lyases were found to have optimal activity between pH 7.5 and 8.5 and at 20 to 25°C, consistent with their use in a marine environment. AlyA, AlyB, AlyD, and AlyE were found to exhibit a turnover number (kcat) for alginate of 0.60 ± 0.02 s−1, 3.7 ± 0.3 s−1, 4.5 ± 0.5 s−1, and 7.1 ± 0.2 s−1, respectively. The Km values of AlyA, AlyB, AlyD, and AlyE toward alginate were 36 ± 7 μM, 22 ± 5 μM, 60 ± 2 μM, and 123 ± 6 μM, respectively. AlyA and AlyB were found principally to cleave the β-1,4 bonds between β-d-mannuronate and α-l-guluronate and subunits; AlyD and AlyE were found to principally cleave the α-1,4 bonds involving α-l-guluronate subunits. The four alginate lyases degrade alginate into longer chains of oligomers. PMID:25556193

  6. Screening of Alginate Lyase-Producing Bacteria and Optimization of Media Compositions for Extracellular Alginate Lyase Production

    PubMed Central

    Tavafi, Hadis; Abdi- Ali, Ahya A; Ghadam, Parinaz; Gharavi, Sara

    2017-01-01

    Background: Alginate is a linear polysaccharide consisting of guluronate (polyG) and mannuronate (polyM) subunits. Methods: In the initial screening of alginate-degrading bacteria from soil, 10 isolates were able to grow on minimal medium containing alginate. The optimization of cell growth and alginate lyase (algL) production was carried out by the addition of 0.8% alginate and 0.2-0.3 M NaCl to the culture medium. Of 10 isolates, one was selected based on its fast growth rate on minimal 9 medium containing 0.4% sodium alginate. The selected bacterium, identified based on morphological and biochemical characteristics, as well as 16S rDNA sequence data, was confirmed to be an isolate belonging to the genus Bacillus and designated as Bacillus sp. TAG8. Results: The results showed the ability of Bacillus sp. TAG8 in utilizing alginate as a sole carbon source. Bacillus sp. TAG8 growth and algL production were augmented with an increase in sodium alginate concentration and also by the addition of 0.2-0.3 M NaCl. Molecular analysis of TAG8 algL gene showed 99% sequence identity with algL of Pseudomonas aeruginosa PAO1. The algL produced by Bacillus sp. TAG8 cleaved both polyM and polyG blocks in alginate molecule, as well as acetylated alginate residues, confirming the bifunctionality of the isolated lyase. Conclusion: The identification of novel algL genes from microbial communities constitutes a new approach for exploring lyases with specific activity against bacterial alginates and may thus contribute to the eradication of persistent biofilms from clinical samples. PMID:27432784

  7. Characterization of AlgMsp, an Alginate Lyase from Microbulbifer sp. 6532A

    PubMed Central

    Swift, Steven M.; Hudgens, Jeffrey W.; Heselpoth, Ryan D.; Bales, Patrick M.; Nelson, Daniel C.

    2014-01-01

    Alginate is a polysaccharide produced by certain seaweeds and bacteria that consists of mannuronic acid and guluronic acid residues. Seaweed alginate is used in food and industrial chemical processes, while the biosynthesis of bacterial alginate is associated with pathogenic Pseudomonas aeruginosa. Alginate lyases cleave this polysaccharide into short oligo-uronates and thus have the potential to be utilized for both industrial and medicinal applications. An alginate lyase gene, algMsp, from Microbulbifer sp. 6532A, was synthesized as an E.coli codon-optimized clone. The resulting 37 kDa recombinant protein, AlgMsp, was expressed, purified and characterized. The alginate lyase displayed highest activity at pH 8 and 0.2 M NaCl. Activity of the alginate lyase was greatest at 50°C; however the enzyme was not stable over time when incubated at 50°C. The alginate lyase was still highly active at 25°C and displayed little or no loss of activity after 24 hours at 25°C. The activity of AlgMsp was not dependent on the presence of divalent cations. Comparing activity of the lyase against polymannuronic acid and polyguluronic acid substrates showed a higher turnover rate for polymannuronic acid. However, AlgMSP exhibited greater catalytic efficiency with the polyguluronic acid substrate. Prolonged AlgMsp-mediated degradation of alginate produced dimer, trimer, tetramer, and pentamer oligo-uronates. PMID:25409178

  8. Characterization of AlgMsp, an alginate lyase from Microbulbifer sp. 6532A.

    PubMed

    Swift, Steven M; Hudgens, Jeffrey W; Heselpoth, Ryan D; Bales, Patrick M; Nelson, Daniel C

    2014-01-01

    Alginate is a polysaccharide produced by certain seaweeds and bacteria that consists of mannuronic acid and guluronic acid residues. Seaweed alginate is used in food and industrial chemical processes, while the biosynthesis of bacterial alginate is associated with pathogenic Pseudomonas aeruginosa. Alginate lyases cleave this polysaccharide into short oligo-uronates and thus have the potential to be utilized for both industrial and medicinal applications. An alginate lyase gene, algMsp, from Microbulbifer sp. 6532A, was synthesized as an E.coli codon-optimized clone. The resulting 37 kDa recombinant protein, AlgMsp, was expressed, purified and characterized. The alginate lyase displayed highest activity at pH 8 and 0.2 M NaCl. Activity of the alginate lyase was greatest at 50°C; however the enzyme was not stable over time when incubated at 50°C. The alginate lyase was still highly active at 25°C and displayed little or no loss of activity after 24 hours at 25°C. The activity of AlgMsp was not dependent on the presence of divalent cations. Comparing activity of the lyase against polymannuronic acid and polyguluronic acid substrates showed a higher turnover rate for polymannuronic acid. However, AlgMSP exhibited greater catalytic efficiency with the polyguluronic acid substrate. Prolonged AlgMsp-mediated degradation of alginate produced dimer, trimer, tetramer, and pentamer oligo-uronates.

  9. Screening of alginate lyase-excreting microorganisms from the surface of brown algae.

    PubMed

    Wang, Mingpeng; Chen, Lei; Zhang, Zhaojie; Wang, Xuejiang; Qin, Song; Yan, Peisheng

    2017-12-01

    Alginate lyase is a biocatalyst that degrades alginate to produce oligosaccharides, which have many bioactive functions and could be used as renewable biofuels. Here we report a simple and sensitive plate assay for screening alginate lyase-excreting microorganisms from brown algae. Brown algae Laminaria japonica, Sargassum horneri and Sargassum siliquatrum were cultured in sterile water. Bacteria growing on the surface of seaweeds were identified and their capacity of excreting alginate lyase was analyzed. A total of 196 strains were recovered from the three different algae samples and 12 different bacterial strains were identified capable of excreting alginate lyases. Sequence analysis of the 16S rRNA gene revealed that these alginate lyase-excreting strains belong to eight genera: Paenibacillus (4/12), Bacillus (2/12), Leclercia (1/12), Isoptericola (1/12), Planomicrobium (1/12), Pseudomonas (1/12), Lysinibacillus (1/12) and Sphingomonas (1/12). Further analysis showed that the LJ-3 strain (Bacillus halosaccharovorans) had the highest enzyme activity. To our best knowledge, this is the first report regarding alginate lyase-excreting strains in Paenibacillus, Planomicrobium and Leclercia. We believe that our method used in this study is relatively easy and reliable for large-scale screening of alginate lyase-excreting microorganisms.

  10. Three alginate lyases from marine bacterium Pseudomonas fluorescens HZJ216: purification and characterization.

    PubMed

    Li, Liyan; Jiang, Xiaolu; Guan, Huashi; Wang, Peng; Guo, Hong

    2011-06-01

    Three alginate lyases (A, B, and C) from an alginate-degrading marine bacterium strain HZJ216 isolated from brown seaweed in the Yellow Sea of China and identified preliminarily as Pseudomonas fluorescens are purified, and their biochemical properties are described. Molecular masses of the three enzymes are determined by SDS-PAGE to be 60.25, 36, and 23 kDa with isoelectric points of 4, 4.36, and 4.59, respectively. Investigations of these enzymes at different pH and temperatures show that they are most active at pH 7.0 and 35 °C. Alginate lyases A and B are stable in the pH range of 5.0-9.0, while alginate lyase C is stable in the pH range of 5.0-7.0. Among the metal ions tested, additions of Na(+), K(+), and Mg(2+) ions can enhance the enzyme activities while Fe(2+), Fe(3+), Ba(2+), and Zn(2+) ions show inhibitory effects. The substrate specificity results demonstrate that alginate lyase C has the specificity for G block while alginate lyases A and B have the activities for both M and G blocks. It is the first report about extracellular alginate lyases with high alginate-degrading activity from P. fluorescens.

  11. Three Alginate Lyases from Marine Bacterium Pseudomonas fluorescens HZJ216: Purification and Characterization

    SciTech Connect

    Liyan, Li; Jiang, Xiaolu; Wang, Peng; Guan, Huashi; Guo, Hong

    2010-01-01

    Three alginate lyases (A, B, and C) from an alginate-degrading marine bacterium strain HZJ216 isolated from brown seaweed in the Yellow Sea of China and identified preliminarily as Pseudomonas fluorescens are purified, and their biochemical properties are described. Molecular masses of the three enzymes are determined by SDS-PAGE to be 60.25, 36, and 23 kDa with isoelectric points of 4, 4.36, and 4.59, respectively. Investigations of these enzymes at different pH and temperatures show that they are most active at pH 7.0 and 35 C. Alginate lyases A and B are stable in the pH range of 5.0 9.0, while alginate lyase C is stable in the pH range of 5.0 7.0. Among the metal ions tested, additions of Na+, K+, and Mg2+ ions can enhance the enzyme activities while Fe2+, Fe3+, Ba2+, and Zn2+ ions show inhibitory effects. The substrate specificity results demonstrate that alginate lyase C has the specificity for G block while alginate lyases A and B have the activities for both M and G blocks. It is the first report about extracellular alginate lyases with high alginate-degrading activity from P. fluorescens.

  12. Optimization of culturing condition and medium composition for the production of alginate lyase by a marine Vibrio sp. YKW-34

    NASA Astrophysics Data System (ADS)

    Fu, Xiaoting; Lin, Hong; Kim, Sang Moo

    2008-02-01

    Carbohydrases secreted by marine Vibrio sp. YKW-34 with strong Laminaria cell wall degrading ability were screened, and among them alginate lyase was found to be dominant. The effects of medium composition and culturing condition on the production of alginate lyase by marine Vibrio sp. YKW-34 in flask were investigated in this study. In the culture medium of marine broth, no alginate lyase was produced. The activity of the alginate lyase, after being induced, reached 5 UmL-1. The best inoculum volume and inoculum age were 10% and 12 h, respectively. The optimal temperature for alginate lyase production was 25°C. The fermentation medium was composed of 0.5% of Laminaria powder and 0.2% of KNO3 with an initial acidity of pH 8.0. Alginate could induce alginate lyase production but not as efficiently as Laminaria powder did. The addition of fucoidan, cellulose and glucose had negative effect on the alginate lyase production. Other kinds of nitrogen sources, such as yeast extract, beef extract and peptone, had positive effect on the growth of the microorganism and negative effect on alginate lyase production. In addition, the time course of alginate lyase production under the optimized condition was described. The optimal harvest time was 48 h.

  13. Maximizing the utilization of Laminaria japonica as biomass via improvement of alginate lyase activity in a two-phase fermentation system.

    PubMed

    Oh, Yuri; Xu, Xu; Kim, Ji Young; Park, Jong Moon

    2015-08-01

    Brown seaweed contains up to 67% of carbohydrates by dry weight and presents high potential as a polysaccharide feedstock for biofuel production. To effectively use brown seaweed as a biomass, degradation of alginate is the major challenge due to its complicated structure and low solubility in water. This study focuses on the isolation of alginate degrading bacteria, determining of the optimum fermentation conditions, as well as comparing the conventional single fermentation system with the two-phase fermentation system which is separately using alginate and mannitol extracted from Laminaria japonica. Maximum yield of organic acids production and volatile solids reduction obtained were 0.516 g/g and 79.7%, respectively, using the two-phase fermentation system in which alginate fermentation was carried out at pH 7 and mannitol fermentation at pH 8. The two-phase fermentation system increased the yield of organic acids production by 1.14 times and led to a 1.45-times reduction of VS when compared to the conventional single fermentation system at pH 8. The results show that the two-phase fermentation system improved the utilization of alginate by separating alginate from mannitol leading to enhanced alginate lyase activity.

  14. Isolation of protoplasts from undaria pinnatifida by alginate lyase digestion

    NASA Astrophysics Data System (ADS)

    Xiaoke, Hu; Xiaolu, Jiang; Huashi, Guan

    2003-04-01

    The aim of this study is to isolate protoplasts from Undaria pinnatifida. Protoplasts of the alga were isolated enzymatically by using alginate lyase, which was prepared by fermenting culture of a strain Vibrio sp. 510. Monofacterial method was applied for optimizing digestion condition. The optimum condition for protoplast preparation is enzymatic digestion at 28°C for 2h using alginate lyase at the concentration of 213.36 U (8 mL) every 0.5g fresh thalline with NaCl 50 and at the shaking speed of 150 r min-1 during digestion. The protoplast yield can reach 2.62±0.09 million per 0.5 g fresh leave under the optimum condition. The enzyme activity is inhibited by Ca2+ and slightly enhanced by Fe2+ and Mn2+ at concentrations of 0.05, 0.08 and 0.10 mol L-1.

  15. Purification and characterization of a bifunctional alginate lyase from Pseudoalteromonas sp. SM0524.

    PubMed

    Li, Jian-Wei; Dong, Sheng; Song, Jie; Li, Chun-Bo; Chen, Xiu-Lan; Xie, Bin-Bin; Zhang, Yu-Zhong

    2011-01-21

    An alginate lyase-producing bacterial strain, Pseudoalteromonas sp. SM0524, was screened from marine rotten kelp. In an optimized condition, the production of alginate lyase from Pseudoalteromonas sp. SM0524 reached 62.6 U/mL, suggesting that strain SM0524 is a good producer of alginate lyases. The bifunctional alginate lyase aly-SJ02 secreted by strain SM0524 was purified. Aly-SJ02 had an apparent molecular mass of 32 kDa. The optimal temperature and pH of aly-SJ02 toward sodium alginate was 50 °C and 8.5, respectively. The half life period of aly-SJ02 was 41 min at 40 °C and 20 min at 50 °C. Aly-SJ02 was most stable at pH 8.0. N-terminal sequence analysis suggested that aly-SJ02 may be an alginate lyase of polysaccharide lyase family 18. Aly-SJ02 showed activities toward both polyG (α-l-guluronic acid) and polyM (β-D-mannuronic acid), indicating that it is a bifunctional alginate lyase. Aly-SJ02 had lower K(m) toward polyG than toward polyM and sodium alginate. Thin layer chromatography and ESI-MS analyses showed that aly-SJ02 mainly released dimers and trimers from polyM and alginate, and trimers and tetramers from polyG, which suggests that aly-SJ02 may be a good tool to produce dimers and trimers from alginate.

  16. Enzymatic Hydrolysis of Alginate to Produce Oligosaccharides by a New Purified Endo-Type Alginate Lyase

    PubMed Central

    Zhu, Benwei; Chen, Meijuan; Yin, Heng; Du, Yuguang; Ning, Limin

    2016-01-01

    Enzymatic hydrolysis of sodium alginate to produce alginate oligosaccharides has drawn increasing attention due to its advantages of containing a wild reaction condition, excellent gel properties and specific products easy for purification. However, the efficient commercial enzyme tools are rarely available. A new alginate lyase with high activity (24,038 U/mg) has been purified from a newly isolated marine strain, Cellulophaga sp. NJ-1. The enzyme was most active at 50 °C and pH 8.0 and maintained stability at a broad pH range (6.0–10.0) and temperature below 40 °C. It had broad substrate specificity toward sodium alginate, heteropolymeric MG blocks (polyMG), homopolymeric M blocks (polyM) and homopolymeric G blocks (polyG), and possessed higher affinity toward polyG (15.63 mM) as well as polyMG (23.90 mM) than polyM (53.61 mM) and sodium alginate (27.21 mM). The TLC and MS spectroscopy analysis of degradation products suggested that it completely hydrolyzed sodium alginate into oligosaccharides of low degrees of polymerization (DPs). The excellent properties would make it a promising tool for full use of sodium alginate to produce oligosaccharides. PMID:27275826

  17. The release of alginate lyase from growing Pseudomonas syringae pathovar phaseolicola

    NASA Technical Reports Server (NTRS)

    Ott, C. M.; Day, D. F.; Koenig, D. W.; Pierson, D. L.

    2001-01-01

    Pseudomonas syringae pathovar phaseolicola, which produces alginate during stationary growth phase, displayed elevated extracellular alginate lyase activity during both mid-exponential and late-stationary growth phases of batch growth. Intracellular activity remained below 22% of the total activity during exponential growth, suggesting that alginate lyase has an extracellular function for this organism. Extracellular enzyme activity in continuous cultures, grown in either nutrient broth or glucose-simple salts medium, peaked at 60% of the washout rate, although nutrient broth-grown cultures displayed more than twice the activity per gram of cell mass. These results imply that growth rate, nutritional composition, or both initiate a release of alginate lyase from viable P. syringae pv. phaseolicola, which could modify its entrapping biofilm.

  18. Alginate Lyase Exhibits Catalysis-Independent Biofilm Dispersion and Antibiotic Synergy

    PubMed Central

    Lamppa, John W.

    2013-01-01

    More than 2 decades of study support the hypothesis that alginate lyases are promising therapeutic candidates for treating mucoid Pseudomonas aeruginosa infections. In particular, the enzymes' ability to degrade alginate, a key component of mucoid biofilm matrix, has been the presumed mechanism by which they disrupt biofilms and enhance antibiotic efficacy. The systematic studies reported here show that, in an in vitro model, alginate lyase dispersion of P. aeruginosa biofilms and enzyme synergy with tobramycin are completely decoupled from catalytic activity. In fact, equivalent antibiofilm effects can be achieved with bovine serum albumin or simple amino acids. These results provide new insights into potential mechanisms of alginate lyase therapeutic activity, and they should motivate a careful reexamination of the fundamental assumptions underlying interest in enzymatic biofilm dispersion. PMID:23070175

  19. Purification and characterization of alginate lyase from locally isolated marine Pseudomonas stutzeri MSEA04.

    PubMed

    Beltagy, Ehab A; El-Borai, Aliaa; Lewiz, Marina; ElAssar, Samy A

    2016-09-01

    An alginate lyase with high specific enzyme activity was purified from Pseudomonas stutzeri MSEA04, isolated from marine brown algae. The alginate lyase was purified by precipitation with ammonium sulphate, acetone and ethanol individually. 70% ethanol fraction showed maximum specific activity (133.3 U/mg). This fraction was re-purified by anion exchange chromatography DEAE- Cellulose A-52. The loaded protein was separated into 3 peaks. The second protein peak was the major one which contained 48.2% of the total protein recovered and 79.4% of the total recovered activity. The collected fractions of this peak were subjected to further purification by re-chromatography on Sephadex G-100. Alginate lyase activity was fractionated in the Sephadex column into one major peak, and the specific activity of this fraction reached 116 U/mg. The optimal substrate concentration, pH and temperature for alginate lyase activity were 8 mg/ml, pH 7.5 and 37 °C, respectively. While, Km and Vmax values were 1.07 mg alginate/ ml and 128.2 U/mg protein, respectively. The enzyme was partially stable below 50 °C, and the activity of the enzyme was strongly enhanced by K(+), and strongly inhibited by Ba(+2), Cd(+2), Fe(+2) and Zn(+2). The purified enzyme yielded a single band on SDS-PAGE with molecular weight (40.0 kDa).

  20. Calcium alginate bead immobilization of cells containing tyrosine ammonia lyase activity for use in the production of p-hydroxycinnamic acid.

    PubMed

    Trotman, Robert J; Camp, Carl E; Ben-Bassat, Arie; DiCosimo, Robert; Huang, Lixuan; Crum, Grace A; Sariaslani, F Sima; Haynie, Sharon L

    2007-01-01

    An Escherichia coli catalyst with tyrosine ammonia lyase activity (TAL) has been stabilized for repeated use in batch conversions of high tyrosine solids to p-hydroxycinnamic acid (pHCA). The TAL biocatalyst was stabilized by controlling the reaction pH to 9.8 +/- 0.1 and immobilizing the cells within a calcium alginate matrix that was cross-linked with glutaraldehyde and polyethyleneimine (GA/PEI). We found a GA range where the bead-encapsulated TAL was not inactivated, and the resulting cross-linking provided the beads with the mechanical stability necessary for repeated use in consecutive batch reactions with catalyst recycle. The GA/PEI calcium alginate TAL catalyst was used in 41 1-L batch reactions where 50 g L(-1) tyrosine was converted to 39 +/- 4 g L(-1) pHCA in each batch. The practical usefulness and ease of this process was demonstrated by scaling up the TAL bead immobilization and using the immobilized TAL catalyst in four 125-L bioconversion reactions to produce over 12 kg of purified pHCA.

  1. Alginate synthesis in Pseudomonas aeruginosa: the role of AlgL (alginate lyase) and AlgX.

    PubMed Central

    Monday, S R; Schiller, N L

    1996-01-01

    Previous studies localized an alginate lyase gene (algL) within the alginate biosynthetic gene cluster at 34 min on the Pseudomonas aeruginosa chromosome. Insertion of a Tn501 polar transposon in a gene (algX) directly upstream of algL in mucoid P. aeruginosa FRD1 inactivated expression of algX, algL, and other downstream genes, including algA. This strain is phenotypically nonmucoid; however, alginate production could be restored by complementation in trans with a plasmid carrying all of the genes inactivated by the insertion, including algL and algX. Alginate production was also recovered when a merodiploid that generated a complete alginate gene cluster on the chromosome was constructed. However, alginate production by merodiploids formed in the algX::Tn501 mutant using an alginate cluster with an algL deletion was not restored to wild-type levels unless algL was provided on a plasmid in trans. In addition, complementation studies of Tn501 mutants using plasmids containing specific deletions in either algL or algX revealed that both genes were required to restore the mucoid phenotype. Escherichia coli strains which expressed algX produced a unique protein of approximately 53 kDa, consistent with the gene product predicted from the DNA sequencing data. These studies demonstrate that AlgX, whose biochemical function remains to be defined, and AlgL, which has alginate lyase activity, are both involved in alginate production by P. aeruginosa. PMID:8550492

  2. Purification and characterisation of a bifunctional alginate lyase from novel Isoptericola halotolerans CGMCC 5336.

    PubMed

    Dou, Wenfang; Wei, Dan; Li, Hui; Li, Heng; Rahman, Muhammad Masfiqur; Shi, Jinsong; Xu, Zhenghong; Ma, Yanhe

    2013-11-06

    A novel halophilic alginate-degrading microorganism was isolated from rotten seaweed and identified as Isoptericola halotolerans CGMCC5336. The lyase from the strain was purified to homogeneity by combining of ammonium sulfate fractionation and anion-exchange chromatography with a specific activity of 8409.19 U/ml and a recovery of 25.07%. This enzyme was a monomer with a molecular mass of approximately 28 kDa. The optimal temperature and pH were 50 °C and pH 7.0, respectively. The lyase maintained stability at neutral pH (7.0-8.0) and temperatures below 50 °C. Metal ions including Na(+), Mg(2+), Mn(2+), and Ca(2+) notably increased the activity of the enzyme. With sodium alginate as the substrate, the Km and Vmax were 0.26 mg/ml and 1.31 mg/ml min, respectively. The alginate lyase had substrate specificity for polyguluronate and polymannuronate units in alginate molecules, indicating its bifunctionality. These excellent characteristics demonstrated the potential applications in alginate oligosaccharides production with low polymerisation degrees.

  3. Purification and Characterization of a New Alginate Lyase from Marine Bacterium Vibrio sp. SY08

    PubMed Central

    Li, Shangyong; Wang, Linna; Hao, Jianhua; Xing, Mengxin; Sun, Jingjing; Sun, Mi

    2016-01-01

    Unsaturated alginate disaccharides (UADs), enzymatically derived from the degradation of alginate polymers, are considered powerful antioxidants. In this study, a new high UAD-producing alginate lyase, AlySY08, has been purified from the marine bacterium Vibrio sp. SY08. AlySY08, with a molecular weight of about 33 kDa and a specific activity of 1070.2 U/mg, showed the highest activity at 40 °C in phosphate buffer at pH 7.6. The enzyme was stable over a broad pH range (6.0–9.0) and retained about 75% activity after incubation at 40 °C for 2 h. Moreover, the enzyme was active in the absence of salt ions and its activity was enhanced by the addition of NaCl and KCl. AlySY08 resulted in an endo-type alginate lyase that degrades both polyM and polyG blocks, yielding UADs as the main product (81.4% of total products). All these features made AlySY08 a promising candidate for industrial applications in the production of antioxidants from alginate polysaccharides. PMID:28025527

  4. Structure of a PL17 Family Alginate Lyase Demonstrates Functional Similarities among Exotype Depolymerases

    PubMed Central

    Park, David; Jagtap, Sujit; Nair, Satish K.

    2014-01-01

    Brown macroalgae represent an ideal source for complex polysaccharides that can be utilized as precursors for cellulosic biofuels. The lack of recalcitrant lignin components in macroalgae polysaccharide reserves provides a facile route for depolymerization of constituent polysaccharides into simple monosaccharides. The most abundant sugars in macroalgae are alginate, mannitol, and glucan, and although several classes of enzymes that can catabolize the latter two have been characterized, studies of alginate-depolymerizing enzymes have lagged. Here, we present several crystal structures of Alg17c from marine bacterium Saccharophagus degradans along with structure-function characterization of active site residues that are suggested to be involved in the exolytic mechanism of alginate depolymerization. This represents the first structural and biochemical characterization of a family 17 polysaccharide lyase enzyme. Despite the lack of appreciable sequence conservation, the structure and β-elimination mechanism for glycolytic bond cleavage by Alg17c are similar to those observed for family 15 polysaccharide lyases and other lyases. This work illuminates the evolutionary relationships among enzymes within this unexplored class of polysaccharide lyases and reinforces the notion of a structure-based hierarchy in the classification of these enzymes. PMID:24478312

  5. Genetically Engineered Alginate Lyase-PEG Conjugates Exhibit Enhanced Catalytic Function and Reduced Immunoreactivity

    PubMed Central

    Lamppa, John W.; Ackerman, Margaret E.; Lai, Jennifer I.; Scanlon, Thomas C.; Griswold, Karl E.

    2011-01-01

    Alginate lyase enzymes represent prospective biotherapeutic agents for treating bacterial infections, particularly in the cystic fibrosis airway. To effectively deimmunize one therapeutic candidate while maintaining high level catalytic proficiency, a combined genetic engineering-PEGylation strategy was implemented. Rationally designed, site-specific PEGylation variants were constructed by orthogonal maleimide-thiol coupling chemistry. In contrast to random PEGylation of the enzyme by NHS-ester mediated chemistry, controlled mono-PEGylation of A1-III alginate lyase produced a conjugate that maintained wild type levels of activity towards a model substrate. Significantly, the PEGylated variant exhibited enhanced solution phase kinetics with bacterial alginate, the ultimate therapeutic target. The immunoreactivity of the PEGylated enzyme was compared to a wild type control using in vitro binding studies with both enzyme-specific antibodies, from immunized New Zealand white rabbits, and a single chain antibody library, derived from a human volunteer. In both cases, the PEGylated enzyme was found to be substantially less immunoreactive. Underscoring the enzyme's potential for practical utility, >90% of adherent, mucoid, Pseudomonas aeruginosa biofilms were removed from abiotic surfaces following a one hour treatment with the PEGylated variant, whereas the wild type enzyme removed only 75% of biofilms in parallel studies. In aggregate, these results demonstrate that site-specific mono-PEGylation of genetically engineered A1-III alginate lyase yielded an enzyme with enhanced performance relative to therapeutically relevant metrics. PMID:21340021

  6. Alginate lyase: Review of major sources and classification, properties, structure-function analysis and applications

    PubMed Central

    Zhu, Benwei; Yin, Heng

    2015-01-01

    Alginate lyases catalyze the degradation of alginate, a complex copolymer of α-L-guluronate and its C5 epimer β-D-mannuronate. The enzymes have been isolated from various kinds of organisms with different substrate specificities, including algae, marine mollusks, marine and terrestrial bacteria, and some viruses and fungi. With the progress of structural biology, many kinds of alginate lyases of different polysaccharide lyases families have been characterized by obtaining crystal structures, and the catalytic mechanism has also been elucidated. Combined with various studies, we summarized the source, classification and properties of the alginate lyases from different polysaccharide lyases families. The relationship between substrate specificity and protein sequence was also investigated. PMID:25831216

  7. Polydopamine-Mediated Immobilization of Alginate Lyase to Prevent P. aeruginosa Adhesion.

    PubMed

    Alves, Diana; Sileika, Tadas; Messersmith, Phillip B; Pereira, Maria Olívia

    2016-09-01

    Given alginate's contribution to Pseudomonas aeruginosa virulence, it has long been considered a promising target for interventional therapies, which have been performed by using the enzyme alginate lyase. In this work, instead of treating pre-established mucoid biofilms, alginate lyase is immobilized onto a surface as a preventive measure against P. aeruginosa adhesion. A polydopamine dip-coating strategy is employed for functionalization of polycarbonate surfaces. Enzyme immobilization is confirmed by surface characterization. Surfaces functionalized with alginate lyase exhibit anti-adhesive properties, inhibiting the attachment of the mucoid strain. Moreover, surfaces modified with this enzyme also inhibit the adhesion of the tested non-mucoid strain. Unexpectedly, treatment with heat-inactivated enzyme also inhibits the attachment of mucoid and non-mucoid P. aeruginosa strains. These findings suggest that the antibacterial performance of alginate lyase functional coatings is catalysis-independent, highlighting the importance of further studies to better understand its mechanism of action against P. aeruginosa strains.

  8. Purification and characterization of a novel alginate lyase from the marine bacterium Cobetia sp. NAP1 isolated from brown algae.

    PubMed

    Yagi, Hisashi; Fujise, Asako; Itabashi, Narumi; Ohshiro, Takashi

    2016-12-01

    The application of marine resources, instead of fossil fuels, for biomass production is important for building a sustainable society. Seaweed is valuable as a source of marine biomass for producing biofuels such as ethanol, and can be used in various fields. Alginate is an anionic polysaccharide that forms the main component of brown algae. Various alginate lyases (e.g. exo- and endo-types and oligoalginate lyase) are generally used to degrade alginate. We herein describe a novel alginate lyase, AlgC-PL7, which belongs to the polysaccharide lyase 7 family. AlgC-PL7 was isolated from the halophilic Gram-negative bacterium Cobetia sp. NAP1 collected from the brown algae Padina arborescens Holmes. The optimal temperature and pH for AlgC-PL7 activity were 45 °C and 8, respectively. Additionally, AlgC-PL7 was thermostable and salt-tolerant, exhibited broad substrate specificity, and degraded alginate into monosaccharides. Therefore, AlgC-PL7 is a promising enzyme for the production of biofuels.

  9. Family 13 carbohydrate-binding module of alginate lyase from Agarivorans sp. L11 enhances its catalytic efficiency and thermostability, and alters its substrate preference and product distribution.

    PubMed

    Li, Shangyong; Yang, Xuemei; Bao, Mengmeng; Wu, Ying; Yu, Wengong; Han, Feng

    2015-05-01

    The carbohydrate-binding module (CBM) in polysaccharide hydrolases plays a key role in the hydrolysis of cellulose, xylan and chitin. However, the function of CBM in alginate lyases has not been elucidated. A new alginate lyase gene, alyL2, was cloned from the marine bacterium Agarivorans sp. L11 by using degenerate and site-finding PCR. The alginate lyase, AlyL2, contained an N-terminal CBM13 and a C-terminal catalytic family 7 polysaccharide lyase (PL7) module. To better understand the function of CBM13 in alginate lyase AlyL2, the full-length enzyme (AlyL2-FL) and its catalytic module (AlyL2-CM) were expressed in Escherichia coli and characterized. The specific activity and catalytic efficiency of AlyL2-FL were approximately twice those of AlyL2-CM. The half-lives of AlyL2-FL were 4.7-6.6 times those of AlyL2-CM at 30-50°C. In addition, the presence of CBM13 in AlyL2 changed its substrate preference and increased the percentage of disaccharides from 50.5% to 64.6% in the total products. This first report of the function of CBM13 in alginate lyase provides new insights into the degradation of alginate by marine microorganisms.

  10. Falsirhodobacter sp. alg1 Harbors Single Homologs of Endo and Exo-Type Alginate Lyases Efficient for Alginate Depolymerization

    PubMed Central

    Takahashi, Mami; Tanaka, Reiji; Miyake, Hideo; Shibata, Toshiyuki; Chow, Seinen; Kuroda, Kouichi; Ueda, Mitsuyoshi; Takeyama, Haruko

    2016-01-01

    Alginate-degrading bacteria play an important role in alginate degradation by harboring highly efficient and unique alginolytic genes. Although the general mechanism for alginate degradation by these bacteria is fairly understood, much is still required to fully exploit them. Here, we report the isolation of a novel strain, Falsirhodobacter sp. alg1, the first report for an alginate-degrading bacterium from the family Rhodobacteraceae. Genome sequencing reveals that strain alg1 harbors a primary alginate degradation pathway with only single homologs of an endo- and exo-type alginate lyase, AlyFRA and AlyFRB, which is uncommon among such bacteria. Subsequent functional analysis showed that both enzymes were extremely efficient to depolymerize alginate suggesting evolutionary interests in the acquirement of these enzymes. The exo-type alginate lyase, AlyFRB in particular could depolymerize alginate without producing intermediate products making it a highly efficient enzyme for the production of 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH). Based on our findings, we believe that the discovery of Falsirhodobacter sp. alg1 and its alginolytic genes hints at the potentiality of a more diverse and unique population of alginate-degrading bacteria. PMID:27176711

  11. cDNA cloning and bacterial expression of a PL-14 alginate lyase from a herbivorous marine snail Littorina brevicula.

    PubMed

    Rahman, Mohammad Matiur; Wang, Ling; Inoue, Akira; Ojima, Takao

    2012-10-01

    Herbivorous marine snails like Littorina species are known to possess alginate lyases in their digestive tracts. The Littorina enzymes have been identified as endolytic polymannuronate (poly(M)) lyases (EC 4.2.2.3); however, it is still unclear which polysaccharide-lyase family (PL) the Littorina enzymes belong to, since no complete primary structure of Littorina enzymes has been determined. Thus, in the present study, we analyzed the primary structure of LbAly28, a 28kDa alginate lyase isozyme of Littorina brevicula, by the cDNA method. LbAly28 cDNAs were amplified by PCR followed by 5'- and 3'-RACE PCRs from the L. brevicula hepatopancreas cDNA. A cDNA covering entire coding region of LbAly28 consisted of 1129bp and encoded an amino-acid sequence of 291 residues. The deduced amino-acid sequence comprised an initiation methionine, a putative signal peptide of 14 residues, a propeptide-like region of 16 residues, and a mature LbAly28 domain of 260 residues. The mature LbAly28 domain showed 43-53% amino-acid identities with other molluscan PL-14 enzymes. The catalytically important residues in PL-14 enzymes, which were identified in the Chlorella virus glucuronate-specific lyase vAL-1 and Aplysia poly(M) lyase AkAly30, were also conserved in LbAly28. Site-directed mutagenesis regarding these residues, that is, replacements of Lys94, Lys97, Thr121, Arg 123, Tyr135, and Tyr137 to Ala, decreased the activity of recombinant LbAly28 to various degrees. From these results we concluded that LbAly28 is a member of PL-14 alginate lyases. Besides the effects of above mutations, we noticed that the replacement of T121 by Ala changed the substrate preference of LbAly28. Namely, the activities toward sodium alginate and poly(MG)-block substrate increased and became comparable with the activity toward poly(M)-block substrate. This suggests that the region including T121 of LbAly28 closely relates to the recognition of poly(MG) region of alginate.

  12. Alginate Lyase Promotes Diffusion of Aminoglycosides through the Extracellular Polysaccharide of Mucoid Pseudomonas aeruginosa

    PubMed Central

    Hatch, Richard A.; Schiller, Neal L.

    1998-01-01

    We demonstrated that a 2% suspension of Pseudomonas aeruginosa alginate completely blocked the diffusion of gentamicin and tobramycin, but not that of carbenicillin, illustrating how alginate production can help protect P. aeruginosa growing within alginate microcolonies in patients with cystic fibrosis (CF) from the effects of aminoglycosides. This aminoglycoside diffusion barrier was degraded with a semipurified preparation of P. aeruginosa alginate lyase, suggesting that this enzyme deserves consideration as an adjunctive agent for CF patients colonized by mucoid strains of P. aeruginosa. PMID:9559826

  13. Discovery of a Novel Alginate Lyase from Nitratiruptor sp. SB155-2 Thriving at Deep-sea Hydrothermal Vents and Identification of the Residues Responsible for Its Heat Stability.

    PubMed

    Inoue, Akira; Anraku, Moe; Nakagawa, Satoshi; Ojima, Takao

    2016-07-22

    Extremophiles are expected to represent a source of enzymes having unique functional properties. The hypothetical protein NIS_0185, termed NitAly in this study, was identified as an alginate lyase-homolog protein in the genomic database of ϵ-Proteobacteria Nitratiruptor sp. SB155-2, which was isolated from deep-sea hydrothermal vents at a water depth of 1,000 m. Among the characterized alginate lyases in the polysaccharide lyase family 7 (PL-7), the amino acid sequence of NitAly showed the highest identity (39%) with that of red alga Pyropia yezoensis alginate lyase PyAly. Recombinant NitAly (rNitAly) was successfully expressed in Escherichia coli Purified rNitAly degraded alginate in an endolytic manner. Among alginate block types, polyM was preferable to polyG and polyMG as a substrate, and its end degradation products were mainly tri-, tetra-, and penta-saccharides. The optimum temperature and pH values were 70 °C and around 6, respectively. A high concentration of NaCl (0.8-1.4 m) was required for maximum activity. In addition, a 50% loss of activity was observed after incubation at 67 °C for 30 min. Heat stability was decreased in the presence of 5 mm DTT, and Cys-80 and Cys-232 were identified as the residues responsible for heat stability but not lyase activity. Introducing two cysteines into PyAly based on homology modeling using Pseudomonas aeruginosa alginate lyase PA1167 as the template enhanced its heat stability. Thus, NitAly is a functional alginate lyase, with its unique optimum conditions adapted to its environment. These insights into the heat stability of NitAly could be applied to improve that of other PL-7 alginate lyases.

  14. Novel Alginate Lyase (Aly5) from a Polysaccharide-Degrading Marine Bacterium, Flammeovirga sp. Strain MY04: Effects of Module Truncation on Biochemical Characteristics, Alginate Degradation Patterns, and Oligosaccharide-Yielding Properties

    PubMed Central

    Han, Wenjun; Gu, Jingyan; Cheng, Yuanyuan; Liu, Huihui; Li, Yuezhong

    2015-01-01

    Alginate lyases are important tools for oligosaccharide preparation, medical treatment, and energy bioconversion. Numerous alginate lyases have been elucidated. However, relatively little is known about their substrate degradation patterns and product-yielding properties, which is a limit to wider enzymatic applications and further enzyme improvements. Herein, we report the characterization and module truncation of Aly5, the first alginate lyase obtained from the polysaccharide-degrading bacterium Flammeovirga. Aly5 is a 566-amino-acid protein and belongs to a novel branch of the polysaccharide lyase 7 (PL7) superfamily. The protein rAly5 is an endolytic enzyme of alginate and associated oligosaccharides. It prefers guluronate (G) to mannuronate (M). Its smallest substrate is an unsaturated pentasaccharide, and its minimum product is an unsaturated disaccharide. The final alginate digests contain unsaturated oligosaccharides that generally range from disaccharides to heptasaccharides, with the tetrasaccharide fraction constituting the highest mass concentration. The disaccharide products are identified as ΔG units. While interestingly, the tri- and tetrasaccharide fractions each contain higher proportions of ΔG to ΔM ends, the larger final products contain only ΔM ends, which constitute a novel oligosaccharide-yielding property of guluronate lyases. The deletion of the noncatalytic region of Aly5 does not alter its M/G preference but significantly decreases the enzymatic activity and enzyme stability. Notably, the truncated protein accumulates large final oligosaccharide products but yields fewer small final products than Aly5, which are codetermined by its M/G preference to and size enlargement of degradable oligosaccharides. This study provides novel enzymatic properties and catalytic mechanisms of a guluronate lyase for potential uses and improvements. PMID:26519393

  15. Novel Alginate Lyase (Aly5) from a Polysaccharide-Degrading Marine Bacterium, Flammeovirga sp. Strain MY04: Effects of Module Truncation on Biochemical Characteristics, Alginate Degradation Patterns, and Oligosaccharide-Yielding Properties.

    PubMed

    Han, Wenjun; Gu, Jingyan; Cheng, Yuanyuan; Liu, Huihui; Li, Yuezhong; Li, Fuchuan

    2015-10-30

    Alginate lyases are important tools for oligosaccharide preparation, medical treatment, and energy bioconversion. Numerous alginate lyases have been elucidated. However, relatively little is known about their substrate degradation patterns and product-yielding properties, which is a limit to wider enzymatic applications and further enzyme improvements. Herein, we report the characterization and module truncation of Aly5, the first alginate lyase obtained from the polysaccharide-degrading bacterium Flammeovirga. Aly5 is a 566-amino-acid protein and belongs to a novel branch of the polysaccharide lyase 7 (PL7) superfamily. The protein rAly5 is an endolytic enzyme of alginate and associated oligosaccharides. It prefers guluronate (G) to mannuronate (M). Its smallest substrate is an unsaturated pentasaccharide, and its minimum product is an unsaturated disaccharide. The final alginate digests contain unsaturated oligosaccharides that generally range from disaccharides to heptasaccharides, with the tetrasaccharide fraction constituting the highest mass concentration. The disaccharide products are identified as ΔG units. While interestingly, the tri- and tetrasaccharide fractions each contain higher proportions of ΔG to ΔM ends, the larger final products contain only ΔM ends, which constitute a novel oligosaccharide-yielding property of guluronate lyases. The deletion of the noncatalytic region of Aly5 does not alter its M/G preference but significantly decreases the enzymatic activity and enzyme stability. Notably, the truncated protein accumulates large final oligosaccharide products but yields fewer small final products than Aly5, which are codetermined by its M/G preference to and size enlargement of degradable oligosaccharides. This study provides novel enzymatic properties and catalytic mechanisms of a guluronate lyase for potential uses and improvements.

  16. Modeling and Re-Engineering of Azotobacter vinelandii Alginate Lyase to Enhance Its Catalytic Efficiency for Accelerating Biofilm Degradation

    PubMed Central

    Jang, Chul Ho; Piao, Yu Lan; Huang, Xiaoqin; Yoon, Eun Jeong; Park, So Hee; Lee, Kyoung; Zhan, Chang-Guo; Cho, Hoon

    2016-01-01

    Alginate is known to prevent elimination of Pseudomonas aeruginosa biofilms. Alginate lyase (AlgL) might therefore facilitate treatment of Pseudomonas aeruginosa-infected cystic fibrosis patients. However, the catalytic activity of wild-type AlgL is not sufficiently high. Therefore, molecular modeling and site-directed mutagenesis of AlgL might assist in enzyme engineering for therapeutic development. AlgL, isolated from Azotobacter vinelandii, catalyzes depolymerization of alginate via a β-elimination reaction. AlgL was modeled based on the crystal structure template of Sphingomonas AlgL species A1-III. Based on this computational analysis, AlgL was subjected to site-directed mutagenesis to improve its catalytic activity. The kcat/Km of the K194E mutant showed a nearly 5-fold increase against the acetylated alginate substrate, as compared to the wild-type. Double and triple mutants (K194E/K245D, K245D/K319A, K194E/K245D/E312D, and K194E/K245D/K319A) were also prepared. The most potent mutant was observed to be K194E/K245D/K319A, which has a 10-fold improved kcat value (against acetylated alginate) compared to the wild-type enzyme. The antibiofilm effect of both AlgL forms was identified in combination with piperacillin/tazobactam (PT) and the disruption effect was significantly higher in mutant AlgL combined with PT than wild-type AlgL. However, for both the wild-type and K194E/K245D/K319A mutant, the use of the AlgL enzyme alone did not show significant antibiofilm effect. PMID:27253324

  17. A fence that eats the weed: Alginate lyase immobilization on ultrafiltration membrane for fouling mitigation and flux recovery.

    PubMed

    Meshram, Pradnya; Dave, Rachna; Joshi, Hiren; Dharani, Gopal; Kirubagaran, Ramalingam; Venugopalan, Vayalam P

    2016-12-01

    Polysaccharide fouling poses a significant challenge in the widespread application of membrane filtration for water purification. In order to mitigate the problem, a polysaccharide-degrading enzyme alginate lyase (Alg L; EC 4.2.2.3) was successfully immobilized on cellulose acetate ultrafiltration membrane using a dead-end filtration unit. Attenuated total reflectance Fourier transform infrared microscopy confirmed covalent linkage of the Alg L to the membrane. HPLC and Alg L activity studies confirmed that Alg L in immobilized form was enzymatically active. Even after 21 d, Alg L in immobilized form retained 80% of its original activity, compared to its free counterpart, which retained only 20% of its original activity. In fouling experiments using tap water containing 50 mg L(-1) alginate, a simple backwash could remove the fouling on Alg L immobilized membrane, but not that on the control membrane. Atomic force microscopic analysis and bright field microscopic images of the fouled test membrane after backwash showed significant removal of fouling, while fouling on the control membrane remained largely intact. The immobilized Alg L remained active even after 10 runs of fouling-backwash cycle. The present antifouling technology using immobilized enzyme is suitable for keeping ultrafiltration membranes clean without the use of toxic chemical biocides.

  18. Characterization of smart auto-degradative hydrogel matrix containing alginate lyase to enhance levofloxacin delivery against bacterial biofilms.

    PubMed

    Islan, German A; Dini, Cecilia; Bartel, Laura C; Bolzán, Alejandro D; Castro, Guillermo R

    2015-12-30

    The aim of the present work is the characterization of smart auto-degradable microspheres composed of calcium alginate/high methoxylated pectin containing an alginate lyase (AL) from Sphingobacterium multivorum and levofloxacin. Microspheres were prepared by ionotropic gelation containing AL in its inactive form at pH 4.0. Incubation of microspheres in Tris-HCl and PBS buffers at pH 7.40 allowed to establish the effect of ion-chelating phosphate on matrix erodability and suggested an intrinsically activation of AL by turning the pH close to neutrality. Scanning electron and optical microscopies revealed the presence of holes and surface changes in AL containing microspheres. Furthermore, texturometric parameters, DSC profiles and swelling properties were showing strong changes in microspheres properties. Encapsulation of levofloxacin into microspheres containing AL showed 70% efficiency and 35% enhancement of antimicrobial activity against Pseudomonas aeruginosa biofilm. Levofloxacin release from microspheres was not changed at acidic pH, but was modified at neutral pH in presence of AL. Advantageously, only gel matrix debris were detectable after overnight incubation, indicating an autodegradative gel process activated by the pH. Absence of matrix cytotoxicity and a reduction of the levofloxacin toxicity after encapsulation were observed in mammalian CHO-K1 cell cultures. These properties make the system a potent and versatile tool for antibiotic oral delivery targeted to intestine, enhancing the drug bioavailability to eradicate bacterial biofilm and avoiding possible intestinal obstructions.

  19. Micellar electrokinetic capillary chromatography determination of alginic acid in pharmaceutical formulations after treatment with alginate lyase and UV detection.

    PubMed

    Volpi, Nicola

    2008-09-01

    A new highly specific and sensitive capillary electrophoresis method (electrokinetic chromatography with SDS) for the determination of the total alginic acid (AA) content in pharmaceutical formulations is described by means of capillary electrophoresis at 230 nm after treatment with alginate lyase [4.2.2.3] and separation of unsaturated products, Delta-oligomers (DeltaHexA-[HexA](n)), in particular, DP3 (DeltaHexA-HexA-HexA) and DP4 (DeltaHexA-HexA-HexA-HexA). Using a buffer constituted with 10 mM sodium borate and 50 mM SDS at pH 9.0, micellar electrokinetic capillary chromatography was able to determine with very high resolution the AA Delta-oligomers produced by the action of the lyase (mainly DP3 and DP4) as one single species. The intra- and inter-day variations (CV%) were between 6.3 and 9.1 for migration time and between 2.5 and 5.7 for peak area, respectively. The calibration curve showed good linearity for the examined concentration range (60-360 ng) with an average correlation coefficient greater than 0.980. The lowest detection limit and the lowest quantitation limit of the method were 15 ng (0.25 mg/mL) and 40 ng (0.67 mg/mL), respectively. The intra- and inter-day variations in terms of CV% were 5.5 and 8.6%, respectively, and the intra- and inter-day accuracy was estimated to range from 4.1 to 8.9%, while the percent recoveries of AA were calculated to be 102, 97 and 93% for different AA amounts. Variations in temperatures, voltage and buffer composition in comparison with adopted conditions within a 10% limit do not modify the electrophoresis results. The evaluation of AA was performed in both solid and liquid pharmaceutical formulations also in the presence of other ingredients, in particular, aluminium, sodium and potassium bicarbonate, and emulsifying and flavouring agents. The quantitative results obtained were 101.2+/-3.4% of AA content in tablets and 98.4+/-2.8% in liquid formulation, in total conformity with the label claims.

  20. Engineered yeast whole-cell biocatalyst for direct degradation of alginate from macroalgae and production of non-commercialized useful monosaccharide from alginate.

    PubMed

    Takagi, Toshiyuki; Yokoi, Takahiro; Shibata, Toshiyuki; Morisaka, Hironobu; Kuroda, Kouichi; Ueda, Mitsuyoshi

    2016-02-01

    Alginate is a major component of brown macroalgae. In macroalgae, an endolytic alginate lyase first degrades alginate into oligosaccharides. These oligosaccharides are further broken down into monosaccharides by an exolytic alginate lyase. In this study, genes encoding various alginate lyases derived from alginate-assimilating marine bacterium Saccharophagus degradans were isolated, and their enzymes were displayed using the yeast cell surface display system. Alg7A-, Alg7D-, and Alg18J-displaying yeasts showed endolytic alginate lyase activity. On the other hand, Alg7K-displaying yeast showed exolytic alginate lyase activity. Alg7A, Alg7D, Alg7K, and Alg18J, when displayed on yeast cell surface, demonstrated both polyguluronate lyase and polymannuronate lyase activities. Additionally, polyguluronic acid could be much easily degraded by Alg7A, Alg7K, and Alg7D than polymannuronic acid. In contrast, polymannuronic acid could be much easily degraded by Alg18J than polyguluronic acid. We further constructed yeasts co-displaying endolytic and exolytic alginate lyases. Degradation efficiency by the co-displaying yeasts were significantly higher than single alginate lyase-displaying yeasts. Alg7A/Alg7K co-displaying yeast had maximum alginate degrading activity, with production of 1.98 g/L of reducing sugars in a 60-min reaction. This system developed, along with our findings, will contribute to the efficient utilization and production of useful and non-commercialized monosaccharides from alginate by Saccharomyces cerevisiae.

  1. Functional Exploration of the Polysaccharide Lyase Family PL6

    PubMed Central

    Mathieu, Sophie; Henrissat, Bernard; Labre, Flavien; Skjåk-Bræk, Gudmund; Helbert, William

    2016-01-01

    Alginate, the main cell-wall polysaccharide of brown algae, is composed of two residues: mannuronic acid (M-residues) and, its C5-epimer, guluronic acid (G-residues). Alginate lyases define a class of enzymes that cleave the glycosidic bond of alginate by β-elimination. They are classified according to their ability to recognize the distribution of M- and G-residues and are named M-, G- or MG-lyases. In the CAZy database, alginate lyases have been grouped by sequence similarity into seven distinct polysaccharide lyase families. The polysaccharide lyase family PL6 is subdivided into three subfamilies. Subfamily PL6_1 includes three biochemically characterized enzymes (two alginate lyases and one dermatan sulfatase lyase). No characterized enzymes have been described in the two other subfamilies (PL6_2 and PL6_3). To improve the prediction of polysaccharide-lyase activity in the PL6 family, we re-examined the classification of the PL6 family and biochemically characterized a set of enzymes reflecting the diversity of the protein sequences. Our results show that subfamily PL6_1 includes two dermatan sulfates lyases and several alginate lyases that have various substrate specificities and modes of action. In contrast, subfamilies PL6_2 and PL6_3 were found to contain only endo-poly-MG-lyases. PMID:27438604

  2. Induced-fit motion of a lid loop involved in catalysis in alginate lyase A1-III.

    PubMed

    Mikami, Bunzo; Ban, Mizuho; Suzuki, Sachiko; Yoon, Hye-Jin; Miyake, Osamu; Yamasaki, Masayuki; Ogura, Kohei; Maruyama, Yukie; Hashimoto, Wataru; Murata, Kousaku

    2012-09-01

    The structures of two mutants (H192A and Y246F) of a mannuronate-specific alginate lyase, A1-III, from Sphingomonas species A1 complexed with a tetrasaccharide substrate [4-deoxy-L-erythro-hex-4-ene-pyranosyluronate-(mannuronate)(2)-mannuronic acid] were determined by X-ray crystallography at around 2.2 Å resolution together with the apo form of the H192A mutant. The final models of the complex forms, which comprised two monomers (of 353 amino-acid residues each), 268-287 water molecules and two tetrasaccharide substrates, had R factors of around 0.17. A large conformational change occurred in the position of the lid loop (residues 64-85) in holo H192A and Y246F compared with that in apo H192A. The lid loop migrated about 14 Å from an open form to a closed form to interact with the bound tetrasaccharide and a catalytic residue. The tetrasaccharide was bound in the active cleft at subsites -3 to +1 as a substrate form in which the glycosidic linkage to be cleaved existed between subsites -1 and +1. In particular, the O(η) atom of Tyr68 in the closed lid loop forms a hydrogen bond to the side chain of a presumed catalytic residue, O(η) of Tyr246, which acts both as an acid and a base catalyst in a syn mechanism.

  3. Alginate-modifying enzymes: biological roles and biotechnological uses

    PubMed Central

    Ertesvåg, Helga

    2015-01-01

    Alginate denotes a group of industrially important 1-4-linked biopolymers composed of the C-5-epimers β-D-mannuronic acid (M) and α-L-guluronic acid (G). The polysaccharide is manufactured from brown algae where it constitutes the main structural cell wall polymer. The physical properties of a given alginate molecule, e.g., gel-strength, water-binding capacity, viscosity and biocompatibility, are determined by polymer length, the relative amount and distribution of G residues and the acetyl content, all of which are controlled by alginate modifying enzymes. Alginate has also been isolated from some bacteria belonging to the genera Pseudomonas and Azotobacter, and bacterially synthesized alginate may be O-acetylated at O-2 and/or O-3. Initially, alginate is synthesized as polymannuronic acid, and some M residues are subsequently epimerized to G residues. In bacteria a mannuronan C-5-epimerase (AlgG) and an alginate acetylase (AlgX) are integral parts of the protein complex necessary for alginate polymerization and export. All alginate-producing bacteria use periplasmic alginate lyases to remove alginate molecules aberrantly released to the periplasm. Alginate lyases are also produced by organisms that utilize alginate as carbon source. Most alginate-producing organisms encode more than one mannuronan C-5 epimerase, each introducing its specific pattern of G residues. Acetylation protects against further epimerization and from most alginate lyases. An enzyme from Pseudomonas syringae with alginate deacetylase activity has been reported. Functional and structural studies reveal that alginate lyases and epimerases have related enzyme mechanisms and catalytic sites. Alginate lyases are now utilized as tools for alginate characterization. Secreted epimerases have been shown to function well in vitro, and have been engineered further in order to obtain enzymes that can provide alginates with new and desired properties for use in medical and pharmaceutical applications

  4. Cloning and characterization of a novel oligoalginate lyase from a newly isolated bacterium Sphingomonas sp. MJ-3.

    PubMed

    Park, Hwan Hee; Kam, Natania; Lee, Eun Yeol; Kim, Hee Sook

    2012-04-01

    A bacterium possessing alginate-degrading activity was isolated from marine brown seaweed soup liquefied by salted and fermented anchovy. The isolated strain was designated as Sphingomonas sp. MJ-3 based on the analyses of 16S ribosomal DNA sequences, 16S-23S internal transcribed spacer region sequences, biochemical characteristics, and cellular fatty acid composition. A novel alginate lyase gene was cloned from genomic DNA library and then expressed in Escherichia coli. When the deduced amino acid sequence was compared with the sequences on the databases, interestingly, the cloned gene product was predicted to consist of AlgL (alginate lyase L)-like and heparinase-like protein domain. The MJ-3 alginate lyase gene shared below 27.0% sequence identity with exolytic alginate lyase of Sphingomonas sp. A1. The optimal pH and temperature for the recombinant MJ-3 alginate lyase were 6.5 and 50°C, respectively. The final degradation products of alginate oligosaccharides were analyzed by electrospray ionization mass spectrometry and proved to be alginate monosaccharides. Based on the results, the recombinant alginate lyase from Sphingomonas sp. MJ-3 is regarded as an oligoalginate lyase that can degrade oligoalginate and alginate into alginate monosaccharides.

  5. In vitro interaction between alginate lyase and amphotericin B against Aspergillus fumigatus biofilm determined by different methods.

    PubMed

    Bugli, Francesca; Posteraro, Brunella; Papi, Massimiliano; Torelli, Riccardo; Maiorana, Alessandro; Paroni Sterbini, Francesco; Posteraro, Patrizia; Sanguinetti, Maurizio; De Spirito, Marco

    2013-03-01

    Aspergillus fumigatus biofilms represent a problematic clinical entity, especially because of their recalcitrance to antifungal drugs, which poses a number of therapeutic implications for invasive aspergillosis, the most difficult-to-treat Aspergillus-related disease. While the antibiofilm activities of amphotericin B (AMB) deoxycholate and its lipid formulations (e.g., liposomal AMB [LAMB]) are well documented, the effectiveness of these drugs in combination with nonantifungal agents is poorly understood. In the present study, in vitro interactions between polyene antifungals (AMB and LAMB) and alginate lyase (AlgL), an enzyme degrading the polysaccharides produced as extracellular polymeric substances (EPSs) within the biofilm matrix, against A. fumigatus biofilms were evaluated by using the checkerboard microdilution and the time-kill assays. Furthermore, atomic force microscopy (AFM) was used to image and quantify the effects of AlgL-antifungal combinations on biofilm-growing hyphal cells. On the basis of fractional inhibitory concentration index values, synergy was found between both AMB formulations and AlgL, and this finding was also confirmed by the time-kill test. Finally, AFM analysis showed that when A. fumigatus biofilms were treated with AlgL or polyene alone, as well as with their combination, both a reduction of hyphal thicknesses and an increase of adhesive forces were observed compared to the findings for untreated controls, probably owing to the different action by the enzyme or the antifungal compounds. Interestingly, marked physical changes were noticed in A. fumigatus biofilms exposed to the AlgL-antifungal combinations compared with the physical characteristics detected after exposure to the antifungals alone, indicating that AlgL may enhance the antibiofilm activity of both AMB and LAMB, perhaps by disrupting the hypha-embedding EPSs and thus facilitating the drugs to reach biofilm cells. Taken together, our results suggest that a combination

  6. S1P lyase in skeletal muscle regeneration and satellite cell activation: exposing the hidden lyase.

    PubMed

    Saba, Julie D; de la Garza-Rodea, Anabel S

    2013-01-01

    Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid whose actions are essential for many physiological processes including angiogenesis, lymphocyte trafficking and development. In addition, S1P serves as a muscle trophic factor that enables efficient muscle regeneration. This is due in part to S1P's ability to activate quiescent muscle stem cells called satellite cells (SCs) that are needed for muscle repair. However, the molecular mechanism by which S1P activates SCs has not been well understood. Further, strategies for harnessing S1P signaling to recruit SCs for therapeutic benefit have been lacking. S1P is irreversibly catabolized by S1P lyase (SPL), a highly conserved enzyme that catalyzes the cleavage of S1P at carbon bond C(2-3), resulting in formation of hexadecenal and ethanolamine-phosphate. SPL enhances apoptosis through substrate- and product-dependent events, thereby regulating cellular responses to chemotherapy, radiation and ischemia. SPL is undetectable in resting murine skeletal muscle. However, we recently found that SPL is dynamically upregulated in skeletal muscle after injury. SPL upregulation occurred in the context of a tightly orchestrated genetic program that resulted in a transient S1P signal in response to muscle injury. S1P activated quiescent SCs via a sphingosine-1-phosphate receptor 2 (S1P2)/signal transducer and activator of transcription 3 (STAT3)-dependent pathway, thereby facilitating skeletal muscle regeneration. Mdx mice, which serve as a model for muscular dystrophy (MD), exhibited skeletal muscle SPL upregulation and S1P deficiency. Pharmacological SPL inhibition raised skeletal muscle S1P levels, enhanced SC recruitment and improved mdx skeletal muscle regeneration. These findings reveal how S1P can activate SCs and indicate that SPL suppression may provide a therapeutic strategy for myopathies. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.

  7. Bacterial community structure and predicted alginate metabolic pathway in an alginate-degrading bacterial consortium.

    PubMed

    Kita, Akihisa; Miura, Toyokazu; Kawata, Satoshi; Yamaguchi, Takeshi; Okamura, Yoshiko; Aki, Tsunehiro; Matsumura, Yukihiko; Tajima, Takahisa; Kato, Junichi; Nishio, Naomichi; Nakashimada, Yutaka

    2016-03-01

    Methane fermentation is one of the effective approaches for utilization of brown algae; however, this process is limited by the microbial capability to degrade alginate, a main polysaccharide found in these algae. Despite its potential, little is known about anaerobic microbial degradation of alginate. Here we constructed a bacterial consortium able to anaerobically degrade alginate. Taxonomic classification of 16S rRNA gene, based on high-throughput sequencing data, revealed that this consortium included two dominant strains, designated HUA-1 and HUA-2; these strains were related to Clostridiaceae bacterium SK082 (99%) and Dysgonomonas capnocytophagoides (95%), respectively. Alginate lyase activity and metagenomic analyses, based on high-throughput sequencing data, revealed that this bacterial consortium possessed putative genes related to a predicted alginate metabolic pathway. However, HUA-1 and 2 did not grow on agar medium with alginate by using roll-tube method, suggesting the existence of bacterial interactions like symbiosis for anaerobic alginate degradation.

  8. Redesign of MST enzymes to target lyase activity instead promotes mutase and dehydratase activities.

    PubMed

    Meneely, Kathleen M; Luo, Qianyi; Lamb, Audrey L

    2013-11-01

    The isochorismate and salicylate synthases are members of the MST family of enzymes. The isochorismate synthases establish an equilibrium for the conversion chorismate to isochorismate and the reverse reaction. The salicylate synthases convert chorismate to salicylate with an isochorismate intermediate; therefore, the salicylate synthases perform isochorismate synthase and isochorismate-pyruvate lyase activities sequentially. While the active site residues are highly conserved, there are two sites that show trends for lyase-activity and lyase-deficiency. Using steady state kinetics and HPLC progress curves, we tested the "interchange" hypothesis that interconversion of the amino acids at these sites would promote lyase activity in the isochorismate synthases and remove lyase activity from the salicylate synthases. An alternative, "permute" hypothesis, that chorismate-utilizing enzymes are designed to permute the substrate into a variety of products and tampering with the active site may lead to identification of adventitious activities, is tested by more sensitive NMR time course experiments. The latter hypothesis held true. The variant enzymes predominantly catalyzed chorismate mutase-prephenate dehydratase activities, sequentially generating prephenate and phenylpyruvate, augmenting previously debated (mutase) or undocumented (dehydratase) adventitious activities.

  9. Redesign of MST enzymes to target lyase activity instead promotes mutase and dehydratase activities

    PubMed Central

    Meneely, Kathleen M.; Luo, Qianyi; Lamb, Audrey L.

    2013-01-01

    The isochorismate and salicylate synthases are members of the MST family of enzymes. The isochorismate synthases establish an equilibrium for the conversion chorismate to isochorismate and the reverse reaction. The salicylate synthases convert chorismate to salicylate with an isochorismate intermediate; therefore, the salicylate synthases perform isochorismate synthase and isochorismate-pyruvate lyase activities sequentially. While the active site residues are highly conserved, there are two sites that show trends for lyase-activity and lyase-deficiency. Using steady state kinetics and HPLC progress curves, we tested the “interchange” hypothesis that interconversion of the amino acids at these sites would promote lyase activity in the isochorismate synthases and remove lyase activity from the salicylate synthases. An alternative, “permute” hypothesis, that chorismate-utilizing enzymes are designed to permute the substrate into a variety of products and tampering with the active site may lead to identification of adventitious activities, is tested by more sensitive NMR time course experiments. The latter hypothesis held true. The variant enzymes predominantly catalyzed chorismate mutase-prephenate dehydratase activities, sequentially generating prephenate and phenylpyruvate, augmenting previously debated (mutase) or undocumented (dehydratase) adventitious activities. PMID:24055536

  10. Impaired 17,20-Lyase Activity in Male Mice Lacking Cytochrome b5 in Leydig Cells

    PubMed Central

    Sondhi, Varun; Owen, Bryn M.; Liu, Jiayan; Chomic, Robert; Kliewer, Steven A.; Hughes, Beverly A.; Arlt, Wiebke; Mangelsdorf, David J.

    2016-01-01

    Androgen and estrogen biosynthesis in mammals requires the 17,20-lyase activity of cytochrome P450 17A1 (steroid 17-hydroxylase/17,20-lyase). Maximal 17,20-lyase activity in vitro requires the presence of cytochrome b5 (b5), and rare cases of b5 deficiency in human beings causes isolated 17,20-lyase deficiency. To study the consequences of conditional b5 removal from testicular Leydig cells in an animal model, we generated Cyb5flox/flox:Sf1-Cre (LeyKO) mice. The LeyKO male mice had normal body weights, testis and sex organ weights, and fertility compared with littermates. Basal serum and urine steroid profiles of LeyKO males were not significantly different than littermates. In contrast, marked 17-hydroxyprogesterone accumulation (100-fold basal) and reduced testosterone synthesis (27% of littermates) were observed after human chorionic gonadotropin stimulation in LeyKO animals. Testis homogenates from LeyKO mice showed reduced 17,20-lyase activity and a 3-fold increased 17-hydroxylase to 17,20-lyase activity ratio, which were restored to normal upon addition of recombinant b5. We conclude that Leydig cell b5 is required for maximal androgen synthesis and to prevent 17-hydroxyprogesterone accumulation in the mouse testis; however, the b5-independent 17,20-lyase activity of mouse steroid 17-hydroxylase/17,20-lyase is sufficient for normal male genital development and fertility. LeyKO male mice are a good model for the biochemistry but not the physiology of isolated 17,20-lyase deficiency in human beings. PMID:26974035

  11. A novel C-S lyase from the latex-producing plant Taraxacum brevicorniculatum displays alanine aminotransferase and l-cystine lyase activity.

    PubMed

    Munt, Oliver; Prüfer, Dirk; Schulze Gronover, Christian

    2013-01-01

    We isolated a novel pyridoxal-5-phosphate-dependent l-cystine lyase from the dandelion Taraxacum brevicorniculatum. Real time qPCR analysis showed that C-S lyase from Taraxacum brevicorniculatum (TbCSL) mRNA is expressed in all plant tissues, although at relatively low levels in the latex and pedicel. The 1251 bp TbCSL cDNA encodes a protein with a calculated molecular mass of 46,127 kDa. It is homologous to tyrosine and alanine aminotransferases (AlaATs) as well as to an Arabidopsis thaliana carbon-sulfur lyase (C-S lyase) (SUR1), which has a role in glucosinolate metabolism. TbCSL displayed in vitrol-cystine lyase and AlaAT activities of 4 and 19nkatmg(-1) protein, respectively. However, we detected no in vitro tyrosine aminotransferase (TyrAT) activity and RNAi knockdown of the enzyme had no effect on phenotype, showing that TbCSL substrates might be channeled into redundant pathways. TbCSL is in vivo localized in the cytosol and functions as a C-S lyase or an aminotransferase in planta, but the purified enzyme converts at least two substrates specifically, and can thus be utilized for further in vitro applications.

  12. Characterization of activity and expression of isocitrate lyase in Mycobacterium avium and Mycobacterium tuberculosis.

    PubMed

    Höner Zu Bentrup, K; Miczak, A; Swenson, D L; Russell, D G

    1999-12-01

    Analysis by two-dimensional gel electrophoresis revealed that Mycobacterium avium expresses several proteins unique to an intracellular infection. One abundant protein with an apparent molecular mass of 50 kDa was isolated, and the N-terminal sequence was determined. It matches a sequence in the M. tuberculosis database (Sanger) with similarity to the enzyme isocitrate lyase of both Corynebacterium glutamicum and Rhodococcus fascians. Only marginal similarity was observed between this open reading frame (ORF) (termed icl) and a second distinct ORF (named aceA) which exhibits a low similarity to other isocitrate lyases. Both ORFs can be found as distinct genes in the various mycobacterial databases recently published. Isocitrate lyase is a key enzyme in the glyoxylate cycle and is essential as an anapleurotic enzyme for growth on acetate and certain fatty acids as carbon source. In this study we express and purify Icl, as well as AceA proteins, and show that both exhibit isocitrate lyase activity. Various known inhibitors for isocitrate lyase were effective. Furthermore, we present evidence that in both M. avium and M. tuberculosis the production and activity of the isocitrate lyase is enhanced under minimal growth conditions when supplemented with acetate or palmitate.

  13. The Active Site of Oligogalacturonate Lyase Provides Unique Insights into Cytoplasmic Oligogalacturonate β-Elimination*

    PubMed Central

    Abbott, D. Wade; Gilbert, Harry J.; Boraston, Alisdair B.

    2010-01-01

    Oligogalacturonate lyases (OGLs; now also classified as pectate lyase family 22) are cytoplasmic enzymes found in pectinolytic members of Enterobacteriaceae, such as the enteropathogen Yersinia enterocolitica. OGLs utilize a β-elimination mechanism to preferentially catalyze the conversion of saturated and unsaturated digalacturonate into monogalacturonate and the 4,5-unsaturated monogalacturonate-like molecule, 5-keto-4-deoxyuronate. To provide mechanistic insights into the specificity of this enzyme activity, we have characterized the OGL from Y. enterocolitica, YeOGL, on oligogalacturonides and determined its three-dimensional x-ray structure to 1.65 Å. The model contains a Mn2+ atom in the active site, which is coordinated by three histidines, one glutamine, and an acetate ion. The acetate mimics the binding of the uronate group of galactourono-configured substrates. These findings, in combination with enzyme kinetics and metal supplementation assays, provide a framework for modeling the active site architecture of OGL. This enzyme appears to contain a histidine for the abstraction of the α-proton in the −1 subsite, a residue that is highly conserved throughout the OGL family and represents a unique catalytic base among pectic active lyases. In addition, we present a hypothesis for an emerging relationship observed between the cellular distribution of pectate lyase folding and the distinct metal coordination chemistries of pectate lyases. PMID:20851883

  14. Methanopyrus kandleri topoisomerase V contains three distinct AP lyase active sites in addition to the topoisomerase active site

    PubMed Central

    Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

    2016-01-01

    Topoisomerase V (Topo-V) is the only topoisomerase with both topoisomerase and DNA repair activities. The topoisomerase activity is conferred by a small alpha-helical domain, whereas the AP lyase activity is found in a region formed by 12 tandem helix-hairpin-helix ((HhH)2) domains. Although it was known that Topo-V has multiple repair sites, only one had been mapped. Here, we show that Topo-V has three AP lyase sites. The atomic structure and Small Angle X-ray Scattering studies of a 97 kDa fragment spanning the topoisomerase and 10 (HhH)2 domains reveal that the (HhH)2 domains extend away from the topoisomerase domain. A combination of biochemical and structural observations allow the mapping of the second repair site to the junction of the 9th and 10th (HhH)2 domains. The second site is structurally similar to the first one and to the sites found in other AP lyases. The 3rd AP lyase site is located in the 12th (HhH)2 domain. The results show that Topo-V is an unusual protein: it is the only known protein with more than one (HhH)2 domain, the only known topoisomerase with dual activities and is also unique by having three AP lyase repair sites in the same polypeptide. PMID:26908655

  15. Methanopyrus kandleri topoisomerase V contains three distinct AP lyase active sites in addition to the topoisomerase active site.

    PubMed

    Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

    2016-04-20

    Topoisomerase V (Topo-V) is the only topoisomerase with both topoisomerase and DNA repair activities. The topoisomerase activity is conferred by a small alpha-helical domain, whereas the AP lyase activity is found in a region formed by 12 tandem helix-hairpin-helix ((HhH)2) domains. Although it was known that Topo-V has multiple repair sites, only one had been mapped. Here, we show that Topo-V has three AP lyase sites. The atomic structure and Small Angle X-ray Scattering studies of a 97 kDa fragment spanning the topoisomerase and 10 (HhH)2 domains reveal that the (HhH)2 domains extend away from the topoisomerase domain. A combination of biochemical and structural observations allow the mapping of the second repair site to the junction of the 9th and 10th (HhH)2 domains. The second site is structurally similar to the first one and to the sites found in other AP lyases. The 3rd AP lyase site is located in the 12th (HhH)2 domain. The results show that Topo-V is an unusual protein: it is the only known protein with more than one (HhH)2 domain, the only known topoisomerase with dual activities and is also unique by having three AP lyase repair sites in the same polypeptide.

  16. Reduced phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities and lignin synthesis in wheat grown under low pressure sodium lamps

    NASA Technical Reports Server (NTRS)

    Guerra, D.; Anderson, A. J.; Salisbury, F. B.

    1985-01-01

    Wheat (Triticum aestivum L. cv Fremont) grown in hydroponic culture under 24-hour continuous irradiation at 560 to 580 micromoles per square meter per second from either metalhalide (MH), high pressure sodium (HPS), or low pressure sodium (LPS) lamps reached maturity in 70 days. Grain yields were similar under all three lamps, although LPS-grown plants lodged at maturity. Phenylalanine ammonia-lyase (PAL) and a tyrosine ammonia lyase (TAL) with lesser activity were detected in all extracts of leaf, inflorescence, and stem. Ammonia-lyase activities increased with age of the plant, and plants grown under the LPS lamp displayed PAL and TAL activities lower than wheat cultured under MH and HPS radiation. Greenhouse solar-grown wheat had the highest PAL and TAL activities. Lignin content of LPS-grown wheat was also significantly reduced from that of plants grown under MH or HPS lamps or in the greenhouse, showing a correlation with the reduced PAL and TAL activities. Ratios of far red-absorbing phytochrome to total phytochrome were similar for all three lamps, but the data do not yet warrant a conclusion about specific wavelengths missing from the LPS lamps that might have induced PAL and TAL activities in plants under the other lamps.

  17. Active site proton delivery and the lyase activity of human CYP17A1

    SciTech Connect

    Khatri, Yogan; Gregory, Michael C.; Grinkova, Yelena V.; Denisov, Ilia G.; Sligar, Stephen G.

    2014-01-03

    Highlights: •The disruption of PREG/PROG hydroxylation activity by T306A showed the participation of Cpd I. •T306A supports the involvement of a nucleophilic peroxo-anion during lyase activity. •The presence of cytochrome b{sub 5} augments C–C lyase activity. •Δ5-Steroids are preferred substrates for CYP17 catalysis. -- Abstract: Cytochrome P450 CYP17A1 catalyzes a series of reactions that lie at the intersection of corticoid and androgen biosynthesis and thus occupies an essential role in steroid hormone metabolism. This multifunctional enzyme catalyzes the 17α-hydroxylation of Δ4- and Δ5-steroids progesterone and pregnenolone to form the corresponding 17α-hydroxy products through its hydroxylase activity, and a subsequent 17,20-carbon–carbon scission of pregnene-side chain produce the androgens androstenedione (AD) and dehydroepiandrosterone (DHEA). While the former hydroxylation reaction is believed to proceed through a conventional “Compound I” rebound mechanism, it has been suggested that the latter carbon cleavage is initiated by an iron-peroxy intermediate. We report on the role of Thr306 in CYP17 catalysis. Thr306 is a member of the conserved acid/alcohol pair thought to be essential for the efficient delivery of protons required for hydroperoxoanion heterolysis and formation of Compound I in the cytochromes P450. Wild type and T306A CYP17A1 self-assembled in Nanodiscs were used to quantitate turnover and coupling efficiencies of CYP17’s physiological Δ4- and Δ5-substrates. We observed that T306A co-incorporated in Nanodiscs with its redox partner cytochrome P450 oxidoreductase, coupled NADPH only by 0.9% and 0.7% compared to the wild type (97% and 22%) during the conversion of pregnenolone and progesterone, respectively, to the corresponding 17-OH products. Despite increased oxidation of pyridine nucleotide, hydroxylase activity was drastically diminished in the T306A mutant, suggesting a high degree of uncoupling in which reducing

  18. Antioxidant activity of low molecular weight alginate produced by thermal treatment.

    PubMed

    Kelishomi, Zahra Habibi; Goliaei, Bahram; Mahdavi, Hossein; Nikoofar, Alireza; Rahimi, Mahmood; Moosavi-Movahedi, Ali Akbar; Mamashli, Fatemeh; Bigdeli, Bahareh

    2016-04-01

    By definition, antioxidants are molecules that inhibit the oxidation of other molecules. Therefore, such compounds have very important clinical roles. In this study alginate polymer was depolymerized by heat treatment. The resulting low molecular weight alginates were investigated by UV-visible spectroscopy, Viscometry, Dynamic light scattering and FT-IR spectroscopy techniques. Antioxidant properties of these heat products were studied by ABTS and superoxide radical scavenging assays. Results showed that heating caused breaks in the polymer chain and so generation of low molecular weight alginates. Antioxidant measurements confirmed antioxidant activity of alginate increased upon a decrease in molecular weight. Therefore, low molecular weight alginate produced by heating could be considered as a stronger antioxidant than alginate polymer. These products could be useful for industrial and biomedical applications.

  19. Rev1 is a base excision repair enzyme with 5′-deoxyribose phosphate lyase activity

    PubMed Central

    Prasad, Rajendra; Poltoratsky, Vladimir; Hou, Esther W.; Wilson, Samuel H.

    2016-01-01

    Rev1 is a member of the Y-family of DNA polymerases and is known for its deoxycytidyl transferase activity that incorporates dCMP into DNA and its ability to function as a scaffold factor for other Y-family polymerases in translesion bypass events. Rev1 also is involved in mutagenic processes during somatic hypermutation of immunoglobulin genes. In light of the mutation pattern consistent with dCMP insertion observed earlier in mouse fibroblast cells treated with a base excision repair-inducing agent, we questioned whether Rev1 could also be involved in base excision repair (BER). Here, we uncovered a weak 5′-deoxyribose phosphate (5′-dRP) lyase activity in mouse Rev1 and demonstrated the enzyme can mediate BER in vitro. The full-length Rev1 protein and its catalytic core domain are similar in their ability to support BER in vitro. The dRP lyase activity in both of these proteins was confirmed by NaBH4 reduction of the Schiff base intermediate and kinetics studies. Limited proteolysis, mass spectrometry and deletion analysis localized the dRP lyase active site to the C-terminal segment of Rev1's catalytic core domain. These results suggest that Rev1 could serve as a backup polymerase in BER and could potentially contribute to AID-initiated antibody diversification through this activity. PMID:27683219

  20. DNA sequences that activate isocitrate lyase gene expression during late embryogenesis and during postgerminative growth.

    PubMed Central

    Zhang, J Z; Santes, C M; Engel, M L; Gasser, C S; Harada, J J

    1996-01-01

    We analyzed DNA sequences that regulate the expression of an isocitrate lyase gene from Brassica napus L. during late embryogenesis and during postgerminative growth to determine whether glyoxysomal function is induced by a common mechanism at different developmental stages. beta-Glucuronidase constructs were used both in transient expression assays in B. napus and in transgenic Arabidopsis thaliana to identify the segments of the isocitrate lyase 5' flanking region that influence promoter activity. DNA sequences that play the principal role in activating the promoter during post-germinative growth are located more than 1,200 bp upstream of the gene. Distinct DNA sequences that were sufficient for high-level expression during late embryogenesis but only low-level expression during postgerminative growth were also identified. Other parts of the 5' flanking region increased promoter activity both in developing seed and in seedlings. We conclude that a combination of elements is involved in regulating the isocitrate lyase gene and that distinct DNA sequences play primary roles in activating the gene in embryos and in seedlings. These findings suggest that different signals contribute to the induction of glyoxysomal function during these two developmental stages. We also showed that some of the constructs were expressed differently in transient expression assays and in transgenic plants. PMID:8934622

  1. A Facile Stable-Isotope Dilution Method for Determination of Sphingosine Phosphate Lyase Activity

    PubMed Central

    Suh, Jung H.; Eltanawy, Abeer; Rangan, Apoorva; Saba, Julie D.

    2015-01-01

    A new technique for quantifying sphingosine phosphate lyase activity in biological samples is described. In this procedure, 2-hydrazinoquinoline is used to convert (2E)-hexadecenal into the corresponding hydrazone derivative to improve ionization efficiency and selectivity of detection. Combined utilization of liquid chromatographic separation and multiple reaction monitoring-mass spectrometry allows for simultaneous quantification of the substrate S1P and product (2E)-hexadecenal. Incorporation of (2E)-d5-hexadecenal as an internal standard improves detection accuracy and precision. A simple one-step derivatization procedure eliminates the need for further extractions. Limits of quantification for (2E)-hexadecenal and sphingosine-1-phosphate are 100 and 50 fmol, respectively. The assay displays a wide dynamic detection range useful for detection of low basal sphingosine phosphate lyase activity in wild type cells, SPL-overexpressing cell lines, and wild type mouse tissues. Compared to current methods, the capacity for simultaneous detection of sphingosine-1-phosphate and (2E)-hexadecenal greatly improves the accuracy of results and shows excellent sensitivity and specificity for sphingosine phosphate lyase activity detection. PMID:26408264

  2. Structure-based engineering of a pectate lyase with improved specific activity for ramie degumming.

    PubMed

    Zhou, Zhanping; Liu, Yang; Chang, Zhenying; Wang, Huilin; Leier, André; Marquez-Lago, Tatiana T; Ma, Yanhe; Li, Jian; Song, Jiangning

    2017-04-01

    Biotechnological applications of microbial pectate lyases (Pels) in plant fiber processing are promising, eco-friendly substitutes for conventional chemical degumming processes. However, to potentiate the enzymes' use for industrial applications, resolving the molecular structure to elucidate catalytic mechanisms becomes necessary. In this manuscript, we report the high resolution (1.45 Å) crystal structure of pectate lyase (pelN) from Paenibacillus sp. 0602 in apo form. Through sequence alignment and structural superposition with other members of the polysaccharide lyase (PL) family 1 (PL1), we determined that pelN shares the characteristic right-handed β-helix and is structurally similar to other members of the PL1 family, while exhibiting key differences in terms of catalytic and substrate binding residues. Then, based on information from structure alignments with other PLs, we engineered a novel pelN. Our rational design yielded a pelN mutant with a temperature for enzymatic activity optimally shifted from 67.5 to 60 °C. Most importantly, this pelN mutant displayed both higher specific activity and ramie fiber degumming ability when compared with the wild-type enzyme. Altogether, our rational design method shows great potential for industrial applications. Moreover, we expect the reported high-resolution crystal structure to provide a solid foundation for future rational, structure-based engineering of genetically enhanced pelNs.

  3. Potato signal molecules that activate pectate lyase synthesis in Pectobacterium atrosepticum SCRI1043.

    PubMed

    Tarasova, Nadezhda; Gorshkov, Vladimir; Petrova, Olga; Gogolev, Yuri

    2013-07-01

    A new type of plant-derived signal molecules that activate extracellular pectate lyase activity in phytopathogenic bacterium Pectobacterium atrosepticum SCRI1043 was revealed. These compounds were characterized and partially purified by means of several approaches including RT-PCR analysis, luminescence bioassay and HPLC fractionation. They were smaller than 1 kDa, thermoresistant, nonproteinaceous, hydrophilic, and slightly negatively charged molecules. Using gene expression analysis and bacterial biosensor assay the mode of activity of revealed compounds was studied. The possibility of their action through quorum sensing- and KdgR-mediated pathways was analyzed.

  4. Activity and Enantioselectivity of the Hydroxynitrile Lyase MeHNL in Dry Organic Solvents

    PubMed Central

    Paravidino, Monica; Sorgedrager, Menno J; Orru, Romano V A; Hanefeld, Ulf

    2010-01-01

    Water concentration affects both the enantioselectivity and activity of enzymes in dry organic media. Its influence has been investigated using the hydrocyanation of benzaldehyde catalyzed by hydroxynitrile lyase cross-linked enzyme aggregate (MeHNL-CLEA) as a model reaction. The enzyme displayed higher enantioselectivity at higher water concentration, thus suggesting a positive effect of enzyme flexibility on selectivity. The activity increased on reducing the solvent water content, but drastic dehydration of the enzyme resulted in a reversible loss of activity. PMID:20486110

  5. Requirements for 5′dRP/AP lyase activity in Ku

    PubMed Central

    Strande, Natasha T.; Carvajal-Garcia, Juan; Hallett, Ryan A.; Waters, Crystal A.; Roberts, Steven A.; Strom, Christina; Kuhlman, Brian; Ramsden, Dale A.

    2014-01-01

    The non-homologous end joining (NHEJ) pathway is used in diverse species to repair chromosome breaks, and is defined in part by a requirement for Ku. We previously demonstrated mammalian Ku has intrinsic 5′ deoxyribosephosphate (5′dRP) and apurinic/apyrimidinic (AP) lyase activity, and showed this activity is important for excising abasic site damage from ends. Here we employ systematic mutagenesis to clarify the protein requirements for this activity. We identify lysine 31 in the 70 kD subunit (Ku70 K31) as the primary candidate nucleophile required for catalysis, but additional mutation of Ku70 K160 and six other lysines within Ku80 were required to eliminate all activity. Ku from Saccharomyces cerevisiae also possesses 5′dRP/AP lyase activity, and robust activity was also reliant on lysines in Ku70 analogous to K31 and K160. By comparison, these lysines are not conserved in Xenopus laevis Ku, and Ku from this species has negligible activity. A role for residues flanking Ku70 K31 in expanding the range of abasic site contexts that can be used as substrate was also identified. Our results suggest an active site well located to provide the substrate specificity required for its biological role. PMID:25200085

  6. Chemical mechanism of the endogenous argininosuccinate lyase activity of duck lens delta2-crystallin.

    PubMed Central

    Wu, C Y; Lee, H J; Wu, S H; Chen, S T; Chiou, S H; Chang, G G

    1998-01-01

    The endogenous argininosuccinate lyase activity of duck delta2-crystallin was specifically inactivated by the histidine-specific reagent, diethyl pyrocarbonate. The protein was protected by l-citrulline or l-arginine from the diethyl pyrocarbonate inactivation. To characterize further the chemical mechanism of the delta2-crystallin-catalysed reaction, deuterium-labelled argininosuccinate was enzymically synthesized from fumarate and l-arginine with delta2-crystallin in 2H2O. The argininosuccinate synthesized contained about 19% of the anhydride form; however, the deuterium was clearly demonstrated to be incorporated enantioselectively. Only the pro-HR atom at C-9 of the succinate moiety was labelled in the [2H]argininosuccinate-9-d synthesized, which indicates an anti-elimination mechanism for the endogenous argininosuccinate lyase activity of delta2-crystallin. The enzymic activity of duck lens delta2-crystallin in the pH range 5.5-8.5 was investigated using both protium- and deuterium-labelled argininosuccinate as the substrate. From the logkcat versus pH plot, two molecular pKa values of 6.18+/-0.02 and 8.75+/-0.03 were detected in the delta2-crystallin-argininosuccinate binary complex. The former must be dehydronated and the latter hydronated to achieve an optimum reaction rate. The logkcat/Km versus pH plot suggested two molecular pKa values of 5.96+/-0.09 and 8.29+/-0.10 for the free delta2-crystallin to be involved in the substrate binding. Small kinetic isotope effects of 1.17+/-0.02 and 1.05+/-0.09 were found for kcat and kcat/Km respectively. Combining results from labelling and kinetic analysis indicates that the endogenous argininosuccinate lyase activity of duck delta2-crystallin is compatible with a stepwise E1cB mechanism, the rate-limiting step probably at the C-N bond-cleavage step. PMID:9657972

  7. Phosphorylation is the major mechanism regulating isocitrate lyase activity in Paracoccidioides brasiliensis yeast cells.

    PubMed

    Cruz, Aline H da Silva; Brock, Matthias; Zambuzzi-Carvalho, Patrícia F; Santos-Silva, Ludier K; Troian, Rogério F; Góes, Alfredo M; Soares, Célia M de Almeida; Pereira, Maristela

    2011-07-01

    The glyoxylate cycle plays an essential role for anaplerosis of oxaloacetate during growth of microorganisms on carbon sources such as acetate or fatty acids and has been shown to contribute to virulence of several pathogens. Here, we investigated the transcriptional and post-translational regulation of the glyoxylate cycle key enzyme isocitrate lyase (PbICL) in the human pathogenic fungus Paracoccidioides brasiliensis. Although sequence analyses on fungal isocitrate lyases revealed a high phylogenetic conservation, their regulation seems to differ significantly. Closely related Aspergillus species regulate the glyoxylate cycle at the transcriptional level, whereas Pbicl was constitutively expressed in yeast cells. However, only low PbICL activity was detected when cells were grown in the presence of glucose. Two-dimensional gel analyses with subsequent antibody hybridization revealed constitutive production of PbICL, but low PbICL activity on glucose coincided with extensive protein phosphorylation. Since an in vitro dephosphorylation of PbICL from glucose grown cells strongly increased ICL activity and resembled the phosphorylation pattern of highly active acetate grown cells, post-translational modification seems the main mechanism regulating PbICL activity in yeast cells. In agreement, a transfer of yeast cells from glucose to acetate medium increased PbICL activity without requirement of de novo protein synthesis. Thus, inactivation of PbICL by phosphorylation is reversible, denoting a new strategy for the rapid adaptation to changing environmental conditions.

  8. Changes in phenylalanine ammonia-lyase activity and gene expression during storage of asparagus spears.

    PubMed

    Bhowmik, Pankaj K; Matsui, Toshiyuki

    2005-01-01

    A cDNA clone coding phenylalanine ammonia-lyase (PAL) was isolated from a cDNA library prepared from asparagus spears (Asparagus officinalis L. cv. Welcome) using the reverse transcription-polymerase chain reaction (RT-PCR). The partial cDNA clone encoded an mRNA of 527 bp and the derived amino acid sequence was found highly homologous to PAL from rice, maize and barley. Northern blot analysis showed an increase of pAS-PAL mRNA until 24 h at 20 degrees C, which coincided well with PAL activity and fiber development, suggesting that the increase is a response to the wounding associated with harvest.

  9. Effects of alginate on frozen-thawed boar spermatozoa quality, lipid peroxidation and antioxidant enzymes activities.

    PubMed

    Hu, Jinghua; Geng, Guoxia; Li, Qingwang; Sun, Xiuzhu; Cao, Hualin; Liu, Yawei

    2014-06-30

    Although alginate was reported to play an important role as free radical scavengers in vitro and could be used as sources of natural antioxidants, there was no study about the cryoprotective effects of alginate on boar spermatozoa freezing. The objective of this research was to evaluate the effects of different concentrations of alginate added to the freezing extenders on boar spermatozoa motility, plasma membrane integrity, acrosomal integrity, mitochondrial activities, lipid peroxidation and antioxidative enzymes activities (SOD and GSH-Px) after thawing. Alginate was added to the TCG extender to yield six different final concentrations: 0, 0.2, 0.4, 0.6, 0.8, and 1.0mg/mL. The semen extender supplemented with various doses of alginate increased (P<0.05) total motility. The spermatozoa plasma membrane integrity and mitochondrial activity were improved at four different concentrations: 0.4, 0.6, 0.8, 1.0mg/mL. The addition of alginate also provided significantly positive effect on post-thaw boar spermatozoa acrosomal integrity at concentrations of 0.6, 0.8, 1.0mg/mL, compared with that of the control (P<0.05). The freezing extenders with the presence of alginate led to higher SOD and GSH-Px activities and lower MDA levels, in comparison to the control (P<0.05). In summary, alginate exhibited a dose-related response on frozen-thawed boar spermatozoa motility, functional integrity and antioxidative capacity at appropriate concentrations. Therefore alginate could be employed as an effective cryoprotectant in boar spermatozoa cryopreservation.

  10. Role of the Arabidopsis DNA glycosylase/lyase ROS1 in active DNA demethylation

    PubMed Central

    Agius, Fernanda; Kapoor, Avnish; Zhu, Jian-Kang

    2006-01-01

    DNA methylation is a stable epigenetic mark for transcriptional gene silencing in diverse organisms including plants and many animals. In contrast to the well characterized mechanism of DNA methylation by methyltransferases, the mechanisms and function of active DNA demethylation have been controversial. Genetic evidence suggested that the DNA glycosylase domain-containing protein ROS1 of Arabidopsis is a putative DNA demethylase, because loss-of-function ros1 mutations cause DNA hypermethylation and enhance transcriptional gene silencing. We report here the biochemical characterization of ROS1 and the effect of its overexpression on the DNA methylation of target genes. Our data suggest that the DNA glycosylase activity of ROS1 removes 5-methylcytosine from the DNA backbone and then its lyase activity cleaves the DNA backbone at the site of 5-methylcytosine removal by successive β- and δ-elimination reactions. Overexpression of ROS1 in transgenic plants led to a reduced level of cytosine methylation and increased expression of a target gene. These results demonstrate that ROS1 is a 5-methylcytosine DNA glycosylase/lyase important for active DNA demethylation in Arabidopsis. PMID:16864782

  11. Enzyme-catalyzed phase transition of alginate gels and gelatin-alginate interpenetrated networks.

    PubMed

    Doumèche, Bastien; Picard, Julien; Larreta-Garde, Véronique

    2007-11-01

    The enzyme-catalyzed gel-sol transition of calcium-alginate obtained by internal gelling strategy with the help of an entrapped alginate lyase is described. We show that alginate molecules and enzyme-produced oligoalginates shorten the gel time of physical gelatin gels (5% and 1.5%), probably due to local protein concentration increase. Interpenetrated networks composed of calcium-alginate and of gelatin were obtained only if elongation of gelatin helices inside a pre-existing calcium-alginate network could occur and only for low gelatin concentration (1.5%). The physical gelatin network is almost reversible inside the alginate one. Both networks can be obtained in the presence of alginate lyase, but gel-sol transition of calcium-alginate cannot be obtained in the presence of gelatin.

  12. Composite active site of chondroitin lyase ABC accepting both epimers of uronic acid

    SciTech Connect

    Shaya, D.; Hahn, Bum-Soo; Bjerkan, Tonje Marita; Kim, Wan Seok; Park, Nam Young; Sim, Joon-Soo; Kim, Yeong-Shik; Cygler, M.

    2008-03-19

    Enzymes have evolved as catalysts with high degrees of stereospecificity. When both enantiomers are biologically important, enzymes with two different folds usually catalyze reactions with the individual enantiomers. In rare cases a single enzyme can process both enantiomers efficiently, but no molecular basis for such catalysis has been established. The family of bacterial chondroitin lyases ABC comprises such enzymes. They can degrade both chondroitin sulfate (CS) and dermatan sulfate (DS) glycosaminoglycans at the nonreducing end of either glucuronic acid (CS) or its epimer iduronic acid (DS) by a {beta}-elimination mechanism, which commences with the removal of the C-5 proton from the uronic acid. Two other structural folds evolved to perform these reactions in an epimer-specific fashion: ({alpha}/{alpha}){sub 5} for CS (chondroitin lyases AC) and {beta}-helix for DS (chondroitin lyases B); their catalytic mechanisms have been established at the molecular level. The structure of chondroitinase ABC from Proteus vulgaris showed surprising similarity to chondroitinase AC, including the presence of a Tyr-His-Glu-Arg catalytic tetrad, which provided a possible mechanism for CS degradation but not for DS degradation. We determined the structure of a distantly related Bacteroides thetaiotaomicron chondroitinase ABC to identify additional structurally conserved residues potentially involved in catalysis. We found a conserved cluster located {approx}12 {angstrom} from the catalytic tetrad. We demonstrate that a histidine in this cluster is essential for catalysis of DS but not CS. The enzyme utilizes a single substrate-binding site while having two partially overlapping active sites catalyzing the respective reactions. The spatial separation of the two sets of residues suggests a substrate-induced conformational change that brings all catalytically essential residues close together.

  13. Effect of pH on the formation of lysosome-alginate beads for antimicrobial activity.

    PubMed

    Park, Hyun Jung; Min, Jiho; Ahn, Joo-Myung; Cho, Sung-Jin; Ahn, Ji-Young; Kim, Yang-Hoon

    2015-02-01

    In this study, we developed lysosome-alginate beads for application as an oral drug delivery system (ODDS). The beads harboring lysosomes, which have antimicrobial activity, and various concentrations of alginate were characterized and optimized. For application as an ODDS, pH-dependent lysosome-alginate beads were generated, and the level of lysosome release was investigated by using antimicrobial tests. At low pH, lysosomes were not released from the lysosome-alginate beads; however, at neutral pH, similar to the pH in the intestine, lysosome release was confirmed, as determined by a high antimicrobial activity. This study shows the potential of such an ODDS for the in vivo treatment of infection with pathogens.

  14. Putative Alginate Assimilation Process of the Marine Bacterium Saccharophagus degradans 2-40 Based on Quantitative Proteomic Analysis.

    PubMed

    Takagi, Toshiyuki; Morisaka, Hironobu; Aburaya, Shunsuke; Tatsukami, Yohei; Kuroda, Kouichi; Ueda, Mitsuyoshi

    2016-02-01

    Quantitative proteomic analysis was conducted to assess the assimilation processes of Saccharophagus degradans cultured with glucose, pectin, and alginate as carbon sources. A liquid chromatography-tandem mass spectrometry approach was used, employing our unique, long monolithic silica capillary column. In an attempt to select candidate proteins that correlated to alginate assimilation, the production of 23 alginate-specific proteins was identified by statistical analyses of the quantitative proteomic data. Based on the analysis, we propose that S. degradans has an alginate-specific gene cluster for efficient alginate utilization. The alginate-specific proteins of S. degradans were comprised of alginate lyases, enzymes related to carbohydrate metabolism, membrane transporters, and transcription factors. Among them, the short-chain dehydrogenase/reductase Sde_3281 annotated in the alginate-specific cluster showed 4-deoxy-L-erythro-5-hexoseulose uronic acid reductase (DehR) activity. Furthermore, we found two different genes (Sde_3280 and Sde_0939) encoding 2-keto-3-deoxy-D-gluconic acid (KDG) kinases (KdgK) that metabolize the KDG derived from alginate and pectin in S. degradans. S. degradans used Sde_3280 to phosphorylate the KDG derived from alginate and Sde_0939 to phosphorylate the KDG derived from pectin. The distinct selection of KdgKs provides an important clue toward the elucidation of how S. degradans recognizes and processes polysaccharides.

  15. Cloning and characterization of the first polysaccharide lyase family 6 oligoalginate lyase from marine Shewanella sp. Kz7

    PubMed Central

    Li, Shangyong; Wang, Linna; Han, Feng; Gong, Qianhong; Yu, Wengong

    2016-01-01

    Alginate, the most abundant carbohydrate in brown macroalgae, is widely used in the food and pharmaceutical industries. Recently, alginate has attracted increasing attention, as it may serve as an alternative biomass for the production of biofuel. The degradation of alginate into monomeric units is the prerequisite for bioethanol production. All known oligoalginate lyases belong to the polysaccharide lyase (PL) family 7, 14, 15 and 17, and most of them preferred to degrade the polyM blocks to yield 4-deoxy-l-erythro-5-hexoseulose uronic acid as the primary product. In this study, we cloned an oligoalginate lyase gene, oalS6, from Shewanella sp. Kz7 and expressed it in Escherichia coli. The PL family 6 oligoalginate lyase (OalS6) has no significant sequence similarity with other known oligoalginate lyases. OalS6 contains a chondroitinase-like domain and was assigned to the PL family 6. This lyase is an exo-type oligoalginate lyase and prefer to depolymerize polyG block into 2, 4, 5, 6-tetrahydroxytetrahydro-2H-pyran-2-carboxylic acid. All of these results indicate that OalS6 is a novel oligoalginate lyase that is structurally and functionally different from other known oligoalginate lyases. This finding provides new insights into the development of biofuel processing biotechnologies from seaweed. PMID:26232404

  16. Cloning and characterization of the first polysaccharide lyase family 6 oligoalginate lyase from marine Shewanella sp. Kz7.

    PubMed

    Li, Shangyong; Wang, Linna; Han, Feng; Gong, Qianhong; Yu, Wengong

    2016-01-01

    Alginate, the most abundant carbohydrate in brown macroalgae, is widely used in the food and pharmaceutical industries. Recently, alginate has attracted increasing attention, as it may serve as an alternative biomass for the production of biofuel. The degradation of alginate into monomeric units is the prerequisite for bioethanol production. All known oligoalginate lyases belong to the polysaccharide lyase (PL) family 7, 14, 15 and 17, and most of them preferred to degrade the polyM blocks to yield 4-deoxy-l-erythro-5-hexoseulose uronic acid as the primary product. In this study, we cloned an oligoalginate lyase gene, oalS6, from Shewanella sp. Kz7 and expressed it in Escherichia coli. The PL family 6 oligoalginate lyase (OalS6) has no significant sequence similarity with other known oligoalginate lyases. OalS6 contains a chondroitinase-like domain and was assigned to the PL family 6. This lyase is an exo-type oligoalginate lyase and prefer to depolymerize polyG block into 2, 4, 5, 6-tetrahydroxytetrahydro-2H-pyran-2-carboxylic acid. All of these results indicate that OalS6 is a novel oligoalginate lyase that is structurally and functionally different from other known oligoalginate lyases. This finding provides new insights into the development of biofuel processing biotechnologies from seaweed.

  17. Activation Mechanism and Cellular Localization of Membrane-Anchored Alginate Polymerase in Pseudomonas aeruginosa.

    PubMed

    Moradali, M Fata; Ghods, Shirin; Rehm, Bernd H A

    2017-03-03

    The exopolysaccharide, alginate, produced by the opportunistic human pathogen Pseudomonas aeruginosa represents a survival advantage by contributing to formation of characteristic biofilms during infection. Membrane anchored proteins Alg8 (catalytic subunit) and Alg44 (co-polymerase) constitute the alginate polymerase which is being activated by the second messenger molecule c-di-GMP, but the mechanism of activation remains elusive. To shed light on the c-di-GMP mediated activation of alginate polymerization in vivo, an in silico structural model of Alg8 fused to the c-di-GMP binding PilZ domain informed by the structure of cellulose synthase, BcsA, was developed. This structural model was probed by site-specific mutagenesis and different cellular levels of c-di-GMP. Results suggested that c-di-GMP-mediated activation of alginate polymerization involves amino acids residing at two loops including H323 (loop A), T457 and E460 (loop B) surrounding the catalytic site in the predicted model. Activity of respective Alg8 variants suggested that c-di-GMP-mediated control of substrate access to the catalytic site of Alg8 is dissimilar to the known activation mechanism of BcsA. Alg8 variants responded differently to various c-di-GMP levels while MucR imparted c-di-GMP for activation of alginate polymerase. Furthermore, we showed that Alg44 co-polymerase constituted a stable dimer, with its periplasmic domains required for protein localization, alginate polymerization and modification. Superfolder GFP fusions of Alg8 and Alg44 showed a non-uniform, punctuate and patchy arrangement of both proteins surrounding the cell. Overall, this study provides insights into the c-di-GMP mediated activation of alginate polymerization while assigning functional roles to Alg8 and Alg44 including their subcellular localization and distribution.IMPORTANCE The exopolysaccharide, alginate, is an important biofilm component of the opportunistic human pathogen P. aeruginosa and the principle

  18. Activation and Stabilization of Olive Recombinant 13-Hydroperoxide Lyase Using Selected Additives.

    PubMed

    Jacopini, Sabrina; Vincenti, Sophie; Mariani, Magali; Brunini-Bronzini de Caraffa, Virginie; Gambotti, Claude; Desjobert, Jean-Marie; Muselli, Alain; Costa, Jean; Tomi, Félix; Berti, Liliane; Maury, Jacques

    2016-12-24

    The stabilization of olive recombinant hydroperoxide lyases (rHPLs) was investigated using selected chemical additives. Two rHPLs were studied: HPL full-length and HPL with its chloroplast transit peptide deleted (matured HPL). Both olive rHPLs are relatively stable at 4 °C, and enzyme activity can be preserved (about 100% of the rHPL activities are maintained) during 5 weeks of storage at -20 or at -80 °C in the presence of glycerol (10%, v/v). Among the additives used in this study, glycine (2.5% w/v), NaCl (0.5 M), and Na2SO4 (0.25 M) provided the highest activation of HPL full-length activity, while the best matured HPL activity was obtained with Na2SO4 (0.25 M) and NaCl (1 M). Although the inactivation rate constants (k) showed that these additives inactivate both rHPLs, their use is still relevant as they strongly increase HPL activity. Results of C6-aldehyde production assays also showed that glycine, NaCl, and Na2SO4 are appropriate additives and that NaCl appears to be the best additive, at least for hexanal production.

  19. Novel vanadyl complexes of alginate saccharides: synthesis, characterization, and biological activities.

    PubMed

    Liu, Shengyi; Liu, Guangyang; Yi, Yuetao

    2015-05-05

    Vanadium compounds present many physiological functions. However, vanadium(IV) and (V) salts are difficult for gastrointestinal absorption and have strong side effects. Therefore organic oxovanadium compounds gain more attention. Vanadyl alginate polysaccharides (VAPS) and vanadyl alginate oligosaccharides (VAOS) were obtained from aqueous solutions of VOSO4 at pH 12. They were characterized by infrared spectroscopy, UV-vis spectroscopy and inductively coupled plasma-mass spectrometry (ICP-MS). The antioxidant activity of oxovanadium(IV) complexes was investigated in hydroxyl and DPPH radical scavenging systems in vitro. The results reveal that activities of VAPS and VAOS in the two systems were stronger than those of alginate polysaccharides (APS) and alginate oligosaccharides (AOS), respectively. In addition, VAPS and VAOS promoted significantly the antiproliferation of ligands of human hepatoma cell line BEL-7402. Oxovanadium(IV) complexes were potent inhibitors of protein tyrosine phosphatase 1B (PTP1B) with IC50 values in the range of 6.4-18.7μg/mL, indicated in biochemical assays. In addition, Vanadyl-alginate had no significant side effects on proliferation and viability of HL-7702 hepatic cells. In the future, they can be added to medicines and ease the growing threat that cancer and diabetes mellitus cause to human health.

  20. Entropic and enthalpic components of catalysis in the mutase and lyase activities of Pseudomonas aeruginosa PchB.

    PubMed

    Luo, Qianyi; Meneely, Kathleen M; Lamb, Audrey L

    2011-05-11

    The isochorismate-pyruvate lyase from Pseudomonas aeruginosa (PchB) catalyzes two pericyclic reactions, demonstrating the eponymous activity and also chorismate mutase activity. The thermodynamic parameters for these enzyme-catalyzed activities, as well as the uncatalyzed isochorismate decomposition, are reported from temperature dependence of k(cat) and k(uncat) data. The entropic effects do not contribute to enzyme catalysis as expected from previously reported chorismate mutase data. Indeed, an entropic penalty for the enzyme-catalyzed mutase reaction (ΔS(++) = -12.1 ± 0.6 cal/(mol K)) is comparable to that of the previously reported uncatalyzed reaction, whereas that of the enzyme-catalyzed lyase reaction (ΔS(++) = -24.3 ± 0.2 cal/(mol K)) is larger than that of the uncatalyzed lyase reaction (-15.77 ± 0.02 cal/(mol K)) documented here. With the assumption that chemistry is rate-limiting, we propose that a reactive substrate conformation is formed upon loop closure of the active site and that ordering of the loop contributes to the entropic penalty for converting the enzyme substrate complex to the transition state.

  1. The effect of methyl-donated hydrogen bonding on active site conformations of hyaluronate lyase

    NASA Astrophysics Data System (ADS)

    Migues, Angela N.; Vergenz, Robert A.; Moore, Kevin B.

    2010-03-01

    Geometric evidence shows a val-A252 methyl-donated (MD) hydrogen bond (HB) in hyaluronate lyase (Streptococcus pneumoniae) interacts with nearby NH--O and OH--O HBs, distorting active-site helical structure. Results for model fragment A248-254 are based on experimental heavy atom positions with ab initio hydrogen atoms. The MDHB, with (H-O distance, donor-H-O angle) = (2.3å; 174^o), exhibits more favorable geometry than thr-A253 OH--O HB (1.8å; 170^o) to the same ala-249 C=O. Consequently, thr-253 N-H--O interaction is forced closer to lys-250 C=O than ala-249 C=O(2.6 versus 2.7å). A novel method has been developed to quantify the effects of atomic diplacements on motions of neighboring helices. A coordinate system was established to track the movement of specific residues and to ascertain the effect of such motions on active site conformations.

  2. Effect of polyphenols on 3-hydroxy-3-methylglutaryl-coenzyme A lyase activity in human hepatoma HepG2 cell extracts.

    PubMed

    Nakagawa, Saori; Kojima, Yuko; Sekino, Koichi; Yamato, Susumu

    2013-01-01

    When carbohydrate metabolism is impaired, fatty acid metabolism is activated. Excess acetyl-coenzyme A (CoA) is generated from fatty acids by β-oxidation and is used for the formation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) and subsequently for acetoacetate. High levels of secreted ketone bodies (acetoacetate and 3β-hydroxybutyrate) lower the pH of blood and urine, resulting in ketoacidosis. HMG-CoA lyase in hepatic cells is a rate-limiting enzyme catalyzing the cleavage of HMG-CoA to acetoacetate, and thus inhibition of this enzyme results in reduced acetoacetate production, in other words, impaired ketoacidosis. Inhibition of HMG-CoA lyase activity possibly prevents ketoacidosis and should be the therapeutic target. Polyphenols are common and abundant dietary constituents with beneficial effects on human health. We examined the inhibitory effects of dietary polyphenols on HMG-CoA lyase activity in cellular extracts of human hepatoma HepG2 cells. Of the nine representative dietary polyphenols tested, (-)-epigallocatechin (EGC), (-)-epigallocatechin gallate (EGCG), and gallic acid (GA) effectively inhibited HMG-CoA lyase activity. Lineweaver-Burk analysis revealed that EGC and EGCG are likely to be mixed-type noncompetitive inhibitors. Pyrogallol with the gallyl structure also inhibited HMG-CoA lyase activity, suggesting that the gallyl moiety of polyphenols is important for the inhibition of HMG-CoA lyase activity.

  3. New Ulvan-Degrading Polysaccharide Lyase Family: Structure and Catalytic Mechanism Suggests Convergent Evolution of Active Site Architecture.

    PubMed

    Ulaganathan, ThirumalaiSelvi; Boniecki, Michal T; Foran, Elizabeth; Buravenkov, Vitaliy; Mizrachi, Naama; Banin, Ehud; Helbert, William; Cygler, Miroslaw

    2017-03-23

    Ulvan is a complex sulfated polysaccharide biosynthesized by green seaweed and contains predominantly rhamnose, xylose, and uronic acid sugars. Ulvan-degrading enzymes have only recently been identified and added to the CAZy ( www.cazy.org ) database as family PL24, but neither their structure nor catalytic mechanism(s) are yet known. Several homologous, new ulvan lyases, have been discovered in Pseudoalteromonas sp. strain PLSV, Alteromonas LOR, and Nonlabens ulvanivorans, defining a new family PL25, with the lyase encoded by the gene PLSV_3936 being one of them. This enzyme cleaves the glycosidic bond between 3-sulfated rhamnose (R3S) and glucuronic acid (GlcA) or iduronic acid (IdoA) via a β-elimination mechanism. We report the crystal structure of PLSV_3936 and its complex with a tetrasaccharide substrate. PLSV_3936 folds into a seven-bladed β-propeller, with each blade consisting of four antiparallel β-strands. Sequence conservation analysis identified a highly conserved region lining at one end of a deep crevice on the protein surface. The putative active site was identified by mutagenesis and activity measurements. Crystal structure of the enzyme with a bound tetrasaccharide substrate confirmed the identity of base and acid residues and allowed determination of the catalytic mechanism and also the identification of residues neutralizing the uronic acid carboxylic group. The PLSV_3936 structure provides an example of a convergent evolution among polysaccharide lyases toward a common active site architecture embedded in distinct folds.

  4. Crystal Structure of Escherichia coli Diaminopropionate Ammonia-lyase Reveals Mechanism of Enzyme Activation and Catalysis*

    PubMed Central

    Bisht, Shveta; Rajaram, Venkatesan; Bharath, Sakshibeedu R.; Kalyani, Josyula Nitya; Khan, Farida; Rao, Appaji N.; Savithri, Handanahal S.; Murthy, Mathur R. N.

    2012-01-01

    Pyridoxal 5′-phosphate (PLP)-dependent enzymes utilize the unique chemistry of a pyridine ring to carry out diverse reactions involving amino acids. Diaminopropionate (DAP) ammonia-lyase (DAPAL) is a prokaryotic PLP-dependent enzyme that catalyzes the degradation of d- and l-forms of DAP to pyruvate and ammonia. Here, we report the first crystal structure of DAPAL from Escherichia coli (EcDAPAL) in tetragonal and monoclinic forms at 2.0 and 2.2 Å resolutions, respectively. Structures of EcDAPAL soaked with substrates were also determined. EcDAPAL has a typical fold type II PLP-dependent enzyme topology consisting of a large and a small domain with the active site at the interface of the two domains. The enzyme is a homodimer with a unique biological interface not observed earlier. Structure of the enzyme in the tetragonal form had PLP bound at the active site, whereas the monoclinic structure was in the apo-form. Analysis of the apo and holo structures revealed that the region around the active site undergoes transition from a disordered to ordered state and assumes a conformation suitable for catalysis only upon PLP binding. A novel disulfide was found to occur near a channel that is likely to regulate entry of ligands to the active site. EcDAPAL soaked with dl-DAP revealed density at the active site appropriate for the reaction intermediate aminoacrylate, which is consistent with the observation that EcDAPAL has low activity under crystallization conditions. Based on the analysis of the structure and results of site-directed mutagenesis, a two-base mechanism of catalysis involving Asp120 and Lys77 is suggested. PMID:22505717

  5. Crystal structure of Escherichia coli diaminopropionate ammonia-lyase reveals mechanism of enzyme activation and catalysis.

    PubMed

    Bisht, Shveta; Rajaram, Venkatesan; Bharath, Sakshibeedu R; Kalyani, Josyula Nitya; Khan, Farida; Rao, Appaji N; Savithri, Handanahal S; Murthy, Mathur R N

    2012-06-08

    Pyridoxal 5'-phosphate (PLP)-dependent enzymes utilize the unique chemistry of a pyridine ring to carry out diverse reactions involving amino acids. Diaminopropionate (DAP) ammonia-lyase (DAPAL) is a prokaryotic PLP-dependent enzyme that catalyzes the degradation of d- and l-forms of DAP to pyruvate and ammonia. Here, we report the first crystal structure of DAPAL from Escherichia coli (EcDAPAL) in tetragonal and monoclinic forms at 2.0 and 2.2 Å resolutions, respectively. Structures of EcDAPAL soaked with substrates were also determined. EcDAPAL has a typical fold type II PLP-dependent enzyme topology consisting of a large and a small domain with the active site at the interface of the two domains. The enzyme is a homodimer with a unique biological interface not observed earlier. Structure of the enzyme in the tetragonal form had PLP bound at the active site, whereas the monoclinic structure was in the apo-form. Analysis of the apo and holo structures revealed that the region around the active site undergoes transition from a disordered to ordered state and assumes a conformation suitable for catalysis only upon PLP binding. A novel disulfide was found to occur near a channel that is likely to regulate entry of ligands to the active site. EcDAPAL soaked with dl-DAP revealed density at the active site appropriate for the reaction intermediate aminoacrylate, which is consistent with the observation that EcDAPAL has low activity under crystallization conditions. Based on the analysis of the structure and results of site-directed mutagenesis, a two-base mechanism of catalysis involving Asp(120) and Lys(77) is suggested.

  6. Sphingosine-1-phosphate lyase downregulation promotes colon carcinogenesis through STAT3-activated microRNAs.

    PubMed

    Degagné, Emilie; Pandurangan, Ashok; Bandhuvula, Padmavathi; Kumar, Ashok; Eltanawy, Abeer; Zhang, Meng; Yoshinaga, Yuko; Nefedov, Mikhail; de Jong, Pieter J; Fong, Loren G; Young, Stephen G; Bittman, Robert; Ahmedi, Yasmin; Saba, Julie D

    2014-12-01

    Growing evidence supports a link between inflammation and cancer; however, mediators of the transition between inflammation and carcinogenesis remain incompletely understood. Sphingosine-1-phosphate (S1P) lyase (SPL) irreversibly degrades the bioactive sphingolipid S1P and is highly expressed in enterocytes but downregulated in colon cancer. Here, we investigated the role of SPL in colitis-associated cancer (CAC). We generated mice with intestinal epithelium-specific Sgpl1 deletion and chemically induced colitis and tumor formation in these animals. Compared with control animals, mice lacking intestinal SPL exhibited greater disease activity, colon shortening, cytokine levels, S1P accumulation, tumors, STAT3 activation, STAT3-activated microRNAs (miRNAs), and suppression of miR-targeted anti-oncogene products. This phenotype was attenuated by STAT3 inhibition. In fibroblasts, silencing SPL promoted tumorigenic transformation through a pathway involving extracellular transport of S1P through S1P transporter spinster homolog 2 (SPNS2), S1P receptor activation, JAK2/STAT3-dependent miR-181b-1 induction, and silencing of miR-181b-1 target cylindromatosis (CYLD). Colon biopsies from patients with inflammatory bowel disease revealed enhanced S1P and STAT3 signaling. In mice with chemical-induced CAC, oral administration of plant-type sphingolipids called sphingadienes increased colonic SPL levels and reduced S1P levels, STAT3 signaling, cytokine levels, and tumorigenesis, indicating that SPL prevents transformation and carcinogenesis. Together, our results suggest that dietary sphingolipids can augment or prevent colon cancer, depending upon whether they are metabolized to S1P or promote S1P metabolism through the actions of SPL.

  7. Sphingosine-1-phosphate lyase downregulation promotes colon carcinogenesis through STAT3-activated microRNAs

    PubMed Central

    Degagné, Emilie; Pandurangan, Ashok; Bandhuvula, Padmavathi; Kumar, Ashok; Eltanawy, Abeer; Zhang, Meng; Yoshinaga, Yuko; Nefedov, Mikhail; de Jong, Pieter J.; Fong, Loren G.; Young, Stephen G.; Bittman, Robert; Ahmedi, Yasmin; Saba, Julie D.

    2014-01-01

    Growing evidence supports a link between inflammation and cancer; however, mediators of the transition between inflammation and carcinogenesis remain incompletely understood. Sphingosine-1-phosphate (S1P) lyase (SPL) irreversibly degrades the bioactive sphingolipid S1P and is highly expressed in enterocytes but downregulated in colon cancer. Here, we investigated the role of SPL in colitis-associated cancer (CAC). We generated mice with intestinal epithelium-specific Sgpl1 deletion and chemically induced colitis and tumor formation in these animals. Compared with control animals, mice lacking intestinal SPL exhibited greater disease activity, colon shortening, cytokine levels, S1P accumulation, tumors, STAT3 activation, STAT3-activated microRNAs (miRNAs), and suppression of miR-targeted anti-oncogene products. This phenotype was attenuated by STAT3 inhibition. In fibroblasts, silencing SPL promoted tumorigenic transformation through a pathway involving extracellular transport of S1P through S1P transporter spinster homolog 2 (SPNS2), S1P receptor activation, JAK2/STAT3-dependent miR-181b-1 induction, and silencing of miR-181b-1 target cylindromatosis (CYLD). Colon biopsies from patients with inflammatory bowel disease revealed enhanced S1P and STAT3 signaling. In mice with chemical-induced CAC, oral administration of plant-type sphingolipids called sphingadienes increased colonic SPL levels and reduced S1P levels, STAT3 signaling, cytokine levels, and tumorigenesis, indicating that SPL prevents transformation and carcinogenesis. Together, our results suggest that dietary sphingolipids can augment or prevent colon cancer, depending upon whether they are metabolized to S1P or promote S1P metabolism through the actions of SPL. PMID:25347472

  8. Alginate/PEO-PPO-PEO composite hydrogels with thermally-active plasticity.

    PubMed

    White, Joseph C; Saffer, Erika M; Bhatia, Surita R

    2013-12-09

    Stimuli-responsive hydrogels with high strength and toughness have received significant interest in recent years. Here, we report thermally active composite hydrogels comprising alginate and one of two poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymers. Temperature-sensitive structural and mechanical changes are probed using calorimetry, neutron scattering, shear rheology, unconfined compression, and fracture. Below the lower gelation temperature, LGT, the mechanical properties are dominated by alginate. As the LGT is reached, the contribution of PEO-PPO-PEO to the mechanical properties is activated, resulting in order-of-magnitude increases in elastic modulus. Under compression, we show the evolution of plasticity for the composite hydrogels as the LGT is approached and surpassed, resulting in dramatic increases in fracture stress compared to neat alginate hydrogels. Plasticity was observed above the LGT and may be attributed to restructuring from the sliding of packed micelles and strain-hardening due to stress concentration on alginate cross-links and junction zones, ultimately leading to fracture.

  9. The roles of active site residues in the catalytic mechanism of methylaspartate ammonia-lyase.

    PubMed

    Raj, Hans; Poelarends, Gerrit J

    2013-01-01

    Methylaspartate ammonia-lyase (MAL; EC 4.3.1.2) catalyzes the reversible addition of ammonia to mesaconate to yield l-threo-(2S,3S)-3-methylaspartate and l-erythro-(2S,3R)-3-methylaspartate as products. In the proposed minimal mechanism for MAL of Clostridium tetanomorphum, Lys-331 acts as the (S)-specific base catalyst and abstracts the 3S-proton from l-threo-3-methylaspartate, resulting in an enolate anion intermediate. This enolic intermediate is stabilized by coordination to the essential active site Mg(2+) ion and hydrogen bonding to the Gln-329 residue. Collapse of this intermediate results in the release of ammonia and the formation of mesaconate. His-194 likely acts as the (R)-specific base catalyst and abstracts the 3R-proton from the l-erythro isomer of 3-methylaspartate, yielding the enolic intermediate. In the present study, we have investigated the importance of the residues Gln-73, Phe-170, Gln-172, Tyr-356, Thr-360, Cys-361 and Leu-384 for the catalytic activity of C. tetanomorphum MAL. These residues, which are part of the enzyme surface lining the substrate binding pocket, were subjected to site-directed mutagenesis and the mutant enzymes were characterized for their structural integrity, ability to catalyze the amination of mesaconate, and regio- and diastereoselectivity. Based on the observed properties of the mutant enzymes, combined with previous structural studies and protein engineering work, we propose a detailed catalytic mechanism for the MAL-catalyzed reaction, in which the side chains of Gln-73, Gln-172, Tyr-356, Thr-360, and Leu-384 provide favorable interactions with the substrate, which are important for substrate binding and activation. This detailed knowledge of the catalytic mechanism of MAL can serve as a guide for future protein engineering experiments.

  10. The role of active site tyrosine 58 in Citrobacter freundii methionine γ-lyase.

    PubMed

    Anufrieva, Natalya V; Faleev, Nicolai G; Morozova, Elena A; Bazhulina, Natalia P; Revtovich, Svetlana V; Timofeev, Vladimir P; Tkachev, Yaroslav V; Nikulin, Alexei D; Demidkina, Tatyana V

    2015-09-01

    In the spatial structure of methionine γ-lyase (MGL, EC 4.4.1.11) from Citrobacter freundii, Tyr58 is located at H-bonding distance to the oxygen atom of the phosphate "handle" of pyridoxal 5'-phosphate (PLP). It was replaced for phenylalanine by site-directed mutagenesis. The X-ray structure of the mutant enzyme was determined at 1.96Å resolution. Comparison of spatial structures and absorption spectra of wild-type and mutant holoenzymes demonstrated that the replacement did not result in essential changes of the conformation of the active site Tyr58Phe MGL. The Kd value of PLP for Tyr58Phe MGL proved to be comparable to the Kd value for the wild-type enzyme. The replacement led to a decrease of catalytic efficiencies in both γ- and β-elimination reactions of about two orders of magnitude as compared to those for the wild-type enzyme. The rates of exchange of C-α- and C-β- protons of inhibitors in D2O catalyzed by the mutant form are comparable with those for the wild-type enzyme. Spectral data on the complexes of the mutant form with the substrates and inhibitors showed that the replacement led to a change of rate the limiting step of the physiological reaction. The results allowed us to conclude that Tyr58 is involved in an optimal positioning of the active site Lys210 at some stages of γ- and β-elimination reactions. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

  11. Adenylosuccinate lyase of Bacillus subtilis regulates the activity of the glutamyl-tRNA synthetase.

    PubMed Central

    Gendron, N; Breton, R; Champagne, N; Lapointe, J

    1992-01-01

    In Bacillus subtilis, the glutamyl-tRNA synthetase [L-glutamate:tRNA(Glu) ligase (AMP-forming), EC 6.1.1.17] is copurified with a polypeptide of M(r) 46,000 that influences its affinity for its substrates and increases its thermostability. The gene encoding this regulatory factor was cloned with the aid of a 41-mer oligonucleotide probe corresponding to the amino acid sequence of an NH2-terminal segment of this factor. The nucleotide sequence of this gene and the physical map of the 1475-base-pair fragment on which it was cloned are identical to those of purB, which encodes the adenylosuccinate lyase (adenylosuccinate AMP-lyase, EC 4.3.2.2), an enzyme involved in the de novo synthesis of purines. This gene complements the purB mutation of Escherichia coli JK268, and its presence on a multicopy plasmid behind the trc promoter in the purB- strain gives an adenylosuccinate lyase level comparable to that in wild-type B. subtilis. A complex between the adenylosuccinate lyase and the glutamyl-tRNA synthetase was detected by centrifugation on a density gradient. The interaction between these enzymes may play a role in the coordination of purine metabolism and protein biosynthesis. Images PMID:1608947

  12. The variability in DMSP content and DMSP lyase activity in marine dinoflagellates

    NASA Astrophysics Data System (ADS)

    Caruana, Amandine M. N.; Malin, Gill

    2014-01-01

    More than 20 years ago Maureen Keller and co-workers published a study that identified dinoflagellates as an important marine phytoplankton group with respect to the production of dimethylsulphoniopropionate (DMSP). Here, we present a synthesis and analysis of all the DMSP and DMSP lyase activity (DLA) measurements currently available for dinoflagellates. The data cover 110 species and strains and reveal over 6 orders of magnitude variability in intracellular DMSP concentrations and substantial variations in DLA in 23 strains. Inter-specific variability was explored with reference to a range of biological characteristics. The presence of a theca did not appear to be related to DMSP concentration but there was a potential relationship with toxicity (P = 0.06) and bioluminescent species produced significantly lower concentrations (P < 0.01) than non-bioluminescent ones. DMSP concentrations were related to plastid types (P < 0.05); dinoflagellates with haptophyte-like plastids contained lower amounts of DMSP than those with peridinin plastids (P < 0.01), whereas those containing cryptomonad-like plastids tended to have higher DMSP concentrations. Heterotrophic dinoflagellates were also considered given their importance in the natural environment. They are the only heterotrophs known to synthesise DMSP and this ability may support the theory that they are of photosynthetic origin. However, the heterotrophic species investigated so far suggest wide variability in DMSP content and the species Oxyrrhis marina had no detectable DMSP. The oceanic province of origin significantly affected the DMSP concentrations (P < 0.05) with higher DMSP content observed in dinoflagellates from the Mediterranean province, the Kuroshio Current province and the East Coastal Australian province. Overall this study supports the concept that DMSP-containing dinoflagellates are an important potential source of DMS to the global atmosphere and highlights current gaps in knowledge.

  13. A Polysaccharide Lyase from Stenotrophomonas maltophilia with a Unique, pH-regulated Substrate Specificity*

    PubMed Central

    MacDonald, Logan C.; Berger, Bryan W.

    2014-01-01

    Polysaccharide lyases (PLs) catalyze the depolymerization of anionic polysaccharides via a β-elimination mechanism. PLs also play important roles in microbial pathogenesis, participating in bacterial invasion and toxin spread into the host tissue via degradation of the host extracellular matrix, or in microbial biofilm formation often associated with enhanced drug resistance. Stenotrophomonas maltophilia is a Gram-negative bacterium that is among the emerging multidrug-resistant organisms associated with chronic lung infections as well as with cystic fibrosis patients. A putative alginate lyase (Smlt1473) from S. maltophilia was heterologously expressed in Escherichia coli, purified in a one-step fashion via affinity chromatography, and activity as well as specificity determined for a range of polysaccharides. Interestingly, Smlt1473 catalyzed the degradation of not only alginate, but poly-β-d-glucuronic acid and hyaluronic acid as well. Furthermore, the pH optimum for enzymatic activity is substrate-dependent, with optimal hyaluronic acid degradation at pH 5, poly-β-d-glucuronic acid degradation at pH 7, and alginate degradation at pH 9. Analysis of the degradation products revealed that each substrate was cleaved endolytically into oligomers comprised predominantly of even numbers of sugar groups, with lower accumulation of trimers and pentamers. Collectively, these results imply that Smlt1473 is a multifunctional PL that exhibits broad substrate specificity, but utilizes pH as a mechanism to achieve selectivity. PMID:24257754

  14. Serine phosphorylation of human P450c17 increases 17,20-lyase activity: implications for adrenarche and the polycystic ovary syndrome.

    PubMed Central

    Zhang, L H; Rodriguez, H; Ohno, S; Miller, W L

    1995-01-01

    Microsomal cytochrome P450c17 catalyzes both steroid 17 alpha-hydroxylase activity and scission of the C17-C20 steroid bond (17,20-lyase) on the same active site. Adrenal 17 alpha-hydroxylase activity is needed to produce cortisol throughout life, but 17,20-lyase activity appears to be controlled independently in a complex, age-dependent pattern. We show that human P450c17 is phosphorylated on serine and threonine residues by a cAMP-dependent protein kinase. Phosphorylation of P450c17 increases 17,20-lyase activity, while dephosphorylation virtually eliminates this activity. Hormonally regulated serine phosphorylation of human P450c17 suggests a possible mechanism for human adrenarche and may be a unifying etiologic link between the hyperandrogenism and insulin resistance that characterize the polycystic ovary syndrome. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:7479852

  15. Kynurenine Aminotransferase III and Glutamine Transaminase L Are Identical Enzymes that have Cysteine S-Conjugate β-Lyase Activity and Can Transaminate l-Selenomethionine*

    PubMed Central

    Pinto, John T.; Krasnikov, Boris F.; Alcutt, Steven; Jones, Melanie E.; Dorai, Thambi; Villar, Maria T.; Artigues, Antonio; Li, Jianyong; Cooper, Arthur J. L.

    2014-01-01

    Three of the four kynurenine aminotransferases (KAT I, II, and IV) that synthesize kynurenic acid, a neuromodulator, are identical to glutamine transaminase K (GTK), α-aminoadipate aminotransferase, and mitochondrial aspartate aminotransferase, respectively. GTK/KAT I and aspartate aminotransferase/KAT IV possess cysteine S-conjugate β-lyase activity. The gene for the former enzyme, GTK/KAT I, is listed in mammalian genome data banks as CCBL1 (cysteine conjugate beta-lyase 1). Also listed, despite the fact that no β-lyase activity has been assigned to the encoded protein in the genome data bank, is a CCBL2 (synonym KAT III). We show that human KAT III/CCBL2 possesses cysteine S-conjugate β-lyase activity, as does mouse KAT II. Thus, depending on the nature of the substrate, all four KATs possess cysteine S-conjugate β-lyase activity. These present studies show that KAT III and glutamine transaminase L are identical enzymes. This report also shows that KAT I, II, and III differ in their ability to transaminate methyl-l-selenocysteine (MSC) and l-selenomethionine (SM) to β-methylselenopyruvate (MSP) and α-ketomethylselenobutyrate, respectively. Previous studies have identified these seleno-α-keto acids as potent histone deacetylase inhibitors. Methylselenol (CH3SeH), also purported to have chemopreventive properties, is the γ-elimination product of SM and the β-elimination product of MSC catalyzed by cystathionine γ-lyase (γ-cystathionase). KAT I, II, and III, in part, can catalyze β-elimination reactions with MSC generating CH3SeH. Thus, the anticancer efficacy of MSC and SM will depend, in part, on the endogenous expression of various KAT enzymes and cystathionine γ-lyase present in target tissue coupled with the ability of cells to synthesize in situ either CH3SeH and/or seleno-keto acid metabolites. PMID:25231977

  16. Comparative study of calcium alginate, activated carbon, and their composite beads on methylene blue adsorption.

    PubMed

    Hassan, A F; Abdel-Mohsen, A M; Fouda, Moustafa M G

    2014-02-15

    Three adsorbents, calcium alginate beads (AB), sodium hydroxide activated carbon based coconut shells (C), and calcium alginate/activated carbon composite beads (ACB) were prepared. Their textural properties were characterized by N2-adsorption at -196°C and scanning electron microscopy. The porosity, surface area and total pore volume of C>ACB>AB, but AB adsorbent was more acidic function groups more than the other adsorbents. Adsorption experiments were conducted to examine the effects of adsorbent dosage, pH, time, temperature and initial concentration of methylene blue. Methylene blue adsorption on C, AB and ACB was observed at pH>6 to avoid the competition of H(+). The amount of dye adsorbed increases as the adsorbent dosage increase. Adsorption of dye follows pseudo-second order mechanism. Thermodynamic studies show spontaneous and endothermic nature of the overall adsorption process.

  17. Gibberellic Acid-Promoted Lignification and Phenylalanine Ammonia-lyase Activity in a Dwarf Pea (Pisum sativum) 1

    PubMed Central

    Cheng, Christina K.-C.; Marsh, H. V.

    1968-01-01

    The effects of gibberellic acid on lignification in seedlings of a dwarf and a tall cultivar of pea (Pisum sativum) grown under red or white light or in the darkness, were studied. Gibberellic acid (10−6-10−4 m) promoted stem elongation in both light and dark and increased the percentage of lignin in the stems of the light-grown dwarf pea. The gibberellin had no effect on the lignin content of the tall pea although high concentrations (10−4 m) promoted growth of the tall plants. Time course studies indicated that the enhanced lignification in the gibberellin-treated dwarf plants occurred only after a lag period of several days. It was concluded that gibberellic acid-enhanced ligmification had no direct relation to gibberellic acid-promoted growth. The activity of phenylalanine ammonia-lyase (E.C. 4.3.1.5) was higher in gibberellin-treated dwarf plants grown under white or red light than in untreated dwarf plants. Gibberellic acid had no detectable effect on the activity of this enzyme when the plants were grown in darkness, just as it had no effect on lignification under dark conditions. The data suggest that in gibberellin-deficient peas the activity of phenylalanine ammonia-lyase is one of the limiting factors in lignification. PMID:16656968

  18. Highly Active and Specific Tyrosine Ammonia-Lyases from Diverse Origins Enable Enhanced Production of Aromatic Compounds in Bacteria and Saccharomyces cerevisiae.

    PubMed

    Jendresen, Christian Bille; Stahlhut, Steen Gustav; Li, Mingji; Gaspar, Paula; Siedler, Solvej; Förster, Jochen; Maury, Jérôme; Borodina, Irina; Nielsen, Alex Toftgaard

    2015-07-01

    Phenylalanine and tyrosine ammonia-lyases form cinnamic acid and p-coumaric acid, which are precursors of a wide range of aromatic compounds of biotechnological interest. Lack of highly active and specific tyrosine ammonia-lyases has previously been a limitation in metabolic engineering approaches. We therefore identified 22 sequences in silico using synteny information and aiming for sequence divergence. We performed a comparative in vivo study, expressing the genes intracellularly in bacteria and yeast. When produced heterologously, some enzymes resulted in significantly higher production of p-coumaric acid in several different industrially important production organisms. Three novel enzymes were found to have activity exclusively for phenylalanine, including an enzyme from the low-GC Gram-positive bacterium Brevibacillus laterosporus, a bacterial-type enzyme from the amoeba Dictyostelium discoideum, and a phenylalanine ammonia-lyase from the moss Physcomitrella patens (producing 230 μM cinnamic acid per unit of optical density at 600 nm [OD600]) in the medium using Escherichia coli as the heterologous host). Novel tyrosine ammonia-lyases having higher reported substrate specificity than previously characterized enzymes were also identified. Enzymes from Herpetosiphon aurantiacus and Flavobacterium johnsoniae resulted in high production of p-coumaric acid in Escherichia coli (producing 440 μM p-coumaric acid OD600 unit(-1) in the medium) and in Lactococcus lactis. The enzymes were also efficient in Saccharomyces cerevisiae, where p-coumaric acid accumulation was improved 5-fold over that in strains expressing previously characterized tyrosine ammonia-lyases.

  19. Mechanistic pathways of mercury removal from the organomercurial lyase active site

    PubMed Central

    Rodrigues, Viviana

    2015-01-01

    Bacterial populations present in Hg-rich environments have evolved biological mechanisms to detoxify methylmercury and other organometallic mercury compounds. The most common resistance mechanism relies on the H+-assisted cleavage of the Hg–C bond of methylmercury by the organomercurial lyase MerB. Although the initial reaction steps which lead to the loss of methane from methylmercury have already been studied experimentally and computationally, the reaction steps leading to the removal of Hg2+ from MerB and regeneration of the active site for a new round of catalysis have not yet been elucidated. In this paper, we have studied the final steps of the reaction catalyzed by MerB through quantum chemical computations at the combined MP2/CBS//B3PW91/6-31G(d) level of theory. While conceptually simple, these reaction steps occur in a complex potential energy surface where several distinct pathways are accessible and may operate concurrently. The only pathway which clearly emerges as forbidden in our analysis is the one arising from the sequential addition of two thiolates to the metal atom, due to the accumulation of negative charges in the active site. The addition of two thiols, in contrast, leads to two feasible mechanistic possibilities. The most straightforward pathway proceeds through proton transfer from the attacking thiol to Cys159 , leading to its removal from the mercury coordination sphere, followed by a slower attack of a second thiol, which removes Cys96. The other pathway involves Asp99 in an accessory role similar to the one observed earlier for the initial stages of the reaction and affords a lower activation enthalpy, around 14 kcal mol−1, determined solely by the cysteine removal step rather than by the thiol ligation step. Addition of one thiolate to the intermediates arising from either thiol attack occurs without a barrier and produces an intermediate bound to one active site cysteine and from which Hg(SCH3)2 may be removed only after

  20. Involvement of the phosphate regulon and the psiD locus in carbon-phosphorus lyase activity of Escherichia coli K-12.

    PubMed Central

    Wackett, L P; Wanner, B L; Venditti, C P; Walsh, C T

    1987-01-01

    Escherichia coli K-12 can readily mutate to use methylphosphonic acid as the sole phosphorus source by a direct carbon-to-phosphorus (C-P) bond cleavage activity that releases methane and Pi. The in vivo C-P lyase activity is both physiologically and genetically regulated as a member of the phosphate regulon. Since psiD::lacZ(Mu d1) mutants cannot metabolize methylphosphonic acid, psiD may be the structural gene(s) for C-P lyase. PMID:3549702

  1. Controlled release of isoproturon, imidacloprid, and cyromazine from alginate-bentonite-activated carbon formulations.

    PubMed

    Garrido-Herrera, F J; Gonzalez-Pradas, E; Fernandez-Pérez, M

    2006-12-27

    Different alginate-based systems of isoproturon, imidacloprid, and cyromazine have been investigated in order to obtain controlled release (CR) properties. The basic formulation [sodium alginate (1.50%), pesticide (0.30%), and water] was modified using different amounts of bentonite and activated carbon. The higher values of encapsulation efficiency corresponded to those formulations prepared with higher percentages of activated carbon, showing higher encapsulation efficiency values for isoproturon and imidacloprid than for cyromazine, which has a higher water solubility. The kinetic experiments of imidacloprid/isoproturon release in water have shown us that the release rate is higher in imidacloprid systems than in those prepared with isoproturon. Moreover, it can be deduced that the use of bentonite and/or activated carbon sorbents reduces the release rate of the isoproturon and imidacloprid in comparison with the technical product and with alginate formulation without modifying agents. The highest decrease in release rate corresponds to the formulations prepared with the highest percentage of activated carbon. The water uptake, permeability, and time taken for 50% of the active ingredient to be released into water, T50, were calculated to compare the formulations. On the basis of a parameter of an empirical equation used to fit the pesticide release data, the release of isoproturon and imidacloprid from the various formulations into water is controlled by a diffusion mechanism. The sorption capacity of the sorbents and the permeability of the formulations were the most important factors modulating pesticide release. Finally, a linear correlation of the T50 values and the content of activated carbon in formulations were obtained.

  2. Enzymatic changes in phenylalanine ammonia-lyase, cinnamic-4-hydroxylase, capsaicin synthase, and peroxidase activities in capsicum under drought stress.

    PubMed

    Phimchan, Paongpetch; Chanthai, Saksit; Bosland, Paul W; Techawongstien, Suchila

    2014-07-23

    Penylalanine ammonia-lyase (PAL), cinnamic-4-hydroxylase (C4H), capsaicin synthase (CS), and peroxidase (POD) are involved in the capsaicinoid biosynthesis pathway and may be altered in cultivars with different pungency levels. This study clarified the action of these enzymes under drought stress for hot Capsicum cultivars with low, medium,and high pungency levels. At the flowering stage, control plants were watered at field capacity, whereas drought-induced plants were subjected to gradual drought stress. Under drought stress, PAL, C4H, CS, and POD enzyme activities increased as compared to the non-drought-stressed plants. A novel discovery was that PAL was the critical enzyme in capsaicinoid biosynthesis under drought stress because its activities and capsaicinoid increased across the different pungency levels of hot pepper cultivars examined.

  3. Effect of inorganic salts, soaps and detergents on dissolution and larvicidal activity of alginate formulation of Bacillus sphaericus.

    PubMed

    Vijayan, V; Balaraman, K

    1995-03-01

    Various inorganic salts and commonly used soaps and detergents were tested in the laboratory for their effect on the dissolution and larvicidal residual activity of a slow-release alginate encapsulated granular formation of Bacillus sphaericus. Fluoride, chloride and sulphate salts and a detergent powder affected the residual activity of this formulation drastically by rupturing it but did not effect its larvicidal activity. Nitrates and phosphates of sodium and potassium also had the same effect but to a moderate level. The safest concentration of these water impurities for effective functioning of the alginate encapsulated B. sphaericus formulation have been determined.

  4. Threonine-124 and phenylalanine-448 in Citrobacter freundii tyrosine phenol-lyase are necessary for activity with L-tyrosine.

    PubMed

    Demidkina, Tatyana V; Barbolina, Maria V; Faleev, Nicolai G; Sundararaju, Bakthavatsalam; Gollnick, Paul D; Phillips, Robert S

    2002-05-01

    Thr-124 and Phe-448 are located in the active site of Citrobacter freundii tyrosine phenol-lyase (TPL) near the phenol ring of a bound substrate analogue, 3-(4'-hydroxyphenyl)propionic acid [Sundararaju, Antson, Phillips, Demidkina, Barbolina, Gollnick, Dodson and Wilson (1997) Biochemistry 36, 6502-6510]. Thr-124 is replaced by Asp and Phe-448 is replaced by His in the crystal structure of a structurally similar enzyme, Proteus vulgaris tryptophan indole-lyase, which has 50% identical residues. Hence, Thr-124 and Phe-448 in TPL were mutated to Ala or Asp, and His, respectively, in order to probe the role of these residues in the reaction specificity for L-Tyr. These mutant enzymes have little or no beta-elimination activity with L-Tyr or 3-fluoro-L-Tyr as a substrate, but retain significant elimination activity with S-(o-nitrophenyl)-L-cysteine, S-alkyl-L-cysteines and beta-chloroalanine. Furthermore, the binding of L-Tyr and other non-substrate amino acids is not significantly affected by the mutations. The mutant TPLs form intermediates in rapid-scanning stopped-flow experiments with L-Phe, L-Tyr and L-Trp, similar to those seen with wild-type TPL. These results demonstrate that Thr-124 and Phe-448 are necessary for the reaction specificity of TPL for L-Tyr, and probably play a role in the elimination stage of the reaction mechanism. Thr-124 is within hydrogen-bonding distance of the phenolic group of the bound substrate, and may help to orientate the ring for beta-elimination to occur. Phe-448 may be important to allow the formation of the closed conformation during the reaction.

  5. Identification of enzymes responsible for extracellular alginate depolymerization and alginate metabolism in Vibrio algivorus.

    PubMed

    Doi, Hidetaka; Tokura, Yuriko; Mori, Yukiko; Mori, Kenichi; Asakura, Yoko; Usuda, Yoshihiro; Fukuda, Hiroo; Chinen, Akito

    2017-02-01

    Alginate is a marine non-food-competing polysaccharide that has potential applications in biorefinery. Owing to its large size (molecular weight >300,000 Da), alginate cannot pass through the bacterial cell membrane. Therefore, bacteria that utilize alginate are presumed to have an enzyme that degrades extracellular alginate. Recently, Vibrio algivorus sp. SA2(T) was identified as a novel alginate-decomposing and alginate-utilizing species. However, little is known about the mechanism of alginate degradation and metabolism in this species. To address this issue, we screened the V. algivorus genomic DNA library for genes encoding polysaccharide-decomposing enzymes using a novel double-layer plate screening method and identified alyB as a candidate. Most identified alginate-decomposing enzymes (i.e., alginate lyases) must be concentrated and purified before extracellular alginate depolymerization. AlyB of V. algivorus heterologously expressed in Escherichia coli depolymerized extracellular alginate without requiring concentration or purification. We found seven homologues in the V. algivorus genome (alyB, alyD, oalA, oalB, oalC, dehR, and toaA) that are thought to encode enzymes responsible for alginate transport and metabolism. Introducing these genes into E. coli enabled the cells to assimilate soluble alginate depolymerized by V. algivorus AlyB as the sole carbon source. The alginate was bioconverted into L-lysine (43.3 mg/l) in E. coli strain AJIK01. These findings demonstrate a simple and novel screening method for identifying polysaccharide-degrading enzymes in bacteria and provide a simple alginate biocatalyst and fermentation system with potential applications in industrial biorefinery.

  6. Adsorption and desorption of Zn(II) and Cu(II) on Ca- alginate immobilized activated rice bran

    NASA Astrophysics Data System (ADS)

    Suratman, A.; Kamalia, N. Z.; Kusumawati, W. A.

    2016-02-01

    Ca-alginate immobilized activated rice bran has been used for adsorption of Zn(II) and Cu(II) from aqueous solution. The effect of the pH, kinetics model, adsorption isotherm and desorption on the adsorption performance was investigated. Activated rice bran was immobilized by the entrapment in alginate beads. The adsorption strength of Ca-alginate immobilized activated rice bran was compared to Ca-alginate and non-immobilized activated rice bran. The concentrations of adsorbed ions were analyzed using Atomic Absorption Spectrophotometer (AAS). The result showed that pH of 4.0 and the contact time of 120 min are the optimum condition for adsorption of Zn(II) and Cu(II). The adsorption kinetic of Zn(II) and Cu(II) followed the pseudo-second-order model with adsorption rate constant 4.9 x 10-2 and 3.14 g.mg-1.min-1, respectively. The both adsorption processes obeyed Langmuir isotherm with adsorption capacity of 2.03 and 2.42 mg.g-1 of adsorbent, respectively. The strength of Zn adsorption on Ca-alginate immobilized activated rice bran (86.63%) was more effective compared to Ca-alginate beads (60.96%) and activated rice bran (43.85%). The strength of Cu adsorption was 80.00%, 61.50% and 22.10%, respectively. The desorption of Zn(II) and Cu(II) showed that recovery percentage of the adsorption was 76.56% and 57.80% with the condition of using HCl 0.1 M as desorption agent for 1 hour.

  7. Improved production of reducing sugars from rice straw using crude cellulase activated with Fe₃O₄/alginate nanocomposite.

    PubMed

    Srivastava, Neha; Singh, Jay; Ramteke, Pramod W; Mishra, P K; Srivastava, Manish

    2015-05-01

    Effect of Fe3O4 nanoparticles (NPs) and Fe3O4/Alginate nanocomposites (NCs) have been investigated on production and thermostability of crude cellulase enzyme system obtained by newly isolated thermotolerant Aspergillus fumigatus AA001. Fe3O4 NPs and Fe3O4/Alginate NCs have been synthesized by co-precipitation method and characterized through various techniques. In presence of Fe3O4 NPs and Fe3O4/Alginate NCs, filter paper activity of crude cellulase was increased about 35% and 40%, respectively in 72 h as compared to control. Fe3O4/Alginate NCs treated crude enzyme was thermally stable up to 8h at 70°C and retained 56% of its relative activity whereas; control samples could retain only 19%. Further, the hydrolysis of 1.0% alkali treated rice straw using Fe3O4/Alginate NCs treated cellulase gave much higher sugar productivity than control at optimal condition. These findings may be utilized in the area of biofuels and biowaste management.

  8. Antimicrobial and anti-inflammatory activity of chitosan-alginate nanoparticles: a targeted therapy for cutaneous pathogens

    PubMed Central

    Friedman, Adam J; Phan, Jenny; Schairer, David; Champer, Jackson; Qin, Min; Pirouz, Aslan; Blecher, Karin; Oren, Ami; Liu, Phil; Modlin, Robert L; Kim, Jenny

    2012-01-01

    Advances in nanotechnology have demonstrated potential application of nanoparticles for effective and targeted drug delivery. Here, we investigated the antimicrobial and immunological properties and the feasibility of using nanoparticles to deliver antimicrobial agents to treat a cutaneous pathogen. Nanoparticles synthesized with chitosan and alginate demonstrated a direct antimicrobial activity in vitro against Propionibacterium acnes, the bacterium linked to the pathogenesis of acne. By electron microscopy imaging, chitosan-alginate nanoparticles were found to induce disruption of the P. acnes cell membrane, providing a mechanism for the bactericidal effect. The chitosan-alginate nanoparticles also exhibited anti-inflammatory properties as they inhibited P. acnes induced inflammatory cytokine production in human monocytes and keratinocytes. Furthermore, benzoyl peroxide, a commonly used anti-acne drug, was effectively encapsulated in the chitosan-alginate nanoparticles and demonstrated superior antimicrobial activity against P. acnes compared to benzoyl peroxide alone while demonstrating less toxicity to eukaryotic cells. Together, these data suggest the potential utility of topical delivery of chitosan-alginate nanoparticle encapsulated drug therapy for the treatment of dermatologic conditions with infectious and inflammatory components. PMID:23190896

  9. Adsorption of arsenic by activated carbon, calcium alginate and their composite beads.

    PubMed

    Hassan, A F; Abdel-Mohsen, A M; Elhadidy, H

    2014-07-01

    The present investigation deals with preparation of three different adsorbent materials namely; potassium hydroxide activated carbon based apricot stone (C), calcium alginate beads (G) and calcium alginate/activated carbon composite beads (GC) were used for the removal of arsenic. The prepared adsorbent materials were characterized by scanning electron microscope (SEM), Fourier transform infrared spectroscopy (FTIR), N2-adsorption at -196°C and point of zero charge. From the obtained results, it was found that the porosity, surface area and total pore volume of the adsorbent material C>GC>G respectively, however, the G adsorbent has more acidic function group than the other adsorbents. The influence of pH, time, temperature and initial concentration of arsenic(V) were studied and optimized. GC exhibits the maximum As(V) adsorption (66.7mg/g at 30°C). The adsorption of arsenic ions was observed to follow pseudo-second order mechanism as well as the thermodynamic parameters confirm also the endothermic spontaneous and a physisorption process.

  10. Dose and time-dependent effects of cyanide on thiosulfate sulfurtransferase, 3-mercaptopyruvate sulfurtransferase, and cystathionine λ-lyase activities.

    PubMed

    Singh, Poonam; Rao, Pooja; Bhattacharya, Rahul

    2013-12-01

    We assessed the dose-dependent effect of potassium cyanide (KCN) on thiosulfate sulfurtransferase (TST), 3-mercaptopyruvate sulfurtransferase (3-MPST), and cystathionine λ-lyase (CST) activities in mice. The time-dependent effect of 0.5 LD50 KCN on cyanide level and cytochrome c oxidase (CCO), TST, 3-MPST, and CST activities was also examined. Furthermore, TST, 3-MPST, and CST activities were measured in stored mice cadavers. Hepatic and renal TST activity increased by 0.5 LD50 KCN but diminished by ≥2.0 LD50. After 0.5 LD50 KCN, the elevated hepatic cyanide level was accompanied by increased TST, 3-MPST, and CST activities, and CCO inhibition. Elevated renal cyanide level was only accompanied by increased 3-MPST activity. No appreciable change in enzyme activities was observed in mice cadavers. The study concludes that high doses of cyanide exert saturating effects on its detoxification enzymes, indicating their exogenous use during cyanide poisoning. Also, these enzymes are not reliable markers of cyanide poisoning in autopsied samples.

  11. Highly Active and Specific Tyrosine Ammonia-Lyases from Diverse Origins Enable Enhanced Production of Aromatic Compounds in Bacteria and Saccharomyces cerevisiae

    PubMed Central

    Stahlhut, Steen Gustav; Li, Mingji; Gaspar, Paula; Siedler, Solvej; Förster, Jochen; Maury, Jérôme; Borodina, Irina

    2015-01-01

    Phenylalanine and tyrosine ammonia-lyases form cinnamic acid and p-coumaric acid, which are precursors of a wide range of aromatic compounds of biotechnological interest. Lack of highly active and specific tyrosine ammonia-lyases has previously been a limitation in metabolic engineering approaches. We therefore identified 22 sequences in silico using synteny information and aiming for sequence divergence. We performed a comparative in vivo study, expressing the genes intracellularly in bacteria and yeast. When produced heterologously, some enzymes resulted in significantly higher production of p-coumaric acid in several different industrially important production organisms. Three novel enzymes were found to have activity exclusively for phenylalanine, including an enzyme from the low-GC Gram-positive bacterium Brevibacillus laterosporus, a bacterial-type enzyme from the amoeba Dictyostelium discoideum, and a phenylalanine ammonia-lyase from the moss Physcomitrella patens (producing 230 μM cinnamic acid per unit of optical density at 600 nm [OD600]) in the medium using Escherichia coli as the heterologous host). Novel tyrosine ammonia-lyases having higher reported substrate specificity than previously characterized enzymes were also identified. Enzymes from Herpetosiphon aurantiacus and Flavobacterium johnsoniae resulted in high production of p-coumaric acid in Escherichia coli (producing 440 μM p-coumaric acid OD600 unit−1 in the medium) and in Lactococcus lactis. The enzymes were also efficient in Saccharomyces cerevisiae, where p-coumaric acid accumulation was improved 5-fold over that in strains expressing previously characterized tyrosine ammonia-lyases. PMID:25911487

  12. Efficiency of uronic acid uptake in marine alginate-degrading fungi

    NASA Astrophysics Data System (ADS)

    Schaumann, K.; Weide, G.

    1995-03-01

    Despite the fact that many marine fungi, including phycomycetes, yeasts, ascomycetes and hyphomycetes, have been recorded from living and/or dead phaeophytes, only a few of these have been shown to be capable of degrading alginic acid or alginates. The degradation is achieved by the action of an exoenzyme complex, comprising alginate lyase, as well as alginate hydrolase activities. The latter was detected only recently by the authors. In this study, the growth of two marine sodiumalginate-degrading deuteromycetes, Asteromyces cruciatus and Dendryphiella salina, was investigated, and the assimilation efficiency of sodiumalginate and its uronic acid degradation products, respectively, was estimated from the economic coefficient (E). E is calculated from the mycelial dry weight, divided by the weight of substrate consumed for this production. The economic coefficient for A. cruciatus was 48.6%, and that of D. salina 38.9%. This indicates that the former species uses the alginate degradation products more efficiently than the latter. The observed E-values for the marine deuteromycetes agree with those from other fungi, e.g. terrestrial species. In general, it is concluded that the marine fungi appear to play a more important role in kelp-based ecosystems than was realized previously.

  13. Cytotoxicity, Bactericidal, and Antioxidant Activity of Sodium Alginate Hydrosols Treated with Direct Electric Current

    PubMed Central

    Król, Żaneta; Marycz, Krzysztof; Kulig, Dominika; Marędziak, Monika; Jarmoluk, Andrzej

    2017-01-01

    The aim of the study was to investigate the effect of using direct electric current (DC) of 0, 200, and 400 mA for five minutes on the physiochemical properties, cytotoxicity, antibacterial, and antioxidant activity of sodium alginate hydrosols with different sodium chloride concentrations. The pH, oxidation-reduction potential (ORP), electrical conductivity (EC), and available chlorine concentration (ACC) were measured. The effect of sodium alginate hydrosols treated with DC on Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, Micrococcus luteus, Escherichia coli, Salmonella enteritidis, Yersinia enterocolitica, Pseudomonas fluorescence, and RAW 264.7 and L929 cells was investigated. Subsequently, the antioxidant properties of hydrosols were evaluated by determining the scavenging ability of 1,1-diphenyl-2-picrylhydrazyl free radical (DPPH) and ferric reducing antioxidant power (FRAP). The results have shown that after applying 400 mA in hydrosol samples with 0.1% and 0.2% NaCl all tested bacteria were inactivated. The ACC concentration of C400 samples with NaCl was equal to 13.95 and 19.71 mg/L, respectively. The cytotoxicity analysis revealed that optimized electric field conditions and the addition of sodium chloride allow for the avoidance of toxicity effects on normal cells without disturbing the antibacterial effects. Due to the presence of oxidizing substances, the DPPH of variants treated with DC was lower than the DPPH of control samples. PMID:28327520

  14. Novel zinc alginate hydrogels prepared by internal setting method with intrinsic antibacterial activity.

    PubMed

    Straccia, Maria Cristina; d'Ayala, Giovanna Gomez; Romano, Ida; Laurienzo, Paola

    2015-07-10

    In this paper, a controlled gelation of alginate was performed for the first time using ZnCO3 and GDL. Uniform and transparent gels were obtained and investigated as potential wound dressings. Homogeneity, water content, swelling capability, water evaporation rate, stability in normal saline solution, mechanical properties and antibacterial activity were assessed as a function of zinc concentration. Gelation rate increased at increasing zinc content, while a decrease in water uptake and an improvement of stability were found. Release of zinc in physiological environments showed that concentration of zinc released in solution lies below the cytotoxicity level. Hydrogels showed antimicrobial activity against Escherichia coli. The hydrogel with highest zinc content was stabilized with calcium by immersion in a calcium chloride solution. The resulting hydrogel preserved homogeneity and antibacterial activity. Furthermore, it showed even an improvement of stability and mechanical properties, which makes it suitable as long-lasting wound dressing.

  15. Relationships between salicylic acid content, phenylalanine ammonia-lyase (PAL) activity, and resistance of barley to aphid infestation.

    PubMed

    Chaman, Mercedes E; Copaja, Sylvia V; Argandoña, Victor H

    2003-04-09

    It has been suggested that salicylic acid (SA) is a signal in acquired resistance to pathogens in several plants. Also, it has been suggested that infestation of plants causes an increase in the activity of phenylalanine ammonia-lyase (PAL), a key phenolic biosynthesis enzyme. The purpose of this work was to investigate whether the induction of SA and PAL activity is related to the susceptibility of barley to aphid infestation. The induction of free and conjugated SA in two barley cultivars that differ in susceptibility to aphids was analyzed. Analyses of several physiological parameters showed that cv. UNA-80 was more susceptible to the aphid Schizaphis graminum than cv. LM-109. Salicylic acid was not detected in noninfested plants. Levels of free and conjugated SA in cv. LM-109 and of conjugated SA in cv. UNA-80 increased with aphid infestation, whereas the levels of free SA in cv. UNA-80 remained high under all infestation degrees. Maximum values reached in both cultivars were not significantly different. With respect to PAL activity, cv. LM-109 showed a significantly higher specific activity than cv. UNA-80, the more susceptible cultivar. The relationship between the susceptibility of a plant to aphid and SA induction and PAL activity is discussed.

  16. Production and characterization of guluronate lyase from Klebsiella pneumoniae for applications in seaweed biotechnology.

    PubMed

    Ostgaard, K; Knutsen, S H; Dyrset, N; Aasen, I M

    1993-09-01

    Cultures of Klebsiella pneumoniae fermenting sodium alginate produce an extracellular guluronate-specific alginate lyase. This enzyme production was studied in stirred-tank fermentors. Different alginate substrates gave moderate differences in growth and enzyme yield. Alginates with low guluronic content gave reduced biomass but favored enzyme production. Low molecular weight (down to DPn approximately 270) also favored enzyme production. Excessive depolymerization of substrates occurred during heat sterilization of culture media. The enzyme was characterized by its specificity and sensitivity to pH, salt, and calcium. Improved yields of viable protoplasts were documented for Laminaria digitata (Huds.) Lamour.

  17. Activation and stabilization of the hydroperoxide lyase enzymatic extract from mint leaves (Mentha spicata) using selected chemical additives.

    PubMed

    Akacha, Najla B; Karboune, Salwa; Gargouri, Mohamed; Kermasha, Selim

    2010-03-01

    The effects of selected lyoprotecting excipients and chemical additives on the specific activity and the thermal stability of the hydroperoxide lyase (HPL) enzymatic extract from mint leaves were investigated. The addition of KCl (5%, w/w) and dextran (2.5%, w/w) to the enzymatic extract, prior to lyophilization, increased the HPL specific activity by 2.0- and 1.2-fold, respectively, compared to the control lyophilized extract. From half-life time (t (1/2)), it can be seen that KCl has enhanced the HPL stability by 1.3- to 2.3-fold, during long-period storage at -20 degrees Celsius and 4 degrees Celsius. Among the selected additives used throughout this study, glycine appeared to be the most effective one. In addition to the activation effect conferred by glycine, it also enhanced the HPL thermal stability. In contrast, polyhydroxyl-containing additives were not effective for stabilizing the HPL enzymatic extract. On the other hand, there was no signification increase in HPL activity and its thermal stability with the presence of Triton X-100. The results also showed that in the presence of glycine (10%), the catalytic efficiency of HPL was increased by 2.45-fold than that without additive.

  18. Structures of almond hydroxynitrile lyase isoenzyme 5 provide a rationale for the lack of oxidoreductase activity in flavin dependent HNLs.

    PubMed

    Pavkov-Keller, Tea; Bakhuis, Janny; Steinkellner, Georg; Jolink, Fenneke; Keijmel, Esther; Birner-Gruenberger, Ruth; Gruber, Karl

    2016-10-10

    Hydroxynitrile lyases (HNLs) catalyze the asymmetric addition of HCN to aldehydes producing enantiomerically pure cyanohydrins. These enzymes can be heterologously expressed in large quantities making them interesting candidates for industrial applications. The HNLs from Rosaceae evolved from flavin dependent dehydrogenase/oxidase structures. Here we report the high resolution X-ray structure of the highly glycosylated Prunus amygdalus HNL isoenzyme5 (PaHNL5 V317A) expressed in Aspergillus niger and its complex with benzyl alcohol. A comparison with the structure of isoenzyme PaHNL1 indicates a higher accessibility to the active site and a larger cavity for PaHNL5. Additionally, the PaHNL5 complex structure with benzyl alcohol was compared with the structurally related aryl-alcohol oxidase (AAO). Even though both enzymes contain an FAD-cofactor and histidine residues at crucial positions in the active site, PaHNL5 lacks the oxidoreductase activity. The structures indicate that in PaHNLs benzyl alcohol is bound too far away from the FAD cofactor in order to be oxidized.

  19. Photo-activated ionic gelation of alginate hydrogel: real-time rheological monitoring of the two-step crosslinking mechanism.

    PubMed

    Higham, Alina K; Bonino, Christopher A; Raghavan, Srinivasa R; Khan, Saad A

    2014-07-21

    We examine the gelation of alginate undergoing ionic crosslinking upon ultraviolet (UV) irradiation using in situ dynamic rheology. Hydrogels are formed by combining alginate with calcium carbonate (CaCO3) particles and a photoacid generator (PAG). The PAG is photolyzed upon UV irradiation, resulting in the release of free calcium ions for ionic crosslinking. The viscous and elastic moduli during gelation are monitored as a function of the UV irradiation intensity, exposure time, alginate concentration, and the ratio between alginate and calcium carbonate. Gel time decreases as irradiation intensity increases because a larger concentration of PAG is photolyzed. Interestingly, dark curing, the continuing growth of microstructure in the absence of UV light, is observed. In some instances, the sample transitions from a solution to a gel during the dark curing phase. Additionally, when exposed to constant UV irradiation after the dark curing phase, samples reach the same plateau modulus as samples exposed to constant UV without dark curing, implying that dark curing does not affect the gelation mechanism. We believe the presence of dark curing is the result of the acidic environment persisting within the sample, allowing CaCO3 to dissociate, thereby releasing free Ca(2+) ions capable of binding with the available appropriate ionic blocks of the polymer chains. The growth of microstructure is then detected if the activation barrier has been crossed to release sufficient calcium ions. In this regard, we calculate a value of 30 J that represents the activation energy required to initiate gelation.

  20. Molecular characterization of plant growth promoting rhizobacteria that enhance peroxidase and phenylalanine ammonia-lyase activities in chile (Capsicum annuum L.) and tomato (Lycopersicon esculentum Mill.).

    PubMed

    Sharma, Alok; Pathak, Ashutosh; Sahgal, Manvika; Meyer, Jean-Marie; Wray, Victor; Johri, Bhavdish N

    2007-11-01

    Pythium and Phytophthora species are associated with damping-off diseases in vegetable nurseries and reduce seedling stand and yield. In this study, bacterial isolates were selected on the basis of in vitro antagonism potential to inhibit mycelial growth of damping-off pathogens along with plant growth properties for field assessment in wet and winter seasons. We demonstrate efficacy of bacterial isolates to protect chile and tomato plants under natural vegetable nursery and artificially created pathogen-infested (Pythium and Phytophthora spp.) nursery conditions. After 21 days of sowing, chile and tomato plants were harvested and analysed for peroxidase and phenylalanine ammonia-lyase activities. Pseudomonas sp. strains FQP PB-3, FQA PB-3 and GRP(3 )were most effective in increasing shoot length (P > 0.05%) in both artificial and natural field sites. For example, Pseudomonas sp. FQA PB-3 treatment increased shoot length by 40% in the artificial Pythium 4746 infested nursery site in chile plants in the wet season. The bacterial treatments significantly increased the activity of peroxidase and phenylalanine ammonia-lyase in chile and tomato plant tissues, which are well known as indicators of an active lignification process. Thus, we conclude that treatment with potential bacterial plant growth promoting agents help plants against pathogen invasion by modulating plant peroxidase and phenylalanine ammonia-lyase activities.

  1. Impact of alginate-producing Pseudomonas aeruginosa on alveolar macrophage apoptotic cell clearance.

    PubMed

    McCaslin, Charles A; Petrusca, Daniela N; Poirier, Christophe; Serban, Karina A; Anderson, Gregory G; Petrache, Irina

    2015-01-01

    Pseudomonas aeruginosa infection is a hallmark of lung disease in cystic fibrosis. Acute infection with P. aeruginosa profoundly inhibits alveolar macrophage clearance of apoptotic cells (efferocytosis) via direct effect of virulence factors. During chronic infection, P. aeruginosa evades host defense by decreased virulence, which includes the production or, in the case of mucoidy, overproduction of alginate. The impact of alginate on innate immunity, in particular on macrophage clearance of apoptotic cells is not known. We hypothesized that P. aeruginosa strains that exhibit reduced virulence impair macrophage clearance of apoptotic cells and we investigated if the polysaccharide alginate produced by mucoid P. aeruginosa is sufficient to inhibit alveolar macrophage efferocytosis. Rat alveolar or human peripheral blood monocyte (THP-1)-derived macrophage cell lines were exposed in vitro to exogenous alginate or to wild type or alginate-overproducing mucoid P. aeruginosa prior to challenge with apoptotic human Jurkat T-lymphocytes. The importance of LPS contamination and that of structural integrity of alginate polymers was tested using alginate of different purities and alginate lyase, respectively. Alginate inhibited alveolar macrophage efferocytosis in a dose- and time-dependent manner. This effect was augmented but not exclusively attributed to lipopolysaccharide (LPS) present in alginates. Alginate-producing P. aeruginosa inhibited macrophage efferocytosis by more than 50%. A mannuronic-specific alginate lyase did not restore efferocytosis inhibited by exogenous guluronic-rich marine alginate, but had a marked beneficial effect on efferocytosis of alveolar macrophages exposed to mucoid P. aeruginosa. Despite decreased virulence, mucoid P. aeruginosa may contribute to chronic airway inflammation through significant inhibition of alveolar clearance of apoptotic cells and debris. The mechanism by which mucoid bacteria inhibit efferocytosis may involve alginate

  2. Preservation of high phenylalanine ammonia lyase activities in roots of Japanese Striped corn: a potential oral therapeutic to treat phenylketonuria.

    PubMed

    López-Villalobos, Arturo; Lücker, Joost; López-Quiróz, Ana Angela; Yeung, Edward C; Palma, Kristoffer; Kermode, Allison R

    2014-06-01

    Phenylketonuria (PKU) is an inherited metabolic disorder caused by deficient phenylalanine hydroxylase (PAH) activity, the enzyme responsible for the disposal of excess amounts of the essential amino acid phenylalanine (Phe). Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) has potential to serve as an enzyme substitution therapy for this human genetic disease. Using 7-day-old Japanese Striped corn seedlings (Japonica Striped maize, Zea mays L. cv. japonica) that contain high activities of PAL, we investigated a number of methods to preserve the roots as an intact food and for long-term storage. The cryoprotectant effects of maple syrup and other edible sugars (mono- and oligosaccharides) were evaluated. Following thawing, the preserved roots were then examined to determine whether the rigid plant cell walls could protect the PAL enzyme from proteolysis during simulated (in vitro) digestion comprised of gastric and intestinal phases. While several treatments led to retention of PAL activity during freezing, upon thawing and in vitro digestion, root tissues that had been previously frozen in the presence of maple syrup exhibited the highest residual PAL activities (∼50% of the initial enzyme activity), in marked contrast to all of the treatments using other edible sugars. The structural integrity of the root cells, and the stability of the functional PAL tetramer were also preserved with the maple syrup protocol. These results have significance for the formulation of oral enzyme/protein therapeutics. When plant tissues are adequately preserved, the rigid cell walls constitute a protective barrier even under harsh (e.g. gastrointestinal-like) conditions.

  3. Anticoagulant and FGF/FGFR signal activating activities of the heparinoid propylene glycol alginate sodium sulfate and its oligosaccharides.

    PubMed

    Wu, Jian; Zhang, Meng; Zhang, Yiran; Zeng, Yangyang; Zhang, Lijuan; Zhao, Xia

    2016-01-20

    Propylene glycol alginate sodium sulfate (PSS), prepared by chemical sulfation of alginate, has been used for treating cardiovascular diseases in China for nearly 30 years. In the current study, the PSS was hydrolyzed partially by an environment-friendly solid phase acid degradation method, and then separated by using a Bio-Gel P6 chromatographic column. Thirteen PSS oligosaccharide fractions were obtained and characterized by ESI-MS. The results of different coagulation assays showed that a high molecular weight and a higher degree of sulfation were essential for the anticoagulant activity of the PSS because the PSS oligosaccharides exhibited no detectable anticoagulant activity. In contrast, not only PSS but also certain oligosaccharides showed significant activities in stimulation of FGF1, 2, 7, 8, 9 or 10 induced cell proliferation in FGFR1c-expressing BaF3 cells. Such properties made the PSS and its oligosaccharides promising compounds in the regulation of FGF-dependent development, treatment of cancer, and wound healing processes.

  4. Disappearance of isocitrate lyase enzyme from cells of Chlorella pyrenoidosa

    PubMed Central

    John, P. C. L.; Thurston, C. F.; Syrett, P. J.

    1970-01-01

    1. When acetate-adapted cells of Chlorella are suspended in nitrogen-free medium and supplied with glucose, isocitrate lyase activity disappears from the cells at a rate of about 9%/h. This loss of activity is shown to be accompanied by loss of isocitrate lyase protein. 2. When isocitrate lyase activity is assayed in intact cells after freezing and thawing, the rate of loss of activity after addition of glucose approaches 20%/h. 3. It is shown, by using 35S, that the rate of turnover of isocitrate lyase protein is somewhat lower than that of other major soluble proteins; general protein turnover during nitrogen starvation, and after glucose addition, is too slow to account for the rate of loss of isocitrate lyase protein. 4. Disappearance of isocitrate lyase activity must result from a mechanism that allows degradation of this specific protein under conditions of limiting nitrogen supply. PMID:5492855

  5. Microbacterium oxydans, a novel alginate- and laminarin-degrading bacterium for the reutilization of brown-seaweed waste.

    PubMed

    Kim, Eun Jung; Fathoni, Ahmad; Jeong, Gwi-Taek; Jeong, Hyun Do; Nam, Taek-Jeong; Kong, In-Soo; Kim, Joong Kyun

    2013-11-30

    There is a growing demand for the efficient treatment of seaweed waste. We identified six bacterial strains from the marine environment for the reutilization of brown-seaweed waste, and the most potentially useful strain, Microbacterium oxydans, was chosen and further investigated. Plate assays indicated that this bacterial isolate possessed both alginate lyase and laminarinase activities. The optimal inoculum size, pH, temperature and substrate concentration for the degradation of brown-seaweed polysaccharides by the isolate were as follows: 20% (v v(-1)), pH 6.0, 37 °C, and 5 g L(-1) for alginate and 20% (v v(-1)), pH 6.0, 30 °C, and 10 g L(-1) for laminarin, respectively. During 6 d in culture under the optimal conditions, the isolate produced 0.17 g L(-1) of reducing sugars from alginate with 11.0 U mL(-1) of maximal alginate lyase activity, and 5.11 and 2.88 g L(-1) of reducing sugars and glucose from laminarin, respectively. In particular, a fair amount of laminarin was degraded to glucose (28.8%) due to the isolate's exolytic laminarinase activity. As a result, the reutilization of brown-seaweed waste by this isolate appears to be possible for the production of reducing sugars as a valuable resource. This is the first study to directly demonstrate the ability of M. oxydans to degrade both alginate and laminarin.

  6. Phenylalanine ammonia-lyase, flavanone 3β-hydroxylase and flavonol synthase enzyme activity by a new in vitro assay method in berry fruits.

    PubMed

    Flores, Gema; De la Peña Moreno, Fernando; Blanch, Gracia Patricia; Del Castillo, Maria Luisa Ruiz

    2014-06-15

    An HPLC method for the determination of phenylalanine ammonia-lyase, flavanone 3β-hydroxylase and flavonol synthase enzyme activity is proposed. This method is based on the determination of the compounds produced and consumed on the enzymatic reaction in just one chromatographic analysis. Optimisation of the method considered kinetic studies to establish the incubation time to perform the assay. The method here described proved to be an interesting approach to measure the activities of the three enzymes simultaneously increasing the rapidity, selectivity and sensitivity over other exiting methods. The enzyme activity method developed was applied to strawberry, raspberry, blackberry, redcurrant and blackcurrant fruits.

  7. A low-temperature-active alkaline pectate lyase from Xanthomonas campestris ACCC 10048 with high activity over a wide pH range.

    PubMed

    Yuan, Peng; Meng, Kun; Wang, Yaru; Luo, Huiying; Shi, Pengjun; Huang, Huoqing; Tu, Tao; Yang, Peilong; Yao, Bin

    2012-11-01

    Alkaline pectate lyases are favorable for the textile industry. Here, we report the gene cloning and expression of a low-temperature-active alkaline pectate lyase (PL D) from Xanthomonas campestris ACCC 10048. Deduced PL D consists of a putative 27-residue signal peptide and a catalytic domain of 320 residues belonging to family PF09492. Recombinant PL D (r-PL D) produced in Escherichia coli was purified to electrophoretic homogeneity with a single step of Ni(2+)-NTA affinity chromatography and showed an apparent molecular weight of ~38 kDa. The pH and temperature optima of r-PL D were found to be 9.0 °C and 30 °C, respectively. Compared with its microbial counterparts, r-PL D had higher activity over a wide pH range (>45 % of the maximum activity at pH 3.0-12.0) and at lower temperatures (>35 % of activity even at 0 °C). The K(m) and V(max) values of r-PL D for polygalacturonic acid were 4.9 gl(-1) and 30.1 μmolmin(-1) mg(-1), respectively. Compared with the commercial compound pectinase from Novozymes, r-PL D showed similar efficacy in reducing the intrinsic viscosity of polygalacturonic acid (35.1 % vs. 36.5 %) and in bioscouring of jute (10.25 % vs. 10.82 %). Thus, r-PL D is a valuable additive candidate for the textile industry.

  8. Antifungal activity against Candida albicans of starch Pickering emulsion with thymol or amphotericin B in suspension and calcium alginate films.

    PubMed

    Cossu, Andrea; Wang, Min S; Chaudhari, Amol; Nitin, Nitin

    2015-09-30

    Conventional antifungal treatments against Candida albicans in the oral cavity often result in increased cytotoxicity. The goal of this study was to determine the potential of starch Pickering emulsion as a delivery vehicle for an antifungal natural phenolic compound such as thymol in simulated saliva fluid (SSF) compared to amphotericin B. An oil-in-water (o/w) emulsion was stabilized using starch particles. Physical stability of the emulsion and disruption induced by α-amylase activity in SSF was evaluated. Encapsulated thymol in o/w emulsion was compared to encapsulated amphotericin B for antifungal activity against C. albicans in suspension using emulsions or zone inhibition assay on agar plates using emulsions dispersed in alginate films. Results showed that the emulsions were stable for at least three weeks. Digestion of the emulsion by α-amylase led to coalescence of emulsion droplets. The antifungal activity of thymol and amphotericin B in emulsion formulation was enhanced upon incubation with α-amylase. Results from the zone inhibition assay demonstrated efficacy of the emulsions dispersed in alginate films. Interestingly, addition of α-amylase to the alginate films resulted in a decreased inhibitory effect. Overall, this study showed that starch Pickering emulsions have a potential to deliver hydrophobic antifungal compounds to treat oral candidiasis.

  9. Pervaporation dehydration of ethanol by hyaluronic acid/sodium alginate two-active-layer composite membranes.

    PubMed

    Gao, Chengyun; Zhang, Minhua; Ding, Jianwu; Pan, Fusheng; Jiang, Zhongyi; Li, Yifan; Zhao, Jing

    2014-01-01

    The composite membranes with two-active-layer (a capping layer and an inner layer) were prepared by sequential spin-coatings of hyaluronic acid (HA) and sodium alginate (NaAlg) on the polyacrylonitrile (PAN) support layer. The SEM showed a mutilayer structure and a distinct interface between the HA layer and the NaAlg layer. The coating sequence of two-active-layer had an obvious influence on the pervaporation dehydration performance of membranes. When the operation temperature was 80 °C and water concentration in feed was 10 wt.%, the permeate fluxes of HA/Alg/PAN membrane and Alg/HA/PAN membrane were similar, whereas the separation factor were 1130 and 527, respectively. It was found that the capping layer with higher hydrophilicity and water retention capacity, and the inner layer with higher permselectivity could increase the separation performance of the composite membranes. Meanwhile, effects of operation temperature and water concentration in feed on pervaporation performance as well as membrane properties were studied.

  10. Anticoagulant and antithrombotic activities of low-molecular-weight propylene glycol alginate sodium sulfate (PSS).

    PubMed

    Xin, Meng; Ren, Li; Sun, Yang; Li, Hai-hua; Guan, Hua-Shi; He, Xiao-Xi; Li, Chun-Xia

    2016-05-23

    Propylene glycol alginate sodium sulfate (PSS), a sulfated polysaccharide derivative, has been used as a heparinoid drug to prevent and treat hyperlipidemia and ischemic cardio-cerebrovascular diseases in China for nearly 30 years. To extend the applications of PSS, a series of low-molecular-weight PSSs (named FPs) were prepared by oxidative-reductive depolymerization, and the antithrombotic activities were investigated thoroughly in vitro and in vivo. The bioactivity evaluation demonstrated a positive correlation between the molecular weight and the anticoagulant and antithrombotic activities of FPs. FPs could prolong the APTT and clotting time and reduce platelet aggregation significantly. FPs could also effectively inhibit factor IIa in the presence of AT-III and HC-II. FPs decreased the wet weights and lengths of the thrombus and increased occlusion times in vivo. FP-6k, a PSS fragment with a molecular weight of 6 kDa, is an optimal antithrombotic candidate for further study and showed little chance for hemorrhagic action.

  11. Insights into the Activity Change of Spore Photoproduct Lyase Induced by Mutations at a Peripheral Glycine Residue

    PubMed Central

    Yang, Linlin; Li, Lei

    2017-01-01

    UV radiation triggers the formation of 5-thyminyl-5,6-dihydrothymine, i.e., the spore photoproduct (SP), in the genomic DNA of bacterial endospores. These SPs, if not repaired in time, may lead to genome instability and cell death. SP is mainly repaired by spore photoproduct lyase (SPL) during spore outgrowth via an unprecedented protein-harbored radical transfer pathway that is composed of at least a cysteine and two tyrosine residues. This mechanism is consistent with the recently solved SPL structure that shows all three residues are located in proximity and thus able to participate in the radical transfer process during the enzyme catalysis. In contrast, an earlier in vivo mutational study identified a glycine to arginine mutation at the position 168 on the B. subtilis SPL that is >15 Å away from the enzyme active site. This mutation appears to abolish the enzyme activity because endospores carrying this mutant were sensitive to UV light. To understand the molecular basis for this rendered enzyme activity, we constructed two SPL mutations G168A and G168R, examined their repair of dinucleotide SP TpT, and found that both mutants exhibit reduced enzyme activity. Comparing with the wildtype (WT) SPL enzyme, the G168A mutant slows down the SP TpT repair by 3~4-fold while the G168R mutant by ~ 80-fold. Both mutants exhibit a smaller apparent (DV) kinetic isotope effect (KIE) but a bigger competitive (DV/K) KIE than that by the WT SPL. Moreover, the G168R mutant also produces a large portion of the abortive repair product TpT-SO2−; the formation of which indicates that cysteine 141 is no longer well positioned as the H-donor to the thymine allylic radical intermediate. All these data imply that the mutation at the remote glycine 168 residue alters the enzyme 3D structure, subsequently reducing the SPL activity by changing the positions of the essential amino acids involved in the radical transfer process.

  12. Efficacy of activated alginate-based nanocomposite films to control Listeria monocytogenes and spoilage flora in rainbow trout slice.

    PubMed

    Alboofetileh, Mehdi; Rezaei, Masoud; Hosseini, Hedayat; Abdollahi, Mehdi

    2016-01-01

    Essential oils of clove, coriander, caraway, marjoram, cinnamon, and cumin were tested for their antilisterial activity by application of agar diffusion assay (experiment 1). Marjoram essential oil (MEO) showed the highest inhibitory effect, followed by clove and cinnamon. Subsequently, these essential oils were incorporated to alginate/clay nanocomposite films and antilisterial effectiveness of the films was studied in a model solid food system during 12 days at 10 °C (experiment 2). The results revealed that the films with MEO were more effective against Listeria monocytogenes in the model step. Finally, alginate-clay film incorporating 1 % MEO was applied to inoculated trout slices during refrigerated storage (4 °C) for 15 days (experiment 3). The control and the wrapped fish samples were analyzed periodically for microbiological (L. monocytogenes, total viable count, psychrotrophic count) and chemical (TVB-N) properties. The results demonstrated that alginate-clay films enriched with 1 % MEO significantly delayed the growth of L. monocytogenes during the 15-day storage with final counts reaching 6.23 log CFU/g while the counts in control samples were significantly higher reaching 7.38 log CFU/g (p < 0.05). Furthermore, active films efficiently reduced total viable count and psychrotrophic count as well as TVB-N in the fish slice during refrigerated storage.

  13. Statins upregulate cystathionine γ-lyase transcription and H2S generation via activating Akt signaling in macrophage.

    PubMed

    Xu, Yuan; Du, Hua-Ping; Li, Jiaojiao; Xu, Ran; Wang, Ya-Li; You, Shou-Jiang; Liu, Huihui; Wang, Fen; Cao, Yong-Jun; Liu, Chun-Feng; Hu, Li-Fang

    2014-09-01

    Hydrogen sulfide (H2S), the third gaseous transmitter, is implicated in various pathophysiologic processes. In the cardiovascular system, H2S exerts effects of cardioprotection, vascular tone regulation, and atherogenesis inhibition. Recent studies demonstrated that atorvastatin, the inhibitor of 3-hydroxyl-3-methyl coenzyme A reductase, affected H2S formation in kidney and other organs. However, the underlying mechanisms are not fully understood. In this study, we examined the effects of three different statins (fluvastatin, atorvastatin and pravastatin) on H2S formation in raw264.7 macrophages. There was a remarkable rise in H2S level in fluvastatin- and atorvastatin-stimulated macrophages, while pravastatin failed to show any significant effect on it. Moreover, fluvastatin and atorvastatin enhanced the mRNA and protein expression of cystathionine γ-lyase (CSE) in dose- and time-dependent manners. Fluvastatin also markedly enhanced the CSE activity. However, fluvastatin did not alter the mRNA or protein expression of another H2S-producing enzyme 3-mercaptopyruvate sulfurtransferase. Blockade of CSE with its inhibitor dl-propargylglycine (PAG) or siRNA markedly reduced the H2S level in fluvastatin-stimulated macrophages. In addition, fluvastatin elevated Akt phosphorylation, which occurred as early as 15 min after treatment, peaked at 1h, and lasted at least 3h. Both PI3K inhibitor LY294002 (10 μM) and Akt inhibitor perifosine (10μM) were able to reverse the increases of CSE mRNA and H2S production in fluvastatin-stimulated macrophages. Last, we showed that fluvastatin reduced the mRNA levels of pro-inflammatory molecules such as IL-1β and MCP-1 in LPS-treated macrophages, which were completely reversed by CSE inhibitor PAG. Taken together, the findings demonstrate that statins may up-regulate CSE expression/activity and subsequently elevate H2S generation by activating Akt signaling pathway and also imply that CSE-H2S pathway plays a critical role in the anti

  14. Insight into the Role of Substrate-binding Residues in Conferring Substrate Specificity for the Multifunctional Polysaccharide Lyase Smlt1473

    PubMed Central

    MacDonald, Logan C.; Berger, Bryan W.

    2014-01-01

    Anionic polysaccharides are of growing interest in the biotechnology industry due to their potential pharmaceutical applications in drug delivery and wound treatment. Chemical composition and polymer length strongly influence the physical and biological properties of the polysaccharide and thus its potential industrial and medical applications. One promising approach to determining monomer composition and controlling the degree of polymerization involves the use of polysaccharide lyases, which catalyze the depolymerization of anionic polysaccharides via a β-elimination mechanism. Utilization of these enzymes for the production of custom-made oligosaccharides requires a high degree of control over substrate specificity. Previously, we characterized a polysaccharide lyase (Smlt1473) from Stenotrophomonas maltophilia k279a, which exhibited significant activity against hyaluronan (HA), poly-β-d-glucuronic acid (poly-GlcUA), and poly-β-d-mannuronic acid (poly-ManA) in a pH-regulated manner. Here, we utilize a sequence structure guided approach based on a homology model of Smlt1473 to identify nine putative substrate-binding residues and examine their effect on substrate specificity via site-directed mutagenesis. Interestingly, single point mutations H221F and R312L resulted in increased activity and specificity toward poly-ManA and poly-GlcUA, respectively. Furthermore, a W171A mutant nearly eliminated HA activity, while increasing poly-ManA and poly-GlcUA activity by at least 35%. The effect of these mutations was analyzed by comparison with the high resolution structure of Sphingomonas sp. A1-III alginate lyase in complex with poly-ManA tetrasaccharide and by taking into account the structural differences between HA, poly-GlcUA, and poly-ManA. Overall, our results demonstrate that even minor changes in active site architecture have a significant effect on the substrate specificity of Smlt1473, whose structural plasticity could be applied to the design of highly

  15. Insight into the role of substrate-binding residues in conferring substrate specificity for the multifunctional polysaccharide lyase Smlt1473.

    PubMed

    MacDonald, Logan C; Berger, Bryan W

    2014-06-27

    Anionic polysaccharides are of growing interest in the biotechnology industry due to their potential pharmaceutical applications in drug delivery and wound treatment. Chemical composition and polymer length strongly influence the physical and biological properties of the polysaccharide and thus its potential industrial and medical applications. One promising approach to determining monomer composition and controlling the degree of polymerization involves the use of polysaccharide lyases, which catalyze the depolymerization of anionic polysaccharides via a β-elimination mechanism. Utilization of these enzymes for the production of custom-made oligosaccharides requires a high degree of control over substrate specificity. Previously, we characterized a polysaccharide lyase (Smlt1473) from Stenotrophomonas maltophilia k279a, which exhibited significant activity against hyaluronan (HA), poly-β-d-glucuronic acid (poly-GlcUA), and poly-β-d-mannuronic acid (poly-ManA) in a pH-regulated manner. Here, we utilize a sequence structure guided approach based on a homology model of Smlt1473 to identify nine putative substrate-binding residues and examine their effect on substrate specificity via site-directed mutagenesis. Interestingly, single point mutations H221F and R312L resulted in increased activity and specificity toward poly-ManA and poly-GlcUA, respectively. Furthermore, a W171A mutant nearly eliminated HA activity, while increasing poly-ManA and poly-GlcUA activity by at least 35%. The effect of these mutations was analyzed by comparison with the high resolution structure of Sphingomonas sp. A1-III alginate lyase in complex with poly-ManA tetrasaccharide and by taking into account the structural differences between HA, poly-GlcUA, and poly-ManA. Overall, our results demonstrate that even minor changes in active site architecture have a significant effect on the substrate specificity of Smlt1473, whose structural plasticity could be applied to the design of highly

  16. Structural and Kinetic Basis of Steroid 17α,20-Lyase Activity in Teleost Fish Cytochrome P450 17A1 and Its Absence in Cytochrome P450 17A2*

    PubMed Central

    Pallan, Pradeep S.; Nagy, Leslie D.; Lei, Li; Gonzalez, Eric; Kramlinger, Valerie M.; Azumaya, Caleigh M.; Wawrzak, Zdzislaw; Waterman, Michael R.; Guengerich, F. Peter; Egli, Martin

    2015-01-01

    Cytochrome P450 (P450) 17A enzymes play a critical role in the oxidation of the steroids progesterone (Prog) and pregnenolone (Preg) to glucocorticoids and androgens. In mammals, a single enzyme, P450 17A1, catalyzes both 17α-hydroxylation and a subsequent 17α,20-lyase reaction with both Prog and Preg. Teleost fish contain two 17A P450s; zebrafish P450 17A1 catalyzes both 17α-hydroxylation and lyase reactions with Prog and Preg, and P450 17A2 is more efficient in pregnenolone 17α-hydroxylation but does not catalyze the lyase reaction, even in the presence of cytochrome b5. P450 17A2 binds all substrates and products, although more loosely than P450 17A1. Pulse-chase and kinetic spectral experiments and modeling established that the two-step P450 17A1 Prog oxidation is more distributive than the Preg reaction, i.e. 17α-OH product dissociates more prior to the lyase step. The drug orteronel selectively blocked the lyase reaction of P450 17A1 but only in the case of Prog. X-ray crystal structures of zebrafish P450 17A1 and 17A2 were obtained with the ligand abiraterone and with Prog for P450 17A2. Comparison of the two fish P450 17A-abiraterone structures with human P450 17A1 (DeVore, N. M., and Scott, E. E. (2013) Nature 482, 116–119) showed only a few differences near the active site, despite only ∼50% identity among the three proteins. The P450 17A2 structure differed in four residues near the heme periphery. These residues may allow the proposed alternative ferric peroxide mechanism for the lyase reaction, or residues removed from the active site may allow conformations that lead to the lyase activity. PMID:25533464

  17. Innate immune-stimulating and immune genes up-regulating activities of three types of alginate from Sargassum siliquosum in Pacific white shrimp, Litopenaeus vannamei.

    PubMed

    Yudiati, Ervia; Isnansetyo, Alim; Murwantoko; Ayuningtyas; Triyanto; Handayani, Christina Retna

    2016-07-01

    The Total Haemocyte Count (THC), phenoloxidase (PO), Superoxide Dismutase (SOD) activity, Phagocytic Activity/Index and Total Protein Plasma (TPP) were examined after feeding the white shrimp Litopenaeus vannamei with diets supplemented with three different types of alginates (acid, calcium and sodium alginates). Immune-related genes expression was evaluated by quantitative Real Time PCR (qRT-PCR). Results indicated that the immune parameters directly increased according to the doses of alginates and time. The 2.0 g kg(-1) of acid and sodium alginate treatments were gave better results. Four immune-related genes expression i.e. LGBP, Toll, Lectin, proPO were up regulated. It is therefore concluded that the supplementation of alginate of Sargassum siliquosum on the diet of L. vannamei enhanced the innate immunity as well as the expression of immune-related genes. It is the first report on the simultaneous evaluation of three alginate types to enhance innate immune parameters and immune-related genes expression in L. vannamei.

  18. Identification of a conserved 5′-dRP lyase activity in bacterial DNA repair ligase D and its potential role in base excision repair

    PubMed Central

    de Ory, Ana; Nagler, Katja; Carrasco, Begoña; Raguse, Marina; Zafra, Olga; Moeller, Ralf; de Vega, Miguel

    2016-01-01

    Bacillus subtilis is one of the bacterial members provided with a nonhomologous end joining (NHEJ) system constituted by the DNA-binding Ku homodimer that recruits the ATP-dependent DNA Ligase D (BsuLigD) to the double-stranded DNA breaks (DSBs) ends. BsuLigD has inherent polymerization and ligase activities that allow it to fill the short gaps that can arise after realignment of the broken ends and to seal the resulting nicks, contributing to genome stability during the stationary phase and germination of spores. Here we show that BsuLigD also has an intrinsic 5′-2-deoxyribose-5-phosphate (dRP) lyase activity located at the N-terminal ligase domain that in coordination with the polymerization and ligase activities allows efficient repairing of 2′-deoxyuridine-containing DNA in an in vitro reconstituted Base Excision Repair (BER) reaction. The requirement of a polymerization, a dRP removal and a final sealing step in BER, together with the joint participation of BsuLigD with the spore specific AP endonuclease in conferring spore resistance to ultrahigh vacuum desiccation suggest that BsuLigD could actively participate in this pathway. We demonstrate the presence of the dRP lyase activity also in the homolog protein from the distantly related bacterium Pseudomonas aeruginosa, allowing us to expand our results to other bacterial LigDs. PMID:26826709

  19. Enhancement of plant growth stimulation activity of irradiated alginate by fractionation

    NASA Astrophysics Data System (ADS)

    Luan, Le Quang; Nagasawa, Naotsugu; Ha, Vo Thi Thu; Hien, Nguyen Quoc; Nakanishi, Tomoko M.

    2009-09-01

    Alginate with the weight-average molecular weight ( Mw) approximately 900 kDa and ratio of M (mannuronate)/G (guluronate) about 1.3 was irradiated by gamma Co-60 in aqueous solution at doses up to 200 kGy. The irradiation dose was shown to be a function for reducing Mw and molecular weight distribution of irradiated alginates. The distribution of oligomer fractions in irradiated products was also investigated by separation using ultrafiltration membranes. The irradiated alginate with Mw approximately 14.2 kDa was found to have a positive influence for growing of barley and soybean. The irradiated oligoalginate fraction with Mw ranging from 1 to 3 kDa displayed the strongest effect on the growth and development of the mentioned plants at low concentration (20 ppm). It is suggested that oligoalginate with Mw in the range 1-3 kDa is a trigger for the growth and development of plants.

  20. Sodium Alginate Based Mucoadhesive System for Gatifloxacin and Its In Vitro Antibacterial Activity

    PubMed Central

    Kesavan, Karthikeyan; Nath, Gopal; Pandit, Jayanta K.

    2010-01-01

    The objective of this study was to formulate sodium alginate based ophthalmic mucoadhesive system of gatifloxacin and its in vitro antibacterial potential on pathogenic microorganisms, Staphylococcus aureus and Escherichia coli. Sodium carboxymethylcellulose (NaCMC) was added to the formulations to enhance the gel bioadhesion properties. The prepared formulations were evaluated for their in vitro drug release, gelation behaviour, rheological behavior, and mucoadhesion force. All formulations in non-physiological and physiological condition showed pseudo plastic behavior. Increase in the concentration of sodium alginate and sodium CMC enhanced the mucoadhesive force significantly. In vitro release of gatifloxacin from the system in simulated tear fluid (STF, pH – 7.4), was influenced significantly by the properties and concentration of sodium alginate, NaCMC. Significant reduction in total bacterial count was observed between control and treatment groups with both the test organisms. PMID:21179327

  1. Lyase activities of heterologous CpcS and CpcT for phycocyanin holo-β-subunit from Arthrospira platensis in Escherichia coli

    NASA Astrophysics Data System (ADS)

    Yi, Junjie; Xu, Di; Zang, Xiaonan; Yuan, Dingyang; Zhao, Bingran; Tang, Li; Tan, Yanning; Zhang, Xuecheng

    2014-06-01

    Arthrospira platensis is an economically important cyanobacterium; and it has been used widely in food and pharmaceutical industries. The phycocyanin (PC) from A. platensis is extremely valuable in medicine and molecular biology due to its antioxidation and anti-tumoring activity and applicability as fluorescence protein tag. In present study, two recombinant plasmids, one contained the phycocyanobilin (PCB)-producing genes ( hox1 and pcyA) while the other contained the phycobiliprotein gene ( cpcB) and the lyase gene (either cpcS/U or cpcT), were constructed and synchronically transferred into E. coli in order to test the the activities of relevant lyases for catalysing PCB addition to CpcB during synthesizing fluorescent PC holo-β-subunit (β-PC) of A. platensis. As was evidenced by the fluorescence emitted at a peak specific for PC, CpcB was successfully synthesized in E. coli, to which co-expressed PCBs attached though at a relatively low efficiency. The results showed that the attachment of PCBs to CpcB were carried out mainly by co-expressed CpcS/U but CpcB also showed some autocatalytic activity. Currently, no CpcT activity was detected in this E. coli expression system. Further studies will be conducted to improve the efficiency of fluorescent PC synthesis in E. coli.

  2. Stress enhances the gene expression and enzyme activity of phenylalanine ammonia-lyase and the endogenous content of salicylic acid to induce flowering in pharbitis.

    PubMed

    Wada, Kaede C; Mizuuchi, Kaori; Koshio, Aya; Kaneko, Kentaro; Mitsui, Toshiaki; Takeno, Kiyotoshi

    2014-07-01

    The involvement of salicylic acid (SA) in the regulation of stress-induced flowering in the short-day plant pharbitis (also called Japanese morning glory) Ipomoea nil (formerly Pharbitis nil) was studied. Pharbitis cv. Violet was induced to flower when grown in 1/100-strength mineral nutrient solution under non-inductive long-day conditions. All fully expanded true leaves were removed from seedlings, leaving only the cotyledons, and flowering was induced under poor-nutrition stress conditions. This indicates that cotyledons can play a role in the regulation of poor-nutrition stress-induced flowering. The expression of the pharbitis homolog of PHENYLALANINE AMMONIA-LYASE, the enzyme activity of phenylalanine ammonia-lyase (PAL; E.C. 4.3.1.5) and the content of SA in the cotyledons were all up-regulated by the stress treatment. The Violet was also induced to flower by low-temperature stress, DNA demethylation and short-day treatment. Low-temperature stress enhanced PAL activity, whereas non-stress factors such as DNA demethylation and short-day treatment decreased the activity. The PAL enzyme activity was also examined in another cultivar, Tendan, obtaining similar results to Violet. The exogenously applied SA did not induce flowering under non-stress conditions but did promote flowering under weak stress conditions in both cultivars. These results suggest that stress-induced flowering in pharbitis is induced, at least partly, by SA, and the synthesis of SA is promoted by PAL.

  3. Hemin-micelles immobilized in alginate hydrogels as artificial enzymes with peroxidase-like activity and substrate selectivity.

    PubMed

    Qu, Rui; Shi, Hejin; Wang, Ruolin; Cheng, Tangjian; Ma, Rujiang; An, Yingli; Shi, Linqi

    2017-02-28

    Artificial enzymes are widely investigated to mimic the active center and the recognition center of natural enzymes. The active center is responsible for the catalytic activity of enzymes, and the recognition center provides enzymes with specificity. Most of the previous studies on artificial enzymes preferred to solve the problem of activity rather than specificity due to the complexity of the enzyme structures related to substrate recognition. Inspired by the multilevel structures of enzymes and the unique net-structures of hydrogels, hemin-micelles immobilized in alginate hydrogels (HM-AH) were constructed by multistep self-assembly. The hemin-micelle was the active center and mimicked the microenvironment of the catalytic site in horseradish peroxidase (HRP). The alginate hydrogel further enhanced the catalytic activity and stability of hemin-micelles and endowed the artificial enzymes with a catalytic capability in harsh water conditions and non-polar organic solvents. The hydrogel also served as the recognition center, which exhibited substrate selectivity owing to the diffusivity differentiations of substrates in hydrogel fibers. It is the first example of constructing a micelle-hydrogel complex system as an artificial enzyme with both catalytic activity and substrate selectivity by the method of multistep self-assembly.

  4. Silver-Carboxylate Ion-Paired Alginate and Carboxymethylated Cotton with Antimicrobial Activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Burn and chronic wounds are highly susceptible to infection. Silver has long been known to have antimicrobial properties and has been used extensively in treating burn wounds. Using a cation exchange technique, it was possible to impregnate commercially available alginate dressings and carboxymethyl...

  5. Molecular Cloning of cpcU and Heterodimeric Bilin Lyase Activity Analysis of CpcU and CpcS for Attachment of Phycocyanobilin to Cys-82 on the β-Subunit of Phycocyanin in Arthrospira platensis FACHB314.

    PubMed

    Wu, Fei; Zang, Xiaonan; Zhang, Xuecheng; Zhang, Ran; Huang, Xiaoyun; Hou, Lulu; Jiang, Minjie; Liu, Chang; Pang, Chunhong

    2016-03-16

    A new bilin lyase gene cpcU was cloned from Arthrospira platensis FACHB314 to study the assembly of the phycocyanin β-Subunit. Two recombinant plasmids, one contained the phycocyanobilin (PCB) producing genes (hoxI and pcyA), while the other contained the gene of the β-Subunit of phycobiliprotein (cpcB) and the lyase gene (cpcU, cpcS, or cpcU/S) were constructed and separately transferred into Escherichia coli in order to test the activities of relevant lyases for catalyzing PCB addition to CpcB during synthesizing fluorescent β-PC of A. platensis FACHB314. The fluorescence intensity examination showed that Cys-82 maybe the active site for the β-Subunit binding to PCBs and the attachment could be carried out by CpcU, CpcS, or co-expressed cpcU/S in A. platensis FACHB314.

  6. Kinetic Parameters and Cytotoxic Activity of Recombinant Methionine γ-Lyase from Clostridium tetani, Clostridium sporogenes, Porphyromonas gingivalis and Citrobacter freundii

    PubMed Central

    Morozova, E. A.; Kulikova, V. V.; Yashin, D. V.; Anufrieva, N. V.; Anisimova, N. Y.; Revtovich, S. V.; Kotlov, M. I.; Belyi, Y. F.; Pokrovsky, V. S.; Demidkina, T. V.

    2013-01-01

    The steady-state kinetic parameters of pyridoxal 5’-phosphate-dependent recombinant methionine γ -lyase from three pathogenic bacteria, Clostridium tetani, Clostridium sporogenes, and Porphyromonas gingivalis, were determined in β- and γ-elimination reactions. The enzyme from C. sporogenes is characterized by the highest catalytic efficiency in the γ-elimination reaction of L-methionine. It was demonstrated that the enzyme from these three sources exists as a tetramer. The N-terminal poly-histidine fragment of three recombinant enzymes influences their catalytic activity and facilitates the aggregation of monomers to yield dimeric forms under denaturing conditions. The cytotoxicity of methionine γ-lyase from C. sporogenes and C. tetani in comparison with Citrobacter freundii was evaluated using K562, PC-3, LnCap, MCF7, SKOV-3, and L5178y tumor cell lines. K562 (IC50=0.4–1.3 U/ml), PC-3 (IC50=0.1–0.4 U/ml), and MCF7 (IC50=0.04–3.2 U/ml) turned out to be the most sensitive cell lines. PMID:24303205

  7. Kinetic Parameters and Cytotoxic Activity of Recombinant Methionine γ-Lyase from Clostridium tetani, Clostridium sporogenes, Porphyromonas gingivalis and Citrobacter freundii.

    PubMed

    Morozova, E A; Kulikova, V V; Yashin, D V; Anufrieva, N V; Anisimova, N Y; Revtovich, S V; Kotlov, M I; Belyi, Y F; Pokrovsky, V S; Demidkina, T V

    2013-07-01

    The steady-state kinetic parameters of pyridoxal 5'-phosphate-dependent recombinant methionine γ -lyase from three pathogenic bacteria, Clostridium tetani, Clostridium sporogenes, and Porphyromonas gingivalis, were determined in β- and γ-elimination reactions. The enzyme from C. sporogenes is characterized by the highest catalytic efficiency in the γ-elimination reaction of L-methionine. It was demonstrated that the enzyme from these three sources exists as a tetramer. The N-terminal poly-histidine fragment of three recombinant enzymes influences their catalytic activity and facilitates the aggregation of monomers to yield dimeric forms under denaturing conditions. The cytotoxicity of methionine γ-lyase from C. sporogenes and C. tetani in comparison with Citrobacter freundii was evaluated using K562, PC-3, LnCap, MCF7, SKOV-3, and L5178y tumor cell lines. K562 (IC50=0.4-1.3 U/ml), PC-3 (IC50=0.1-0.4 U/ml), and MCF7 (IC50=0.04-3.2 U/ml) turned out to be the most sensitive cell lines.

  8. Mucin degradation in the human colon: production of sialidase, sialate O-acetylesterase, N-acetylneuraminate lyase, arylesterase, and glycosulfatase activities by strains of fecal bacteria.

    PubMed Central

    Corfield, A P; Wagner, S A; Clamp, J R; Kriaris, M S; Hoskins, L C

    1992-01-01

    Oligosaccharide side chains of human colonic mucins contain O-acetylated sialic acids and glycosulfate esters. Although these substituents are considered to protect the chains against degradation by bacterial glycosidases, sialate O-acetylesterase, N-acetylneuraminate lyase, and glycosulfatase activities have been found in fecal extracts. To better define the source of these activities, we measured extracellular and cell-bound sialidase, sialate O-acetylesterase, N-acetylneuraminate lyase, arylesterase, and glycosulfatase activities produced by 23 isolates of human fecal bacteria grown anaerobically in a hog gastric mucin culture medium; these represented dominant populations of fecal anaerobes, facultative anaerobes, and the subset of mucin oligosaccharide-degrading bacteria. Every strain produced sialidase and high levels of arylesterase, and all but five facultative anaerobes produced sialate O-acetylesterase. Sialic acids containing 2 mol or more of O-acetyl ester per mol of sialic acid were cleaved from mucin glycoproteins more slowly by sialidases of mucin oligosaccharide-degrading stains than were sialic acids containing 1 or 0 mol, and only N-acetyl- and mono-O-acetylated sialic acids were recovered from enzyme digests of a mucin containing di-O-acetylated sialic acids. No detectable N-acetylneuraminate lyase activity was produced by any strain, but low activity was induced by increasing the glycoprotein-bound sialic acid concentration in the culture medium of six Escherichia coli strains. Using lactitol-6-sulfate as a substrate, we found weak glycosulfatase activity in the partially purified, concentrated enzyme mixture in the culture supernatants of four mucin oligosaccharide-degrading strains but in none of the unconcentrated culture fractions. We conclude that the presence of two or more O-acetyl groups on sialic acids inhibits enteric bacterial sialidases but that production of sialate O-acetylesterases by several populations of enteric bacteria

  9. Different utilization of alginate and other algal polysaccharides by marine Alteromonas macleodii ecotypes.

    PubMed

    Neumann, Anna M; Balmonte, John P; Berger, Martine; Giebel, Helge-Ansgar; Arnosti, Carol; Voget, Sonja; Simon, Meinhard; Brinkhoff, Thorsten; Wietz, Matthias

    2015-10-01

    The marine bacterium Alteromonas macleodii is a copiotrophic r-strategist, but little is known about its potential to degrade polysaccharides. Here, we studied the degradation of alginate and other algal polysaccharides by A. macleodii strain 83-1 in comparison to other A. macleodii strains. Cell densities of strain 83-1 with alginate as sole carbon source were comparable to those with glucose, but the exponential phase was delayed. The genome of 83-1 was found to harbour an alginolytic system comprising five alginate lyases, whose expression was induced by alginate. The alginolytic system contains additional CAZymes, including two TonB-dependent receptors, and is part of a 24 kb genomic island unique to the A. macleodii 'surface clade' ecotype. In contrast, strains of the 'deep clade' ecotype contain only a single alginate lyase in a separate 7 kb island. This difference was reflected in an eightfold greater efficiency of surface clade strains to grow on alginate. Strain 83-1 furthermore hydrolysed laminarin, pullulan and xylan, and corresponding polysaccharide utilization loci were detected in the genome. Alteromonas macleodii alginate lyases were predominantly detected in Atlantic Ocean metagenomes. The demonstrated hydrolytic capacities are likely of ecological relevance and represent another level of adaptation among A. macleodii ecotypes.

  10. Comparative study on the in vitro effects of Pseudomonas aeruginosa and seaweed alginates on human gut microbiota.

    PubMed

    Bai, Shaofeng; Chen, Huahai; Zhu, Liying; Liu, Wei; Yu, Hongwei D; Wang, Xin; Yin, Yeshi

    2017-01-01

    Alginates pertain to organic polysaccharides that have been extensively used in food- and medicine-related industries. The present study obtained alginates from an alginate overproducing Pseudomonas aeruginosa PAO1 mutant by screening transposon mutagenesis libraries. The interaction between bacterial and seaweed alginates and gut microbiota were further studied by using an in vitro batch fermentation system. Thin-layer chromatography (TLC) analysis indicated that both bacterial and seaweed alginates can be completely degraded by fecal bacteria isolated from study volunteers, indicating that a minor structural difference between bacterial and seaweed alginates (O-acetylation and lack of G-G blocks) didn't affect the digestion of alginates by human microbiota. Although, the digestion of bacterial and seaweed alginates was attributed to different Bacteroides xylanisolvens strains, they harbored similar alginate lyase genes. Genus Bacteroides with alginate-degrading capability were enriched in growth medium containing bacterial or seaweed alginates after in vitro fermentation. Short-chain fatty acid (SCFA) production in both bacterial and seaweed alginates was also comparable, but was significantly higher than the same medium using starch. In summary, the present study has isolated an alginate-overproducing P. aeruginosa mutant strain. Both seaweed and bacterial alginates were degraded by human gut microbiota, and their regulatory function on gut microbiota was similar.

  11. Comparative study on the in vitro effects of Pseudomonas aeruginosa and seaweed alginates on human gut microbiota

    PubMed Central

    Bai, Shaofeng; Chen, Huahai; Zhu, Liying; Liu, Wei; Yu, Hongwei D.; Wang, Xin; Yin, Yeshi

    2017-01-01

    Alginates pertain to organic polysaccharides that have been extensively used in food- and medicine-related industries. The present study obtained alginates from an alginate overproducing Pseudomonas aeruginosa PAO1 mutant by screening transposon mutagenesis libraries. The interaction between bacterial and seaweed alginates and gut microbiota were further studied by using an in vitro batch fermentation system. Thin-layer chromatography (TLC) analysis indicated that both bacterial and seaweed alginates can be completely degraded by fecal bacteria isolated from study volunteers, indicating that a minor structural difference between bacterial and seaweed alginates (O-acetylation and lack of G-G blocks) didn’t affect the digestion of alginates by human microbiota. Although, the digestion of bacterial and seaweed alginates was attributed to different Bacteroides xylanisolvens strains, they harbored similar alginate lyase genes. Genus Bacteroides with alginate-degrading capability were enriched in growth medium containing bacterial or seaweed alginates after in vitro fermentation. Short-chain fatty acid (SCFA) production in both bacterial and seaweed alginates was also comparable, but was significantly higher than the same medium using starch. In summary, the present study has isolated an alginate-overproducing P. aeruginosa mutant strain. Both seaweed and bacterial alginates were degraded by human gut microbiota, and their regulatory function on gut microbiota was similar. PMID:28170428

  12. Different responses in the expression of alginases, alginate polymerase and acetylation genes during alginate production by Azotobacter vinelandii under oxygen-controlled conditions.

    PubMed

    Díaz-Barrera, Alvaro; Maturana, Nataly; Pacheco-Leyva, Ivette; Martínez, Irene; Altamirano, Claudia

    2017-02-28

    Alginate production and gene expression of genes involved in alginate biosynthesis were evaluated in continuous cultures under dissolved oxygen tension (DOT) controlled conditions. Chemostat at 8% DOT showed an increase in the specific oxygen uptake rate [Formula: see text] from 10.9 to 45.3 mmol g(-1) h(-1) by changes in the dilution rate (D) from 0.06 to 0.10 h(-1), whereas under 1% DOT the [Formula: see text] was not affected. Alginate molecular weight was not affected by DOT. However, chemostat at 1% DOT showed a downregulation up to 20-fold in genes encoding both the alginate polymerase (alg8, alg44), alginate acetylases (algV, algI) and alginate lyase AlgL. alyA1 and algE7 lyases gene expressions presented an opposite behavior by changing the DOT, suggesting that A. vinelandii can use specific depolymerases depending on the oxygen level. Overall, the DOT level have a differential effect on genes involved in alginate synthesis, thus a gene expression equilibrium determines the production of alginates of similar molecular weight under DOT controlled.

  13. Pb(II) adsorption by a novel activated carbon - alginate composite material. A kinetic and equilibrium study.

    PubMed

    Cataldo, Salvatore; Gianguzza, Antonio; Milea, Demetrio; Muratore, Nicola; Pettignano, Alberto

    2016-11-01

    The adsorption capacity of an activated carbon - calcium alginate composite material (ACAA-Ca) has been tested with the aim of developing a new and more efficient adsorbent material to remove Pb(II) ion from aqueous solution. The study was carried out at pH=5, in NaCl medium and in the ionic strength range 0.1-0.75molL(-1). Differential Pulse Anodic Stripping Voltammetry (DP-ASV) technique was used to check the amount of Pb(II) ion removed during kinetic and equilibrium experiments. Different kinetic (pseudo first order, pseudo second order and Vermuelen) and equilibrium (Langmuir and Freundlich) models were used to fit experimental data, and were statistically compared. Calcium alginate (AA-Ca) improves the adsorption capacity (qm) of active carbon (AC) in the ACAA-Ca adsorbent material (e.g., qm=15.7 and 10.5mgg(-1) at I=0.25molL(-1), for ACAA-Ca and AC, respectively). SEM-EDX and thermogravimetric (TGA) measurements were carried out in order to characterize the composite material. The results of the speciation study on the Pb(II) solution and of the characterization of the ACAA-Ca and of the pristine AA-Ca and AC were evaluated in order to explain the specific contribution of AC and AA-Ca to the adsorption of the metal ion.

  14. A Catalase-related Hemoprotein in Coral Is Specialized for Synthesis of Short-chain Aldehydes: DISCOVERY OF P450-TYPE HYDROPEROXIDE LYASE ACTIVITY IN A CATALASE.

    PubMed

    Teder, Tarvi; Lõhelaid, Helike; Boeglin, William E; Calcutt, Wade M; Brash, Alan R; Samel, Nigulas

    2015-08-07

    In corals a catalase-lipoxygenase fusion protein transforms arachidonic acid to the allene oxide 8R,9-epoxy-5,9,11,14-eicosatetraenoic acid from which arise cyclopentenones such as the prostanoid-related clavulones. Recently we cloned two catalase-lipoxygenase fusion protein genes (a and b) from the coral Capnella imbricata, form a being an allene oxide synthase and form b giving uncharacterized polar products (Lõhelaid, H., Teder, T., Tõldsepp, K., Ekins, M., and Samel, N. (2014) PloS ONE 9, e89215). Here, using HPLC-UV, LC-MS, and NMR methods, we identify a novel activity of fusion protein b, establishing its role in cleaving the lipoxygenase product 8R-hydroperoxy-eicosatetraenoic acid into the short-chain aldehydes (5Z)-8-oxo-octenoic acid and (3Z,6Z)-dodecadienal; these primary products readily isomerize in an aqueous medium to the corresponding 6E- and 2E,6Z derivatives. This type of enzymatic cleavage, splitting the carbon chain within the conjugated diene of the hydroperoxide substrate, is known only in plant cytochrome P450 hydroperoxide lyases. In mechanistic studies using (18)O-labeled substrate and incubations in H2(18)O, we established synthesis of the C8-oxo acid and C12 aldehyde with the retention of the hydroperoxy oxygens, consistent with synthesis of a short-lived hemiacetal intermediate that breaks down spontaneously into the two aldehydes. Taken together with our initial studies indicating differing gene regulation of the allene oxide synthase and the newly identified catalase-related hydroperoxide lyase and given the role of aldehydes in plant defense, this work uncovers a potential pathway in coral stress signaling and a novel enzymatic activity in the animal kingdom.

  15. Role of active-site residues Tyr55 and Tyr114 in catalysis and substrate specificity of Corynebacterium diphtheriae C-S lyase.

    PubMed

    Astegno, Alessandra; Allegrini, Alessandra; Piccoli, Stefano; Giorgetti, Alejandro; Dominici, Paola

    2015-01-01

    In recent years, there has been increased interest in bacterial methionine biosynthesis enzymes as antimicrobial targets because of their pivotal role in cell metabolism. C-S lyase from Corynebacterium diphtheriae is a pyridoxal 5'-phosphate-dependent enzyme in the transsulfuration pathway that catalyzes the α,β-elimination of sulfur-containing amino acids, such as L-cystathionine, to generate ammonia, pyruvate, and homocysteine, the immediate precursor of L-methionine. In order to gain deeper insight into the functional and dynamic properties of the enzyme, mutants of two highly conserved active-site residues, Y55F and Y114F, were characterized by UV-visible absorbance, fluorescence, and CD spectroscopy in the absence and presence of substrates and substrate analogs, as well as by steady-state kinetic studies. Substitution of Tyr55 with Phe apparently causes a 130-fold decrease in K(d)(PLP) at pH 8.5 providing evidence that Tyr55 plays a role in cofactor binding. Moreover, spectral data show that the mutant accumulates the external aldimine intermediate suggesting that the absence of interaction between the hydroxyl moiety and PLP-binding residue Lys222 causes a decrease in the rate of substrate deprotonation. Mutation of Tyr114 with Phe slightly influences hydrolysis of L-cystathionine, and causes a change in substrate specificity towards L-serine and O-acetyl-L-serine compared to the wild type enzyme. These findings, together with computational data, provide useful insights in the substrate specificity of C-S lyase, which seems to be regulated by active-site architecture and by the specific conformation in which substrates are bound, and will aid in development of inhibitors.

  16. TALE-induced bHLH transcription factors that activate a pectate lyase contribute to water soaking in bacterial spot of tomato

    PubMed Central

    Schwartz, Allison R.; Morbitzer, Robert; Lahaye, Thomas; Staskawicz, Brian J.

    2017-01-01

    AvrHah1 [avirulence (avr) gene homologous to avrBs3 and hax2, no. 1] is a transcription activator-like (TAL) effector (TALE) in Xanthomonas gardneri that induces water-soaked disease lesions on fruits and leaves during bacterial spot of tomato. We observe that water from outside the leaf is drawn into the apoplast in X. gardneri-infected, but not X. gardneriΔavrHah1 (XgΔavrHah1)-infected, plants, conferring a dark, water-soaked appearance. The pull of water can facilitate entry of additional bacterial cells into the apoplast. Comparing the transcriptomes of tomato infected with X. gardneri vs. XgΔavrHah1 revealed the differential up-regulation of two basic helix–loop–helix (bHLH) transcription factors with predicted effector binding elements (EBEs) for AvrHah1. We mined our RNA-sequencing data for differentially up-regulated genes that could be direct targets of the bHLH transcription factors and therefore indirect targets of AvrHah1. We show that two pectin modification genes, a pectate lyase and pectinesterase, are targets of both bHLH transcription factors. Designer TALEs (dTALEs) for the bHLH transcription factors and the pectate lyase, but not for the pectinesterase, complement water soaking when delivered by XgΔavrHah1. By perturbing transcriptional networks and/or modifying the plant cell wall, AvrHah1 may promote water uptake to enhance tissue damage and eventual bacterial egression from the apoplast to the leaf surface. Understanding how disease symptoms develop may be a useful tool for improving the tolerance of crops from damaging disease lesions. PMID:28100489

  17. Correlation of rutin accumulation with 3-O-glucosyl transferase and phenylalanine ammonia-lyase activities during the ripening of tomato fruit.

    PubMed

    Capanoglu, Esra; Beekwilder, Jules; Matros, Andrea; Boyacioglu, Dilek; Hall, Robert D; Mock, Hans Peter

    2012-12-01

    In tomato, the predominant flavonoid is quercetin-3-rutinoside (rutin). In this study, we aim to investigate the phenylalanine ammonia-lyase (PAL) and the quercetin-3-O-glucosyl transferase (3-GT) reactions in the formation of rutin during tomato fruit ripening. Tomatoes of the Moneymaker variety at different development stages (green, breaker, turning, pink, red, and deep red) were divided into flesh and peel fractions. In each sample, both the content of rutin and the enzymatic activities for PAL and 3-GT were recorded. The highest activities of PAL were recorded in the peel of turning fruit (3,000 μkat/mg fresh weight). In fruit flesh, maximal activity was observed in red fruit (917.3 μkat/mg). For both tissues, PAL activity strongly decreased at the final (deep red) fruit stage. The activity of 3-GT in peel peaked in the turning fruit stage (50.7 pkat/mg), while in flesh maximal activity (33.4 pkat/mg) was observed in green fruit, which rapidly declined at the turning stage. Higher levels of rutin were detected in the tomato peel compared to the flesh part with the highest level being found at the green stage. The relation of PAL and 3-GT activities to rutin content is also evaluated.

  18. Panax Notoginseng Saponins Ameliorates Coxsackievirus B3-Induced Myocarditis by Activating the Cystathionine-γ-Lyase/Hydrogen Sulfide Pathway.

    PubMed

    Pan, Lulu; Zhang, Yuanhai; Lu, Jiacheng; Geng, Zhimin; Jia, Lianhong; Rong, Xing; Wang, Zhenquan; Zhao, Qifeng; Wu, Rongzhou; Chu, Maoping; Zhang, Chunxiang

    2015-12-01

    This study is to determine the therapeutic effects of Panax notoginseng saponins (PNSs) on coxsackievirus B3 (CVB3)-induced myocarditis, and whether cystathionine-γ-lyase (CSE)/hydrogen sulfide (H2S) pathway is involved. Mouse model of myocarditis was induced by CVB3 infection, and the mice were subjected to vehicle (saline) or drug treatments (sodium bisulfide (NaHS), propargylglycine (PAG), or PNSs). The results showed that there were inflammatory cell infiltrations, interstitial edemas, and elevated inflammatory cytokines, in CVB3-induced myocarditis. PAG administration increased, whereas NaHS treatment decreased the severity of the myocarditis. PNS treatment dramatically alleviated these myocardial injuries and decreased the viral messenger RNA (mRNA) expression by the enhanced expression of CSE/H2S pathway. Moreover, the therapeutic effects of PNSs on myocarditis were stronger than those of NaHS. Finally, the effect of PNSs on CSE/H2S pathway and cardiac cell protection were verified in cultured cardiac cells. PNSs may be a promising medication for viral myocarditis therapy.

  19. [Lactate as competitive inhibitor of Pinus pinea isocitrate lyase].

    PubMed

    Ranaldi, F; Iacoviello, C; Vanni, P

    1995-01-01

    We studied the effect of L-lactate on both the cleavage and the condensation reactions of Pinus pinea isocitrate lyase. This compound is a competitive of Pinus pinea isocitrate lyase towards both isocitrate and glyoxylate, whereas is a mixed type inhibitor towards succinate. Assuming that L-lactate acts as a glyoxylate analogue, our finding agrees with an uni-bi ordered mechanism of isocitrate lyase, with glyoxylate first substrate to enter the active site in the condensation reaction. Results are discussed and compared with those known in the literature about other structurally related metabolites.

  20. Conversion of citrate synthase into citryl-CoA lyase as a result of mutation of the active-site aspartic acid residue to glutamic acid.

    PubMed Central

    Man, W J; Li, Y; O'Connor, C D; Wilton, D C

    1991-01-01

    The active-site aspartic acid residue, Asp-362, of Escherichia coli citrate synthase was changed by site-directed mutagenesis to Glu-362, Asn-362 or Gly-362. Only very low catalytic activity could be detected with the Asp----Asn and Asp----Gly mutations. The Asp----Glu mutation produced an enzyme that expressed about 0.8% of the overall catalytic rate, and the hydrolysis step in the reaction, monitored as citryl-CoA hydrolysis, was inhibited to a similar extent. However, the condensation reaction, measured in the reverse direction as citryl-CoA cleavage to oxaloacetate and acetyl-CoA, was not affected by the mutation, and this citryl-CoA lyase activity was the major catalytic activity of the mutant enzyme. This high condensation activity in an enzyme in which the subsequent hydrolysis step was about 98% inhibited permitted considerable exchange of the methyl protons of acetyl-CoA during catalysis by the mutant enzyme. The Km for oxaloacetate was not significantly altered in the D362E mutant enzyme, whereas the Km for acetyl-CoA was about 5 times lower. A mechanism is proposed in which Asp-362 is involved in the hydrolysis reaction of this enzyme, and not as a base in the deprotonation of acetyl-CoA as recently suggested by others. [Karpusas, Branchaud & Remington (1990) Biochemistry 29, 2213-2219; Alter, Casazza, Zhi, Nemeth, Srere & Evans, (1990) Biochemistry 29, 7557-7563]. PMID:1684105

  1. Enhancement of solubility, purification and inclusion-bodies-refolding of an active pectin lyase from Penicillium occitanis expressed in Escherichia coli.

    PubMed

    Hadj Sassi, Azza; Trigui-Lahiani, Hèla; Abdeljalil, Salma; Gargouri, Ali

    2017-02-01

    Pectin lyase (pnl) is the only pectinase able to hydrolyze directly the highly methylated pectin without liberating the toxic methanol and without disturbing ester content responsible for specific aroma of juices. The cDNA of Penicillium occitanis pnl (mature form) was cloned into pET-21a as expression vector and over-expressed into Esherichia coli. Most of recombinant pnl was expressed as inclusion bodies. Pnl activity was confirmed by colorimetric assay. To enhance the solubility yield of the expressed pnl, the effects of induction temperature, host strain and expression level were optimized. Maximal production of functional pnl was obtained after induction by 0.4mM IPTG at 30°C and 150rpm for 16h. Interestingly, the use of Origami host strain, having an oxidized cytoplasm favoring disulfide bonds formation required for the active conformation of the enzyme, has significantly improved the yield of the soluble active form of recombinant pnl. This pnl was successfully purified through a single step purification using His-Trap affinity column chromatography. This work is the first to report pnl expression into Origami strain. Alternatively, the inclusion bodies were isolated, denatured by high concentration of urea and gradually refolded by successive dialysis, leading to their transformation into soluble and active form.

  2. Expression and enzymatic activity of phenylalanine ammonia-lyase and p-coumarate 3-hydroxylase in mango (Mangifera indica 'Ataulfo') during ripening.

    PubMed

    Palafox-Carlos, H; Contreras-Vergara, C A; Muhlia-Almazán, A; Islas-Osuna, M A; González-Aguilar, G A

    2014-05-16

    Phenylalanine ammonia lyase (PAL) and p-coumarate 3-hydroxylase (C3H) are key enzymes in the phenylpropanoid pathway. The relative expression of PAL and C3H was evaluated in mango fruit cultivar 'Ataulfo' in four ripening stages (RS1, RS2, RS3, and RS4) by quantitative polymerase chain reaction. In addition, enzyme activity of PAL and C3H was determined in mango fruits during ripening. The PAL levels were downregulated at the RS2 and RS3 stages, while C3H levels were upregulated in fruits only at RS3. The enzyme activity of PAL followed a pattern that was different from that of the PAL expression, thus suggesting regulation at several levels. For C3H, a regulation at the transcriptional level is suggested because a similar pattern was revealed by its activity and transcript level. In this study, the complexity of secondary metabolite biosynthesis regulation is emphasized because PAL and C3H enzymes are involved in the biosynthesis of several secondary metabolites that are active during all mango ripening stages.

  3. Type III secretion system expression in oxygen-limited Pseudomonas aeruginosa cultures is stimulated by isocitrate lyase activity

    PubMed Central

    Chung, Jade C. S.; Rzhepishevska, Olena; Ramstedt, Madeleine; Welch, Martin

    2013-01-01

    Pseudomonas aeruginosa is an opportunistic human pathogen and a common cause of chronic infections in individuals with cystic fibrosis (CF). Oxygen limitation was recently reported to regulate the expression of a major virulence determinant in P. aeruginosa, the type III secretion system (T3SS). Here, we show that expression of the T3SS in oxygen-limited growth conditions is strongly dependent on the glyoxylate shunt enzyme, isocitrate lyase (ICL; encoded by aceA), which was previously shown to be highly expressed in CF isolates. ICL-dependent regulation of the T3SS did not alter the expression level of the master transcriptional regulator, ExsA, but did affect expression of the T3 structural proteins, effectors and regulators (ExsC, ExsD and ExsE). An aceA mutant displayed enhanced biofilm formation during anaerobic growth, which suggested that AceA-dependent modulation of type III secretion might impinge upon the RetS/LadS signalling pathways. Indeed, our data suggest that RetS is able to mediate some of its effects through AceA, as expression of aceA in trans partially restored T3SS expression in a retS mutant. Our findings indicate that AceA is a key player in the metabolic regulation of T3SS expression during oxygen-limited growth of P. aeruginosa. To the best of our knowledge, this is the first demonstration that the T3SS can be regulated by factors that do not affect ExsA expression levels. PMID:23363478

  4. The activity of HYDROPEROXIDE LYASE 1 regulates accumulation of galactolipids containing 12-oxo-phytodienoic acid in Arabidopsis

    PubMed Central

    Nilsson, Anders K.; Fahlberg, Per; Johansson, Oskar N.; Hamberg, Mats; Andersson, Mats X.; Ellerström, Mats

    2016-01-01

    Arabidopsis produces galactolipids containing esters of 12-oxo-phytodienoic acid (OPDA) and dinor-12-oxo-phytodienoic acid (dnOPDA). These lipids are referred to as arabidopsides and accumulate in response to abiotic and biotic stress. We explored the natural genetic variation found in 14 different Arabidopsis accessions to identify genes involved in the formation of arabidopsides. The accession C24 was identified as a poor accumulator of arabidopsides whereas the commonly used accession Col-0 was found to accumulate comparably large amounts of arabidopsides in response to tissue damage. A quantitative trait loci analysis of an F2 population created from a cross between C24 and Col-0 located a region on chromosome four strongly linked to the capacity to form arabidopsides. Expression analysis of HYDROPEROXIDE LYASE 1 (HPL1) showed large differences in transcript abundance between accessions. Transformation of Col-0 plants with the C24 HPL1 allele under transcriptional regulation of the 35S promoter revealed a strong negative correlation between HPL1 expression and arabidopside accumulation after tissue damage, thereby strengthening the view that HPL1 competes with ALLENE OXIDE SYNTHASE (AOS) for lipid-bound hydroperoxide fatty acids. We further show that the last step in the synthesis of galactolipid-bound OPDA and dnOPDA from unstable allene oxides is exclusively enzyme-catalyzed and not the result of spontaneous cyclization. Thus, the results presented here together with previous studies suggest that all steps in arabidopside biosynthesis are enzyme-dependent and apparently all reactions can take place with substrates being esterified to galactolipids. PMID:27422994

  5. Involvement of nitric oxide in oxidative burst, phenylalanine ammonia-lyase activation and Taxol production induced by low-energy ultrasound in Taxus yunnanensis cell suspension cultures.

    PubMed

    Wang, Jian Wen; Zheng, Li Ping; Wu, Jian Yong; Tan, Ren Xiang

    2006-12-01

    This work was to characterize the generation of nitric oxide (NO) in Taxus yunnanensis cells exposed to low-energy ultrasound (US) and the signal role of NO in elicitation of plant defense responses and secondary metabolite accumulation. The US sonication (3.5-55.6 mW/cm(3) at 40 kHz fixed frequency) for 2 min induced a rapid and dose-dependent NO production in the Taxus cell culture, which exhibited a biphasic time course, reaching the first plateau within 1.5 h and the second within 7 h after US sonication. The NO donor sodium nitroprusside (SNP) potentiated US-induced H(2)O(2) production and cell death. Inhibition of nitric oxide synthase (NOS) activity by N(omega)-nitro-L-arginine (L-NNA) or scavenging NO by 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxyde (PTIO) partially blocked the US-induced H(2)O(2) production and cell death. Moreover, the NO inhibitors suppressed US-induced activation of phenylalanine ammonium-lyase (PAL) and accumulation of diterpenoid taxanes (Taxol and baccatin III). These results suggest that NO plays a signal role in the US-induced responses and secondary metabolism activities in the Taxus cells.

  6. Pseudomonas aeruginosa AlgG is a polymer level alginate C5-mannuronan epimerase.

    PubMed Central

    Franklin, M J; Chitnis, C E; Gacesa, P; Sonesson, A; White, D C; Ohman, D E

    1994-01-01

    Alginate is a viscous extracellular polymer produced by mucoid strains of Pseudomonas aeruginosa that cause chronic pulmonary infections in patients with cystic fibrosis. Alginate is polymerized from GDP-mannuronate to a linear polymer of beta-1-4-linked residues of D-mannuronate and its C5-epimer, L-guluronate. We previously identified a gene called algG in the alginate biosynthetic operon that is required for incorporation of L-guluronate residues into alginate. In this study, we tested the hypothesis that the product of algG is a C5-epimerase that directly converts D-mannuronate to L-guluronate. The DNA sequence of algG was determined, and an open reading frame encoding a protein (AlgG) of approximately 60 kDa was identified. The inferred amino terminus of AlgG protein contained a putative signal sequence of 35 amino acids. Expression of algG in Escherichia coli demonstrated both 60-kDa pre-AlgG and 55-kDa mature AlgG proteins, the latter of which was localized to the periplasm. An N-terminal analysis of AlgG showed that the signal sequence was removed in the mature form. Pulse-chase experiments in both E. coli and P. aeruginosa provided evidence for conversion of the 60- to the 55-kDa size in vivo. Expression of algG from a plasmid inan algG (i.e., polymannuronate-producing) mutant of P. aeruginosa restored production of an alginate containing L-guluronate residues. The observation that AlgG is apparently processed and exported from the cytoplasm suggested that it may act as a polymer-level mannuronan C5-epimerase. An in vitro assay for mannuronan C5 epimerization was developed wherein extracts of E. coli expressing high levels of AlgG were incubated with polymannuronate. Epimerization of D-mannuronate to L-guluronate residues in the polymer was detected enzymatically, using a L-guluronate-specific alginate lyase of Klebsiella aerogenes. Epimerization was also detected in the in vitro reaction between recombinant AlgG and poly-D-mannuronate, using high

  7. Xylella fastidiosa esterase rather than hydroxynitrile lyase.

    PubMed

    Torrelo, Guzman; Ribeiro de Souza, Fayene Zeferino; Carrilho, Emanuel; Hanefeld, Ulf

    2015-03-02

    In 2009, we reported that the product of the gene SCJ21.16 (XFa0032) from Xylella fastidiosa, a xylem-restricted plant pathogen that causes a range of diseases in several important crops, encodes a protein (XfHNL) with putative hydroxynitrile lyase activity. Sequence analysis and activity tests indicated that XfHNL exhibits an α/β-hydrolase fold and could be classified as a member of the family of FAD-independent HNLs. Here we provide a more detailed sequence analysis and new experimental data. Using pure heterologously expressed XfHNL we show that this enzyme cannot catalyse the cleavage/synthesis of mandelonitrile and that this protein is in fact a non-enantioselective esterase. Homology modelling and ligand docking simulations were used to study the active site and support these results. This finding could help elucidate the common ancestor of esterases and hydroxynitrile lyases with an α/β -hydrolase fold.

  8. Rhodotorula glutinis Phenylalanine/Tyrosine Ammonia Lyase Enzyme Catalyzed Synthesis of the Methyl Ester of para-Hydroxycinnamic Acid and its Potential Antibacterial Activity

    PubMed Central

    MacDonald, Marybeth C.; Arivalagan, Pugazhendhi; Barre, Douglas E.; MacInnis, Judith A.; D’Cunha, Godwin B.

    2016-01-01

    Biotransformation of L-tyrosine methyl ester (L-TM) to the methyl ester of para- hydroxycinnamic acid (p-HCAM) using Rhodotorula glutinis yeast phenylalanine/tyrosine ammonia lyase (PTAL; EC 4.3.1.26) enzyme was successfully demonstrated for the first time; progress of the reaction was followed by spectrophotometric determination at 315 nm. The following conditions were optimized for maximal formation of p-HCAM: pH (8.5), temperature (37°C), speed of agitation (50 rpm), enzyme concentration (0.080 μM), and substrate concentration (0.50 mM). Under these conditions, the yield of the reaction was ∼15% in 1 h incubation period and ∼63% after an overnight (∼18 h) incubation period. The product (p-HCAM) of the reaction of PTAL with L-TM was confirmed using Nuclear Magnetic Resonance spectroscopy (NMR). Fourier Transform Infra-Red spectroscopy (FTIR) was carried out to rule out potential hydrolysis of p-HCAM during overnight incubation. Potential antibacterial activity of p-HCAM was tested against several strains of Gram-positive and Gram-negative bacteria. This study describes a synthetically useful transformation, and could have future clinical and industrial applications. PMID:27014206

  9. Biocompatibility of mannuronic acid-rich alginates.

    PubMed

    Klöck, G; Pfeffermann, A; Ryser, C; Gröhn, P; Kuttler, B; Hahn, H J; Zimmermann, U

    1997-05-01

    Highly purified algin preparations free of adverse contaminants with endotoxins and other mitogens recently became available by a new purification process (Klöck et al., Appl. Microbiol. Biotechnol., 1994, 40, 638-643). An advantage of this purification protocol is that it can be applied to alginates with various ratios of mannuronic acid to guluronic acid. High mannuronic acid alginate capsules are of particular practical interest for cell transplantation and for biohybrid organs, because mannuronate-rich alginates are usually less viscous, allowing one to make gels with a higher alginate content. This will increase their stability and reduce the diffusion permeability and could therefore protect immobilized cells more efficiently against the host immune system. Here we report the biocompatibility of purified, mannuronic acid-rich alginate (68% mannuronate residues) in a series of in vitro, as well as in vivo, assays. In contrast to raw alginate extracts, the purified product showed no mitogenic activity towards murine lymphocytes in vitro. Its endotoxin content was reduced to the level of the solvent. Animal studies with these new, purified algin formulations revealed the absence of a mitogen-induced foreign body reaction, even when the purified material (after cross-linking with Ba2+ ions) is implanted into animal models with elevated macrophage activity (diabetes-prone BB/OK rat). Thus, alginate capsules with high mannuronic acid content become available for applications such as implantation. In addition to the utilization as implantable cell reactors in therapy and biotechnology, these purified algins have broad application potential as ocular fillings, tissue replacements, microencapsulated growth factors and/or interleukins or slow-release dosage forms of antibodies, surface coatings of sensors and other invasive medical devices, and in encapsulation of genetically engineered cells for gene therapy.

  10. Role of Calcium Alginate and Mannitol in Protecting Bifidobacterium

    PubMed Central

    Dianawati, Dianawati; Mishra, Vijay

    2012-01-01

    Fourier transform infrared (FTIR) spectroscopy was carried out to ascertain the mechanism of Ca-alginate and mannitol protection of cell envelope components and secondary proteins of Bifidobacterium animalis subsp. lactis Bb12 after freeze-drying and after 10 weeks of storage at room temperature (25°C) at low water activities (aw) of 0.07, 0.1, and 0.2. Preparation of Ca-alginate and Ca-alginate-mannitol as microencapsulants was carried out by dropping an alginate or alginate-mannitol emulsion containing bacteria using a burette into CaCl2 solution to obtain Ca-alginate beads and Ca-alginate-mannitol beads, respectively. The wet beads were then freeze-dried. The aw of freeze-dried beads was then adjusted to 0.07, 0.1, and 0.2 using saturated salt solutions; controls were prepared by keeping Ca-alginate and Ca-alginate-mannitol in aluminum foil without aw adjustment. Mannitol in the Ca-alginate system interacted with cell envelopes during freeze-drying and during storage at low aws. In contrast, Ca-alginate protected cell envelopes after freeze-drying but not during 10-week storage. Unlike Ca-alginate, Ca-alginate-mannitol was effective in retarding the changes in secondary proteins during freeze-drying and during 10 weeks of storage at low aws. It appears that Ca-alginate-mannitol is more effective than Ca-alginate in preserving cell envelopes and proteins after freeze-drying and after 10 weeks of storage at room temperature (25°C). PMID:22843535

  11. ATP citrate lyase activity is post-translationally regulated by sink strength and impacts the wax, cutin and rubber biosynthetic pathways.

    PubMed

    Xing, Shufan; van Deenen, Nicole; Magliano, Pasqualina; Frahm, Lea; Forestier, Edith; Nawrath, Christiane; Schaller, Hubert; Gronover, Christian S; Prüfer, Dirk; Poirier, Yves

    2014-07-01

    Cytosolic acetyl-CoA is involved in the synthesis of a variety of compounds, including waxes, sterols and rubber, and is generated by the ATP citrate lyase (ACL). Plants over-expressing ACL were generated in an effort to understand the contribution of ACL activity to the carbon flux of acetyl-CoA to metabolic pathways occurring in the cytosol. Transgenic Arabidopsis plants synthesizing the polyester polyhydroxybutyrate (PHB) from cytosolic acetyl-CoA have reduced growth and wax content, consistent with a reduction in the availability of cytosolic acetyl-CoA to endogenous pathways. Increasing the ACL activity via the over-expression of the ACLA and ACLB subunits reversed the phenotypes associated with PHB synthesis while maintaining polymer synthesis. PHB production by itself was associated with an increase in ACL activity that occurred in the absence of changes in steady-state mRNA or protein level, indicating a post-translational regulation of ACL activity in response to sink strength. Over-expression of ACL in Arabidopsis was associated with a 30% increase in wax on stems, while over-expression of a chimeric homomeric ACL in the laticifer of roots of dandelion led to a four- and two-fold increase in rubber and triterpene content, respectively. Synthesis of PHB and over-expression of ACL also changed the amount of the cutin monomer octadecadien-1,18-dioic acid, revealing an unsuspected link between cytosolic acetyl-CoA and cutin biosynthesis. Together, these results reveal the complexity of ACL regulation and its central role in influencing the carbon flux to metabolic pathways using cytosolic acetyl-CoA, including wax and polyisoprenoids.

  12. Novel sulfated glucomannan-barium-alginate microcapsules in islet transplantation: significantly decreased the secretion of monocyte chemotactic protein 1 and improved the activity of islet in rats.

    PubMed

    Chen, X; Zhang, L; Qi, Z; Guo, B; Zhong, L; Shen, B; Yan, Z; Zhang, J

    2009-12-01

    The sulfated glucomannan can be used to filter the heparin-binding properties of cytokines. In this study, novel sulfated glucomannan-barium-alginate (SGA) microcapsules were prepared to encapsulate islets with barium-alginate (ABa) and calcium alginate-poly-l-lysine (APA) microcapsules as controls. SD rat islets were purified as donor cells to Lewis rats that had been treated with streptozotocin. Intraperitoneal transplantation was performed with about 3000 islet equivalent (IEQ) rat. At week three after transplantation, the concentrations of monocyte chemotactic protein-1 (MCP-1), interleukin (IL)-1 beta, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha in intraperitoneal fluid were determined using ELISA. At week 8, the islet cell mass in the abdominal microcapsules was excised to test insulin release. The EB-FDA fluorescence staining method was used to observe the functional activity of the islet cells. Compared with ABa and APA microcapsules, SGA microcapsules showed significantly decreased MCP-1 secretion by beta-cells. Also, the concentrations of cytokines IL-1beta, IFN-gamma, and TNF-alpha were decreased significantly. The activity of the transplanted islets was significantly improved in SGA microcapsules, which shielded against cytokines better than ABa or APA microcapsules and may serve as novel method.

  13. Molecular cloning of cDNA for rat argininosuccinate lyase and its expression in rat hepatoma cell lines.

    PubMed Central

    Lambert, M A; Simard, L R; Ray, P N; McInnes, R R

    1986-01-01

    Using antibody and plaque hybridization screening, we isolated rat argininosuccinate lyase (AS lyase) cDNA clones from a liver cDNA library prepared in the phage expression vector lambda gt11. Five overlapping cDNAs covering 1.7 kilobases of the estimated 2.0-kilobase AS lyase mRNA were characterized and confirmed as AS lyase sequences by hybrid selection. We examined the differential expression of AS lyase in rat liver and four rat hepatoma cell lines (7800C1, H4, HTC, and MH1C1). These cells exhibited a 60-fold range of AS lyase enzyme activity, with a direct correlation between activity, amount of AS lyase immunoreactive protein, and quantity of specific AS lyase mRNA. These observations suggest that the differences in AS lyase expression between rat liver and the hepatoma cell lines result from variations in AS lyase transcriptional activity or alterations in nuclear processing of AS lyase RNA. Images PMID:3785176

  14. Degradation of Argininosuccinate Lyase by a Protease Synthesized in Soybean Cell Suspension Cultures 1

    PubMed Central

    Shargool, P. D.

    1975-01-01

    Suspension cultures of soybean (Glycine max L.) were shown to contain protease activity which could be inhibited by the addition of protease inhibitors such as p-hydroxymercuribenzoate and ethylenediaminetetraacetic acid. The use of these inhibitors, coupled with studies of the rate of degradation of argininosuccinate lyase (argininosuccinate-lyase = l-arginino-succinate arginine-lyase, EC 4.3.2.1) in extracts of cell cultures grown for 24 hours led to the hypothesis that a metal-dependent protease is synthesized by the cells after 24 hours of growth, to remove the lyase enzyme. PMID:16659138

  15. Alginate Oligosaccharides Inhibit Fungal Cell Growth and Potentiate the Activity of Antifungals against Candida and Aspergillus spp

    PubMed Central

    Tøndervik, Anne; Sletta, Håvard; Klinkenberg, Geir; Emanuel, Charlotte; Powell, Lydia C.; Pritchard, Manon F.; Khan, Saira; Craine, Kieron M.; Onsøyen, Edvar; Rye, Phil D.; Wright, Chris; Thomas, David W.; Hill, Katja E.

    2014-01-01

    The oligosaccharide OligoG, an alginate derived from seaweed, has been shown to have anti-bacterial and anti-biofilm properties and potentiates the activity of selected antibiotics against multi-drug resistant bacteria. The ability of OligoG to perturb fungal growth and potentiate conventional antifungal agents was evaluated using a range of pathogenic fungal strains. Candida (n = 11) and Aspergillus (n = 3) spp. were tested using germ tube assays, LIVE/DEAD staining, scanning electron microscopy (SEM), atomic force microscopy (AFM) and high-throughput minimum inhibition concentration assays (MICs). In general, the strains tested showed a significant dose-dependent reduction in cell growth at ≥6% OligoG as measured by optical density (OD600; P<0.05). OligoG (>0.5%) also showed a significant inhibitory effect on hyphal growth in germ tube assays, although strain-dependent variations in efficacy were observed (P<0.05). SEM and AFM both showed that OligoG (≥2%) markedly disrupted fungal biofilm formation, both alone, and in combination with fluconazole. Cell surface roughness was also significantly increased by the combination treatment (P<0.001). High-throughput robotic MIC screening demonstrated the potentiating effects of OligoG (2, 6, 10%) with nystatin, amphotericin B, fluconazole, miconazole, voriconazole or terbinafine with the test strains. Potentiating effects were observed for the Aspergillus strains with all six antifungal agents, with an up to 16-fold (nystatin) reduction in MIC. Similarly, all the Candida spp. showed potentiation with nystatin (up to 16-fold) and fluconazole (up to 8-fold). These findings demonstrate the antifungal properties of OligoG and suggest a potential role in the management of fungal infections and possible reduction of antifungal toxicity. PMID:25409186

  16. Multisite inhibition of Pinus pinea isocitrate lyase by phosphate.

    PubMed

    Ranaldi, F; Vanni, P; Giachetti, E

    2000-11-01

    Our results show that the phosphate ion is a nonlinear competitive inhibitor of Pinus pinea isocitrate lyase. In addition, this compound induces a sigmoidal response of the enzyme, which usually exhibits standard Michaelis-Menten kinetics. This peculiar behavior of P. pinea isocitrate lyase could be explained by a dimer (two-site) model, in which phosphate binds cooperatively, but the affinity of the vacant site for substrate (the magnesium-isocitrate complex) remains the same. As a result, the interaction of phosphate with free enzyme produces an inhibitor-enzyme-inhibitor species that is of significant importance in determining reaction rate; a possible regulatory role of the glyoxylate cycle by inorganic phosphate is suggested. The mode of phosphate inhibition is consistent with both the mechanism for magnesium ion activation of P. pinea isocitrate lyase and its site heterogeneity. Our results explain the cooperative effects observed by some authors in kinetic studies of isocitrate lyase carried out in phosphate buffers and also account for the higher K(m) values determined by using such assay systems. Phosphate buffer should be avoided in performing isocitrate lyase kinetics.

  17. Antibacterial performance of alginic acid coating on polyethylene film.

    PubMed

    Karbassi, Elika; Asadinezhad, Ahmad; Lehocký, Marian; Humpolíček, Petr; Vesel, Alenka; Novák, Igor; Sáha, Petr

    2014-08-21

    Alginic acid coated polyethylene films were examined in terms of surface properties and bacteriostatic performance against two most representative bacterial strains, that is, Escherichia coli and Staphylococcus aureus. Microwave plasma treatment followed by brush formation in vapor state from three distinguished precursors (allylalcohol, allylamine, hydroxyethyl methacrylate) was carried out to deposit alginic acid on the substrate. Surface analyses via various techniques established that alginic acid was immobilized onto the surface where grafting (brush) chemistry influenced the amount of alginic acid coated. Moreover, alginic acid was found to be capable of bacterial growth inhibition which itself was significantly affected by the brush type. The polyanionic character of alginic acid as a carbohydrate polymer was assumed to play the pivotal role in antibacterial activity. The cell wall composition of two bacterial strains along with the substrates physicochemical properties accounted for different levels of bacteriostatic performance.

  18. L-alanine-glyoxylate aminotransferase II of rat kidney and liver mitochondria possesses cysteine S-conjugate beta-lyase activity: a contributing factor to the nephrotoxicity/hepatotoxicity of halogenated alkenes?

    PubMed Central

    Cooper, Arthur J L; Krasnikov, Boris F; Okuno, Etsuo; Jeitner, Thomas M

    2003-01-01

    Several halogenated alkenes are metabolized in part to cysteine S-conjugates, which are mitochondrial toxicants of kidney and, to a lesser extent, other organs. Toxicity is due to cysteine S-conjugate beta-lyases, which convert the cysteine S-conjugate into pyruvate, ammonia and a reactive sulphur-containing fragment. A section of the human population is exposed to halogenated alkenes. To understand the health effects of such exposure, it is important to identify cysteine S-conjugate beta-lyases that contribute to mitochondrial damage. Mitochondrial aspartate aminotransferase [Cooper, Bruschi, Iriarte and Martinez-Carrion (2002) Biochem. J. 368, 253-261] and mitochondrial branched-chain aminotransferase [Cooper, Bruschi, Conway and Hutson (2003) Biochem. Pharmacol. 65, 181-192] exhibit beta-lyase activity toward S -(1,2-dichlorovinyl)-L-cysteine (the cysteine S-conjugate of trichloroethylene) and S -(1,1,2,2-tetrafluoroethyl)-L-cysteine (the cysteine S-conjugate of tetrafluoroethylene). Turnover leads to eventual inactivation of these enzymes. Here we report that mitochondrial L-alanine-glyoxylate aminotransferase II, which, in the rat, is most active in kidney, catalyses cysteine S-conjugate beta-lyase reactions with S -(1,1,2,2-tetrafluoroethyl)-L-cysteine, S -(1,2-dichlorovinyl)-L-cysteine and S -(benzothiazolyl-L-cysteine); turnover leads to inactivation. Previous workers showed that the reactive-sulphur-containing fragment released from S -(1,1,2,2-tetrafluoroethyl)-L-cysteine and S -(1,2-dichlorovinyl)-L-cysteine is toxic by acting as a thioacylating agent - particularly of lysine residues in nearby proteins. Toxicity, however, may also involve 'self-inactivation' of key enzymes. The present findings suggest that alanine-glyoxylate aminotransferase II may be an important factor in the well-established targeting of rat kidney mitochondria by toxic halogenated cysteine S-conjugates. Previous reports suggest that alanine-glyoxylate aminotransferase II is absent

  19. Comparison of some biochemical properties of artichoke polyphenol oxidase entrapped in alginate-carrageenan and alginate gels.

    PubMed

    Yagar, Hulya; Kocaturk, Selin

    2014-08-01

    Polyphenol oxidase (PPO, EC.1.14.18.1) isolated from artichoke (Cynara scolymus) was entrapped within alginate and alginate+ carrageenan beads, and the catecholase and cresolase activities of both entrapped enzymes were determined. Some properties of these immobilized enzymes such as optimum pH and temperature, kinetic parameters (Km and Vmax), thermal, and storage stability were determined and compared to each other. The highest catecholase activity was observed in alginate gel (370 U/g bead) while the highest cresolase activity was in alginate+ carrageenan gel (90 U/g bead). For catecholase and cresolase activities, optimum pHs of alginate and alginate+ carrageenan beads were determined to be 7.0 and 4.0, respectively. Optimum temperatures for catecholase activity were determined to be 40°C for both entrapped enzymes. These values for cresolase activity were 30°C and 20°C, respectively. Immobilized artichoke PPOs greatly preserved their thermal stability which exists anyway. The catalytic efficiency value (Vmax/Km) of the alginate beads is approximately high as two-and-a-half folds of that of alginate+κ-carrageenan beads for cresolase activity. These values were very close for catecholase activity. Immobilized beads saved their both activities after 30 days of storage at 4°C.

  20. Preparation methods of alginate nanoparticles.

    PubMed

    Paques, Jerome P; van der Linden, Erik; van Rijn, Cees J M; Sagis, Leonard M C

    2014-07-01

    This article reviews available methods for the formation of alginate nano-aggregates, nanocapsules and nanospheres. Primarily, alginate nanoparticles are being prepared by two methods. In the "complexation method", complex formation on the interface of an oil droplet is used to form alginate nanocapsules, and complex formation in an aqueous solution is used to form alginate nano-aggregates. In a second method w/o emulsification coupled with gelation of the alginate emulsion droplet can be used to form alginate nanospheres. We review advantages and disadvantages of these methods, and give an overview of the properties of the alginate particles produced with these methods.

  1. Radiolytically depolymerized sodium alginate improves physiological activities, yield attributes and composition of essential oil of Eucalyptus citriodora Hook.

    PubMed

    Ali, Akbar; Khan, M Masroor A; Uddin, Moin; Naeem, M; Idrees, Mohd; Hashmi, Nadeem; Dar, Tariq Ahmad; Varshney, Lalit

    2014-11-04

    Eucalyptus citriodora Hook. is highly valued for its citronellal-rich essential oil (EO) extracted from its leaves. Hence, escalated EO production of eucalyptus is the need of hour. Marine polysaccharides (sodium alginate) are processed through gamma radiation of particular intensity, to obtain the irradiated sodium alginate (ISA). A pot experiment was conducted to study the effect of foliar application of ISA on growth, biochemical, physiological, EO yield and composition of E. citriodora. The treatments were applied as: foliar spray of deionized water only (control), seed soaked with ISA (90 mg L(-1)) and foliar spray of ISA with 30, 60, 120 and 240 mg L(-1). The treatment 6 (spray of ISA at 120 mg L(-1)) showed the highest value for most of the parameters studied. It also enhanced the EO content (33.3%), EO yield (86.7%), citronellal content (63.4%) and citronellal yield (205.5%) as compared to the control.

  2. Bioactive apatite incorporated alginate microspheres with sustained drug-delivery for bone regeneration application.

    PubMed

    Li, Haibin; Jiang, Fei; Ye, Song; Wu, Yingying; Zhu, Kaiping; Wang, Deping

    2016-05-01

    The strontium-substituted hydroxyapatite microspheres (SrHA) incorporated alginate composite microspheres (SrHA/Alginate) were prepared via adding SrHA/alginate suspension dropwise into calcium chloride solution, in which the gel beads were formed by means of crosslinking reaction. The structure, morphology and in vitro bioactivity of the composite microspheres were studied by using XRD, SEM and EDS methods. The biological behaviors were characterized and analyzed through inductively coupled plasma optical emission spectroscopy (ICP-OES), CCK-8, confocal laser microscope and ALP activity evaluations. The experimental results indicated that the synthetic SrHA/Alginate showed similar morphology to the well-known alginate microspheres (Alginate) and both of them possessed a great in vitro bioactivity. Compared with the control Alginate, the SrHA/Alginate enhanced MC3T3-E1 cell proliferation and ALP activity by releasing osteoinductive and osteogenic Sr ions. Furthermore, vancomycin was used as a model drug to investigate the drug release behaviors of the SrHA/Alginate, Alginate and SrHA. The results suggested that the SrHA/Alginate had a highest drug-loading efficiency and best controlled drug release properties. Additionally, the SrHA/Alginate was demonstrated to be pH-sensitive as well. The increase of the pH value in phosphate buffer solution (PBS) accelerated the vancomycin release. Accordingly, the multifunctional SrHA/Alginate can be applied in the field of bioactive drug carriers and bone filling materials.

  3. Calcium alginate gels as stem cell matrix-making paracrine stem cell activity available for enhanced healing after surgery.

    PubMed

    Schmitt, Andreas; Rödel, Philipp; Anamur, Cihad; Seeliger, Claudine; Imhoff, Andreas B; Herbst, Elmar; Vogt, Stephan; van Griensven, Martijn; Winter, Gerhard; Engert, Julia

    2015-01-01

    Regeneration after surgery can be improved by the administration of anabolic growth factors. However, to locally maintain these factors at the site of regeneration is problematic. The aim of this study was to develop a matrix system containing human mesenchymal stem cells (MSCs) which can be applied to the surgical site and allows the secretion of endogenous healing factors from the cells. Calcium alginate gels were prepared by a combination of internal and external gelation. The gelling behaviour, mechanical stability, surface adhesive properties and injectability of the gels were investigated. The permeability of the gels for growth factors was analysed using bovine serum albumin and lysozyme as model proteins. Human MSCs were isolated, cultivated and seeded into the alginate gels. Cell viability was determined by AlamarBlue assay and fluorescence microscopy. The release of human VEGF and bFGF from the cells was determined using an enzyme-linked immunoassay. Gels with sufficient mechanical properties were prepared which remained injectable through a syringe and solidified in a sufficient time frame after application. Surface adhesion was improved by the addition of polyethylene glycol 300,000 and hyaluronic acid. Humans MSCs remained viable for the duration of 6 weeks within the gels. Human VEGF and bFGF was found in quantifiable concentrations in cell culture supernatants of gels loaded with MSCs and incubated for a period of 6 weeks. This work shows that calcium alginate gels can function as immobilization matrices for human MSCs.

  4. Calcium Alginate Gels as Stem Cell Matrix – Making Paracrine Stem Cell Activity Available for Enhanced Healing after Surgery

    PubMed Central

    Schmitt, Andreas; Rödel, Philipp; Anamur, Cihad; Seeliger, Claudine; Imhoff, Andreas B.; Herbst, Elmar; Vogt, Stephan; van Griensven, Martijn; Winter, Gerhard; Engert, Julia

    2015-01-01

    Regeneration after surgery can be improved by the administration of anabolic growth factors. However, to locally maintain these factors at the site of regeneration is problematic. The aim of this study was to develop a matrix system containing human mesenchymal stem cells (MSCs) which can be applied to the surgical site and allows the secretion of endogenous healing factors from the cells. Calcium alginate gels were prepared by a combination of internal and external gelation. The gelling behaviour, mechanical stability, surface adhesive properties and injectability of the gels were investigated. The permeability of the gels for growth factors was analysed using bovine serum albumin and lysozyme as model proteins. Human MSCs were isolated, cultivated and seeded into the alginate gels. Cell viability was determined by AlamarBlue assay and fluorescence microscopy. The release of human VEGF and bFGF from the cells was determined using an enzyme-linked immunoassay. Gels with sufficient mechanical properties were prepared which remained injectable through a syringe and solidified in a sufficient time frame after application. Surface adhesion was improved by the addition of polyethylene glycol 300,000 and hyaluronic acid. Humans MSCs remained viable for the duration of 6 weeks within the gels. Human VEGF and bFGF was found in quantifiable concentrations in cell culture supernatants of gels loaded with MSCs and incubated for a period of 6 weeks. This work shows that calcium alginate gels can function as immobilization matrices for human MSCs. PMID:25793885

  5. The ppuI-rsaL-ppuR quorum-sensing system regulates cellular motility, pectate lyase activity, and virulence in potato opportunistic pathogen Pseudomonas sp. StFLB209.

    PubMed

    Kato, Taro; Morohoshi, Tomohiro; Someya, Nobutaka; Ikeda, Tsukasa

    2015-01-01

    Pseudomonas sp. StFLB209 was isolated from potato leaf as an N-acylhomoserine lactone (AHL)-producing bacterium and showed a close phylogenetic relationship with P. cichorii, a known plant pathogen. Although there are no reports of potato disease caused by pseudomonads in Japan, StFLB209 was pathogenic to potato leaf. In this study, we reveal the complete genome sequence of StFLB209, and show that the strain possesses a ppuI-rsaL-ppuR quorum-sensing system, the sequence of which shares a high similarity with that of Pseudomonas putida. Disruption of ppuI results in a loss of AHL production as well as remarkable reduction in motility. StFLB209 possesses strong pectate lyase activity and causes maceration on potato tuber and leaf, which was slightly reduced in the ppuI mutant. These results suggest that the quorum-sensing system is well conserved between StFLB209 and P. putida and that the system is essential for motility, full pectate lyase activity, and virulence in StFLB209.

  6. Evaluation of the antimicrobial activity and dimensional alterations of alginate impression disinfectants.

    PubMed

    Semensato, Ana Paula Nocentini; Crosariol, Sonia Khouri; Marchini, Leonardo

    2009-09-01

    This paper offers a quantitative evaluation of the antimicrobial efficacy of eight different disinfection procedures for irreversible hydrocolloid impressions and the dimensional changes induced by them. Samples were collected immediately after impressions, after the disinfection procedures and over casts and analyzed for bacterial growth. Control, enzyme solutions, acetic acid and ultraviolet irradiation samples showed bacterial growth. Chlorhexidine and 1% sodium hypochlorite presented adequate antimicrobial activity, while 2% sodium hypochlorite solution showed the best results. Dimensional changes were similar to those of the controls in all the tested agents. The results indicated 2% hypochlorite was the most appropriate disinfectant tested.

  7. Isocitrate lyase and the glyoxylate cycle. Progress report, February 15, 1989--February 15, 1990

    SciTech Connect

    McFadden, B.A.

    1990-12-31

    Active site modifications of isocitrate lyase (icl) from Escherichia coli are described. In addition directed mutagenesis of icl gene are detailed aimed at varying the charge yet conserving the structure of the enzymes active site.

  8. Molecular characterization of a Penicillium chrysogenum exo-rhamnogalacturonan lyase that is structurally distinct from other polysaccharide lyase family proteins.

    PubMed

    Iwai, Marin; Kawakami, Takuya; Ikemoto, Takeshi; Fujiwara, Daisuke; Takenaka, Shigeo; Nakazawa, Masami; Ueda, Mitsuhiro; Sakamoto, Tatsuji

    2015-10-01

    We previously described an endo-acting rhamnogalacturonan (RG) lyase, termed PcRGL4A, of Penicillium chrysogenum 31B. Here, we describe a second RG lyase, called PcRGLX. We determined the cDNA sequence of the Pcrglx gene, which encodes PcRGLX. Based on analyses using a BLAST search and a conserved domain search, PcRGLX was found to be structurally distinct from known RG lyases and might belong to a new polysaccharide lyase family together with uncharacterized fungal proteins of Nectria haematococca, Aspergillus oryzae, and Fusarium oxysporum. The Pcrglx cDNA gene product (rPcRGLX) expressed in Escherichia coli demonstrated specific activity against RG but not against homogalacturonan. Divalent cations were not essential for the enzymatic activity of rPcRGLX. rPcRGLX mainly released unsaturated galacturonosyl rhamnose (ΔGR) from RG backbones used as the substrate from the initial stage of the reaction, indicating that the enzyme can be classified as an exo-acting RG lyase (EC 4.2.2.24). This is the first report of an RG lyase with this mode of action in Eukaryota. rPcRGLX acted synergistically with PcRGL4A to degrade soybean RG and released ΔGR. This ΔGR was partially decorated with galactose (Gal) residues, indicating that rPcRGLX preferred oligomeric RGs to polymeric RGs, that the enzyme did not require Gal decoration of RG backbones for degradation, and that the enzyme bypassed the Gal side chains of RG backbones. These characteristics of rPcRGLX might be useful in the determination of complex structures of pectins.

  9. Characterization of ATP citrate lyase from Chlorobium limicola.

    PubMed Central

    Antranikian, G; Herzberg, C; Gottschalk, G

    1982-01-01

    ATP citrate lyase (EC 4.1.3.8) from Chlorobium limicola was partially purified. It was established that the consumption of substrates and the formation of products proceeded stoichiometrically and that citrate cleavage was of the si-type. ADP and oxaloacetate inhibited enzyme activity. Oxaloacetate also inhibited the growth of C. limicola. PMID:7142107

  10. Characterization of free and alginate-polylysine-alginate microencapsulated Erwinia herbicola for the conversion of ammonia, pyruvate, and phenol into L-tyrosine

    SciTech Connect

    Lloyd-George, I.; Chang, T.M.S.

    1995-12-20

    The whole cell tyrosine phenol-lyase activity of Erwinia herbicola was microencapsulated. The authors studied the use of this for the conversion of ammonia and pyruvate along with phenol or catechol, respectively, into L-tyrosine or dihydroxyphenyl-L-alanine (L-dopa). The reactions are relevant to the development of new methods for the production of L-tyrosine and L-dopa. The growth of E. herbicola at temperatures from 22 C to 32 C is stable, since at these temperatures the cells grow up to the stationary phase and remain there for at least 10 h. At 37 C the cells grow rapidly, but they also enter the death phase rapidly. There is only limited growth of E. herbicola at 42 C. Whole cells of E. herbicola were encapsulated within alginate-polylysine-alginate microcapsules (916 {+-} 100 {micro}m, mean {+-} std. dev.). The TPL activity of the cells catalyzed the production of L-tyrosine or dihydroxyphenol-L-alanine (L-dopa) from ammonia, pyruvate, and phenol or catechol, respectively. In the production of tyrosine, an integrated equation based on an ordered ter-uni rapid equilibrium mechanism can be used to find the kinetic parameters of TPL. In an adequately stirred system, the apparent values of the kinetic parameters of whole cell TPL are equal whether the cells are free or encapsulated. The apparent K{sub M} of tyrosine varies with the amount of whole cells in the system, ranging from 0.2 to 0.3 mM. The apparent K{sub M} for phenol is 0.5 mM. The apparent K{sub M} values for pyruvate and ammonia are an order of magnitude greater for whole cells than they are for the cell free enzyme.

  11. Method for quantifying alginate and determining release from a food vehicle in gastrointestinal digesta.

    PubMed

    Houghton, David; Wilcox, Matthew D; Brownlee, Iain A; Chater, Peter; Seal, Chris J; Pearson, Jeffrey P

    2014-05-15

    To assess the efficacy of alginate as a modifier of enzyme activity, a suitable method to quantify its release must be developed. This paper develops and assesses the ability of the Periodic Acid Schiffs (PAS) assay to quantify alginate, and its release from bread during digestion in a model gut. Control and alginate enriched (4% w/w wet dough) bread were used. A model gut replicating the mouth, stomach and small intestines was used. Standard curves were created for alginate in deionised H2O and model gut solutions using a modified PAS to remove interference. The PAS assay quantified alginate with excellent linearity (R(2)=0.99), and optical density range (0.02-0.5). There was a significant difference in alginate release at 180 min compared to 0 and 60 min. The data indicate the modified PAS assay is a simple method for quantifying alginate release and release rate from alginate enriched products.

  12. Tyrosine phenol-lyase and tryptophan indole-lyase encapsulated in wet nanoporous silica gels: Selective stabilization of tertiary conformations

    PubMed Central

    Pioselli, Barbara; Bettati, Stefano; Demidkina, Tatyana V.; Zakomirdina, Lyudmila N.; Phillips, Robert S.; Mozzarelli, Andrea

    2004-01-01

    The pyridoxal 5′-phosphate-dependent enzymes tyrosine phenol-lyase and tryptophan indole-lyase were encapsulated in wet nanoporous silica gels, a powerful method to selectively stabilize tertiary and quaternary protein conformations and to develop bioreactors and biosensors. A comparison of the enzyme reactivity in silica gels and in solution was carried out by determining equilibrium and kinetic parameters, exploiting the distinct spectral properties of catalytic intermediates and reaction products. The encapsulated enzymes exhibit altered distributions of ketoenamine and enolimine tautomers, increased values of inhibitors dissociation constants, slow attaining of steady-state in the presence of substrate and substrate analogs, modified steady-state distribution of catalytic intermediates, and a sixfold–eightfold decrease of specific activities. This behavior can be rationalized by a reduced conformational flexibility for the encapsulated enzymes and a selective stabilization of either the open (inactive) or the closed (active) form of the enzymes. Despite very similar structures and catalytic mechanisms, the influence of encapsulation is more pronounced for tyrosine phenol-lyase than tryptophan indole-lyase. This finding indicates that subtle structural and dynamic differences can lead to distinct interactions of the protein with the gel matrix. PMID:15044726

  13. Tyrosine phenol-lyase and tryptophan indole-lyase encapsulated in wet nanoporous silica gels: Selective stabilization of tertiary conformations.

    PubMed

    Pioselli, Barbara; Bettati, Stefano; Demidkina, Tatyana V; Zakomirdina, Lyudmila N; Phillips, Robert S; Mozzarelli, Andrea

    2004-04-01

    The pyridoxal 5'-phosphate-dependent enzymes tyrosine phenol-lyase and tryptophan indole-lyase were encapsulated in wet nanoporous silica gels, a powerful method to selectively stabilize tertiary and quaternary protein conformations and to develop bioreactors and biosensors. A comparison of the enzyme reactivity in silica gels and in solution was carried out by determining equilibrium and kinetic parameters, exploiting the distinct spectral properties of catalytic intermediates and reaction products. The encapsulated enzymes exhibit altered distributions of ketoenamine and enolimine tautomers, increased values of inhibitors dissociation constants, slow attaining of steady-state in the presence of substrate and substrate analogs, modified steady-state distribution of catalytic intermediates, and a sixfold-eightfold decrease of specific activities. This behavior can be rationalized by a reduced conformational flexibility for the encapsulated enzymes and a selective stabilization of either the open (inactive) or the closed (active) form of the enzymes. Despite very similar structures and catalytic mechanisms, the influence of encapsulation is more pronounced for tyrosine phenol-lyase than tryptophan indole-lyase. This finding indicates that subtle structural and dynamic differences can lead to distinct interactions of the protein with the gel matrix.

  14. A Novel Aldo-Keto Reductase, HdRed, from the Pacific Abalone Haliotis discus hannai, Which Reduces Alginate-derived 4-Deoxy-l-erythro-5-hexoseulose Uronic Acid to 2-Keto-3-deoxy-d-gluconate*

    PubMed Central

    Mochizuki, Shogo; Nishiyama, Ryuji; Inoue, Akira; Ojima, Takao

    2015-01-01

    Abalone feeds on brown seaweeds and digests seaweeds' alginate with alginate lyases (EC 4.2.2.3). However, it has been unclear whether the end product of alginate lyases (i.e. unsaturated monouronate-derived 4-deoxy-l-erythro-5-hexoseulose uronic acid (DEH)) is assimilated by abalone itself, because DEH cannot be metabolized via the Embden-Meyerhof pathway of animals. Under these circumstances, we recently noticed the occurrence of an NADPH-dependent reductase, which reduced DEH to 2-keto-3-deoxy-d-gluconate, in hepatopancreas extract of the pacific abalone Haliotis discus hannai. In the present study, we characterized this enzyme to some extent. The DEH reductase, named HdRed in the present study, could be purified from the acetone-dried powder of hepatopancreas by ammonium sulfate fractionation followed by conventional column chromatographies. HdRed showed a single band of ∼40 kDa on SDS-PAGE and reduced DEH to 2-keto-3-deoxy-d-gluconate with an optimal temperature and pH at around 50 °C and 7.0, respectively. HdRed exhibited no appreciable activity toward 28 authentic compounds, including aldehyde, aldose, ketose, α-keto-acid, uronic acid, deoxy sugar, sugar alcohol, carboxylic acid, ketone, and ester. The amino acid sequence of 371 residues of HdRed deduced from the cDNA showed 18–60% identities to those of aldo-keto reductase (AKR) superfamily enzymes, such as human aldose reductase, halophilic bacterium reductase, and sea hare norsolorinic acid (a polyketide derivative) reductase-like protein. Catalytic residues and cofactor binding residues known in AKR superfamily enzymes were fairly well conserved in HdRed. Phylogenetic analysis for HdRed and AKR superfamily enzymes indicated that HdRed is an AKR belonging to a novel family. PMID:26555267

  15. An ancient relative of cyclooxygenase in cyanobacteria is a linoleate 10S-dioxygenase that works in tandem with a catalase-related protein with specific 10S-hydroperoxide lyase activity.

    PubMed

    Brash, Alan R; Niraula, Narayan P; Boeglin, William E; Mashhadi, Zahra

    2014-05-09

    In the course of exploring the scope of catalase-related hemoprotein reactivity toward fatty acid hydroperoxides, we detected a novel candidate in the cyanobacterium Nostoc punctiforme PCC 73102. The immediate neighboring upstream gene, annotated as "cyclooxygenase-2," appeared to be a potential fatty acid heme dioxygenase. We cloned both genes and expressed the cDNAs in Escherichia coli, confirming their hemoprotein character. Oxygen electrode recordings demonstrated a rapid (>100 turnovers/s) reaction of the heme dioxygenase with oleic and linoleic acids. HPLC, including chiral column analysis, UV, and GC-MS of the oxygenated products, identified a novel 10S-dioxygenase activity. The catalase-related hemoprotein reacted rapidly and specifically with linoleate 10S-hydroperoxide (>2,500 turnovers/s) with a hydroperoxide lyase activity specific for the 10S-hydroperoxy enantiomer. The products were identified by NMR as (8E)10-oxo-decenoic acid and the C8 fragments, 1-octen-3-ol and 2Z-octen-1-ol, in ∼3:1 ratio. Chiral HPLC analysis established strict enzymatic control in formation of the 3R alcohol configuration (99% enantiomeric excess) and contrasted with racemic 1-octen-3-ol formed in reaction of linoleate 10S-hydroperoxide with hematin or ferrous ions. The Nostoc linoleate 10S-dioxygenase, the sequence of which contains the signature catalytic sequence of cyclooxygenases and fungal linoleate dioxygenases (YRWH), appears to be a heme dioxygenase ancestor. The novel activity of the lyase expands the known reactions of catalase-related proteins and functions in Nostoc in specific transformation of the 10S-hydroperoxylinoleate.

  16. Hits identified in library screening demonstrate selective CYP17A1 lyase inhibition.

    PubMed

    Krug, Sebastian J; Hu, Qingzhong; Hartmann, Rolf W

    2013-03-01

    A screening of structurally different steroid hormone synthesis inhibitors was performed in order to find a starting point for the development of a new inhibitor of the bifunctional steroidogenic enzyme CYP17A1. Emphasis was placed on determination of selectivity between the two catalytic steps, namely 17α-hydroxylase and C(17,20)-lyase. For that purpose a new inhibition assay has been developed. Hits identified within this novel assay demonstrated selective inhibition of CYP17A1 lyase activity, and thus mark the basis for the development of selective C(17,20)-lyase inhibitors for the treatment of prostate cancer.

  17. Ethylene-enhanced Synthesis of Phenylalanine Ammonia-Lyase in Pea Seedlings 1

    PubMed Central

    Hyodo, Hiroshi; Yang, Shang Fa

    1971-01-01

    The effect of ethylene on the development of phenylalanine ammonia-lyase activity in segments excised from the epicotyl apex of pea seedling was studied. Although there was some increase in phenylalanine ammonia-lyase activity in segments not treated with ethylene, a marked increase in phenylalanine ammonia-lyase activity occurred in ethylene-treated tissues during the incubation. The induction period was estimated to be about 6 hours. The activity reached a maxmum at 30 hours and then declined. On withdrawal of ethylene, the increase was sustained for a short period and then stopped. After retreatment with ethylene, the increase was resumed. Addition of CO2 reduced the effect of ethylene. Administration of cycloheximide or actinomycin D at an early period almost completely suppressed the increase in phenylalanine ammonia-lyase activity. However, if these inhibitors were administered at a later period, while phenylalanine ammonia-lyase activity was approaching a maximum, they not only failed to reduce but rather stimulated the activity. These results are consistent with the view that there exist both phenylalanine ammonia-lyase-synthesizing and -inactivating systems, and that the development of both systems may involve de novo synthesis of protein. PMID:16657701

  18. Enhancer-like activity of A1gR1-binding site in alginate gene activation: positional, orientational, and sequence specificity.

    PubMed Central

    Fujiwara, S; Zielinski, N A; Chakrabarty, A M

    1993-01-01

    Significant activation of promoters of alginate genes such as algD or algC occurs in mucoid Pseudomonas aeruginosa during its proliferation in the lungs of cystic fibrosis patients. These promoters have been shown to be responsive to environmental signals such as high osmolarity. The signaling is mediated by a so-called two-component signal transduction system, in which a soluble protein, AlgR2, undergoes autophosphorylation and transfers the phosphate to a DNA-binding response regulator protein, AlgR1. The phosphorylated form of AlgR1 has a high affinity for binding at upstream sequences of both the algC and algD promoters. Two AlgR1-binding sites (ABS) have been reported upstream of the algC gene. One of the two ABSs (algC-ABS1, located at -94 to -81) is critical for the algC activation process, while the second ABS (algC-ABS2, located at +161 to +174) is only weakly active. We now report the presence of a third ABS within the structural gene of algC, and this ABS (algC-ABS3) is also important for algC promoter activation. algC-ABS1 can be replaced functionally by algC-ABS2, algD-ABS1, or algD-ABS2 and somewhat weakly by algD-ABS3. Introduction of a half-integral turn in the DNA helix between the algC site of transcription initiation and algC-ABS1 allowed only slight reduction of promoter activity, suggesting that the binding site could be appreciably functional even when present in the opposite face of the helix. Activation of the algC promoter is independent of the relative location (upstream or downstream of the mRNA start site), the number of copies, or the orientation of algC-ABS1, suggesting that it behaves like a eukaryotic enhancer element in promoting transcription from the algC promoter. Images PMID:8366031

  19. Identification of 2-keto-3-deoxy-d-Gluconate Kinase and 2-keto-3-deoxy-d-Phosphogluconate Aldolase in an Alginate-Assimilating Bacterium, Flavobacterium sp. Strain UMI-01.

    PubMed

    Nishiyama, Ryuji; Inoue, Akira; Ojima, Takao

    2017-02-14

    Recently, we identified an alginate-assimilating gene cluster in the genome of Flavobacterium sp. strain UMI-01, a member of Bacteroidetes. Alginate lyase genes and a 4-deoxy-l-erythro-5-hexoseulose uronic acid (DEH) reductase gene in the cluster have already been characterized; however, 2-keto-3-deoxy-d-gluconate (KDG) kinase and 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase genes, i.e., flkin and flald, still remained uncharacterized. The amino acid sequences deduced from flkin and flald showed low identities with those of corresponding enzymes of Saccharophagus degradans 2-40(T), a member of Proteobacteria (Kim et al., Process Biochem., 2016). This led us to consider that the DEH-assimilating enzymes of Bacteroidetes species are somewhat deviated from those of Proteobacteria species. Thus, in the present study, we first assessed the characteristics in the primary structures of KDG kinase and KDG aldolase of the strain UMI-01, and then investigated the enzymatic properties of recombinant enzymes, recFlKin and recFlAld, expressed by an Escherichia coli expression system. Multiple-sequence alignment among KDG kinases and KDG aldolases from several Proteobacteria and Bacteroidetes species indicated that the strain UMI-01 enzymes showed considerably low sequence identities (15%-25%) with the Proteobacteria enzymes, while they showed relatively high identities (47%-68%) with the Bacteroidetes enzymes. Phylogenetic analyses for these enzymes indicated the distant relationship between the Proteobacteria enzymes and the Bacteroidetes enzymes, i.e., they formed distinct clusters in the phylogenetic tree. recFlKin and recFlAld produced with the genes flkin and flald, respectively, were confirmed to show KDG kinase and KDPG aldolase activities. Namely, recFlKin produced 1.7 mM KDPG in a reaction mixture containing 2.5 mM KDG and 2.5 mM ATP in a 90-min reaction, while recFlAld produced 1.2 mM pyruvate in the reaction mixture containing 5 mM KDPG at the equilibrium state

  20. Identification of 2-keto-3-deoxy-d-Gluconate Kinase and 2-keto-3-deoxy-d-Phosphogluconate Aldolase in an Alginate-Assimilating Bacterium, Flavobacterium sp. Strain UMI-01

    PubMed Central

    Nishiyama, Ryuji; Inoue, Akira; Ojima, Takao

    2017-01-01

    Recently, we identified an alginate-assimilating gene cluster in the genome of Flavobacterium sp. strain UMI-01, a member of Bacteroidetes. Alginate lyase genes and a 4-deoxy-l-erythro-5-hexoseulose uronic acid (DEH) reductase gene in the cluster have already been characterized; however, 2-keto-3-deoxy-d-gluconate (KDG) kinase and 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase genes, i.e., flkin and flald, still remained uncharacterized. The amino acid sequences deduced from flkin and flald showed low identities with those of corresponding enzymes of Saccharophagus degradans 2-40T, a member of Proteobacteria (Kim et al., Process Biochem., 2016). This led us to consider that the DEH-assimilating enzymes of Bacteroidetes species are somewhat deviated from those of Proteobacteria species. Thus, in the present study, we first assessed the characteristics in the primary structures of KDG kinase and KDG aldolase of the strain UMI-01, and then investigated the enzymatic properties of recombinant enzymes, recFlKin and recFlAld, expressed by an Escherichia coli expression system. Multiple-sequence alignment among KDG kinases and KDG aldolases from several Proteobacteria and Bacteroidetes species indicated that the strain UMI-01 enzymes showed considerably low sequence identities (15%–25%) with the Proteobacteria enzymes, while they showed relatively high identities (47%–68%) with the Bacteroidetes enzymes. Phylogenetic analyses for these enzymes indicated the distant relationship between the Proteobacteria enzymes and the Bacteroidetes enzymes, i.e., they formed distinct clusters in the phylogenetic tree. recFlKin and recFlAld produced with the genes flkin and flald, respectively, were confirmed to show KDG kinase and KDPG aldolase activities. Namely, recFlKin produced 1.7 mM KDPG in a reaction mixture containing 2.5 mM KDG and 2.5 mM ATP in a 90-min reaction, while recFlAld produced 1.2 mM pyruvate in the reaction mixture containing 5 mM KDPG at the equilibrium

  1. Promotive effects of alginate-derived oligosaccharides on the inducing drought resistance of tomato

    NASA Astrophysics Data System (ADS)

    Liu, Ruizhi; Jiang, Xiaolu; Guan, Huashi; Li, Xiaoxia; Du, Yishuai; Wang, Peng; Mou, Haijin

    2009-09-01

    In order to determine the role of alginate-derived oligosaccharides (ADO) in drought stress resistance of tomato ( Lycopersicon esculentum Miller) seedlings, the leaves were exposed to different concentrations of ADO (0.05%, 0.10%, 0.20%, 0.30% and 0.50%) after drought stress was simulated by exposing the roots to 0.6 molL-1 PEG-6000 solution for 6 h. Changes in biomass, electrolyte leakage and malondialdehyde (MDA), free proline, total soluble sugars (TSS) and abscisic acid (ABA), the enzyme activities of catalase (CAT), superoxide dismutase (SOD), peroxidase (POD) and phenylalanine ammonia-lyase (PAL) were measured to investigate the effects of ADO treatment. The results showed that the treatment with an ADO concentration of 0.20% exhibited the highest performance of drought stress resistance in the tomato seedlings by decreasing the electrolyte leakage and the concentration of MDA, increasing the contents of free proline, TSS and ABA, and increasing the activities of CAT, SOD, POD and PAL after treatment with ADO. It is suggested that changes in electrolyte leakage, MDA, osmotic solutes, ABA, anti-oxidative enzyme and PAL activities were responsible for the increased drought stress resistance in tomato seedlings. To our best knowledge, this is the first report of the effect of ADO treatment on enhancing the drought stress resistance of tomato seedlings.

  2. Development of a morphogenetically active scaffold for three-dimensional growth of bone cells: biosilica-alginate hydrogel for SaOS-2 cell cultivation.

    PubMed

    Müller, Werner E G; Schröder, Heinz C; Feng, Qingling; Schlossmacher, Ute; Link, Thorben; Wang, Xiaohong

    2015-11-01

    Polymeric silica is formed from ortho-silicate during a sol-gel formation process, while biosilica is the product of an enzymatically driven bio-polycondensation reaction. Both polymers have recently been described as a template that induces an increased expression of the genes encoding bone morphogenetic protein 2 (BMP-2) and osteoprotegerin in osteoblast-related SaOS-2 cells; simultaneously or subsequently the cells respond with enhanced hydroxyapatite formation. In order to assess whether the biocompatible polymeric silica/biosilica can serve as a morphogenetically active matrix suitable for three-dimensional (3D) cell growth, or even for 3D cell bioprinting, SaOS-2 cells were embedded into a Na-alginate-based hydrogel. Four different gelatinous hydrogel matrices were used for suspending SaOS-2 cells: (a) the hydrogel alone; (b) the hydrogel with 400 μM ortho-silicate; (c) the hydrogel supplemented with 400 μM ortho-silicate and recombinant silicatein to allow biosilica synthesis to occur; and (d) the hydrogel with ortho-silicate and BSA. The SaOS-2 cells showed an increased growth if silica/biosilica components were present in the hydrogel. Likewise intensified was the formation of hydroxyapatite nodules in the silica-containing hydrogels. After an incubation period of 2 weeks, cells present in silica-containing hydrogels showed a significantly higher expression of the genes encoding the cytokine BMP-2, the major fibrillar structural protein collagen 1 and likewise of carbonic anhydrase. It is concluded that silica, and to a larger extent biosilica, retains its morphogenetic/osteogenic potential after addition to Na-alginate-based hydrogels. This property might qualify silica hydrogels to be also used as a matrix for 3D cell printing.

  3. Alginic Acid Accelerates Calcite Dissolution

    NASA Astrophysics Data System (ADS)

    Perry, T. D.; Duckworth, O. W.; McNamara, C. J.; Martin, S. T.; Mitchell, R.

    2003-12-01

    Accelerated carbonate weathering through biological activity affects both geochemical cycling and the local pH and alkalinity of terrestrial and marine waters. Microbes affect carbonate dissolution through metabolic activity, production of acidic or chelating exudates, and cation binding by cell walls. Dissolution occurs within microbial biofilms - communities of microorganisms attached to stone in an exopolymer matrix. We investigated the effect of alginic acid, a common biological polymer produced by bacteria and algae, on calcite dissolution using a paired atomic force microscopy/flow-through reactor apparatus. The alginic acid caused up to an order of magnitude increase in dissolution rate at 3 < pH < 12. Additionally, the polymer preferentially binds to the obtuse pit steps and increases step velocity. We propose that the polymer is actively chelating surficial cations reducing the activation energy and increasing dissolution rate. The role of biologically produced polymers in mineral weathering is important in the protection of cultural heritage materials and understanding of marine and terrestrial systems.

  4. Characterization of Saccharomycopsis lipolytica mutants that express temperature-sensitive synthesis of isocitrate lyase.

    PubMed Central

    Matsuoka, M; Himeno, T; Aiba, S

    1984-01-01

    Four mutants specifically deficient in the activity of isocitrate lyase were independently isolated in the alkane yeast Saccharomycopsis lipolytica. Genetic analysis by means of protoplast fusion and mitotic haploidization revealed that the mutations were recessive and non-complementary at a single genetic locus, icl. icl is a structural gene for isocitrate lyase, because some revertants from icl-1 and icl-3 mutants produced thermolabile isocitrate lyase in comparison with the wild-type enzyme, and also because the gene dosage effect was observed on the specific activity of isocitrate lyase in icl+/icl-1 and icl+/icl-3 heterozygotes. The icl-3 mutation also gave rise to temperature-sensitive revertants that could grow on acetate at 23 degrees C but not at 33 degrees C, exhibiting temperature-sensitive synthesis as well as thermostable activity of isocitrate lyase. Studies on purified isocitrate lyase showed that this enzyme is tetrameric and that the enzyme synthesized at 23 degrees C by a temperature-sensitive synthesis mutant was indistinguishable from the wild-type enzyme with respect to the subunit molecular weight (59,000), the isoelectric pH (5.3), the thermostability, and the Km value for threo-Ds-isocitrate (0.2 mM). When induced by acetate at 33 degrees C, the temperature-sensitive synthesis mutant did not express isocitrate lyase activity but did synthesize polypeptides whose electrophoretic mobilities were equal to that of the purified mutant enzyme. Hence, the temperature-sensitive mutation assumed in the structural gene for isocitrate lyase might have prevented the maturation of the polypeptide chains synthesized at the restrictive temperature. Images PMID:6698940

  5. Reasons for reduced activities of 17 alpha-hydroxylase and C17-C20 lyase in spite of increased contents of cytochrome P-450 in mature rat testis fetally irradiated with 60Co.

    PubMed

    Inano, H; Ishii-Ohba, H; Suzuki, K; Ikeda, K

    1990-05-01

    Pregnant rats received whole body irradiation with 2.6 Gy gamma-ray from a 60Co source at Day 20 of gestation. When pups were 4 months old, activities of electron transport system and steroid monooxygenase in tests were assayed. The content of total cytochrome P-450 in the irradiated testes had increased to 170% of that in non-irradiated rats, but NADPH-cytochrome P-450 reductase activity had reduced to 36% of the control. Also, amounts of cytochrome b5 in testicular microsomal fraction were decreased markedly after irradiation, but no significant change of NADH-cytochrome b5 reductase activity was observed in the treated pups. Because both 17 alpha-hydroxylase and C17-C20 lyase activities tended to be decreased by fetal irradiation, testosterone production from progesterone and 17 alpha-hydroxyprogesterone was reduced to about 30% of the control. From these results, it has been suggested that the testicular cytochrome P-450 is radioresistant but steroid monooxygenase activities are reduced after the fetal irradiation. We propose that the discrepancy arises from the marked decrement of NADPH-cytochrome P-450 reductase activity.

  6. 21 CFR 184.1187 - Calcium alginate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Calcium alginate. 184.1187 Section 184.1187 Food... Specific Substances Affirmed as GRAS § 184.1187 Calcium alginate. (a) Calcium alginate (CAS Reg. No. 9005.... Calcium alginate is prepared by the neutralization of purified alginic acid with appropriate pH...

  7. 21 CFR 184.1610 - Potassium alginate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Potassium alginate. 184.1610 Section 184.1610 Food... GRAS § 184.1610 Potassium alginate. (a) Potassium alginate (CAS Reg. No. 9005-36-1) is the potassium salt of alginic acid, a natural polyuronide constituent of certain brown algae. Potassium alginate...

  8. 21 CFR 184.1724 - Sodium alginate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Sodium alginate. 184.1724 Section 184.1724 Food and... Substances Affirmed as GRAS § 184.1724 Sodium alginate. (a) Sodium alginate (CAS Reg. No. 9005-38-3) is the sodium salt of alginic acid, a natural polyuronide constituent of certain brown algae. Sodium alginate...

  9. 21 CFR 184.1724 - Sodium alginate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Sodium alginate. 184.1724 Section 184.1724 Food... GRAS § 184.1724 Sodium alginate. (a) Sodium alginate (CAS Reg. No. 9005-38-3) is the sodium salt of alginic acid, a natural polyuronide constituent of certain brown algae. Sodium alginate is prepared by...

  10. Purification of L-glutamate-dependent citrate lyase from Clostridium sphenoides and electron microscopic analysis of citrate lyase isolated from Rhodopseudomonas gelatinosa, Streptococcus diacetilactis and C. sphenoides.

    PubMed

    Antranikian, G; Klinner, C; Kümmel, A; Schwanitz, D; Zimmermann, T; Mayer, F; Gottschalk, G

    1982-08-01

    Citrate lyase from Clostridium sphenoides was purified 72-fold with a yield of 11%. In contrast to citrate lyase from other sources the activity of this enzyme was strictly dependent on the presence of L-glutamate. The purified enzyme was only stable in the presence of 150 mM L-glutamate or 7 mM L-glutamate plus glycerol, sucrose or bovine serum albumin. Changes of the L-glutamate pool and of enzyme activity in growing cells of C. sphenoides indicated that citrate lyase activity in this organism was regulated by the intracellular L-glutamate concentration. Citrate lyase isolated from C. sphenoides, Rhodopseudomonas gelatinosa and Streptococcus diacetilactis was investigated by electron microscopy using the negative staining technique. Three different projections of enzyme molecules were observed: 'star' form, 'ring' form and 'triangle' form. In samples from R. gelatinosa and S. diacetilactis, star and ring forms occurred in a ratio of about 1:9. Using the enzyme from S. diacetilactis it was demonstrated that this ratio could be altered in favour of the star form by the addition of citrate or tricarballylate. The triangle form was observed in less than 1% of all evaluated molecules and may represent a transition form. In lyase samples from C. sphenoides there existed a correlation between enzyme activity and the proportion of stars and rings at varying concentrations of L-glutamate.

  11. An insulin-sensitive cytosolic protein kinase accounts for the regulation of ATP citrate-lyase phosphorylation.

    PubMed Central

    Yu, K T; Benjamin, W B; Ramakrishna, S; Khalaf, N; Czech, M P

    1990-01-01

    Purified rat liver ATP citrate-lyase is phosphorylated on serine residues by an insulin-stimulated cytosolic kinase activity partially purified from rat adipocytes [Yu, Khalaf & Czech (1987) J. Biol. Chem. 262, 16677-16685]. The Km for lyase phosphorylation by this hormone-sensitive kinase activity is approx. 3 microM. Two-dimensional tryptic-peptide mapping of the 32P-labelled lyase reveals that the kinase-catalysed phosphorylation occurs primarily on a specific peptide. In intact 32P-labelled adipocytes, insulin enhances the serine phosphorylation of ATP citrate-lyase by 2-3-fold. Tryptic digestion of the 32P-labelled lyase immunopurified from insulin-treated adipocytes also yields one major phosphopeptide. 32P-labelled lyase tryptic peptides derived from labelling experiments in vitro and in vivo exhibit identical electrophoretic and chromatographic migration profiles. Furthermore, radio-sequencing of the phosphopeptide from lyase 32P-labelled in vitro indicates that serine-3 from the N-terminus is phosphorylated by the insulin-stimulated cytosolic kinase, in agreement with previous studies on the position of the phosphoserine residue in ATP citrate-lyase isolated from insulin-treated cells. Taken together, the similarity in site-specific phosphorylation of ATP citrate-lyase from insulin-treated adipocytes to that catalysed by the hormone-activated cytosolic kinase in vitro strongly suggests that this kinase mediates insulin action on lyase phosphorylation in intact cells. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:2114095

  12. Design of pectin-sodium alginate based films for potential healthcare application: Study of chemico-physical interactions between the components of films and assessment of their antimicrobial activity.

    PubMed

    Nešić, Aleksandra; Onjia, Antonije; Davidović, Sladjana; Dimitrijević, Suzana; Errico, Maria Emanuela; Santagata, Gabriella; Malinconico, Mario

    2017-02-10

    In this study, pectin based films including different amounts of sodium alginate were prepared by casting method. All the films, with and without polyglycerol as plasticizer, were crosslinked with zinc ions in order to extend their potential functionality. The development of junction points, occurring during the crosslinking process with zinc ions, induced the increasing of free volume with following changing in chemico-physical properties of films. The inclusion of alginate in pectin based formulations improved the strength of zinc ions crosslinking network, whereas the addition of polyglycerol significantly improved mechanical performance. Finally, zinc-crosslinked films evidenced antimicrobial activity against the most common exploited pathogens: Staphylococcus Aureus, Escherichia Coli and Candida Albicans. These results suggest that zinc-crosslinked based films can be exploitable as novel bio-active biomaterials for protection and disinfection of medical devices.

  13. Purification and characterization of tyrosine phenol lyase from Citrobacter freundii.

    PubMed

    Chandel, Meenakshi; Azmi, Wamik

    2013-12-01

    The purification and characterization of intracellular tyrosine phenol lyase from Citrobacter freundii has been carried out. The enzyme was purified 35-fold to homogeneity by ammonium sulphate precipitation and hydrophobic interaction chromatography. Its subunit molecular weight was found to be 52 kDa on sodium dodecyl sulphate polyacrylamide gel electrophoresis. The purified tyrosine phenol lyase showed maximum activity in borate buffer (0.05 M at pH 8.5) at 45 °C after 20 min of incubation. The Km and Vmax values of purified enzyme were found to be 0.446 mm and 0.342 mM/min/mg. This enzyme exhibits t1/2 of 10, 52 and 130 min at 55, 45 and 35 °C, respectively. The N-terminal amino acid sequence was determined as MET-ASN-TYR-PRO-ALA-GLU-PRO-PHE-ARG-ILETRP- TRP-VAL-GLY.

  14. Genetics Home Reference: adenylosuccinate lyase deficiency

    MedlinePlus

    ... analysis of five disease-associated human adenylosuccinate lyase mutants. Biochemistry. 2009 Jun 16;48(23):5291-302. ... J, Kmoch S. Biochemical and structural analysis of 14 mutant adsl enzyme complexes and correlation to phenotypic heterogeneity ...

  15. Preparation, characterisation and viability of encapsulated Trichoderma harzianum UPM40 in alginate-montmorillonite clay.

    PubMed

    Adzmi, Fariz; Meon, Sariah; Musa, Mohamed Hanafi; Yusuf, Nor Azah

    2012-01-01

    Microencapsulation is a process by which tiny parcels of an active ingredient are packaged within a second material for the purpose of shielding the active ingredient from the surrounding environment. This study aims to determine the ability of the microencapsulation technique to improve the viability of Trichoderma harzianum UPM40 originally isolated from healthy groundnut roots as effective biological control agents (BCAs). Alginate was used as the carrier for controlled release, and montmorillonite clay (MMT) served as the filler. The encapsulated Ca-alginate-MMT beads were characterised using Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA) and scanning electron microscopy (SEM). The FTIR results showed the interaction between the functional groups of alginate and MMT in the Ca-alginate-MMT beads. Peaks at 1595, 1420 and 1020 cm(-1) characterised alginate, and peaks at 1028 and 453 cm(-1) characterised MMT; both sets of peaks appeared in the Ca-alginate-MMT FTIR spectrum. The TGA analysis showed an improvement in the thermal stability of the Ca-alginate-MMT beads compared with the alginate beads alone. SEM analysis revealed a homogeneous distribution of the MMT particles throughout the alginate matrix. T. harzianum UPM40 was successfully encapsulated in the Ca-alginate-MMT beads. Storage analysis of the encapsulated T. harzianum UPM40 showed that the low storage temperature of 5°C resulted in significantly (p < 0.05) better storage compared with room temperature (30°C).

  16. Efficacy of acidic pretreatment for the saccharification and fermentation of alginate from brown macroalgae.

    PubMed

    Wang, Damao; Yun, Eun Ju; Kim, Sooah; Kim, Do Hyoung; Seo, Nari; An, Hyun Joo; Kim, Jae-Han; Cheong, Nam Yong; Kim, Kyoung Heon

    2016-06-01

    This study was performed to evaluate the effectiveness of acidic pretreatment in increasing the enzymatic digestibility of alginate from brown macroalgae. Pretreatment with 1 % (w/v) sulfuric acid at 120 °C for 30 min produced oligosaccharides, mannuronic acid, and guluronic acid. Enzymatic saccharification of pretreated alginate by alginate lyases produced 52.2 % of the theoretical maximal sugar yield, which was only 7.5 % higher than the sugar yield obtained with unpretreated alginate. Mass spectrometric analyses of products of the two reactions revealed that acidic pretreatment and enzymatic saccharification produced saturated monomers (i.e., mannuronic and guluronic acid) with saturated oligosaccharides and unsaturated monomers (i.e., 4-deoxy-L-erythro-5-hexoseulose uronic acid; DEH), respectively. While DEH is further metabolized by microorganisms, mannuronic acid and guluronic acid are not metabolizable. Because of the poor efficacy in increasing enzymatic digestibility and owing to the formation of non-fermentable saturated monomers, acidic pretreatment cannot be recommended for enzymatic saccharification and fermentation of alginate.

  17. Alginate-based hybrid aerogel microparticles for mucosal drug delivery.

    PubMed

    Gonçalves, V S S; Gurikov, P; Poejo, J; Matias, A A; Heinrich, S; Duarte, C M M; Smirnova, I

    2016-10-01

    The application of biopolymer aerogels as drug delivery systems (DDS) has gained increased interest during the last decade since these structures have large surface area and accessible pores allowing for high drug loadings. Being biocompatible, biodegradable and presenting low toxicity, polysaccharide-based aerogels are an attractive carrier to be applied in pharmaceutical industry. Moreover, some polysaccharides (e.g. alginate and chitosan) present mucoadhesive properties, an important feature for mucosal drug delivery. This feature allows to extend the contact of DDS with biological membranes, thereby increasing the absorption of drugs through the mucosa. Alginate-based hybrid aerogels in the form of microparticles (<50μm) were investigated in this work as carriers for mucosal administration of drugs. Low methoxyl pectin and κ-carrageenan were co-gelled with alginate and further dried with supercritical CO2 (sc-CO2). Spherical mesoporous aerogel microparticles were obtained for alginate, hybrid alginate/pectin and alginate/κ-carrageenan aerogels, presenting high specific surface area (370-548m(2)g(-1)) and mucoadhesive properties. The microparticles were loaded with ketoprofen via adsorption from its solution in sc-CO2, and with quercetin via supercritical anti-solvent precipitation. Loading of ketoprofen was in the range between 17 and 22wt% whereas quercetin demonstrated loadings of 3.1-5.4wt%. Both the drugs were present in amorphous state. Loading procedure allowed the preservation of antioxidant activity of quercetin. Release of both drugs from alginate/κ-carrageenan aerogel was slightly faster compared to alginate/pectin. The results indicate that alginate-based aerogel microparticles can be viewed as promising matrices for mucosal drug delivery applications.

  18. Immobilization of Cd in river sediments by sodium alginate modified nanoscale zero-valent iron: Impact on enzyme activities and microbial community diversity.

    PubMed

    Huang, Danlian; Xue, Wenjing; Zeng, Guangming; Wan, Jia; Chen, Guomin; Huang, Chao; Zhang, Chen; Cheng, Min; Xu, Piao

    2016-12-01

    This paper investigated how sodium alginate (SA)-modified nanoscale zero-valent iron (NZVI), play a constructive role in the remediation of cadmium (Cd) contaminated river sediments. The changes of the fraction of Cd, enzyme activities (urease, catalase, dehydrogenase) and bacterial community structures with the treatment by SNZVI were observed. The sequential extraction experiments demonstrated that most mobile fractions of Cd were transformed into residues (the maximum residual percentage of Cd increases from 15.49% to 57.28% after 30 days of incubation at 0.1 wt% SA), with the decrease of bioavailability of Cd. Exclusive of dehydrogenase, the activities of the other two enzymes tested were enhanced with the increase of incubation time, which indicated that dehydrogenase might be inhibited by ferric ions formed from SNZVI whereas no obvious inhibition was found for other enzymes. Polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) analyses were used for the detection of microbial community changes, and the results showed that SNZVI and NZVI could increase bacterial taxa and improve bacterial abundance. All the experimental findings of this study provide new insights into the potential consequences of SNZVI treatments on the metal Cd immobilization in contaminated river sediments.

  19. Fabrication of granular activated carbons derived from spent coffee grounds by entrapment in calcium alginate beads for adsorption of acid orange 7 and methylene blue.

    PubMed

    Jung, Kyung-Won; Choi, Brian Hyun; Hwang, Min-Jin; Jeong, Tae-Un; Ahn, Kyu-Hong

    2016-11-01

    Biomass-based granular activated carbon was successfully prepared by entrapping activated carbon powder derived from spent coffee grounds into calcium-alginate beads (SCG-GAC) for the removal of acid orange 7 (AO7) and methylene blue (MB) from aqueous media. The dye adsorption process is highly pH-dependent and essentially independent of ionic effects. The adsorption kinetics was satisfactorily described by the pore diffusion model, which revealed that pore diffusion was the rate-limiting step during the adsorption process. The equilibrium isotherm and isosteric heat of adsorption indicate that SCG-GAC possesses an energetically heterogeneous surface and operates via endothermic process in nature. The maximum adsorption capacities of SCG-GAC for AO7 (pH 3.0) and MB (pH 11.0) adsorption were found to be 665.9 and 986.8mg/g at 30°C, respectively. Lastly, regeneration tests further confirmed that SCG-GAC has promising potential in its reusability, showing removal efficiency of more than 80% even after seven consecutive cycles.

  20. Staphylococcal Hyaluronate Lyase: Purification and Characterization Studies

    PubMed Central

    Abramson, Carl; Friedman, Herman

    1968-01-01

    Staphylococcal hyaluronate lyase (hyaluronidase) derived from a pathogenic strain of staphylococcus was purified by means of salt fractionation with ammonium sulfate and gel filtration through Sephadex G-100. Most of the enzyme activity from concentrated culture supernatant fluids of staphylococci was obtained in a fraction precipitated by 90 to 100% saturation with ammonium sulfate. A small amount of enzyme was also precipitated by 80 to 90% saturation with the salt. The hyaluronidase-rich fractions did not contain other staphylococcal enzymes, such as coagulase, protease, lipase, and staphylokinase. These enzymes were present in the original concentrates. Molecular sieving chromatography of the partially purified enzyme by filtration through Sephadex G-100 resulted in a further increase in specific enzyme activity. However, more than one active peak was obtained after gel filtration, thus suggesting that there may be more than one molecular form of the enzyme. Immunodiffusion in agar gel of the chromatographically purified enzyme fraction, with immune serum from rabbits injected with concentrated staphylococcal culture supernatant fluids, indicated that there was one major antigen. A similar antigen, giving reactions of identity with the purified material, was present in the original culture supernatant fluid. Images PMID:4301047

  1. Effects of extraction methods on molecular characteristics, antioxidant properties and immunomodulation of alginates from Sargassum angustifolium.

    PubMed

    Borazjani, Niloofar Jokar; Tabarsa, Mehdi; You, SangGuan; Rezaei, Masoud

    2017-03-29

    The relationship between molecular structure and bioactivity was evaluated for alginates obtained under different extraction methods (water, acid, alcalase and cellulase) from Sargassum angustifolium. The use of enzymes considerably reduced protein (from 14.58% to <0.4%) and polyphenol (from 16.0% to <1.7mg GA/g sample) contaminations of alginates compared to those of water and acid. The FT-IR spectrum revealed that extraction method did not affect the structure of the recovered alginates. The highest molecular weight (Mw) (557.1×10(3)g/mol) was found in acid treated alginate while the Mw of cellulase assistant alginate (356.2×10(3)g/mol) was the minimum. The SVg values varied from 2.79-5.17cm(3)/g revealing the loosed conformational structures of alcalase and cellulase assistant alginates. Alcalase assistant alginate stimulated RAW264.7 cells to release nitric oxide and inflammatory cytokines TNF-α, IL-1, IL-6, IL-10 and IL-12. Enzyme treated alginates showed maximum DPPH radical scavenging activity and reducing power. Therefore, the present results showed the determinant effect of pretreatment during the extraction process of alginate and the beneficial influence of enzymatic process when biological functions of alginates are of high interest in the industry.

  2. CyanoLyase: a database of phycobilin lyase sequences, motifs and functions

    PubMed Central

    Bretaudeau, Anthony; Coste, François; Humily, Florian; Garczarek, Laurence; Le Corguillé, Gildas; Six, Christophe; Ratin, Morgane; Collin, Olivier; Schluchter, Wendy M.; Partensky, Frédéric

    2013-01-01

    CyanoLyase (http://cyanolyase.genouest.org/) is a manually curated sequence and motif database of phycobilin lyases and related proteins. These enzymes catalyze the covalent ligation of chromophores (phycobilins) to specific binding sites of phycobiliproteins (PBPs). The latter constitute the building bricks of phycobilisomes, the major light-harvesting systems of cyanobacteria and red algae. Phycobilin lyases sequences are poorly annotated in public databases. Sequences included in CyanoLyase were retrieved from all available genomes of these organisms and a few others by similarity searches using biochemically characterized enzyme sequences and then classified into 3 clans and 32 families. Amino acid motifs were computed for each family using Protomata learner. CyanoLyase also includes BLAST and a novel pattern matching tool (Protomatch) that allow users to rapidly retrieve and annotate lyases from any new genome. In addition, it provides phylogenetic analyses of all phycobilin lyases families, describes their function, their presence/absence in all genomes of the database (phyletic profiles) and predicts the chromophorylation of PBPs in each strain. The site also includes a thorough bibliography about phycobilin lyases and genomes included in the database. This resource should be useful to scientists and companies interested in natural or artificial PBPs, which have a number of biotechnological applications, notably as fluorescent markers. PMID:23175607

  3. Inducible thermoalkalophilic polygalacturonate lyase from Thermomonospora fusca.

    PubMed Central

    Stutzenberger, F J

    1987-01-01

    A thermostable polygalacturonate lyase (PL; EC 4.2.2.2) was secreted by Thermomonospora fusca during stationary phase in pectin-mineral salts medium at 52 degrees C. Biosynthesis was induced by addition of pectic substances to cultures growing on glucose or cellulose but not cellobiose; the disaccharide repressed enzyme synthesis and triggered inactivation of enzyme previously secreted. The PL, purified to electrophoretic and serologic homogeneity, had a molecular size of 56 kilodaltons and an isoelectric point at pH 4.16. The amino acid composition closely resembled that of the major extracellular endoglucanases of the actinomycete. The enzyme had six cystine residues but no detectable sulfhydryl groups. It was inactivated by mild reducing agents and activated by oxygenation, indicating the necessity for disulfide bond maintenance. Temperature and pH optima for the PL reaction were 60 degrees C and 10.45, respectively. Calcium was essential for activity but not stability; calcium dependence curves were altered by low concentrations of toxic metals. The Km for pectin increased 30,000-fold as the percent esterification (methoxylation) of that substrate was increased from 0 to 60%. The size of the minimal susceptible site for PL attack on the pectin molecule was calculated as being equivalent to 10 unesterified residues, based on the correlation of Km values at various degrees of esterification with the percentage of cleavable bonds predicted by a random-number-generating computer program. Images PMID:3584069

  4. Structural and Biochemical Characterization of a Copper-Binding Mutant of the Organomercurial Lyase MerB: Insight into the Key Role of the Active Site Aspartic Acid in Hg-Carbon Bond Cleavage and Metal Binding Specificity.

    PubMed

    Wahba, Haytham M; Lecoq, Lauriane; Stevenson, Michael; Mansour, Ahmed; Cappadocia, Laurent; Lafrance-Vanasse, Julien; Wilkinson, Kevin J; Sygusch, Jurgen; Wilcox, Dean E; Omichinski, James G

    2016-02-23

    In bacterial resistance to mercury, the organomercurial lyase (MerB) plays a key role in the detoxification pathway through its ability to cleave Hg-carbon bonds. Two cysteines (C96 and C159; Escherichia coli MerB numbering) and an aspartic acid (D99) have been identified as the key catalytic residues, and these three residues are conserved in all but four known MerB variants, where the aspartic acid is replaced with a serine. To understand the role of the active site serine, we characterized the structure and metal binding properties of an E. coli MerB mutant with a serine substituted for D99 (MerB D99S) as well as one of the native MerB variants containing a serine residue in the active site (Bacillus megaterium MerB2). Surprisingly, the MerB D99S protein copurified with a bound metal that was determined to be Cu(II) from UV-vis absorption, inductively coupled plasma mass spectrometry, nuclear magnetic resonance, and electron paramagnetic resonance studies. X-ray structural studies revealed that the Cu(II) is bound to the active site cysteine residues of MerB D99S, but that it is displaced following the addition of either an organomercurial substrate or an ionic mercury product. In contrast, the B. megaterium MerB2 protein does not copurify with copper, but the structure of the B. megaterium MerB2-Hg complex is highly similar to the structure of the MerB D99S-Hg complexes. These results demonstrate that the active site aspartic acid is crucial for both the enzymatic activity and metal binding specificity of MerB proteins and suggest a possible functional relationship between MerB and its only known structural homologue, the copper-binding protein NosL.

  5. 21 CFR 184.1610 - Potassium alginate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Potassium alginate. 184.1610 Section 184.1610 Food... Specific Substances Affirmed as GRAS § 184.1610 Potassium alginate. (a) Potassium alginate (CAS Reg. No. 9005-36-1) is the potassium salt of alginic acid, a natural polyuronide constituent of certain...

  6. 21 CFR 184.1610 - Potassium alginate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Potassium alginate. 184.1610 Section 184.1610 Food... Specific Substances Affirmed as GRAS § 184.1610 Potassium alginate. (a) Potassium alginate (CAS Reg. No. 9005-36-1) is the potassium salt of alginic acid, a natural polyuronide constituent of certain...

  7. 21 CFR 184.1610 - Potassium alginate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Potassium alginate. 184.1610 Section 184.1610 Food... Specific Substances Affirmed as GRAS § 184.1610 Potassium alginate. (a) Potassium alginate (CAS Reg. No. 9005-36-1) is the potassium salt of alginic acid, a natural polyuronide constituent of certain...

  8. 21 CFR 184.1610 - Potassium alginate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Potassium alginate. 184.1610 Section 184.1610 Food... Specific Substances Affirmed as GRAS § 184.1610 Potassium alginate. (a) Potassium alginate (CAS Reg. No. 9005-36-1) is the potassium salt of alginic acid, a natural polyuronide constituent of certain...

  9. 21 CFR 184.1724 - Sodium alginate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Sodium alginate. 184.1724 Section 184.1724 Food... Specific Substances Affirmed as GRAS § 184.1724 Sodium alginate. (a) Sodium alginate (CAS Reg. No. 9005-38-3) is the sodium salt of alginic acid, a natural polyuronide constituent of certain brown...

  10. 21 CFR 184.1724 - Sodium alginate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Sodium alginate. 184.1724 Section 184.1724 Food... Specific Substances Affirmed as GRAS § 184.1724 Sodium alginate. (a) Sodium alginate (CAS Reg. No. 9005-38-3) is the sodium salt of alginic acid, a natural polyuronide constituent of certain brown...

  11. 21 CFR 184.1724 - Sodium alginate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Sodium alginate. 184.1724 Section 184.1724 Food... Specific Substances Affirmed as GRAS § 184.1724 Sodium alginate. (a) Sodium alginate (CAS Reg. No. 9005-38-3) is the sodium salt of alginic acid, a natural polyuronide constituent of certain brown...

  12. Cysteine S-conjugate β-lyases

    PubMed Central

    Cooper, Arthur J. L.; Krasnikov, Boris F.; Pinto, John T.; Bruschi, Sam A.

    2010-01-01

    Cysteine S-conjugate β-lyases are pyridoxal 5′-phosphate (PLP)-containing enzymes that catalyze the conversion of cysteine S-conjugates [RSCH2CH(NH3+)CO2−] and selenium Se-conjugates [RSeCH2CH(NH3+)CO2−] that contain a leaving group in the β position to pyruvate, ammonium and a sulfur-containing fragment (RSH) or selenium-containing fragment (RSeH), respectively. At least ten PLP enzymes catalyze β-elimination reactions with such cysteine S-conjugates. All are enzymes involved in amino acid metabolism that do not normally catalyze a β-lyase reaction, but catalyze a non-physiological β-lyase side reaction that depends on the electron-withdrawing properties of the –SR or –SeR moiety. In the case of the cysteine S-conjugates, if the eliminated RSH is stable the compound may be S-thiomethylated and excreted (thiomethyl shunt) or S-glucuronidated and harmlessly excreted [the possibility that RSeH compounds may be similarly metabolized has not been extensively studied]. If, however, RSH is chemically reactive the cysteine S-conjugate may be toxic as a result of the β-lyase reaction. The cysteine S-conjugate β-lyase pathway is of particular interest to toxicologists because it is involved in the bioactivation (toxification) of halogenated alkenes and certain drugs. PMID:20949433

  13. Enzyme immobilization in novel alginate-chitosan core-shell microcapsules.

    PubMed

    Taqieddin, Ehab; Amiji, Mansoor

    2004-05-01

    Alginate-chitosan core-shell microcapsules were prepared in order to develop a biocompatible matrix for enzyme immobilization, where the protein is retained either in a liquid or solid core and the shell allows permeability control over substrates and products. The permeability coefficients of different molecular weight compounds (vitamin B2, vitamin B12, and myoglobin) were determined through sodium tripolyphosphate (Na-TPP)-crosslinked chitosan membrane. The microcapsule core was formed by crosslinking sodium alginate with either calcium or barium ions. The crosslinked alginate core was uniformly coated with a chitosan layer and crosslinked with Na-TPP. In the case of calcium alginate, the phosphate ions of Na-TPP were able to extract the calcium ions from alginate and liquefy the core. A model enzyme, beta-galactosidase, was immobilized in the alginate core and the catalytic activity was measured with o-nitrophenyl-beta-D-galactopyranoside (ONPG). Change in the activity of free and immobilized enzyme was determined at three different temperatures. Na-TPP crosslinked chitosan membranes were found to be permeable to solutes of up to 17,000Da molecular weight. The enzyme loading efficiency was higher in the barium alginate core (100%) as compared to the calcium alginate core (60%). The rate of ONPG conversion to o-nitrophenol was faster in the case of calcium alginate-chitosan microcapsules as compared to barium alginate-chitosan microcapsules. Barium alginate-chitosan microcapsules, however, did improve the stability of the enzyme at 37 degrees C relative to calcium alginate-chitosan microcapsules or free enzyme. This study illustrates a new method of enzyme immobilization for biotechnology applications using liquid or solid core and shell microcapsule technology.

  14. Binding sequences for RdgB, a DNA damage-responsive transcriptional activator, and temperature-dependent expression of bacteriocin and pectin lyase genes in Pectobacterium carotovorum subsp. carotovorum.

    PubMed

    Yamada, Kazuteru; Kaneko, Jun; Kamio, Yoshiyuki; Itoh, Yoshifumi

    2008-10-01

    Pectobacterium carotovorum subsp. carotovorum strain Er simultaneously produces the phage tail-like bacteriocin carotovoricin (Ctv) and pectin lyase (Pnl) in response to DNA-damaging agents. The regulatory protein RdgB of the Mor/C family of proteins activates transcription of pnl through binding to the promoter. However, the optimal temperature for the synthesis of Ctv (23 degrees C) differs from that for synthesis of Pnl (30 degrees C), raising the question of whether RdgB directly activates ctv transcription. Here we report that RdgB directly regulates Ctv synthesis. Gel mobility shift assays demonstrated RdgB binding to the P(0), P(1), and P(2) promoters of the ctv operons, and DNase I footprinting determined RdgB-binding sequences (RdgB boxes) on these and on the pnl promoters. The RdgB box of the pnl promoter included a perfect 7-bp inverted repeat with high binding affinity to the regulator (K(d) [dissociation constant] = 150 nM). In contrast, RdgB boxes of the ctv promoters contained an imperfect inverted repeat with two or three mismatches that consequently reduced binding affinity (K(d) = 250 to 350 nM). Transcription of the rdgB and ctv genes was about doubled at 23 degrees C compared with that at 30 degrees C. In contrast, the amount of pnl transcription tripled at 30 degrees C. Thus, the inverse synthesis of Ctv and Pnl as a function of temperature is apparently controlled at the transcriptional level, and reduced rdgB expression at 30 degrees C obviously affected transcription from the ctv promoters with low-affinity RdgB boxes. Pathogenicity toward potato tubers was reduced in an rdgB knockout mutant, suggesting that the RdgAB system contributes to the pathogenicity of this bacterium, probably by activating pnl expression.

  15. Alginate/bacterial cellulose nanocomposite beads prepared using Gluconacetobacter xylinus and their application in lipase immobilization.

    PubMed

    Kim, Ji Hyun; Park, Saerom; Kim, Hyungsup; Kim, Hyung Joo; Yang, Yung-Hun; Kim, Yong Hwan; Jung, Sang-Kyu; Kan, Eunsung; Lee, Sang Hyun

    2017-02-10

    Alginate/bacterial cellulose nanocomposite beads, with well-controlled size and regular spherical shapes, were prepared in a simple manner by entrapping Gluconacetobacter xylinus in barium alginate hydrogel beads, followed by cultivation of the entrapped cells in culture media with a low sodium ion concentration. The entire surface of the alginate hydrogel beads containing the cells was covered with cellulose fibers (∼30nm) after 36h of cultivation. The cellulose crystallinity index of the alginate/bacterial cellulose beads was 0.7, which was slightly lower than that of bacterial cellulose prepared by cultivating dispersed cells. The water vapor sorption capacity of the alginate/bacterial cellulose beads increased significantly from 0.07 to 38.00 (g/g dry bead) as cultivation time increased. These results clearly indicate that alginate/bacterial cellulose beads have a much higher surface area, crystallinity, and water-holding capacity than alginate beads. The immobilization of lipase on the surface of the nanocomposite beads was also investigated as a potential application of this system. The activity and specific activity of lipase immobilized on alginate/bacterial cellulose beads were 2.6- and 3.8-fold higher, respectively, than that of lipase immobilized on cellulose beads. The alginate/bacterial cellulose nanocomposite beads prepared in this study have several potential applications in the biocatalytic, biomedical, and pharmaceutical fields because of their biocompatibility, biodegradability, high crystallinity, and large surface area.

  16. Understanding Alginate Gel Development for Bioclogging and Biogeophysical Experiments

    NASA Astrophysics Data System (ADS)

    Brown, I.; Atekwana, E. A.; Abdel Aal, G. Z.; Atekwana, E. A.; Sarkisova, S.; Patrauchan, M.

    2012-12-01

    Bioremediation strategies to mitigate the transport of heavy metals and radionuclides in subsurface sediments have largely targeted to increase the mobility and/or solubility of these compounds by the stimulation of biogeochemical activity of the metal- and sulfate-reducing bacteria. The latter secrete and/or release out diverse biochemical molecule including, first of all, organic acids and biopolymers such as alginic acid, proteins and DNA. Alginate gel is one of the major components determining the structure of biofilm which causes clogging in porous media. Biopolymers composing biofilm having, at least, two main functions: to be a scaffold for a microbial biofilm, and to regulate the exchange of metabolites and ions between an environment and bacterial cells. Additionally, the accumulation of biopolymers and a matured biofilm within porous media was shown to contribute to a detectable biogeophysical signal, spectral induced polarization (SIP), in particular. Our objective is to understand the role of different biofilm components on the SIP response as the latter has been proposed as a non-invasive tool to monitor biofilm development and rate of clogging in the subsurface. Understanding the process of alginate gel development may aid in the understanding of the fate and transport of mineralized heavy metals and radionuclides in contaminated soils. Here we describe the reciprocal relationship between environmental chemistry and alginate gel development. Commercial (Sigma) alginic acid (AA) was used as a substratum for the preparation of a model gel. AA was solubilized by adjusting solutions with pH up to 4 with 0.1 NaOH. Both Ca(OH)2 or CaCl2 were used to initiate the gelation of alginate. pH, fluid conductivity, soluble Ca2+ concentration, and a yield of gelated alginate were monitored in both liquid and porous media after the interaction of calcium compounds with alginate. This study confirms the critical role of Ca2+ for alginate gelation, biofilm development

  17. Overexpression of Cystathionine γ-Lyase Suppresses Detrimental Effects of Spinocerebellar Ataxia Type 3

    PubMed Central

    Snijder, Pauline M; Baratashvili, Madina; Grzeschik, Nicola A; Leuvenink, Henri G D; Kuijpers, Lucas; Huitema, Sippie; Schaap, Onno; Giepmans, Ben N G; Kuipers, Jeroen; Miljkovic, Jan Lj; Mitrovic, Aleksandra; Bos, Eelke M; Szabó, Csaba; Kampinga, Harm H; Dijkers, Pascale F; den Dunnen, Wilfred F A; Filipovic, Milos R; van Goor, Harry; Sibon, Ody C M

    2015-01-01

    Spinocerebellar ataxia type 3 (SCA3) is a polyglutamine (polyQ) disorder caused by a CAG repeat expansion in the ataxin-3 (ATXN3) gene resulting in toxic protein aggregation. Inflammation and oxidative stress are considered secondary factors contributing to the progression of this neurodegenerative disease. There is no cure that halts or reverses the progressive neurodegeneration of SCA3. Here we show that overexpression of cystathionine γ-lyase, a central enzyme in cysteine metabolism, is protective in a Drosophila model for SCA3. SCA3 flies show eye degeneration, increased oxidative stress, insoluble protein aggregates, reduced levels of protein persulfidation and increased activation of the innate immune response. Overexpression of Drosophila cystathionine γ-lyase restores protein persulfidation, decreases oxidative stress, dampens the immune response and improves SCA3-associated tissue degeneration. Levels of insoluble protein aggregates are not altered; therefore, the data implicate a modifying role of cystathionine γ-lyase in ameliorating the downstream consequence of protein aggregation leading to protection against SCA3-induced tissue degeneration. The cystathionine γ-lyase expression is decreased in affected brain tissue of SCA3 patients, suggesting that enhancers of cystathionine γ-lyase expression or activity are attractive candidates for future therapies. PMID:26467707

  18. Effect of alginate composition on profile release and characteristics of chitosan-alginate microparticles loaded with mangosteen extract

    NASA Astrophysics Data System (ADS)

    Mulia, Kamarza; Halimah, Nur; Krisanti, Elsa

    2017-03-01

    Preparation of mangostin-loaded chitosan-alginate microparticles, chemical and physical characterization of the particles, and mangostin release profiles, are described herein. Mangostin rich fraction was obtained from Garcinia mangostana L. pericarp by extraction followed by fractionation. Mangostin-loaded chitosan-alginate microparticles were prepared by ionic gelation method using tripolyphosphate as the linking agent and various concentration of alginate. Mangostin was effectively loaded in all microparticle formulations, resulting in ˜97% encapsulation efficiencies. The loading of mangostin and the in-vitro release profiles in simulated gastrointestinal fluids were affected by the chitosan to alginate ratios used in the preparation of the microparticles. Increased alginate concentration resulted in lowered release of mangostin from microparticles immersed in simulated gastric fluid (pH 1.2) up to two hours. Low release of mangostin in acidic fluid but high release in simulated colon fluid, indicated that the chitosan-alginate microparticles are prospective carrier for extended release of active compound in gastrointestinal system.

  19. Host-Pathogen interactions. 25. Endopolygalacturonic acid lyase from Erwinia carotovora elicits phytoalexin accumulation by releasing plant cell wall fragments

    SciTech Connect

    Davis, K.R.; Lyon, G.D.; Darvill, A.G.; Albersheim, P.

    1984-01-01

    Heat-labile elicitors of phytoalexin accumulation in soybeans (Glycine max L. Merr. cv Wayne) were detected in culture filtrates of Erwinia carotovora grown on a defined medium containing citrus pectin as the sole carbon source. The heat-labile elicitors were highly purified by cation-exchange chromatography on a CM-Sephadex (C-50) column, followed by agarose-affinity chromatography on a Bio-Gel A-0.5m gel filtration column. The heat-labile elicitor activity co-purified with two ..cap alpha..-1,4-endopolygalacturonic acid lyases (EC 4 x 2 x 2 x 2). Endopolygalacturonic acid lyase activity appeared to be necessary for elicitor activity because heat-inactivated enzyme preparations did not elicit phytoalexins. The purified endopolygalacturonic acid lyases elicited pterocarpan phytoalexins at microbial-inhibitory concentrations in the soybean-cotyledon bioassay when applied at a concentration of 55 nanograms per milliliter (1 x 10/sup -9/ molar). One of these lyases released heat-stable elicitors from soybean cell walls, citrus pectin, and sodium polypectate. The heat-stable elicitor-active material solubilized from soybean cell walls by the lyase was composed of at least 90% (w/v) uronosyl residues. These results demonstrate that endopolygalacturonic acid lyase elicits phytoalexin accumulation by releasing fragments from pectic polysaccharides in plant cell walls.

  20. Alginate: properties and biomedical applications

    PubMed Central

    Lee, Kuen Yong; Mooney, David J.

    2011-01-01

    Alginate is a biomaterial that has found numerous applications in biomedical science and engineering due to its favorable properties, including biocompatibility and ease of gelation. Alginate hydrogels have been particularly attractive in wound healing, drug delivery, and tissue engineering applications to date, as these gels retain structural similarity to the extracellular matrices in tissues and can be manipulated to play several critical roles. This review will provide a comprehensive overview of general properties of alginate and its hydrogels, their biomedical applications, and suggest new perspectives for future studies with these polymers. PMID:22125349

  1. Improving the quality of Laminaria japonica-based diet for Apostichopus japonicus through degradation of its algin content with Bacillus amyloliquefaciens WB1.

    PubMed

    Wang, Xitao; Wang, Lili; Che, Jian; Li, Zhen; Zhang, Jiancheng; Li, Xiaoyu; Hu, Weiqing; Xu, Yongping

    2015-07-01

    Laminaria japonica feedstuff is used as a substitute for Sargassum thunbergii in the small-scale culturing of Apostichopus japonicus (sea cucumber) because of its abundant sources and low price in China. However, the difficulty associated with the degradation of algin by A. japonicus and, hence, its utilization have limited the practical value of L. japonica feedstuff in sea cucumber farming. In this study, A. japonicus individuals were fed with L. japonica feedstuff pretreated, via fermentation with the algin-degrading bacterial strain, Bacillus amyloliquefaciens WB1, and their growth performance, nonspecific immune responses, and resistance against Vibrio infection were then determined over a 60-day period. Growth performance of these individuals was similar to those fed with a commercial feedstuff made from S. thunbergii (mean weight gain of 5.79 versus 5.69 g on day 60), but was significantly (P < 0.05) increased compared to those fed with untreated L. japonica feedstuff (mean weight gain of 1.31 g). At the same time, they also showed significantly higher levels of amylase, protease, and alginate lyase activities than the other groups. These individuals and those fed with the commercial feedstuff or heat-inactivated but B. amyloliquefaciens WB1-treated L. japonicas feedstuff showed enhanced levels of activities for the immune enzymes nitric oxide synthase, lysozyme, peroxidase, and acid phosphatase, compared to those fed with nontreated L. japonica feedstuff. Furthermore, A. japonicus individuals fed with B. amyloliquefaciens WB1-treated L. japonica feedstuff exhibited greater resistance to disease following Vibrio splendidus challenge, as shown by the much lower cumulative symptom (10 %) compared to the rest, which showed as much as 73 % in the case of individuals fed with the untreated L. japonica feedstuff. Analysis of their intestinal tract revealed a much lower number of total Vibrio sp. These results demonstrated that L. japonica in which the algin

  2. Oxygen-dependent regulation of c-di-GMP synthesis by SadC controls alginate production in Pseudomonas aeruginosa.

    PubMed

    Schmidt, Annika; Hammerbacher, Anna Silke; Bastian, Mike; Nieken, Karen Jule; Klockgether, Jens; Merighi, Massimo; Lapouge, Karine; Poschgan, Claudia; Kölle, Julia; Acharya, K Ravi; Ulrich, Martina; Tümmler, Burkhard; Unden, Gottfried; Kaever, Volkhard; Lory, Stephen; Haas, Dieter; Schwarz, Sandra; Döring, Gerd

    2016-10-01

    Pseudomonas aeruginosa produces increased levels of alginate in response to oxygen-deprived conditions. The regulatory pathway(s) that links oxygen limitation to increased synthesis of alginate has remained elusive. In the present study, using immunofluorescence microscopy, we show that anaerobiosis-induced alginate production by planktonic PAO1 requires the diguanylate cyclase (DGC) SadC, previously identified as a regulator of surface-associated lifestyles. Furthermore, we found that the gene products of PA4330 and PA4331, located in a predicted operon with sadC, have a major impact on alginate production: deletion of PA4330 (odaA, for oxygen-dependent alginate synthesis activator) caused an alginate production defect under anaerobic conditions, whereas a PA4331 (odaI, for oxygen-dependent alginate synthesis inhibitor) deletion mutant produced alginate also in the presence of oxygen, which would normally inhibit alginate synthesis. Based on their sequence, OdaA and OdaI have predicted hydratase and dioxygenase reductase activities, respectively. Enzymatic assays using purified protein showed that unlike OdaA, which did not significantly affect DGC activity of SadC, OdaI inhibited c-di-GMP production by SadC. Our data indicate that SadC, OdaA and OdaI are components of a novel response pathway of P. aeruginosa that regulates alginate synthesis in an oxygen-dependent manner.

  3. Encapsulation optimization of lemon balm antioxidants in calcium alginate hydrogels.

    PubMed

    Najafi-Soulari, Samira; Shekarchizadeh, Hajar; Kadivar, Mahdi

    2016-11-01

    Calcium alginate hydrogel beads were used to encapsulate lemon balm extract. Chitosan layer was used to investigate the effect of hydrogel coating. To determine the interactions of antioxidant compounds of extract with encapsulation materials and its stability, microstructure of hydrogel beads was thoroughly monitored using scanning electron microscopy and Fourier transform infrared (FTIR). Total polyphenols content and antiradical activity of lemon balm extract were also evaluated before and after encapsulation. Three significant parameters (lemon balm extract, sodium alginate, and calcium chloride concentrations) were optimized by response surface methodology to obtain maximum encapsulation efficiency. The FTIR spectra showed no interactions between extract and polymers as there were no new band in spectra of alginate hydrogel after encapsulation of active compounds of lemon balm extract. The antioxidant activity of lemon balm extract did not change after encapsulation. Therefore, it was found that alginate is a suitable material for encapsulation of natural antioxidants. Sodium alginate solution concentration, 1.84%, lemon balm extract concentration, 0.4%, and calcium chloride concentration, 0.2% was determined to be the optimum condition to reach maximum encapsulation efficiency.

  4. Characterization of a mutant Bacillus subtilis adenylosuccinate lyase equivalent to a mutant enzyme found in human adenylosuccinate lyase deficiency: asparagine 276 plays an important structural role.

    PubMed

    Palenchar, Jennifer Brosius; Colman, Roberta F

    2003-02-25

    Adenylosuccinate lyase, an enzyme catalyzing two reactions in purine biosynthesis (the cleavage of either adenylosuccinate or succinylaminoimidazole carboxamide ribotide), has been implicated in a human disease arising from point mutations in the gene encoding the enzyme. Asn(276) of Bacillus subtilis adenylosuccinate lyase, a residue corresponding to the location of a human enzyme mutation, was replaced by Cys, Ser, Ala, Arg, and Glu. The mutant enzymes exhibit decreased V(max) values (2-400-fold lower) for both substrates compared to the wild-type enzyme and some changes in the pH dependence of V(max) but no loss in affinity for adenylosuccinate. Circular dichroism reveals no difference in secondary structure between the wild-type and mutant enzymes. We show here for the first time that wild-type adenylosuccinate lyase exhibits a protein concentration dependence of molecular weight, secondary structure, and specific activity. An equilibrium constant between the dimer and tetramer was measured by light scattering for the wild-type and mutant enzymes. The equilibrium is somewhat shifted toward the tetramer in the mutant enzymes. The major difference between the wild-type and mutant enzymes appears to be in quaternary structure, with many mutant enzymes exhibiting marked thermal instability relative to the wild-type enzyme. We propose that mutations at position 276 result in structurally impaired adenylosuccinate lyases which are assembled into defective tetramers.

  5. Stereospecificity of isotopic exchange of C-α-protons of glycine catalyzed by three PLP-dependent lyases: the unusual case of tyrosine phenol-lyase.

    PubMed

    Koulikova, Vitalia V; Zakomirdina, Lyudmila N; Gogoleva, Olga I; Tsvetikova, Marina A; Morozova, Elena A; Komissarov, Vsevolod V; Tkachev, Yaroslav V; Timofeev, Vladimir P; Demidkina, Tatyana V; Faleev, Nicolai G

    2011-11-01

    A comparative study of the kinetics and stereospecificity of isotopic exchange of the pro-2R- and pro-2S protons of glycine in (2)H(2)O under the action of tyrosine phenol-lyase (TPL), tryptophan indole-lyase (TIL) and methionine γ-lyase (MGL) was undertaken. The kinetics of exchange was monitored using both (1)H- and (13)C-NMR. In the three compared lyases the stereospecificities of the main reactions with natural substrates dictate orthogonal orientation of the pro-2R proton of glycine with respect to the cofactor pyridoxal 5'-phosphate (PLP) plane. Consequently, according to Dunathan's postulate with all the three enzymes pro-2R proton should exchange faster than does the pro-2S one. In fact the found ratios of 2R:2S reactivities are 1:20 for TPL, 108:1 for TIL, and 1,440:1 for MGL. Thus, TPL displays an unprecedented inversion of stereospecificity. A probable mechanism of the observed phenomenon is suggested, which is based on the X-ray data for the quinonoid intermediate, formed in the reaction of TPL with L-alanine. The mechanism implies different conformational changes in the active site upon binding of glycine and alanine. These changes can lead to relative stabilization of either the neutral amino group, accepting the α-proton, or the respective ammonium group, which is formed after the proton abstraction.

  6. Tissue and method specificities of phenylalanine ammonia-lyase assay.

    PubMed

    Kováčik, Jozef; Klejdus, Bořivoj

    2012-09-01

    A large number of studies have estimated phenylalanine ammonia-lyase (PAL) activity because it strongly reacts to various stimuli. Activity of this enzyme has been assayed mainly by means of spectrophotometry, but the precision of this method is poorly known. We compared assays of PAL activity using spectrophotometry and high performance liquid chromatography (HPLC) in two species (Matricaria chamomilla and Arabidopsis thaliana). Additionally, copper-exposed M. chamomilla plants and buffer with additive were also tested. Our data indicate that spectrophotometry both overestimates (leaves of M. chamomilla) and underestimates (leaves and roots of A. thaliana) PAL activity in comparison with HPLC, suggesting interference of UV-absorbing metabolites. HPLC also showed more accurate detection of cinnamic acid in Cu-exposed chamomile roots. Addition of dithiothreitol to the extraction buffer enhanced PAL activity but reduced proteins, indicating an artificial negative effect. A comparison of PAL activity in selected species is also provided.

  7. Mechanically reinforced cell-laden scaffolds formed using alginate-based bioink printed onto the surface of a PCL/alginate mesh structure for regeneration of hard tissue.

    PubMed

    Kim, Yong Bok; Lee, Hyeongjin; Yang, Gi-Hoon; Choi, Chang Hyun; Lee, DaeWeon; Hwang, Heon; Jung, Won-Kyo; Yoon, Hyeon; Kim, Geun Hyung

    2016-01-01

    Cell-printing technology has provided a new paradigm for biofabrication, with potential to overcome several shortcomings of conventional scaffold-based tissue regeneration strategies via controlled delivery of various cell types in well-defined target regions. Here we describe a cell-printing method to obtain mechanically reinforced multi-layered cell-embedded scaffolds, formed of micron-scale poly(ε-caprolactone) (PCL)/alginate struts coated with alginate-based bioink. To compare the physical and cellular activities, we used a scaffold composed of pure alginate (without cells) coated PCL/alginate struts as a control. We systematically varied the ratio of alginate cross-linking agent, and determined the optimal cell-coating conditions to form the PCL/alginate struts. Following fabrication of the cell (MG63)-laden PCL/alginate scaffold, the bioactivity was evaluated in vitro. The laden cells exhibited a substantially more developed cytoskeleton compared with those on a control scaffold consisting of the same material composition. Based on these results, the printed cells exhibited a significantly more homogenous distribution within the scaffold compared with the control. Cell proliferation was determined via MTT assays at 1, 3, 7, and 14 days of culture, and the proliferation of the cell-printed scaffold was substantially in excess (∼2.4-fold) of that on the control. Furthermore, the osteogenic activity such as ALP was measured, and the cell-laden scaffold exhibited significantly greater activity (∼3.2-fold) compared with the control scaffold.

  8. Oxidized alginate hydrogels for bone morphogenetic protein-2 delivery in long bone defects.

    PubMed

    Priddy, Lauren B; Chaudhuri, Ovijit; Stevens, Hazel Y; Krishnan, Laxminarayanan; Uhrig, Brent A; Willett, Nick J; Guldberg, Robert E

    2014-10-01

    Autograft treatment of large bone defects and fracture non-unions is complicated by limited tissue availability and donor site morbidity. Polymeric biomaterials such as alginate hydrogels provide an attractive tissue engineering alternative due to their biocompatibility, injectability, and tunable degradation rates. Irradiated RGD-alginate hydrogels have been used to deliver proteins such as bone morphogenetic protein-2 (BMP-2), to promote bone regeneration and restoration of function in a critically sized rat femoral defect model. However, slow degradation of irradiated alginate hydrogels may impede integration and remodeling of the regenerated bone to its native architecture. Oxidation of alginate has been used to promote degradation of alginate matrices. The objective of this study was to evaluate the effects of alginate oxidation on BMP-2 release and bone regeneration. We hypothesized that oxidized-irradiated alginate hydrogels would elicit an accelerated release of BMP-2, but degrade faster in vivo, facilitating the formation of higher quality, more mature bone compared to irradiated alginate. Indeed, oxidation of irradiated alginate did accelerate in vitro BMP-2 release. Notably, the BMP-2 retained within both constructs was bioactive at 26days, as observed by induction of alkaline phosphatase activity and positive Alizarin Red S staining of MC3T3-E1 cells. From the in vivo study, robust bone regeneration was observed in both groups through 12weeks by radiography, micro-computed tomography analyses, and biomechanical testing. Bone mineral density was significantly greater for the oxidized-irradiated alginate group at 8weeks. Histological analyses of bone defects revealed enhanced degradation of oxidized-irradiated alginate and suggested the presence of more mature bone after 12weeks of healing.

  9. Gene Deletion Strategy To Examine the Involvement of the Two Chondroitin Lyases in Flavobacterium columnare Virulence

    PubMed Central

    Li, Nan; Qin, Ting; Zhang, Xiao Lin; Huang, Bei; Liu, Zhi Xin; Xie, Hai Xia; Zhang, Jin; McBride, Mark J.

    2015-01-01

    Flavobacterium columnare is an important bacterial pathogen of freshwater fish that causes high mortality of infected fish and heavy economic losses in aquaculture. The pathogenesis of this bacterium is poorly understood, in part due to the lack of efficient methods for genetic manipulation. In this study, a gene deletion strategy was developed and used to determine the relationship between the production of chondroitin lyases and virulence. The F. johnsoniae ompA promoter (PompA) was fused to sacB to construct a counterselectable marker for F. columnare. F. columnare carrying PompA-sacB failed to grow on media containing 10% sucrose. A suicide vector carrying PompA-sacB was constructed, and a gene deletion strategy was developed. Using this approach, the chondroitin lyase-encoding genes, cslA and cslB, were deleted. The ΔcslA and ΔcslB mutants were both partially deficient in digestion of chondroitin sulfate A, whereas a double mutant (ΔcslA ΔcslB) was completely deficient in chondroitin lyase activity. Cells of F. columnare wild-type strain G4 and of the chondroitin lyase-deficient ΔcslA ΔcslB mutant exhibited similar levels of virulence toward grass carp in single-strain infections. Coinfections, however, revealed a competitive advantage for the wild type over the chondroitin lyase mutant. The results indicate that chondroitin lyases are not essential virulence factors of F. columnare but may contribute to the ability of the pathogen to compete and cause disease in natural infections. The gene deletion method developed in this study may be employed to investigate the virulence factors of this bacterium and may have wide application in many other members of the phylum Bacteroidetes. PMID:26253667

  10. Gene deletion strategy to examine the involvement of the two chondroitin lyases in Flavobacterium columnare virulence.

    PubMed

    Li, Nan; Qin, Ting; Zhang, Xiao Lin; Huang, Bei; Liu, Zhi Xin; Xie, Hai Xia; Zhang, Jin; McBride, Mark J; Nie, Pin

    2015-11-01

    Flavobacterium columnare is an important bacterial pathogen of freshwater fish that causes high mortality of infected fish and heavy economic losses in aquaculture. The pathogenesis of this bacterium is poorly understood, in part due to the lack of efficient methods for genetic manipulation. In this study, a gene deletion strategy was developed and used to determine the relationship between the production of chondroitin lyases and virulence. The F. johnsoniae ompA promoter (PompA) was fused to sacB to construct a counterselectable marker for F. columnare. F. columnare carrying PompA-sacB failed to grow on media containing 10% sucrose. A suicide vector carrying PompA-sacB was constructed, and a gene deletion strategy was developed. Using this approach, the chondroitin lyase-encoding genes, cslA and cslB, were deleted. The ΔcslA and ΔcslB mutants were both partially deficient in digestion of chondroitin sulfate A, whereas a double mutant (ΔcslA ΔcslB) was completely deficient in chondroitin lyase activity. Cells of F. columnare wild-type strain G4 and of the chondroitin lyase-deficient ΔcslA ΔcslB mutant exhibited similar levels of virulence toward grass carp in single-strain infections. Coinfections, however, revealed a competitive advantage for the wild type over the chondroitin lyase mutant. The results indicate that chondroitin lyases are not essential virulence factors of F. columnare but may contribute to the ability of the pathogen to compete and cause disease in natural infections. The gene deletion method developed in this study may be employed to investigate the virulence factors of this bacterium and may have wide application in many other members of the phylum Bacteroidetes.

  11. Biochemical Characterization and Overexpression of an Endo-rhamnogalacturonan Lyase from Penicillium chrysogenum.

    PubMed

    Iwai, Marin; Yamada, Hiroyuki; Ikemoto, Takeshi; Matsumoto, Shotaro; Fujiwara, Daisuke; Takenaka, Shigeo; Sakamoto, Tatsuji

    2015-06-01

    Rhamnogalacturonan lyase (PcRGL4A) was purified from the culture supernatant of Penicillium chrysogenum 31B. PcRGL4A optimal activity occurred between pH 7-8 and at 40 °C. Conserved Domain Search analysis identified PcRGL4A as a member of Polysaccharide Lyase family 4. PcRGL4A contains two conserved catalytic and four conserved substrate-binding residues as determined by X-ray crystallography of the Aspergillus aculeatus RG lyase. Recombinant PcRGL4A (rPcRGL4A) expressed in Escherichia coli demonstrated specific activity against rhamnogalacturonan (RG) but not homogalacturonan. Analysis of the RG reaction products by high-performance anion-exchange chromatography revealed that rPcRGL4A cleaved the substrate in an endo-manner and that the major final product was an RG tetrasaccharide with 4-deoxy-4,5-unsaturated galacturonic acid at the nonreducing end. Based on these results, PcRGL4A was classified as an endo-acting RG lyase (EC 4.2.2.23). Divalent cations were not essential for the enzymatic activity of rPcRGL4A, but addition of calcium ions to the reaction mixture increased enzymatic activity. rPcRGL4A demonstrated a preference for RG lacking galactose decoration.

  12. Structural (betaalpha)8 TIM barrel model of 3-hydroxy-3-methylglutaryl-coenzyme A lyase.

    PubMed

    Casals, Núria; Gómez-Puertas, Paulino; Pié, Juan; Mir, Cecilia; Roca, Ramón; Puisac, Beatriz; Aledo, Rosa; Clotet, Josep; Menao, Sebastián; Serra, Dolors; Asins, Guillermina; Till, Jacqueline; Elias-Jones, Alun C; Cresto, Juan C; Chamoles, Nestor A; Abdenur, Jose E; Mayatepek, Ertan; Besley, Guy; Valencia, Alfonso; Hegardt, Fausto G

    2003-08-01

    This study describes three novel homozygous missense mutations (S75R, S201Y, and D204N) in the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase gene, which caused 3-hydroxy-3-methylglutaric aciduria in patients from Germany, England, and Argentina. Expression studies in Escherichia coli show that S75R and S201Y substitutions completely abolished the HMG-CoA lyase activity, whereas D204N reduced catalytic efficiency to 6.6% of the wild type. We also propose a three-dimensional model for human HMG-CoA lyase containing a (betaalpha)8 (TIM) barrel structure. The model is supported by the similarity with analogous TIM barrel structures of functionally related proteins, by the localization of catalytic amino acids at the active site, and by the coincidence between the shape of the substrate (HMG-CoA) and the predicted inner cavity. The three novel mutations explain the lack of HMG-CoA lyase activity on the basis of the proposed structure: in S75R and S201Y because the new amino acid residues occlude the substrate cavity, and in D204N because the mutation alters the electrochemical environment of the active site. We also report the localization of all missense mutations reported to date and show that these mutations are located in the beta-sheets around the substrate cavity.

  13. Isocitrate lyase and the glyoxylate cycle. Progress report, February 16, 1992--February 15, 1993

    SciTech Connect

    McFadden, B.A.

    1992-12-31

    This progress report describes efforts directed at the active-site modification of isocitrate lyase (icl) of Escherichia coli. Studies are reported that describe the results of several amino acid substitutions gained by directed mutagenesis of the icl gene. Preliminary studies are also related in cloning, sequencing and expression of icl of watermelon.

  14. Phototriggered formation and repair of DNA containing a site-specific single strand break of the type produced by ionizing radiation or AP lyase activity.

    PubMed

    Zhang, K; Taylor, J S

    2001-01-09

    DNA strand breaks are produced by a variety of agents and processes such as ionizing radiation, xenobiotics, oxidative metabolism, and enzymatic processing of DNA base damage. One of the major types of strand breaks produced by these processes is a single nucleotide gap terminating in 5'- and 3'-phosphates. Previously, we had developed a method for sequence-specifically producing such phosphate-terminated strand breaks in an oligodeoxynucleotide by way of two photochemically activated (caged) building blocks placed in tandem. We now report the design and synthesis of a single caged building block consisting of 1,3-(2-nitrophenyl)-1,3-propanediol, for producing phosphate-terminated strand breaks, and its use producing such a break at a specific site in a double-stranded circular DNA vector. To produce the site-specific break in a duplex vector, a primer containing the caged single strand break was extended opposite the single strand form of a circular DNA vector followed by enzymatic ligation and purification. The single strand break could then be formed in quantitative yield by irradiation of the vector with 365 nm light. In contrast to a previous study, it was found that the strand break can be repaired by Escherichia coli DNA polymerase I and E. coli DNA ligase alone, though less efficiently than in the presence of the 3'-phosphate processing enzyme E. coli endonuclease IV. Repair in the absence of endonuclease IV could be attributed to hydrolysis of the 3'-phosphate in the presence of dNTP and to a lesser extent to exonucleolytic removal of the 3'-phosphate-bearing terminal nucleotide by way of the 3' --> 5' exonuclease activity of polymerase I. This work demonstrates that specialized 3'-end processing enzymes such as endonuclease IV or exonuclease III are not absolutely required for repair of phosphate-terminated gaps. In addition to preparing single strand breaks, the caged building block described should also be useful for preparing double strand breaks and

  15. Alginate Nanoparticles as a Promising Adjuvant and Vaccine Delivery System

    PubMed Central

    Sarei, F.; Dounighi, N. Mohammadpour; Zolfagharian, H.; Khaki, P.; Bidhendi, S. Moradi

    2013-01-01

    During last decades, diphtheria has remained as a serious disease that still outbreaks and can occur worldwide. Recently, new vaccine delivery systems have been developed by using the biodegradable and biocompatible polymers such as alginate. Alginate nanoparticles as a carrier with adjuvant and prolong release properties that enhance the immunogenicity of vaccines. In this study diphtheria toxoid loaded nanoparticles were prepared by ionic gelation technique and characterized with respect to size, zeta potential, morphology, encapsulation efficiency, release profile, and immunogenicity. Appropriate parameters (calcium chloride and sodium alginate concentration, homogenization rate and homogenization time) redounded to the formation of suitable nanoparticles with a mean diameter of 70±0.5 nm. The loading studies of the nanoparticles resulted in high loading capacities (>90%) and subsequent release studies showed prolong profile. The stability and antigenicity of toxoid were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and ouchterlony test and proved that the encapsulation process did not affect the antigenic integrity and activity. Guinea pigs immunized with the diphtheria toxoid-loaded alginate nanoparticles showed highest humoral immune response than conventional vaccine. It is concluded that, with regard to the desirable properties of nanoparticles and high immunogenicity, alginate nanoparticles could be considered as a new promising vaccine delivery and adjuvant system. PMID:24302799

  16. Microencapsulation of bioactives in cross-linked alginate matrices by spray drying.

    PubMed

    Santa-Maria, Monica; Scher, Herbert; Jeoh, Tina

    2012-01-01

    Microencapsulation of biomolecules, cells and chemicals is widely used in the food and pharmaceutical industries to improve stability, delivery and to control the release of encapsulated moieties. Among encapsulation matrices, alginate is preferred due to its low cost, biodegradability and biocompatibility. Current methods for producing stable alginate gels involve dropping alginate suspensions into divalent cation solutions. This procedure is difficult to scale-up and produces undesirably large alginate beads. In our novel encapsulation method, alginate gelation occurs during spray drying upon volatilisation of a base and rapid release of otherwise unavailable calcium ions. The resulting particles, with median particle sizes in the range 15-120 µm, are insoluble in solution. Cellulase and hemicellulase activities encapsulated by this method were not compromised during spray drying and remained stable over prolonged storage. The procedure described here offers a one-step alternative to other encapsulation methods that are costly and difficult to scale-up.

  17. Functions and cellular localization of cysteine desulfurase and selenocysteine lyase in Trypanosoma brucei.

    PubMed

    Poliak, Pavel; Van Hoewyk, Douglas; Oborník, Miroslav; Zíková, Alena; Stuart, Kenneth D; Tachezy, Jan; Pilon, Marinus; Lukes, Julius

    2010-01-01

    Nfs-like proteins have cysteine desulfurase (CysD) activity, which removes sulfur (S) from cysteine, and provides S for iron-sulfur cluster assembly and the thiolation of tRNAs. These proteins also have selenocysteine lyase activity in vitro, and cleave selenocysteine into alanine and elemental selenium (Se). It was shown previously that the Nfs-like protein called Nfs from the parasitic protist Trypanosoma brucei is a genuine CysD. A second Nfs-like protein is encoded in the nuclear genome of T. brucei. We called this protein selenocysteine lyase (SCL) because phylogenetic analysis reveals that it is monophyletic with known eukaryotic selenocysteine lyases. The Nfs protein is located in the mitochondrion, whereas the SCL protein seems to be present in the nucleus and cytoplasm. Unexpectedly, downregulation of either Nfs or SCL protein leads to a dramatic decrease in both CysD and selenocysteine lyase activities concurrently in the mitochondrion and the cytosolic fractions. Because loss of Nfs causes a growth phenotype but loss of SCL does not, we propose that Nfs can fully complement SCL, whereas SCL can only partially replace Nfs under our growth conditions.

  18. Deficiency of sphingosine-1-phosphate lyase impairs lysosomal metabolism of the amyloid precursor protein.

    PubMed

    Karaca, Ilker; Tamboli, Irfan Y; Glebov, Konstantin; Richter, Josefine; Fell, Lisa H; Grimm, Marcus O; Haupenthal, Viola J; Hartmann, Tobias; Gräler, Markus H; van Echten-Deckert, Gerhild; Walter, Jochen

    2014-06-13

    Progressive accumulation of the amyloid β protein in extracellular plaques is a neuropathological hallmark of Alzheimer disease. Amyloid β is generated during sequential cleavage of the amyloid precursor protein (APP) by β- and γ-secretases. In addition to the proteolytic processing by secretases, APP is also metabolized by lysosomal proteases. Here, we show that accumulation of intracellular sphingosine-1-phosphate (S1P) impairs the metabolism of APP. Cells lacking functional S1P-lyase, which degrades intracellular S1P, strongly accumulate full-length APP and its potentially amyloidogenic C-terminal fragments (CTFs) as compared with cells expressing the functional enzyme. By cell biological and biochemical methods, we demonstrate that intracellular inhibition of S1P-lyase impairs the degradation of APP and CTFs in lysosomal compartments and also decreases the activity of γ-secretase. Interestingly, the strong accumulation of APP and CTFs in S1P-lyase-deficient cells was reversed by selective mobilization of Ca(2+) from the endoplasmic reticulum or lysosomes. Intracellular accumulation of S1P also impairs maturation of cathepsin D and degradation of Lamp-2, indicating a general impairment of lysosomal activity. Together, these data demonstrate that S1P-lyase plays a critical role in the regulation of lysosomal activity and the metabolism of APP.

  19. Characterization of C-S lyase from Lactobacillus delbrueckii subsp. bulgaricus ATCC BAA-365 and its potential role in food flavour applications.

    PubMed

    Allegrini, Alessandra; Astegno, Alessandra; La Verde, Valentina; Dominici, Paola

    2016-12-21

    Volatile thiols have substantial impact on the aroma of many beverages and foods. Thus, the control of their formation, which has been linked to C-S lyase enzymatic activities, is of great significance in industrial applications involving food flavours. Herein, we have carried out a spectroscopic and functional characterization of a putative pyridoxal 5'-phosphate (PLP)-dependent C-S lyase from the lactic acid bacterium Lactobacillus delbrueckii subsp. bulgaricus ATCC BAA-365 (LDB C-S lyase). Recombinant LDB C-S lyase exists as a tetramer in solution and shows spectral properties of enzymes containing PLP as cofactor. The enzyme has a broad substrate specificity toward sulphur-containing amino acids with aminoethyl-L-cysteine and L-cystine being the most effective substrates over L-cysteine and L-cystathionine. Notably, the protein also reveals cysteine-S-conjugate β-lyase activity in vitro, and is able to cleave a cysteinylated substrate precursor into the corresponding flavour-contributing thiol, with a catalytic efficiency higher than L-cystathionine. Contrary to similar enzymes of other lactic acid bacteria however, LDB C-S lyase is not capable of α,γ-elimination activity towards L-methionine to produce methanethiol, which is a significant compound in flavour development. Based on our results, future developments can be expected regarding the flavour-forming potential of Lactobacillus C-S lyase and its use in enhancing food flavours.

  20. 21 CFR 582.7187 - Calcium alginate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Calcium alginate. 582.7187 Section 582.7187 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Calcium alginate. (a) Product. Calcium alginate. (b) Conditions of use. This substance is...

  1. 21 CFR 582.7133 - Ammonium alginate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Ammonium alginate. 582.7133 Section 582.7133 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Ammonium alginate. (a) Product. Ammonium alginate. (b) Conditions of use. This substance is...

  2. 21 CFR 582.7610 - Potassium alginate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Potassium alginate. 582.7610 Section 582.7610 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Potassium alginate. (a) Product. Potassium alginate. (b) Conditions of use. This substance is...

  3. 21 CFR 582.7610 - Potassium alginate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Potassium alginate. 582.7610 Section 582.7610 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Potassium alginate. (a) Product. Potassium alginate. (b) Conditions of use. This substance is...

  4. 21 CFR 582.7610 - Potassium alginate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Potassium alginate. 582.7610 Section 582.7610 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Potassium alginate. (a) Product. Potassium alginate. (b) Conditions of use. This substance is...

  5. 21 CFR 582.7610 - Potassium alginate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Potassium alginate. 582.7610 Section 582.7610 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Potassium alginate. (a) Product. Potassium alginate. (b) Conditions of use. This substance is...

  6. 21 CFR 582.7610 - Potassium alginate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Potassium alginate. 582.7610 Section 582.7610 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Potassium alginate. (a) Product. Potassium alginate. (b) Conditions of use. This substance is...

  7. 21 CFR 582.7724 - Sodium alginate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Sodium alginate. 582.7724 Section 582.7724 Food... DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Stabilizers § 582.7724 Sodium alginate. (a) Product. Sodium alginate. (b) Conditions of use. This substance is generally recognized...

  8. 21 CFR 582.7724 - Sodium alginate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Sodium alginate. 582.7724 Section 582.7724 Food... DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Stabilizers § 582.7724 Sodium alginate. (a) Product. Sodium alginate. (b) Conditions of use. This substance is generally recognized...

  9. 21 CFR 582.7724 - Sodium alginate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Sodium alginate. 582.7724 Section 582.7724 Food... DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Stabilizers § 582.7724 Sodium alginate. (a) Product. Sodium alginate. (b) Conditions of use. This substance is generally recognized...

  10. 21 CFR 582.7724 - Sodium alginate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Sodium alginate. 582.7724 Section 582.7724 Food... DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Stabilizers § 582.7724 Sodium alginate. (a) Product. Sodium alginate. (b) Conditions of use. This substance is generally recognized...

  11. 21 CFR 582.7724 - Sodium alginate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Sodium alginate. 582.7724 Section 582.7724 Food... DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Stabilizers § 582.7724 Sodium alginate. (a) Product. Sodium alginate. (b) Conditions of use. This substance is generally recognized...

  12. 21 CFR 184.1011 - Alginic acid.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Alginic acid. 184.1011 Section 184.1011 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN... Substances Affirmed as GRAS § 184.1011 Alginic acid. (a) Alginic acid is a colloidal,...

  13. 21 CFR 184.1011 - Alginic acid.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Alginic acid. 184.1011 Section 184.1011 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DIRECT FOOD....1011 Alginic acid. (a) Alginic acid is a colloidal, hydrophilic polysaccharide obtained from...

  14. 21 CFR 184.1011 - Alginic acid.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Alginic acid. 184.1011 Section 184.1011 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN... Substances Affirmed as GRAS § 184.1011 Alginic acid. (a) Alginic acid is a colloidal,...

  15. 21 CFR 184.1011 - Alginic acid.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Alginic acid. 184.1011 Section 184.1011 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN... Substances Affirmed as GRAS § 184.1011 Alginic acid. (a) Alginic acid is a colloidal,...

  16. 21 CFR 184.1011 - Alginic acid.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Alginic acid. 184.1011 Section 184.1011 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN... Substances Affirmed as GRAS § 184.1011 Alginic acid. (a) Alginic acid is a colloidal,...

  17. Efficient functionalization of alginate biomaterials.

    PubMed

    Dalheim, Marianne Ø; Vanacker, Julie; Najmi, Maryam A; Aachmann, Finn L; Strand, Berit L; Christensen, Bjørn E

    2016-02-01

    Peptide coupled alginates obtained by chemical functionalization of alginates are commonly used as scaffold materials for cells in regenerative medicine and tissue engineering. We here present an alternative to the commonly used carbodiimide chemistry, using partial periodate oxidation followed by reductive amination. High and precise degrees of substitution were obtained with high reproducibility, and without formation of by-products. A protocol was established using l-Tyrosine methyl ester as a model compound and the non-toxic pic-BH3 as the reducing agent. DOSY was used to indirectly verify covalent binding and the structure of the product was further elucidated using NMR spectroscopy. The coupling efficiency was to some extent dependent on alginate composition, being most efficient on mannuronan. Three different bioactive peptide sequences (GRGDYP, GRGDSP and KHIFSDDSSE) were coupled to 8% periodate oxidized alginate resulting in degrees of substitution between 3.9 and 6.9%. Cell adhesion studies of mouse myoblasts (C2C12) and human dental stem cells (RP89) to gels containing various amounts of GRGDSP coupled alginate demonstrated the bioactivity of the material where RP89 cells needed higher peptide concentrations to adhere.

  18. Nonlinear elasticity of alginate gels

    NASA Astrophysics Data System (ADS)

    Hashemnejad, Seyed Meysam; Kundu, Santanu

    Alginate is a naturally occurring anionic polysaccharide extracted from brown algae. Because of biocompatibility, low toxicity, and simple gelation process, alginate gels are used in biomedical and food applications. Here, we report the rheological behavior of ionically crosslinked alginate gels, which are obtained by in situ gelation of alginates with calcium salts, in between two parallel plates of a rheometer. Strain stiffening behavior was captured using large amplitude oscillatory shear (LAOS) experiments. In addition, negative normal stress was observed for these gels, which has not been reported earlier for any polysaccharide networks. The magnitude of negative normal stress increases with applied strain and can exceed that of the shear stress at large strain. Rheological results fitted with a constitutive model that considers both stretching and bending of chains indicate that nonlinearity is likely related to the stretching of the chains between the crosslink junctions. The results provide an improved understanding of the deformation mechanism of ionically crosslinked alginate gel and the results will be important in developing synthetic extracellular matrix (ECM) from these materials.

  19. Crystal structure of Proteus vulgaris chondroitin sulfate ABC lyase I at 1.9A resolution.

    PubMed

    Huang, Weijun; Lunin, Vladimir V; Li, Yunge; Suzuki, Sakaru; Sugiura, Nobuo; Miyazono, Hirofumi; Cygler, Miroslaw

    2003-05-02

    Chondroitin Sulfate ABC lyase I from Proteus vulgaris is an endolytic, broad-specificity glycosaminoglycan lyase, which degrades chondroitin, chondroitin-4-sulfate, dermatan sulfate, chondroitin-6-sulfate, and hyaluronan by beta-elimination of 1,4-hexosaminidic bond to unsaturated disaccharides and tetrasaccharides. Its structure revealed three domains. The N-terminal domain has a fold similar to that of carbohydrate-binding domains of xylanases and some lectins, the middle and C-terminal domains are similar to the structures of the two-domain chondroitin lyase AC and bacterial hyaluronidases. Although the middle domain shows a very low level of sequence identity with the catalytic domains of chondroitinase AC and hyaluronidase, the residues implicated in catalysis of the latter enzymes are present in chondroitinase ABC I. The substrate-binding site in chondroitinase ABC I is in a wide-open cleft, consistent with the endolytic action pattern of this enzyme. The tryptophan residues crucial for substrate binding in chondroitinase AC and hyaluronidases are lacking in chondroitinase ABC I. The structure of chondroitinase ABC I provides a framework for probing specific functions of active-site residues for understanding the remarkably broad specificity of this enzyme and perhaps engineering a desired specificity. The electron density map showed clearly that the deposited DNA sequence for residues 495-530 of chondroitin ABC lyase I, the segment containing two putative active-site residues, contains a frame-shift error resulting in an incorrectly translated amino acid sequence.

  20. Overexpression of the plg1 gene encoding pectin lyase in Penicillium griseoroseum.

    PubMed

    Cardoso, Patrícia Gomes; Ribeiro, João Batista; Teixeira, Janaina Aparecida; de Queiroz, Marisa Vieira; de Araújo, Elza Fernandes

    2008-03-01

    The pectin lyase (PL) is an industrially important enzyme since it is used for maceration and clarification in the process of fruit juice production in food industries. In order to increase the yields of pectin lyase we cloned the plg1 (pectin lyase 1) from Penicillium griseoroseum gene under the control of the strong constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) and the terminator region of the tryptophan synthetase (trpC) gene from Aspergillus nidulans (plasmid pAN52-Plg1) and transformed this construct into the P. griseoroseum strain PG63. One of the pAN52-Plg1 multi-copy transformants (strain 105) grown in culture medium containing glucose or sugar cane juice showed PL activities of 4,804 or 5,202 U ml(-1) respectively, which represented 57- and 132-fold increases. In addition, the apparent specific activity of PL produced by this strain was much higher than the one observed for a commercial pectinase preparation. Evaluation of the extracellular proteins in the culture supernatant of strain 105 by SDS-PAGE showed the presence of a clear and strong band of approximately 40 kDa that probably corresponds to PL. The enzyme yields reported here demonstrate that the system we developed is able to express pectin lyase at levels comparable to, or exceeding, previously reported data.

  1. Purification and properties of malyl-coenzyme A lyase from Pseudomonas AM1

    PubMed Central

    Hacking, A. J.; Quayle, J. R.

    1974-01-01

    1. Malyl-CoA lyase was purified 20-fold from extracts of methanol-grown Pseudomonas AM1. 2. Preparations of the enzyme were essentially homogeneous by electrophoretic and ultracentrifugal criteria. 3. Malyl-CoA lyase has a molecular weight of 190000 determined from sedimentation-equilibrium data. 4. Within the range of compounds tested, malyl-CoA lyase is specific for (2S)-4-malyl-CoA or glyoxylate and acetyl-CoA or propionyl-CoA. 5. A bivalent cation is essential for activity, Mg2+ or Co2+ being most effective. 6. Malyl-CoA lyase is inhibited by (2R)-4-malyl-CoA and by some buffers, but thiol-group inhibitors are without effect. 7. Optimal activity was recorded at pH7.8. 8. An equilibrium constant of 4.7×10−4m was determined for the malyl-CoA cleavage reaction. 9. The Michaelis constants for the enzyme are: 4-malyl-CoA, 6.6×10−5m; acetyl-CoA, 1.5×10−5m; glyoxylate, 1.7×10−3m; Mg2+, 1.2×10−3m. ImagesFig. 1. PMID:4447618

  2. Pectin Lyase Production by a Penicillium italicum Strain

    PubMed Central

    Alaña, Aitor; Gabilondo, Ane; Hernando, Fernando; Moragues, Maria D.; Dominguez, Juan B.; Llama, Maria J.; Serra, Juan L.

    1989-01-01

    Growth and concomitant production of an extracellular pectin lyase (PL) [poly(methoxylgalactosiduronate) endolyase; EC 4.2.2.10] were investigated in a group of 16 fungi grown in liquid medium containing pectin as a supplementary carbon source. Culture filtrates of both Penicillium italicum (CECT 2294) and P. expansum (CECT 2275) showed the highest PL activity and contained polygalacturonase but not pectinesterase activity. The effect of the inoculum size, the carbon source (sucrose and glucose syrup), and the presence of pectin on the production of PL by P. italicum was studied. The presence of 2.6 mM glycerophosphate in the culture medium enhanced the appearance of PL but was not inhibitory for the in vitro activity. However, glycerol inhibited the enzyme nearly 50% at such a concentration. PMID:16347954

  3. Dodecenyl succinylated alginate as a novel material for encapsulation and hyperactivation of lipases.

    PubMed

    Falkeborg, Mia; Paitaid, Pattarapon; Shu, Allen Ndonwi; Pérez, Bianca; Guo, Zheng

    2015-11-20

    Alginate was modified with dodecenyl succinic anhydride (SAC12) in an aqueous reaction medium at neutral pH. The highest degree of succinylation (33.9±3.5%) was obtained after 4h at 30°C, using four mole SAC12 per mol alginate monomer. Alginate was modified with succinic anhydride (SAC0) for comparison, and the structures and thermal properties of alg-SAC0 and alg-SAC12 were evaluated using FTIR, (1)H NMR, and DSC. Calcium-hydrogel beads were formed from native and modified alginates, in which lipases were encapsulated with a load of averagely 76μg lipase per mg alginate, irrespective of the type of alginate. Lipases with a "lid", which usually are dependent on interfacial activation, showed a 3-fold increase in specific activity toward water-soluble substrates when encapsulated in alg-SAC12, compared to the free lipase. Such hyperactivation was not observed for lipases independent of interfacial activation, or for lipases encapsulated in native alginate or alg-SAC0 hydrogels.

  4. Identification and characterization of a methionine γ-lyase in the calicheamicin biosynthetic cluster of Micromonospora echinospora.

    PubMed

    Song, Haigang; Xu, Ri; Guo, Zhihong

    2015-01-02

    CalE6 is a previously uncharacterized protein involved in the biosynthesis of calicheamicins in Micromonospora echinospora. It is a pyridoxal-5'-phosphate-dependent enzyme and exhibits high sequence homology to cystathionine γ-lyases and cystathionine γ-synthases. However, it was found to be active towards methionine and to convert this amino acid into α-ketobutyrate, ammonium, and methanethiol. The crystal structure of the cofactor-bound holoenzyme was resolved at 2.0 Å; it contains two active site residues, Gly105 and Val322, specific for methionine γ-lyases. Modeling of methionine into the active site allows identification of the active site residues responsible for substrate recognition and catalysis. These findings support that CalE6 is a putative methionine γ-lyase producing methanethiol as a building block in biosynthesis of calicheamicins.

  5. Proteolytic regulation of alginate overproduction in Pseudomonas aeruginosa.

    PubMed

    Damron, F Heath; Goldberg, Joanna B

    2012-05-01

    Pseudomonas aeruginosa, a Gram-negative bacterium, is a significant opportunistic pathogen associated with skin and soft tissue infections, nosocomial pneumonia and sepsis. In addition, it can chronically colonize the lungs of cystic fibrosis (CF) patients. Overproduction of the exopolysaccharide called alginate provides P. aeruginosa with a selective advantage and facilitates survival in the CF lung. The in vitro phenotype of alginate overproduction observed on solid culture media is referred to as mucoid. Expression of the alginate machinery and biosynthetic enzymes are controlled by the extracytoplasmic sigma factor, σ(22) (AlgU/T). The key negative regulator of both σ(22) activity and the mucoid phenotype is the cognate anti-sigma factor MucA. MucA sequesters σ(22) to the inner membrane inhibiting the sigma factor's transcriptional activity. The well-studied mechanism for transition to the mucoid phenotype is mutation of mucA, leading to loss of MucA function and therefore activation of σ(22) . Recently, regulated intramembrane proteolysis (RIP) has been recognized as a mechanism whereby proteolysis of the anti-sigma factor MucA leads to active σ(22) allowing P. aeruginosa to respond to environmental stress conditions by overproduction of alginate. The goal of this review is to illuminate the pathways leading to RIP that have been identified and proposed.

  6. 3D Cell Culture in Alginate Hydrogels

    PubMed Central

    Andersen, Therese; Auk-Emblem, Pia; Dornish, Michael

    2015-01-01

    This review compiles information regarding the use of alginate, and in particular alginate hydrogels, in culturing cells in 3D. Knowledge of alginate chemical structure and functionality are shown to be important parameters in design of alginate-based matrices for cell culture. Gel elasticity as well as hydrogel stability can be impacted by the type of alginate used, its concentration, the choice of gelation technique (ionic or covalent), and divalent cation chosen as the gel inducing ion. The use of peptide-coupled alginate can control cell–matrix interactions. Gelation of alginate with concomitant immobilization of cells can take various forms. Droplets or beads have been utilized since the 1980s for immobilizing cells. Newer matrices such as macroporous scaffolds are now entering the 3D cell culture product market. Finally, delayed gelling, injectable, alginate systems show utility in the translation of in vitro cell culture to in vivo tissue engineering applications. Alginate has a history and a future in 3D cell culture. Historically, cells were encapsulated in alginate droplets cross-linked with calcium for the development of artificial organs. Now, several commercial products based on alginate are being used as 3D cell culture systems that also demonstrate the possibility of replacing or regenerating tissue. PMID:27600217

  7. Alginate Production by Plant-Pathogenic Pseudomonads

    PubMed Central

    Fett, William F.; Osman, Stanley F.; Fishman, Marshall L.; Siebles, T. S.

    1986-01-01

    Eighteen plant-pathogenic and three non-plant-pathogenic pseudomonads were tested for the ability to produce alginic acid as an exopolysaccharide in vitro. Alginate production was demonstrated for 10 of 13 fluorescent plant-pathogenic pseudomonads tested with glucose or gluconate as the carbon source, but not for all 5 nonfluorescent plant pathogens and all 3 non-plant pathogens tested. With sucrose as the carbon source, some strains produced alginate while others produced both polyfructan (levan) and alginate. Alginates ranged from <1 to 28% guluronic acid, were acetylated, and had number-average molecular weights of 11.3 × 103 to 47.1 × 103. Polyfructans and alginates were not elicitors of the soybean phytoalexin glyceollin when applied to wounded cotyledon surfaces and did not induce prolonged water soaking of soybean leaf tissues. All or most pseudomonads in rRNA-DNA homology group I may be capable of synthesizing alginate as an exopolysaccharide. PMID:16347146

  8. Entrapment of cross-linked cellulase colloids in alginate beads for hydrolysis of cellulose.

    PubMed

    Nguyen, Le Truc; Lau, Yun Song; Yang, Kun-Lin

    2016-09-01

    Entrapment of enzymes in calcium alginate beads is a popular enzyme immobilization method. However, leaching of immobilized enzymes from the alginate beads is a common problem because enzyme molecules are much smaller than the pore size of alginate beads (∼200nm). To address this issue, we employ a millifluidic reactor to prepare cross-linked cellulase aggregate (XCA) colloids with a uniform size (∼300nm). Subsequently, these colloids are immobilized in calcium alginate beads as biocatalysts to hydrolyze cellulose substrates. By using fluorescent microscopy, we conclude that the immobilized XCA colloids distribute uniformly inside the beads and do not leach out from the beads after long-term incubation. Meanwhile, the pore size of the alginate beads is big enough for the cellulose substrates and fibers to diffuse into the beads for hydrolysis. For example, palm oil fiber and microcrystalline cellulose can be hydrolyzed within 48h and release reducing sugar concentrations up to 2.48±0.08g/l and 4.99±0.09g/l, respectively. Moreover, after 10 cycles of hydrolysis, 96.4% of the XCA colloids remain inside the alginate beads and retain 67% of the original activity. In contrast, free cellulase immobilized in the alginate beads loses its activity completely after 10 cycles. The strategy can also be used to prepare other types of cross-linked enzyme aggregates with high uniformity.

  9. Alginate Hydrogels Coated with Chitosan for Wound Dressing

    PubMed Central

    Straccia, Maria Cristina; Gomez d’Ayala, Giovanna; Romano, Ida; Oliva, Adriana; Laurienzo, Paola

    2015-01-01

    In this work, a coating of chitosan onto alginate hydrogels was realized using the water-soluble hydrochloride form of chitosan (CH-Cl), with the dual purpose of imparting antibacterial activity and delaying the release of hydrophilic molecules from the alginate matrix. Alginate hydrogels with different calcium contents were prepared by the internal setting method and coated by immersion in a CH-Cl solution. Structural analysis by cryo-scanning electron microscopy was carried out to highlight morphological alterations due to the coating layer. Tests in vitro with human mesenchymal stromal cells (MSC) were assessed to check the absence of toxicity of CH-Cl. Swelling, stability in physiological solution and release characteristics using rhodamine B as the hydrophilic model drug were compared to those of relative uncoated hydrogels. Finally, antibacterial activity against Escherichia coli was tested. Results show that alginate hydrogels coated with chitosan hydrochloride described here can be proposed as a novel medicated dressing by associating intrinsic antimicrobial activity with improved sustained release characteristics. PMID:25969981

  10. Alginate hydrogels coated with chitosan for wound dressing.

    PubMed

    Straccia, Maria Cristina; d'Ayala, Giovanna Gomez; Romano, Ida; Oliva, Adriana; Laurienzo, Paola

    2015-05-11

    In this work, a coating of chitosan onto alginate hydrogels was realized using the water-soluble hydrochloride form of chitosan (CH-Cl), with the dual purpose of imparting antibacterial activity and delaying the release of hydrophilic molecules from the alginate matrix. Alginate hydrogels with different calcium contents were prepared by the internal setting method and coated by immersion in a CH-Cl solution. Structural analysis by cryo-scanning electron microscopy was carried out to highlight morphological alterations due to the coating layer. Tests in vitro with human mesenchymal stromal cells (MSC) were assessed to check the absence of toxicity of CH-Cl. Swelling, stability in physiological solution and release characteristics using rhodamine B as the hydrophilic model drug were compared to those of relative uncoated hydrogels. Finally, antibacterial activity against Escherichia coli was tested. Results show that alginate hydrogels coated with chitosan hydrochloride described here can be proposed as a novel medicated dressing by associating intrinsic antimicrobial activity with improved sustained release characteristics.

  11. Characterization of holmium loaded alginate microspheres for multimodality imaging and therapeutic applications.

    PubMed

    Zielhuis, S W; Seppenwoolde, J H; Bakker, C J G; Jahnz, U; Zonnenberg, B A; van het Schip, A D; Hennink, W E; Nijsen, J F W

    2007-09-15

    In this paper the preparation and characterization of holmium-loaded alginate microspheres is described. The rapid development of medical imaging techniques offers new opportunities for the visualisation of (drug-loaded) microparticles. Therefore, suitable imaging agents have to be incorporated into these particles. For this reason, the element holmium was used in this study in order to utilize its unique imaging characteristics. The paramagnetic behaviour of this element allows visualisation with MRI and holmium can also be neutron-activated resulting in the emission of gamma-radiation, allowing visualisation with gamma cameras, and beta-radiation, suitable for therapeutic applications. Almost monodisperse alginate microspheres were obtained by JetCutter technology where alginate droplets of a uniform size were hardened in an aqueous holmium chloride solution. Ho(3+) binds via electrostatic interactions to the carboxylate groups of the alginate polymer and as a result alginate microspheres loaded with holmium were obtained. The microspheres had a mean size of 159 microm and a holmium loading of 1.3 +/- 0.1% (w/w) (corresponding with a holmium content based on dry alginate of 18.3 +/- 0.3% (w/w)). The binding capacity of the alginate polymer for Ho(3+) (expressed in molar amounts) is equal to that for Ca(2+), which is commonly used for the hardening of alginate. This indicates that Ho(3+) has the same binding affinity as Ca(2+). In line herewith, dynamic mechanical analyses demonstrated that alginate gels hardened with Ca(2+) or Ho(3+) had similar viscoelastic properties. The MRI relaxation properties of the microspheres were determined by a MRI phantom experiment, demonstrating a strong R(2)* effect of the particles. Alginate microspheres could also be labelled with radioactive holmium by adding holmium-166 to alginate microspheres, previously hardened with calcium (labelling efficiency 96%). The labelled microspheres had a high radiochemical stability (94% after

  12. Characterization and Immunological Evaluation of Low-Molecular- Weight Alginate Derivatives.

    PubMed

    Xu, Xu; Bi, Decheng; Wan, Min

    2016-01-01

    Alginate is a naturally occurring acidic linear polysaccharide obtained from marine brown seaweed. Low molecular weight structurally diverse derivatives and oligosaccharides derived from alginate have shown various tremendous biological and pharmacological activities. It has been demonstrated that immuno-inflammation is involved in many prevalent human diseases, such as cancer, severe infection and neurodegeneration. Given the activities of marine natural products in the regulation of immune responses, increasing efforts are being made toward the development of lowmolecular- weight natural compounds that aid in the prevention and treatment of immune- and inflammatory-related diseases. In this review, we describe the development of chemical modification and molecular depolymerization methods that modify the physicochemical and biological characteristics of alginate. Additionally, current progress in research on immuno-inflammatory, anti-neurodegenerative and anti-tumor activities of alginate derivatives is highlighted.

  13. Quantification of alginate by aggregation induced by calcium ions and fluorescent polycations.

    PubMed

    Zheng, Hewen; Korendovych, Ivan V; Luk, Yan-Yeung

    2016-01-01

    For quantification of polysaccharides, including heparins and alginates, the commonly used carbazole assay involves hydrolysis of the polysaccharide to form a mixture of UV-active dye conjugate products. Here, we describe two efficient detection and quantification methods that make use of the negative charges of the alginate polymer and do not involve degradation of the targeted polysaccharide. The first method utilizes calcium ions to induce formation of hydrogel-like aggregates with alginate polymer; the aggregates can be quantified readily by staining with a crystal violet dye. This method does not require purification of alginate from the culture medium and can measure the large amount of alginate that is produced by a mucoid Pseudomonas aeruginosa culture. The second method employs polycations tethering a fluorescent dye to form suspension aggregates with the alginate polyanion. Encasing the fluorescent dye in the aggregates provides an increased scattering intensity with a sensitivity comparable to that of the conventional carbazole assay. Both approaches provide efficient methods for monitoring alginate production by mucoid P. aeruginosa.

  14. Pneumococcal galactose catabolism is controlled by multiple regulators acting on pyruvate formate lyase

    PubMed Central

    Al-Bayati, Firas A. Y.; Kahya, Hasan F. H.; Damianou, Andreas; Shafeeq, Sulman; Kuipers, Oscar P.; Andrew, Peter W.; Yesilkaya, Hasan

    2017-01-01

    Catabolism of galactose by Streptococcus pneumoniae alters the microbe’s metabolism from homolactic to mixed acid fermentation, and this shift is linked to the microbe’s virulence. However, the genetic basis of this switch is unknown. Pyruvate formate lyase (PFL) is a crucial enzyme for mixed acid fermentation. Functional PFL requires the activities of two enzymes: pyruvate formate lyase activating enzyme (coded by pflA) and pyruvate formate lyase (coded by pflB). To understand the genetic basis of mixed acid fermentation, transcriptional regulation of pflA and pflB was studied. By microarray analysis of ΔpflB, differential regulation of several transcriptional regulators were identified, and CcpA, and GlnR’s role in active PFL synthesis was studied in detail as these regulators directly interact with the putative promoters of both pflA and pflB, their mutation attenuated pneumococcal growth, and their expression was induced on host-derived sugars, indicating that these regulators have a role in sugar metabolism, and multiple regulators are involved in active PFL synthesis. We also found that the influence of each regulator on pflA and pflB expression was distinct in terms of activation and repression, and environmental condition. These results show that active PFL synthesis is finely tuned, and feed-back inhibition and activation are involved. PMID:28240278

  15. Ammonia lyases and aminomutases as biocatalysts for the synthesis of α-amino and β-amino acids.

    PubMed

    Turner, Nicholas J

    2011-04-01

    Ammonia lyases catalyse the reversible addition of ammonia to cinnamic acid (1: R=H) and p-hydroxycinnamic (1: R=OH) to generate L-phenylalanine (2: R=H) and L-tyrosine (2: R=OH) respectively (Figure 1a). Both phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL) are widely distributed in plants, fungi and prokaryotes. Recently there has been interest in the use of these enzymes for the synthesis of a broader range of L-arylalanines. Aminomutases catalyse a related reaction, namely the interconversion of α-amino acids to β-amino acids (Figure 1b). In the case of L-phenylalanine, this reaction is catalysed by phenylalanine aminomutase (PAM) and proceeds stereospecifically via the intermediate cinnamic acid to generate β-Phe 3. Ammonia lyases and aminomutases are related in sequence and structure and share the same active site cofactor 4-methylideneimidazole-5-one (MIO). There is currently interest in the possibility of using these biocatalysts to prepare a wide range of enantiomerically pure l-configured α-amino and β-amino acids. Recent reviews have focused on the mechanism of these MIO containing enzymes. The aim of this review is to review recent progress in the application of ammonia lyase and aminomutase enzymes to prepare enantiomerically pure α-amino and β-amino acids.

  16. Non-Invasive Evaluation of Alginate/Poly-L-lysine/Alginate Microcapsules by Magnetic Resonance Microscopy

    PubMed Central

    Constantinidis, Ioannis; Grant, Samuel C.; Celper, Susanne; Gauffin-Holmberg, Isabel; Agering, Kristina; Oca-Cossio, Jose A.; Bui, Jonathan D.; Flint, Jeremy; Hamaty, Christine; Simpson, Nicholas E.; Blackband, Stephen J.

    2007-01-01

    In this report, we present data to demonstrate the utility of 1H MR microscopy to noninvasively examine alginate/poly-L-lysine/alginate (APA) microcapsules. Specifically, high-resolution images were used to visualize and quantify the poly-L-lysine (PLL) layer, and monitor temporal changes in the alginate gel microstructure during a month long in vitro culture. The thickness of the alginate/PLL layer was quantified to be 40.6±6.2 μm regardless of the alginate composition used to generate the beads or the time of alginate/PLL interaction (2, 6, or 20 minutes). However, there was a notable difference in the contrast of the PLL layer that depended upon the guluronic content of the alginate and the alginate/PLL interaction time. The T2 relaxation time and the apparent diffusion coefficient (ADC) of the alginate matrix were measured periodically throughout the month long culture period. Alginate beads generated with a high guluronic content alginate demonstrated a temporal decrease in T2 over the duration of the experiment, while ADC was unaffected. This decrease in T2 is attributed to a reorganization of the alginate microstructure due to periodic media exchanges that mimicked a regular feeding regiment for cultured cells. In beads coated with a PLL layer, this temporal decrease in T2 was less pronounced suggesting that the PLL layer helped maintain the integrity of the initial alginate microstructure. Conversely, alginate beads generated with a high mannuronic content alginate (with or without a PLL layer) did not display temporal changes in either T2 or ADC. This observation suggests that the microstructure of high mannuronic content alginate beads is less susceptible to culture conditions. PMID:17239948

  17. Heparin-like properties of sulfated alginates with defined sequences and sulfation degrees.

    PubMed

    Arlov, Øystein; Aachmann, Finn Lillelund; Sundan, Anders; Espevik, Terje; Skjåk-Bræk, Gudmund

    2014-07-14

    Sulfated glycosaminoglycans have a vast range of protein interactions relevant to the development of new biomaterials and pharmaceuticals, but their characterization and application is complicated mainly due to a high structural variability and the relative difficulty to isolate large quantities of structurally homogeneous samples. Functional and versatile analogues of heparin/heparan sulfate can potentially be created from sulfated alginates, which offer structure customizability through targeted enzymatic epimerization and precise tuning of the sulfation degree. Alginates are linear polysaccharides consisting of β-D-mannuronic acid (M) and α-L-guluronic acid (G), derived from brown algae and certain bacteria. The M/G ratio and distribution of blocks are critical parameters for the physical properties of alginates and can be modified in vitro using mannuronic-C5-epimerases to introduce sequence patterns not found in nature. Alginates with homogeneous sequences (poly-M, poly-MG, and poly-G) and similar molecular weights were chemically sulfated and structurally characterized by the use of NMR and elemental analysis. These sulfated alginates were shown to bind and displace HGF from the surface of myeloma cells in a manner similar to heparin. We observed dependence on the sulfation degree (DS) as well as variation in efficacy based on the alginate monosaccharide sequence, relating to relative flexibility and charge density in the polysaccharide chains. Co-incubation with human plasma showed complement compatibility of the alginates and lowering of soluble terminal complement complex levels by sulfated alginates. The sulfated polyalternating (poly-MG) alginate proved to be the most reproducible in terms of precise sulfation degrees and showed the greatest relative degree of complement inhibition and HGF interaction, maintaining high activity at low DS values.

  18. Kefiran-alginate gel microspheres for oral delivery of ciprofloxacin.

    PubMed

    Blandón, Lina M; Islan, German A; Castro, Guillermo R; Noseda, Miguel D; Thomaz-Soccol, Vanete; Soccol, Carlos R

    2016-09-01

    Ciprofloxacin is a broad-spectrum antibiotic associated with gastric and intestinal side effects after extended oral administration. Alginate is a biopolymer commonly employed in gel synthesis by ionotropic gelation, but unstable in the presence of biological metal-chelating compounds and/or under dried conditions. Kefiran is a microbial biopolymer able to form gels with the advantage of displaying antimicrobial activity. In the present study, kefiran-alginate gel microspheres were developed to encapsulate ciprofloxacin for antimicrobial controlled release and enhanced bactericidal effect against common pathogens. Scanning electron microscopy (SEM) analysis of the hybrid gel microspheres showed a spherical structure with a smoother surface compared to alginate gel matrices. In vitro release of ciprofloxacin from kefiran-alginate microspheres was less than 3.0% and 5.0% at pH 1.2 (stomach), and 5.0% and 25.0% at pH 7.4 (intestine) in 3 and 21h, respectively. Fourier transform infrared spectroscopy (FTIR) of ciprofloxacin-kefiran showed the displacement of typical bands of ciprofloxacin and kefiran, suggesting a cooperative interaction by hydrogen bridges between both molecules. Additionally, the thermal analysis of ciprofloxacin-kefiran showed a protective effect of the biopolymer against ciprofloxacin degradation at high temperatures. Finally, antimicrobial assays of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhymurium, and Staphylococcus aureus demonstrated the synergic effect between ciprofloxacin and kefiran against the tested microorganisms.

  19. Bacterial carbon-phosphorus lyase: products, rates, and regulation of phosphonic and phosphinic acid metabolism.

    PubMed Central

    Wackett, L P; Shames, S L; Venditti, C P; Walsh, C T

    1987-01-01

    Carbon-phosphorus bond cleavage activity, found in bacteria that utilize alkyl- and phenylphosphonic acids, has not yet been obtained in a cell-free system. Given this constraint, a systematic examination of in vivo C-P lyase activity has been conducted to develop insight into the C-P cleavage reaction. Six bacterial strains were obtained by enrichment culture, identified, and characterized with respect to their phosphonic acid substrate specificity. One isolate, Agrobacterium radiobacter, was shown to cleave the carbon-phosphorus bond of a wide range of substrates, including fosfomycin, glyphosate, and dialkyl phosphinic acids. Furthermore, this organism processed vinyl-, propenyl-, and propynylphosphonic acids, a previously uninvestigated group, to ethylene, propene, and propyne, respectively. A determination of product stoichiometries revealed that both C-P bonds of dimethylphosphinic acid are cleaved quantitatively to methane and, furthermore, that the extent of C-P bond cleavage correlated linearly with the specific growth rate for a range of substrates. The broad substrate specificity of Agrobacterium C-P lyase and the comprehensive characterization of the in vivo activity make this an attractive system for further biochemical and mechanistic experiments. In addition, the failure to observe the activity in a group of gram-positive bacteria holds open the possibility that a periplasmic component may be required for in vivo expression of C-P lyase activity. PMID:3804975

  20. Engineering alginate for intervertebral disc repair.

    PubMed

    Bron, Johannes L; Vonk, Lucienne A; Smit, Theodoor H; Koenderink, Gijsje H

    2011-10-01

    Alginate is frequently studied as a scaffold for intervertebral disc (IVD) repair, since it closely mimics mechanical and cell-adhesive properties of the nucleus pulposus (NP) of the IVD. The aim of this study was to assess the relation between alginate concentration and scaffold stiffness and find preparation conditions where the viscoelastic behaviour mimics that of the NP. In addition, we measured the effect of variations in scaffold stiffness on the expression of extracellular matrix molecules specific to the NP (proteoglycans and collagen) by native NP cells. We prepared sample discs of different concentrations of alginate (1%-6%) by two different methods, diffusion and in situ gelation. The stiffness increased with increasing alginate concentration, while the loss tangent (dissipative behaviour) remained constant. The diffusion samples were ten-fold stiffer than samples prepared by in situ gelation. Sample discs prepared from 2% alginate by diffusion closely matched the stiffness and loss tangent of the NP. The stiffness of all samples declined upon prolonged incubation in medium, especially for samples prepared by diffusion. The biosynthetic phenotype of native cells isolated from NPs was preserved in alginate matrices up to 4 weeks of culturing. Gene expression levels of extracellular matrix components were insensitive to alginate concentration and corresponding matrix stiffness, likely due to the poor adhesiveness of the cells to alginate. In conclusion, alginate can mimic the viscoelastic properties of the NP and preserve the biosynthetic phenotype of NP cells but certain limitations like long-term stability still have to be addressed.

  1. Engineering alginate as bioink for bioprinting

    PubMed Central

    Jia, Jia; Richards, Dylan J.; Pollard, Samuel; Tan, Yu; Rodriguez, Joshua; Visconti, Richard P.; Trusk, Thomas C.; Yost, Michael J.; Yao, Hai; Markwald, Roger R.; Mei, Ying

    2015-01-01

    Recent advances in 3D printing offer an excellent opportunity to address critical challenges faced by current tissue engineering approaches. Alginate hydrogels have been extensively utilized as bioinks for 3D bioprinting. However, most previous research has focused on native alginates with limited degradation. The application of oxidized alginates with controlled degradation in bioprinting has not been explored. Here, we prepared a collection of 30 different alginate hydrogels with varied oxidation percentages and concentrations to develop a bioink platform that can be applied to a multitude of tissue engineering applications. We systematically investigated the effects of two key material properties (i.e. viscosity and density) of alginate solutions on their printabilities to identify a suitable range of material properties of alginates to be applied to bioprinting. Further, four alginate solutions with varied biodegradability were printed with human adipose-derived stem cells (hADSCs) into lattice-structured, cell-laden hydrogels with high accuracy. Notably, these alginate-based bioinks were shown to be capable of modulating proliferation and spreading of hADSCs without affecting structure integrity of the lattice structures (except the highly degradable one) after 8 days in culture. This research lays a foundation for the development of alginate-based bioink for tissue-specific tissue engineering applications. PMID:24998183

  2. Engineering alginate as bioink for bioprinting.

    PubMed

    Jia, Jia; Richards, Dylan J; Pollard, Samuel; Tan, Yu; Rodriguez, Joshua; Visconti, Richard P; Trusk, Thomas C; Yost, Michael J; Yao, Hai; Markwald, Roger R; Mei, Ying

    2014-10-01

    Recent advances in three-dimensional (3-D) printing offer an excellent opportunity to address critical challenges faced by current tissue engineering approaches. Alginate hydrogels have been used extensively as bioinks for 3-D bioprinting. However, most previous research has focused on native alginates with limited degradation. The application of oxidized alginates with controlled degradation in bioprinting has not been explored. Here, a collection of 30 different alginate hydrogels with varied oxidation percentages and concentrations was prepared to develop a bioink platform that can be applied to a multitude of tissue engineering applications. The authors systematically investigated the effects of two key material properties (i.e. viscosity and density) of alginate solutions on their printabilities to identify a suitable range of material properties of alginates to be applied to bioprinting. Further, four alginate solutions with varied biodegradability were printed with human adipose-derived stem cells (hADSCs) into lattice-structured, cell-laden hydrogels with high accuracy. Notably, these alginate-based bioinks were shown to be capable of modulating proliferation and spreading of hADSCs without affecting the structure integrity of the lattice structures (except the highly degradable one) after 8days in culture. This research lays a foundation for the development of alginate-based bioink for tissue-specific tissue engineering applications.

  3. Influence of hydrophobic modification in alginate-based hydrogels for biomedical applications

    NASA Astrophysics Data System (ADS)

    Choudhary, Soumitra

    Alginate has been exploited commercially for decades in foods, textiles, paper, pharmaceutical industries, and also as a detoxifier for removing heavy metals. Alginate is also popular in cell encapsulation because of its relatively mild gelation protocol and simple chemistry with which biological active entities can be immobilized. Surface modification of alginate gels has been explored to induce desired cell interactions with the gel matrix. These modifications alter the bulk properties, which strongly determine on how cells feel and response to the three-dimensional microenvironment. However, there is a need to develop strategies to engineer functionalities into bulk alginate hydrogels that not only preserve their inherent qualities but are also less toxic. In this thesis, our main focus was to optimize the mechanical properties of alginate-based hydrogels, and by doing so control the performance of the biomaterials. In the first scheme, we used alginate and hydrophobically modified ethyl hydroxy ethyl cellulose as components in interpenetrating polymer network (IPN) gels. The second network was used to control gelation time and rheological properties. We believe these experiments also may provide insight into the mechanical and structural properties of more complex biopolymer gels and naturally-occurring IPNs. Next, we worked on incorporating a hydrophobic moiety directly into the alginate chain, resulting in materials for extended release of hydrophobic drugs. We successfully synthesized hydrophobically modified alginate (HMA) by attaching octylamine groups onto the alginate backbone by standard carbodiimide based amide coupling reaction. Solubility of several model hydrophobic drugs in dilute HMA solutions was found to be increased by more than an order of magnitude. HMA hydrogels, prepared by crosslinking the alginate chains with calcium ions, were found to exhibit excellent mechanical properties (modulus ˜100 kPa) with release extended upto 5 days. Ability

  4. Arg²³⁵ is an essential catalytic residue of Bacillus pumilus DKS1 pectate lyase to degum ramie fibre.

    PubMed

    Basu, Snehasish; Roy, Arunava; Ghosh, Abhrajyoti; Bera, Amit; Chattopadhyay, Dhrubajyoti; Chakrabarti, Krishanu

    2011-02-01

    After 24 h of incubation with only purified pectate lyase isolated from Bacillus pumilus DKS1 (EF467045), the weight loss of the ramie fibre was found to be 25%. To know the catalytic residue of pectate lyase the pel gene encoding a pectate lyase from the strain Bacillus pumilus DKS1 was cloned in E. coli XL1Blue and expressed in E. coli BL21 (DE3) pLysS. The pel gene was sequenced and showed 1032 bp length. After purification using CM-Sepharose the enzyme showed molecular weight of 35 kDa and maximal enzymatic activity was observed at 60°C and a pH range of 8.5-9.0. Both Ca²(+) and Mn²(+) ions were required for activity on Na-pectate salt substrates, while the enzyme was strongly inhibited by Zn²(+) and EDTA. The deduced nucleotide sequence of the DKS1 pectate lyase (EU652988) showed 90% homology to pectate lyases from Bacillus pumilus SAFR-032 (CP000813). The 3D structure as well as the catalytic residues was predicted using EasyPred software and Catalytic Site Atlas (CSA), respectively. Site directed mutagenesis confirmed that arginine is an essential catalytic residue of DKS1 pectate lyase.

  5. Novel copper (II) alginate hydrogels and their potential for use as anti-bacterial wound dressings.

    PubMed

    Klinkajon, Wimonwan; Supaphol, Pitt

    2014-08-01

    The incorporation of a metal ion, with antimicrobial activity, into an alginate dressing is an attractive approach to minimize infection in a wound. In this work, copper (II) cross-linked alginate hydrogels were successfully prepared using a two-step cross-linking procedure. In the first step, solid alginate films were prepared using a solvent-casting method from soft gels of alginate solutions that had been lightly cross-linked using a copper (II) (Cu(2+)) sulfate solution. In the second step, the films were further cross-linked in a corresponding Cu(2+) sulfate solution using a dipping method to further improve their dimensional stability. Alginate solution (at 2%w/v) and Cu(2+) sulfate solution (at 2%w/v) in acetate buffer at a low pH provided soft films with excellent swelling behavior. An increase in either Cu(2+) ion concentration or cross-linking time led to hydrogels with more densely-cross-linked networks that limited water absorption. The hydrogels clearly showed antibacterial activity against Escherichia coli, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis and Streptococcus pyogenes, which was proportional to the Cu(2+) ion concentration. Blood coagulation studies showed that the tested copper (II) cross-linked alginate hydrogels had a tendency to coagulate fibrin, and possibly had an effect on pro-thrombotic coagulation and platelet activation. Conclusively, the prepared films are likely candidates as antibacterial wound dressings.

  6. Alginate cryogel based glucose biosensor

    NASA Astrophysics Data System (ADS)

    Fatoni, Amin; Windy Dwiasi, Dian; Hermawan, Dadan

    2016-02-01

    Cryogel is macroporous structure provides a large surface area for biomolecule immobilization. In this work, an alginate cryogel based biosensor was developed to detect glucose. The cryogel was prepared using alginate cross-linked by calcium chloride under sub-zero temperature. This porous structure was growth in a 100 μL micropipette tip with a glucose oxidase enzyme entrapped inside the cryogel. The glucose detection was based on the colour change of redox indicator, potassium permanganate, by the hydrogen peroxide resulted from the conversion of glucose. The result showed a porous structure of alginate cryogel with pores diameter of 20-50 μm. The developed glucose biosensor was showed a linear response in the glucose detection from 1.0 to 5.0 mM with a regression of y = 0.01x+0.02 and R2 of 0.994. Furthermore, the glucose biosensor was showed a high operational stability up to 10 times of uninterrupted glucose detections.

  7. Preparation of carbon nanotube-alginate nanocomposite gel for tissue engineering.

    PubMed

    Kawaguchi, Minoru; Fukushima, Tadao; Hayakawa, Toru; Nakashima, Naotoshi; Inoue, Yusuke; Takeda, Shoji; Okamura, Kazuhiko; Taniguchi, Kunihisa

    2006-12-01

    A novel scaffold material based on an alginate hydrogel which contained carbon nanotubes (CNTs) was prepared, and its mechanical property and biocompatibility evaluated. Soluble CNTs were prepared with acid treatment and dispersed in sodium alginate solution as a cross-linker. After which, the mechanical property (elastic deformation), saline sorption, histological reaction, and cell viability of the resultant nanocomposite gel (CNT-Alg gel) were evaluated. The CNT-Alg gel showed faster gelling and higher mechanical strength than the conventional alginate gel. Saline sorption amount of freeze-dried CNT-Alg gel was equal to that of the alginate gel. In terms of histological evaluation and cell viability assay, CNT-Alg gel exhibited a mild inflammatory response and non-cytotoxicity. These results thus suggested that CNT-Alg gel could be useful as a scaffold material in tissue engineering with the sidewalls of CNTs acting as active sites for chemical functionalization.

  8. Optimization of polyphenol oxidase immobilization in copper alginate beads.

    PubMed

    Kocaturk, Selin; Yagar, Hulya

    2010-05-01

    Polyphenol oxidase (PPO, EC 1.14.18.1) was isolated from artichoke head (Cynara scolymus L.) by using 0.1 M Tris-HCl buffer (pH 7.0), concentrated by (NH4)2SO4 precipitation, and immobilized in copper-alginate beads. Immobilization yield was determined to be 70%. The cresolase and catecholase activities of enzyme immobilized at optimum immobilization conditions were found to be 13.3 and 670 U g beads min(-1), respectively. Effects of immobilization conditions such as alginate concentration, CaCl2 concentration, amount of loading enzyme, bead size, and amount of beads on enzymatic activity were investigated. Optimum alginate and CuCl2 concentration were found to be 2 % and 3 % (w/v), respectively. Using bead (diameter 3 mm) amount of 0.25 g maximum enzyme activities were observed for both polyphenol activities. The initial concentrations of loading free enzyme were 6.5 U mL(-1) and 5815 U mL(-1) for cresolase activity and catecholase activities, respectively. Beads prepared at optimum immobilization conditions were suitable for up to 8 repeated uses.

  9. Alginate as a protease inhibitor in vitro and in a model gut system; selective inhibition of pepsin but not trypsin.

    PubMed

    Chater, Peter Ian; Wilcox, Mathew D; Brownlee, Iain A; Pearson, Jeffrey P

    2015-10-20

    Alginates are widely used in the food and medical industries, including as a Gastro-Oesophagul Reflux treatment. This work investigates the inhibitory effects of alginate on the reflux aggressors trypsin and pepsin and the role of alginate-substrate binding, pH and alginate structure on inhibition. Alginates were shown to reduce pepsin activity by up to 53.9% (±9.5SD) in vitro. Strong positive correlation between alginate mannuronate residue frequency and levels of pepsin inhibition was observed. Limited inhibition of trypsin was shown. Viscometric observations of pH dependent interactions between alginate and protein suggest a mechanism whereby pH dependent ionic interactions reduce substrate availability to enzyme at acidic pH. To understand how dietary protein digestion is affected by alginate, proteolytic digestion was investigated in an in vitro model of the upper digestive tract. Significant inhibition of proteolysis was shown in the gastric phase of digestion, but not the small intestinal phase.

  10. Encapsulation of urease enzyme in xanthan-alginate spheres.

    PubMed

    Elçin, Y M

    1995-10-01

    Urease-containing xanthan-alginate spheres were prepared by a two-step process which involved the Ca2+ coupling of the polysaccharides, followed by gentle glutaraldehyde cross-linking with amine groups of gelatin present in the initial mixture. This second step caused a slight decrease in the enzymatic activity but increased the stability. The water content and size distribution of the spheres were examined together with the sphere morphology. The effect of polymer ratio and enzyme loading on urease activity was investigated. An increase in xanthan content was found to affect the water uptake of the spheres. Temperature and pH stability of encapsulated urease was found to be higher than the free form. The xanthan-alginate spheres showed 75% of maximum urease activity even after 20 repeated uses under optimal conditions.

  11. Fungal and Plant Phenylalanine Ammonia-lyase

    PubMed Central

    Hyun, Min Woo; Yun, Yeo Hong; Kim, Jun Young

    2011-01-01

    L-Phenylalanine is one of the essential amino acids that cannot be synthesized in mammals in adequate amounts to meet the requirements for protein synthesis. Fungi and plants are able to synthesize phenylalanine via the shikimic acid pathway. L-Phenylalanine, derived from the shikimic acid pathway, is used directly for protein synthesis in plants or metabolized through the phenylpropanoid pathway. This phenylpropanoid metabolism leads to the biosynthesis of a wide array of phenylpropanoid secondary products. The first step in this metabolic sequence involves the action of phenylalanine ammonia-lyase (PAL). The discovery of PAL enzyme in fungi and the detection of 14CO2 production from 14C-ring-labeled phenylalanine and cinnamic acid demonstrated that certain fungi can degrade phenylalanine by a pathway involving an initial deamination to cinnamic acid, as happens in plants. In this review, we provide background information on PAL and a recent update on the presence of PAL genes in fungi. PMID:22783113

  12. Alginate Biosynthesis Factories in Pseudomonas fluorescens: Localization and Correlation with Alginate Production Level.

    PubMed

    Maleki, Susan; Almaas, Eivind; Zotchev, Sergey; Valla, Svein; Ertesvåg, Helga

    2015-12-11

    Pseudomonas fluorescens is able to produce the medically and industrially important exopolysaccharide alginate. The proteins involved in alginate biosynthesis and secretion form a multiprotein complex spanning the inner and outer membranes. In the present study, we developed a method by which the porin AlgE was detected by immunogold labeling and transmission electron microscopy. Localization of the AlgE protein was found to depend on the presence of other proteins in the multiprotein complex. No correlation was found between the number of alginate factories and the alginate production level, nor were the numbers of these factories affected in an algC mutant that is unable to produce the precursor needed for alginate biosynthesis. Precursor availability and growth phase thus seem to be the main determinants for the alginate production rate in our strain. Clustering analysis demonstrated that the alginate multiprotein complexes were not distributed randomly over the entire outer cell membrane surface.

  13. Alginate Biosynthesis Factories in Pseudomonas fluorescens: Localization and Correlation with Alginate Production Level

    PubMed Central

    Maleki, Susan; Almaas, Eivind; Zotchev, Sergey; Valla, Svein

    2015-01-01

    Pseudomonas fluorescens is able to produce the medically and industrially important exopolysaccharide alginate. The proteins involved in alginate biosynthesis and secretion form a multiprotein complex spanning the inner and outer membranes. In the present study, we developed a method by which the porin AlgE was detected by immunogold labeling and transmission electron microscopy. Localization of the AlgE protein was found to depend on the presence of other proteins in the multiprotein complex. No correlation was found between the number of alginate factories and the alginate production level, nor were the numbers of these factories affected in an algC mutant that is unable to produce the precursor needed for alginate biosynthesis. Precursor availability and growth phase thus seem to be the main determinants for the alginate production rate in our strain. Clustering analysis demonstrated that the alginate multiprotein complexes were not distributed randomly over the entire outer cell membrane surface. PMID:26655760

  14. The visualisation and speed of kill of wound isolates on a silver alginate dressing.

    PubMed

    Hooper, Samuel J; Percival, Steven L; Hill, Katja E; Thomas, David W; Hayes, A J; Williams, David W

    2012-12-01

    In chronic wound management, alginate dressings are used to absorb exudate and reduce the microbial burden. Silver alginate offers the added benefit of an additional antimicrobial pressure on contaminating microorganisms. This present study compares the antimicrobial activity of a RESTORE silver alginate dressing with a silver-free control dressing using a combination of in vitro culture and imaging techniques. The wound pathogens examined included Candida albicans, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, β-haemolytic Streptococcus, and strictly anaerobic bacteria. The antimicrobial efficacy of the dressings was assessed using log(10) reduction and 13-day corrected zone of inhibition (CZOI) time-course assays. Confocal laser scanning microscopy (CLSM) was used to visualise the relative proportions of live/dead microorganisms sequestered into the dressings over 24 hours and estimate the comparative speed of kill. The RESTORE silver alginate dressing showed significantly greater log(10) reductions and CZOIs for all microorganisms compared with the control, indicating the antimicrobial effect of ionic silver. Antimicrobial activity was evident against all test organisms for up to 5 days and, in some cases, up to 12 days following an on-going microbial challenge. Imaging bacteria sequestered in the silver-free dressing showed that each microbial species aggregated in the dressing and remained viable for more than 20 hours. Growth was not observed inside of the dressing, indicating a possible microbiostatic effect of the alginate fibres. In comparison, organisms in the RESTORE silver alginate dressing were seen to lose viability at a considerably greater rate. After 16 hours of contact with the RESTORE silver alginate dressing, >90% of cells of all bacteria and yeast were no longer viable. In conclusion, collectively, the data highlights the rapid speed of kill and antimicrobial suitability of this RESTORE silver alginate dressing on wound

  15. Syntheses of L-tyrosine-related amino acids by tyrosine phenol-lyase of Citrobacter intermedius.

    PubMed

    Nagasawa, T; Utagawa, T; Goto, J; Kim, C J; Tani, Y; Kumagai, H; Yamada, H

    1981-06-01

    Degradation of tyrosine to phenol, pyruvate and ammonia by tyrosine phenol-lyase from Citrobacter intermedius (formerly named Escherichia intermedia) is readily reversible at high concentrations of pyruvate and ammonia. Spectrophotometric studies indicate that ammonia is the first substrate which interacts with bound pyridoxal 5'-phosphate. Kinetic results show that pyruvate is the second substrate bound, hence phenol must be the third. When an appropriate phenol derivative is substituted for phenol, the corresponding tyrosine analogue can be synthesized. 3-Fluoro-, 2-fluoro-, 3-chloro-, 2-chloro-, 3-bromo-, 2-bromo-, 2-iodo-, 3-methyl-, 2-methyl- and 2-methoxy-L-tyrosines have been synthesized by this reaction. By using various phenol derivatives or tyrosine analogues as substrates, the substrate specificity of tyrosine phenol-lyase is investigated and the situation of its active site is discussed.

  16. 21 CFR 184.1187 - Calcium alginate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... alginic acid, a natural polyuronide constituent of certain brown algae. Calcium alginate is prepared by... Do. Fats and oils, § 170.3(n)(12) of this chapter 0.5 Do. Gelatins, puddings, § 170.3(n)(22) of...

  17. Scaling law and microstructure of alginate hydrogel.

    PubMed

    Liu, Sijun; Li, Huijun; Tang, Bijun; Bi, Shuguang; Li, Lin

    2016-01-01

    The gelation of alginate in aqueous solution was studied as a function of Ca(2+) concentration. At each given concentration of alginate, a critical gel concentration [Formula: see text] , was successfully determined for the first time using the Winter-Chambon criterion. The critical gel concentration [Formula: see text] was found to increase linearly with alginate concentration. At the same time, the critical relaxation exponent n decreased and the critical gel strength Sg increased linearly with alginate concentration. An improved egg-box model was proposed to describe the change in gel junction and gel network. In the stable gel state, the plateau modulus Ge of alginate gel depended on Ca(2+) concentration according to a power-law scaling, Ge=kɛ(1.5), where ɛ is the relative distance of a gelling variable (Ca(2+) concentration in this case) from the gel point ( [Formula: see text] ). The FESEM images verified the microstructure of alginate gel in which alginate chains associated into fibrils in the presence of Ca(2+) ions. The fibrillar diameter and network density increased with increasing Ca(2+) ion concentration while alginate concentration had a weak influence on fibrillar diameter.

  18. Cloning and heterologous expression of a thermostable pectate lyase from Penicillium occitanis in Escherichia coli.

    PubMed

    Damak, Naourez; Abdeljalil, Salma; Koubaa, Aida; Trigui, Sameh; Ayadi, Malika; Trigui-Lahiani, Hèla; Kallel, Emna; Turki, Nadia; Djemal, Lamia; Belghith, Hafeth; Taieb, Noomen Hadj; Gargouri, Ali

    2013-11-01

    The entire pectate lyase cDNA (Pel1) of Penicillium occitanis was cloned from a cDNA bank and sequenced. The ORF exhibited a great homology to Penicillium marneffei and conservation of all features of fungal pectate lyases such as the barrel structure with "eight right-handed parallel β-helix" architecture. The structure modeling also showed the interesting resemblance with thermostable pectate lyases since several specific residues were also shared by Pel1 and these thermostable enzymes. Having shown that the enzyme retains its activity after endoH-mediated deglycosylation, we investigated its expression in Escherichia coli BL21 using the pET28-a vector. This expression was shown to be optimum when cells were induced at room temperature in 2YT medium rather than at 37 °C and LB medium. In such conditions, the recombinant protein was apparently produced more in soluble form than as inclusion bodies. The effect of NaCl concentration was investigated during the binding and elution steps of recombinant His-tagged enzyme on MagneHis Ni-particles. The purified enzyme was shown to retain its thermo-activity as well as a great tolerance to high concentration of NaCl and imidazole.

  19. Engineering disease resistance with pectate lyase-like genes

    DOEpatents

    Vogel, John; Somerville, Shauna

    2005-03-08

    A mutant gene coding for pectate lyase and homologs thereof is provided, which when incorporated in transgenic plants effect an increased level disease resistance in such plants. Also is provided the polypeptide sequence for the pectate lyase of the present invention. Methods of obtaining the mutant gene, producing transgenic plants which include the nucleotide sequence for the mutant gene and producing improved disease resistance in a crop of such transgenic plants are also provided.

  20. Membrane-anchored MucR mediates nitrate-dependent regulation of alginate production in Pseudomonas aeruginosa.

    PubMed

    Wang, Yajie; Hay, Iain D; Rehman, Zahid U; Rehm, Bernd H A

    2015-09-01

    Alginates exhibit unique material properties suitable for medical and industrial applications. However, if produced by Pseudomonas aeruginosa, it is an important virulence factor in infection of cystic fibrosis patients. The alginate biosynthesis machinery is activated by c-di-GMP imparted by the inner membrane protein, MucR. Here, it was shown that MucR impairs alginate production in response to nitrate in P. aeruginosa. Subsequent site-specific mutagenesis of MucR revealed that the second MHYT sensor motif (MHYT II, amino acids 121-124) of MucR sensor domain was involved in nitrate sensing. We also showed that both c-di-GMP synthesizing and degrading active sites of MucR were important for alginate production. Although nitrate and deletion of MucR impaired alginate promoter activity and global c-di-GMP levels, alginate yields were not directly correlated with alginate promoter activity or c-di-GMP levels, suggesting that nitrate and MucR modulate alginate production at a post-translational level through a localized pool of c-di-GMP. Nitrate increased pel promoter activity in the mucR mutant while in the same mutant the psl promoter activity was independent of nitrate. Nitrate and deletion of mucR did not impact on swarming motility but impaired attachment to solid surfaces. Nitrate and deletion of mucR promoted the formation of biofilms with increased thickness, cell density, and survival. Overall, this study provided insight into the functional role of MucR with respect to nitrate-mediated regulation of alginate biosynthesis.

  1. Characterization of a novel HMG-CoA lyase enzyme with a dual location in endoplasmic reticulum and cytosol.

    PubMed

    Arnedo, María; Menao, Sebastián; Puisac, Beatriz; Teresa-Rodrigo, María E; Gil-Rodríguez, María C; López-Viñas, Eduardo; Gómez-Puertas, Paulino; Casals, Nuria; Casale, César H; Hegardt, Fausto G; Pié, Juan

    2012-10-01

    A novel lyase activity enzyme is characterized for the first time: HMG-CoA lyase-like1 (er-cHL), which is a close homolog of mitochondrial HMG-CoA lyase (mHL). Initial data show that there are nine mature transcripts for the novel gene HMGCLL1, although none of them has all its exons. The most abundant transcript is called "variant b," and it lacks exons 2 and 3. Moreover, a three-dimensional model of the novel enzyme is proposed. Colocalization studies show a dual location of the er-cHL in the endoplasmic reticulum (ER) and cytosol, but not in mitochondria or peroxisomes. Furthermore, the dissociation experiment suggests that it is a nonendoplasmic reticulum integral membrane protein. The kinetic parameters of er-cHL indicate that it has a lower V(max) and a higher substrate affinity than mHL. Protein expression and lyase activity were found in several tissues, and were particularly strong in lung and kidney. The occurrence of er-cHL in brain is surprising, as mHL has not been found there. Although mHL activity is clearly associated with energy metabolism, the results suggest that er-cHL is more closely related to another metabolic function, mostly at the pulmonary and brain level.

  2. Osteogenic Differentiation of Human Mesenchymal Stem Cells in Mineralized Alginate Matrices

    PubMed Central

    Westhrin, Marita; Xie, Minli; Olderøy, Magnus Ø.; Sikorski, Pawel

    2015-01-01

    Mineralized biomaterials are promising for use in bone tissue engineering. Culturing osteogenic cells in such materials will potentially generate biological bone grafts that may even further augment bone healing. Here, we studied osteogenic differentiation of human mesenchymal stem cells (MSC) in an alginate hydrogel system where the cells were co-immobilized with alkaline phosphatase (ALP) for gradual mineralization of the microenvironment. MSC were embedded in unmodified alginate beads and alginate beads mineralized with ALP to generate a polymer/hydroxyapatite scaffold mimicking the composition of bone. The initial scaffold mineralization induced further mineralization of the beads with nanosized particles, and scanning electron micrographs demonstrated presence of collagen in the mineralized and unmineralized alginate beads cultured in osteogenic medium. Cells in both types of beads sustained high viability and metabolic activity for the duration of the study (21 days) as evaluated by live/dead staining and alamar blue assay. MSC in beads induced to differentiate in osteogenic direction expressed higher mRNA levels of osteoblast-specific genes (RUNX2, COL1AI, SP7, BGLAP) than MSC in traditional cell cultures. Furthermore, cells differentiated in beads expressed both sclerostin (SOST) and dental matrix protein-1 (DMP1), markers for late osteoblasts/osteocytes. In conclusion, Both ALP-modified and unmodified alginate beads provide an environment that enhance osteogenic differentiation compared with traditional 2D culture. Also, the ALP-modified alginate beads showed profound mineralization and thus have the potential to serve as a bone substitute in tissue engineering. PMID:25769043

  3. Radiation synthesis of PVP/alginate hydrogel containing nanosilver as wound dressing.

    PubMed

    Singh, Rita; Singh, Durgeshwer

    2012-11-01

    Hydrogels with polyvinyl pyrrolidone (PVP) and alginate were synthesized and silver nanoparticles were incorporated in hydrogel network using gamma radiation. PVP (10 and 15 %) in combination with 0.5 and 1 % alginate was gamma irradiated at different doses of 25 and 40 kGy. Maximum gel percent was obtained with 15 % PVP in combination with 0.5 % alginate. The fluid absorption capacity for the PVP/alginate hydrogels was about 1881-2361 % at 24 h. Moisture vapour transmission rate (MVTR) of hydrogels containing nanosilver at 24 h was 278.44 g/(m(2)h). The absorption capacity and moisture permeability of the PVP/alginate-nanosilver composite hydrogel dressings show the ability of the hydrogels to prevent fluid accumulation in exudating wound. The hydrogels containing nanosilver demonstrated strong antimicrobial effect and complete inhibition of microbial growth was observed with 70 ppm nanosilver dressings. PVP/alginate hydrogels containing nanosilver with efficient fluid handling capacity and antimicrobial activity was found suitable for use as wound dressing.

  4. Effects of the biologically produced polymer alginic acid on macroscopic and microscopic calcite dissolution rates.

    PubMed

    Perry, Thomas D; Duckworth, Owen W; McNamara, Christopher J; Martin, Scot T; Mitchell, Ralph

    2004-06-01

    Dissolution of carbonate minerals has significant environmental effects. Microorganisms affect carbonate dissolution rates by producing extracellular metabolites, including complex polysaccharides such as alginic acid. Using a combined atomic force microscopy (AFM)/flowthrough reactor apparatus, we investigated the effects of alginic acid on calcite dissolution. Macroscopic dissolution rates, derived from the aqueous metal ion concentrations, are 10(-5.5) mol m(-2) s(-1) for 5 < pH < 12 in the absence of alginic acid compared to 10(-4.8) mol m(-2) s(-1) in its presence. The AFM images demonstrate that alginic acid preferentially attacks the obtuse steps of dissolution pits on the calcite surface. In pure water, the obtuse and acute steps retreat at similar rates, and the pits are nearly isotropic except under highly acidic conditions. In alginic acid, the acute step retreat rate is nearly unchanged in comparison to water, whereas the obtuse step retreat rate increases with decreasing pH values. As a result, the pits remain rhombohedral but propagate faster in the obtuse direction. To explain these observations, we propose that alginic acid preferentially forms dissolution active surface complexes with calcium atoms on the obtuse step, which results in anisotropic ligand-promoted dissolution.

  5. Inhibition of testicular microsomal cytochrome P-450 (17 alpha-hydroxylase/C-17,20-lyase) by estrogens.

    PubMed

    Onoda, M; Hall, P F

    1981-09-01

    Highly purified cytochrome P-450 from neonatal pig testicular microsomes is capable of catalyzing both 17 alpha-hydroxylation and C-17,20-lyase activity. Estradiol was found to inhibit both activities of the purified enzyme with delta 4 and with delta 5 substrates (progesterone, pregnenolone, and the corresponding 17 alpha-hydroxysteroids). For the delta 4 series, inhibition of lyase is competitive and that of 17 alpha-hydroxylase is noncompetitive; for the delta 5 series, inhibition was noncompetitive for both activities. Ki values for lyase activity were determined from enzyme kinetics (5.0 microM for the delta 4 substrate and 20 microM for the delta 5 substrate). Estradiol produces a typical type I spectral shift with the pure enzyme (Ks = 3.0 microM where Ks is the concentration of steroid required to give half maximal spectral shift), so that Ki values were also determined directly from binding studies by using substrate-induced difference spectroscopy. Fifty per cent inhibition of the maximal spectral shift induced by the 17 alpha-hydroxysubstrates (Ki) are 3.8 and 7.6 microM for the delta 4 and delta 5 substrates, respectively. Values for Ki are higher with the substrates of 17 alpha-hydroxylase (progesterone and pregnenolone), by either method, than the corresponding Ki values for the lyase substrates. The concentration of estradiol in Leydig cells of neonatal pig testis is approximately 1.5 nmol/g. It is proposed that estradiol may influence testicular steroidogensis under physiological conditions by competitive inhibition of lyase activity.

  6. 17α-hydroxylase/17,20-lyase deficiency in congenital adrenal hyperplasia: A case report

    PubMed Central

    Xu, Simiao; Hu, Shuhong; Yu, Xuefeng; Zhang, Muxun; Yang, Yan

    2016-01-01

    Congenital adrenal hyperplasia (CAH) is a rare autosomal recessive disorder caused by mutations in the cytochrome P450 family 17 subfamily A member 1 (CYP17A1) gene located on chromosome 10q24.3, which leads to a deficiency in 17α-hydroxylase/17,20-lyase. The disorder is characterized by low blood levels of estrogens, androgens and cortisol, which leads to a compensatory increase in adrenocorticotropic hormone levels that stimulate the production of mineralocorticoid precursors. This subsequently leads to hypertension, hypokalemia, primary amenorrhea and sexual infantilism. Over 90 distinct genetic lesions have been identified in patients with this disorder. The prevalence of common mutation of CYP17A1 gene differs among ethnic groups. Treatment of this disorder involves replacement of glucocorticoids and sex steroids. Estrogen alone is prescribed for patients who are biologically male with 17α-hydroxylase deficiencies that identify as female. However, genetically female patients may receive estrogen and progesterone supplementation. In the present study, a 17-year-old female with 17α-hydroxylase/17,20-lyase deficiency that presented with primary amenorrhea and sexual infantilism and no hypertension, was examined. The karyotype of the patient was 46, XX, and genetic analysis revealed the presence of a compound heterozygous mutation in exons 6 and 8, leading to the complete absence of 17α-hydroxylase/17,20-lyase activity. The patient was treated with prednisolone and ethinyl estradiol. In addition, a summary of the recent literature regarding CAH is presented. PMID:27959413

  7. Benzaldehyde lyase, a novel thiamine PPi-requiring enzyme, from Pseudomonas fluorescens biovar I.

    PubMed Central

    González, B; Vicuña, R

    1989-01-01

    Pseudomonas fluorescens biovar I can grow on benzoin as the sole carbon and energy source. This ability is due to benzaldehyde lyase, a new type of enzyme that irreversibly cleaves the acyloin linkage of benzoin, producing two molecules of benzaldehyde. Benzaldehyde lyase was purified 70-fold and found to require catalytic amounts of thiamine PPi (TPP) and a divalent cation as cofactors. Optimal activity was obtained with a 1.0 mM concentration of Mn2+, Mg2+, or Ca2+. Gel permeation chromatography indicated a native molecular weight of 80,000, whereas the enzyme migrated in sodium dodecyl sulfate-containing polyacrylamide gels as a single polypeptide with a molecular weight of 53,000. Benzaldehyde lyase is highly specific; of a variety of structurally related compounds tested, only benzoin and anisoin (4,4'-dimethoxybenzoin) acted as substrates, their apparent Kms being 9.0 x 10(-3) and 3.25 x 10(-2) mM, respectively. A catalytic mechanism for the enzyme is proposed. Images PMID:2496105

  8. Structural insights into recognition and repair of UV-DNA damage by Spore Photoproduct Lyase, a radical SAM enzyme

    PubMed Central

    Benjdia, Alhosna; Heil, Korbinian; Barends, Thomas R. M.; Carell, Thomas; Schlichting, Ilme

    2012-01-01

    Bacterial spores possess an enormous resistance to ultraviolet (UV) radiation. This is largely due to a unique DNA repair enzyme, Spore Photoproduct Lyase (SP lyase) that repairs a specific UV-induced DNA lesion, the spore photoproduct (SP), through an unprecedented radical-based mechanism. Unlike DNA photolyases, SP lyase belongs to the emerging superfamily of radical S-adenosyl-l-methionine (SAM) enzymes and uses a [4Fe–4S]1+ cluster and SAM to initiate the repair reaction. We report here the first crystal structure of this enigmatic enzyme in complex with its [4Fe–4S] cluster and its SAM cofactor, in the absence and presence of a DNA lesion, the dinucleoside SP. The high resolution structures provide fundamental insights into the active site, the DNA lesion recognition and binding which involve a β-hairpin structure. We show that SAM and a conserved cysteine residue are perfectly positioned in the active site for hydrogen atom abstraction from the dihydrothymine residue of the lesion and donation to the α-thyminyl radical moiety, respectively. Based on structural and biochemical characterizations of mutant proteins, we substantiate the role of this cysteine in the enzymatic mechanism. Our structure reveals how SP lyase combines specific features of radical SAM and DNA repair enzymes to enable a complex radical-based repair reaction to take place. PMID:22761404

  9. Immobilization of a Plant Lipase from Pachira aquatica in Alginate and Alginate/PVA Beads

    PubMed Central

    Bonine, Bárbara M.; Polizelli, Patricia Peres; Bonilla-Rodriguez, Gustavo O.

    2014-01-01

    This study reports the immobilization of a new lipase isolated from oleaginous seeds of Pachira aquatica, using beads of calcium alginate (Alg) and poly(vinyl alcohol) (PVA). We evaluated the morphology, number of cycles of reuse, optimum temperature, and temperature stability of both immobilization methods compared to the free enzyme. The immobilized enzymes were more stable than the free enzyme, keeping 60% of the original activity after 4 h at 50°C. The immobilized lipase was reused several times, with activity decreasing to approximately 50% after 5 cycles. Both the free and immobilized enzymes were found to be optimally active between 30 and 40°C. PMID:24818012

  10. Controlled antiseptic release by alginate polymer films and beads.

    PubMed

    Liakos, Ioannis; Rizzello, Loris; Bayer, Ilker S; Pompa, Pier Paolo; Cingolani, Roberto; Athanassiou, Athanassia

    2013-01-30

    Biodegradable polymeric materials based on blending aqueous dispersions of natural polymer sodium alginate (NaAlg) and povidone iodine (PVPI) complex, which allow controlled antiseptic release, are presented. The developed materials are either free standing NaAlg films or Ca(2+)-cross-linked alginate beads, which properly combined with PVPI demonstrate antibacterial and antifungal activity, suitable for therapeutic applications, such as wound dressing. Glycerol was used as the plasticizing agent. Film morphology was studied by optical and atomic force microscopy. It was found that PVPI complex forms well dispersed circular micro-domains within the NaAlg matrix. The beads were fabricated by drop-wise immersion of NaAlg/PVPI/glycerol solutions into aqueous calcium chloride solutions to form calcium alginate beads encapsulating PVPI solution (CaAlg/PVPI). Controlled release of PVPI was possible when the composite films and beads were brought into direct contact with water or with moist media. Bactericidal and fungicidal properties of the materials were tested against Escherichia coli bacteria and Candida albicans fungi. The results indicated very efficient antibacterial and antifungal activity within 48 h. Controlled release of PVPI into open wounds is highly desired in clinical applications to avoid toxic doses of iodine absorption by the wound. A wide variety of applications are envisioned such as external and internal wound dressings with controlled antiseptic release, hygienic and protective packaging films for medical devices, and polymer beads as water disinfectants.

  11. Stabilization of Aspergillus parasiticus cytosine deaminase by immobilization on calcium alginate beads improved enzyme operational stability.

    PubMed

    Zanna, H; Nok, A J; Ibrahim, S; Inuwa, H M

    2013-12-01

    Cytosine deaminase (CD) from Aspergillus parasiticus, which has half-life of 1.10 h at 37°C, was stabilized by immobilization on calcium alginate beads. The immobilized CD had pH and temperature optimum of 5 and 50°C respectively. The immobilized enzyme also stoichiometrically deaminated Cytosine and 5-fluorocytosine (5-FC) with the apparent K(M) values of 0.60 mM and 0.65 mM respectively, displaying activation energy of 10.72 KJ/mol. The immobilization of native CD on calcium alginate beads gave the highest yield of apparent enzymatic activity of 51.60% of the original activity and the enzymatic activity was lost exponentially at 37°C over 12 h with a half-life of 5.80 h. Hence, the operational stability of native CD can be improved by immobilization on calcium alginate beads.

  12. Purification and Characterization of a Unique Pectin Lyase from Aspergillus giganteus Able to Release Unsaturated Monogalacturonate during Pectin Degradation

    PubMed Central

    Carmona, Eleonora Cano

    2014-01-01

    A pectin lyase, named PLIII, was purified to homogeneity from the culture filtrate of Aspergillus giganteus grown in submerged culture containing orange peel waste as carbon source. PLIII was able to digest apple pectin and citrus pectins with different degrees of methyl esterification. Interestingly, the PLIII activity was stimulated in the presence of some divalent cations including Pb2+ and was not significantly affected by Hg2+. Like other pectin lyases, PLIII is stimulated by but is not dependent on Ca2+. The main soluble product released during the degradation of pectic substances promoted by the PLIII is compatible with an unsaturated monogalacturonate. PLIII is a unique enzyme able to release unsaturated monogalacturonate as the only soluble product during the degradation of pectic substances; therefore, PLIII was classified as an exo-pectin lyase. To our knowledge, this is the first characterization of an exo-pectin lyase. The PLIII described in this work is potentially useful for ethanol production from pectin-rich biomass, besides other common applications for alkaline pectinases like preparation of textile fibers, coffee and tea fermentation, vegetable oil extraction, and the treatment of pulp in papermaking. PMID:25610636

  13. Mammalian selenocysteine lyase is involved in selenoprotein biosynthesis.

    PubMed

    Kurokawa, Suguru; Takehashi, Masanori; Tanaka, Hiromitsu; Mihara, Hisaaki; Kurihara, Tatsuo; Tanaka, Seigo; Hill, Kristina; Burk, Raymond; Esaki, Nobuyoshi

    2011-01-01

    Selenocysteine lyase (SCL) catalyzes the decomposition of L-selenocysteine to yield L-alanine and selenium by acting exclusively on l-selenocysteine. The X-ray structural analysis of rat SCL has demonstrated how SCL discriminates L-selenocysteine from L-cysteine on the molecular basis. SCL has been proposed to function in the recycling of the micronutrient selenium from degraded selenoproteins containing selenocysteine residues, but the role of SCL in selenium metabolism in vivo remains unclear. We here demonstrate that the (75)Se-labeling efficiency of selenoproteins with (75)Se-labeled selenoprotein P (Sepp1) as a selenium source was decreased in HeLa cells transfected with SCL siRNA as compared to the cells transfected with control siRNA. Immunocytochemical analyses showed high SCL expression in kidney and liver cells, where selenocysteine is recovered from selenoproteins. Mature testes of mice exhibited a specific staining pattern of SCL in spermatids that actively produce selenoproteins. However, SCL was weakly expressed in Sertoli cells, which receive Sepp1 and supply selenium to germ cells. These demonstrate that SCL occurs in the cells requiring selenoproteins, probably to recycle selenium derived from selenoproteins such as Sepp1.

  14. Cystathionine γ-lyase deficiency mediates neurodegeneration in Huntington's disease.

    PubMed

    Paul, Bindu D; Sbodio, Juan I; Xu, Risheng; Vandiver, M Scott; Cha, Jiyoung Y; Snowman, Adele M; Snyder, Solomon H

    2014-05-01

    Huntington's disease is an autosomal dominant disease associated with a mutation in the gene encoding huntingtin (Htt) leading to expanded polyglutamine repeats of mutant Htt (mHtt) that elicit oxidative stress, neurotoxicity, and motor and behavioural changes. Huntington's disease is characterized by highly selective and profound damage to the corpus striatum, which regulates motor function. Striatal selectivity of Huntington's disease may reflect the striatally selective small G protein Rhes binding to mHtt and enhancing its neurotoxicity. Specific molecular mechanisms by which mHtt elicits neurodegeneration have been hard to determine. Here we show a major depletion of cystathionine γ-lyase (CSE), the biosynthetic enzyme for cysteine, in Huntington's disease tissues, which may mediate Huntington's disease pathophysiology. The defect occurs at the transcriptional level and seems to reflect influences of mHtt on specificity protein 1, a transcriptional activator for CSE. Consistent with the notion of loss of CSE as a pathogenic mechanism, supplementation with cysteine reverses abnormalities in cultures of Huntington's disease tissues and in intact mouse models of Huntington's disease, suggesting therapeutic potential.

  15. Structural insights into the bacterial carbon-phosphorus lyase machinery

    PubMed Central

    Seweryn, Paulina; Van, Lan Bich; Kjeldgaard, Morten; Russo, Christopher J.; Passmore, Lori A.; Hove-Jensen, Bjarne; Jochimsen, Bjarne; Brodersen, Ditlev E.

    2015-01-01

    Summary Phosphorous is required for all life and microorganisms can extract it from their environment through several metabolic pathways. When phosphate is in limited supply, some bacteria are able to use organic phosphonate compounds, which require specialised enzymatic machinery for breaking the stable carbon-phosphorus (C-P) bond. Despite its importance, the details of how this machinery catabolises phosphonate remain unknown. Here we determine the crystal structure of the 240 kDa Escherichia coli C-P lyase core complex (PhnGHIJ) and show that it is a two-fold symmetric hetero-octamer comprising an intertwined network of subunits with unexpected self-homologies. It contains two potential active sites that likely couple organic phosphonate compounds to ATP and subsequently hydrolyse the C-P bond. We map the binding site of PhnK on the complex using electron microscopy and show that it binds to PhnJ via a conserved insertion domain. Our results provide a structural basis for understanding microbial phosphonate breakdown. PMID:26280334

  16. Structural insights into the bacterial carbon-phosphorus lyase machinery.

    PubMed

    Seweryn, Paulina; Van, Lan Bich; Kjeldgaard, Morten; Russo, Christopher J; Passmore, Lori A; Hove-Jensen, Bjarne; Jochimsen, Bjarne; Brodersen, Ditlev E

    2015-09-03

    Phosphorus is required for all life and microorganisms can extract it from their environment through several metabolic pathways. When phosphate is in limited supply, some bacteria are able to use phosphonate compounds, which require specialized enzymatic machinery to break the stable carbon-phosphorus (C-P) bond. Despite its importance, the details of how this machinery catabolizes phosphonates remain unknown. Here we determine the crystal structure of the 240-kilodalton Escherichia coli C-P lyase core complex (PhnG-PhnH-PhnI-PhnJ; PhnGHIJ), and show that it is a two-fold symmetric hetero-octamer comprising an intertwined network of subunits with unexpected self-homologies. It contains two potential active sites that probably couple phosphonate compounds to ATP and subsequently hydrolyse the C-P bond. We map the binding site of PhnK on the complex using electron microscopy, and show that it binds to a conserved insertion domain of PhnJ. Our results provide a structural basis for understanding microbial phosphonate breakdown.

  17. Surface modification of cotton fabrics for antibacterial application by coating with AgNPs-alginate composite.

    PubMed

    Zahran, M K; Ahmed, Hanan B; El-Rafie, M H

    2014-08-08

    In recent years nano-sized particles have been focused on bacteriostasis. We investigated antimicrobial activities by applying AgNPs-alginate composite on cotton fabric, using a simple one-step rapid synthetic route by reduction of silver nitrate using alkali hydrolyzed alginate solution which acts as both reducing and capping agent. FTIR spectra, color coordinates, silver content, silver release percent and SEM images of treated fabric samples confirmed the successful physical deposition of AgNPs-alginate composite on the fabric. The treated fabrics demonstrated an excellent antibacterial activity against the tested bacteria, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. A slight decrease in the antibacterial feature of the cotton fabrics was observed after successive washings. However, an efficient antibacterial activity still remained on the fabrics.

  18. Preparation and characterization of. beta. -D-glucosidase immobilized in calcium alginate

    SciTech Connect

    Krasniak, S. R.; Smith, R. D.

    1982-01-01

    Enzymatic hydrolysis of biomass to produce glucose may become feasible if an inexpensive method to reuse the enzyme can be found. This study investigated one such method whereby ..beta..-D-glucosidase (E.C. 3.2.1.21) was immobilized in calcium alginate gel spheres, which were shown to catalyze the hydrolysis of cellobiose to glucose. There was a loss of 49% of the enzyme from the alginate slurry during gelation. After gelation, in the stable gel spheres, there was a 37% retention of the enzyme activity that was actually immobilized. The reason for the loss in activity was investigated and may be caused by inhibition of the enzyme within the sphere by the calcium cations and the alginate anions also present. Mass transfer effects were minimal in this system and were not responsible for the activity loss.

  19. Relevance of rheological properties of sodium alginate in solution to calcium alginate gel properties.

    PubMed

    Fu, Shao; Thacker, Ankur; Sperger, Diana M; Boni, Riccardo L; Buckner, Ira S; Velankar, Sachin; Munson, Eric J; Block, Lawrence H

    2011-06-01

    The purpose of this study is to determine whether sodium alginate solutions' rheological parameters are meaningful relative to sodium alginate's use in the formulation of calcium alginate gels. Calcium alginate gels were prepared from six different grades of sodium alginate (FMC Biopolymer), one of which was available in ten batches. Cylindrical gel samples were prepared from each of the gels and subjected to compression to fracture on an Instron Universal Testing Machine, equipped with a 1-kN load cell, at a cross-head speed of 120 mm/min. Among the grades with similar % G, (grades 1, 3, and 4), there is a significant correlation between deformation work (L(E)) and apparent viscosity (η(app)). However, the results for the partial correlation analysis for all six grades of sodium alginate show that L(E) is significantly correlated with % G, but not with the rheological properties of the sodium alginate solutions. Studies of the ten batches of one grade of sodium alginate show that η(app) of their solutions did not correlate with L(E) while tan δ was significantly, but minimally, correlated to L(E). These results suggest that other factors--polydispersity and the randomness of guluronic acid sequencing--are likely to influence the mechanical properties of the resultant gels. In summary, the rheological properties of solutions for different grades of sodium alginate are not indicative of the resultant gel properties. Inter-batch differences in the rheological behavior for one specific grade of sodium alginate were insufficient to predict the corresponding calcium alginate gel's mechanical properties.

  20. Comparative analysis of the five major Erwinia chrysanthemi pectate lyases: enzyme characteristics and potential inhibitors.

    PubMed Central

    Tardy, F; Nasser, W; Robert-Baudouy, J; Hugouvieux-Cotte-Pattat, N

    1997-01-01

    In Erwinia chrysanthemi 3937, pectate lyase activity mainly results from the cumulative action of five major isoenzymes, PelA to PelE. Comparison of their amino acid sequences revealed two families, PelB-C and PelA-D-E. Molecular cloning permitted expression of the different pel genes in Escherichia coli and the isolation of each Pel independently from the other isoenzymes. We used similar experimental conditions to overproduce and purify the five Pels in a one-step chromatography method. We analyzed some of the basic enzymatic properties of these five isoenzymes. PelA has a low specific activity compared to the other four enzymes. PelB and PelC have a high affinity for their substrate: about 10-fold higher than the enzymes of the PelA-D-E group. The optimum pH is more alkaline for PelB and PelC (about 9.2) than for PelA, PelD, and PelE (from 8 to 8.8). Below pH 7, activity was negligible for PelB and PelC, while PelA, PelD, and PelE retained 25 to 30% of their activities. The temperature optima were determined to be 50 degrees C for PelD and PelE, 55 degrees C for PelA, and 60 degrees C for PelB and PelC. Enzymes of the PelB-C group are more stable than those of the PelA-D-E group. Use of substrates presenting various degrees of methylation revealed that PelA, PelD, and PelE are active only for very low levels of methylation, while PelB and PelC are more active on partially methylated pectins (up to 22% for PelC and up to 45% for PelB). Pectate lyases have an absolute requirement for Ca2+ ions. For the five isoenzymes, maximal activity was obtained at a Ca2+ concentration of 0.1 mM. None of the tested cations (Ba2+, Co2+, Cu2+, Mg2+, Mn2+, Sr2+, Zn2+) can substitute for Ca2+. At a high concentration (1 mM), most of the divalent cations inhibited pectate lyase activity. In addition, we demonstrated that two compounds present in plant tissues, epicatechin and salicylic acid, inhibit the pectate lyases at a concentration of 0.2 mM. PMID:9098045

  1. Molecular cloning and characterization of l-methionine γ-lyase from Streptomyces avermitilis.

    PubMed

    Kudou, Daizou; Yasuda, Eri; Hirai, Yoshiyuki; Tamura, Takashi; Inagaki, Kenji

    2015-10-01

    A pyridoxal 5'-phosphate-dependent methionine γ-lyase (MGL) was cloned from Streptomyces avermitilis catalyzed the degradation of methionine to α-ketobutyrate, methanethiol, and ammonia. The sav7062 gene (1,242 bp) was corresponded to 413 amino acid residues with a molecular mass of 42,994 Da. The deduced amino acid sequence showed a high degree of similarity to those of other MGL enzymes. The sav7062 gene was overexpressed in Escherichia coli. The enzyme was purified to homogeneity and exhibited the MGL catalytic activities. We cloned the enzyme that has the MGL activity in Streptomyces for the first time.

  2. Lead removal in rats using calcium alginate.

    PubMed

    Savchenko, Olga V; Sgrebneva, Marina N; Kiselev, Vladimir I; Khotimchenko, Yuri S

    2015-01-01

    Lead (Pb) exposure, even at low levels, causes a variety of health problems. The aims of this study were to investigate the tissue distribution of lead in the bodies of rats, to evaluate lead removal from the internal organs and bones using calcium alginate in doses of 500, 200 and 100 mg/kg per day for 28 days and to assess the impact of calcium alginate on the level of essential elements. Lead (Pb), calcium (Ca), manganese (Mn), iron (Fe), copper (Cu) and zinc (Zn) levels in the blood, hearts, kidneys, livers and femurs of the experimental animals were measured using mass spectrometry with inductively coupled plasma. The results revealed that lead acetate exposure increased the levels of Pb in the blood and organs of the animals and significantly reduced contents of Ca, Mn, Fe, Cu and Zn. Treatment with calcium alginate in dose 500 mg/kg contributed to significant decreases in the amount of lead in the kidney, heart and bones of animals and a slight increase in the content of essential elements in the liver, kidneys and heart, although these changes were not significant. Decreasing of lead was not significant in the internal organs, bones and blood of animals treated with calcium alginate 200 and 100 mg/kg. Consequently, calcium alginate dose of 500 mg/kg more efficiently removes lead accumulated in the body. Calcium alginate does not have negative effect on level of essential elements quite the contrary; reducing the levels of lead, calcium alginate helps normalize imbalances of Ca, Mn, Fe, Cu and Zn. The results of this study suggest that calcium alginate may potentially be useful for the treatment and prevention of heavy metal intoxications.

  3. Altered fermentative metabolism in Chlamydomonas reinhardtii mutants lacking pyruvate formate lyase and both pyruvate formate lyase and alcohol dehydrogenase.

    PubMed

    Catalanotti, Claudia; Dubini, Alexandra; Subramanian, Venkataramanan; Yang, Wenqiang; Magneschi, Leonardo; Mus, Florence; Seibert, Michael; Posewitz, Matthew C; Grossman, Arthur R

    2012-02-01

    Chlamydomonas reinhardtii, a unicellular green alga, often experiences hypoxic/anoxic soil conditions that activate fermentation metabolism. We isolated three Chlamydomonas mutants disrupted for the pyruvate formate lyase (PFL1) gene; the encoded PFL1 protein catalyzes a major fermentative pathway in wild-type Chlamydomonas cells. When the pfl1 mutants were subjected to dark fermentative conditions, they displayed an increased flux of pyruvate to lactate, elevated pyruvate decarboxylation, ethanol accumulation, diminished pyruvate oxidation by pyruvate ferredoxin oxidoreductase, and lowered H(2) production. The pfl1-1 mutant also accumulated high intracellular levels of lactate, succinate, alanine, malate, and fumarate. To further probe the system, we generated a double mutant (pfl1-1 adh1) that is unable to synthesize both formate and ethanol. This strain, like the pfl1 mutants, secreted lactate, but it also exhibited a significant increase in the levels of extracellular glycerol, acetate, and intracellular reduced sugars and a decrease in dark, fermentative H(2) production. Whereas wild-type Chlamydomonas fermentation primarily produces formate and ethanol, the double mutant reroutes glycolytic carbon to lactate and glycerol. Although the metabolic adjustments observed in the mutants facilitate NADH reoxidation and sustained glycolysis under dark, anoxic conditions, the observed changes could not have been predicted given our current knowledge of the regulation of fermentation metabolism.

  4. Altered Fermentative Metabolism in Chlamydomonas reinhardtii Mutants Lacking Pyruvate Formate Lyase and Both Pyruvate Formate Lyase and Alcohol Dehydrogenase

    SciTech Connect

    Catalanotti, C.; Dubini, A.; Subramanian, V.; Yang, W. Q.; Magneschi, L.; Mus, F.; Seibert, M.; Posewitz, M. C.; Grossman, A. R.

    2012-02-01

    Chlamydomonas reinhardtii, a unicellular green alga, often experiences hypoxic/anoxic soil conditions that activate fermentation metabolism. We isolated three Chlamydomonas mutants disrupted for the pyruvate formate lyase (PFL1) gene; the encoded PFL1 protein catalyzes a major fermentative pathway in wild-type Chlamydomonas cells. When the pfl1 mutants were subjected to dark fermentative conditions, they displayed an increased flux of pyruvate to lactate, elevated pyruvate decarboxylation, ethanol accumulation, diminished pyruvate oxidation by pyruvate ferredoxin oxidoreductase, and lowered H2 production. The pfl1-1 mutant also accumulated high intracellular levels of lactate, succinate, alanine, malate, and fumarate. To further probe the system, we generated a double mutant (pfl1-1 adh1) that is unable to synthesize both formate and ethanol. This strain, like the pfl1 mutants, secreted lactate, but it also exhibited a significant increase in the levels of extracellular glycerol, acetate, and intracellular reduced sugars and a decrease in dark, fermentative H2 production. Whereas wild-type Chlamydomonas fermentation primarily produces formate and ethanol, the double mutant reroutes glycolytic carbon to lactate and glycerol. Although the metabolic adjustments observed in the mutants facilitate NADH reoxidation and sustained glycolysis under dark, anoxic conditions, the observed changes could not have been predicted given our current knowledge of the regulation of fermentation metabolism.

  5. Purification, characterization, and immunological cross-reactivity of alginates produced by mucoid Pseudomonas aeruginosa from patients with cystic fibrosis.

    PubMed

    Pedersen, S S; Espersen, F; Høiby, N; Shand, G H

    1989-04-01

    Alginates from nine mucoid Pseudomonas aeruginosa isolates from patients with cystic fibrosis were purified by repeated ethanol precipitation, nuclease digestion, anion-exchange chromatography, dialysis, and lyophilization. Uronic acid constituted 72% of the dry weight when mannuronolactone was used as the internal standard in the carbazole-borate assay for uronic acids. The average degree of acetylation was 16%, and the ratio of mannuronic acid to gluluronic acid was 4.7. No homopolymeric blocks of guluronic acid were found when analyzed by nuclear magnetic resonance spectroscopy. Contaminating proteins were denatured by heating, and during purification the content of protein relative to alginate fell from 566 to 0.9%. The content of lipopolysaccharide was 0.012%. No immunological or biological activity was attributable to the protein or lipopolysaccharide content as estimated by immunoblotting, enzyme-linked immunosorbent assay (ELISA), and a neutrophil chemotaxis assay. Rabbits were hyperimmunized with P. aeruginosa alginates and alginate from the seaweed Laminaria hyperborea, and an ELISA that detected alginate-specific antibodies was developed. Antibodies to P. aeruginosa alginate were detected by ELISA in 1:4,000 dilutions of serum from patients with cystic fibrosis with chronic P. aeruginosa lung infection. The serological cross-reactions between serum from the nine patients with cystic fibrosis and the corresponding P. aeruginosa alginates were investigated and showed considerable heterogeneity. This finding indicates that P. aeruginosa alginate from more than one P. aeruginosa strain should be used in serological tests. There was no serological cross-reactivity between P. aeruginosa and Laminaria hyperborea alginate in either rabbits or patients with cystic fibrosis.

  6. Phycobilin:cystein-84 biliprotein lyase, a near-universal lyase for cysteine-84-binding sites in cyanobacterial phycobiliproteins.

    PubMed

    Zhao, Kai-Hong; Su, Ping; Tu, Jun-Ming; Wang, Xing; Liu, Hui; Plöscher, Matthias; Eichacker, Lutz; Yang, Bei; Zhou, Ming; Scheer, Hugo

    2007-09-04

    Phycobilisomes, the light-harvesting complexes of cyanobacteria and red algae, contain two to four types of chromophores that are attached covalently to seven or more members of a family of homologous proteins, each carrying one to four binding sites. Chromophore binding to apoproteins is catalyzed by lyases, of which only few have been characterized in detail. The situation is complicated by nonenzymatic background binding to some apoproteins. Using a modular multiplasmidic expression-reconstitution assay in Escherichia coli with low background binding, phycobilin:cystein-84 biliprotein lyase (CpeS1) from Anabaena PCC7120, has been characterized as a nearly universal lyase for the cysteine-84-binding site that is conserved in all biliproteins. It catalyzes covalent attachment of phycocyanobilin to all allophycocyanin subunits and to cysteine-84 in the beta-subunits of C-phycocyanin and phycoerythrocyanin. Together with the known lyases, it can thereby account for chromophore binding to all binding sites of the phycobiliproteins of Anabaena PCC7120. Moreover, it catalyzes the attachment of phycoerythrobilin to cysteine-84 of both subunits of C-phycoerythrin. The only exceptions not served by CpeS1 among the cysteine-84 sites are the alpha-subunits from phycocyanin and phycoerythrocyanin, which, by sequence analyses, have been defined as members of a subclass that is served by the more specialized E/F type lyases.

  7. Ulvan Lyases Isolated from the Flavobacteria Persicivirga ulvanivorans Are the First Members of a New Polysaccharide Lyase Family*

    PubMed Central

    Nyvall Collén, Pi; Sassi, Jean-François; Rogniaux, Hélène; Marfaing, Hélène; Helbert, William

    2011-01-01

    Ulvans are complex sulfated polysaccharides found in the cell walls of green algae belonging to the genus Ulva. These polysaccharides are composed of disaccharide repetition moieties made up of sulfated rhamnose linked to either glucuronic acid, iduronic acid, or xylose. Two ulvan lyases of 30 and 46 kDa were purified from the culture supernatant of Persicivirga ulvanivorans. Based on peptide sequencing, the gene encoding the 46-kDa ulvan lyase was cloned. Sequence analysis revealed that the protein is modular and possesses a catalytic module similar to that of the 30-kDa ulvan lyase along with a module of unknown function. The ulvan-degrading function of the gene was confirmed by expression of the catalytic module in a heterologous system. The gene encoding the catalytic module has no sequence homolog in sequence databases and is likely to be the first member of a novel polysaccharide lyase family. Analysis of degradation products showed that both the 30- and 46-kDa ulvan lyases are endolytic and cleave the glycosidic bond between the sulfated rhamnose and a glucuronic or iduronic acid. PMID:22009751

  8. Controlled release of carbofuran from an alginate-bentonite formulation: water release kinetics and soil mobility.

    PubMed

    Fernández-Pérez, M; Villafranca-Sánchez, M; González-Pradas, E; Martinez-López, F; Flores-Céspedes, F

    2000-03-01

    The insecticide-nematicide carbofuran was incorporated in alginate-based granules to obtain controlled-release (CR) properties. The basic formulation [sodium alginate (1.61%)-carbofuran (0. 59%)-water] was modified by addition of sorbents. The effect on carbofuran release rate, caused by the incorporation of natural and acid-treated bentonite (0.5 and 1.0 M H(2)SO(4)) in alginate formulation, was studied by immersion of the granules in water under shaking. The time taken for 50% of the active ingredient to be released into water, t(50), was longer for those formulations containing natural bentonite (6.1 h) or acid-treated bentonite (9.0 and 11.7 h for 0.5 and 1.0 M H(2)SO(4) treatments, respectively) than for the preparation without bentonite (4.7 h). It appears from the results that the release of carbofuran from the various formulations is controlled by a diffusion mechanism according to the n values obtained, which were close to 0.5 in all cases. The mobility of carbofuran from alginate-based CR formulations was investigated by using soil columns packed with a clay soil (53% clay and 0.08% organic matter). Two alginate-based CR formulations containing natural bentonite or acid-treated bentonite (0.5 M H(2)SO(4)) were compared to technical grade carbofuran. The use of alginate-based CR formulations resulted in a reduction of the leached amount of carbofuran compared with the total amount of pesticide leached using the technical product (50 and 75% for CR granules containing natural and acid-treated bentonite, respectively). Alginate-bentonite CR formulations might be efficient systems for reducing carbofuran leaching in clay soils, which would reduce the risk of groundwater pollution.

  9. Preparation of in situ hardening composite microcarriers: calcium phosphate cement combined with alginate for bone regeneration.

    PubMed

    Park, Jung-Hui; Lee, Eun-Jung; Knowles, Jonathan C; Kim, Hae-Won

    2014-03-01

    Novel microcarriers consisting of calcium phosphate cement and alginate were prepared for use as three-dimensional scaffolds for the culture and expansion of cells that are effective for bone tissue engineering. The calcium phosphate cement-alginate composite microcarriers were produced by an emulsification of the composite aqueous solutions mixed at varying ratios (calcium phosphate cement powder/alginate solution = 0.8-1.2) in an oil bath and the subsequent in situ hardening of the compositions during spherodization. Moreover, a porous structure could be easily created in the solid microcarriers by soaking the produced microcarriers in water and a subsequent freeze-drying process. Bone mineral-like apatite nanocrystallites were shown to rapidly develop on the calcium phosphate cement-alginate microcarriers under moist conditions due to the conversion of the α-tricalcium phosphate phase in the calcium phosphate cement into a carbonate-hydroxyapatite. Osteoblastic cells cultured on the microspherical scaffolds were proven to be viable, with an active proliferative potential during 14 days of culture, and their osteogenic differentiation was confirmed by the determination of alkaline phosphatase activity. The in situ hardening calcium phosphate cement-alginate microcarriers developed herein may be used as potential three-dimensional scaffolds for cell delivery and tissue engineering of bone.

  10. Preparation of in situ hardening composite microcarriers: Calcium phosphate cement combined with alginate for bone regeneration

    PubMed Central

    Park, Jung-Hui; Lee, Eun-Jung; Knowles, Jonathan C

    2014-01-01

    Novel microcarriers consisting of calcium phosphate cement and alginate were prepared for use as three-dimensional scaffolds for the culture and expansion of cells that are effective for bone tissue engineering. The calcium phosphate cement-alginate composite microcarriers were produced by an emulsification of the composite aqueous solutions mixed at varying ratios (calcium phosphate cement powder/alginate solution = 0.8–1.2) in an oil bath and the subsequent in situ hardening of the compositions during spherodization. Moreover, a porous structure could be easily created in the solid microcarriers by soaking the produced microcarriers in water and a subsequent freeze-drying process. Bone mineral-like apatite nanocrystallites were shown to rapidly develop on the calcium phosphate cement–alginate microcarriers under moist conditions due to the conversion of the α-tricalcium phosphate phase in the calcium phosphate cement into a carbonate–hydroxyapatite. Osteoblastic cells cultured on the microspherical scaffolds were proven to be viable, with an active proliferative potential during 14 days of culture, and their osteogenic differentiation was confirmed by the determination of alkaline phosphatase activity. The in situ hardening calcium phosphate cement–alginate microcarriers developed herein may be used as potential three-dimensional scaffolds for cell delivery and tissue engineering of bone. PMID:23836845

  11. Control of Alginate Core Size in Alginate-Poly (Lactic-Co-Glycolic) Acid Microparticles

    NASA Astrophysics Data System (ADS)

    Lio, Daniel; Yeo, David; Xu, Chenjie

    2016-01-01

    Core-shell alginate-poly (lactic-co-glycolic) acid (PLGA) microparticles are potential candidates to improve hydrophilic drug loading while facilitating controlled release. This report studies the influence of the alginate core size on the drug release profile of alginate-PLGA microparticles and its size. Microparticles are synthesized through double-emulsion fabrication via a concurrent ionotropic gelation and solvent extraction. The size of alginate core ranges from approximately 10, 50, to 100 μm when the emulsification method at the first step is homogenization, vortexing, or magnetic stirring, respectively. The second step emulsification for all three conditions is performed with magnetic stirring. Interestingly, although the alginate core has different sizes, alginate-PLGA microparticle diameter does not change. However, drug release profiles are dramatically different for microparticles comprising different-sized alginate cores. Specifically, taking calcein as a model drug, microparticles containing the smallest alginate core (10 μm) show the slowest release over a period of 26 days with burst release less than 1 %.

  12. Quantitation of heparosan with heparin lyase III and spectrophotometry.

    PubMed

    Huang, Haichan; Zhao, Yingying; Lv, Shencong; Zhong, Weihong; Zhang, Fuming; Linhardt, Robert J

    2014-02-15

    Heparosan is Escherichia coli K5 capsule polysaccharide, which is the key precursor for preparing bioengineered heparin. A rapid and effective quantitative method for detecting heparosan is important in the large-scale production of heparosan. Heparin lyase III (Hep III) effectively catalyzes the heparosan depolymerization, forming unsaturated disaccharides that are measurable using a spectrophotometer at 232 nm. We report a new method for the quantitative detection of heparosan with heparin lyase III and spectrophotometry that is safer and more specific than the traditional carbazole assay. In an optimized detection system, heparosan at a minimum concentration of 0.60 g/L in fermentation broth can be detected.

  13. Alginate/polyoxyethylene and alginate/gelatin hydrogels: preparation, characterization, and application in tissue engineering.

    PubMed

    Aroguz, Ayse Z; Baysal, Kemal; Adiguzel, Zelal; Baysal, Bahattin M

    2014-05-01

    Hydrogels are attractive biomaterials for three-dimensional cell culture and tissue engineering applications. The preparation of hydrogels using alginate and gelatin provides cross-linked hydrophilic polymers that can swell but do not dissolve in water. In this work, we first reinforced pure alginate by using polyoxyethylene as a supporting material. In an alginate/PEO sample that contains 20 % polyoxyethylene, we obtained a stable hydrogel for cell culture experiments. We also prepared a stable alginate/gelatin hydrogel by cross-linking a periodate-oxidized alginate with another functional component such as gelatin. The hydrogels were found to have a high fluid uptake. In this work, preparation, characterization, swelling, and surface properties of these scaffold materials were described. Lyophilized scaffolds obtained from hydrogels were used for cell viability experiments, and the results were presented in detail.

  14. Salicylanilide pyrazinoates inhibit in vitro multidrug-resistant Mycobacterium tuberculosis strains, atypical mycobacteria and isocitrate lyase.

    PubMed

    Krátký, Martin; Vinšová, Jarmila; Novotná, Eva; Stolaříková, Jiřina

    2014-03-12

    The development of antimicrobial agents represents an up-to-date topic. This study investigated in vitro antimycobacterial activity, mycobacterial isocitrate lyase inhibition and cytotoxicity of salicylanilide pyrazinoates. They may be considered being mutual prodrugs of both antimycobacterial active salicylanilides and pyrazinoic acid (POA), an active metabolite of pyrazinamide, in which these esters are likely hydrolysed without presence of pyrazinamidase/nicotinamidase. Minimum inhibitory concentrations (MICs) of the esters were within the range 0.5-8 μmol/l for Mycobacterium tuberculosis and 1-32 μmol/l for nontuberculous mycobacteria (Mycobacterium avium, Mycobacterium kansasii). All esters showed a weak inhibition (8-17%) of isocitrate lyase at the concentration of 10 μmol/l. The most active pyrazinoates showed MICs for multidrug-resistant tuberculosis strains in the range of 0.125-2 μmol/l and no cross-resistance with clinically used drugs, thus being the most in vitro efficacious salicylanilide esters with 4-chloro-2-{[4-(trifluoromethyl)phenyl]carbamoyl}phenyl pyrazine-2-carboxylate superiority (MICs⩽0.25 μmol/l). This promising activity is likely due to an additive or synergistic effect of released POA and salicylanilides. Selectivity indexes for the most active salicylanilide pyrazinoates ranged up to 64, making some derivatives being attractive candidates for the next research; 4-bromo-2-{[4-(trifluoromethyl)phenyl]carbamoyl}phenyl pyrazine-2-carboxylate showed the most convenient toxicity profile.

  15. Differential HMG-CoA lyase expression in human tissues provides clues about 3-hydroxy-3-methylglutaric aciduria

    PubMed Central

    Puisac, Beatriz; Arnedo, María; Casale, Cesar H.; Ribate, María Pilar; Castiella, Tomás; Ramos, Feliciano J.; Ribes, Antonia; Pérez-Cerdá, Celia; Casals, Nuria; Hegardt, Fausto G.

    2010-01-01

    3-Hydroxy-3-methylglutaric aciduria is a rare human autosomal recessive disorder caused by deficiency of 3-hydroxy-3-methylglutaryl CoA lyase (HL). This mitochondrial enzyme catalyzes the common final step of leucine degradation and ketogenesis. Acute symptoms include vomiting, seizures and lethargy, accompanied by metabolic acidosis and hypoketotic hypoglycaemia. Such organs as the liver, brain, pancreas, and heart can also be involved. However, the pathophysiology of this disease is only partially understood. We measured mRNA levels, protein expression and enzyme activity of human HMG-CoA lyase from liver, kidney, pancreas, testis, heart, skeletal muscle, and brain. Surprisingly, the pancreas is, after the liver, the tissue with most HL activity. However, in heart and adult brain, HL activity was not detected in the mitochondrial fraction. These findings contribute to our understanding of the enzyme function and the consequences of its deficiency and suggest the need for assessment of pancreatic damage in these patients. PMID:20532825

  16. Crystallization and preliminary X-ray analysis of the rhamnogalacturonan lyase YesW from Bacillus subtilis strain 168, a member of polysaccharide lyase family 11

    SciTech Connect

    Ochiai, Akihito; Yamasaki, Masayuki; Itoh, Takafumi; Mikami, Bunzo; Hashimoto, Wataru; Murata, Kousaku

    2006-05-01

    The crystallization and preliminary X-ray characterization of the polysaccharide lyase family 11 rhamnogalacturonan lyase are presented. Rhamnogalacturonan lyases degrade rhamnogalacturonan I, a major component of pectin, through a β-elimination reaction. YesW from Bacillus subtilis strain 168 is a novel rhamnogalacturonan lyase classified into polysaccharide lyase family 11 (PL-11). The enzyme was crystallized at 293 K using the sitting-drop vapour-diffusion method with 2-methyl-2,4-pentanediol (MPD) as a precipitant. Preliminary X-ray analysis revealed that the YesW crystals belong to space group P2{sub 1} and diffract to 2.40 Å resolution, with unit-cell parameters a = 56.7, b = 105.6, c = 101.4 Å, β = 94.9°. This is the first report on the crystallization and preliminary X-ray analysis of a family PL-11 rhamnogalacturonan lyase.

  17. Purification and Characterization of l-Methionine γ-Lyase from Brevibacterium linens BL2†

    PubMed Central

    Dias, Benjamin; Weimer, Bart

    1998-01-01

    l-Methionine γ-lyase (EC 4.4.1.11) was purified to homogeneity from Brevibacterium linens BL2, a coryneform bacterium which has been used successfully as an adjunct bacterium to improve the flavor of Cheddar cheese. The enzyme catalyzes the α,γ elimination of methionine to produce methanethiol, α-ketobutyrate, and ammonia. It is a pyridoxal phosphate-dependent enzyme, with a native molecular mass of approximately 170 kDa, consisting of four identical subunits of 43 kDa each. The purified enzyme had optimum activity at pH 7.5 and was stable at pHs ranging from 6.0 to 8.0 for 24 h. The pure enzyme had its highest activity at 25°C but was active between 5 and 50°C. Activity was inhibited by carbonyl reagents, completely inactivated by dl-propargylglycine, and unaffected by metal-chelating agents. The pure enzyme had catalytic properties similar to those of l-methionine γ-lyase from Pseudomonas putida. Its Km for the catalysis of methionine was 6.12 mM, and its maximum rate of catalysis was 7.0 μmol min−1 mg−1. The enzyme was active under salt and pH conditions found in ripening Cheddar cheese but susceptible to degradation by intracellular proteases. PMID:9726878

  18. Purification and characterization of L-methionine gamma-lyase from brevibacterium linens BL2

    PubMed

    Dias; Weimer

    1998-09-01

    L-Methionine gamma-lyase (EC 4.4.1.11) was purified to homogeneity from Brevibacterium linens BL2, a coryneform bacterium which has been used successfully as an adjunct bacterium to improve the flavor of Cheddar cheese. The enzyme catalyzes the alpha,gamma elimination of methionine to produce methanethiol, alpha-ketobutyrate, and ammonia. It is a pyridoxal phosphate-dependent enzyme, with a native molecular mass of approximately 170 kDa, consisting of four identical subunits of 43 kDa each. The purified enzyme had optimum activity at pH 7.5 and was stable at pHs ranging from 6.0 to 8.0 for 24 h. The pure enzyme had its highest activity at 25 degreesC but was active between 5 and 50 degreesC. Activity was inhibited by carbonyl reagents, completely inactivated by DL-propargylglycine, and unaffected by metal-chelating agents. The pure enzyme had catalytic properties similar to those of L-methionine gamma-lyase from Pseudomonas putida. Its Km for the catalysis of methionine was 6.12 mM, and its maximum rate of catalysis was 7.0 &mgr;mol min-1 mg-1. The enzyme was active under salt and pH conditions found in ripening Cheddar cheese but susceptible to degradation by intracellular proteases.

  19. Performance of alginate films for retention of L-(+)-ascorbic acid.

    PubMed

    De'Nobili, M D; Curto, L M; Delfino, J M; Soria, M; Fissore, E N; Rojas, A M

    2013-06-25

    In view of acting as controlled delivery systems for nutritional supplementation, therapy or antioxidant activity at interfaces, alginate films of different copolymer composition and glycerol plasticizer levels were developed in the presence of Ca(2+) for achieving higher stability of L-(+)-ascorbic acid (AA). The ability of the alginate network to preserve AA from hydrolysis, tested by storage under vacuum at 25 °C, only decreased with the relative humidity (RH) increase when alginates were mainly constituted by guluronic-guluronic acid blocks (GG), whereas also decreased with the glycerol level increase when mannuronic-mannuronic acid (MM) and/or alternating guluronic-mannuronic (GM+MG) flexible blocks were present in higher proportions. This result could be probably related to the lower capability of the latter alginate block compositions to immobilize water in the network as they are not able to constitute Ca(2+) mediated junction zones where water molecules are highly retained. Films also studied under air storage showed that even at less favorable conditions of RH and glycerol levels, both GG and GM+MG enriched alginate networks in general preserved AA from oxidation. It also demonstrated that hydrolysis is the principal way by which AA is lost when supported in films.

  20. Affinity-based growth factor delivery using biodegradable, photocrosslinked heparin-alginate hydrogels

    PubMed Central

    Jeon, Oju; Powell, Caitlin; Solorio, Loran D.; Krebs, Melissa D.; Alsberg, Eben

    2013-01-01

    Photocrosslinkable biomaterials are promising for tissue engineering applications due to their capacity to be injected and form hydrogels in situ in a minimally invasive manner. Our group recently reported on the development of photocrosslinked alginate hydrogels with controlled biodegradation rates, mechanical properties, and cell adhesive properties. In this study, we present an affinity-based growth factor delivery system by incorporating heparin into photocrosslinkable alginate hydrogels (HP-ALG), which allows for controlled, prolonged release of therapeutic proteins. Heparin modification had minimal effect on the biodegradation profiles, swelling ratios, and elastic moduli of the hydrogels in media. The release profiles of growth factors from this affinity-based platform were sustained for 3 weeks with no initial burst release, and the released growth factors retained their biological activity. Implantation of bone morphogenetic protein-2 (BMP-2)-loaded photocrosslinked alginate hydrogels induced moderate bone formation around the implant periphery. Importantly, BMP-2-loaded photocrosslinked HP-ALG hydrogels induced significantly more osteogenesis than BMP-2-loaded photocrosslinked unmodified alginate hydrogels, with 1.9-fold greater peripheral bone formation and 1.3-fold greater calcium content in the BMP-2-loaded photocrosslinked HP-ALG hydrogels compared to the BMP-2-loaded photocrosslinked unmodified alginate hydrogels after 8 weeks implantation. This sustained and controllable growth factor delivery system, with independently controllable physical and cell adhesive properties, may provide a powerful modality for a variety of therapeutic applications. PMID:21745508

  1. Cloning and expression of hyaluronate lyase genes of Streptococcus intermedius and Streptococcus constellatus subsp. constellatus(1).

    PubMed

    Takao, Ayuko

    2003-02-14

    Hyaluronate lyase (HAase) genes of Streptococcus intermedius and Streptococcus constellatus subsp. constellatus were isolated. In S. constellatus subsp. constellatus, the deduced amino acid sequence of HAase was most similar to that of S. intermedius (68%), whereas the enzyme of S. intermedius was most similar to that of S. pneumoniae (72%). Upstream of the HAase gene on the opposite strands, an open reading frame of a putative glutathione peroxidase started in S. intermedius, and this arrangement was similar to that in S. pneumoniae but unlike that in S. constellatus subsp. constellatus. Cell lysates of Escherichia coli carrying each streptococcal gene showed HAase activity, demonstrating that each cloned gene actually coded for HAase.

  2. Bacterial Anabaena variabilis phenylalanine ammonia lyase: a biocatalyst with broad substrate specificity.

    PubMed

    Lovelock, Sarah L; Turner, Nicholas J

    2014-10-15

    Phenylalanine ammonia lyases (PALs) catalyse the regio- and stereoselective hydroamination of cinnamic acid analogues to yield optically enriched α-amino acids. Herein, we demonstrate that a bacterial PAL from Anabaena variabilis (AvPAL) displays significantly higher activity towards a series of non-natural substrates than previously described eukaryotic PALs. Biotransformations performed on a preparative scale led to the synthesis of the 2-chloro- and 4-trifluoromethyl-phenylalanine derivatives in excellent ee, highlighting the enormous potential of bacterial PALs as biocatalysts for the synthesis of high value, non-natural amino acids.

  3. Synthesis of cadmium, lead and copper alginate nanobeads as immunosensing probes for the detection of AFP, CEA and PSA.

    PubMed

    Wang, Zifeng; Liu, Na; Feng, Feng; Ma, Zhanfang

    2015-08-15

    A double-water-in-oil-emulsion procedure was designed to synthesize cadmium, lead and copper alginate nanobeads less than 200n m diameter under mild conditions. The cadmium, lead and copper alginate nanobeads can be activated to immobilize biomacromolecules and can directly produce distinctive electrochemical signals. Using the novel alginate nanobeads labeled with antibodies as electrochemical probes, a sandwich-type immunosensor was constructed using AFP, CEA and PSA as model analytes. This proposed immunosensor shows wide linear range with detection limits of 0.01, 0.0086 and 0.0075 ng mL(-1) for AFP, CEA and PSA, respectively. Analysis of clinical serum samples using this immunosensor was well consistent with the data determined by the enzyme-linked immunosorbent assay (ELISA). It suggested that the alginate nanobeads electrochemical probes could be generally extended to other multiple analytes detection.

  4. Characterization of recombinant pectate lyase refolded from inclusion bodies generated in E. coli BL21(DE3).

    PubMed

    Kumar, Sandeep; Jain, Kavish Kumar; Singh, Anupam; Panda, Amulya K; Kuhad, Ramesh Chander

    2015-06-01

    Pectate lyase (EC 4.2.2.2) gene from Bacillus subtilis RCK was cloned and expressed in Escherichia coli to maximize its production. In addition to soluble fraction, bioactive pectate lyase was also obtained from inclusion body aggregates by urea solubilization and refolding under in vitro conditions. Enzyme with specific activity ∼3194IU/mg and ∼1493IU/mg were obtained from soluble and inclusion bodies (IBs) fraction with recovery of 56% and 74% in terms of activity, respectively. The recombinant enzyme was moderately thermostable (t1/2 60min at 50°C) and optimally active in wider alkaline pH range (7.0-10.5). Interaction of protein with its cofactor CaCl2 was found to stimulate the change in tertiary structure as revealed by near UV CD spectra. Intrinsic tryptophan fluorescence spectra indicated that tryptophan is involved in substrate binding and there might be independent binding of Ca(2+) and polygalacturonic acid to the active site. The recombinant enzyme was found to be capable of degrading pectin and polygalacturonic acid. The work reports novel conditions for refolding to obtain active recombinant pectate lyase from inclusion bodies and elucidates the effect of ligand and substrate binding on protein conformation by circular dichroism (CD) and fluorescence spectrofluorometry.

  5. Sargassum filipendula alginate from Brazil: seasonal influence and characteristics.

    PubMed

    Bertagnolli, Caroline; Espindola, Ana Paula D M; Kleinübing, Sirlei Jaiana; Tasic, Ljubica; da Silva, Meuris Gurgel Carlos

    2014-10-13

    The aim of this work is focused on the extraction and characterization of the Brazilian seaweed Sargassum filipendula alginate. Alginates obtained at different seasons were characterized by liquid state nuclear magnetic resonance spectroscopy and scanning electron microscopy. The alginate extraction efficiency was about 20%. Different seasons of the year and different stages in the life cycle of Sargassum sp. in southeastern Brazil influenced the M/G and, consequently, the technological properties of extracted alginates.

  6. Dietary administration of sodium alginate enhances the immune ability of white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus.

    PubMed

    Cheng, Winton; Liu, Chun-Hung; Kuo, Ching-Ming; Chen, Jiann-Chu

    2005-01-01

    Haemocyte count, phenoloxidase activity, respiratory burst (release of superoxide anion), superoxide dismutase (SOD) activity, glutathione peroxidase (GPX) activity, phagocytic activity and clearance efficiency to the pathogen Vibrio alginolyticus were measured in white shrimp Litopenaeus vannamei juveniles (12.3 +/- 1.2 g) which had been fed diets containing sodium alginate at 0.5, 1.0, 2.0 g kg(-1) after five months. L. vannamei fed a diet containing 2.0 g kg(-1) sodium alginate had increased phenoloxidase activity, respiratory burst and SOD activity, but decreased GPX activity significantly. L. vannamei fed a diet containing 2.0 g kg(-1) sodium alginate had increased phagocytic activity and the shrimp fed a diet containing sodium alginate at 0.5, 1.0 or 2.0 g kg(-1) had increased clearance efficiency to V. alginolyticus. In another experiment, L. vannamei, which had been fed control diet, or sodium alginate-containing diets after 5 months, were challenged with V. alginolyticus at 2 x 10(6) colony-forming units (CFU) shrimp(-1) and then placed in seawater of 15 per thousand. The survival of shrimp fed a diet containing 2.0 g kg(-1) after one day, and the survival of shrimp fed diets containing sodium alginate at 0.5 and 1.0 g kg(-1) after 2-4 days increased significantly, as compared to that of shrimp fed control diet. It is therefore concluded that administration of sodium alginate in the diet at 2.0 g kg(-1) or less could enhance the immune ability of L. vannamei and increase its resistance to V. alginolyticus infection.

  7. In Silico Characterization of Pectate Lyase Protein Sequences from Different Source Organisms

    PubMed Central

    Dubey, Amit Kumar; Yadav, Sangeeta; Kumar, Manish; Singh, Vinay Kumar; Sarangi, Bijaya Ketan; Yadav, Dinesh

    2010-01-01

    A total of 121 protein sequences of pectate lyases were subjected to homology search, multiple sequence alignment, phylogenetic tree construction, and motif analysis. The phylogenetic tree constructed revealed different clusters based on different source organisms representing bacterial, fungal, plant, and nematode pectate lyases. The multiple accessions of bacterial, fungal, nematode, and plant pectate lyase protein sequences were placed closely revealing a sequence level similarity. The multiple sequence alignment of these pectate lyase protein sequences from different source organisms showed conserved regions at different stretches with maximum homology from amino acid residues 439–467, 715–816, and 829–910 which could be used for designing degenerate primers or probes specific for pectate lyases. The motif analysis revealed a conserved Pec_Lyase_C domain uniformly observed in all pectate lyases irrespective of variable sources suggesting its possible role in structural and enzymatic functions. PMID:21048874

  8. 21 CFR 172.858 - Propylene glycol alginate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Propylene glycol alginate. 172.858 Section 172.858... Propylene glycol alginate. The food additive propylene glycol alginate (CAS Reg. No. 9005-37-2) may be used... the act: (1) The name of the additive, “propylene glycol alginate” or “propylene glycol ester...

  9. Preparation, characterization, and sensing behavior of polydiacetylene liposomes embedded in alginate fibers.

    PubMed

    Kauffman, Jennifer S; Ellerbrock, Brett M; Stevens, Kathryn A; Brown, Philip J; Pennington, William T; Hanks, Timothy W

    2009-06-01

    Polydiacetylene (PDA)-doped calcium alginate fibers were created by the solution blending of polymerized 10,12-pentacosadiynoic acid liposomes with sodium alginate in water prior to extrusion. The liposomes maintained their blue color during wet spinning and drying of the fibers but changed to red with exposure to specific external stimuli (heat, solvent, and chemical). In the latter case, the color change only occurred when the fibers were sufficiently permeable for the reacting species to reach the interior. A parameter termed the "Raman response" (RR) has been developed to quantify the amount of PDA liposomes in each of two critical conformations within the fibers. The RR attributes a quantitative measure of PDA response to individual stimuli. This method provides advantages over the commonly used "colorimetric response" in systems where sample limitations and chromophore activity make UV-vis spectroscopic measurements difficult or inaccurate. PDA liposomes are shown to effectively add a versatile sensing component to alginate fibers.

  10. Assessment of the Behavior of Mesenchymal Stem Cells Immobilized in Biomimetic Alginate Microcapsules.

    PubMed

    Garate, Ane; Ciriza, Jesús; Casado, Javier G; Blazquez, Rebeca; Pedraz, José Luis; Orive, Gorka; Hernandez, Rosa Maria

    2015-11-02

    The combination of mesenchymal stem cells (MSCs) and biomimetic matrices for cell-based therapies has led to enormous advances, including the field of cell microencapsulation technology. In the present work, we have evaluated the potential of genetically modified MSCs from mice bone marrow, D1-MSCs, immobilized in alginate microcapsules with different RGD (Arg-Gly-Asp) densities. Results demonstrated that the microcapsules represent a suitable platform for D1-MSC encapsulation since cell immobilization into alginate matrices does not affect their main characteristics. The in vitro study showed a higher activity of D1-MSCs when they are immobilized in RGD-modified alginate microcapsules, obtaining the highest therapeutic factor secretion with low and intermediate densities of the bioactive molecule. In addition, the inclusion of RGD increased the differentiation potential of immobilized cells upon specific induction. However, subcutaneous implantation did not induce differentiation of D1-MSCs toward any lineage remaining at an undifferentiated state in vivo.

  11. Antimicrobial Effects of Silver Nanoparticles Stabilized in Solution by Sodium Alginate

    PubMed Central

    Kubyshkin, Anatoliy; Chegodar, Denis; Katsev, Andrew; Petrosyan, Armen; Krivorutchenko, Yuri; Postnikova, Olga

    2016-01-01

    Background/purpose To investigate the effect of nanosilver particles in solution stabilized in a matrix of sodium alginate on the growth and development of pathogenic bacteria such as Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Proteus vulgaris, Enterobacter cloacae, the antibiotic-resistant strain of Pseudomonas aeruginosa, the yeast-like fungus Candida albicans, and the luminescent bacteria Photobacterium leiognathi Sh1. Methods Isolates of pathogenic bacteria obtained from bronchoalveolar and peritoneal lavage samples from Wistar rats with experimental pneumonia and peritonitis were tested for their susceptibility to silver nanoparticles in solution with an alginate stabilizer. The antifungal activity of silver nanoparticles in sodium alginate was studied for C. albicans (strain CCM885) using the Sabouraud agar method. The biocidal impact of silver nanoparticles in solution with a sodium alginate matrix on the luminescent bacteria P. leiognathi Sh1 was investigated using a BLM 8801 luminometer. Results It was observed that a 0.02-0.05% nanosilver solution with an alginate stabilizer limits the growth and development of pathogenic bacteria within the first 24 hours of exposure. If the concentration of nanosilver solution is 0.0005-0.05%, it inhibits the viability of the fungus C. albicans. A nanosilver solution at a concentration of 0.05-0.2 μg/mL represses bioluminescence in the bacteria P. leiognathi Sh1. From these results, it appears that the biocidal effect of nanosilver is related either to the presence of ions that are formed during dissolution, or to the availability of nanoparticles that interrupt the membrane permeability of bacterial cells. Conclusion Silver nanoparticles stabilized in a solution of sodium alginate possess significant in vitro antimicrobial activity, which is manifested by inhibition of the bioluminescence of P. leiognathi Sh1, and inhibition of the growth and development of the pathogenic bacteria S. aureus, E

  12. Chondroitin Lyase from a Marine Arthrobacter sp. MAT3885 for the Production of Chondroitin Sulfate Disaccharides.

    PubMed

    Kale, Varsha; Friðjónsson, Ólafur; Jónsson, Jón Óskar; Kristinsson, Hörður G; Ómarsdóttir, Sesselja; Hreggviðsson, Guðmundur Ó

    2015-08-01

    Chondroitin sulfate (CS) saccharides from cartilage tissues have potential application in medicine or as dietary supplements due to their therapeutic bioactivities. Studies have shown that depolymerized CS saccharides may display enhanced bioactivity. The objective of this study was to isolate a CS-degrading enzyme for an efficient production of CS oligo- or disaccharides. CS-degrading bacteria from marine environments were enriched using in situ artificial support colonization containing CS from shark cartilage as substrate. Subsequently, an Arthrobacter species (strain MAT3885) efficiently degrading CS was isolated from a CS enrichment culture. The genomic DNA from strain MAT3885 was pyro-sequenced by using the 454 FLX sequencing technology. Following assembly and annotation, an orf, annotated as family 8 polysaccharide lyase genes, was identified, encoding an amino acid sequence with a similarity to CS lyases according to NCBI blastX. The gene, designated choA1, was cloned in Escherichia coli and expressed downstream of and in frame with the E. coli malE gene for obtaining a high yield of soluble recombinant protein. Applying a dual-tag system (MalE-Smt3-ChoA1), the MalE domain was separated from ChoA1 with proteolytic cleavage using Ulp1 protease. ChoA1 was defined as an AC-type enzyme as it degraded chondroitin sulfate A, C, and hyaluronic acid. The optimum activity of the enzyme was at pH 5.5-7.5 and 40 °C, running a 10-min reaction. The native enzyme was estimated to be a monomer. As the recombinant chondroitin sulfate lyase (designated as ChoA1R) degraded chondroitin sulfate efficiently compared to a benchmark enzyme, it may be used for the production of chondroitin sulfate disaccharides for the food industry or health-promoting products.

  13. Sphingosine-1-Phosphate Lyase Deficient Cells as a Tool to Study Protein Lipid Interactions

    PubMed Central

    Gerl, Mathias J.; Bittl, Verena; Kirchner, Susanne; Sachsenheimer, Timo; Brunner, Hanna L.; Lüchtenborg, Christian; Özbalci, Cagakan; Wiedemann, Hannah; Wegehingel, Sabine; Nickel, Walter; Haberkant, Per; Schultz, Carsten; Krüger, Marcus; Brügger, Britta

    2016-01-01

    Cell membranes contain hundreds to thousands of individual lipid species that are of structural importance but also specifically interact with proteins. Due to their highly controlled synthesis and role in signaling events sphingolipids are an intensely studied class of lipids. In order to investigate their metabolism and to study proteins interacting with sphingolipids, metabolic labeling based on photoactivatable sphingoid bases is the most straightforward approach. In order to monitor protein-lipid-crosslink products, sphingosine derivatives containing a reporter moiety, such as a radiolabel or a clickable group, are used. In normal cells, degradation of sphingoid bases via action of the checkpoint enzyme sphingosine-1-phosphate lyase occurs at position C2-C3 of the sphingoid base and channels the resulting hexadecenal into the glycerolipid biosynthesis pathway. In case the functionalized sphingosine looses the reporter moiety during its degradation, specificity towards sphingolipid labeling is maintained. In case degradation of a sphingosine derivative does not remove either the photoactivatable or reporter group from the resulting hexadecenal, specificity towards sphingolipid labeling can be achieved by blocking sphingosine-1-phosphate lyase activity and thus preventing sphingosine derivatives to be channeled into the sphingolipid-to-glycerolipid metabolic pathway. Here we report an approach using clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated nuclease Cas9 to create a sphingosine-1-phosphate lyase (SGPL1) HeLa knockout cell line to disrupt the sphingolipid-to-glycerolipid metabolic pathway. We found that the lipid and protein compositions as well as sphingolipid metabolism of SGPL1 knock-out HeLa cells only show little adaptations, which validates these cells as model systems to study transient protein-sphingolipid interactions. PMID:27100999

  14. Sphingosine-1-Phosphate Lyase Deficient Cells as a Tool to Study Protein Lipid Interactions.

    PubMed

    Gerl, Mathias J; Bittl, Verena; Kirchner, Susanne; Sachsenheimer, Timo; Brunner, Hanna L; Lüchtenborg, Christian; Özbalci, Cagakan; Wiedemann, Hannah; Wegehingel, Sabine; Nickel, Walter; Haberkant, Per; Schultz, Carsten; Krüger, Marcus; Brügger, Britta

    2016-01-01

    Cell membranes contain hundreds to thousands of individual lipid species that are of structural importance but also specifically interact with proteins. Due to their highly controlled synthesis and role in signaling events sphingolipids are an intensely studied class of lipids. In order to investigate their metabolism and to study proteins interacting with sphingolipids, metabolic labeling based on photoactivatable sphingoid bases is the most straightforward approach. In order to monitor protein-lipid-crosslink products, sphingosine derivatives containing a reporter moiety, such as a radiolabel or a clickable group, are used. In normal cells, degradation of sphingoid bases via action of the checkpoint enzyme sphingosine-1-phosphate lyase occurs at position C2-C3 of the sphingoid base and channels the resulting hexadecenal into the glycerolipid biosynthesis pathway. In case the functionalized sphingosine looses the reporter moiety during its degradation, specificity towards sphingolipid labeling is maintained. In case degradation of a sphingosine derivative does not remove either the photoactivatable or reporter group from the resulting hexadecenal, specificity towards sphingolipid labeling can be achieved by blocking sphingosine-1-phosphate lyase activity and thus preventing sphingosine derivatives to be channeled into the sphingolipid-to-glycerolipid metabolic pathway. Here we report an approach using clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated nuclease Cas9 to create a sphingosine-1-phosphate lyase (SGPL1) HeLa knockout cell line to disrupt the sphingolipid-to-glycerolipid metabolic pathway. We found that the lipid and protein compositions as well as sphingolipid metabolism of SGPL1 knock-out HeLa cells only show little adaptations, which validates these cells as model systems to study transient protein-sphingolipid interactions.

  15. Evidence for a link between histone deacetylation and Ca²+ homoeostasis in sphingosine-1-phosphate lyase-deficient fibroblasts.

    PubMed

    Ihlefeld, Katja; Claas, Ralf Frederik; Koch, Alexander; Pfeilschifter, Josef M; Meyer Zu Heringdorf, Dagmar

    2012-11-01

    Embryonic fibroblasts from S1P (sphingosine-1-phosphate) lyase-deficient mice [Sgpl1-/- MEFs (mouse embryonic fibroblasts)] are characterized by intracellular accumulation of S1P, elevated cytosolic [Ca2+]i and enhanced Ca2+ storage. Since S1P, produced by sphingosine kinase 2 in the nucleus of MCF-7 cells, inhibited HDACs (histone deacetylases) [Hait, Allegood, Maceyka, Strub, Harikumar, Singh, Luo, Marmorstein, Kordula, Milstein et al. (2009) Science 325, 1254-1257], in the present study we analysed whether S1P accumulated in the nuclei of S1P lyase-deficient MEFs and caused HDAC inhibition. Interestingly, nuclear concentrations of S1P were disproportionally elevated in Sgpl1-/- MEFs. HDAC activity was reduced, acetylation of histone 3-Lys9 was increased and the HDAC-regulated gene p21 cyclin-dependent kinase inhibitor was up-regulated in these cells. Furthermore, the expression of HDAC1 and HDAC3 was reduced in Sgpl1-/- MEFs. In wild-type MEFs, acetylation of histone 3-Lys9 was increased by the S1P lyase inhibitor 4-deoxypyridoxine. The non-specific HDAC inhibitor trichostatin A elevated basal [Ca2+]i and enhanced Ca2+ storage, whereas the HDAC1/2/3 inhibitor MGCD0103 elevated basal [Ca2+]i without influence on Ca2+ storage in wild-type MEFs. Overexpression of HDAC1 or HDAC2 reduced the elevated basal [Ca2+]i in Sgpl1-/- MEFs. Taken together, S1P lyase-deficiency was associated with elevated nuclear S1P levels, reduced HDAC activity and down-regulation of HDAC isoenzymes. The decreased HDAC activity in turn contributed to the dysregulation of Ca2+ homoeostasis, particularly to the elevated basal [Ca2+]i, in Sgpl1-/- MEFs.

  16. Mutational Analyses of Glucose Dehydrogenase and Glucose-6-Phosphate Dehydrogenase Genes in Pseudomonas fluorescens Reveal Their Effects on Growth and Alginate Production.

    PubMed

    Maleki, Susan; Mærk, Mali; Valla, Svein; Ertesvåg, Helga

    2015-05-15

    The biosynthesis of alginate has been studied extensively due to the importance of this polymer in medicine and industry. Alginate is synthesized from fructose-6-phosphate and thus competes with the central carbon metabolism for this metabolite. The alginate-producing bacterium Pseudomonas fluorescens relies on the Entner-Doudoroff and pentose phosphate pathways for glucose metabolism, and these pathways are also important for the metabolism of fructose and glycerol. In the present study, the impact of key carbohydrate metabolism enzymes on growth and alginate synthesis was investigated in P. fluorescens. Mutants defective in glucose-6-phosphate dehydrogenase isoenzymes (Zwf-1 and Zwf-2) or glucose dehydrogenase (Gcd) were evaluated using media containing glucose, fructose, or glycerol. Zwf-1 was shown to be the most important glucose-6-phosphate dehydrogenase for catabolism. Both Zwf enzymes preferred NADP as a coenzyme, although NAD was also accepted. Only Zwf-2 was active in the presence of 3 mM ATP, and then only with NADP as a coenzyme, indicating an anabolic role for this isoenzyme. Disruption of zwf-1 resulted in increased alginate production when glycerol was used as the carbon source, possibly due to decreased flux through the Entner-Doudoroff pathway rendering more fructose-6-phosphate available for alginate biosynthesis. In alginate-producing cells grown on glucose, disruption of gcd increased both cell numbers and alginate production levels, while this mutation had no positive effect on growth in a non-alginate-producing strain. A possible explanation is that alginate synthesis might function as a sink for surplus hexose phosphates that could otherwise be detrimental to the cell.

  17. New insights into Pseudomonas fluorescens alginate biosynthesis relevant for the establishment of an efficient production process for microbial alginates.

    PubMed

    Maleki, Susan; Mærk, Mali; Hrudikova, Radka; Valla, Svein; Ertesvåg, Helga

    2017-07-25

    Alginate denotes a family of linear polysaccharides with a wide range of industrial and pharmaceutical applications. Presently, all commercially available alginates are manufactured from brown algae. However, bacterial alginates have advantages with regard to compositional homogeneity and reproducibility. In order to be able to design bacterial strains that are better suited for industrial alginate production, defining limiting factors for alginate biosynthesis is of vital importance. Our group has been studying alginate biosynthesis in Pseudomonas fluorescens using several complementary approaches. Alginate is synthesised and transported out of the cell by a multiprotein complex spanning from the inner to the outer membrane. We have developed an immunogold labelling procedure in which the porin AlgE, as a part of this alginate factory, could be detected by transmission electron microscopy. No time-dependent correlation between the number of such factories on the cell surface and alginate production level was found in alginate-producing strains. Alginate biosynthesis competes with the central carbon metabolism for the key metabolite fructose 6-phosphate. In P. fluorescens, glucose, fructose and glycerol, are metabolised via the Entner-Doudoroff and pentose phosphate pathways. Mutational analysis revealed that disruption of the glucose 6-phosphate dehydrogenase gene zwf-1 resulted in increased alginate production when glycerol was used as carbon source. Furthermore, alginate-producing P. fluorescens strains cultivated on glucose experience acid stress due to the simultaneous production of alginate and gluconate. The combined results from our studies strongly indicate that the availability of fructose 6-phosphate and energy requires more attention in further research aimed at the development of an optimised alginate production process.

  18. Delaying cluster growth of ionotropic induced alginate gelation by oligoguluronate.

    PubMed

    Padoł, Anna Maria; Maurstad, Gjertrud; Draget, Kurt Ingar; Stokke, Bjørn Torger

    2015-11-20

    Alginates form gels in the presence of various divalent ions, such as Ca(2+) that mediate lateral association of chain segments. Various procedures exist that introduce Ca(2+) to yield alginate hydrogels with overall homogeneous or controlled gradients in the concentration profiles. In the present study, the effect of adding oligomers of α-l-guluronic acid (oligoGs) to gelling solutions of alginate was investigated by determination of the cluster growth stimulated by in situ release of Ca(2+). Three different alginate samples varying in fraction of α-l-guluronic acid and molecular weights were employed. The cluster growth was determined for both pure alginates and alginates with two different concentrations of the oligoGs employing dynamic light scattering. The results show that addition of oligoG slows down the cluster growth, the more efficient for the alginates with higher fraction of α-l-guluronic acid, and the higher molecular weight. The efficiency in delaying and slowing the cluster growth induced by added oligoG were discussed in view of the molecular parameters of the alginates. These results show that oligoG can be added to alginate solutions to control the cluster growth and eventually also transition to the gel state. Quantitative relation between the concentration of added oligoG, type and molecular weight of the alginate, and concentration, can be employed as guidelines in tuning alginate cluster growth with specific properties.

  19. Modulation of Central Carbon Metabolism by Acetylation of Isocitrate Lyase in Mycobacterium tuberculosis

    PubMed Central

    Bi, Jing; Wang, Yihong; Yu, Heguo; Qian, Xiaoyan; Wang, Honghai; Liu, Jun; Zhang, Xuelian

    2017-01-01

    Several enzymes involved in central carbon metabolism such as isocitrate lyase and phosphoenolpyruvate carboxykinase are key determinants of pathogenesis of Mycobacterium tuberculosis (M. tb). In this study, we found that lysine acetylation plays an important role in the modulation of central carbon metabolism in M. tb. Mutant of M. tb defective in sirtuin deacetylase exhibited improved growth in fatty acid-containing media. Global analysis of lysine acetylome of M. tb identified three acetylated lysine residues (K322, K331, and K392) of isocitrate lyase (ICL1). Using a genetically encoding system, we demonstrated that acetylation of K392 increased the enzyme activity of ICL1, whereas acetylation of K322 decreased its activity. Antibodies that specifically recognized acetyllysine at 392 and 322 of ICL1 were used to monitor the levels of ICL1 acetylation in M. tb cultures. The physiological significance of ICL1 acetylation was demonstrated by the observation that M. tb altered the levels of acetylated K392 in response to changes of carbon sources, and that acetylation of K392 affected the abundance of ICL1 protein. Our study has uncovered another regulatory mechanism of ICL1. PMID:28322251

  20. Legionella pneumophila S1P-lyase targets host sphingolipid metabolism and restrains autophagy

    PubMed Central

    Rolando, Monica; Escoll, Pedro; Nora, Tamara; Botti, Joëlle; Boitez, Valérie; Daniels, Craig; Abraham, Gilu; Stogios, Peter J.; Skarina, Tatiana; Christophe, Charlotte; Dervins-Ravault, Delphine; Cazalet, Christel; Hilbi, Hubert; Rupasinghe, Thusitha W. T.; Tull, Dedreia; McConville, Malcolm J.; Ong, Sze Ying; Hartland, Elizabeth L.; Codogno, Patrice; Levade, Thierry; Naderer, Thomas; Savchenko, Alexei; Buchrieser, Carmen

    2016-01-01

    Autophagy is an essential component of innate immunity, enabling the detection and elimination of intracellular pathogens. Legionella pneumophila, an intracellular pathogen that can cause a severe pneumonia in humans, is able to modulate autophagy through the action of effector proteins that are translocated into the host cell by the pathogen’s Dot/Icm type IV secretion system. Many of these effectors share structural and sequence similarity with eukaryotic proteins. Indeed, phylogenetic analyses have indicated their acquisition by horizontal gene transfer from a eukaryotic host. Here we report that L. pneumophila translocates the effector protein sphingosine-1 phosphate lyase (LpSpl) to target the host sphingosine biosynthesis and to curtail autophagy. Our structural characterization of LpSpl and its comparison with human SPL reveals high structural conservation, thus supporting prior phylogenetic analysis. We show that LpSpl possesses S1P lyase activity that was abrogated by mutation of the catalytic site residues. L. pneumophila triggers the reduction of several sphingolipids critical for macrophage function in an LpSpl-dependent and -independent manner. LpSpl activity alone was sufficient to prevent an increase in sphingosine levels in infected host cells and to inhibit autophagy during macrophage infection. LpSpl was required for efficient infection of A/J mice, highlighting an important virulence role for this effector. Thus, we have uncovered a previously unidentified mechanism used by intracellular pathogens to inhibit autophagy, namely the disruption of host sphingolipid biosynthesis. PMID:26831115

  1. [Characterization and properties of two dehydroquinate hydro-lyases in higher plants].

    PubMed

    Boudet, A M; Lécussan, R; Boudet, A

    1975-01-01

    Two dehydroquinate hydro-lyases (E.C. 4.2.1.10) have been routinely separated from different organs of Zea mays L. by chromatography on Cellex-D Bio-Rad or hydroxypatite using linear salt gradients. Dehydroquinate hydro-lyase 1 is associated with shikimate: NADP(+) oxidoreductase (E.C. 1.1.1.25). DHQase 2 is a free constitutive enzyme; in this respect it differs from the inducible enzyme of microorganisms which appears only when dehydroquinate or quinate is the principal carbon source. DHQase 1 and DHQase 2 have a similar apparent Michaelis constant and pH optimum, but they differ in their molecular weight, thermal stability and sensitivity to metabolic effectors. DHQase 2 is specifically activated by shikimic acid. This strong activation and the channeling properties of the complex involved in the shikimate pathway can provide an effective means of control in the utilization of dehydroquinate between two different pathways. The significance of such a system involving both a specific regulation of isoenzymes and a molecular compartmentation by means of an enzymatic complex is discussed.

  2. Abundance and Genetic Diversity of Microbial Polygalacturonase and Pectate Lyase in the Sheep Rumen Ecosystem

    PubMed Central

    Wang, Yaru; Luo, Huiying; Huang, Huoqing; Shi, Pengjun; Bai, Yingguo; Yang, Peilong; Yao, Bin

    2012-01-01

    Background Efficient degradation of pectin in the rumen is necessary for plant-based feed utilization. The objective of this study was to characterize the diversity, abundance, and functions of pectinases from microorganisms in the sheep rumen. Methodology/Principal Findings A total of 103 unique fragments of polygalacturonase (PF00295) and pectate lyase (PF00544 and PF09492) genes were retrieved from microbial DNA in the rumen of a Small Tail Han sheep, and 66% of the sequences of these fragments had low identities (<65%) with known sequences. Phylogenetic tree building separated the PF00295, PF00544, and PF09492 sequences into five, three, and three clades, respectively. Cellulolytic and noncellulolytic Butyrivibrio, Prevotella, and Fibrobacter species were the major sources of the pectinases. The two most abundant pectate lyase genes were cloned, and their protein products, expressed in Escherichia coli, were characterized. Both enzymes probably act extracellularly as their nucleotide sequences contained signal sequences, and they had optimal activities at the ruminal physiological temperature and complementary pH-dependent activity profiles. Conclusion/Significance This study reveals the specificity, diversity, and abundance of pectinases in the rumen ecosystem and provides two additional ruminal pectinases for potential industrial use under physiological conditions. PMID:22815874

  3. Utilization of glyphosate as phosphate source: biochemistry and genetics of bacterial carbon-phosphorus lyase.

    PubMed

    Hove-Jensen, Bjarne; Zechel, David L; Jochimsen, Bjarne

    2014-03-01

    After several decades of use of glyphosate, the active ingredient in weed killers such as Roundup, in fields, forests, and gardens, the biochemical pathway of transformation of glyphosate phosphorus to a useful phosphorus source for microorganisms has been disclosed. Glyphosate is a member of a large group of chemicals, phosphonic acids or phosphonates, which are characterized by a carbon-phosphorus bond. This is in contrast to the general phosphorus compounds utilized and metabolized by microorganisms. Here phosphorus is found as phosphoric acid or phosphate ion, phosphoric acid esters, or phosphoric acid anhydrides. The latter compounds contain phosphorus that is bound only to oxygen. Hydrolytic, oxidative, and radical-based mechanisms for carbon-phosphorus bond cleavage have been described. This review deals with the radical-based mechanism employed by the carbon-phosphorus lyase of the carbon-phosphorus lyase pathway, which involves reactions for activation of phosphonate, carbon-phosphorus bond cleavage, and further chemical transformation before a useful phosphate ion is generated in a series of seven or eight enzyme-catalyzed reactions. The phn genes, encoding the enzymes for this pathway, are widespread among bacterial species. The processes are described with emphasis on glyphosate as a substrate. Additionally, the catabolism of glyphosate is intimately connected with that of aminomethylphosphonate, which is also treated in this review. Results of physiological and genetic analyses are combined with those of bioinformatics analyses.

  4. Legionella pneumophila S1P-lyase targets host sphingolipid metabolism and restrains autophagy.

    PubMed

    Rolando, Monica; Escoll, Pedro; Nora, Tamara; Botti, Joëlle; Boitez, Valérie; Bedia, Carmen; Daniels, Craig; Abraham, Gilu; Stogios, Peter J; Skarina, Tatiana; Christophe, Charlotte; Dervins-Ravault, Delphine; Cazalet, Christel; Hilbi, Hubert; Rupasinghe, Thusitha W T; Tull, Dedreia; McConville, Malcolm J; Ong, Sze Ying; Hartland, Elizabeth L; Codogno, Patrice; Levade, Thierry; Naderer, Thomas; Savchenko, Alexei; Buchrieser, Carmen

    2016-02-16

    Autophagy is an essential component of innate immunity, enabling the detection and elimination of intracellular pathogens. Legionella pneumophila, an intracellular pathogen that can cause a severe pneumonia in humans, is able to modulate autophagy through the action of effector proteins that are translocated into the host cell by the pathogen's Dot/Icm type IV secretion system. Many of these effectors share structural and sequence similarity with eukaryotic proteins. Indeed, phylogenetic analyses have indicated their acquisition by horizontal gene transfer from a eukaryotic host. Here we report that L. pneumophila translocates the effector protein sphingosine-1 phosphate lyase (LpSpl) to target the host sphingosine biosynthesis and to curtail autophagy. Our structural characterization of LpSpl and its comparison with human SPL reveals high structural conservation, thus supporting prior phylogenetic analysis. We show that LpSpl possesses S1P lyase activity that was abrogated by mutation of the catalytic site residues. L. pneumophila triggers the reduction of several sphingolipids critical for macrophage function in an LpSpl-dependent and -independent manner. LpSpl activity alone was sufficient to prevent an increase in sphingosine levels in infected host cells and to inhibit autophagy during macrophage infection. LpSpl was required for efficient infection of A/J mice, highlighting an important virulence role for this effector. Thus, we have uncovered a previously unidentified mechanism used by intracellular pathogens to inhibit autophagy, namely the disruption of host sphingolipid biosynthesis.

  5. Utilization of Glyphosate as Phosphate Source: Biochemistry and Genetics of Bacterial Carbon-Phosphorus Lyase

    PubMed Central

    Zechel, David L.; Jochimsen, Bjarne

    2014-01-01

    SUMMARY After several decades of use of glyphosate, the active ingredient in weed killers such as Roundup, in fields, forests, and gardens, the biochemical pathway of transformation of glyphosate phosphorus to a useful phosphorus source for microorganisms has been disclosed. Glyphosate is a member of a large group of chemicals, phosphonic acids or phosphonates, which are characterized by a carbon-phosphorus bond. This is in contrast to the general phosphorus compounds utilized and metabolized by microorganisms. Here phosphorus is found as phosphoric acid or phosphate ion, phosphoric acid esters, or phosphoric acid anhydrides. The latter compounds contain phosphorus that is bound only to oxygen. Hydrolytic, oxidative, and radical-based mechanisms for carbon-phosphorus bond cleavage have been described. This review deals with the radical-based mechanism employed by the carbon-phosphorus lyase of the carbon-phosphorus lyase pathway, which involves reactions for activation of phosphonate, carbon-phosphorus bond cleavage, and further chemical transformation before a useful phosphate ion is generated in a series of seven or eight enzyme-catalyzed reactions. The phn genes, encoding the enzymes for this pathway, are widespread among bacterial species. The processes are described with emphasis on glyphosate as a substrate. Additionally, the catabolism of glyphosate is intimately connected with that of aminomethylphosphonate, which is also treated in this review. Results of physiological and genetic analyses are combined with those of bioinformatics analyses. PMID:24600043

  6. Characterization of C-S Lyase from C. diphtheriae: A Possible Target for New Antimicrobial Drugs

    PubMed Central

    2013-01-01

    The emergence of antibiotic resistance in microbial pathogens requires the identification of new antibacterial drugs. The biosynthesis of methionine is an attractive target because of its central importance in cellular metabolism. Moreover, most of the steps in methionine biosynthesis pathway are absent in mammals, lowering the probability of unwanted side effects. Herein, detailed biochemical characterization of one enzyme required for methionine biosynthesis, a pyridoxal-5′-phosphate (PLP-) dependent C-S lyase from Corynebacterium diphtheriae, a pathogenic bacterium that causes diphtheria, has been performed. We overexpressed the protein in E. coli and analyzed substrate specificity, pH dependence of steady state kinetic parameters, and ligand-induced spectral transitions of the protein. Structural comparison of the enzyme with cystalysin from Treponema denticola indicates a similarity in overall folding. We used site-directed mutagenesis to highlight the importance of active site residues Tyr55, Tyr114, and Arg351, analyzing the effects of amino acid replacement on catalytic properties of enzyme. Better understanding of the active site of C. diphtheriae C-S lyase and the determinants of substrate and reaction specificity from this work will facilitate the design of novel inhibitors as antibacterial therapeutics. PMID:24106714

  7. Synergistic effects in semidilute mixed solutions of alginate and lactose-modified chitosan (chitlac).

    PubMed

    Donati, Ivan; Haug, Ingvild J; Scarpa, Tommaso; Borgogna, Massimiliano; Draget, Kurt I; Skjåk-Braek, Gudmund; Paoletti, Sergio

    2007-03-01

    The present study specifically aimed at preparing and characterizing semidilute binary polymer mixtures of alginate and chitlac which might find an application in the field of cell encapsulation. A polyanion, alginate, and a polycation, a lactose-modified chitosan, were mixed under physiological conditions (pH 7.4 and NaCl 0.15) and at a semidilute concentration avoiding associative phase separation. The mutual solubility was found to be dependent on the charge screening effect of the added NaCl salt, being prevented below 0.05 M NaCl. A comparison with the behavior of the polyanion (alginate) under the same experimental conditions revealed that both the viscosity and the relaxation times of the binary polymer solutions are strongly affected by the presence of the polycation. In particular, the occurrence of electrostatic interactions between the two oppositely charged polysaccharides led to a synergistic effect on the zero-shear viscosity of the solution, which showed a 4.2-fold increase with respect to that of the main component of the solution, i.e., alginate. Moreover, the relaxation time, calculated as the reciprocal of the critical share rate, markedly increased upon reducing the alginate fraction in the binary polysaccharide solution. However, the formation of the soluble complexes and the synergistic effect are reduced upon increasing the concentration of the 1:1 electrolyte. By containing a gel-forming polyanion (alginate, e.g., with Ca(2+) ions) and a bioactive polycation (chitlac, bearing a beta-linked D-galactose), the present system can be regarded as a first step toward the development of biologically active scaffold from polysaccharide mixtures.

  8. The Refined Three-Dimensional Structure of Pectate Lyase E from Erwinia chrysanthemi at 2.2 A Resolution.

    PubMed Central

    Lietzke, S. E.; Scavetta, R. D.; Yoder, M. D.; Jurnak, F.

    1996-01-01

    The crystal structure of pectate lyase E (PelE; EC 4.2.2.2) from the enterobacteria Erwinia chrysanthemi has been refined by molecular dynamics techniques to a resolution of 2.2 A and an R factor (an agreement factor between observed structure factor amplitudes) of 16.1%. The final model consists of all 355 amino acids and 157 water molecules. The root-mean-square deviation from ideality is 0.009 A for bond lengths and 1.721[deg] for bond angles. The structure of PelE bound to a lanthanum ion, which inhibits the enzymatic activity, has also been refined and compared to the metal-free protein. In addition, the structures of pectate lyase C (PelC) in the presence and absence of a lutetium ion have been refined further using an improved algorithm for identifying waters and other solvent molecules. The two putative active site regions of PelE have been compared to those in the refined structure of PelC. The analysis of the atomic details of PelE and PelC in the presence and absence of lanthanide ions provides insight into the enzymatic mechanism of pectate lyases. PMID:12226275

  9. Alginate hydrogel-mediated crystallization of calcium carbonate

    SciTech Connect

    Ma, Yufei; Feng, Qingling

    2011-05-15

    We documented a specific method for combining calcium ions and alginate molecules slowly and continuously in the mineralization system for the purpose of understanding the mediating function of alginate on the crystallization of calcium carbonate. The alginate was involved in the nucleation and the growth process of CaCO{sub 3}. The crystal size, morphology and roughness of crystal surface were significantly influenced by the type of the alginate, which could be accounted for by the length of the G blocks in alginate. A combination of Fourier transform infrared spectroscopy and thermogravimetric analysis showed that there were the chemical interactions between the alginate and the mineral phase. This strategic approach revealed the biologically controlled CaCO{sub 3} mineralization within calcium alginate hydrogels via the selective nucleation and the confined crystallization of CaCO{sub 3}. The results presented here could contribute to the understanding of the mineralization process in hydrogel systems. -- Graphical abstract: Schematic illustration of the growth of calcite aggregates with different morphologies obtained from (a) Low G alginate gels and (b) High G alginate gels. Display Omitted highlights: > We use a specific method for combining calcium ions and alginate molecules slowly and continuously in the mineralization system to understand the mediating function of alginate on the crystallization of CaCO{sub 3} crystals. > The crystal size, morphology and crystal surface roughness are influenced by the length of G blocks in alginate. There are chemical interactions between the alginate and the mineral phase. > We propose a potential mechanism of CaCO{sub 3} crystallization within High G and Low G calcium alginate hydrogel.

  10. Carboxymethyl starch/alginate microspheres containing diamine oxidase for intestinal targeting

    PubMed Central

    Blemur, Lindsay; Le, Tien Canh; Marcocci, Lucia; Pietrangeli, Paola

    2015-01-01

    Abstract The association of carboxymethyl starch (CMS) and alginate is proposed as a novel matrix for the entrapment of bioactive agents in microspheres affording their protection against gastrointestinal degradation. In this study, the enzyme diamine oxidase (DAO) from white pea (Lathyrus sativus) was immobilized by inclusion in microspheres formed by ionotropic gelation of CMS/alginate by complexation with Ca2+. The association of CMS to alginate generated a more compact structure presenting a lesser porosity, thus decreasing the access of gastric fluid inside the microspheres and preventing the loss of entrapped enzyme. Moreover, the immobilized enzyme remained active and was able to oxidize the polyamine substrates even in the presence of degrading proteases of pancreatin. The inclusion yield in terms of entrapped protein was of about 82%–95%. The DAO entrapped in calcium CMS/alginate beads retained up to 70% of its initial activity in simulated gastric fluid (pH 2.0). In simulated intestinal fluid (pH 7.2) with pancreatin, an overall retention of 65% of activity for the immobilized DAO was observed over 24 H, whereas in similar conditions the free enzyme was totally inactivated. Our project proposes the vegetal DAO as an antihistaminic agent orally administered to treat food histaminosis and colon inflammation. PMID:25779356

  11. Carboxymethyl starch/alginate microspheres containing diamine oxidase for intestinal targeting.

    PubMed

    Blemur, Lindsay; Le, Tien Canh; Marcocci, Lucia; Pietrangeli, Paola; Mateescu, Mircea Alexandru

    2016-05-01

    The association of carboxymethyl starch (CMS) and alginate is proposed as a novel matrix for the entrapment of bioactive agents in microspheres affording their protection against gastrointestinal degradation. In this study, the enzyme diamine oxidase (DAO) from white pea (Lathyrus sativus) was immobilized by inclusion in microspheres formed by ionotropic gelation of CMS/alginate by complexation with Ca(2+) . The association of CMS to alginate generated a more compact structure presenting a lesser porosity, thus decreasing the access of gastric fluid inside the microspheres and preventing the loss of entrapped enzyme. Moreover, the immobilized enzyme remained active and was able to oxidize the polyamine substrates even in the presence of degrading proteases of pancreatin. The inclusion yield in terms of entrapped protein was of about 82%-95%. The DAO entrapped in calcium CMS/alginate beads retained up to 70% of its initial activity in simulated gastric fluid (pH 2.0). In simulated intestinal fluid (pH 7.2) with pancreatin, an overall retention of 65% of activity for the immobilized DAO was observed over 24 H, whereas in similar conditions the free enzyme was totally inactivated. Our project proposes the vegetal DAO as an antihistaminic agent orally administered to treat food histaminosis and colon inflammation.

  12. Effect of nutrients on alginate synthesis in Azotobacter vinelandii and characterization of the produced alginate.

    PubMed

    Sabry, S A; Ghanem, K M; Sabra, W A

    1996-12-01

    The role of nutrients on alginate production by Azotobacter vinelandii was studied in batch cultures. The largest amount of bacterial alginate was obtained in presence of: 0.3 g/l MgSO4.7H2O. 0.4 g/l NaCl, 42 mg/l CaCl2.2H2O,.4 mg/l KH2PO4, 16 mg/l K2HPO4, 2.5 mg/l FeSO4.7H2O, 2.9 mg/l H3BO3, 2 mg/l ZnSO4.7H2O, 2 mg/l Na2MoO4.2H2O, 0.3 mg/l CuSO4.5H2O, 0.2 mg/l MnCl2.4H2O. Alginate production was not enhanced by natural additives or inducing agents, except for acetate, which increased alginate yield. The pure alginate contained 0.36% ash and 0.4% protein. It is similar to algal alginate, but it has an extra acetyl group. It contains 69.5% M-M block, 27.5% M-G block and 3% G-G block.

  13. Mutation R96W in cytochrome P450c17 gene causes combined 17{alpha}-hydroxylase/17-20-lyase deficiency in two french canadian patients

    SciTech Connect

    LaFlamme, N.; Leblanc, J.F.; Mailloux, J.

    1996-01-01

    Congenital adrenal hyperplasia (CAH) is the most frequent cause of adrenal insufficiency and ambiguous genitalia in newborn children. In contrast to CAH caused by 21{alpha}-hydroxylase and 11{beta}-hydroxylase deficiencies, which impairs steroid formation in the adrenal exclusively, 17{alpha}-hydroxylase/17,20-lyase deficiency impairs steroid biosynthesis in the adrenals and gonads. The sequence of CYP17 gene was determined by direct sequencing of asymmetric PCR products in two French-Canadian 46,XY pseudohermaphrodite siblings suffering from combined 17{alpha}-hydroxylase/17,20-lyase deficiency. The two patients are homozygous for the novel missense mutation R96W caused by a C to T transition converting codon Arg{sup 96} (CGG) into a Trp (TGG) in exon 1. Both parents are heterozygous for this missense mutation. We assessed the effect of the R96W mutation on 17{alpha}-hydroxylase/17,20-lyase activity by analysis of mutant enzyme, generated by site-directed mutagenesis, expressed in COS-1 cells. The presence of R96W substitution almost completely abolished the activity of the mutant protein. The present findings provide a molecular explanation for the signs and symptoms of combined 17 {alpha}-hydroxylase/17,20-lyase deficiency in these two patients and provide useful information on the structure-activity relationships of the P450c17 enzyme. 31 refs., 4 figs., 1 tab.

  14. Characterization of a novel HMG-CoA lyase enzyme with a dual location in endoplasmic reticulum and cytosol[S

    PubMed Central

    Arnedo, María; Menao, Sebastián; Puisac, Beatriz; Teresa-Rodrigo, María E.; Gil-Rodríguez, María C.; López-Viñas, Eduardo; Gómez-Puertas, Paulino; Casals, Nuria; Casale, César H.; Hegardt, Fausto G.; Pié, Juan

    2012-01-01

    A novel lyase activity enzyme is characterized for the first time: HMG-CoA lyase-like1 (er-cHL), which is a close homolog of mitochondrial HMG-CoA lyase (mHL). Initial data show that there are nine mature transcripts for the novel gene HMGCLL1, although none of them has all its exons. The most abundant transcript is called “variant b,” and it lacks exons 2 and 3. Moreover, a three-dimensional model of the novel enzyme is proposed. Colocalization studies show a dual location of the er-cHL in the endoplasmic reticulum (ER) and cytosol, but not in mitochondria or peroxisomes. Furthermore, the dissociation experiment suggests that it is a nonendoplasmic reticulum integral membrane protein. The kinetic parameters of er-cHL indicate that it has a lower Vmax and a higher substrate affinity than mHL. Protein expression and lyase activity were found in several tissues, and were particularly strong in lung and kidney. The occurrence of er-cHL in brain is surprising, as mHL has not been found there. Although mHL activity is clearly associated with energy metabolism, the results suggest that er-cHL is more closely related to another metabolic function, mostly at the pulmonary and brain level. PMID:22847177

  15. 21 CFR 184.1187 - Calcium alginate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... use in food (as served) (percent) Functional use Baked goods, § 170.3(n)(1) of this chapter 0.002... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Calcium alginate. 184.1187 Section 184.1187 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD...

  16. 21 CFR 184.1133 - Ammonium alginate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...: Category of food Maximum level of use in food (as served) (percent) Functional use Confections, frostings... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Ammonium alginate. 184.1133 Section 184.1133 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD...

  17. 21 CFR 184.1133 - Ammonium alginate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... prepared by the neutralization of purified alginic acid with appropriate pH control agents. (b) The ingredient meets the specifications of the Food Chemicals Codex, 3d Ed. (1981), p. 18, which is incorporated... food Maximum level of use in food (as served) (percent) Functional use Confections, frostings, §...

  18. 21 CFR 184.1187 - Calcium alginate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...-35-0) is the calcium salt of alginic acid, a natural polyuronide constituent of certain brown algae... this chapter 0.6 Do. Fats and oils, § 170.3(n)(12) of this chapter 0.5 Do. Gelatins, puddings, §...

  19. 21 CFR 184.1133 - Ammonium alginate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...-34-9) is the ammonium salt of alginic acid, a natural polyuronide constituent of certain brown algae..., § 170.3(n)(9) of this chapter 0.4 Stabilizer, thickener, § 170.3(o)(28) of this chapter. Fats and...

  20. 21 CFR 184.1187 - Calcium alginate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...-35-0) is the calcium salt of alginic acid, a natural polyuronide constituent of certain brown algae... this chapter 0.6 Do. Fats and oils, § 170.3(n)(12) of this chapter 0.5 Do. Gelatins, puddings, §...

  1. 21 CFR 184.1133 - Ammonium alginate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...-34-9) is the ammonium salt of alginic acid, a natural polyuronide constituent of certain brown algae..., § 170.3(n)(9) of this chapter 0.4 Stabilizer, thickener, § 170.3(o)(28) of this chapter. Fats and...

  2. 21 CFR 184.1133 - Ammonium alginate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...: Category of food Maximum level of use in food (as served) (percent) Functional use Confections, frostings... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Ammonium alginate. 184.1133 Section 184.1133 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD...

  3. Enantioselective Synthesis of Various Cyanohydrins Using Covalently Immobilized Preparations of Hydroxynitrile Lyase from Prunus dulcis.

    PubMed

    Alagöz, Dilek; Tükel, S Seyhan; Yildirim, Deniz

    2015-11-01

    The carrier-based and carrier-free (cross-linked enzyme aggregate) covalent immobilizations of Prunus dulcis hydroxynitrile lyase were investigated. The immobilized preparations were tested for enantioselective carbon-carbon bond formation activity in the biphasic medium. Of the tested preparations, only cross-linked enzyme aggregate of P. dulcis hydroxynitrile lyase (PdHNL-CLEA) achieved the synthesis of (R)-mandelonitrile with 93% yield and 99% enantiopurity. PdHNL-CLEA was also used in the synthesis of various (R)-cyanohydrins from corresponding aldehydes/ketones and hydrocyanic acid. When 4-methoxybenzaldehyde, 4-methyl benzaldehyde, and 4-hydroxybenzaldehyde were used as substrates, the yield-enantiomeric excess of corresponding (R)-cyanohydrins were obtained as 95-95, 85-79, and 2-25%, respectively, after 96 h at pH 4.0 and 5 °C. For acetophenone, 4-fluoroacetophenone, 4-chloroacetophenone, 4-bromoacetophenone, and 4-iodoacetophenone, the yield-enantiomeric excess of corresponding (R)-cyanohydrins were 1-99, 20-84, 11-95, 5-99, and 3-24%, respectively at the same conditions. The results demonstrate PdHNL-CLEA can be effectively used in the synthesis of (R)-mandelonitrile.

  4. Biochemical and structural characterization of a novel bacterial manganese-dependent hydroxynitrile lyase.

    PubMed

    Hajnal, Ivan; Lyskowski, Andrzej; Hanefeld, Ulf; Gruber, Karl; Schwab, Helmut; Steiner, Kerstin

    2013-11-01

    Hydroxynitrile lyases (HNLs), which catalyse the decomposition of cyanohydrins, are found mainly in plants. In vitro, they are able to catalyse the synthesis of enantiopure cyanohydrins, which are versatile building blocks in the chemical industry. Recently, HNLs have also been discovered in bacteria. Here, we report on the detailed biochemical and structural characterization of a hydroxynitrile lyase from Granulicella tundricola (GtHNL), which was successfully heterologously expressed in Escherichia coli. The crystal structure was solved at a crystallographic resolution of 2.5 Å and exhibits a cupin fold. As GtHNL does not show any sequence or structural similarity to any other HNL and does not contain conserved motifs typical of HNLs, cupins represent a new class of HNLs. GtHNL is metal-dependent, as confirmed by inductively coupled plasma/optical emission spectroscopy, and in the crystal structure, manganese is bound to three histidine and one glutamine residue. GtHNL displayed a specific activity of 1.74 U·mg(-1) at pH 6 with (R)-mandelonitrile, and synthesized (R)-mandelonitrile with 90% enantiomeric excess at 80% conversion using 0.5 m benzaldehyde in a biphasic reaction system with methyl tertiary butyl ether.

  5. Improving the thermoactivity and thermostability of pectate lyase from Bacillus pumilus for ramie degumming.

    PubMed

    Liang, Chaoning; Gui, Xiwu; Zhou, Cheng; Xue, Yanfen; Ma, Yanhe; Tang, Shuang-Yan

    2015-03-01

    Thermostable alkaline pectate lyases can be potentially used for enzymatically degumming ramie in an environmentally sustainable manner and as an alternative to the currently used chemical-based ramie degumming processes. To assess its potential applications, pectate lyase from Bacillus pumilus (ATCC 7061) was cloned and expressed in Escherichia coli. Evolutionary strategies were applied to generate efficient ramie degumming enzymes. Obtained from site-saturation mutagenesis and random mutagenesis, the best performing mutant enzyme M3 exhibited a 3.4-fold higher specific activity on substrate polygalacturonic acid, compared with the wild-type enzyme. Furthermore, the half-life of inactivation at 50 °C for M3 mutant extended to over 13 h. In contrast, the wild-type enzyme was completely inactivated in less than 10 min under the same conditions. An upward shift in the optimal reaction temperature of M3 mutant, to 75 °C, was observed, which was 10 °C higher than that of the wild-type enzyme. Kinetic parameter data revealed that the catalysis efficiency of M3 mutant was higher than that of the wild-type enzyme. Ramie degumming with M3 mutant was also demonstrated to be more efficient than that with the wild-type enzyme. Collectively, our results suggest that the M3 mutant, with remarkable improvements in thermoactivity and thermostability, has potential applications for ramie degumming in the textile industry.

  6. Overexpression of isocitrate lyase-glyoxylate bypass influence on metabolism in Aspergillus niger.

    PubMed

    Meijer, S; Otero, J; Olivares, R; Andersen, M R; Olsson, L; Nielsen, J

    2009-03-01

    In order to improve the production of succinate and malate by the filamentous fungus Aspergillus niger the activity of the glyoxylate bypass pathway was increased by over-expression of the isocitrate lyase (icl) gene. The hypothesis was that when isocitrate lyase was up-regulated the flux towards glyoxylate would increase, leading to excess formation of malate and succinate compared to the wild-type. However,metabolic network analysis showed that an increased icl expression did not result in an increased glyoxylate bypass flux. The analysis did show a global response with respect to gene expression, leading to an increased flux through the oxidative part of the TCA cycle. Instead of an increased production of succinate and malate, a major increase in fumarate production was observed. The effect of malonate, a competitive inhibitor of succinate dehydrogenase (SDH), on the physiological behaviour of the cells was investigated. Inhibition of SDH was expected to lead to succinate production, but this was not observed. There was an increase in citrate and oxalate production in the wild-type strain. Further more, in the strain with over-expression of icl the organic acid production shifted from fumarate towards malate production when malonate was added to the cultivation medium. Overall,the icl over-expression and malonate addition had a significant impact on metabolism and on organic acid production profiles. Although the expected succinate and malate formation was not observed, a distinct and interesting production of fumarate and malate was found.

  7. The mechanism of action of phenylalanine ammonia-lyase: the role of prosthetic dehydroalanine.

    PubMed Central

    Schuster, B; Rétey, J

    1995-01-01

    Phenylalanine ammonia-lyase (EC 4.3.1.5) from parsley is posttranslationally modified by dehydrating its Ser-202 to the catalytically essential dehydroalanine prosthetic group. The codon of Ser-202 was changed to those of alanine and threonine by site-directed mutagenesis. These mutants and the recombinant wild-type enzyme, after treatment with sodium borohydride, were virtually inactive with L-phenylalanine as substrate but catalyzed the deamination of L-4-nitrophenylalanine, which is also a substrate for the wild-type enzyme. Although the mutants reacted about 20 times slower with L-4-nitrophenylalanine than the wild-type enzyme, their Vmax for L-4-nitrophenylalanine was two orders of magnitude higher than for L-phenylalanine. In contrast to L-tyrosine, which was a poor substrate, DL-3-hydroxyphenylalanine (DL-m-tyrosine) was converted by phenylalanine ammonia-lyase at a rate comparable to that of L-phenylalanine. These results suggest a mechanism in which the crucial step is an electrophilic attack of the prosthetic group at position 2 or 6 of the phenyl group. In the resulting carbenium ion, the beta-HSi atom is activated in a similar way as it is in the nitro analogue. Subsequent elimination of ammonia, concomitant with restoration of both the aromatic ring and the prosthetic group, completes the catalytic cycle. Images Fig. 1 PMID:7667307

  8. Expression and Bioinformatics Analysis of Pectate Lyase Gene from Bacillus subtilis521

    NASA Astrophysics Data System (ADS)

    Xiao, Jing; Lu, Fu-Ping; Li, Yu; Li, Jin-Ting

    In order to exploit new genetic resources, Pectate lyase(PEL) gene was amplified by PCR using the genome DNA from an alkaline Bacillus subtilis521. The PCR product was inserted into pET22b(+) vector. The recombinant plasmids were cloned in E.coli DH5α and then expressed in E.coli BL21. When cultured in the optimized medium, the positive clones E.coli BL21(pET22b(+)pel)showed intracellular pectate lyase activity of 90.0 U/mL. It was indicated that we had obtained the correct PEL gene. The pel has an open reading frame of 1263 nucleotides and codes for a product of 420 amino acids with a calculated molecular mass of 45.5 kD. Based on computer assisted analysis, a signal peptides and two conserved domains were revealed. The sequence analysis for PEL showed that it shares 26-82% homology with other strains in GenBank. In addition, the advanced structure of PEL were also predicted and analysed. This study will help to the experimental design of PEL fermentation and production purification and enzyme evolution.

  9. Mechanistic studies of a novel C-S lyase in ergothioneine biosynthesis: the involvement of a sulfenic acid intermediate

    PubMed Central

    Song, Heng; Hu, Wen; Naowarojna, Nathchar; Her, Ampon Sae; Wang, Shu; Desai, Rushil; Qin, Li; Chen, Xiaoping; Liu, Pinghua

    2015-01-01

    Ergothioneine is a histidine thio-derivative isolated in 1909. In ergothioneine biosynthesis, the combination of a mononuclear non-heme iron enzyme catalyzed oxidative C-S bond formation reaction and a PLP-mediated C-S lyase (EgtE) reaction results in a net sulfur transfer from cysteine to histidine side-chain. This demonstrates a new sulfur transfer strategy in the biosynthesis of sulfur-containing natural products. Due to difficulties associated with the overexpression of Mycobacterium smegmatis EgtE protein, the proposed EgtE functionality remained to be verified biochemically. In this study, we have successfully overexpressed and purified M. smegmatis EgtE enzyme and evaluated its activities under different in vitro conditions: C-S lyase reaction using either thioether or sulfoxide as a substrate in the presence or absence of reductants. Results from our biochemical characterizations support the assignment of sulfoxide 4 as the native EgtE substrate and the involvement of a sulfenic acid intermediate in the ergothioneine C-S lyase reaction. PMID:26149121

  10. Structure of Methylobacterium extorquens malyl-CoA lyase: CoA-substrate binding correlates with domain shift.

    PubMed

    González, Javier M; Marti-Arbona, Ricardo; Chen, Julian C H; Unkefer, Clifford J

    2017-02-01

    Malyl-CoA lyase (MCL) is an Mg(2+)-dependent enzyme that catalyzes the reversible cleavage of (2S)-4-malyl-CoA to yield acetyl-CoA and glyoxylate. MCL enzymes, which are found in a variety of bacteria, are members of the citrate lyase-like family and are involved in the assimilation of one- and two-carbon compounds. Here, the 1.56 Å resolution X-ray crystal structure of MCL from Methylobacterium extorquens AM1 with bound Mg(2+) is presented. Structural alignment with the closely related Rhodobacter sphaeroides malyl-CoA lyase complexed with Mg(2+), oxalate and CoA allows a detailed analysis of the domain motion of the enzyme caused by substrate binding. Alignment of the structures shows that a simple hinge motion centered on the conserved residues Phe268 and Thr269 moves the C-terminal domain by about 30° relative to the rest of the molecule. This domain motion positions a conserved aspartate residue located in the C-terminal domain in the active site of the adjacent monomer, which may serve as a general acid/base in the catalytic mechanism.

  11. Structure of Methylobacterium extorquens malyl-CoA lyase: CoA-substrate binding correlates with domain shift

    DOE PAGES

    Gonzalez, Javier M.; Marti-Arbona, Ricardo; Chen, Julian C. -H.; ...

    2017-01-27

    Malyl-CoA lyase (MCL) is an Mg2+-dependent enzyme that catalyzes the reversible cleavage of (2S)-4-malyl-CoA to yield acetyl-CoA and glyoxylate. MCL enzymes, which are found in a variety of bacteria, are members of the citrate lyase-like family and are involved in the assimilation of one- and two-carbon compounds. Here, the 1.56 Å resolution X-ray crystal structure of MCL from Methylobacterium extorquens AM1 with bound Mg2+is presented. Structural alignment with the closely related Rhodobacter sphaeroides malyl-CoA lyase complexed with Mg2+, oxalate and CoA allows a detailed analysis of the domain motion of the enzyme caused by substrate binding. Alignment of the structuresmore » shows that a simple hinge motion centered on the conserved residues Phe268 and Thr269 moves the C-terminal domain by about 30° relative to the rest of the molecule. Furthermore, this domain motion positions a conserved aspartate residue located in the C-terminal domain in the active site of the adjacent monomer, which may serve as a general acid/base in the catalytic mechanism.« less

  12. Calcium-Alginate-Inulin Microbeads as Carriers for Aqueous Carqueja Extract.

    PubMed

    Balanč, Bojana; Kalušević, Ana; Drvenica, Ivana; Coelho, Maria Teresa; Djordjević, Verica; Alves, Vitor D; Sousa, Isabel; Moldão-Martins, Margarida; Rakić, Vesna; Nedović, Viktor; Bugarski, Branko

    2016-01-01

    Carqueja (Pterospartum tridentatum) is an endemic species and various bioactive compounds have been identified in its aqueous extract. The aim of this study was to protect the natural antioxidants from the aqueous extract of carqueja by encapsulation in Ca-alginate microbeads and Ca-alginate microbeads containing 10% and 20% (w/v) of inulin. The microbeads produced by electrostatic extrusion technique had an average diameter from 625 μm to 830 μm depending on the portion of inulin. The sphericity factor of the hydrogel microbeads had values between 0.014 and 0.026, while freeze dried microbeads had irregular shape, especially those with no excipient. The reduction in microbeads size after freeze drying process (expressed as shrinkage factor) ranged from 0.338 (alginate microbeads with 20% (w/v) of inulin) to 0.523 (plain alginate microbeads). The expressed radical scavenging activity against ABTS and DPPH radicals was found to be between 30% and 40% for encapsulated extract, while the fresh extract showed around 47% and 57% of radical scavenging activity for ABTS and DPPH radicals, respectively. The correlation between antioxidant activity and the total phenolic content were found to be positive (in both assay methods, DPPH and ABTS), which indicate that the addition of inulin didn't have influence on antioxidant activity. The presence of inulin reduced stiffness of the hydrogel, and protected bead structure from collapse upon freeze-drying. Alginate-inulin beads are envisaged to be used for delivery of aqueous P. tridentatum extract in functional food products.

  13. Dye removal from aqueous solution by magnetic alginate beads crosslinked with epichlorohydrin.

    PubMed

    Rocher, Vincent; Bee, Agnès; Siaugue, Jean-Michel; Cabuil, Valérie

    2010-06-15

    Innovative magnetic alginate beads are used to remove organic pollutants from aqueous solution under different experimental conditions. These alginate beads (EpiMAB) are prepared by an extrusion technique and crosslinked with epichlorohydrin. They contain both magnetic nanoparticles and activated carbon (AC). With the addition of magnetic properties, the beads can be easily recovered or manipulated with an external magnetic field. Their capacity to adsorb pollutants is linked to encapsulated AC and to active sites coming from both magnetic nanoparticles and alginate. The efficiency of the beads as biosorbent for the removal of dyes is assessed using methyl orange (MO) and methylene blue (MB) as model molecules. The dye uptake is found to vary with the initial concentration and the charge of the adsorbed molecule. The Langmuir equation fits well the adsorption data with maximum adsorption capacities of 0.02 mmol/g for MO and 0.7 mmol/g for MB. Kinetics experiments are performed to evaluate the equilibrium time; the pseudo-second-order kinetic model adequately describes the experimental data. The influence of the pH of the solution on adsorption is also investigated and a comparison with alginate beads crosslinked by calcium ions is made.

  14. Encapsulation of cardiac stem cells in superoxide dismutase-loaded alginate prevents doxorubicin-mediated toxicity.

    PubMed

    Liu, Ting Chu Ken; Ismail, Siti; Brennan, Orlaith; Hastings, Conn; Duffy, Garry P

    2013-04-01

    Anthracyclines are powerful drugs available for the treatment of neoplastic diseases. Unfortunately, these chemotherapy agents cause cardiomyopathy and congestive heart failure. Doxorubicin (DOX) is a widely used anthracycline and evidence indicates that DOX-induced cardiotoxicity can be viewed as a stem cell disease, whereby the formation of reactive oxygen species (ROS) by DOX is seen to predominantly hinder cardiac stem cell (CSC) regenerative capability. Acute, early-onset and late-onset cardiotoxicity have been described and this may be reversible by the local administration of CSCs, which regenerate myocardial tissue and rescue the failing heart. CSCs are, however, particularly sensitive to oxidative stress and die rapidly by apoptosis in such adverse conditions. Therefore, this study aims to enhance CSC survival by encapsulation in an alginate hydrogel formulation containing superoxide dismutase (SOD), a reactive oxygen species scavenger. Cell survival was qualitatively and quantitatively assessed by fluorescent microscopy and assays measuring metabolic activity, cell viability, cytotoxicity and apoptosis. CSCs were cultured in DOX-conditioned cell culture medium and displayed reduced live cell numbers as well as high levels of apoptosis. Encapsulation of CSCs in alginate alone failed to prevent apoptosis. Encapsulation in SOD-loaded alginate reduced apoptosis to near-normal levels, whilst metabolic activity was returned to baseline. In conclusion, this study demonstrates that encapsulation of CSCs in SOD-loaded alginate hydrogel enhances CSC survival in the presence of DOX, raising the possibility of its application as a novel therapy for the treatment of acute and early onset DOX-induced cardiotoxicity.

  15. l-Phenylalanine Ammonia-lyase (Maize)

    PubMed Central

    Reid, Philip D.; Havir, Evelyn A.; Marsh, Herbert V.

    1972-01-01

    Extracts of maize leaf sheath tissue deaminate both l-phenylalanine and l-tyrosine. The activities with both substrates are enhanced by treating the plant with gibberellic acid. Both activities decrease rapidly at the same rate when tissue is incubated in a moist atmosphere, and this decrease can be slowed by treatment with cycloheximide. The ratio of the activities was constant throughout a series of purification steps which included acetone and ammonium sulfate precipitation, and passage through an agarose column. The two activities could not be separated by isoelectric focusing. These results support our earlier conclusion that both activities occur at the same catalytic site. PMID:16658200

  16. Alginate composites for bone tissue engineering: a review.

    PubMed

    Venkatesan, Jayachandran; Bhatnagar, Ira; Manivasagan, Panchanathan; Kang, Kyong-Hwa; Kim, Se-Kwon

    2015-01-01

    Bone is a complex and hierarchical tissue consisting of nano hydroxyapatite and collagen as major portion. Several attempts have been made to prepare the artificial bone so as to replace the autograft and allograft treatment. Tissue engineering is a promising approach to solve the several issues and is also useful in the construction of artificial bone with materials including polymer, ceramics, metals, cells and growth factors. Composites consisting of polymer-ceramics, best mimic the natural functions of bone. Alginate, an anionic polymer owing enormous biomedical applications, is gaining importance particularly in bone tissue engineering due to its biocompatibility and gel forming properties. Several composites such as alginate-polymer (PLGA, PEG and chitosan), alginate-protein (collagen and gelatin), alginate-ceramic, alginate-bioglass, alginate-biosilica, alginate-bone morphogenetic protein-2 and RGD peptides composite have been investigated till date. These alginate composites show enhanced biochemical significance in terms of porosity, mechanical strength, cell adhesion, biocompatibility, cell proliferation, alkaline phosphatase increase, excellent mineralization and osteogenic differentiation. Hence, alginate based composite biomaterials will be promising for bone tissue regeneration. This review will provide a broad overview of alginate preparation and its applications towards bone tissue engineering.

  17. Adsorption of CO2 by alginate immobilized zeolite beads

    NASA Astrophysics Data System (ADS)

    Suratman, A.; Kunarti, E. S.; Aprilita, N. H.; Pamurtya, I. C.

    2017-03-01

    Immobilized zeolit in alginate beads for adsorption of CO2 was developed. Alginate immobilized zeolit beads was generated by dropping the mixture of Na-alginate and zeolite solution into Ca2+ solution. The adsorption efficacy such as the influence of contact time, mass of zeolite, flowrate of CO2, and mass of adsorbent was evaluated. The adsorption of CO2 onto alginate immobilized zeolit beads was investigated by performing both equilibrium and kinetic batch test. Bead was characterized by FTIR and SEM. Alginate immobilized zeolit beads demonstrated significantly higher sorption efficacy compared to plain alginate beads and zeolite with 0.25 mmol CO2 adsorbed /g adsorbent. Optimum condition was achieved with mass composition of alginate:zeolite (3:1), flowrate 50 mL/min for 20 minutes. The alginate immobilized zeolit beads showed that adsorption of CO2 followed Freundlich isotherm and pseudo second order kinetic model. Adsorption of CO2 onto alginate immobilized zeolite beads is a physisorption with adsorption energy of 6.37 kJ/mol. This results indicates that the alginate immobilized zeolit beads can be used as promising adsorbents for CO2.

  18. Effect of substituting arginine and lysine with alanine on antimicrobial activity and the mechanism of action of a cationic dodecapeptide (CL(14-25)), a partial sequence of cyanate lyase from rice.

    PubMed

    Taniguchi, Masayuki; Takahashi, Nobuteru; Takayanagi, Tomohiro; Ikeda, Atsuo; Ishiyama, Yohei; Saitoh, Eiichi; Kato, Tetsuo; Ochiai, Akihito; Tanaka, Takaaki

    2014-01-01

    The antimicrobial activity of analogs obtained by substituting arginine and lysine in CL(14-25), a cationic α-helical dodecapeptide, with alanine against Porphyromonas gingivalis, a periodontal pathogen, varied significantly depending on the number and position of cationic amino acids. The alanine-substituted analogs had no hemolytic activity, even at a concentration of 1 mM. The antimicrobial activities of CL(K20A) and CL(K20A, K25A) were 3.8-fold and 9.1-fold higher, respectively, than that of CL(14-25). The antimicrobial activity of CL(R15A) was slightly lower than that of CL(14-25), suggesting that arginine at position 15 is not essential but is important for the antimicrobial activity. The experiments in which the alanine-substituted analogs bearing the replacement of arginine at position 24 and/or lysine at position 25 were used showed that arginine at position 24 was crucial for the antimicrobial activity whenever lysine at position 25 was substituted with alanine. Helical wheel projections of the alanine-substituted analogs indicate that the hydrophobicity in the vicinity of leucine at position 16 and alanines at positions 18 and/or 21 increased by substituting lysine at positions 20 and 25 with alanine, respectively. The degrees of diSC3 -5 release from P. gingivalis cells and disruption of GUVs induced by the alanine-substituted analogs with different positive charges were not closely related to their antimicrobial activities. The enhanced antimicrobial activities of the alanine-substituted analogs appear to be mainly attributable to the changes in properties such as hydrophobicity and amphipathic propensity due to alanine substitution and not to their extents of positive charge (cationicity).

  19. An acidic pectin lyase from Aspergillus niger with favourable efficiency in fruit juice clarification.

    PubMed

    Xu, S X; Qin, X; Liu, B; Zhang, D Q; Zhang, W; Wu, K; Zhang, Y H

    2015-02-01

    The pectin lyase gene pnl-zj5a from Aspergillus niger ZJ5 was identified and expressed in Pichia pastoris. PNL-ZJ5A was purified by ultrafiltration, anion exchange and gel chromatography. The Km and Vmax values determined using citrus pectin were 0.66 mg ml(-1) and 32.6 μmol min(-1) mg(-1) , respectively. PNL-ZJ5A exhibited optimal activity at 43°C and retained activity over 25-50°C. PNL-ZJ5A was optimally active at pH 5 and effective in apple juice clarification. Compared with controls, PNL-ZJ5A increased the fruit juice yield significantly. Furthermore, PNL-ZJ5A reduced the viscosity of apple juice by 38.8% and increased its transmittance by 86.3%. PNL-ZJ5A combined with a commercial pectin esterase resulted in higher juice volume.

  20. Cloning and bacterial expression of the CYS3 gene encoding cystathionine gamma-lyase of Saccharomyces cerevisiae and the physicochemical and enzymatic properties of the protein.

    PubMed Central

    Yamagata, S; D'Andrea, R J; Fujisaki, S; Isaji, M; Nakamura, K

    1993-01-01

    By screening a yeast genomic library, we isolated and characterized a gene rescuing the cysteine requirement in a "cys1" strain of Saccharomyces cerevisiae. Except for four residues in the open reading frame composed of 1,182 nucleotides, the DNA sequence was the same as that for the CYS3 (CYI1) gene, encoding cystathionine gamma-lyase (EC 4.4.1.1), and isolated previously as a cycloheximide-induced gene (B. Ono, K. Tanaka, K. Naito, C. Heike, S. Shinoda, S. Yamamoto, S. Ohmori, T. Oshima, and A. Toh-e, J. Bacteriol. 174:pp.3339-3347, 1992). S. cerevisiae "cys1" strains carry two closely linked mutations; one (cys1) causes a defect in serine O-acetyltransferase (EC 2.3.1.30), and another, designated cys3, impairs cystathionine gamma-lyase activity. Rescue of the cysteine requirement by the gene encoding cystathionine gamma-lyase is consistent with both defects being responsible for the cysteine auxotrophy. In an effort to further determine the physicochemical and enzymatic properties of this enzyme, a coding fragment was cloned into an Escherichia coli expression plasmid, and the protein was produced in the bacteria. The induced protein was extracted by sonication and purified to homogeneity through one course of DEAE-cellulose column chromatography. The yield of the protein was approximately 150 mg from cells cultured in 1 liter of L broth. The protein showed molecular weights of approximately 194,000 and 48,000 (for the subunit), suggesting a tetrameric structure. An s20,w value of 8.8 was estimated by centrifugation in a sucrose concentration gradient. No sulfhydryl groups were detected, which is consistent with the absence of cysteine residues in the coding sequence. The isoelectric point was at pH 5.2. The protein showed a number of cystathionine-related activities, i.e., cystathionine beta-lyase (EC 4.4.1.8), cystathionine gamma-lyase, and cystathionine gamma-synthase (EC 4.2.99.9) with L-homoserine as substrate. In addition, we demonstrated L

  1. Impact of preharvest and postharvest alginate treatments enriched with vanillin on postharvest decay, biochemical properties, quality and sensory attributes of table grapes.

    PubMed

    Konuk Takma, Dilara; Korel, Figen

    2017-04-15

    Alginate solution enriched with vanillin as a bioactive compound was investigated for improving preharvest and postharvest quality and safety of table grapes. Alginate treatments with or without vanillin as preharvest spray and postharvest coating were implemented on table grapes of Alphonse Lavalleé and Razaki cultivars. Fungal decay, biochemical properties, quality and sensory attributes were evaluated at day of preharvest treatment, at harvesting and during 35days of storage at 4±2°C. Alginate treatments with or without vanillin were effective in preventing weight and firmness losses. Total soluble solids, titratable acidity, and color of grapes coated with alginate coatings with or without vanillin showed minor changes compared to control grapes. Alginate coating incorporating vanillin provided significant reduction (1.73log CFU/g) in yeast-mold growth. Moreover, the coatings maintained greater total phenolic content and antioxidant activity compared to others during postharvest storage. In terms of sensory attributes, appearance was ranked as the highest for alginate coating without vanillin due to glossiness of alginate.

  2. The crystal structure of novel chondroitin lyase ODV-E66, a baculovirus envelope protein.

    PubMed

    Kawaguchi, Yoshirou; Sugiura, Nobuo; Kimata, Koji; Kimura, Makoto; Kakuta, Yoshimitsu

    2013-12-11

    Chondroitin lyases have been known as pathogenic bacterial enzymes that degrade chondroitin. Recently, baculovirus envelope protein ODV-E66 was identified as the first reported viral chondroitin lyase. ODV-E66 has low sequence identity with bacterial lyases at <12%, and unique characteristics reflecting the life cycle of baculovirus. To understand ODV-E66's structural basis, the crystal structure was determined and it was found that the structural fold resembled that of polysaccharide lyase 8 proteins and that the catalytic residues were also conserved. This structure enabled discussion of the unique substrate specificity and the stability of ODV-E66 as well as the host specificity of baculovirus.

  3. The crystal structure of novel chondroitin lyase ODV-E66, a baculovirus envelope protein.

    PubMed

    Kawaguchi, Yoshirou; Sugiura, Nobuo; Kimata, Koji; Kimura, Makoto; Kakuta, Yoshimitu

    2013-10-25

    Chondroitin lyases have been known as pathogenic bacterial enzymes that degrade chondroitin. Recently, baculovirus envelope protein ODV-E66 was identified as the first reported viral chondroitin lyase. ODV-E66 has low sequence identity with bacterial lyases at <12%, and unique characteristics reflecting the life cycle of baculovirus. To understand ODV-E66's structural basis, the crystal structure was determined and it was found that the structural fold resembled that of polysaccharide lyase 8 proteins and that the catalytic residues were also conserved. This structure enabled discussion of the unique substrate specificity and the stability of ODV-E66 as well as the host specificity of baculovirus.

  4. Lysine succinylation of Mycobacterium tuberculosis isocitrate lyase (ICL) fine-tunes the microbial resistance to antibiotics.

    PubMed

    Zhou, Mingliang; Xie, Longxiang; Yang, Zhaozhen; Zhou, Jiahai; Xie, Jianping

    2017-04-01

    Lysine succinylation (Ksucc) is a newly identified protein posttranslational modification (PTM), which may play an important role in cellular physiology. However, the role of lysine succinylation in antibiotic resistance remains elusive. Isocitrate lyase (ICL) is crucial for broad-spectrum antibiotics tolerance in Mycobacterium tuberculosis (Mtb). We previously found that MtbICL (Rv0467) has at least three succinylated lysine residues, namely K189, K322, and K334.To explore the effect of succinylation on the activity of MtbICL, mutants' mimicry of the lysine succinylation were generated by site-directed mutagenesis. ICL-K189E mutant strain is more sensitive than the wild-type to rifampicin and streptomycin, but not isoniazid. For the in vitro activity of the purified isocitrate lyase, only K189E mutant showed significantly decreased activity. Crystal structure analysis showed that Lys189 Glu dramatically increased the pKa of Glu188 and decreased the pKa of Lys190, whereas had negligible effect on other residues within 5 Å as well as disruption of the electrostatic interaction between Lys189 and Glu182, which might prevent the closure of the active site loop and cause severe reduction of the enzyme activity. Considering the genetic, biochemical, and crystallographical evidences together, the succinylation of specific ICL residue can fine-tune the bacterial resistance to selected antibiotics. The decreased enzymatic activity resulting from the succinylation-changed electrostatic interaction might underlie this phenotype. This study provided the first insight into the link between lysine succinylation and antibiotic resistance.

  5. Inter-grade and inter-batch variability of sodium alginate used in alginate-based matrix tablets.

    PubMed

    Fu, Shao; Buckner, Ira S; Block, Lawrence H

    2014-10-01

    The purpose of this study is to characterize the inter-grade and inter-batch variability of sodium alginate used in the formulation of matrix tablets. Four different grades and three batches of one grade of sodium alginate were used to prepare matrix tablets. Swelling, erosion, and drug release tests of sodium alginate matrix tablets were conducted in a USP dissolution apparatus. Substantial differences in swelling and erosion behavior of sodium alginate matrix tablets were evident among different viscosity grades. Even different batches of the same grade exhibit substantial differences in the swelling and erosion behavior of their matrix tablets. The erosion behavior of sodium alginate matrix tablets can be partly explained by their rheological properties (both apparent viscosity and viscoelasticity) in solution. Sodium alginate with higher apparent viscosity and viscoelasticity in solution show slower erosion rate and higher swelling rate. Compacts prepared from grades or batches with higher viscosity and higher viscoelasticity show slower drug release. For grades or batches with similar apparent viscosities, apparent viscosities of sodium alginate solution at low concentration alone are not sufficient to predict the functionality of sodium alginate in matrix tablets. Viscoelastic properties of sodium alginate solutions at one high concentration corresponding to the polymer gel state, may be suitable indicia of the extended release behavior of sodium alginate matrix tablets.

  6. Performance and Biocompatibility of Extremely Tough Alginate/Polyacrylamide Hydrogels

    PubMed Central

    Darnell, Max; Sun, Jeong-Yun; Mehta, Manav; Johnson, Chris; Arany, Praveen; Suo, Zhigang

    2013-01-01

    Although hydrogels now see widespread use in a host of applications, low fracture toughness and brittleness have limited their more broad use. As a recently described interpenetrating network (IPN) of alginate and polyacrylamide demonstrated a fracture toughness of ∼9000 J/m2, we sought to explore the biocompatibility and maintenance of mechanical properties of these hydrogels in cell culture and in vivo conditions. These hydrogels can sustain a compressive strain of over 90% with minimal loss of Young's Modulus as well as minimal swelling for up to 50 days of soaking in culture conditions. Mouse mesenchymal stem cells exposed to the IPN gel-conditioned media maintain high viability, and although cells exposed to conditioned media demonstrate slight reductions in proliferation and metabolic activity (WST assay), these effects are abrogated in a dose-dependent manner. Implantation of these IPN hydrogels into subcutaneous tissue of rats for 8 weeks led to mild fibrotic encapsulation and minimal inflammatory response. These results suggest the further exploration of extremely tough alginate/PAAM IPN hydrogels as biomaterials. PMID:23896005

  7. Alpha-1,4-glucan lyase, a new class of starch/glycogen degrading enzyme. I. Efficient purification and characterization from red seaweeds.

    PubMed

    Yu, S; Kenne, L; Pedersén, M

    1993-03-21

    This study presents the first purification and characterization of an alpha-1,4-glucan lyase. The enzyme degraded alpha-1,4-glucan to produce 1,5-anhydrofructose. A simple and efficient purification procedure has been developed and the enzyme has been purified to homogeneity from two red seaweeds Gracilariopsis lemaneiformis and Gracilaria verrucosa. alpha-1,4-Glucan lyase was apparently a single polypeptide as a molecular weight of 111,000 was observed in SDS-gel electrophoresis, and 98,000 by gel filtration chromatography on Sephacryl S-200. Amino acid composition analysis of the enzyme showed high amounts of Asp/Asn, Gly and Glu/Gln. The isoelectric point of the enzyme was 3.9, as revealed by isoelectrofocusing. The concentrations of maltotriose, maltose and amylopectin that yield half of the maximum activity were 798 micrograms ml-1 (1.58 mM), 1,418 micrograms ml-1 (4.14 mM) and 1,600 micrograms ml-1, respectively. alpha-1,4-Glucan lyase exhibited a wide pH optimum range from pH 2.5 to 7.0 for maltose and from pH 3.5 to 7.5 for amylopectin. The optimal temperature for activity of the algal lyase was 50 degrees C when maltose or amylopectin was used as a substrate under the assay conditions. The Arrhenius activation energies were 45.8 and 44.0 kJ mol-1 for maltose and amylopectin as substrate, respectively. Only one form of alpha-1,4-glucan lyase was found in cell-free extracts of the two red seaweeds.

  8. Silk sericin-alginate-chitosan microcapsules: hepatocytes encapsulation for enhanced cellular functions.

    PubMed

    Nayak, Sunita; Dey, Sanchareeka; Kundu, Subhas C

    2014-04-01

    The encapsulation based technology permits long-term delivery of desired therapeutic products in local regions of body without the need of immunosuppressant drugs. In this study microcapsules composed of sericin and alginate micro bead as inner core and with an outer chitosan shell are prepared. This work is proposed for live cell encapsulation for potential therapeutic applications. The sericin protein is obtained from cocoons of non-mulberry silkworm Antheraea mylitta. The sericin-alginate micro beads are prepared via ionotropic gelation under high applied voltage. The beads further coated with chitosan and crosslinked with genipin. The microcapsules developed are nearly spherical in shape with smooth surface morphology. Alamar blue assay and confocal microscopy indicate high cell viability and uniform encapsulated cell distribution within the sericin-alginate-chitosan microcapsules indicating that the microcapsules maintain favourable microenvironment for the cells. The functional analysis of encapsulated cells demonstrates that the glucose consumption, urea secretion rate and intracellular albumin content increased in the microcapsules. The study suggests that the developed sericin-alginate-chitosan microcapsule contributes towards the development of cell encapsulation model. It also offers to generate enriched population of metabolically and functionally active cells for the future therapeutics especially for hepatocytes transplantation in acute liver failure.

  9. Mobility of isoproturon from an alginate-bentonite controlled release formulation in layered soil.

    PubMed

    Fernández-Pérez, M; González-Pradas, E; Villafranca-Sánchez, M; Flores-Céspedes, F

    2000-11-01

    The mobility of isoproturon [3-(4-isopropylphenyl)-1,1-dimethylurea] from an alginate-based controlled release (CR) formulation was investigated by using soil columns. A layered bed system simulating the typical arrangement under a plastic greenhouse, which is composed of sand, peat, amended soil and native soil was used. The CR formulation was based on sodium alginate (1.87%), isoproturon (1.19%), natural bentonite (3.28%), and water (93.66%), and was compared to technical grade isoproturon. The use of the alginate-bentonite CR formulation produced less vertical mobility of the active ingredient as compared to the technical product. There was no presence of herbicide in the leachate when the alginate-bentonite CR formulation was used. However, 0.90% of isoproturon appeared when the treatment was carried out with technical grade material. Isoproturon mobility was modelled using the programme CMLS, which showed the peat layer to retard pesticide leaching. Analysis of the soil columns showed the highest isoproturon concentration in the peat layer.

  10. Preparation and characterization of alginate/silver/nicotinamide nanocomposites for treating diabetic wounds.

    PubMed

    Montaser, A S; Abdel-Mohsen, A M; Ramadan, M A; Sleem, A A; Sahffie, N M; Jancar, J; Hebeish, A

    2016-11-01

    Silver/Alginate/Nicotinamide nanoparticles composite (Ag/ALG/Nic) was prepared and used for the first time to fabricate wound dressing material. Sodium alginate (ALG) was used as reducing and stabilizing agents for preparation of silver nanoparticles (Ag-NPs). Effect of concentrations of alginate (ALG) on the particle size of silver were studied and confirmed by different techniques like UV/vis spectroscopy, transmission electron microscope (TEM) and dynamic light scattering (DLS). Nonwoven viscous fabrics were used as a carrier for silver/alginate/nanoparticles composite by impregnated the nonwoven fabrics as per the padding-curing technique. Nicotinamide (Nic) as anti-inflammatory drug was entrapped into Ag-NPS/ALG/nonwoven fabrics. Scanning electron microscope and energy dispersive x-ray (SEM-EDX) were used to evaluate the presence of Ag/ALG/Nic nanoparticles composite anchored the nonwoven fabrics. The antibacterial activity of the Ag/ALG/Nic wound dressing material was evaluated against Escherichia coli (E. coli) and Staphylococcus Aureus (St. Aureus). The wound healing and histological studied were evaluated by using burn diabetic rat animals.

  11. Alginate copper oxide nano-biocomposite as a novel material for amperometric glucose biosensing.

    PubMed

    Buk, Vuslat; Emregul, Emel; Emregul, Kaan Cebesoy

    2017-05-01

    A novel amperometric glucose biosensor based on alginate-CuO nano-biocomposite and glucose oxidase (GOD) film was developed and characterized. The properties of the alginate-CuO-GOD film were characterized using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Amperometric measurements were employed to characterize the analytical performance of the biosensor. Several parameters including amount of alginate, concentration of GOD and cross-linkers, amount of CuO nanoparticles, and effect of pH were studied and optimized. Under optimal conditions, the developed alginate-CuO-GOD biosensor was shown to have two linear ranges; from 0.04mM to 3mM (with a correlation coefficient of 0.9996 and the sensitivity of 30.443μAmM(-1)cm(-2)) and from 4mM to 35mM (with a correlation coefficient of 0.9994 and the sensitivity of 7.205μAmM(-1)cm(-2)). The overall detection limit was estimated to be 1.6μM (signal-to-noise ratio of 3) and the Km value of 2.82mM. The biosensor exhibited rather good performance with long-term stability (remainder of activity is 78% after 15days) and significant specificity for glucose when compared to possible interfering molecules such as ascorbic acid, uric acid and acetaminophen.

  12. Structure of a Bacterial ABC Transporter Involved in the Import of an Acidic Polysaccharide Alginate.

    PubMed

    Maruyama, Yukie; Itoh, Takafumi; Kaneko, Ai; Nishitani, Yu; Mikami, Bunzo; Hashimoto, Wataru; Murata, Kousaku

    2015-09-01

    The acidic polysaccharide alginate represents a promising marine biomass for the microbial production of biofuels, although the molecular and structural characteristics of alginate transporters remain to be clarified. In Sphingomonas sp. A1, the ATP-binding cassette transporter AlgM1M2SS is responsible for the import of alginate across the cytoplasmic membrane. Here, we present the substrate-transport characteristics and quaternary structure of AlgM1M2SS. The addition of poly- or oligoalginate enhanced the ATPase activity of reconstituted AlgM1M2SS coupled with one of the periplasmic solute-binding proteins, AlgQ1 or AlgQ2. External fluorescence-labeled oligoalginates were specifically imported into AlgM1M2SS-containing proteoliposomes in the presence of AlgQ2, ATP, and Mg(2+). The crystal structure of AlgQ2-bound AlgM1M2SS adopts an inward-facing conformation. The interaction between AlgQ2 and AlgM1M2SS induces the formation of an alginate-binding tunnel-like structure accessible to the solvent. The translocation route inside the transmembrane domains contains charged residues suitable for the import of acidic saccharides.

  13. The signaling protein MucG negatively affects the production and the molecular mass of alginate in Azotobacter vinelandii.

    PubMed

    Ahumada-Manuel, Carlos Leonel; Guzmán, Josefina; Peña, Carlos; Quiroz-Rocha, Elva; Espín, Guadalupe; Núñez, Cinthia

    2017-02-01

    Azotobacter vinelandii is a soil bacterium that produces the polysaccharide alginate. In this work, we identified a miniTn5 mutant, named GG9, which showed increased alginate production of higher molecular mass, and increased expression of the alginate biosynthetic genes algD and alg8 when compared to its parental strain. The miniTn5 was inserted within ORF Avin07920 encoding a hypothetical protein. Avin07910, located immediately downstream and predicted to form an operon with Avin07920, encodes an inner membrane multi-domain signaling protein here named mucG. Insertional inactivation of mucG resulted in a phenotype of increased alginate production of higher molecular mass similar to that of mutant GG9. The MucG protein contains a periplasmic and putative HAMP and PAS domains, which are linked to GGDEF and EAL domains. The last two domains are potentially involved in the synthesis and degradation, respectively, of bis-(3'-5')-cyclic dimeric GMP (c-di-GMP), a secondary messenger that has been reported to be essential for alginate production. Therefore, we hypothesized that the negative effect of MucG on the production of this polymer could be explained by the putative phosphodiesterase activity of the EAL domain. Indeed, we found that alanine replacement mutagenesis of the MucG EAL motif or deletion of the entire EAL domain resulted in increased alginate production of higher molecular mass similar to the GG9 and mucG mutants. To our knowledge, this is the first reported protein that simultaneous affects the production of alginate and its molecular mass.

  14. Evaluation of the Effect of Psyllium on the Viability of Lactobacillus Acidophilus in Alginate-Polyl Lysine Beads

    PubMed Central

    Esmaeilzadeh, Jaleh; Nazemiyeh, Hossein; Maghsoodi, Maryam; Lotfipour, Farzaneh

    2016-01-01

    Purpose: Psylliumseeds are used in traditional herbal medicine to treat various disorders. Moreover, as a soluble fiber, psyllium has potential to stimulate bacterial growth in digestive system. We aimed to substitute alkali-extractable polysaccharides of psyllium for alginate in beads with second coat of poly-l-lysine to coat Lactobacillus acidophilus. Methods: Beads were prepared using extrusion technique. Poly-l-lysine as second coat was incorporated on optimum alginate/psyllium beads using immersion technique. Beads were characterized in terms of size, encapsulation efficiency, integrity and bacterial survival in harsh conditions. Results: Beads with narrow size distribution ranging from 1.85 ± 0.05 to 2.40 ± 0.18 mm with encapsulation efficiency higher than 96% were achieved. Psyllium concentrations in beads did not produce constant trend in bead sizes. Surface topography by SEM showed that substitution of psyllium enhanced integrity of obtained beads. Psyllium successfully protected the bacteria against acidic condition and lyophilization equal to alginate in the beads. Better survivability with beads of alginate/psyllium-poly-l-lysine was achieved with around 2 log rise in bacterial count in acid condition compared to the corresponding single coat beads. Conclusion: Alginate/psyllium (1:2) beads with narrow size distribution and high encapsulation efficiency of the bacteria have been achieved. Presence of psyllium produced a much smoother and integrated surface texture for the beads with sufficient protection of the bacteria against acidic condition as much as alginate. Considering the health benefits of psyllium and its prebiotic activity, psyllium can be beneficially replaced in part for alginate in probiotic coating. PMID:27766217

  15. Highly stable and magnetically separable alginate/Fe3O4 composite for the removal of strontium (Sr) from seawater.

    PubMed

    Hong, Hye-Jin; Jeong, Hyeon Su; Kim, Byoung-Gyu; Hong, Jeongsik; Park, In-Su; Ryu, Taegong; Chung, Kang-Sup; Kim, Hyuncheol; Ryu, Jungho

    2016-12-01

    In this study, a highly stable alginate/Fe3O4 composite was synthesized, and systematically investigated for the practical application of strontium (Sr) removal in complex media, such as seawater and radioactive wastewater. To overcome the drawbacks of the use of alginate microspheres, high contents of alginic acid and Fe3O4 were used to provide a more rigid structure with little swelling and facile separation, respectively. The synthesized composite was optimized for particle sizes of <400 μm and 1% content of Fe3O4. The alginate/Fe3O4 composite showed excellent Sr uptake (≈400.0 mg/g) and exhibited outstanding selectivity for Sr among various cations (Na, Mg, Ca and K). However, in diluted Sr condition (50 mg/L), Ca significantly affected Sr adsorption, resulting in a decrease of Kd value from 3.7 to 2.4 at the 0.01 M Ca. The alginate/Fe3O4 composite could be completely regenerated using 0.1 M HCl and CaCl2. In real seawater spiked with 50 mg/L of Sr, the alginate/Fe3O4 composite showed 12.5 mg/g of Sr uptake, despite the highly concentrated ions in seawater. The adsorption experiment for radio-active (90)Sr revealed a removal efficiency of 67% in real seawater, demonstrating the reliability of the alginate/Fe3O4 composite.

  16. Three-dimensional structures of noncovalent complexes of Citrobacter freundii methionine γ-lyase with substrates.

    PubMed

    Revtovich, S V; Morozova, E A; Khurs, E N; Zakomirdina, L N; Nikulin, A D; Demidkina, T V; Khomutov, R M

    2011-05-01

    Crystal structures of Citrobacter freundii methionine γ-lyase complexes with the substrates of γ- (L-1-amino-3-methylthiopropylphosphinic acid) and β- (S-ethyl-L-cysteine) elimination reactions and the competitive inhibitor L-norleucine have been determined at 1.45, 1.8, and 1.63 Å resolution, respectively. All three amino acids occupy the active site of the enzyme but do not form a covalent bond with pyridoxal 5'-phosphate. Hydrophobic interactions between the active site residues and the side groups of the substrates and the inhibitor are supposed to cause noncovalent binding. Arg374 and Ser339 are involved in the binding of carboxyl groups of the substrates and the inhibitor. The hydroxyl of Tyr113 is a potential acceptor of a proton from the amino groups of the amino acids.

  17. Multifaceted regulations of gateway enzyme phenylalanine ammonia-lyase in the biosynthesis of phenylpropanoids

    DOE PAGES

    Zhang, Xuebin; Liu, Chang-Jun

    2014-12-11

    Phenylpropanoid biosynthesis in plants engenders a vast variety of aromatic metabolites critically important for their growth, development, and environmental adaptation. Some of these aromatic compounds have high economic value. Phenylalanine ammonia-lyase (PAL) is the first committed enzyme in the pathway; it diverts the central flux of carbon from primary metabolism to the synthesis of myriad phenolics. Over the decades, many studies have shown that exquisite regulatory mechanisms at multiple levels control the transcription and the enzymatic activity of PALs. In this review, we present a current overview on our understanding of the complicated regulatory mechanisms governing PAL's activity; we particularlymore » highlight recent progresses in unraveling its post-translational modifications, its metabolite feedback regulation, and its enzyme organization.« less

  18. Multifaceted regulations of gateway enzyme phenylalanine ammonia-lyase in the biosynthesis of phenylpropanoids

    SciTech Connect

    Zhang, Xuebin; Liu, Chang-Jun

    2014-12-11

    Phenylpropanoid biosynthesis in plants engenders a vast variety of aromatic metabolites critically important for their growth, development, and environmental adaptation. Some of these aromatic compounds have high economic value. Phenylalanine ammonia-lyase (PAL) is the first committed enzyme in the pathway; it diverts the central flux of carbon from primary metabolism to the synthesis of myriad phenolics. Over the decades, many studies have shown that exquisite regulatory mechanisms at multiple levels control the transcription and the enzymatic activity of PALs. In this review, we present a current overview on our understanding of the complicated regulatory mechanisms governing PAL's activity; we particularly highlight recent progresses in unraveling its post-translational modifications, its metabolite feedback regulation, and its enzyme organization.

  19. Engineering methylaspartate ammonia lyase for the asymmetric synthesis of unnatural amino acids

    NASA Astrophysics Data System (ADS)

    Raj, Hans; Szymański, Wiktor; de Villiers, Jandré; Rozeboom, Henriëtte J.; Veetil, Vinod Puthan; Reis, Carlos R.; de Villiers, Marianne; Dekker, Frank J.; de Wildeman, Stefaan; Quax, Wim J.; Thunnissen, Andy-Mark W. H.; Feringa, Ben L.; Janssen, Dick B.; Poelarends, Gerrit J.

    2012-06-01

    The redesign of enzymes to produce catalysts for a predefined transformation remains a major challenge in protein engineering. Here, we describe the structure-based engineering of methylaspartate ammonia lyase (which in nature catalyses the conversion of 3-methylaspartate to ammonia and 2-methylfumarate) to accept a variety of substituted amines and fumarates and catalyse the asymmetric synthesis of aspartic acid derivatives. We obtained two single-active-site mutants, one exhibiting a wide nucleophile scope including structurally diverse linear and cyclic alkylamines and one with broad electrophile scope including fumarate derivatives with alkyl, aryl, alkoxy, aryloxy, alkylthio and arylthio substituents at the C2 position. Both mutants have an enlarged active site that accommodates the new substrates while retaining the high stereo- and regioselectivity of the wild-type enzyme. As an example, we demonstrate a highly enantio- and diastereoselective synthesis of threo-3-benzyloxyaspartate (an important inhibitor of neuronal excitatory glutamate transporters in the brain).

  20. Comparison of the cryoprotective effects of trehalose, alginate, and its oligosaccharides on peeled shrimp (Litopenaeus vannamei) during frozen storage.

    PubMed

    Ma, Lu-kai; Zhang, Bin; Deng, Shang-gui; Xie, Chao

    2015-03-01

    The cryoprotective effects of trehalose, alginate, and its oligosaccharides on peeled shrimp (Litopenaeus vannamei) during frozen storage was investigated by monitoring thawing loss, color, texture, myofibrillar protein content, Ca2+ -ATPase activity, and performing microscopic structural analysis. Data revealed significant (p < 0.05) inhibitory effects on thawing loss and textural variables (springiness and chewiness) in trehalose-, alginate oligosaccharides-, and sodium pyrophosphate-treated shrimp compared with the control and alginate-treated batches. L* values revealed that these saccharides had a positive effect on color stability during frozen storage. In addition, the results of chemical analyses showed that trehalose and alginate oligosaccharide treatments effectively maintained an increased myofibrillar protein content and Ca2+ -ATPase activity in frozen shrimp. In addition, hematoxylin & eosin staining and SDS-PAGE confirmed that these cryoprotective saccharides slowed the degradation of muscle proteins and the damage to muscle tissue structures. Overall, the application of trehalose and alginate oligosaccharides to peeled frozen shrimp might maintain better quality and extend the commercialization of these refrigerated products.

  1. Preparation of microfibrillated cellulose/chitosan-benzalkonium chloride biocomposite for enhancing antibacterium and strength of sodium alginate films.

    PubMed

    Liu, Kai; Lin, Xinxing; Chen, Lihui; Huang, Liulian; Cao, Shilin; Wang, Huangwei

    2013-07-03

    The nonantibacterial and low strength properties of sodium alginate films negatively impact their application for food packaging. In order to improve these properties, a novel chitosan-benzalkonium chloride (C-BC) complex was prepared by ionic gelation using tripolyphosphate (TPP) as a coagulant, and a biocomposite obtained through the adsorption of C-BC complex on microfibrillated cellulose, MFC/C-BC, was then incorporated into a sodium alginate film. The TEM image showed that the C-BC nanoparticles were spherical in shape with a diameter of about 30 nm, and the adsorption equilibrium time of these nanoparticles on the surface of MFC was estimated to be 6 min under the driving forces of hydrogen bonds and electrostatic interactions. According to the disc diffusion method, the MFC/C-BC biocomposite-incorporated sodium alginate film exhibited remarkable antibacterial activity against Staphylococcus aureus and certain antibacterial activity against Escherichia coli . The strength tests indicated that the tensile strength of the composite sodium alginate film increased about 225% when the loading of MFC/C-BC biocomposite was 10 wt %. These results suggested that the MFC/C-BC biocomposite-incorporated sodium alginate film with excellent antibacterial and strength properties would be a promising material for food packaging, and the MFC/C-BC may also be a potential multifunctional biocomposite for other biodegradable materials.

  2. Exploration of swapping enzymatic function between two proteins: a simulation study of chorismate mutase and isochorismate pyruvate lyase.

    PubMed

    Choutko, Alexandra; Eichenberger, Andreas P; van Gunsteren, Wilfred F; Dolenc, Jožica

    2013-06-01

    The enzyme chorismate mutase EcCM from Escherichia coli catalyzes one of the few pericyclic reactions in biology, the transformation of chorismate to prephenate. The isochorismate pyruvate lyase PchB from Pseudomonas aeroginosa catalyzes another pericyclic reaction, the isochorismate to salicylate transformation. Interestingly, PchB possesses weak chorismate mutase activity as well thus being able to catalyze two distinct pericyclic reactions in a single active site. EcCM and PchB possess very similar folds, despite their low sequence identity. Using molecular dynamics simulations of four combinations of the two enzymes (EcCM and PchB) with the two substrates (chorismate and isochorismate) we show that the electrostatic field due to EcCM at atoms of chorismate favors the chorismate to prephenate transition and that, analogously, the electrostatic field due to PchB at atoms of isochorismate favors the isochorismate to salicylate transition. The largest differences between EcCM and PchB in electrostatic field strengths at atoms of the substrates are found to be due to residue side chains at distances between 0.6 and 0.8 nm from particular substrate atoms. Both enzymes tend to bring their non-native substrate in the same conformation as their native substrate. EcCM and to a lower extent PchB fail in influencing the forces on and conformations of the substrate such as to favor the other chemical reaction (isochorismate pyruvate lyase activity for EcCM and chorismate mutase activity for PchB). These observations might explain the difficulty of engineering isochorismate pyruvate lyase activity in EcCM by solely mutating active site residues.

  3. Versatile click alginate hydrogels crosslinked via tetrazine-norbornene chemistry.

    PubMed

    Desai, Rajiv M; Koshy, Sandeep T; Hilderbrand, Scott A; Mooney, David J; Joshi, Neel S

    2015-05-01

    Alginate hydrogels are well-characterized, biologically inert materials that are used in many biomedical applications for the delivery of drugs, proteins, and cells. Unfortunately, canonical covalently crosslinked alginate hydrogels are formed using chemical strategies that can be biologically harmful due to their lack of chemoselectivity. In this work we introduce tetrazine and norbornene groups to alginate polymer chains and subsequently form covalently crosslinked click alginate hydrogels capable of encapsulating cells without damaging them. The rapid, bioorthogonal, and specific click reaction is irreversible and allows for easy incorporation of cells with high post-encapsulation viability. The swelling and mechanical properties of the click alginate hydrogel can be tuned via the total polymer concentration and the stoichiometric ratio of the complementary click functional groups. The click alginate hydrogel can be modified after gelation to display cell adhesion peptides for 2D cell culture using thiol-ene chemistry. Furthermore, click alginate hydrogels are minimally inflammatory, maintain structural integrity over several months, and reject cell infiltration when injected subcutaneously in mice. Click alginate hydrogels combine the numerous benefits of alginate hydrogels with powerful bioorthogonal click chemistry for use in tissue engineering applications involving the stable encapsulation or delivery of cells or bioactive molecules.

  4. PLGA/alginate composite microspheres for hydrophilic protein delivery.

    PubMed

    Zhai, Peng; Chen, X B; Schreyer, David J

    2015-11-01

    Poly(lactic-co-glycolic acid) (PLGA) microspheres and PLGA/alginate composite microspheres were prepared by a novel double emulsion and solvent evaporation technique and loaded with bovine serum albumin (BSA) or rabbit anti-laminin antibody protein. The addition of alginate and the use of a surfactant during microsphere preparation increased the encapsulation efficiency and reduced the initial burst release of hydrophilic BSA. Confocal laser scanning microcopy (CLSM) of BSA-loaded PLGA/alginate composite microspheres showed that PLGA, alginate, and BSA were distributed throughout the depths of microspheres; no core/shell structure was observed. Scanning electron microscopy revealed that PLGA microspheres erode and degrade more quickly than PLGA/alginate composite microspheres. When loaded with anti-laminin antibody, the function of released antibody was well preserved in both PLGA and PLGA/alginate composite microspheres. The biocompatibility of PLGA and PLGA/alginate microspheres were examined using four types of cultured cell lines, representing different tissue types. Cell survival was variably affected by the inclusion of alginate in composite microspheres, possibly due to the sensitivity of different cell types to excess calcium that may be released from the calcium cross-linked alginate.

  5. Stability testing of alginate-chitosan films.

    PubMed

    Rabisková, Miloslava; Dvorácková, Katerina; Kofronvá, Lenka

    2012-02-01

    Pellets containing rutin prepared by the extrusion/spheronization method were coated with sodium alginate-chitosan film. Important quality parameters in the pellets before coating were determined, and after coating the dissolution profiles of the drug were evaluated in dissolution media of the pH corresponding to the conditions in the gastrointestinal tract. Samples of coated pellets were located in the boxes for stability testing under different conditions, i.e. 25 degrees C and 60% of relative humidity (RH); 30 degrees C and 65% RH and 40 degrees C and 75% RH. After 1, 3, 6, 9 and 12 months (or 1, 3 and 6 months), the dissolution test was repeated and compared with the original profiles using similarity factors. All similarity factor values above 50 indicate excellent stability of alginate-chitosan films.

  6. Cloning, expression and characterization of a pectate lyase from Paenibacillus sp. 0602 in recombinant Escherichia coli

    PubMed Central

    2014-01-01

    Background Biotechnological applications of microbial pectate lyases (Pels) in plant fiber processing are considered as environmentally friendly. As such, they become promising substitutes for conventional chemical degumming process. Since applications of Pels in various fields are widening, it is necessary to explore new pectolytic microorganisms and enzymes for efficient and effective usage. Here, we describe the cloning, expression, characterization and application of the recombinant Pel protein from a pectolytic bacterium of the genus Paenibacillus in Escherichia coli. Results A Pel gene (pelN) was cloned using degenerate PCR and inverse PCR from the chromosomal DNA of Paenibacillus sp. 0602. The open reading frame of pelN encodes a 30 amino acid signal peptide and a 445 amino acid mature protein belonging to the polysaccharide lyase family 1. The maximum Pel activity produced by E. coli in shake flasks reached 2,467.4 U mL−1, and the purified recombinant enzyme exhibits a specific activity of 2,060 U mg−1 on polygalacturonic acid (PGA). The maximum activity was observed in a buffer with 5 mM Ca2+ at pH 9.8 and 65°C. PelN displays a half-life of around 9 h and 42 h at 50°C and 45°C, respectively. The biochemical treatment achieved the maximal reduction of percentage weight (30.5%) of the ramie bast fiber. Conclusions This work represents the first study that describes the extracellular expression of a Pel gene from Paenibacillus species in E. coli. The high yield of the extracellular overexpression, relevant thermostability and efficient degumming using combined treatments indicate its strong potential for large-scale industrial production. PMID:24612647

  7. CYP17 inhibitors--abiraterone, C17,20-lyase inhibitors and multi-targeting agents.

    PubMed

    Yin, Lina; Hu, Qingzhong

    2014-01-01

    As the first in class steroid 17α-hydroxylase/C17,20-lyase (CYP17) inhibitor, abiraterone acetate (of which the active metabolite is abiraterone) has been shown to improve overall survival in patients with castration-resistant prostate cancer (CRPC)--in those who are chemotherapy-naive and those previously treated with docetaxel. Furthermore, the clinical success of abiraterone demonstrated that CRPC, which has previously been regarded as an androgen-independent disease, is still driven, at least in part, by androgens. More importantly, abiraterone is a 'promiscuous' drug that interacts with a number of targets, which dictate its clinical benefits and adverse effects profile. Besides CYP17 inhibition, abiraterone acts as an antagonist to the androgen receptor and inhibits 3β-hydroxysteroid dehydrogenase--two effects that potentially contribute to its antitumour effects. However, the inhibition of the 17α-hydroxylase activity of CYP17, CYP11B1 and a panel of hepatic CYP enzymes leads to adverse effects and toxicities that include secondary mineralocorticoid excess. Abiraterone is also associated with increased incidence of cardiac disorders. Under such circumstances, development of new CYP17 inhibitors as an additional line of defence is urgently needed. To achieve enhanced clinical benefits, new strategies are being explored that include selective inhibition of the C17,20-lyase activity of CYP17 and multi-targeting strategies that affect androgen synthesis and signalling at different points. Some of these strategies-including the drugs orteronel, VT-464 and galeterone--are supported by preclinical data and are being explored in the clinic.

  8. Magnetite-alginate beads for purification of some starch degrading enzymes.

    PubMed

    Teotia, Sunita; Gupta, M N

    2002-03-01

    Starch degrading enzymes, viz., beta-amylase, glucoamylase, and pullulanase, were purified using magnetite-alginate beads. In each case, the enzyme activity was eluted by using 1.0 M maltose. beta-Amylase (sweet potato), glucoamylase (Aspergillus niger), and pullulanase (Bacillus acidopullulyticus) from their crude preparations were purified 37-, 31-, and 49-fold with 86, 87, and 95% activity recovery, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed single band in each case.

  9. L-Phenylalanine ammonia-lyase from Phaseolus vulgaris. Characterisation and differential induction of multiple forms from elicitor-treated cell suspension cultures.

    PubMed

    Bolwell, G P; Bell, J N; Cramer, C L; Schuch, W; Lamb, C J; Dixon, R A

    1985-06-03

    -fold increase in phenylalanine ammonia-lyase extractable activity within 8 h, and chromatofocussing analysis indicated that this was associated with differential increased appearance of the high-pI, low-Km forms as compared to the two higher Km forms. This differential induction was further confirmed by immune blotting of crude extracts subjected to isoelectric focussing.

  10. New Family of Ulvan Lyases Identified in Three Isolates from the Alteromonadales Order*

    PubMed Central

    Kopel, Moran; Helbert, William; Belnik, Yana; Buravenkov, Vitaliy; Herman, Asael; Banin, Ehud

    2016-01-01

    Ulvan is the main polysaccharide component of the Ulvales (green seaweed) cell wall. It is composed of disaccharide building blocks comprising 3-sulfated rhamnose linked to d-glucuronic acid (GlcUA), l-iduronic acid (IdoUA), or d-xylose (Xyl). The degradation of ulvan requires ulvan lyase, which catalyzes the endolytic cleavage of the glycoside bond between 3-sulfated rhamnose and uronic acid according to a β-elimination mechanism. The first characterized ulvan lyase was identified in Nonlabens ulvanivorans, an ulvanolytic bacterial isolate. In the current study, we have identified and biochemically characterized novel ulvan lyases from three Alteromonadales isolated bacteria. Two homologous ulvan lyases (long and short) were found in each of the bacterial genomes. The protein sequences have no homology to the previously reported ulvan lyases and therefore are the first representatives of a new family of polysaccharide lyases. The enzymes were heterologously expressed in Escherichia coli to determine their mode of action. The heterologous expressed enzymes were secreted into the milieu subsequent to their signal sequence cleavage. An endolytic mode of action was observed and studied using gel permeation chromatography and 1H NMR. In contrast to N. ulvanivorans ulvan lyase, cleavage occurred specifically at the GlcUA residues. In light of the genomic context and modular structure of the ulvan lyase families identified to date, we propose that two ulvan degradation pathways evolved independently. PMID:26763234

  11. Biocompatibility of microcapsules for cell immobilization elaborated with different type of alginates.

    PubMed

    Orive, G; Ponce, S; Hernández, R M; Gascón, A R; Igartua, M; Pedraz, J L

    2002-09-01

    The biocompatibility of alginate-PLL-alginate (APA) microcapsules has been evaluated with respect to impurity levels. The impurity content of three different alginates (a raw high M-alginate, a raw high G-alginate and a purified high G-alginate) has been determined and the in vivo antigenic response of APA beads made with each alginate assessed. Results show that purification of the alginate not only reduces the total amount of impurities (63% less in polyphenols, 91.45% less in endotoxins and 68.5% less in protein in relation to raw high M-alginate), but also avoids an antibody response when microcapsules of this material are implanted in mice. In contrast, raw alginates produced a detectable antibody response though the differences in their impurity content. Consequently, this work revealed that purity of the alginate rather than their chemical composition, is probably of greater importance in determining microcapsule biocompatibility.

  12. A three-dimensional culture system using alginate hydrogel prolongs hatched cattle embryo development in vitro.

    PubMed

    Zhao, Shuan; Liu, Zhen-Xing; Gao, Hui; Wu, Yi; Fang, Yuan; Wu, Shuai-Shuai; Li, Ming-Jie; Bai, Jia-Hua; Liu, Yan; Evans, Alexander; Zeng, Shen-Ming

    2015-07-15

    No successful method exists to maintain the three-dimensional architecture of hatched embryos in vitro. Alginate, a linear polysaccharide derived from brown algae, has characteristics that make it an ideal material as a three-dimensional (3D) extracellular matrix for in vitro cell, tissue, or embryo culture. In this study, alginate hydrogel was used for IVC of posthatched bovine embryos to observe their development under the 3D system. In vitro-fertilized and parthenogenetically activated posthatched bovine blastocysts were cultured in an alginate encapsulation culture system (AECS), an alginate overlay culture system (AOCS), or control culture system. After 18 days of culture, the survival rate of embryos cultured in AECS was higher than that in the control group (P < 0.05), and the embryos were expanded and elongated in AECS with the maximal length of 1.125 mm. When the AECS shrinking embryos were taken out of the alginate beads on Day 18 and cultured in the normal culture system, 9.09% of them attached to the bottoms of the plastic wells and grew rapidly, with the largest area of an attached embryo being 66.00 mm(2) on Day 32. The embryos cultured in AOCS developed monovesicular or multivesicular morphologies. Total cell number of the embryos cultured in AECS on Day 19 was significantly higher than that of embryos on Day 8. Additionally, AECS and AOCS supported differentiation of the embryonic cells. Binuclear cells were visible in Day-26 adherent embryos, and the messenger RNA expression patterns of Cdx2 and Oct4 in AOCS-cultured embryos were similar to those in vivo embryos, whereas IFNT and ISG15 messenger RNA were still expressed in Day-26 and Day-32 prolong-cultured embryos. In conclusion, AECS and AOCS did support cell proliferation, elongation, and differentiation of hatched bovine embryos during prolonged IVC. The culture system will be useful to further investigate the molecular mechanisms controlling ruminant embryo elongation and implantation.

  13. Preparation and detection of calcium alginate/bone powder hybrid microbeads for in vitro culture of ADSCs.

    PubMed

    Song, Kedong; Yan, Xinyu; Li, Shixiao; Zhang, Yu; Wang, Hong; Wang, Ling; Lim, Mayasari; Liu, Tianqing

    2015-01-01

    Calcium alginate microbeads have been widely used in tissue engineering application, due to their excellent biocompatibility, biodegradability, enhanced mechanical strength and toughness. Bone powder containing abundant hydroxylapatite, type I collagen and growth factors such as BMP2 and BMP4, possesses good osteoinductive activity. Herein, a hybrid calcium alginate/bone powder microbead was therefore prepared. Afterwards, different seeding density of adipose-derived stem cells (ADSCs) in these hybrid microbeads was discussed systematically for further in vitro expansion. Optimised microbeads suitable for in vitro expansion and differentiation of ADSCs were prepared using the droplet method under overall considering suitable concentrations of calcium alginate and calcium chloride as well as the density of bone powder through an orthogonal experiment. The results showed that the concentration of sodium alginate had the most influence on inside mass transfer and mechanical strength of the hybrid microbeads, secondly the calcium chloride, then the density of bone powder. The hybrid microbeads could be optimally performed while the concentrations of sodium alginate and calcium chloride were 2.5% and 4.5%, as well as 5.0 mg/mL bone powder, respectively. Live/Dead assay showed that the expanded ADSCs differentiated well with an initial embedding density of 5 × 10(6) cells/mL.

  14. Spore Photoproduct Lyase: The Known, the Controversial, and the Unknown*

    PubMed Central

    Yang, Linlin; Li, Lei

    2015-01-01

    Spore photoproduct lyase (SPL) repairs 5-thyminyl-5,6-dihydrothymine, a thymine dimer that is also called the spore photoproduct (SP), in germinating endospores. SPL is a radical S-adenosylmethionine (SAM) enzyme, utilizing the 5′-deoxyadenosyl radical generated by SAM reductive cleavage reaction to revert SP to two thymine residues. Here we review the current progress in SPL mechanistic studies. Protein radicals are known to be involved in SPL catalysis; however, how these radicals are quenched to close the catalytic cycle is under debate. PMID:25477522

  15. Enhancing Production of Alkaline Polygalacturonate Lyase from Bacillus subtilis by Fed-Batch Fermentation

    PubMed Central

    Zou, Mouyong; Guo, Fenfen; Li, Xuezhi; Zhao, Jian; Qu, Yinbo

    2014-01-01

    Alkaline polygalacturonate lyase (PGL, EC 4.2.2.2) is an enzyme used in many industries. We developed a fed-batch fermentation process that combines the enzymatic pretreatment of the carbon source with controlling the pH of the fermentative broth to enhance the PGL production from Bacillus subtilis 7-3-3 to decrease the production cost. Maintaining the fermentation broth at pH 6.5 prior to feeding with ammonia and at pH 6.0 after feeding significantly improved PGL activity (743.5 U mL−1) compared with the control (202.5 U mL−1). The average PGL productivity reached 19.6 U mL−1 h−1 after 38 h of fermentation. The crude PGL was suitable for environmentally friendly ramie enzymatic degumming. PMID:24603713

  16. Sphingosine 1-phosphate lyase enzyme assay using a BODIPY-labeled substrate

    SciTech Connect

    Bandhuvula, Padmavathi; Li Zaiguo; Bittman, Robert; Saba, Julie D.

    2009-03-06

    Sphingosine 1-phosphate lyase (SPL) is responsible for the irreversible catabolism of sphingosine 1-phosphate, which signals through five membrane receptors to mediate cell stress responses, angiogenesis, and lymphocyte trafficking. The standard assay for SPL activity utilizes a radioactive dihydrosphingosine 1-phosphate substrate and is expensive and cumbersome. In this study, we describe an SPL assay that employs an {omega}-labeled BODIPY-sphingosine 1-phosphate substrate, allowing fluorescent product detection by HPLC and incorporating advantages of the BODIPY fluorophore. The major aldehyde product is confirmed by reaction with 2,4-dinitrophenylhydrazine. The SPL-catalyzed reaction is linear over a 30 min time period and yields a K{sub m} of 35 {mu}M for BODIPY-sphingosine 1-phosphate.

  17. Identification and functional analysis of the gene encoding methionine-gamma-lyase in Brevibacterium linens.

    PubMed

    Amarita, Felix; Yvon, Mireille; Nardi, Michele; Chambellon, Emilie; Delettre, Jerôme; Bonnarme, Pascal

    2004-12-01

    The enzymatic degradation of L-methionine and subsequent formation of volatile sulfur compounds (VSCs) is believed to be essential for flavor development in cheese. L-methionine-gamma-lyase (MGL) can convert L-methionine to methanethiol (MTL), alpha-ketobutyrate, and ammonia. The mgl gene encoding MGL was cloned from the type strain Brevibacterium linens ATCC 9175 known to produce copious amounts of MTL and related VSCs. The disruption of the mgl gene, achieved in strain ATCC 9175, resulted in a 62% decrease in thiol-producing activity and a 97% decrease in total VSC production in the knockout strain. Our work shows that L-methionine degradation via gamma-elimination is a key step in the formation of VSCs in B. linens.

  18. Phenylalanine ammonia lyase catalyzed synthesis of amino acids by an MIO-cofactor independent pathway.

    PubMed

    Lovelock, Sarah L; Lloyd, Richard C; Turner, Nicholas J

    2014-04-25

    Phenylalanine ammonia lyases (PALs) belong to a family of 4-methylideneimidazole-5-one (MIO) cofactor dependent enzymes which are responsible for the conversion of L-phenylalanine into trans-cinnamic acid in eukaryotic and prokaryotic organisms. Under conditions of high ammonia concentration, this deamination reaction is reversible and hence there is considerable interest in the development of PALs as biocatalysts for the enantioselective synthesis of non-natural amino acids. Herein the discovery of a previously unobserved competing MIO-independent reaction pathway, which proceeds in a non-stereoselective manner and results in the generation of both L- and D-phenylalanine derivatives, is described. The mechanism of the MIO-independent pathway is explored through isotopic-labeling studies and mutagenesis of key active-site residues. The results obtained are consistent with amino acid deamination occurring by a stepwise E1 cB elimination mechanism.

  19. Production optimization of invertase by Lactobacillus brevis Mm-6 and its immobilization on alginate beads.

    PubMed

    Awad, Ghada E A; Amer, Hassan; El-Gammal, Eman W; Helmy, Wafaa A; Esawy, Mona A; Elnashar, Magdy M M

    2013-04-02

    A sequential optimization strategy, based on statistical experimental designs, was employed to enhance the production of invertase by Lactobacillus brevis Mm-6 isolated from breast milk. First, a 2-level Plackett-Burman design was applied to screen the bioprocess parameters that significantly influence the invertase production. The second optimization step was performed using fractional factorial design in order to optimize the amounts of variables have the highest positive significant effect on the invertase production. A maximal enzyme activity of 1399U/ml was more than five folds the activity obtained using the basal medium. Invertase was immobilized onto grafted alginate beads to improve the enzyme's stability. Immobilization process increased the operational temperature from 30 to 60°C compared to the free enzyme. The reusability test proved the durability of the grafted alginate beads for 15 cycles with retention of 100% of the immobilized enzyme activity to be more convenient for industrial uses.

  20. A C⋅As lyase for degradation of environmental organoarsenical herbicides and animal husbandry growth promoters

    PubMed Central

    Yoshinaga, Masafumi; Rosen, Barry P.

    2014-01-01

    Arsenic is the most widespread environmental toxin. Substantial amounts of pentavalent organoarsenicals have been used as herbicides, such as monosodium methylarsonic acid (MSMA), and as growth enhancers for animal husbandry, such as roxarsone (4-hydroxy-3-nitrophenylarsonic acid) [Rox(V)]. These undergo environmental degradation to more toxic inorganic arsenite [As(III)]. We previously demonstrated a two-step pathway of degradation of MSMA to As(III) by microbial communities involving sequential reduction to methylarsonous acid [MAs(III)] by one bacterial species and demethylation from MAs(III) to As(III) by another. In this study, the gene responsible for MAs(III) demethylation was identified from an environmental MAs(III)-demethylating isolate, Bacillus sp. MD1. This gene, termed arsenic inducible gene (arsI), is in an arsenic resistance (ars) operon and encodes a nonheme iron-dependent dioxygenase with C⋅As lyase activity. Heterologous expression of ArsI conferred MAs(III)-demethylating activity and MAs(III) resistance to an arsenic-hypersensitive strain of Escherichia coli, demonstrating that MAs(III) demethylation is a detoxification process. Purified ArsI catalyzes Fe2+-dependent MAs(III) demethylation. In addition, ArsI cleaves the C⋅As bond in trivalent roxarsone and other aromatic arsenicals. ArsI homologs are widely distributed in prokaryotes, and we propose that ArsI-catalyzed organoarsenical degradation has a significant impact on the arsenic biogeocycle. To our knowledge, this is the first report of a molecular mechanism for organoarsenic degradation by a C⋅As lyase. PMID:24821808

  1. A C⋅As lyase for degradation of environmental organoarsenical herbicides and animal husbandry growth promoters.

    PubMed

    Yoshinaga, Masafumi; Rosen, Barry P

    2014-05-27

    Arsenic is the most widespread environmental toxin. Substantial amounts of pentavalent organoarsenicals have been used as herbicides, such as monosodium methylarsonic acid (MSMA), and as growth enhancers for animal husbandry, such as roxarsone (4-hydroxy-3-nitrophenylarsonic acid) [Rox(V)]. These undergo environmental degradation to more toxic inorganic arsenite [As(III)]. We previously demonstrated a two-step pathway of degradation of MSMA to As(III) by microbial communities involving sequential reduction to methylarsonous acid [MAs(III)] by one bacterial species and demethylation from MAs(III) to As(III) by another. In this study, the gene responsible for MAs(III) demethylation was identified from an environmental MAs(III)-demethylating isolate, Bacillus sp. MD1. This gene, termed arsenic inducible gene (arsI), is in an arsenic resistance (ars) operon and encodes a nonheme iron-dependent dioxygenase with C ⋅ As lyase activity. Heterologous expression of ArsI conferred MAs(III)-demethylating activity and MAs(III) resistance to an arsenic-hypersensitive strain of Escherichia coli, demonstrating that MAs(III) demethylation is a detoxification process. Purified ArsI catalyzes Fe(2+)-dependent MAs(III) demethylation. In addition, ArsI cleaves the C ⋅ As bond in trivalent roxarsone and other aromatic arsenicals. ArsI homologs are widely distributed in prokaryotes, and we propose that ArsI-catalyzed organoarsenical degradation has a significant impact on the arsenic biogeocycle. To our knowledge, this is the first report of a molecular mechanism for organoarsenic degradation by a C ⋅ As lyase.

  2. Substrate and Product Complexes of Escherichia Coli Adenylosuccinate Lyase Provide New Insights into the Enzymatic Mechanism

    SciTech Connect

    Tsai,M.; Koo, J.; Yip, P.; Colman, R.; Segall, M.; Howell, P.

    2007-01-01

    Adenylosuccinate lyase (ADL) catalyzes the breakdown of 5-aminoimidazole- (N-succinylocarboxamide) ribotide (SAICAR) to 5-aminoimidazole-4-carboxamide ribotide (AICAR) and fumarate, and of adenylosuccinate (ADS) to adenosine monophosphate (AMP) and fumarate in the de novo purine biosynthetic pathway. ADL belongs to the argininosuccinate lyase (ASL)/fumarase C superfamily of enzymes. Members of this family share several common features including: a mainly {alpha}-helical, homotetrameric structure; three regions of highly conserved amino acid residues; and a general acid-base catalytic mechanism with the overall {beta}-elimination of fumarate as a product. The crystal structures of wild-type Escherichia coli ADL (ec-ADL), and mutant-substrate (H171A-ADS) and -product (H171N-AMP{center_dot}FUM) complexes have been determined to 2.0, 1.85, and 2.0 {angstrom} resolution, respectively. The H171A-ADS and H171N-AMP{center_dot}FUM structures provide the first detailed picture of the ADL active site, and have enabled the precise identification of substrate binding and putative catalytic residues. Contrary to previous suggestions, the ec-ADL structures implicate S295 and H171 in base and acid catalysis, respectively. Furthermore, structural alignments of ec-ADL with other superfamily members suggest for the first time a large conformational movement of the flexible C3 loop (residues 287-303) in ec-ADL upon substrate binding and catalysis, resulting in its closure over the active site. This loop movement has been observed in other superfamily enzymes, and has been proposed to be essential for catalysis. The ADL catalytic mechanism is re-examined in light of the results presented here.

  3. Uncovering divergent evolution of α/β-hydrolases: a surprising residue substitution needed to convert Hevea brasiliensis hydroxynitrile lyase into an esterase

    PubMed Central

    Nedrud, David M.; Lin, Hui; Lopez, Gilsinia; Padhi, Santosh K.; Legatt, Graig A.

    2014-01-01

    Hevea brasiliensis hydroxynitrile lyase (HbHNL) and salicylic acid binding protein 2 (SABP2, an esterase) share 45% amino acid sequence identity, the same protein fold, and even the same catalytic triad of Ser-His-Asp. However, they catalyze different reactions: cleavage of hydroxynitriles and hydrolysis of esters, respectively. To understand how other active site differences in the two enzymes enable the same catalytic triad to catalyze different reactions, we substituted amino acid residues in HbHNL with the corresponding residues from SABP2, expecting hydroxynitrile lyase activity to decrease and esterase activity to increase. Previous mechanistic studies and x-ray crystallography suggested that esterase activity requires removal of an active site lysine and threonine from the hydroxynitrile lyase. The Thr11Gly Lys236Gly substitutions in HbHNL reduced hydroxynitrile lyase activity for cleavage of mandelonitrile 100-fold, but increased esterase activity only threefold to kcat ~ 0.1 min−1 for hydrolysis of p-nitrophenyl acetate. Adding a third substitution – Glu79His – increased esterase activity more than tenfold to kcat ~ 1.6 min−1. The specificity constant (kcat/KM) for this triple substitution variant versus wild type HbHNL shifted more than one million-fold from hydroxynitrile lyase activity (acetone cyanohydrin substrate) to esterase activity (p-nitrophenyl acetate substrate). The contribution of Glu79His to esterase activity was surprising since esterases and lipases contain many different amino acids at this position, including glutamate. Saturation mutagenesis at position 79 showed that 13 of 19 possible amino acid substitutions increased esterase activity, suggesting that removal of glutamate, not addition of histidine, increased esterase activity. Molecular modeling indicates that Glu79 disrupts esterase activity in HbHNL when its negatively charged side chain distorts the orientation of the catalytic histidine. Naturally occurring glutamate at

  4. Evaluation of microbeads of calcium alginate as a fluidized bed medium for affinity chromatography of Aspergillus niger Pectinase.

    PubMed

    Roy, Ipsita; Jain, Sulakshana; Teotia, Sunita; Gupta, Munishwar Nath

    2004-01-01

    Calcium alginate microbeads (212-425 microm) were prepared by spraying 2% (w/v) alginate solution into 1 M CaCl2 solution. The fluidization behavior of these beads was studied, and the bed expansion index and terminal velocity were found to be 4.3 and 1808 cm h(-1), respectively. Residence time distribution curves showed that the dispersion of the protein was much less with these microbeads than with conventionally prepared calcium alginate macrobeads when both kinds of beads were used for chromatography in a fluidized bed format. The fluidized bed of these beads was used for the purification of pectinase from a commercial preparation. The media performed well even with diluted feedstock; 90% activity recovery with 211-fold purification was observed.

  5. Small-angle X-ray scattering and rheological characterization of alginate gels. 3. Alginic acid gels.

    PubMed

    Draget, Kurt Ingar; Stokke, Bjørn T; Yuguchi, Yoshiaki; Urakawa, Hiroshi; Kajiwara, Kanji

    2003-01-01

    Alginic acid gels were studied by small-angle X-ray scattering and rheology to elucidate the influence of alginate chemical composition and molecular weight on the gel elasticity and molecular structure. The alginic acid gels were prepared by homogeneous pH reduction throughout the sample. Three alginates with different chemical composition and sequence, and two to three different molecular weights of each sample were examined. Three alginate samples with fractions of guluronic acid residues of 0.39 (LoG), 0.50 (InG), and 0.68 (HiG), covering the range of commercially available alginates, were employed. The excess scattering intensity I of the alginic acid gels was about 1 order of magnitude larger and exhibited a stronger curvature toward low q compared to ionically cross-linked alginate. The I(q) were decomposed into two components by assuming that the alginic acid gel is composed of aggregated multiple junctions and single chains. Time-resolved experiments showed a large increase in the average size of aggregates and their weight fraction within the first 2 h after onset of gelling, which also coincides with the most pronounced rheological changes. At equilibrium, little or no effect of molecular weight was observed, whereas at comparable molecular weights, an increased scattering intensity with increasing content of guluronic acid residues was recorded, probably because of a larger apparent molecular mass of domains. The results suggest a quasi-ordered junction zone is formed in the initial stage, followed by subsequent assembling of such zones, forming domains in the order of 50 A. The average length of the initial junction zones, being governed by the relative fraction of stabilizing G-blocks and destabilizing alternating (MG) blocks, determines the density of the final random aggregates. Hence, high-G alginates give alginic acid gels of a higher aggregate density compared to domains composed of loosely packed shorter junction zones in InG or LoG system.

  6. Phenylalanine Ammonia-Lyase from Loblolly Pine 1

    PubMed Central

    Whetten, Ross W.; Sederoff, Ronald R.

    1992-01-01

    Phenylalanine ammonia-lyase (EC 4.3.1.5) has been purified from differentiating secondary xylem of loblolly pine (Pinus taeda L.). Native molecular weight of the enzyme was estimated to be 280,000, with a subunit molecular weight of 74,000; isoelectric point, 5.8; and Michaelis constant for i-phenylalanine, 27 micromolar. No evidence was obtained for the existence of isoforms of the enzyme, nor for negative cooperativity of substrate binding. Polyclonal antibodies were raised against the phenylalanine ammonia-lyase subunit and used to identify a pal clone in an expression library of xylem complementary DNA (cDNA). Polymerase chain reaction, using oligonucleotide primers made from N-terminal amino acid sequence and from the 5′ end of the clone isolated from the expres