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Sample records for alginate lyase production

  1. 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.

  2. Cultivable alginate lyase-excreting bacteria associated with the Arctic brown alga Laminaria.

    PubMed

    Dong, Sheng; Yang, Jie; Zhang, Xi-Ying; Shi, Mei; Song, Xiao-Yan; Chen, Xiu-Lan; Zhang, Yu-Zhong

    2012-11-01

    Although some alginate lyases have been isolated from marine bacteria, alginate lyases-excreting bacteria from the Arctic alga have not yet been investigated. Here, the diversity of the bacteria associated with the brown alga Laminaria from the Arctic Ocean was investigated for the first time. Sixty five strains belonging to nine genera were recovered from six Laminaria samples, in which Psychrobacter (33/65), Psychromonas (10/65) and Polaribacter (8/65) were the predominant groups. Moreover, 21 alginate lyase-excreting strains were further screened from these Laminaria-associated bacteria. These alginate lyase-excreting strains belong to five genera. Psychromonas (8/21), Psedoalteromonas (6/21) and Polaribacter (4/21) are the predominant genera, and Psychrobacter, Winogradskyella, Psychromonas and Polaribacter were first found to produce alginate lyases. The optimal temperatures for the growth and algiante lyase production of many strains were as low as 10–20 °C, indicating that they are psychrophilic bacteria. The alginate lyases produced by 11 strains showed the highest activity at 20–30 °C, indicating that these enzymes are cold-adapted enzymes. Some strians showed high levels of extracellular alginate lyase activity around 200 U/mL. These results suggest that these algiante lyase-excreting bacteria from the Arctic alga are good materials for studying bacterial cold-adapted alginate lyases. PMID:23203272

  3. 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

  4. 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

  5. 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

  6. 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.

  7. 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. PMID:26490549

  8. 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

  9. Cloning of alginate lyase gene (alxM) and expression in Escherichia coli.

    PubMed Central

    Brown, B J; Preston, J F; Ingram, L O

    1991-01-01

    The alxM gene encoding a D-mannuronan-specific alginate lyase has been cloned from a marine bacterium isolated as an epiphyte on the brown alga, Sargassum fluitans. Expression of this gene in Escherichia coli provides a source of this enzyme for probing alginate structure and modifying the mannuronan-rich alginate polymers produced by bacterial pathogens. Images PMID:1872617

  10. 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

  11. 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. PMID:27198822

  12. 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.

  13. 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.

  14. 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.

  15. Characterization of alginate lyase gene using a metagenomic library constructed from the gut microflora of abalone.

    PubMed

    Sim, Su-Jung; Baik, Keun Sik; Park, Seong Chan; Choe, Han Na; Seong, Chi Nam; Shin, Tai-Sun; Woo, Hee Chul; Cho, Jeong-Yong; Kim, Duwoon

    2012-04-01

    A metagenomic fosmid library was constructed using a genomic DNA mixture extracted from the gut microflora of abalone. The library gave an alginate lyase positive clone (AlyDW) harboring a 31.7-kbp insert. The AlyDW insert consisted of 22 open reading frames (ORFs). The deduced amino acid sequences of ORFs 11-13 were similar to those of known alginate lyase genes, which are found adjacent in the genome of Klebsiella pneumoniae subsp. aerogenes, Vibrio splendidus, and Vibrio sp. belonging to the phylum Gammaproteobacteria. Among the three recombinant proteins expressed from the three ORFs, alginate lyase activity was only observed in the recombinant protein (AlyDW11) coded by ORF 11. The expressed protein (AlyDW11) had the highest alginate lyase activity at pH 7.0 and 45°C in the presence of 1 mM AgNO(3). The alginate lyase activity of ORF 11 was confirmed to be endolytic by thin-layer chromatography. AlyDW11 preferred poly(β-D: -mannuronate) as a substrate over poly(α-L: -guluronate). AlyDW11 contained three highly conserved regions, RSEL, QIH, and YFKAGVYNQ, which may act to stabilize the three-dimensional conformation and function of the alginate lyase.

  16. Crystallization and preliminary X-ray analysis of an exotype alginate lyase Atu3025 from Agrobacterium tumefaciens strain C58, a member of polysaccharide lyase family 15

    SciTech Connect

    Ochiai, Akihito; Yamasaki, Masayuki; Mikami, Bunzo; Hashimoto, Wataru; Murata, Kousaku

    2006-05-01

    The crystallization and preliminary X-ray characterization of a family PL-15 exotype alginate lyase are presented. Almost all alginate lyases depolymerize alginate in an endolytical fashion via a β-elimination reaction. The alginate lyase Atu3025 from Agrobacterium tumefaciens strain C58, consisting of 776 amino-acid residues, is a novel exotype alginate lyase classified into polysaccharide lyase family 15. The enzyme was crystallized at 293 K by sitting-drop vapour diffusion with polyethylene glycol 4000 as a precipitant. Preliminary X-ray analysis showed that the Atu3025 crystal belonged to space group P2{sub 1} and diffracted to 2.8 Å resolution, with unit-cell parameters a = 107.7, b = 108.3, c = 149.5 Å, β = 91.5°.

  17. 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). PMID:27630053

  18. 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

  19. Molecular insight into the role of the N-terminal extension in the maturation, substrate recognition, and catalysis of a bacterial alginate lyase from polysaccharide lyase family 18.

    PubMed

    Dong, Sheng; Wei, Tian-Di; Chen, Xiu-Lan; Li, Chun-Yang; Wang, Peng; Xie, Bin-Bin; Qin, Qi-Long; Zhang, Xi-Ying; Pang, Xiu-Hua; Zhou, Bai-Cheng; Zhang, Yu-Zhong

    2014-10-24

    Bacterial alginate lyases, which are members of several polysaccharide lyase (PL) families, have important biological roles and biotechnological applications. The mechanisms for maturation, substrate recognition, and catalysis of PL18 alginate lyases are still largely unknown. A PL18 alginate lyase, aly-SJ02, from Pseudoalteromonas sp. 0524 displays a β-jelly roll scaffold. Structural and biochemical analyses indicated that the N-terminal extension in the aly-SJ02 precursor may act as an intramolecular chaperone to mediate the correct folding of the catalytic domain. Molecular dynamics simulations and mutational assays suggested that the lid loops over the aly-SJ02 active center serve as a gate for substrate entry. Molecular docking and site-directed mutations revealed that certain conserved residues at the active center, especially those at subsites +1 and +2, are crucial for substrate recognition. Tyr(353) may function as both a catalytic base and acid. Based on our results, a model for the catalysis of aly-SJ02 in alginate depolymerization is proposed. Moreover, although bacterial alginate lyases from families PL5, 7, 15, and 18 adopt distinct scaffolds, they share the same conformation of catalytic residues, reflecting their convergent evolution. Our results provide the foremost insight into the mechanisms of maturation, substrate recognition, and catalysis of a PL18 alginate lyase.

  20. 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.

  1. 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. PMID:27231344

  2. 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

  3. 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

  4. 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.

  5. Crystallization and preliminary X-ray analysis of alginate lyase, a member of family PL-7, from Pseudomonas aeruginosa.

    PubMed

    Yamasaki, Masayuki; Moriwaki, Satoko; Hashimoto, Wataru; Mikami, Bunzo; Murata, Kousaku

    2003-08-01

    Alginate lyase depolymerizes alginate, a heteropolysaccharide consisting of alpha-L-guluronate and beta-D-mannuronate, through a beta-elimination reaction. A protein PA1167 with a molecular mass of 25 kDa produced by Pseudomonas aeruginosa is an alginate lyase classified into polysaccharide lyase family PL-7. The enzyme was crystallized at 293 K in a drop solution comprising 1.4 M sodium chloride, 0.1 M potassium sodium phosphate and 0.1 M 2-morpholinoethanesulfonate-sodium hydroxide pH 6.5 by means of the vapor-diffusion method. The crystals were monoclinic and belonged to space group P2(1), with unit-cell parameters a = 43.4, b = 70.3, c = 67.4 A, beta = 94.5 degrees. Diffraction data were collected to 2.0 A from a single crystal. PMID:12876365

  6. Depolymerization of alginate into a monomeric sugar acid using Alg17C, an exo-oligoalginate lyase cloned from Saccharophagus degradans 2-40.

    PubMed

    Kim, Hee Taek; Chung, Jae Hyuk; Wang, Damao; Lee, Jieun; Woo, Hee Chul; Choi, In-Geol; Kim, Kyoung Heon

    2012-03-01

    Macroalgae are considered to be promising biomass for fuels and chemicals production. To utilize brown macroalgae as biomass, the degradation of alginate, which is the main carbohydrate of brown macroalgae, into monomeric units is a critical prerequisite step. Saccharophagus degradans 2-40 is capable of degrading more than ten different polysaccharides including alginate, and its genome sequence demonstrated that this bacterium contains several putative alginate lyase genes including alg17C. The gene for Alg17C, which is classified into the PL-17 family, was cloned and overexpressed in Escherichia coli. The recombinant Alg17C was found to preferentially act on oligoalginates with degrees of polymerization higher than 2 to produce the alginate monomer, 4-deoxy-L: -erythro-5-hexoseulose uronic acid. The optimal pH and temperature for Alg17C were found to be 6 and 40 °C, respectively. The K (M) and V (max) of Alg17C were 35.2 mg/ml and 41.7 U/mg, respectively. Based on the results of this study, Alg17C could be used as the key enzyme to produce alginate monomers in the process of utilizing alginate for biofuels and chemicals production. PMID:22281843

  7. Cloning, sequencing and overexpression in Escherichia coli of the alginatelyase-encoding aly gene of Pseudomonas alginovora: identification of three classes of alginate lyases.

    PubMed Central

    Chavagnat, F; Duez, C; Guinand, M; Potin, P; Barbeyron, T; Henrissat, B; Wallach, J; Ghuysen, J M

    1996-01-01

    A gene of Pseudomonas alginovora, called aly, has been cloned in Escherichia coli using a battery of PCR techniques and sequenced. It encodes a 210-amino-acid alginate lyase (EC 4.2.2.3), Aly, in the form of a 233-amino-acid precursor. P. alginovora Aly has been overproduced in E. coli with a His-tag sequence fused at the C-terminal end under conditions in which the formation of inclusion bodies is avoided. His-tagged P. alginovora Aly has the same enzymic properties as the wild-type enzyme and has the specificity of a mannuronate lyase. It can be purified in a one-step procedure by affinity chromatography on Ni(2+)-nitriloacetate resin. The yield is of 5 mg of enzyme per litre of culture. The amplification factor is 12.5 compared with the level of production by wild-type P. alginovora. The six alginate lyases of known primary structure fall into three distinct classes, one of which comprises the pair P. alginovora Aly and Klebsiella pneumoniae Aly. PMID:8912697

  8. 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. PMID:22940178

  9. Evaluation of Gene Expression and Alginate Production in Response to Oxygen Transfer in Continuous Culture of Azotobacter vinelandii

    PubMed Central

    Díaz-Barrera, Alvaro; Martínez, Fabiola; Guevara Pezoa, Felipe; Acevedo, Fernando

    2014-01-01

    Alginates are polysaccharides used as food additives and encapsulation agents in biotechnology, and their functional properties depend on its molecular weight. In this study, different steady-states in continuous cultures of A. vinelandii were established to determine the effect of the dilution rate (D) and the agitation rate on alginate production and expression of genes involved in alginate polymerization and depolymerization. Both, the agitation and dilution rates, determined the partitioning of the carbon utilization from sucrose into alginate and CO2 under oxygen-limiting conditions. A low D (0.07 h−1) and 500 rpm resulted in the highest carbon utilization into alginate (25%). Quantitative real-time polymerase chain reaction was used to determine the transcription level of six genes involved in alginate polymerization and depolymerization. In chemostat cultures at 0.07 h−1, the gene expression was affected by changes in the agitation rate. By increasing the agitation rate from 400 to 600 rpm, the algE7 gene expression decreased tenfold, whereas alyA1, algL and alyA2 gene expression increased between 1.5 and 2.8 times under similar conditions evaluated. Chemostat at 0.07 h−1 showed a highest alginate molecular weight (580 kDa) at 500 rpm whereas similar molecular weights (480 kDa) were obtained at 400 and 600 rpm. The highest molecular weight was not explained by changes in the expression of alg8 and alg44 (genes involved in alginate polymerization). Nonetheless, a different expression pattern observed for lyases could explain the highest alginate molecular weight obtained. Overall, the results suggest that the control of alginate molecular weight in A. vinelandii cells growing in continuous mode is determined by a balance between the gene expression of intracellular and extracellular lyases in response to oxygen availability. These findings better our understanding of the biosynthesis of bacterial alginate and help us progress toward obtain tailor

  10. Evaluation of gene expression and alginate production in response to oxygen transfer in continuous culture of Azotobacter vinelandii.

    PubMed

    Díaz-Barrera, Alvaro; Martínez, Fabiola; Pezoa, Felipe Guevara; Acevedo, Fernando

    2014-01-01

    Alginates are polysaccharides used as food additives and encapsulation agents in biotechnology, and their functional properties depend on its molecular weight. In this study, different steady-states in continuous cultures of A. vinelandii were established to determine the effect of the dilution rate (D) and the agitation rate on alginate production and expression of genes involved in alginate polymerization and depolymerization. Both, the agitation and dilution rates, determined the partitioning of the carbon utilization from sucrose into alginate and CO2 under oxygen-limiting conditions. A low D (0.07 h(-1)) and 500 rpm resulted in the highest carbon utilization into alginate (25%). Quantitative real-time polymerase chain reaction was used to determine the transcription level of six genes involved in alginate polymerization and depolymerization. In chemostat cultures at 0.07 h(-1), the gene expression was affected by changes in the agitation rate. By increasing the agitation rate from 400 to 600 rpm, the algE7 gene expression decreased tenfold, whereas alyA1, algL and alyA2 gene expression increased between 1.5 and 2.8 times under similar conditions evaluated. Chemostat at 0.07 h(-1) showed a highest alginate molecular weight (580 kDa) at 500 rpm whereas similar molecular weights (480 kDa) were obtained at 400 and 600 rpm. The highest molecular weight was not explained by changes in the expression of alg8 and alg44 (genes involved in alginate polymerization). Nonetheless, a different expression pattern observed for lyases could explain the highest alginate molecular weight obtained. Overall, the results suggest that the control of alginate molecular weight in A. vinelandii cells growing in continuous mode is determined by a balance between the gene expression of intracellular and extracellular lyases in response to oxygen availability. These findings better our understanding of the biosynthesis of bacterial alginate and help us progress toward obtain tailor

  11. 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

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

    PubMed

    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

  13. 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.

  14. Comparative Characterization of Two Marine Alginate Lyases from Zobellia galactanivorans Reveals Distinct Modes of Action and Exquisite Adaptation to Their Natural Substrate*

    PubMed Central

    Thomas, François; Lundqvist, Lena C. E.; Jam, Murielle; Jeudy, Alexandra; Barbeyron, Tristan; Sandström, Corine; Michel, Gurvan; Czjzek, Mirjam

    2013-01-01

    Cell walls of brown algae are complex supramolecular assemblies containing various original, sulfated, and carboxylated polysaccharides. Among these, the major marine polysaccharide component, alginate, represents an important biomass that is successfully turned over by the heterotrophic marine bacteria. In the marine flavobacterium Zobellia galactanivorans, the catabolism and uptake of alginate are encoded by operon structures that resemble the typical Bacteroidetes polysaccharide utilization locus. The genome of Z. galactanivorans contains seven putative alginate lyase genes, five of which are localized within two clusters comprising additional carbohydrate-related genes. This study reports on the detailed biochemical and structural characterization of two of these. We demonstrate here that AlyA1PL7 is an endolytic guluronate lyase, and AlyA5 cleaves unsaturated units, α-l-guluronate or β-d-manuronate residues, at the nonreducing end of oligo-alginates in an exolytic fashion. Despite a common jelly roll-fold, these striking differences of the mode of action are explained by a distinct active site topology, an open cleft in AlyA1PL7, whereas AlyA5 displays a pocket topology due to the presence of additional loops partially obstructing the catalytic groove. Finally, in contrast to PL7 alginate lyases from terrestrial bacteria, both enzymes proceed according to a calcium-dependent mechanism suggesting an exquisite adaptation to their natural substrate in the context of brown algal cell walls. PMID:23782694

  15. 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

  16. 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.

  17. 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.

  18. Microbial alginate production, modification and its applications

    PubMed Central

    Hay, Iain D; Rehman, Zahid Ur; Moradali, M Fata; Wang, Yajie; Rehm, Bernd H A

    2013-01-01

    Alginate is an important polysaccharide used widely in the food, textile, printing and pharmaceutical industries for its viscosifying, and gelling properties. All commercially produced alginates are isolated from farmed brown seaweeds. These algal alginates suffer from heterogeneity in composition and material properties. Here, we will discuss alginates produced by bacteria; the molecular mechanisms involved in their biosynthesis; and the potential to utilize these bacterially produced or modified alginates for high-value applications where defined material properties are required. PMID:24034361

  19. Maximization of volatile fatty acids production from alginate in acidogenesis.

    PubMed

    Pham, Hong Duc; Seon, Jiyun; Lee, Seong Chan; Song, Minkyung; Woo, Hee-Chul

    2013-11-01

    In this study, the response surface methodology (RSM) was applied to determine the optimum fermentative condition of alginate with the respect to the simultaneous effects of alginate concentration and initial pH to maximize the production of total volatile fatty acids (TVFAs) and alcohols. The results showed that the alginate fermentation was significantly affected by initial pH than by alginate concentration and there was no interaction between the two variables. The optimum condition was 6.2g alginate/L and initial pH 7.6 with a maximum TVFAs yield of 37.1%. Acetic acids were the main constituents of the TVFAs mixtures (i.e., 71.9-95.5%), while alcohols (i.e., ethanol, butanol, and propanol) were not detected.

  20. In Vitro Interaction between Alginate Lyase and Amphotericin B against Aspergillus fumigatus Biofilm Determined by Different Methods

    PubMed Central

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

    2013-01-01

    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

  1. Inactivating effects of the lactoperoxidase system on bacterial lyases involved in oral malodour production.

    PubMed

    Nakano, Manabu; Shin, Kouichirou; Wakabayashi, Hiroyuki; Yamauchi, Koji; Abe, Fumiaki; Hironaka, Shouji

    2015-10-01

    The main components of oral malodour have been identified as volatile sulfur compounds (VSCs), including hydrogen sulfide (H(2)S) and methyl mercaptan (CH(3)SH). The lactoperoxidase (LPO) system (consisting of LPO, glucose oxidase, glucose and thiocyanate) was previously shown to exhibit antimicrobial activities against some oral bacteria in vitro and suppressive effects on VSCs in mouth air in a clinical trial. Here, we examined the in vitro effects of the LPO system on the activities of the bacterial lyases involved in the production of VSCs by oral anaerobes. The exposure of crude bacterial extracts of Fusobacterium nucleatum and Porphyromonas gingivalis or purified methionine γ-lyase to the LPO system resulted in the inactivation of their lyase activities through l-cysteine and l-methionine, which was linked to the production of H(2)S and CH(3)SH, respectively. The exposure of living F. nucleatum and P. gingivalis cells to the LPO system resulted in the suppression of cell numbers and lyase activities. The inactivation of the crude bacterial extracts of F. nucleatum and purified methionine γ-lyase by the LPO system was partly recovered by the addition of DTT. Therefore, the LPO system may inactivate bacterial lyases including methionine γ-lyase by reacting with the free cysteine residues of lyases. These results suggested that the LPO system suppresses the production of VSCs not only through its antimicrobial effects, but also by its inactivating effects on the bacterial lyases of F. nucleatum and P. gingivalis.

  2. Cinnamic acid production using Streptomyces lividans expressing phenylalanine ammonia lyase.

    PubMed

    Noda, Shuhei; Miyazaki, Takaya; Miyoshi, Takanori; Miyake, Michiru; Okai, Naoko; Tanaka, Tsutomu; Ogino, Chiaki; Kondo, Akihiko

    2011-05-01

    Cinnamic acid production was demonstrated using Streptomyces as a host. A gene encoding phenylalanine ammonia lyase (PAL) from Streptomyces maritimus was introduced into Streptomyces lividans, and its expression was confirmed by Western blot analysis. After 4 days cultivation using glucose as carbon source, the maximal level of cinnamic acid reached 210 mg/L. When glycerol (30 g/L) was used as carbon source, the maximal level of produced cinnamic acid reached 450 mg/L. In addition, using raw starch, xylose or xylan as carbon source, the maximal level of cinnamic acid reached 460, 300, and 130 mg/L, respectively. We demonstrated that S. lividans has great potential to produce cinnamic acid as well as other aromatic compounds.

  3. Role of Azotobacter vinelandii mucA and mucC Gene Products in Alginate Production

    PubMed Central

    Núñez, Cinthia; León, Renato; Guzmán, Josefina; Espín, Guadalupe; Soberón-Chávez, Gloria

    2000-01-01

    Azotobacter vinelandii produces the exopolysaccharide alginate, which is essential for its differentiation to desiccation-resistant cysts. In different bacterial species, the alternative sigma factor ςE regulates the expression of functions related to the extracytoplasmic compartments. In A. vinelandii and Pseudomonas aeruginosa, the ςE factor (AlgU) is essential for alginate production. In both bacteria, the activity of this sigma factor is regulated by the product of the mucA, mucB, mucC, and mucD genes. In this work, we studied the transcriptional regulation of the A. vinelandii algU-mucABCD gene cluster, as well as the role of the mucA and mucC gene products in alginate production. Our results show the existence of AlgU autoregulation and show that both MucA and MucC play a negative role in alginate production. PMID:11073894

  4. 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

  5. Polygalacturonase production by calcium alginate immobilized Enterobacter aerogenes NBO2 cells.

    PubMed

    Darah, I; Nisha, M; Lim, Sheh-Hong

    2015-03-01

    Bacterial cells of Enterobacter aerogenes NBO2 were entrapped in calcium alginate beads in order to enhance polygalacturonase production compared to free cells. The optimized condition of 5 % (w/v) sodium alginate concentration, agitation speed of 250 rpm, and 15 beads of calcium alginate with inoculum size of 4 % (v/v; 5.4 × 10(7) cells/ml) produced 23.48 U/mL of polygalacturonase compared to free cells of 18.54 U/ml. There was about 26.6 % increment in polygalaturonase production. However, in this study, there was 296.6 % of increment in polygalacturonase production after improvement parameters compared to before improvement parameters of calcium alginate bead immobilization cells (5.92 U/ml). This research has indicated that optimized physical parameters of calcium alginate bead immobilization cells have significantly enhanced the production of polygalacturonase.

  6. Hydroperoxide lyase products, hexanal, hexenal and nonenal, inhibit soybean seedling growth

    SciTech Connect

    Gardner, H.W.; Dornbos, D.L. Jr. )

    1989-04-01

    Hexanal, a product of hydroperoxide lyase, inhibited the germination and growth of soybean seeds. Hexanal was continuously delivered to germinating seeds as a vapor dissolved in air with a flow-through system (100 ml/min). Only 0.8 {mu}g hexanal/ml air was required to inhibit seedling growth by 50%; nearly 100% inhibition occurred with a dose of 1.8 {mu}g hexanal/ml air. In the absence of hexanal brown spots were often visible on the seedlings, but at sublethal doses of hexanal, the seedlings were largely devoid of these spots. The relative toxicity of three hydroperoxide lyase products, hexanal, trans-2-hexanal and trans-2-nonenal, were compared with a Petri-dish bioassay. The order of toxicity against seedling growth was hexenal>hexanal>nonenal.

  7. 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

  8. 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.

  9. Immobilization of Bacillus amyloliquefaciens MBL27 cells for enhanced antimicrobial protein production using calcium alginate beads.

    PubMed

    Kumaravel, Vijayalakshmi; Gopal, Suseela Rajakumar

    2010-12-01

    Cell immobilization is one of the common techniques for increasing the overall cell concentration and productivity. Bacillus amyloliquefaciens MBL27 cells were immobilized in calcium alginate beads and it is a promising method for repeated AMP (antimicrobial protein) production. The present study aimed at determining the optimal conditions for immobilization of B. amyloliquefaciens MBL27 cells in calcium alginate beads and the operational stability for enhanced production of the AMP. AMP production with free and immobilized cells was also done. In batch fermentation, maximum AMP production (7300 AU (arbitrary units)/ml against Staphylococcus aureus) was obtained with immobilized cells in shake flasks under optimized parameters such as 3% (w/v) sodium alginate, 136 mM CaCl2 with 350 alginate beads/flask of 2.7-3.0 mm diameter. In repeated cultivation, the highest activity was obtained after the second cycle of use and approx. 94% production was noted up to the fifth cycle. The immobilized cells of B. amyloliquefaciens MBL27 in alginate beads are more efficient for the production of AMP and had good stability. The potential application of AMP as a wound healant and the need for development of economical methods for improved production make whole cell immobilization an excellent alternative method for enhanced AMP production.

  10. Correlation between nitrogen fixation rate and alginate productivity of an indigenous Azotobacter vinelandii from Iran

    PubMed Central

    Nosrati, R; Owlia, P; Saderi, H; Olamaee, M; Rasooli, I; Akhavian, Tehrani A

    2012-01-01

    Background and Objectives Azotobacter vinelandii, a gamma-proteobacterium, is an obligate aerobic free-living gram-negative soil bacterium capable of fixing nitrogen. Oxygen transfer rate into the cell is reduced by the increase of alginate concentrations during the course of A. vinelandii cultivation. This phenomenon provides a low intracellular oxygen concentration needed for nitrogenase activity. The aim of this study was to design a simple strategy to explain the alginate production, cell growth and nitrogenase activity correlation in A. vinelandii under aerobic conditions. Material and Methods Thirty-five different soil samples were taken from the rhizosphere of agricultural crops of Iran. Enrichment and isolation strategies were employed for microbial isolation. Physiological and biochemical characteristics were determined. Molecular identification was performed using selective nifH-g1 primers. Alginate production and nitrogenase activity assay by each isolate of Azotobacter were carried out. Bacterial growth, alginate production and Nitrogenase activity were conducted by time-coursed quantitative measurements. Results Total of 26 isolates were selected after enrichment, isolation, and screening. The isolate was identified by molecular tests as A. vinelandii. The highest alginate productions of 1.02 g/l and 0.91g/l were noted after 4 days in 8 isolates, cell biomass of which were estimated 4.88-5.26 g/l. Six of 8 isolates were able to fix atmospheric N2 on nitrogen-free medium. Rates obtained in isolates were in the range of 12.1 to 326.4 nmol C2H4 h-1 vial-1. Conclusions Nitrogen fixation and alginate production yielded significant and positive Pearson's correlation coefficient of R2 = 0.760, p ∼ 0.02. Finally association between bacterial growth, alginate production and nitrogenase activity almost noticeable yielded significant and positive Pearson's correlation coefficient R2= 0.723, p ∼ 0.04. PMID:23066492

  11. Molecular and bioengineering strategies to improve alginate and polydydroxyalkanoate production by Azotobacter vinelandii

    PubMed Central

    Galindo, Enrique; Peña, Carlos; Núñez, Cinthia; Segura, Daniel; Espín, Guadalupe

    2007-01-01

    Several aspects of alginate and PHB synthesis in Azotobacter vinelandii at a molecular level have been elucidated in articles published during the last ten years. It is now clear that alginate and PHB synthesis are under a very complex genetic control. Genetic modification of A. vinelandii has produced a number of very interesting mutants which have particular traits for alginate production. One of these mutants has been shown to produce the alginate with the highest mean molecular mass so far reported. Recent work has also shed light on the factors determining molecular mass distribution; the most important of these being identified as; dissolved oxygen tension and specific growth rate. The use of specific mutants has been very useful for the correct analysis and interpretation of the factors affecting polymerization. Recent scale-up/down work on alginate production has shown that oxygen limitation is crucial for producing alginate of high molecular mass, a condition which is optimized in shake flasks and which can now be reproduced in stirred fermenters. It is clear that the phenotypes of mutants grown on plates are not necessarily reproducible when the strains are tested in lab or bench scale fermenters. In the case of PHB, A. vinelandii has shown itself able to produce relatively large amounts of this polymer of high molecular weight on cheap substrates, even allowing for simple extraction processes. The development of fermentation strategies has also shown promising results in terms of improving productivity. The understanding of the regulatory mechanisms involved in the control of PHB synthesis, and of its metabolic relationships, has increased considerably, making way for new potential strategies for the further improvement of PHB production. Overall, the use of a multidisciplinary approach, integrating molecular and bioengineering aspects is a necessity for optimizing alginate and PHB production in A. vinelandii. PMID:17306024

  12. Production of methionine γ- lyase in recombinant Citrobacter freundii bearing the hemoglobin gene.

    PubMed

    Kahraman, Huseyin; Aytan, Emel; Kurt, Ash Giray

    2011-09-01

    The production of antileukemic enzyme methionine γ-lyase (MGL) in distinctly related bacteria, Citrobacter freundii and in their recombinants expressing the Vitresocilla hemoglobin (VHb) has been studied. This study concerns the potential of Citrobacter freundii expressing the Vitreoscilla hemoglobin gene (vgb) for the methionine γ- liyase production. Methionine γ- liyase production by Citrobacter freundii and its vgb(-) and vgb(+) bearing recombinant strain was studied in shake-flasks under 200 rpm agitation, culture medium and 30 °C in a time-course manner. The vgb(+) and especially the carbon type had a dramatic effect on methionine γ- liyase production. The vgb(+) strain of C. freundii had about 2-fold and 3.1-fold higher levels of MGL than the host and vgb(-) strain, respectively.

  13. Production of a calcium silicate cement material from alginate impression material.

    PubMed

    Washizawa, Norimasa; Narusawa, Hideaki; Tamaki, Yukimichi; Miyazaki, Takashi

    2012-01-01

    The purpose of this study was to synthesize biomaterials from daily dental waste. Since alginate impression material contains silica and calcium salts, we aimed to synthesize calcium silicate cement from alginate impression material. Gypsum-based investment material was also investigated as control. X-ray diffraction analyses revealed that although firing the set gypsum-based and modified investment materials at 1,200°C produced calcium silicates, firing the set alginate impression material did not. However, we succeeded when firing the set blend of pre-fired set alginate impression material and gypsum at 1,200°C. SEM observations of the powder revealed that the featured porous structures of diatomite as an alginate impression material component appeared useful for synthesizing calcium silicates. Experimentally fabricated calcium silicate powder was successfully mixed with phosphoric acid solution and set by depositing the brushite. Therefore, we conclude that the production of calcium silicate cement material is possible from waste alginate impression material.

  14. Paraffin as oxygen vector modulates tyrosine phenol lyase production by Citrobacter freundii MTCC 2424.

    PubMed

    Azmi, Wamik; Kumar, Ajay; Dev, Varun

    2013-06-01

    The efficiency of three oxygen-vectors liquid paraffin, silicone oil and n-dodecane in the production of tyrosine phenol lyase (TPL) by Citrobacter freundii MTCC 2424 was evaluated at 4% (v/v) concentration. The liquid paraffin as oxygenvectors was found to exhibit a stimulatory effect on TPL synthesis. The liquid paraffin at 6% (v/v) resulted in 34% increase in the TPL synthesis accompanied by a 13% increase in the production of cell mass at a 10 L scale. This improvement in TPL and cell mass production in the presence of liquid paraffin can be related to the fact that liquid paraffin was capable of maintaining dissolved O2 concentration above 28% throughout the course of the fermentation. Maintenance of the dissolved O2 concentration above 28% could be viewed in terms of an adequate oxygen supply to the rapidly dividing cells of the bacterium, which in turn resulted in enhanced synthesis of TPL and cell mass.

  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. Iron-Regulated Expression of Alginate Production, Mucoid Phenotype, and Biofilm Formation by Pseudomonas aeruginosa

    PubMed Central

    Wiens, Jacinta R.; Vasil, Adriana I.; Schurr, Michael J.; Vasil, Michael L.

    2014-01-01

    ABSTRACT Pseudomonas aeruginosa strains of non-cystic fibrosis (non-CF) origin do not produce significant amounts of extracellular alginate and are nonmucoid. In CF, such isolates can become mucoid through mutation of one of the genes (mucA, mucB, mucC, or mucD) that produce regulatory factors that sequester AlgU, required for increased expression of alginate genes. Mutation of the muc genes in the nonmucoid PAO1, PA14, PAKS-1, and Ps388 strains led to increased levels of extracellular alginate and an obvious mucoid phenotype, but only under iron-limiting growth conditions (≤5 µM), not under iron-replete conditions (≥10 µM). In contrast, >50% of P. aeruginosa isolates from chronic CF pulmonary infections expressed increased levels of alginate and mucoidy both under iron-limiting and iron-replete conditions (i.e., iron-constitutive phenotype). No single iron regulatory factor (e.g., Fur, PvdS) was associated with this loss of iron-regulated alginate expression and mucoidy in these CF isolates. However, the loss of only pyoverdine production, or its uptake, abrogated the ability of P. aeruginosa to produce a robust biofilm that represents the Psl-type of biofilm. In contrast, we show that mutation of the pyoverdine and pyochelin biosynthesis genes and the pyoverdine receptor (FpvA) lead to iron-constitutive expression of the key alginate biosynthesis gene, algD, and an explicitly mucoid phenotype in both iron-limiting and iron-replete conditions. These data indicate that alginate production and mucoidy, in contrast to other types of biofilms produced by P. aeruginosa, are substantially enhanced under iron limitation. These results also have compelling implications in relation to the use of iron chelators in the treatment of P. aeruginosa CF infections. PMID:24496793

  17. 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.

  18. Cloning and expression in Pseudomonas aeruginosa of a gene involved in the production of alginate.

    PubMed Central

    Goldberg, J B; Ohman, D E

    1984-01-01

    Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis commonly produce a capsule-like exopolysaccharide called alginate. The alginate-producing (Alg+) phenotype results in a mucoid colony morphology and is an unstable trait. A mutant of P. aeruginosa FRD (a cystic fibrosis isolate) was obtained which was temperature sensitive for alginate production ( Algts ). At elevated growth temperatures (41 degrees C), no alginate was detected in culture supernatants of the Algts mutant, but yields of alginate increased as the temperature of incubation was reduced. The mutation responsible for the Algts phenotype, alg-50(Ts), has been mapped to a region of the FRD chromosome closely linked to trp-2. The alg-50(Ts) marker did not map near the met-l-linked chromosomal mutations responsible for the instability of the Alg+ phenotype. A broad host range cosmid cloning system based upon derivatives of plasmid RK2 was used to construct a P. aeruginosa clone bank. After transfer of the clone bank to the Algts mutant, hybrid plasmids were obtained which complemented the Algts defect. Deletion mapping of the original 20.3 kilobases of P. aeruginosa DNA cloned showed that a 4.7-kilobase fragment would complement the alg-50(Ts) mutation. PMID:6427188

  19. Characterization of an encapsulation device for the production of monodisperse alginate beads for cell immobilization.

    PubMed

    Serp, D; Cantana, E; Heinzen, C; Von Stockar, U; Marison, I W

    2000-10-01

    An encapsulation device, designed on the basis of the laminar jet break-up technique, is characterized for cell immobilization with different types of alginate. The principle of operation of the completely sterilizable encapsulator, together with techniques for the continuous production of beads from 250 microm to 1 mm in diameter, with a size distribution below 5%, at a flow rate of 1-15 mL/min, is described. A modification of the device, to incorporate an electrostatic potential between the alginate droplets and an internal electrode, results in enhanced monodispersity with no adverse effects on cell viability. The maximum cell loading capacity of the beads strongly depends on the nozzle diameter as well as the cells used. For the yeast Phaffia rhodozyma, it is possible to generate 700 microm alginate beads with an initial cell concentration of 1 x 10(8) cells/mL of alginate whereas only 1 x 10(6) cells/ml could be entrapped within 400 microm beads. The alginate beads have been characterized with respect to mechanical resistance and size distribution immediately after production and as a function of storage conditions. The beads remain stable in the presence of acetic acid, hydrochloric acid, water, basic water, and sodium ions. The latter stability applies when the ratio of sodium: calcium ions is less than 1/5. Complexing agents such as sodium citrate result in the rapid solubilization of the beads due to calcium removal. The presence of cells does not affect the mechanical resistance of the beads. Finally, the mechanical resistance of alginate beads can be doubled by treatment with 5-10 kDa chitosan, resulting in reduced leaching of cells.

  20. High-level extracellular production of alkaline polygalacturonate lyase in Bacillus subtilis with optimized regulatory elements.

    PubMed

    Zhang, Junjiao; Kang, Zhen; Ling, Zhenmin; Cao, Wenlong; Liu, Long; Wang, Miao; Du, Guocheng; Chen, Jian

    2013-10-01

    The present work aims to construct a robust recombinant Bacillus subtilis to achieve secretory production of alkaline polygalacturonate lyase (PGL). First, 6 signal peptides (amyX, bpr, vpr, yvgO, wapA and nprE) were screened with a semi-rational approach and comparatively investigated their effects on the production of PGL. The signal peptide bpr directed efficient PGL secretory expression and increased PGL titer to 313.7 U mL(-1). By optimizing and applying strong promoter P43 and Shine-Dalgarno sequence, higher titer of 446.3 U mL(-1) PGL was achieved. Finally, the capacity of the recombinant B. subtilis WB43CB was evaluated with a fed-batch strategy in 3 L fermentor. The PGL titer reached 632.6 U mL(-1) with a productivity of 17.6 U mL(-1) h(-1), which was the highest secretory production of PGL by the B. subtilis system. The recombinant B. subtilis strain WB43CB constructed in the present work has great potential in production of alkaline PGL.

  1. Degumming of ramie fiber and the production of reducing sugars from waste peels using nanoparticle supplemented pectate lyase.

    PubMed

    Mukhopadhyay, Arka; Dutta, Nalok; Chattopadhyay, Dhrubajyoti; Chakrabarti, Krishanu

    2013-06-01

    Banana, citrus and potato peels were subjected to treatment with hydroxyapatite nanoparticle (NP) supplemented purified pectate lyase (NP-PL), isolated from Bacillus megaterium AK2 to produce reducing sugar (RS). At both 50 and 90°C production of RS by NP-PL was almost twofold greater than that by untreated pectate lyase (PL) from each of the three peels. The optimal production of RS from banana and citrus peels were after 24 and 6h of incubation while it was 24 and 4h for potato peels at 50 and 90°C, respectively, on NP-PL treatment. NP-PL could degum raw, decorticated ramie fibers as well as enhance fiber tenacity and fineness. The weight loss of the fibers were 24% and 31% better (compared to PL treatment) after 24 and 48 h of processing. These findings have potential implications for the bio-ethanol, bio-fuel and textile industries. PMID:23587821

  2. Production of alginate by Azotobacter vinelandii grown at two bioreactor scales under oxygen-limited conditions.

    PubMed

    Díaz-Barrera, Alvaro; Gutierrez, Javiera; Martínez, Fabiola; Altamirano, Claudia

    2014-06-01

    The oxygen transfer rate (OTR) was evaluated as a scale-up criterion for alginate production in 3- and 14-L stirred fermentors. Batch cultures were performed at different agitation rates (200, 300, and 600 rpm) and airflow rates (0.25, 0.5, and 1 vvm), resulting in different maximum OTR levels (OTRmax). Although the two reactors had a similar OTRmax (19 mmol L(-1) h(-1)) and produced the same alginate concentration (3.8 g L(-1)), during the cell growth period the maximum molecular weight of the alginate was 1,250 kDa in the 3-L stirred fermentor and 590 kDa in 14-L stirred fermentor. The results showed for the first time the evolution of the molecular weight of alginate and OTR profiles for two different scales of stirred fermentors. There was a different maximum specific oxygen uptake rate between the two fermenters, reaching 8.3 mmol g(-1) h(-1) in 3-L bioreactor and 10.6 mmol g(-1) h(-1) in 14-L bioreactor, which could explain the different molecular weights observed. These findings open the possibility of using [Formula: see text] instead of OTRmax as a scaling criterion to produce polymers with similar molecular weights during fermentation.

  3. The release of fluoride from two products of alginate impression materials.

    PubMed

    Hattab, F; Frostell, G

    1980-01-01

    The purpose of this study was to determine the fluoride content of two products of alginate and the possible fluoride transfer to the teeth, saliva and blood. The total fluoride content of Zelgan normal-set and Kerr alginate fastset powder was assayed by direct diffusion and diffusion of the ash. The soluble fluoride leaching out in water over 24 hour was also determined. The results show that the fluoride contents of Zelgan and Kerr alginate powders are about 1.9% and 1.5% fluoride, respectively. Of the fluoride present in Zelgan and Kerr approximately 6.5% and 5.8%, respectively, leached out in 400 ml deionized water. The fluoride uptake was estimated in two adjacent enamel layers each approximately 7 micrometers thick, using 10 teeth exposed for 5 minutes and 18 h to the alginate gel (Zelgan). The results of acid etch microsamplings indicate a significant increase in the fluoride concentration of the first enamel layer after both 5 min and 18 h exposure. Fluoride uptake within the second enamel layer was insignificant, however. Fluoride transfer to the oral saliva and to the blood was evident after impression taking.

  4. Controlled microfluidic production of alginate beads for in situ encapsulation of microbes

    SciTech Connect

    Kalyanaraman, Meenaa; Retterer, Scott T; McKnight, Timothy E; Ericson, Milton Nance; Allman, Steve L; Elkins, James G; Palumbo, Anthony Vito; Keller, Martin; Doktycz, Mitchel John

    2009-01-01

    The development and refinement of a microfluidic-based alginate bead generator system for bacterial encapsulation is presented. The resulting microgels have application for the encapsulation of single cells, and can allow for small scale, clonal expansion of thousands of isolated cells in parallel. PDMS based microfluidic chips were fabricated using conventional lithography techniques to produce both externally gelled and directly gelled alginate microspheres using a controlled, water-in-oil emulsion system. The production of directly gelled beads, formed by the in-chip mixing of aqueous alginate and calcium chloride solutions dispersed within an organic carrier flowstream is qualitatively compared to a system, which produces beads and relies on diffusion of a crosslinking agent from the carrier fluid to cause gelation (external gelation). While the direct gelation scheme allows the use of biocompatible oils as the organic carrier, it also has a detrimental effect on device stability often resulting in clogging and gel-streaming at the microfluidic interface of these solutions. A design for the continuous production of directly gelled beads was evaluated in terms of the threshold flow conditions and reagent concentrations that did not result in clogging or streaming. Monodisperse alginate microgels of 30 mum diameter were produced at frequencies of over 500 beads per second. The beads could be completely dispersed into aqueous media using an off-chip washing protocol to remove the organic phase. The microgels effectively encapsulated individual or small numbers of GFP-expressing Escherichia. coli, which could be subsequently clonally expanded. The described microfluidic platform is a robust front-end sample preparation technology that shows strong potential for use in drug delivery systems, biosensors, and other cell-based microcompartmentalization applications. The co-culturing of microbial colonies in a large population of alginate beads will allow for functional

  5. Increased erythromycin production by alginate as a medium ingredient or immobilization support in cultures of Saccharopolyspora erythraea.

    PubMed

    Hamedi, J; Khodagholi, F; Hassani-Nasab, A

    2005-05-01

    Erythromycin production by Saccharopolyspora erythraea immobilized in 2% (w/v) calcium alginate or grown in medium containing 20 g sodium alginate/l inoculated with free cells was almost twice more than that of the control. S. erythraea did not consume alginate, agar, dextran, silicon antifoaming agent or cyclodextrin as a carbon source, although, all of these increased the production of erythromycin. Highest titer of erythromycin (2.3 times more than that of the control) was achieved in medium containing 1 g agar/l.

  6. Contribution of alginate and levan production to biofilm formation by Pseudomonas syringae.

    PubMed

    Laue, Heike; Schenk, Alexander; Li, Hongqiao; Lambertsen, Lotte; Neu, Thomas R; Molin, Søren; Ullrich, Matthias S

    2006-10-01

    Exopolysaccharides (EPSs) play important roles in the attachment of bacterial cells to a surface and/or in building and maintaining the three-dimensional, complex structure of bacterial biofilms. To elucidate the spatial distribution and function of the EPSs levan and alginate during biofilm formation, biofilms of Pseudomonas syringae strains with different EPS patterns were compared. The mucoid strain PG4180.muc, which produces levan and alginate, and its levan- and/or alginate-deficient derivatives all formed biofilms in the wells of microtitre plates and in flow chambers. Confocal laser scanning microscopy with fluorescently labelled lectins was applied to investigate the spatial distribution of levan and an additional as yet unknown EPS in flow-chamber biofilms. Concanavalin A (ConA) bound specifically to levan and accumulated in cell-depleted voids in the centres of microcolonies and in blebs. No binding of ConA was observed in biofilms of the levan-deficient mutants or in wild-type biofilms grown in the absence of sucrose as confirmed by an enzyme-linked lectin-sorbent assay using peroxidase-linked ConA. Time-course studies revealed that expression of the levan-forming enzyme, levansucrase, occurred mainly during early exponential growth of both planktonic and sessile cells. Thus, accumulation of levan in biofilm voids hints to a function as a nutrient storage source for later stages of biofilm development. The presence of a third EPS besides levan and alginate was indicated by binding of the lectin from Naja mossambica to a fibrous structure in biofilms of all P. syringae derivatives. Production of the as yet uncharacterized additional EPS might be more important for biofilm formation than the syntheses of levan and alginate.

  7. Production of polyhydroxybutyrate and alginate from glycerol by Azotobacter vinelandii under nitrogen-free conditions.

    PubMed

    Yoneyama, Fuminori; Yamamoto, Mayumi; Hashimoto, Wataru; Murata, Kousaku

    2015-01-01

    Glycerol is an interesting feedstock for biomaterials such as biofuels and bioplastics because of its abundance as a by-product during biodiesel production. Here we demonstrate glycerol metabolism in the nitrogen-fixing species Azotobacter vinelandii through metabolomics and nitrogen-free bacterial production of biopolymers, such as poly-d-3-hydroxybutyrate (PHB) and alginate, from glycerol. Glycerol-3-phosphate was accumulated in A. vinelandii cells grown on glycerol to the exponential phase, and its level drastically decreased in the cells grown to the stationary growth phase. A. vinelandii also overexpressed the glycerol-3-phosphate dehydrogenase gene when it was grown on glycerol. These results indicate that glycerol was first converted to glycerol-3-phosphate by glycerol kinase. Other molecules with industrial interests, such as lactic acid and amino acids including γ-aminobutyric acid, have also been accumulated in the bacterial cells grown on glycerol. Transmission electron microscopy revealed that glycerol-grown A. vinelandii stored PHB within the cells. The PHB production level reached 33% per dry cell weight in nitrogen-free glycerol medium. When grown on glycerol, alginate-overproducing mutants generated through chemical mutagenesis produced 2-fold the amount of alginate from glycerol than the parental wild-type strain. To the best of our knowledge, this is the first report on bacterial production of biopolymers from glycerol without addition of any nitrogen source. PMID:25880041

  8. Production of polyhydroxybutyrate and alginate from glycerol by Azotobacter vinelandii under nitrogen-free conditions

    PubMed Central

    Yoneyama, Fuminori; Yamamoto, Mayumi; Hashimoto, Wataru; Murata, Kousaku

    2015-01-01

    Glycerol is an interesting feedstock for biomaterials such as biofuels and bioplastics because of its abundance as a by-product during biodiesel production. Here we demonstrate glycerol metabolism in the nitrogen-fixing species Azotobacter vinelandii through metabolomics and nitrogen-free bacterial production of biopolymers, such as poly-d-3-hydroxybutyrate (PHB) and alginate, from glycerol. Glycerol-3-phosphate was accumulated in A. vinelandii cells grown on glycerol to the exponential phase, and its level drastically decreased in the cells grown to the stationary growth phase. A. vinelandii also overexpressed the glycerol-3-phosphate dehydrogenase gene when it was grown on glycerol. These results indicate that glycerol was first converted to glycerol-3-phosphate by glycerol kinase. Other molecules with industrial interests, such as lactic acid and amino acids including γ-aminobutyric acid, have also been accumulated in the bacterial cells grown on glycerol. Transmission electron microscopy revealed that glycerol-grown A. vinelandii stored PHB within the cells. The PHB production level reached 33% per dry cell weight in nitrogen-free glycerol medium. When grown on glycerol, alginate-overproducing mutants generated through chemical mutagenesis produced 2-fold the amount of alginate from glycerol than the parental wild-type strain. To the best of our knowledge, this is the first report on bacterial production of biopolymers from glycerol without addition of any nitrogen source. PMID:25880041

  9. Production of protocatechuic acid by Corynebacterium glutamicum expressing chorismate-pyruvate lyase from Escherichia coli.

    PubMed

    Okai, Naoko; Miyoshi, Takanori; Takeshima, Yasunobu; Kuwahara, Hiroaki; Ogino, Chiaki; Kondo, Akihiko

    2016-01-01

    Protocatechuic acid (3,4-dihydroxybenzoic acid; PCA) serves as a building block for polymers and pharmaceuticals. In this study, the biosynthetic pathway for PCA from glucose was engineered in Corynebacterium glutamicum. The pathway to PCA-employed elements of the chorismate pathway by using chorismate-pyruvate lyase (CPL) and 4-hydroxybenzoate hydroxylase (4-HBA hydroxylase). As C. glutamicum has the potential to synthesize the aromatic amino acid intermediate chorismate and possesses 4-HBA hydroxylase, we focused on expressing Escherichia coli CPL in a phenylalanine-producing strain of C. glutamicum ATCC21420. To secrete PCA, the gene (ubiC) encoding CPL from E. coli was expressed in C. glutamicum ATCC 21420 (strain F(UbiC)). The formation of 28.8 mg/L of extracellular 4-HBA (36 h) and 213 ± 29 mg/L of extracellular PCA (80 h) was obtained by the C. glutamicum strain F(UbiC) from glucose. The strain ATCC21420 was also found to produce extracellular PCA. PCA fermentation was performed using C. glutamicum strain F(UbiC) in a bioreactor at the optimized pH of 7.5. C. glutamicum F(UbiC) produced 615 ± 2.1 mg/L of PCA from 50 g/L of glucose after 72 h. Further, fed-batch fermentation of PCA by C. glutamicum F(UbiC) was performed with feedings of glucose every 24 h. The maximum production of PCA (1140.0 ± 11.6 mg/L) was achieved when 117.0 g/L of glucose was added over 96 h of fed-batch fermentation. PMID:26392137

  10. ATP citrate lyase mediated cytosolic acetyl-CoA biosynthesis increases mevalonate production in Saccharomyces cerevisiae

    DOE PAGES

    Rodriguez, Sarah; Denby, Charles M.; Van Vu, T.; Baidoo, Edward E. K.; Wang, George; Keasling, Jay D.

    2016-03-03

    With increasing concern about the environmental impact of a petroleum based economy, focus has shifted towards greener production strategies including metabolic engineering of microbes for the conversion of plant-based feedstocks to second generation biofuels and industrial chemicals. Saccharomyces cerevisiae is an attractive host for this purpose as it has been extensively engineered for production of various fuels and chemicals. Many of the target molecules are derived from the central metabolite and molecular building block, acetyl-CoA. To date, it has been difficult to engineer S. cerevisiae to continuously convert sugars present in biomass-based feedstocks to acetyl-CoA derived products due to intrinsicmore » physiological constraints—in respiring cells, the precursor pyruvate is directed away from the endogenous cytosolic acetyl-CoA biosynthesis pathway towards the mitochondria, and in fermenting cells pyruvate is directed towards the byproduct ethanol. In this study we incorporated an alternative mode of acetyl-CoA biosynthesis mediated by ATP citrate lyase (ACL) that may obviate such constraints. We characterized the activity of several heterologously expressed ACLs in crude cell lysates, and found that ACL from Aspergillus nidulans demonstrated the highest activity. We employed a push/pull strategy to shunt citrate towards ACL by deletion of the mitochondrial NAD+-dependent isocitrate dehydrogenase (IDH1) and engineering higher flux through the upper mevalonate pathway. We demonstrated that combining the two modifications increases accumulation of mevalonate pathway intermediates, and that both modifications are required to substantially increase production. Finally, we incorporated a block strategy by replacing the native ERG12 (mevalonate kinase) promoter with the copper-repressible CTR3 promoter to maximize accumulation of the commercially important molecule mevalonate. In conclusion, by combining the push/pull/block strategies, we significantly

  11. Production of endothelial cell-enclosing alginate-based hydrogel fibers with a cell adhesive surface through simultaneous cross-linking by horseradish peroxidase-catalyzed reaction in a hydrodynamic spinning process.

    PubMed

    Liu, Yang; Sakai, Shinji; Taya, Masahito

    2012-09-01

    We developed an alginate-based hydrogel fiber enabling to enclose endothelial cells, degradable on-demand by alginate lyase, and having a cell adhesive surface. The hydrogel fiber was obtained by extruding an aqueous solution of 4% (w/v) alginate derivative possessing phenolic hydroxyl moieties (Alg-Ph) and horseradish peroxidase (HRP) into a flow of aqueous solution containing 0.3 mM H(2)O(2) and gelatin derivative possessing Ph moieties (Gelatin-Ph). In the process, cross-linking of Alg-Ph resulting in a hydrogel fiber and immobilization of Gelatin-Ph on the surface of the hydrogel fiber were simultaneously accomplished by an HRP-catalyzed cross-linking reaction between Ph moieties. The diameter of the hydrogel fiber and the quantity of immobilized Gelatin-Ph on the fiber were controllable by changing the flow rates of the solutions and the concentration of HRP in the Alg-Ph-containing solution, respectively. The viability of the human endothelial cells enclosed in the hydrogel fibers obtained by 10 s of flowing in the H(2)O(2)-containing solution was 87.1%. In addition, the cells harvested from the hydrogel fibers through degradation using alginate lyase grew on tissue culture dishes in the same fashion as the cells seeded by a conventional subculture protocol. Human smooth muscle cells adhered, grew and achieved confluence on the surface of the hydrogel fibers. By degrading the hydrogel fibers using alginate lyase, a tubular cell construct was successfully obtained.

  12. 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

  13. 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.

  14. 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. PMID:25740000

  15. Enhancement of cell viability and alkaline polygalacturonate lyase production by sorbitol co-feeding with methanol in Pichia pastoris fermentation.

    PubMed

    Wang, Zhihao; Wang, Yun; Zhang, Dongxu; Li, Jianghua; Hua, Zhaozhe; Du, Guocheng; Chen, Jian

    2010-02-01

    Alkaline polygalacturonate lyase (PGL) production by Pichia pastoris GS115 was used as a model to study the mechanism and strategy for enhancing heterologous protein production. In order to enhance cell viability and volumetric recombinant protein productivity, sorbitol, which had been confirmed to be a non-repressive carbon source, was added together with methanol during the induction phase. The resultant PGL activity was up to 1593 U mL(-1), which was enhanced 1.85-fold compared to the control (863 U mL(-1)) cultured with sorbitol added at a constant rate of 3.6 g h(-1)L(-1) after an induction period of 100 h. Further results revealed that an appropriate sorbitol co-feeding strategy not only decreased the cell mortality to 8.8% (the control is about 23.1%) in the end of fermentation, but also reduced the proteolytic degradation of PGL.

  16. Isomaltulose production from sucrose by Protaminobacter rubrum immobilized in calcium alginate.

    PubMed

    de Oliva-Neto, P; Menão, Paula T P

    2009-09-01

    Different culture conditions for Protaminobacter rubrum and enzymatic reaction parameters were evaluated with the goal of improving isomaltulose production. P. rubrum was grown in a medium with 1% (w/v) cane molasses and 0.5% yeast extract and achieved a maximum cell yield Y(x/s) of 0.295 g of cells/g sucrose and a specific growth rate (mu) of 0.192 h(-1). The immobilization of P. rubrum cells was carried out with calcium alginate, glutaraldehyde and polyethyleneimine. Stabile immobilized cell pellets were obtained and used 24 times in batch processes. Enzymatic conversion was carried out at different sucrose concentrations and in pH 6 medium with 70% (w/v) sucrose at 30 degrees C an isomaltulose yield of 89-94% (w/v) was obtained. The specific activity of the P. rubrum immobilized pellets in calcium alginate at 30 degrees C ranged from 1.6 to 4.0 g isomaltulose g(-1) pellet h(-1), respectively with 70% and 65% sucrose solution, while in lower sucrose concentration had higher specific activities presumably due to substrate inhibition of the isomaltulose synthase in higher sucrose concentrations.

  17. Biomedical-grade, high mannuronic acid content (BioMVM) alginate enhances the proteoglycan production of primary human meniscal fibrochondrocytes in a 3-D microenvironment

    PubMed Central

    Rey-Rico, Ana; Klich, Angelique; Cucchiarini, Magali; Madry, Henning

    2016-01-01

    Alginates are important hydrogels for meniscus tissue engineering as they support the meniscal fibrochondrocyte phenotype and proteoglycan production, the extracellular matrix (ECM) component chiefly responsible for its viscoelastic properties. Here, we systematically evaluated four biomedical- and two nonbiomedical-grade alginates for their capacity to provide the best three-dimensional (3-D) microenvironment and to support proteoglycan synthesis of encapsulated human meniscal fibrochondrocytes in vitro. Biomedical-grade, high mannuronic acid alginate spheres (BioLVM, BioMVM) were the most uniform in size, indicating an effect of the purity of alginate on the shape of the spheres. Interestingly, the purity of alginates did not affect cell viability. Of note, only fibrochondrocytes encapsulated in BioMVM alginate produced and retained significant amounts of proteoglycans. Following transplantation in an explant culture model, the alginate spheres containing fibrochondrocytes remained in close proximity with the meniscal tissue adjacent to the defect. The results reveal a promising role of BioMVM alginate to enhance the proteoglycan production of primary human meniscal fibrochondrocytes in a 3-D hydrogel microenvironment. These findings have significant implications for cell-based translational studies aiming at restoring lost meniscal tissue in regions containing high amounts of proteoglycans. PMID:27302206

  18. 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

  19. Dimeric c-di-GMP is required for post-translational regulation of alginate production in Pseudomonas aeruginosa

    SciTech Connect

    Whitney, John C.; Robinson, Howard; Whitfield, Gregory B.; Marmont, Lindsey S.; Yip, Patrick; Neculai, A. Mirela; Lobsanov, Yuri D.; Ohman, Dennis E.; Howell, P. Lynne

    2015-05-15

    Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZ domain fold with a dimerization mode not previously observed for this family of proteins. Moreover, calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. Our results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa.

  20. Dimeric c-di-GMP is required for post-translational regulation of alginate production in Pseudomonas aeruginosa

    DOE PAGES

    Whitney, John C.; Robinson, Howard; Whitfield, Gregory B.; Marmont, Lindsey S.; Yip, Patrick; Neculai, A. Mirela; Lobsanov, Yuri D.; Ohman, Dennis E.; Howell, P. Lynne

    2015-05-15

    Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZmore » domain fold with a dimerization mode not previously observed for this family of proteins. Moreover, calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. Our results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa.« less

  1. Dimeric c-di-GMP is required for post-translational regulation of alginate production in Pseudomonas aeruginosa.

    PubMed

    Whitney, John C; Whitfield, Gregory B; Marmont, Lindsey S; Yip, Patrick; Neculai, A Mirela; Lobsanov, Yuri D; Robinson, Howard; Ohman, Dennis E; Howell, P Lynne

    2015-05-15

    Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZ domain fold with a dimerization mode not previously observed for this family of proteins. Calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. These results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa.

  2. Dimeric c-di-GMP Is Required for Post-translational Regulation of Alginate Production in Pseudomonas aeruginosa*

    PubMed Central

    Whitney, John C.; Whitfield, Gregory B.; Marmont, Lindsey S.; Yip, Patrick; Neculai, A. Mirela; Lobsanov, Yuri D.; Robinson, Howard; Ohman, Dennis E.; Howell, P. Lynne

    2015-01-01

    Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3′,5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZ domain fold with a dimerization mode not previously observed for this family of proteins. Calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. These results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa. PMID:25817996

  3. Bacterial pectate lyases, structural and functional diversity.

    PubMed

    Hugouvieux-Cotte-Pattat, Nicole; Condemine, Guy; Shevchik, Vladimir E

    2014-10-01

    Pectate lyases are enzymes involved in plant cell wall degradation. They cleave pectin using a β-elimination mechanism, specific for acidic polysaccharides. They are mainly produced by plant pathogens and plant-associated organisms, and only rarely by animals. Pectate lyases are also commonly produced in the bacterial world, either by bacteria living in close proximity with plants or by gut bacteria that find plant material in the digestive tract of their hosts. The role of pectate lyases is essential for plant pathogens, such as Dickeya dadantii, that use a set of pectate lyases as their main virulence factor. Symbiotic bacteria produce their own pectate lyases, but they also induce plant pectate lyases to initiate the symbiosis. Pectin degradation products may act as signals affecting the plant–bacteria interactions. Bacterial pectate lyases are also essential for using the pectin of dead or living plants as a carbon source for growth. In the animal gut, Bacteroides pectate lyases degrade the pectin of ingested food, and this is particularly important for herbivores that depend on their microflora for the digestion of pectin. Some human pathogens, such as Yersinia enterocolitica, produce a few intracellular pectate lyases that can facilitate their growth in the presence of highly pectinolytic bacteria, at the plant surface, in the soil or in the animal gut. PMID:25646533

  4. Efficient induction of formate hydrogen lyase of aerobically grown Escherichia coli in a three-step biohydrogen production process.

    PubMed

    Yoshida, Akihito; Nishimura, Taku; Kawaguchi, Hideo; Inui, Masayuki; Yukawa, Hideaki

    2007-03-01

    A three-step biohydrogen production process characterized by efficient anaerobic induction of the formate hydrogen lyase (FHL) of aerobically grown Escherichia coli was established. Using E. coli strain SR13 (fhlA (++), DeltahycA) at a cell density of 8.2 g/l medium in this process, a specific hydrogen productivity (28.0 +/- 5.0 mmol h(-1) g(-1) dry cell) of one order of magnitude lower than we previously reported was realized after 8 h of anaerobic incubation. The reduced productivity was attributed partly to the inhibitory effects of accumulated metabolites on FHL induction. To avoid this inhibition, strain SR14 (SR13 DeltaldhA DeltafrdBC) was constructed and used to the effect that specific hydrogen productivity increased 1.3-fold to 37.4 +/- 6.9 mmol h(-1) g(-1). Furthermore, a maximum hydrogen production rate of 144.2 mmol h(-1) g(-1) was realized when a metabolite excretion system that achieved a dilution rate of 2.0 h(-1) was implemented. These results demonstrate that by avoiding anaerobic cultivation altogether, more economical harvesting of hydrogen-producing cells for use in our biohydrogen process was made possible.

  5. 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.

  6. Effect of production variables on the physicochemical characteristics of celecoxib-loaded stearic and alginic acids-based microparticles.

    PubMed

    Shunmugaperumal, Tamilvanan; Sharma, Deepak; Thakur, Ashutosh; Vinaykumar

    2013-12-01

    The objectives of the current investigation were (1) to prepare the microparticles based on stearic and alginic acids from an aqueous system by hot (melt) dispersion method, (2) to achieve a higher drug entrapment efficiency and process yield (%) by changing the production variables such as stirring speed, concentration of stabilizer in aqueous dispersion medium, volume of aqueous dispersion medium, and stirring time, and (3) to see whether or not a retardation in drug release profile was attained from the celecoxib-loaded stearic and alginic acids-based microparticles compared to that of the celecoxib alone. The addition of alginic acid into stearic acid produced spherical-shaped particles with an almost smooth surface. Higher drug entrapment efficiency and process yield (%) values were obtained when the microparticles were prepared at 1000 r/min using 0.1% w/v polyvinyl alcohol in 100 mL aqueous dispersion medium and 30 min stirring time. The in vitro dissolution study in 900 mL of 2% sodium lauryl sulfate (SLS) solution at 75 r/min, however, showed only around 10% retardation in drug release from microparticles compared to the drug release from pure celecoxib alone. This indicated that the gel-like network formed by the alginic acid around the microparticles could not prevent the drug leakage from the microparticles.

  7. Ethanol production from sugar beet molasses by S. cerevisiae entrapped in an alginate-maize stem ground tissue matrix.

    PubMed

    Razmovski, R; Vučurović, V

    2011-04-01

    A new alginate-maize stem ground tissue matrix was developed as a Saccharomyces cerevisiae carrier for ethanol fermentation from sugar beet molasses. There were several fermentation procedures in the present study: using free cells and alginate-entrapped cells with and without maize stem ground tissue supplementation (F; F+C; AB; AB+C), and using a new combined alginate-maize stem ground tissue carrier (ABC). It was found that addition of maize stem ground tissue meal (C), with honeycomb configuration, provided high surface areas for cell attachment and biofilm growth, and also increased alginate matrix porosity, enabling better mass transfer characteristic, better physical strength and stability of beads. The highest values of process parameters were obtained in the case of new carrier (ABC): the ethanol concentration of 60.36 g/l, percentage of the theoretical ethanol yield of 96.56%, ethanol yield of 0.493 g/g and the volumetric ethanol productivity of 2.51 g/lh. The medium supplementation with maize stem ground tissue significantly decreased acetaldehyde and acetic acid content, did not affect fusel alcohol and ethylacetate content of the distillate.

  8. 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...

  9. 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...

  10. 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...

  11. 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...

  12. 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...

  13. Influence of immobilization parameters on growth and lactic acid production by Streptococcus thermophilus and Lactobacillus bulgaricus co-immobilized in calcium alginate gel beads.

    PubMed

    Garbayo, I; Vílchez, C; Vega, J M; Nava-Saucedo, J E; Barbotin, J N

    2004-12-01

    Streptococcus thermophilus and Lactobacillus bulgaricus were co-immobilized in different systems with varying calcium (0.1-1.5 M) and alginate (1-2%, w/v) concentrations. Highest lactic acid production was 35 g l(-1) when both bacteria were in high viscosity beads (1%, w/v alginate) hardened in 0.1 M CaCl2 . The gel bead composition affected size and distribution of entrapped lactic acid bacteria.

  14. High pressure studies on hesperitin production with hesperidinase free and immobilized in calcium alginate beads

    NASA Astrophysics Data System (ADS)

    Furtado, Andreia; Rosário, Pedro M.; Calado, António R. T.; Alfaia, António J. I.; Ribeiro, Maria H. L.

    2012-03-01

    The use of high pressure for the enzymatic synthesis of pharmacologically interesting molecules is a very important tool. Hesperidin and hesperitin exhibit anti-inflammatory, antimicrobial, antioxidant, and anticarcinogenic properties and prevent bone loss. However, hesperidin has a low bioavailability compared with hesperitin, due to the rutinoside moiety attached to the flavonoid. The aim of this work was the enzymatic production of hesperitin from hesperidin (soluble and insoluble) with hesperidinase free and immobilized in Ca-alginate beads, under high pressure conditions. The work was focused on the optimization of enzyme activity, studying the effects: pressure (50-150 MPa), temperature (35-75 °C), concentration of substrate (100-800 mg/L), and immobilization of hesperidinase. An 18-fold increase in hesperidinase residual activity was observed under high pressure conditions of 100 MPa compared to 0.1 MPa. A higher specificity of the hydrolytic reaction under high pressure (100 MPa) with a two-and three-fold increase in the ratio K cat/K M (specificity constant) at 55 °C and 75 °C was observed. A two-fold increase in the maximum activity at 100 MPa was observed with immobilized hesperinase compared to 0.1 MPa. In the second reutilization, almost a four-fold increase was obtained under high pressure conditions in comparison to atmospheric pressure.

  15. 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...

  16. 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...

  17. 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...

  18. 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...

  19. 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...

  20. 21 CFR 582.7187 - Calcium alginate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 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 582.7187 - Calcium alginate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 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...

  2. 21 CFR 582.7187 - Calcium alginate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 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...

  3. 21 CFR 582.7187 - Calcium alginate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 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...

  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 582.7133 - 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...

  6. 21 CFR 582.7133 - Ammonium alginate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 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...

  7. Improvement of bioethanol productivity of immobilized Saccharomyces bayanus with using sodium alginate-graft-poly(N-vinyl-2-pyrrolidone) matrix.

    PubMed

    İnal, Murat; Yiğitoğlu, Mustafa

    2012-09-01

    In this study, immobilization conditions and bioethanol production characteristics of immobilized Saccharomyces bayanus were investigated into sodium alginate-graft-poly(N-vinyl-2-pyrrolidone; NaAlg-g-PVP) matrix. The matrix that crosslinked with calcium clorid was used for immobilization of S. bayanus. Bioethanol productivity of the NaAlg-g-PVP matrix was found to increase from 4.21 to 4.84 gL(-1) h(-1) when compared with the convential sodium alginate matrix. The production of bioethanol was affected by initial glucose concentration and percentage of immobilized cell beads in fermentation medium. Bioethanol productivity was increased from 3.62 to 4.84 gL(-1) h(-1) while the glucose concentration increasing from 50 to 100 gL(-1). Due to the increase in percentage from 10 to 20 % of immobilized cell beads in the fermentation medium, bioethanol productivity was increased from 4.84 to 8.68 gL(-1) h(-1). The cell immobilized NaAlg-g-PVP beads were protected 92 % of initial activity after six repeated fermentation.

  8. 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.

  9. Application of an active alginate coating to control the growth of Listeria monocytogenes on poached and deli turkey products.

    PubMed

    Juck, Greg; Neetoo, Hudaa; Chen, Haiqiang

    2010-09-01

    The relatively high prevalence of Listeria monocytogenes in ready-to-eat (RTE) turkey products is of great concern. The overall objective of this study was to develop antimicrobial edible coating formulations to effectively control the growth of this pathogen. The antimicrobials studied were nisin (500IU/g), Novagard CB 1 (0.25%), Guardian NR100 (500ppm), sodium lactate (SL, 2.4%), sodium diacetate (SD, 0.25%), and potassium sorbate (PS, 0.3%). These were incorporated alone or in binary combinations into five edible coatings: alginate, kappa-carrageenan, pectin, xanthan gum, and starch. The coatings were applied onto the surface of home-style poached and processed deli turkey discs inoculated with ~3log CFU/g of L. monocytogenes. The turkey samples were then stored at 22 degrees C for 7days. For poached and processed deli turkey, the coatings were found to be equally effective, with pectin being slightly less effective than the others. The most effective poached turkey treatments seemed to be SL (2.4%)/SD (0.25%) and Nisin (500IU/g)/SL (2.4%), which yielded final populations of 3.0 and 4.9log CFU/g respectively compared to the control which was 7.9log CFU/g. For processed deli turkey, the most effective antimicrobial treatments seemed to be Nisin (500IU/g)/SD (0.25%) and Nisin (500IU/g)/SL (2.4%) with final populations of 1.5 and 1.7log CFU/g respectively compared to the control which was 6.5log CFU/g. In the second phase of the study, home-style poached and store-purchased roasted (deli) turkey inoculated with the pathogen at a level of ~3log CFU/g were coated with alginate incorporating selected antimicrobial combinations and stored for 8weeks at 4 degrees C. Alginate coatings supplemented with SL (2.4%)/PS (0.3%) delayed the growth of L. monocytogenes with final counts reaching 4.3log CFU/g (home-style poached turkey) and 6.5log CFU/g (roasted deli turkey) respectively while the counts in their untreated counterparts were significantly higher (P<0.05) reaching 9

  10. Application of an active alginate coating to control the growth of Listeria monocytogenes on poached and deli turkey products.

    PubMed

    Juck, Greg; Neetoo, Hudaa; Chen, Haiqiang

    2010-09-01

    The relatively high prevalence of Listeria monocytogenes in ready-to-eat (RTE) turkey products is of great concern. The overall objective of this study was to develop antimicrobial edible coating formulations to effectively control the growth of this pathogen. The antimicrobials studied were nisin (500IU/g), Novagard CB 1 (0.25%), Guardian NR100 (500ppm), sodium lactate (SL, 2.4%), sodium diacetate (SD, 0.25%), and potassium sorbate (PS, 0.3%). These were incorporated alone or in binary combinations into five edible coatings: alginate, kappa-carrageenan, pectin, xanthan gum, and starch. The coatings were applied onto the surface of home-style poached and processed deli turkey discs inoculated with ~3log CFU/g of L. monocytogenes. The turkey samples were then stored at 22 degrees C for 7days. For poached and processed deli turkey, the coatings were found to be equally effective, with pectin being slightly less effective than the others. The most effective poached turkey treatments seemed to be SL (2.4%)/SD (0.25%) and Nisin (500IU/g)/SL (2.4%), which yielded final populations of 3.0 and 4.9log CFU/g respectively compared to the control which was 7.9log CFU/g. For processed deli turkey, the most effective antimicrobial treatments seemed to be Nisin (500IU/g)/SD (0.25%) and Nisin (500IU/g)/SL (2.4%) with final populations of 1.5 and 1.7log CFU/g respectively compared to the control which was 6.5log CFU/g. In the second phase of the study, home-style poached and store-purchased roasted (deli) turkey inoculated with the pathogen at a level of ~3log CFU/g were coated with alginate incorporating selected antimicrobial combinations and stored for 8weeks at 4 degrees C. Alginate coatings supplemented with SL (2.4%)/PS (0.3%) delayed the growth of L. monocytogenes with final counts reaching 4.3log CFU/g (home-style poached turkey) and 6.5log CFU/g (roasted deli turkey) respectively while the counts in their untreated counterparts were significantly higher (P<0.05) reaching 9

  11. Molecular Characterization of a Recombinant Zea mays Phenylalanine Ammonia-Lyase (ZmPAL2) and Its Application in trans-Cinnamic Acid Production from L-Phenylalanine.

    PubMed

    Zang, Ying; Jiang, Ting; Cong, Ying; Zheng, Zhaojuan; Ouyang, Jia

    2015-06-01

    Phenylalanine ammonia-lyase (PAL) is one of the most extensively studied enzymes with its crucial role in secondary phenylpropanoid metabolism of plants. Recently, its demand has been increased for aromatic chemical production, but its applications in trans-cinnamic acid production were not much explored. In the present study, a putative PAL gene from Zea mays designated as ZmPAL2 was expressed and characterized in Escherichia coli BL21 (DE3). The recombinant ZmPAL2 exhibited a high PAL activity (7.14 U/mg) and a weak tyrosine ammonia-lyase activity. The optimal temperature of ZmPAL2 was 55 °C, and the thermal stability results showed that about 50 % of enzyme activity remained after a treatment at 60 °C for 6 h. The recombinant ZmPAL2 is a good candidate for the production of trans-cinnamic acid. The vitro conversion indicated that the recombinant ZmPAL2 could effectively catalyze the L-phenylalanine to trans-cinnamic acid, and the trans-cinnamic acid concentration can reach up to 5 g/l.

  12. 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

  13. 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.

  14. 3D Cell Culture in Alginate Hydrogels.

    PubMed

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

    2015-03-24

    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.

  15. Improved production of isomaltulose by a newly isolated mutant of Serratia sp. cells immobilized in calcium alginate.

    PubMed

    Kim, Yonghwan; Koo, Bong-Seong; Lee, Hyeon-Cheol; Yoon, Youngdae

    2015-03-01

    Isomaltulose, also known as palatinose, is produced by sucrose isomerase and has been highlighted as a sugar substitute due to a number of advantageous properties. For the massive production of isomaltulose, high resistance to sucrose and stability of sucrose isomerase as well as sucrose conversion yields would be critical factors. We describe a series of screening procedures to isolate the mutant strain of Serratia sp. possessing enhanced isomaltulose production with improved stability. The new Serratia sp. isolated from a series of screening procedures allowed us to produce isomaltulose from 60% sucrose solution, with over 90% conversion yield. Moreover, when this strain was immobilized in calcium alginate beads and placed in a medium containing 60% sucrose, it showed over 70% sucrose conversion yields for 30 cycles of repeated-batch reactions. Thus, improved conversion activity and stability of the newly isolated Serratia sp. strain in the present study would be highly valuable for industries related to isomaltulose production.

  16. Microwave assisted step-by-step process for the production of fucoidan, alginate sodium, sugars and biochar from Ascophyllum nodosum through a biorefinery concept.

    PubMed

    Yuan, Yuan; Macquarrie, Duncan J

    2015-12-01

    The biorefinery is an important concept for the development of alternative routes to a range of interesting and important materials from renewable resources. It ensures that the resources are used fully and that all parts of them are valorized. This paper develops this concept, using brown macroalgae Ascophyllum nodosum as an example, by assistance of microwave technology. A step-by-step process was designed to obtain fucoidan, alginates, sugars and biochar (alga residue) consecutively. The yields of fucoidan, alginates, sugars and biochar were 14.09%, 18.24%, 10.87% and 21.44%, respectively. To make an evaluation of the biorefinery process, seaweed sample was also treated for fucoidan extraction only, alginate extraction only and hydrothermal treatment for sugars and biochar only. The chemical composition and properties of each product were also analyzed. The results indicated that A. nodosum could be potentially used as feedstock for a biorefinery process to produce valuable chemicals and fuels.

  17. Microwave assisted step-by-step process for the production of fucoidan, alginate sodium, sugars and biochar from Ascophyllum nodosum through a biorefinery concept.

    PubMed

    Yuan, Yuan; Macquarrie, Duncan J

    2015-12-01

    The biorefinery is an important concept for the development of alternative routes to a range of interesting and important materials from renewable resources. It ensures that the resources are used fully and that all parts of them are valorized. This paper develops this concept, using brown macroalgae Ascophyllum nodosum as an example, by assistance of microwave technology. A step-by-step process was designed to obtain fucoidan, alginates, sugars and biochar (alga residue) consecutively. The yields of fucoidan, alginates, sugars and biochar were 14.09%, 18.24%, 10.87% and 21.44%, respectively. To make an evaluation of the biorefinery process, seaweed sample was also treated for fucoidan extraction only, alginate extraction only and hydrothermal treatment for sugars and biochar only. The chemical composition and properties of each product were also analyzed. The results indicated that A. nodosum could be potentially used as feedstock for a biorefinery process to produce valuable chemicals and fuels. PMID:26454369

  18. Construction and characterization of Pseudomonas aeruginosa algB mutants: role of algB in high-level production of alginate.

    PubMed Central

    Goldberg, J B; Ohman, D E

    1987-01-01

    The algB gene, which is involved in the production of alginate in Pseudomonas aeruginosa, was localized to approximately 2.2 kilobases of DNA from strain FRD by using transposon Tn501 insertion mutagenesis, subcloning, and complementation techniques. The previously reported alg-50(Ts) mutation, which confers the phenotype of temperature-sensitive alginate production, was here designated as an algB allele. A transduction-mediated gene replacement technique was used for site-directed mutagenesis to isolate and characterize algB::Tn501 mutants of P. aeruginosa FRD. Although algB::Tn501 mutants had a nonmucoid phenotype (indicating an alginate deficiency), they still produced about 1 to 5% of wild-type levels of alginate in most growth media and up to 16% in very rich media. The algB::Tn501 mutations had no apparent effect on growth rate or growth requirements. Using another gene replacement technique called excision marker rescue, we constructed a chromosomal algB deletion (delta algB) mutant of P. aeruginosa FRD. The delta algB mutant also produced low levels of alginate as did the algB::Tn501 mutants. The alginate produced by algB::Tn501 mutants resembled wild-type alginate by all criteria studied: molecular weight, acetylation, and proportion of mannuronic and guluronic acids. Thus, the algB gene product is apparently involved in the high-level production of alginate by P. aeruginosa and is not directly involved in the pathway leading to its biosynthesis. Chromosomal mapping of an algB::Tn501 insertion showed linkage to the trp-2 marker on the FRD chromosome as does the algB50(Ts) mutation. The excision marker rescue technique was also used to place the algB::Tn501 marker on the chromosome of characterized strains of P. aeruginosa PAO. The algB::Tn501 mutation mapped near 21 min on the PAO chromosome. Images PMID:3031015

  19. Production of BCG alginate-PLL microcapsules by emulsification/internal gelation.

    PubMed

    Esquisabel, A; Hernández, R M; Igartua, M; Gascón, A R; Calvo, B; Pedraz, J L

    1997-01-01

    A biocompatible emulsification method for microencapsulation of live cells and enzymes within a calcium alginate matrix applied to Bacillus Calmette-Guérin (BCG) has been developed. Small-diameter alginate beads (microcapsules) were formed via internal gelation of an alginate solution emulsified within vegetable oil. Five different oils (sesame, sweet almond, perhydrosqualene, camomile and jojoba) were used. The rheological analysis of the oils showed a Newtonian behaviour, with viscosities = 30.0, 37.7, 51.2, 59.3 and 67.1 mPa.s for perhydrosqualene, jojoba, camomile, sesame and sweet almond oil respectively. The particle size of the microcapsules obtained ranged from 30.3 microns for the microcapsules prepared with sweet almond oil to 57.0 microns for those made with perhydrosqualene. The mean particle diameter obtained was found to be dependent on the viscosity of the oil employed, according to the equation: phi (micron) = 76.6-0.628 eta (mPa.s) (r2 = 0.943). The encapsulated BCG was identified by the Difco TB stain set K, followed by observation under optical microscopy. Freeze-drying of the microcapsules was carried out to ensure their stability during storage. Two batches of microcapsules (those prepared with sesame and jojoba oil) and four types of cryoprotectors (glucose, trehalose, mannitol and sorbitol), at three concentration levels (5, 10 and 20% w/v) were studied. The parameters evaluated were particle size, physical appearance, reconstitution of lyophilizates and microscopical evaluation. For both batches of microcapsules the best results were obtained with trehalose 5%, showing particle sizes of 42.1 microns in the case of the microcapsules prepared with sesame oil, and of 45.3 microns for those prepared with jojoba.

  20. Growth and by-product profiles of Kluyveromyces marxianus cells immobilized in foamed alginate.

    PubMed

    Wilkowska, Agnieszka; Kregiel, Dorota; Guneser, Onur; Karagul Yuceer, Yonca

    2015-01-01

    The aim of this research was to study how the yeast cell immobilization technique influences the growth and fermentation profiles of Kluyveromyces marxianus cultivated on apple/chokeberry and apple/cranberry pomaces. Encapsulation of the cells was performed by droplet formation from a foamed alginate solution. The growth and metabolic profiles were evaluated for both free and immobilized cells. Culture media with fruit waste produced good growth of free as well as immobilized yeast cells. The fermentation profiles of K. marxianus were different with each waste material. The most varied aroma profiles were noted for immobilized yeast cultivated on apple/chokeberry pomace. PMID:25277269

  1. Evaluation of various parameters of calcium-alginate immobilization method for enhanced alkaline protease production by Bacillus licheniformis NCIM-2042 using statistical methods.

    PubMed

    Potumarthi, Ravichandra; Subhakar, Ch; Pavani, A; Jetty, Annapurna

    2008-04-01

    Calcium-alginate immobilization method for the production of alkaline protease by Bacillus licheniformis NCIM-2042 was optimized statistically. Four variables, such as sodium-alginate concentration, calcium chloride concentration, inoculum size and agitation speed were optimized by 2(4) full factorial central composite design and subsequent analysis and model validation by a second-order regression equation. Eleven carbon, 11 organic nitrogen and seven inorganic nitrogen sources were screened by two-level Plackett-Burman design for maximum alkaline protease production by using optimized immobilized conditions. The levels of four variables, such as Na-alginate 2.78%; CaCl(2), 2.15%; inoculum size, 8.10% and agitation, 139 rpm were found to be optimum for maximal production of protease. Glucose, soybean meal and ammonium sulfate were resulted in maximum protease production at 644 U/ml, 720 U/ml, and 806 U/ml when screened for carbon, organic nitrogen and inorganic nitrogen sources, respectively, using optimized immobilization conditions. Repeated fed batch mode of operation, using optimized immobilized conditions, resulted in continuous operation for 12 cycles without disintegration of beads. Cross-sectional scanning electron microscope images have shown the growth pattern of B. licheniformis in Ca-alginate immobilized beads.

  2. Quantitative study of the production and properties of alginate/poly-L-lysine microcapsules.

    PubMed

    Gugerli, R; Cantana, E; Heinzen, C; von Stockar, U; Marison, I W

    2002-01-01

    Alginate-polylysine-alginate (APA) microcapsules are of particular interest for their application as implants or for bioreactor cultures. Although their formation has been widely studied, there is still a lack of quantitative data describing resistance, membrane thickness and permeability. In this study, the quantitative application of a Texture Analyser for the measurement of capsule deformation yielded important results that permit comparison with other polymer systems used for encapsulation. Furthermore, single-membrane and multi-membrane capsules were formed in order to improve the modulation of the capsule properties. For single-membrane capsules, resistance was mostly affected by the incubation time in poly-L-lysine (PLL), the PLL molecular weight and concentration. The increase in resistance from 0.1 +/- 0.01 g/capsules to 2 +/- 0.2 g/capsules was linked to a membrane thickening (35-120 microm) and a decrease in permeability (150 to 40 kD). Thus, it was not possible to modify resistance and membrane permeability independently. Multi-membrane capsules with a resistance comparable to single-membrane capsules could be formed using various combinations of PLL molecular weights, and enabled uncoupling of permeability and resistance properties. PMID:12433301

  3. Production of volatiles in fresh-cut apple: effect of applying alginate coatings containing linoleic acid or isoleucine.

    PubMed

    Maya-Meraz, Irma O; Espino-Díaz, Miguel; Molina-Corral, Francisco J; González-Aguilar, Gustavo A; Jacobo-Cuellar, Juan L; Sepulveda, David R; Olivas, Guadalupe I

    2014-11-01

    One of the main quality parameters in apples is aroma, its main precursors are fatty acids (FA) and amino acids (AA). In this study, alginate edible coatings were used as carriers of linoleic acid or isoleucine to serve as precursors for the production of aroma in cut apples. Apple wedges were immersed in a CaCl2 solution and coated with one of the following formulations: alginate solution (Alg-Ca), Alg-Ca-low-level linoleic acid (0.61 g/Lt), (LFA), Alg-Ca-high-level linoleic acid (2.44 g/L; HFA), Alg-Ca-low-level isoleucine (0.61 g/L; LAA), and Alg-Ca-high-level isoleucine (2.44 g/L; HAA). Apple wedges were stored at 3 °C and 85% relative humidity for 21 d and key volatiles were studied during storage. Addition of precursors, mainly isoleucine, showed to increase the production of some key volatiles on coated fresh-cut apples during storage. The concentration of 2-methyl-1-butanol was 4 times higher from day 12 to day 21 in HAA, while 2-methyl butyl acetate increased from day 12 to day 21 in HAA. After 21 d, HAA-apples presented a 40-fold value of 2-methyl-butyl acetate, compared to Alg-Ca cut apples. Values of hexanal increased during cut apple storage when the coating carried linoleic acid, mainly on HFA, from 3 to 12 d. The ability of apples to metabolize AA and FA depends on the concentration of precursors, but also depends on key enzymes, previous apple storage, among others. Further studies should be done to better clarify the behavior of fresh-cut apples as living tissue to metabolize precursors contained in edible coatings for the production of volatiles. PMID:25296624

  4. Fragment-based de novo design of a cystathionine γ-lyase selective inhibitor blocking hydrogen sulfide production

    PubMed Central

    Corvino, Angela; Severino, Beatrice; Fiorino, Ferdinando; Frecentese, Francesco; Magli, Elisa; Perissutti, Elisa; Santagada, Vincenzo; Bucci, Mariarosaria; Cirino, Giuseppe; Kelly, Geoff; Servillo, Luigi; Popowicz, Grzegorz; Pastore, Annalisa; Caliendo, Giuseppe

    2016-01-01

    Hydrogen sulfide is an essential catabolite that intervenes in the pathophysiology of several diseases from hypertension to stroke, diabetes and pancreatitis. It is endogenously synthesized mainly by two pyridoxal-5′-phosphate-dependent enzymes involved in L-cysteine metabolism: cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE). Research in this field is currently impaired by the lack of pharmacological tools such as selective enzymatic inhibitors that could target specifically only one of these pathways. We used a novel approach based on a hybrid method that includes drug design, synthetic biology, metabolomics and pharmacological assays to rationally design a new inhibitor selective for the CSE enzyme. The identification of this compound opens new frontiers towards a better understanding of the role of CSE over CBS in the pathophysiology of diseases where a role for the H2S pathway has been proposed and the development of new lead compounds that could target the CSE enzyme. PMID:27708394

  5. Genetic linkage in Pseudomonas aeruginosa of algT and nadB: mutation in nadB does not affect NAD biosynthesis or alginate production.

    PubMed

    DeVries, C A; Hassett, D J; Flynn, J L; Ohman, D E

    1995-04-14

    The 68-min region of the chromosome of Pseudomonas aeruginosa (Pa) contains the gene algT, encoding a putative alternate sigma factor similar to sigma E in Escherichia coli, that is required for the expression of several genes in the alginate biosynthetic regulon. Sequences immediately upstream from algT were found to contain a divergently expressed open reading frame encoding a 60-kDa protein with 64 and 36% identity to the nadB gene products of E. coli and Bacillus subtilis, respectively. The nadB gene encodes L-aspartate oxidase and has been shown in several bacteria to be essential for de novo nicotinamide-adenine dinucleotide (NAD) biosynthesis. Pa nadB complemented the growth requirement for nicotinic acid in a nadB mutant strain of E. coli, suggesting that this gene encodes a functional homologue of L-aspartate oxidase. A nadB::Tn501 mutant was constructed by gene replacement in the alginate-producing strain, Pa FRD. This NadB- mutant still produced alginate and appeared normal with respect to the regulation of alginate synthesis. Interestingly, the NadB- mutant did not have an auxotrophic phenotype for nicotinic acid, indicating that this nadB was not essential for NAD biosynthesis in Pa. These results suggest the possibility that Pa has an alternate mechanism for de novo NAD biosynthesis.

  6. Optimization of culture variables for improving glucoamylase production by alginate-entrapped Thermomucor indicae-seudaticae using statistical methods.

    PubMed

    Kumar, Pardeep; Satyanarayana, T

    2007-04-01

    Alginate-entrapped sporangiospores of Thermomucor indicae-seudaticae were used for the production of glucoamylase. The critical variables that affected glucoamylase production were identified by Plackett-Burman design (sucrose, yeast-extract, K(2)HPO(4) and asparagine) and further optimized by using a four factor central composite design (CCD) of response surface methodology (RSM). Immobilized sporangiospores secreted 41% and 60% higher glucoamylase titers in shake flasks and airlift fermenter, respectively, when the variables were used at their optimum levels (sucrose 3.0%, yeast-extract 0.2%, K(2)HPO(4) 0.1% and asparagine 0.35%). Glucoamylase production (26.3 U ml(-1)) in the optimized medium was in good agreement with the values predicted by the quadratic model (26.7 U ml(-1)), thereby confirming its validity. The enzyme production was sustainable in flasks of higher volume and also airlift fermenter, and attained a peak within 32 h in the fermenter as compared to that of 48 h in shake flasks. PMID:16806908

  7. Biophotolytic H2 production using alginate-immobilized chloroplasts, enzymes and synthetic catalysts

    NASA Astrophysics Data System (ADS)

    Gisby, P. E.; Hall, D. O.

    1980-09-01

    Hydrogen can be produced by illumination of an aqueous mixture of chloroplasts and hydrogenase, in the presence of an electron carrier. This system may have potential for development of a solar converter to produce hydrogen from water if it can be stabilized or constructed as a completely synthetic system. The immobilization of the chloroplasts, or membrane analogues, would make possible a one-stage reactor with all the components in one chamber, or a two-stage reactor if the electron carrier was passed to another chamber to react with an immobilized hydrogen-producing catalyst. However, techniques for immobilizing enzymes tend to yield immobilized chloroplasts that are not very active, and other methods must be used. This paper describes the immobilization of chloroplasts using calcium alginate gels on reinforcing grids of nylon and stainless steel. Chloroplasts thus immobilized are fully active and can be used to produce hydrogen gas. Strengthened films of this sort could provide a good, solid, rigid matrix for a solar converter.

  8. Enhanced production of alkaline thermostable keratinolytic protease from calcium alginate immobilized cells of thermoalkalophilic Bacillus halodurans JB 99 exhibiting dehairing activity.

    PubMed

    Shrinivas, Dengeti; Kumar, Raghwendra; Naik, G R

    2012-01-01

    The thermoalkalophilic Bacillus halodurans JB 99 cells known for production of novel thermostable alkaline keratinolytic protease were immobilized in calcium alginate matrix. Batch and repeated batch cultivation using calcium alginate immobilized cells were studied for alkaline protease production in submerged fermentation. Immobilized cells with 2.5% alginate and 350 beads/flask of initial cell loading showed enhanced production of alkaline protease by 23.2% (5,275 ± 39.4 U/ml) as compared to free cells (4,280 ± 35.4 U/ml) after 24 h. In the semicontinuous mode of cultivation, immobilized cells under optimized conditions produced an appreciable level of alkaline protease in up to nine cycles and reached a maximal value of 5,975 U/ml after the seventh cycle. The enzyme produced from immobilized cells efficiently degraded chicken feathers in the presence of a reducing agent which can help the poultry industry in the management of keratin-rich waste and obtaining value-added products.

  9. The production of sulfonated chitosan-sodium alginate found in brown algae (Sargassum sp.) composite membrane as proton exchange membrane fuel cell (PEMFC)

    NASA Astrophysics Data System (ADS)

    Wafiroh, Siti; Pudjiastuti, Pratiwi; Sari, Ilma Indana

    2016-03-01

    The majority of energy was used in this period is from fossil fuel, which getting decreased in the future. The objective of this research is production and characterization of sulfonated chitosan-sodium alginate found in brown algae (Sargassum sp.) composite membrane as Proton Exchange Membrane Fuel Cell (PEMFC) for alternative energy. PEMFC was produced with 4 variations (w/w) ratio between chitosan and sodium alginate, 8 : 0, 8 : 1, 8 : 2, 8 : 4 (w/w). The production of membrane was mixed sodium alginate solution into chitosan solution and sulfonated with H2SO4 0.72 N. The characterization of the PEM was uses Modulus Young analysis, water swelling, ion exchange capacity, FTIR, SEM, DTA, methanol permeability and proton conductivity. The result of the research, showed that the optimum membrane was with ratio 8 : 2 (w/w) that the Modulus Young 8564 kN/m2, water swelling 31.86%, ion exchange capacity 1.020 meq/g, proton conductivity 8,8 × 10-6 S/cm, methanol permeability 1.90 × 10-8 g/cm2s and glass transition temperature (Tg) 100.9 °C, crystalline temperature (Tc) 227.6 °C, and the melting temperature (Tm) 267.9 °C.

  10. Production, partial characterization, and immobilization in alginate beads of an alkaline protease from a new thermophilic fungus Myceliophthora sp.

    PubMed

    Zanphorlin, Letícia Maria; Facchini, Fernanda Dell Antonio; Vasconcelos, Filipe; Bonugli-Santos, Rafaella Costa; Rodrigues, André; Sette, Lara Durães; Gomes, Eleni; Bonilla-Rodriguez, Gustavo Orlando

    2010-06-01

    Thermophilic fungi produce thermostable enzymes which have a number of applications, mainly in biotechnological processes. In this work, we describe the characterization of a protease produced in solidstate (SSF) and submerged (SmF) fermentations by a newly isolated thermophilic fungus identified as a putative new species in the genus Myceliophthora. Enzyme-production rate was evaluated for both fermentation processes, and in SSF, using a medium composed of a mixture of wheat bran and casein, the proteolytic output was 4.5-fold larger than that obtained in SmF. Additionally, the peak of proteolytic activity was obtained after 3 days for SSF whereas for SmF it was after 4 days. The crude enzyme obtained by both SSF and SmF displayed similar optimum temperature at 50 degrees C, but the optimum pH shifted from 7 (SmF) to 9(SSF). The alkaline protease produced through solid-state fermentation (SSF), was immobilized on beads of calcium alginate, allowing comparative analyses of free and immobilized proteases to be carried out. It was observed that both optimum temperature and thermal stability of the immobilized enzyme were higher than for the free enzyme. Moreover, the immobilized enzyme showed considerable stability for up to 7 reuses.

  11. 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

  12. Protein Crystal Isocitrate Lyase

    NASA Technical Reports Server (NTRS)

    1998-01-01

    The comparison of protein crystal, Isocitrate Lyase earth-grown (left) and space-grown (right). This is a target enzyme for fungicides. A better understanding of this enzyme should lead to the discovery of more potent fungicides to treat serious crop diseases such as rice blast; it regulates the flow of metabolic intermediates required for cell growth. Principal Investigator is Larry DeLucas.

  13. Identification of an L-methionine γ-lyase involved in the production of hydrogen sulfide from L-cysteine in Fusobacterium nucleatum subsp. nucleatum ATCC 25586.

    PubMed

    Suwabe, Kyosuke; Yoshida, Yasuo; Nagano, Keiji; Yoshimura, Fuminobu

    2011-10-01

    Fusobacterium nucleatum produces an abundance of hydrogen sulfide (H(2)S) in the oral cavity that is mediated by several enzymes. The identification and characterization of three distinct enzymes (Fn0625, Fn1055 and Fn1220) in F. nucleatum that catalyse the production of H(2)S from l-cysteine have been reported. In the current study, a novel enzyme involved in the production of H(2)S in F. nucleatum ATCC 25586, whose molecular mass had been estimated to be approximately 130 kDa, was identified by two-dimensional electrophoresis combined with MALDI-TOF MS. The enzyme, Fn1419, has previously been characterized as an l-methionine γ-lyase. SDS-PAGE and gel-filtration chromatography indicated that Fn1419 has a molecular mass of 43 kDa and forms tetramers in solution. Unlike other enzymes associated with H(2)S production in F. nucleatum, the quaternary structure of Fn1419 was not completely disrupted by exposure to SDS. The purified recombinant enzyme exhibited a K(m) of 0.32±0.02 mM and a k(cat) of 0.69±0.01 s(-1). Based on current and published data, the enzymic activity for H(2)S production from l-cysteine in F. nucleatum is ranked as follows: Fn1220>Fn1055>Fn1419>Fn0625. Based on kinetic values and relative mRNA levels of the respective genes, as determined by real-time quantitative PCR, the amount of H(2)S produced by Fn1419 was estimated to be 1.9 % of the total H(2)S produced from l-cysteine in F. nucleatum ATCC 25586. In comparison, Fn1220 appeared to contribute significantly to H(2)S production (87.6 %).

  14. Genetic Evidence that Loss of Virulence Associated with gacS or gacA Mutations in Pseudomonas syringae B728a Does Not Result from Effects on Alginate Production

    PubMed Central

    Willis, David K.; Holmstadt, Jeremy J.; Kinscherf, Thomas G.

    2001-01-01

    Mutations in the global regulatory genes gacS and gacA render Pseudomonas syringae pv. syringae strain B728a completely nonpathogenic in foliar infiltration assays on bean plants. It had been previously demonstrated that gac genes regulate alginate production in Pseudomonas species, while other published work indicated that alginate is involved in the pathogenic interaction of P. syringae on bean plants. Together, these results suggested that the effects of gacS and gacA mutations on virulence in B728a might stem directly from a role in regulating alginate. In this report, we confirm a role for gac genes in both algD expression and alginate production in B728a. However, B728a mutants completely devoid of detectable alginate were as virulent as the wild-type strain in our assay. Thus, factors other than, or in addition to, a deficiency of alginate must be involved in the lack of pathogenicity observed with gacS and gacA mutants. PMID:11229941

  15. High-Level Expression, Purification and Large-Scale Production of l-Methionine γ-Lyase from Idiomarina as a Novel Anti-Leukemic Drug

    PubMed Central

    Huang, Kui-Ying; Hu, Hai-Yan; Tang, Yan-Lai; Xia, Feng-Geng; Luo, Xue-Qun; Liu, Jian-Zhong

    2015-01-01

    l-Methionine γ-lyase (MGL), a pyridoxal 5′-phosphate-dependent enzyme, possesses anti-tumor activity. However, the low activity of MGL blocks the anti-tumor effect. This study describes an efficient production process for the recombinant MGL (rMGL) from Idiomarina constructed using the overexpression plasmid in Escherichia coli BL21 (DE3), purification, and large-scale production. The enzyme produced by the transformants accounted for 53% of the total proteins and accumulated at 1.95 mg/mL using a 500 L fermentor. The enzyme was purified to approximately 99% purity using a high-pressure mechanical homogenizer and nickel (Ni) Sepharose 6 Fast Flow (FF) chromatography. Then, the enzyme was polished by gel filtration, the endotoxins were removed using diethyl-aminoethanol (DEAE) Sepharose FF, and the final product was lyophilized with a vacuum freeze dryer at −35 °C. The specific activity of rMGL in the lyophilized powder was up to 108 U/mg. Compared to the control, the enzyme significantly inhibited cellular proliferation in a concentration-dependent manner as tested using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay and induced cellular apoptosis as analyzed by Annexin V-fluorescein isothiocyanate (FITC) with fluorescence-activated cell sorting (FACS) in leukemia cells. This paper demonstrated the cloning, overexpression, and large-scale production protocols for rMGL, which enabled rMGL to be used as a novel anti-leukemic drug. PMID:26308011

  16. In Vivo Validation of In Silico Predicted Metabolic Engineering Strategies in Yeast: Disruption of α-Ketoglutarate Dehydrogenase and Expression of ATP-Citrate Lyase for Terpenoid Production

    PubMed Central

    Gruchattka, Evamaria; Kayser, Oliver

    2015-01-01

    Background Engineering of the central carbon metabolism of Saccharomyces cerevisiae to redirect metabolic flux towards cytosolic acetyl-CoA has become a central topic in yeast biotechnology. A cell factory with increased flux into acetyl-CoA can be used for heterologous production of terpenoids for pharmaceuticals, biofuels, fragrances, or other acetyl-CoA derived compounds. In a previous study, we identified promising metabolic engineering targets in S. cerevisiae using an in silico stoichiometric metabolic network analysis. Here, we validate selected in silico strategies in vivo. Results Patchoulol was produced by yeast via a heterologous patchoulol synthase of Pogostemon cablin. To increase the metabolic flux from acetyl-CoA towards patchoulol, a truncated HMG-CoA reductase was overexpressed and farnesyl diphosphate synthase was fused with patchoulol synthase. The highest increase in production could be achieved by modifying the carbon source; sesquiterpenoid titer increased from glucose to ethanol by a factor of 8.4. Two strategies predicted in silico were chosen for validation in this work. Disruption of α-ketoglutarate dehydrogenase gene (KGD1) was predicted to redirect the metabolic flux via the pyruvate dehydrogenase bypass towards acetyl-CoA. The metabolic flux was redirected as predicted, however, the effect was dependent on cultivation conditions and the flux was interrupted at the level of acetate. High amounts of acetate were produced. As an alternative pathway to synthesize cytosolic acetyl-CoA, ATP-citrate lyase was expressed as a polycistronic construct, however, in vivo performance of the enzyme needs to be optimized to increase terpenoid production. Conclusions Stoichiometric metabolic network analysis can be used successfully as a metabolic prediction tool. However, this study highlights that kinetics, regulation and cultivation conditions may interfere, resulting in poor in vivo performance. Main sites of regulation need to be released and

  17. High-Level Expression, Purification and Large-Scale Production of l-Methionine γ-Lyase from Idiomarina as a Novel Anti-Leukemic Drug.

    PubMed

    Huang, Kui-Ying; Hu, Hai-Yan; Tang, Yan-Lai; Xia, Feng-Geng; Luo, Xue-Qun; Liu, Jian-Zhong

    2015-08-01

    l-Methionine γ-lyase (MGL), a pyridoxal 5'-phosphate-dependent enzyme, possesses anti-tumor activity. However, the low activity of MGL blocks the anti-tumor effect. This study describes an efficient production process for the recombinant MGL (rMGL) from Idiomarina constructed using the overexpression plasmid in Escherichia coli BL21 (DE3), purification, and large-scale production. The enzyme produced by the transformants accounted for 53% of the total proteins and accumulated at 1.95 mg/mL using a 500 L fermentor. The enzyme was purified to approximately 99% purity using a high-pressure mechanical homogenizer and nickel (Ni) Sepharose 6 Fast Flow (FF) chromatography. Then, the enzyme was polished by gel filtration, the endotoxins were removed using diethyl-aminoethanol (DEAE) Sepharose FF, and the final product was lyophilized with a vacuum freeze dryer at -35 °C. The specific activity of rMGL in the lyophilized powder was up to 108 U/mg. Compared to the control, the enzyme significantly inhibited cellular proliferation in a concentration-dependent manner as tested using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay and induced cellular apoptosis as analyzed by Annexin V-fluorescein isothiocyanate (FITC) with fluorescence-activated cell sorting (FACS) in leukemia cells. This paper demonstrated the cloning, overexpression, and large-scale production protocols for rMGL, which enabled rMGL to be used as a novel anti-leukemic drug.

  18. [Use of a preparation from fungal pectin lyase in the food industry].

    PubMed

    Semenova, M V; Sinitsyna, O A; Morozova, V V; Fedorova, E A; Gusakov, A V; Okunev, O N; Sokolova, L M; Koshelev, A V; Bubnova, Iu P; Sinitsyn, A P

    2006-01-01

    A new enzyme preparation of fungal pectin lyase (EC 4.2.2.10) was shown to be useful for the production of cranberry juice and clarification of apple juice in the food industry. A comparative study showed that the preparation of pectin lyase is competitive with commercial pectinase products. The molecular weight of homogeneous pectin lyase was 38 kDa. Properties of the homogeneous enzyme were studied. This enzyme was most efficient in removing highly esterified pectin.

  19. Antibiotic resistance in Streptomyces lividans: fluorescence assay for streptogramin B lyase.

    PubMed

    Bateman, K P; Armstrong, S M; White, R L; Ross, N W

    1997-06-01

    A fluorescence assay for streptogramin B lyase, an enzyme that confers resistance to streptogramin B antibiotics, has been developed. The antibiotic substrates are fluorescent and the linear peptide products formed in the lyase-catalyzed reaction are relatively nonfluorescent. The assay has potential for assessing bacterial resistance to streptogramin B antibiotics and will be utilized to direct the purification of streptogramin B lyase from bacterial extracts.

  20. Xylella fastidiosa esterase rather than hydroxynitrile lyase.

    PubMed

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

    2015-03-01

    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. PMID:25684099

  1. Study of the potential of the air lift bioreactor for xylitol production in fed-batch cultures by Debaryomyces hansenii immobilized in alginate beads.

    PubMed

    Pérez-Bibbins, Belinda; de Souza Oliveira, Ricardo Pinheiro; Torrado, Ana; Aguilar-Uscanga, María Guadalupe; Domínguez, José Manuel

    2014-01-01

    Cell immobilization has shown to be especially adequate for xylitol production. This work studies the suitability of the air lift bioreactor for xylitol production by Debaryomyces hansenii immobilized in Ca-alginate operating in fed-batch cultures to avoid substrate inhibition. The results showed that the air lift bioreactor is an adequate system since the minimum air flow required for fluidization was even lower than that leading to the microaerobic conditions that trigger xylitol accumulation by this yeast, also maintaining the integrity of the alginate beads and the viability of the immobilized cells until 3 months of reuses. Maximum productivities and yields of 0.43 g/l/h and 0.71 g/g were achieved with a xylose concentration of 60 g/l after each feeding. The xylose feeding rate, the air flow, and the biomass concentration at the beginning of the fed-batch operation have shown to be critical parameters for achieving high productivities and yields. Although a maximum xylitol production of 139 g/l was obtained, product inhibition was evidenced in batch experiments, which allowed estimating at 200 and 275 g/l the IC50 for xylitol productivity and yield, respectively. The remarkable production of glycerol in the absence of glucose was noticeable, which could not only be attributed to the osmoregulatory function of this polyol in conditions of high osmotic pressure caused by high xylitol concentrations but also to the role of the glycerol synthesis pathway in the regeneration of NAD(+) in conditions of suboptimal microaeration caused by insufficient aeration or high oxygen demand when high biomass concentrations were achieved.

  2. Factors affecting protein release from alginate-chitosan coacervate microcapsules during production and gastric/intestinal simulation.

    PubMed

    Vandenberg, G W; Drolet, C; Scott, S L; de la Noüe, J

    2001-12-13

    A series of experiments was performed to evaluate the influence of a number of physico-chemical factors on the diffusion of a model protein, bovine serum albumin (BSA), from dried chitosan-coated alginate microcapsules. Diffusion of BSA was quantified during the microcapsule manufacture processes (gelation, washing, rinsing) and during incubation in conditions simulating the pH encountered during the gastric (0.1 N HCl; pH 1.5) and intestinal (200 mM Tris-HCl; pH 7.5) phases of digestion. Factors tested included alginate and chitosan concentration, calcium chloride (CaCl2) concentration in the gelation medium, loading rate, chitosan molecular mass and pH of the gelation medium. Microcapsule size and gelation time were altered in order to determine their effects on protein retention. Alginate and chitosan concentration significantly influenced BSA retention during microcapsule manufacture and acid incubation, as did calcium chloride concentration in the gelation medium (P<0.05). BSA retention during manufacture was not significantly altered by protein loading rate or pH of the encapsulation medium, however, protein retention during acid incubation decreased significantly with increasing protein loading rate and encapsulation medium pH (P<0.05). Microcapsules that were washed with acetone following manufacture demonstrated significantly increased protein retention during acid incubation (P<0.05). In microcapsules that had been acetone-dried to a point whereby their mass was reduced to 10% of that immediately following encapsulation, protein retention was over 80% following 24-h acid incubation vs. only 20% protein retention from non acetone-dried microcapsules. The presence of calcium in the neutral buffer medium significantly reduced BSA diffusion in a concentration-dependent manner (P<0.05).

  3. Crystal Structure of Heparinase II from Pedobacter Heparinus and its Complex with a Disaccharide Product

    SciTech Connect

    Shaya,D.; Tocilj, A.; Li, Y.; Myette, J.; Venkatarman, G.; Sasisekharan, R.; Cygler, M.

    2006-01-01

    Heparinase II depolymerizes heparin and heparan sulfate glycosaminoglycans, yielding unsaturated oligosaccharide products through an elimination degradation mechanism. This enzyme cleaves the oligosaccharide chain on the nonreducing end of either glucuronic or iduronic acid, sharing this characteristic with a chondroitin ABC lyase. We have determined the first structure of a heparin-degrading lyase, that of heparinase II from Pedobacter heparinus (formerly Flavobacterium heparinum), in a ligand-free state at 2.15Angstroms resolution and in complex with a disaccharide product of heparin degradation at 2.30Angstroms resolution. The protein is composed of three domains: an N-terminal {alpha}-helical domain, a central two-layered {beta}-sheet domain, and a C-terminal domain forming a two-layered {beta}-sheet. Heparinase II shows overall structural similarities to the polysaccharide lyase family 8 (PL8) enzymes chondroitin AC lyase and hyaluronate lyase. In contrast to PL8 enzymes, however, heparinase II forms stable dimers, with the two active sites formed independently within each monomer. The structure of the N-terminal domain of heparinase II is also similar to that of alginate lyases from the PL5 family. A Zn2+ ion is bound within the central domain and plays an essential structural role in the stabilization of a loop forming one wall of the substrate-binding site. The disaccharide binds in a long, deep canyon formed at the top of the N-terminal domain and by loops extending from the central domain. Based on structural comparison with the lyases from the PL5 and PL8 families having bound substrates or products, the disaccharide found in heparinase II occupies the '+1' and '+2' subsites. The structure of the enzyme-product complex, combined with data from previously characterized mutations, allows us to propose a putative chemical mechanism of heparin and heparan-sulfate degradation.

  4. Influence of various alginate brands on the redifferentiation of dedifferentiated bovine articular chondrocytes in alginate bead culture under high and low oxygen tension.

    PubMed

    Domm, C; Schünke, M; Steinhagen, J; Freitag, S; Kurz, B

    2004-01-01

    We examined the influence of various alginates on the redifferentiation of dedifferentiated articular chondrocytes in alginate bead culture under low (5%) and (21%) high oxygen supply. Isolated bovine articular chondrocytes were dedifferentiated and multiplied by 2-week monolayer culture under 21% oxygen. They were subcultured at a density of 10(7) cells/mL in six different commercially available sodium alginates (1.2%, w/v) and held under 21 or 5% oxygen for 3 weeks. Proliferation (DNA measurement on days 0 and 21 of culture), collagen type II production (immunocytochemistry and Western blotting), and [(3)H]proline and [(35)S]sulfate incorporation were monitored. Collagen type II production was significantly stronger under 5% oxygen compared with 21% oxygen in two alginates (three other alginates nearly reached the significance level). However, alginate-based differences proved not to be significant. [(3)H]Proline incorporation was not influenced by alginate but showed strong oxygen dependency (up to 3-fold higher under 5% oxygen). For [(35)S]sulfate incorporation oxygen dependency was even stronger (up to 8-fold higher under 5% oxygen) and significant alginate-dependent differences were found for several alginates. The effects of the different alginates did not correlate with their pH, viscosity, or guluronic:mannuronic acid ratio. Thus, the type of alginate and even more, the oxygen supply, influence the redifferentiation and matrix production of dedifferentiated bovine articular chondrocytes. PMID:15684688

  5. 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

  6. The action of exogenous gibberellic acid on isocitrate lyase -mRNA in germinating castor bean seeds.

    PubMed

    Martin, C; Northcote, D H

    1982-03-01

    Gibberellic acid (GA3) stimulates isocitrate lyase activity of the endosperm during germination of castor bean seeds. Isocitrate lyase from castor bean was purified and an antibody to it was prepared from rabbit serum. This antibody was used to measure the amounts of isocitrate lyase-mRNA using an in vitro translation system. No specific stimulation of isocitrate lyase-mRNA by application of GA3 was detected. The stimulation of isocitrate lyase activity by exogenous GA3 may be accounted for by the action of the growth substance in advancing the overall production of rRNA and mRNA which accelerates the rate of total protein synthesis during germination. The application of Amo 1618 retards the production of isocitrate lyase activity but also retards protein synthesis in general. This suggests that endogenous gibberellins also act non-specifically in the regulation of protein synthesis during castor bean germination.

  7. 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.

  8. Bioacetylation of Seaweed Alginate

    PubMed Central

    Lee, J. W.; Day, D. F.

    1995-01-01

    Seaweed alginate was acetylated by intact, resting cells of Pseudomonas syringae ATCC 19304. Maximum acetylation of this polymer occurred at a pH of 6.0 and a temperature of 25 deg C. Aeration and gluconic acid were required for an optimal reaction. A reactor which contained carbon-immobilized cells was constructed to continuously acetylate alginate. The maximal yield of acetylation was about 90%, and the half-life of this system was 6.5 days. PMID:16534934

  9. Statistical optimization of alpha-amylase production by Streptomyces erumpens MTCC 7317 cells in calcium alginate beads using response surface methodology.

    PubMed

    Kar, Shaktimay; Ray, Ramesh C

    2008-01-01

    Alpha-amylase has a wide range of applications in starch industries, i.e. baking, brewing, distillery, etc. The alpha-amylase production from Streptomyces erumpens MTCC 7317 immobilized cells was compared with that of free cells. The immobilized cells of S. erumpens in calcium alginate beads were more effective for production of alpha-amylase (12.2% more yield) than free cells. Response surface methodology (RSM) was used to evaluate the effect of main variables, i.e. incubation period, pH and temperature on enzyme production with immobilized cells. A full factorial Central Composite Design (CCD) was applied to study these main factors that affected alpha-amylase production. The experimental results showed that the optimum incubation period, pH and temperature were 36 h, 6.0 and 50 degrees C, respectively for immobilized cells. Repeated batch fermentation of immobilized cells in shake flasks carried out in starch-beef extract medium showed that S. erumpens cells were physiologically active on the support even after four cycles of fermentation.

  10. A stable mercury-containing complex of the organomercurial lyase MerB: catalysis, product release, and direct transfer to MerA.

    PubMed

    Benison, Gregory C; Di Lello, Paola; Shokes, Jacob E; Cosper, Nathaniel J; Scott, Robert A; Legault, Pascale; Omichinski, James G

    2004-07-01

    Bacteria isolated from organic mercury-contaminated sites have developed a system of two enzymes that allows them to efficiently convert both ionic and organic mercury compounds to the less toxic elemental mercury. Both enzymes are encoded on the mer operon and require sulfhydryl-bound substrates. The first enzyme is an organomercurial lyase (MerB), and the second enzyme is a mercuric ion reductase (MerA). MerB catalyzes the protonolysis of the carbon-mercury bond, resulting in the formation of a reduced carbon compound and inorganic ionic mercury. Of several mercury-containing MerB complexes that we attempted to prepare, the most stable was a complex consisting of the organomercurial lyase (MerB), a mercuric ion, and a molecule of the MerB inhibitor dithiothreitol (DTT). Nuclear magnetic resonance (NMR) spectroscopy and extended X-ray absorption fine structure spectroscopy of the MerB/Hg/DTT complex have shown that the ligands to the mercuric ion in the complex consist of both sulfurs from the DTT molecule and one cysteine ligand, C96, from the protein. The stability of the MerB/Hg/DTT complex, even in the presence of a large excess of competing cysteine, has been demonstrated by NMR and dialysis. We used an enzyme buffering test to determine that the MerB/Hg/DTT complex acts as a substrate for the mercuric reductase MerA. The observed MerA activity is higher than the expected activity assuming free diffusion of the mercuric ion from MerB to MerA. This suggests that the mercuric ion can be transferred between the two enzymes by a direct transfer mechanism. PMID:15222746

  11. 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

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

    2015-12-25

    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

  12. 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

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

    2015-12-25

    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.

  13. Enhanced lipid production by co-cultivation and co-encapsulation of oleaginous yeast Trichosporonoides spathulata with microalgae in alginate gel beads.

    PubMed

    Kitcha, Suleeporn; Cheirsilp, Benjamas

    2014-05-01

    This study attempted to enhance biomass and lipid productivity of an oleaginous yeast Trichosporonoides spathulata by co-culturing with microalgae Chlorella spp., optimizing culture conditions, and encapsulating them in alginate gel beads. The co-culture of the yeast with microalgae Chlorella vulgaris var. vulgaris TISTR 8261 most enhanced overall biomass and lipid productivity by 1.6-fold of the yeast pure culture at 48 h and by 1.1-fold at 72 h. After optimization and scale-up in a bioreactor, this co-culture produced the highest biomass of 12.2 g/L with a high lipid content of 47 %. The dissolved oxygen monitoring system in the bioreactor showed that the microalgae worked well as an oxygen supplier to the yeast. This study also showed that the co-encapsulated yeast and microalgae could grow and produce lipid as same as their free cells did. Therefore, it is possible to apply this encapsulation technique for lipid production and simplification of downstream harvesting process. This co-culture system also produced the lipid with high content of saturated fatty acids, indicating its potential use as biodiesel feedstock with high oxidative stability.

  14. Factors influencing alginate gel biocompatibility.

    PubMed

    Tam, Susan K; Dusseault, Julie; Bilodeau, Stéphanie; Langlois, Geneviève; Hallé, Jean-Pierre; Yahia, L'Hocine

    2011-07-01

    Alginate remains the most popular polymer used for cell encapsulation, yet its biocompatibility is inconsistent. Two commercially available alginates were compared, one with 71% guluronate (HiG), and the other with 44% (IntG). Both alginates were purified, and their purities were verified. After 2 days in the peritoneal cavity of C57BL/6J mice, barium (Ba)-gel and calcium (Ca)-gel beads of IntG alginate were clean, while host cells were adhered to beads of HiG alginate. IntG gel beads, however, showed fragmentation in vivo while HiG gel beads stayed firm. The physicochemical properties of the sodium alginates and their gels were thoroughly characterized. The intrinsic viscosity of IntG alginate was 2.5-fold higher than that of HiG alginate, suggesting a greater molecular mass. X-ray photoelectron spectroscopy indicated that both alginates were similar in elemental composition, including low levels of counterions in all gels. The wettabilities of the alginates and gels were also identical, as measured by contact angles of water on dry films. Ba-gel beads of HiG alginate resisted swelling and degradation when immersed in water, much more than the other gel beads. These results suggest that the main factors contributing to the biocompatibility of gels of purified alginate are the mannuronate/guluronate content and/or intrinsic viscosity.

  15. Hydrophobic modification of sodium alginate and its application in drug controlled release.

    PubMed

    Yao, Bolong; Ni, Caihua; Xiong, Cheng; Zhu, Changping; Huang, Bo

    2010-05-01

    Sodium alginate was hydrophobically modified by coupling of polybutyl methacrylate onto the alginate. The polybutyl methacrylate was previously prepared through polymerization of butyl methacrylate in the presence of 2-amino-ethanethiol as a chain transfer agent. The structure of the product was characterized by Fourier-transformed infrared spectrometry, nuclear magnetic resonance ((1)HNMR) and thermogravimetry. The result of fluorescence analysis showed that the hydrophobicity of the modified alginate was obviously increased. The modified alginate conjugate was used for immobilization of bovine serum albumin in the presence of calcium chloride. In addition, the release behavior of the drug-loaded alginate in deionized water and Tris-HCl buffer solution (pH 7.2) was investigated. It was found that the modified sodium alginate possessed prolonged release behavior compared to unmodified sodium alginate, and it had potential application in controlled release as a drug carrier.

  16. 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

  17. Lipoxygenase and Hydroperoxide Lyase in Germinating Watermelon Seedlings 1

    PubMed Central

    Vick, Brady A.; Zimmerman, Don C.

    1976-01-01

    Lipoxygenase (EC 1.13.1.13) was found in seedlings of Citrullus lanatus (Thunb.) Matsum. and Nakai (watermelon). The enzyme has pH optima of 4.4 and 5.5 and is inhibited by 0.2 mM nordihydroguaiaretic acid. It is present in two functional units with estimated molecular weights of 120,000 and 240,000, respectively. A new enzyme, tentatively termed hydroperoxide lyase, has been partially purified from watermelon seedlings. The enzyme, located principally in the region of the hypocotyl-root junction, catalyzes the conversion of 13-l-hydroperoxy-cis-9-trans-11-octadecadienoic acid to 12-oxo-trans-10-dodecenoic acid and hexanal. The hydroperoxide lyase enzyme from watermelon has a molecular weight in excess of 250,000, a pH optimum in the range of 6 to 6.5, and is inhibited by p-chloromercuribenzoic acid. Its presence has also been demonstrated in other cucurbits. The maximum activity of both enzymes occurs on the 6th day of germination. The identification of the products of the hydroperoxide lyase reaction suggests that lipoxygenase and hydroperoxide lyase may be involved in the conversion of certain polyunsaturated fatty acids to traumatic acid (trans-2-dodecenedioic acid). PMID:16659569

  18. Expression Analysis of Phenylalanine Ammonia Lyase Gene and Rosmarinic Acid Production in Salvia officinalis and Salvia virgata Shoots Under Salicylic Acid Elicitation.

    PubMed

    Ejtahed, Roghayeh Sadat; Radjabian, Tayebeh; Hoseini Tafreshi, Sayed Ali

    2015-08-01

    Partial fragments of phenylalanine ammonia lyase (PAL) genes were cloned and characterized from Salvia officinalis (SoPAL) and Salvia virgata (SvPAL). Different concentrations (250 and 500 μM) of exogenous salicylic acid (SA) were used when correlation between PAL expression and rosmarinic acid (RA) accumulation was compared. The results showed that the deduced cDNA sequences of the partial genes had high similarities with those of known PAL gene from other plant species. Semi-quantitative reverse transcription PCR (RT-PCR) analysis revealed that exogenous application of SA led to up-regulating of the PAL expression. Further analysis showed that in S. virgata, at higher concentration of SA, higher accumulation of RA was achieved, while in S. officinalis, the higher RA accumulation was observed at lower concentration of SA. It was concluded that there was no positive correlation between the intensity of PAL transcription and the RA accumulation in the studied species. Therefore, despite of the increase in transcription rate of the PAL at the higher concentration of SA, the lower amounts of RA were accumulated in the case of S. officinalis. Consequently, the hypothesis that PAL is the rate-determining step in RA biosynthesis is not always valid and probably some other unknown factors participate in the synthesis of phenolics.

  19. Bio-based barium alginate film: Preparation, flame retardancy and thermal degradation behavior.

    PubMed

    Liu, Yun; Zhang, Chuan-Jie; Zhao, Jin-Chao; Guo, Yi; Zhu, Ping; Wang, De-Yi

    2016-03-30

    A bio-based barium alginate film was prepared via a facile ionic exchange and casting approach. Its flammability, thermal degradation and pyrolysis behaviors, thermal degradation mechanism were studied systemically by limiting oxygen index (LOI), vertical burning (UL-94), microscale combustion calorimetry (MCC), thermogravimetric analysis (TGA) coupled with Fourier transform infrared analysis (FTIR) and pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS). It showed that barium alginate film had much higher LOI value (52.0%) than that of sodium alginate film (24.5%). Moreover, barium alginate film passed the UL-94 V-0 rating, while the sodium alginate film showed no classification. Importantly, peak of heat release rate (PHRR) of barium alginate film in MCC test was much lower than that of sodium alginate film, suggested that introduction of barium ion into alginate film significantly decreased release of combustible gases. TG-FTIR and Py-GC-MS results indicated that barium alginate produced much less flammable products than that of sodium alginate in whole thermal degradation procedure. Finally, a possible degradation mechanism of barium alginate had been proposed.

  20. Bio-based barium alginate film: Preparation, flame retardancy and thermal degradation behavior.

    PubMed

    Liu, Yun; Zhang, Chuan-Jie; Zhao, Jin-Chao; Guo, Yi; Zhu, Ping; Wang, De-Yi

    2016-03-30

    A bio-based barium alginate film was prepared via a facile ionic exchange and casting approach. Its flammability, thermal degradation and pyrolysis behaviors, thermal degradation mechanism were studied systemically by limiting oxygen index (LOI), vertical burning (UL-94), microscale combustion calorimetry (MCC), thermogravimetric analysis (TGA) coupled with Fourier transform infrared analysis (FTIR) and pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS). It showed that barium alginate film had much higher LOI value (52.0%) than that of sodium alginate film (24.5%). Moreover, barium alginate film passed the UL-94 V-0 rating, while the sodium alginate film showed no classification. Importantly, peak of heat release rate (PHRR) of barium alginate film in MCC test was much lower than that of sodium alginate film, suggested that introduction of barium ion into alginate film significantly decreased release of combustible gases. TG-FTIR and Py-GC-MS results indicated that barium alginate produced much less flammable products than that of sodium alginate in whole thermal degradation procedure. Finally, a possible degradation mechanism of barium alginate had been proposed. PMID:26794953

  1. 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...

  2. 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...

  3. 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...

  4. 21 CFR 184.1187 - Calcium alginate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Calcium alginate. 184.1187 Section 184.1187 Food... GRAS § 184.1187 Calcium alginate. (a) Calcium alginate (CAS Reg. No. 9005-35-0) is the calcium salt of alginic acid, a natural polyuronide constituent of certain brown algae. Calcium alginate is prepared...

  5. Improving the controlled delivery formulations of caffeine in alginate hydrogel beads combined with pectin, carrageenan, chitosan and psyllium.

    PubMed

    Belščak-Cvitanović, Ana; Komes, Draženka; Karlović, Sven; Djaković, Senka; Spoljarić, Igor; Mršić, Gordan; Ježek, Damir

    2015-01-15

    Alginate-based blends consisting of carrageenan, pectin, chitosan or psyllium husk powder were prepared for assessment of the best formulation aimed at encapsulation of caffeine. Alginate-pectin blend exhibited the lowest viscosity and provided the smallest beads. Alginate-psyllium husk blend was characterised with higher viscosity, yielding the largest bead size and the highest caffeine encapsulation efficiency (83.6%). The release kinetics of caffeine indicated that the porosity of alginate hydrogel was not reduced sufficiently to retard the diffusion of caffeine from the beads. Chitosan coated alginate beads provided the most retarded release of caffeine in water. Morphological characteristics of beads encapsulating caffeine were adversely affected by freeze drying. Bitterness intensity of caffeine-containing beads in water was the lowest for alginate-psyllium beads and chitosan coated alginate beads. Higher sodium alginate concentration (3%) for production of hydrogel beads in combination with psyllium or chitosan coating would present the most favourable carrier systems for immobilization of caffeine.

  6. Physiological characterization of ATP-citrate lyase in Aspergillus niger.

    PubMed

    Chen, Hong; He, Xihong; Geng, Hongran; Liu, Hao

    2014-04-01

    Acetyl-CoA, an important molecule in cellular metabolism, is generated in multiple subcellular compartments and mainly used for energy production, biosynthesis of a diverse set of molecules, and protein acetylation. In eukaryotes, cytosolic acetyl-CoA is derived mainly from the conversion of citrate and CoA by ATP-citrate lyase. Here, we describe the targeted deletions of acl1 and acl2, two tandem divergently transcribed genes encoding subunits of ATP-citrate lyase in Aspergillus niger. We show that loss of acl1 or/and acl2 results in a significant decrease of acetyl-CoA and citric acid levels in these mutants, concomitant with diminished vegetative growth, decreased pigmentation, reduced asexual conidiogenesis, and delayed conidial germination. Exogenous addition of acetate repaired the defects of acl-deficient strains in growth and conidial germination but not pigmentation and conidiogenesis. We demonstrate that both Acl1 and Acl2 subunits are required to form a functional ATP-citrate lyase in A. niger. First, deletion of acl1 or/and acl2 resulted in similar defects in growth and development. Second, enzyme activity assays revealed that loss of either acl1 or acl2 gene resulted in loss of ATP-citrate lyase activity. Third, in vitro enzyme assays using bacterially expressed 6His-tagged Acl protein revealed that only the complex of Acl1 and Acl2 showed ATP-citrate lyase activity, no enzyme activities were detected with the individual protein. Fourth, EGFP-Acl1 and mCherry-Acl2 proteins were co-localized in the cytosol. Thus, acl1 and acl2 coordinately modulate the cytoplasmic acetyl-CoA levels to regulate growth, development, and citric acid synthesis in A. niger.

  7. [Cystathionine γ-lyase].

    PubMed

    Jurkowska, Halina; Kaczor-Kamińska, Marta; Bronowicka-Adamska, Patrycja; Wróbel, Maria

    2014-01-01

    γ-Cystathionase (CTH, EC: 4.4.1.1), an enzyme widely distributed in the world of prokaryotic and eukaryotic organisms, catalyzes the formation and transformations of sulfane sulfur-containing compounds and plays a pivotal role in the L-cysteine desulfuration pathway. Human, tetrameric CTH is composed of two dimers and each monomer binds pyridoxal phosphate (PLP). The gene, located on the short arm of chromosome 1, consists of 13 exons and 12 introns. As a result of alternative splicing, three isoforms of human CTH arise. Analysis of genetic variations of the CTH encoding gene showed a large number of polymorphisms. A decrease of the expression of CTH entails a drop in the level of cysteine , glutathione (GSH), taurine and hydrogen sulfide (H2S) in the cells and, more importantly, leads to cystathioninuria. H2S, endogenously formed by CTH, affects the vasodilation and regulation of blood pressure. CTH knockout mice have decreased levels of H2S, hypertension, and reduced capacity for vascular endothelium relaxation. Overexpression of the gene encoding CTH in the cells leads to increased production of H2S. H2S plays a role in protection of neurons against oxidative stress, and stimulates an increase in γ-glutamylcysteine synthetase and thereby an increase in the level of GSH. Sulfurtransferases, including CTH, can locally prevent oxidative stress due to reversible oxidation of - SH groups in the presence of increased levels of reactive oxygen species, and reduction in the presence of GSH and/or reduced thioredoxin.

  8. 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.

  9. Multiple Genes in a Single Host: Cost-Effective Production of Bacterial Laccase (cotA), Pectate Lyase (pel), and Endoxylanase (xyl) by Simultaneous Expression and Cloning in Single Vector in E. coli

    PubMed Central

    Kumar, Sandeep; Jain, Kavish Kumar; Bhardwaj, Kailash N.; Chakraborty, Subhojit; Kuhad, Ramesh Chander

    2015-01-01

    This study attempted to reduce the enzyme production cost for exploiting lignocellulosic materials by expression of multiple genes in a single host. Genes for bacterial laccase (CotA), pectate lyase (Pel) and endoxylanase (Xyl), which hold significance in lignocellulose degradation, were cloned in pETDuet-1 vector containing two independent cloning sites (MCS). CotA and xyl genes were cloned in MCS1 and MCS 2, respectively. Pel gene was cloned by inserting complete cassette (T7 promoter, ribosome binding site, pel gene, His tag and complete gene ORF) preceded by cotA open reading frame in the MCS1. IPTG induction of CPXpDuet-1 construct in E. coli BL21(DE3) resulted in expression of all three heterologous proteins of ~65 kDa (CotA), ~45 kDa (Pel) and ~25 kDa (Xyl), confirmed by SDS-PAGE and western blotting. Significant portions of the enzymes were also found in culture supernatant (~16, ~720 and ~370 IU/ml activities of CotA, Pel and Xyl, respectively). Culture media optimization resulted in 2, 3 and 7 fold increased secretion of recombinant CotA, Pel and Xyl, respectively. Bioreactor level optimization of the recombinant cocktail expression resulted in production of 19 g/L dry cell biomass at OD600nm 74 from 1 L induced culture after 15 h of cultivation, from which 9, 627 and 1090 IU/ml secretory enzyme activities of CotA, Xyl and Pel were obtained, respectively. The cocktail was also found to increase the saccharification of orange peel in comparison to the xylanase alone. Thus, simultaneous expression as well as extra cellular secretion of these enzymes as cocktail can reduce the enzyme production cost which increases their applicability specially for exploiting lignocellulosic materials for their conversion to value added products like alcohol and animal feed. PMID:26642207

  10. Novel alginate based nanocomposite hydrogels with incorporated silver nanoparticles.

    PubMed

    Obradovic, Bojana; Stojkovska, Jasmina; Jovanovic, Zeljka; Miskovic-Stankovic, Vesna

    2012-01-01

    Alginate colloid solution containing electrochemically synthesized silver nanoparticles (AgNPs) was investigated regarding the nanoparticle stabilization and possibilities for production of alginate based nanocomposite hydrogels in different forms. AgNPs were shown to continue to grow in alginate solutions for additional 3 days after the synthesis by aggregative mechanism and Ostwald ripening. Thereafter, the colloid solution remains stable for 30 days and could be used alone or in mixtures with aqueous solutions of poly(vinyl alcohol) (PVA) and poly(N-vinyl-2-pyrrolidone) (PVP) while preserving AgNPs as verified by UV-Vis spectroscopy studies. We have optimized techniques for production of Ag/alginate microbeads and Ag/alginate/PVA beads, which were shown to efficiently release AgNPs decreasing the Escherichia coli concentration in suspensions for 99.9% over 24 h. Furthermore, Ag/hydrogel discs based on alginate, PVA and PVP were produced by freezing-thawing technique allowing adjustments of hydrogel composition and mechanical properties as demonstrated in compression studies performed in a biomimetic bioreactor. PMID:22203513

  11. Microfluidic generation of hollow Ca-alginate microfibers.

    PubMed

    Meng, Zhi-Jun; Wang, Wei; Xie, Rui; Ju, Xiao-Jie; Liu, Zhuang; Chu, Liang-Yin

    2016-07-01

    This work reports on an efficient microfluidic approach for continuous production of hollow Ca-alginate microfibers with controllable structures and functions. A coaxial microcapillary microfluidic device combined with a rotator is constructed to produce a cylindrical flow jet with four aqueous solutions as templates for continuous fabrication and collection of microfibers. A four-aqueous-phase flow jet with an intermediate buffer flow between the Ca(2+)-containing and alginate-containing flows is used as the template for microfiber fabrication. The buffer flow efficiently controls the diffusion of Ca(2+) into the alginate-containing flow as well as the crosslinking reaction, thus ensuring the continuous fabrication of hollow Ca-alginate microfibers under relatively low flow rates without clogging of the microchannel. The structure of the hollow microfibers can be flexibly adjusted by changing the flow rates and device dimensions. Meanwhile, the continuous fabrication process of the microfibers allows flexible incorporation of a functional component into the sheath flow for functionalization and addition of active substances in the core flow for encapsulation. This is demonstrated by fabricating hollow Ca-alginate microfibers with a wall containing magnetic nanoparticles for magnetic functionalization and with hollow internals containing Chlorella pyrenoidosa cells for confined growth. This work provides an efficient strategy for continuous fabrication of functional hollow Ca-alginate microfibers with controllable structures and functions.

  12. Microfluidic generation of hollow Ca-alginate microfibers.

    PubMed

    Meng, Zhi-Jun; Wang, Wei; Xie, Rui; Ju, Xiao-Jie; Liu, Zhuang; Chu, Liang-Yin

    2016-07-01

    This work reports on an efficient microfluidic approach for continuous production of hollow Ca-alginate microfibers with controllable structures and functions. A coaxial microcapillary microfluidic device combined with a rotator is constructed to produce a cylindrical flow jet with four aqueous solutions as templates for continuous fabrication and collection of microfibers. A four-aqueous-phase flow jet with an intermediate buffer flow between the Ca(2+)-containing and alginate-containing flows is used as the template for microfiber fabrication. The buffer flow efficiently controls the diffusion of Ca(2+) into the alginate-containing flow as well as the crosslinking reaction, thus ensuring the continuous fabrication of hollow Ca-alginate microfibers under relatively low flow rates without clogging of the microchannel. The structure of the hollow microfibers can be flexibly adjusted by changing the flow rates and device dimensions. Meanwhile, the continuous fabrication process of the microfibers allows flexible incorporation of a functional component into the sheath flow for functionalization and addition of active substances in the core flow for encapsulation. This is demonstrated by fabricating hollow Ca-alginate microfibers with a wall containing magnetic nanoparticles for magnetic functionalization and with hollow internals containing Chlorella pyrenoidosa cells for confined growth. This work provides an efficient strategy for continuous fabrication of functional hollow Ca-alginate microfibers with controllable structures and functions. PMID:27302737

  13. Genetics Home Reference: adenylosuccinate lyase deficiency

    MedlinePlus

    ... of five disease-associated human adenylosuccinate lyase mutants. Biochemistry. 2009 Jun 16;48(23):5291-302. doi: ... ADSL) and the R303C ADSL deficiency-associated mutation. Biochemistry. 2012 Aug 21;51(33):6701-13. doi: ...

  14. 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...

  15. 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...

  16. 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...

  17. 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...

  18. 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...

  19. 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...

  20. 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. 9005-35-0) is the calcium salt of alginic acid, a natural polyuronide constituent of certain brown...

  1. 21 CFR 184.1187 - Calcium alginate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Calcium alginate. 184.1187 Section 184.1187 Food... Specific Substances Affirmed as GRAS § 184.1187 Calcium alginate. (a) Calcium alginate (CAS Reg. No. 9005-35-0) is the calcium salt of alginic acid, a natural polyuronide constituent of certain brown...

  2. 21 CFR 184.1187 - Calcium alginate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Calcium alginate. 184.1187 Section 184.1187 Food... Specific Substances Affirmed as GRAS § 184.1187 Calcium alginate. (a) Calcium alginate (CAS Reg. No. 9005-35-0) is the calcium salt of alginic acid, a natural polyuronide constituent of certain brown...

  3. 21 CFR 184.1187 - Calcium alginate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Calcium alginate. 184.1187 Section 184.1187 Food... Specific Substances Affirmed as GRAS § 184.1187 Calcium alginate. (a) Calcium alginate (CAS Reg. No. 9005-35-0) is the calcium salt of alginic acid, a natural polyuronide constituent of certain brown...

  4. 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...

  5. [Phenylalanine ammonia-lyase of pigmented yeasts].

    PubMed

    Mushi, N Iu; Kupletskaia, M B; Bab'eva, I P; Egorov, N S

    1980-01-01

    116 pigmented yeast cultures were tested for the presence of L-phenylalanine-ammonia lyase transforming L-phenylalanine into trans-cinnamic acid. The enzyme was found in 54 strains. Most of these strains belonged to the genera Rhodotorula and Sporobolomyces. Toluene, along with acetone, was successfully used to increase cellular permeability of the yeast cultures while determining the activity of phenylalanine-ammonia lyase.

  6. Effect of concentration and substrate flow rate on isomaltulose production from sucrose by Erwinia sp. cells immobilized in calcium-alginate using packed bed reactor.

    PubMed

    Kawaguti, Haroldo Yukio; Harumi Sato, Hélia

    2010-09-01

    Isomaltulose was obtained from sucrose solution by immobilized cells of Erwinia sp. D12 using a batch and a continuous process. Parameters for sucrose conversion into isomaltulose were evaluated using both experimental design and response surface methodology. Erwinia sp. D12 cells were immobilized in different alginates, and the influence of substrate flow rate and concentration parameters to produce isomaltulose from sucrose were observed. Response surface methodology demonstrated that packed bed columns containing cells immobilized in low-viscosity sodium alginate (250 cP) presented a mean isomaltulose conversion rate of 47%. In a continuous process, both sucrose substrate concentration and substrate flow rate parameters had a significant effect (p < 0.05) and influenced the conversion of sucrose into isomaltulose. Higher conversion rates of sucrose into isomaltulose, from 53-75% were obtained using 75 g of immobilized cells at a substrate flow rate of 0.6 mL/min.

  7. 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.

  8. Crystal structures of halohydrin hydrogen-halide-lyases from Corynebacterium sp. N-1074.

    PubMed

    Watanabe, Fumiaki; Yu, Fujio; Ohtaki, Akashi; Yamanaka, Yasuaki; Noguchi, Keiichi; Yohda, Masafumi; Odaka, Masafumi

    2015-12-01

    Halohydrin hydrogen-halide-lyase (H-Lyase) is a bacterial enzyme that is involved in the degradation of halohydrins. This enzyme catalyzes the intramolecular nucleophilic displacement of a halogen by a vicinal hydroxyl group in halohydrins to produce the corresponding epoxides. The epoxide products are subsequently hydrolyzed by an epoxide hydrolase, yielding the corresponding 1, 2-diol. Until now, six different H-Lyases have been studied. These H-Lyases are grouped into three subtypes (A, B, and C) based on amino acid sequence similarities and exhibit different enantioselectivity. Corynebacterium sp. strain N-1074 has two different isozymes of H-Lyase, HheA (A-type) and HheB (B-type). We have determined their crystal structures to elucidate the differences in enantioselectivity among them. All three groups share a similar structure, including catalytic sites. The lack of enantioselectivity of HheA seems to be due to the relatively wide size of the substrate tunnel compared to that of other H-Lyases. Among the B-type H-Lyases, HheB shows relatively high enantioselectivity compared to that of HheBGP1 . This difference seems to be due to amino acid replacements at the active site tunnel. The binding mode of 1, 3-dicyano-2-propanol at the catalytic site in the crystal structure of the HheB-DiCN complex suggests that the product should be (R)-epichlorohydrin, which agrees with the enantioselectivity of HheB. Comparison with the structure of HheC provides a clue for the difference in their enantioselectivity.

  9. Crystal structures of halohydrin hydrogen-halide-lyases from Corynebacterium sp. N-1074.

    PubMed

    Watanabe, Fumiaki; Yu, Fujio; Ohtaki, Akashi; Yamanaka, Yasuaki; Noguchi, Keiichi; Yohda, Masafumi; Odaka, Masafumi

    2015-12-01

    Halohydrin hydrogen-halide-lyase (H-Lyase) is a bacterial enzyme that is involved in the degradation of halohydrins. This enzyme catalyzes the intramolecular nucleophilic displacement of a halogen by a vicinal hydroxyl group in halohydrins to produce the corresponding epoxides. The epoxide products are subsequently hydrolyzed by an epoxide hydrolase, yielding the corresponding 1, 2-diol. Until now, six different H-Lyases have been studied. These H-Lyases are grouped into three subtypes (A, B, and C) based on amino acid sequence similarities and exhibit different enantioselectivity. Corynebacterium sp. strain N-1074 has two different isozymes of H-Lyase, HheA (A-type) and HheB (B-type). We have determined their crystal structures to elucidate the differences in enantioselectivity among them. All three groups share a similar structure, including catalytic sites. The lack of enantioselectivity of HheA seems to be due to the relatively wide size of the substrate tunnel compared to that of other H-Lyases. Among the B-type H-Lyases, HheB shows relatively high enantioselectivity compared to that of HheBGP1 . This difference seems to be due to amino acid replacements at the active site tunnel. The binding mode of 1, 3-dicyano-2-propanol at the catalytic site in the crystal structure of the HheB-DiCN complex suggests that the product should be (R)-epichlorohydrin, which agrees with the enantioselectivity of HheB. Comparison with the structure of HheC provides a clue for the difference in their enantioselectivity. PMID:26422370

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

    PubMed

    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.

  11. 3-Hydroxy-3-methylglutaric and 3-methylglutaric acids impair redox status and energy production and transfer in rat heart: relevance for the pathophysiology of cardiac dysfunction in 3-hydroxy-3-methylglutaryl-coenzyme A lyase deficiency.

    PubMed

    da Rosa, Mateus Struecker; Seminotti, Bianca; Ribeiro, César Augusto João; Parmeggiani, Belisa; Grings, Mateus; Wajner, Moacir; Leipnitz, Guilhian

    2016-09-01

    3-Hydroxy-3-methylglutaryl-coenzyme A lyase (HL) deficiency is characterized by tissue accumulation of 3-hydroxy-3-methylglutaric (HMG), and 3-methylglutaric (MGA) acids. Affected patients present cardiomyopathy, whose pathomechanisms are not yet established. We investigated the effects of HMG and MGA on energy and redox homeostasis in rat heart using in vivo and in vitro models. In vivo experiments showed that intraperitoneal administration of HMG and MGA decreased the activities of the respiratory chain complex II and creatine kinase (CK), whereas HMG also decreased the activity of complex II-III. Furthermore, HMG and MGA injection increased reactive species production and carbonyl formation, and decreased glutathione concentrations. Regarding the enzymatic antioxidant defenses, HMG and MGA increased glutathione peroxidase (GPx) and glutathione reductase (GR) activities, while only MGA diminished the activities of superoxide dismutase (SOD) and catalase, as well as the protein content of SOD1. Pre-treatment with melatonin (MEL) prevented MGA-induced decrease of CK activity and SOD1 levels. In vitro results demonstrated that HMG and MGA increased reactive species formation, induced lipid peroxidation and decreased glutathione. We also verified that reactive species overproduction and glutathione decrease provoked by HMG and MGA were abrogated by MEL and lipoic acid (LA), while only MEL prevented HMG- and MGA-induced lipoperoxidation. Allopurinol (ALP) also prevented reactive species overproduction caused by both metabolites. Our data provide solid evidence that bioenergetics dysfunction and oxidative stress are induced by HMG and MGA in heart, which may explain the cardiac dysfunction observed in HL deficiency, and also suggest that antioxidant supplementation could be considered as adjuvant therapy for affected patients.

  12. Cysteine S-conjugate β-lyases: Important roles in the metabolism of naturally occurring sulfur and selenium-containing compounds, xenobiotics and anticancer agents

    PubMed Central

    Cooper, Arthur J. L.; Krasnikov, Boris F.; Niatsetskaya, Zoya V.; Pinto, John T.; Callery, Patrick S.; Villar, Maria T.; Artigues, Antonio; Bruschi, Sam A.

    2010-01-01

    Summary Cysteine S-conjugate β-lyases are pyridoxal 5′-phosphate-containing enzymes that catalyze β-elimination reactions with cysteine S-conjugates that possess a good leaving group in the β-position. The end products are aminoacrylate and a sulfur-containing fragment. The aminoacrylate tautomerizes and hydrolyzes to pyruvate and ammonia. The mammalian cysteine S-conjugate β-lyases thus far identified are enzymes involved in amino acid metabolism that catalyze β-lyase reactions as non-physiological side reactions. Most are aminotransferases. In some cases the lyase is inactivated by reaction products. The cysteine S-conjugate β-lyases are of much interest to toxicologists because they play an important key role in the bioactivation (toxication) of halogenated alkenes, some of which are produced on an industrial scale and are environmental contaminants. The cysteine S-conjugate β-lyases have been reviewed in this journal previously [Cooper and Pinto, 2006]. Here we focus on more recent findings regarding: 1) the identification of enzymes associated with high-Mr cysteine S-conjugate β-lyases in the cytosolic and mitochondrial fractions of rat liver and kidney; 2) the mechanism of syncatalytic inactivation of rat liver mitochondrial aspartate aminotransferase by the nephrotoxic β-lyase substrate S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (the cysteine S-conjugate of tetrafluoroethylene); 3) toxicant channeling of reactive fragments from the active site of mitochondrial aspartate aminotransferase to susceptible proteins in the mitochondria; 4) the involvement of cysteine S-conjugate β-lyases in the metabolism/bioactivation of drugs and natural products; and 5) the role of cysteine S-conjugate β-lyases in the metabolism of selenocysteine Se-conjugates. This review emphasizes the fact that the cysteine S-conjugate β-lyases are biologically more important than hitherto appreciated. PMID:20306345

  13. Cysteine S-conjugate β-lyases: important roles in the metabolism of naturally occurring sulfur and selenium-containing compounds, xenobiotics and anticancer agents.

    PubMed

    Cooper, Arthur J L; Krasnikov, Boris F; Niatsetskaya, Zoya V; Pinto, John T; Callery, Patrick S; Villar, Maria T; Artigues, Antonio; Bruschi, Sam A

    2011-06-01

    Cysteine S-conjugate β-lyases are pyridoxal 5'-phosphate-containing enzymes that catalyze β-elimination reactions with cysteine S-conjugates that possess a good leaving group in the β-position. The end products are aminoacrylate and a sulfur-containing fragment. The aminoacrylate tautomerizes and hydrolyzes to pyruvate and ammonia. The mammalian cysteine S-conjugate β-lyases thus far identified are enzymes involved in amino acid metabolism that catalyze β-lyase reactions as non-physiological side reactions. Most are aminotransferases. In some cases the lyase is inactivated by reaction products. The cysteine S-conjugate β-lyases are of much interest to toxicologists because they play an important key role in the bioactivation (toxication) of halogenated alkenes, some of which are produced on an industrial scale and are environmental contaminants. The cysteine S-conjugate β-lyases have been reviewed in this journal previously (Cooper and Pinto in Amino Acids 30:1-15, 2006). Here, we focus on more recent findings regarding: (1) the identification of enzymes associated with high-M(r) cysteine S-conjugate β-lyases in the cytosolic and mitochondrial fractions of rat liver and kidney; (2) the mechanism of syncatalytic inactivation of rat liver mitochondrial aspartate aminotransferase by the nephrotoxic β-lyase substrate S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (the cysteine S-conjugate of tetrafluoroethylene); (3) toxicant channeling of reactive fragments from the active site of mitochondrial aspartate aminotransferase to susceptible proteins in the mitochondria; (4) the involvement of cysteine S-conjugate β-lyases in the metabolism/bioactivation of drugs and natural products; and (5) the role of cysteine S-conjugate β-lyases in the metabolism of selenocysteine Se-conjugates. This review emphasizes the fact that the cysteine S-conjugate β-lyases are biologically more important than hitherto appreciated.

  14. 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

  15. A modern view of phenylalanine ammonia lyase.

    PubMed

    MacDonald, M Jason; D'Cunha, Godwin B

    2007-06-01

    Phenylalanine ammonia lyase (PAL; E.C.4.3.1.5), which catalyses the biotransformation of L-phenylalanine to trans-cinnamic acid and ammonia, was first described in 1961 by Koukol and Conn. Since its discovery, much knowledge has been gathered with reference to the enzyme's catabolic role in microorganisms and its importance in the phenyl propanoid pathway of plants. The 3-dimensional structure of the enzyme has been characterized using X-ray crystallography. This has led to a greater understanding of the mechanism of PAL-catalyzed reactions, including the discovery of a recently described cofactor, 3,5-dihydro-5-methyldiene-4H-imidazol-4-one. In the past 3 decades, PAL has gained considerable significance in several clinical, industrial, and biotechnological applications. The reversal of the normal physiological reaction can be effectively employed in the production of optically pure L-phenylalanine, which is a precursor of the noncalorific sweetener aspartame (L-phenylalanyl-L-aspartyl methyl ester). The enzyme's natural ability to break down L-phenylalanine makes PAL a reliable treatment for the genetic condition phenylketonuria. In this mini-review, we discuss prominent details relating to the physiological role of PAL, the mechanism of catalysis, methods of determination and purification, enzyme kinetics, and enzyme activity in nonaqueous media. Two topics of current study on PAL, molecular biology and crystal structure, are also discussed. PMID:17612622

  16. A modern view of phenylalanine ammonia lyase.

    PubMed

    MacDonald, M Jason; D'Cunha, Godwin B

    2007-06-01

    Phenylalanine ammonia lyase (PAL; E.C.4.3.1.5), which catalyses the biotransformation of L-phenylalanine to trans-cinnamic acid and ammonia, was first described in 1961 by Koukol and Conn. Since its discovery, much knowledge has been gathered with reference to the enzyme's catabolic role in microorganisms and its importance in the phenyl propanoid pathway of plants. The 3-dimensional structure of the enzyme has been characterized using X-ray crystallography. This has led to a greater understanding of the mechanism of PAL-catalyzed reactions, including the discovery of a recently described cofactor, 3,5-dihydro-5-methyldiene-4H-imidazol-4-one. In the past 3 decades, PAL has gained considerable significance in several clinical, industrial, and biotechnological applications. The reversal of the normal physiological reaction can be effectively employed in the production of optically pure L-phenylalanine, which is a precursor of the noncalorific sweetener aspartame (L-phenylalanyl-L-aspartyl methyl ester). The enzyme's natural ability to break down L-phenylalanine makes PAL a reliable treatment for the genetic condition phenylketonuria. In this mini-review, we discuss prominent details relating to the physiological role of PAL, the mechanism of catalysis, methods of determination and purification, enzyme kinetics, and enzyme activity in nonaqueous media. Two topics of current study on PAL, molecular biology and crystal structure, are also discussed.

  17. The purification and physicochemical characterization of maize (Zea mays L.) isocitrate lyase.

    PubMed

    Khan, A S; Van Driessche, E; Kanarek, L; Beeckmans, S

    1992-08-15

    A purification scheme is described for the glyoxylate cycle enzyme isocitrate lyase from maize scutella. Purification involves an acetone precipitation and a heat denaturation step, followed by ammonium sulfate precipitation and chromatography on DEAE-cellulose and on blue-Sepharose. The latter step results in the removal of the remaining malate dehydrogenase activity, and of a high molecular mass (62 kDa) but inactive degradation product of isocitrate lyase. Catalase can be completely removed by performing the DEAE-cellulose chromatography in the presence of Triton X-100. Pure isocitrate lyase can be stored without appreciable loss of activity at -70 degrees C in 5 mM triethanolamine buffer containing 6 mM MgCl2, 7 mM 2-mercaptoethanol, and 50% (v/v) glycerol, pH 7.6. Maize isocitrate lyase is a tetrameric protein with a subunit molecular mass of 64 kDa. Purity of the enzyme preparation was demonstrated by polyacrylamide gel electrophoresis in the presence of dodecylsulfate, in acid (pH 3.2) urea and by isoelectric focusing (pI = 5.1). Maize isocitrate lyase is devoid of covalently linked sugar residues. From circular dichroism measurements we estimate that its structure comprises 30% alpha-helical and 15% beta-pleated sheet segments. The enzyme requires Mg2+ ions for activity, and only Mn2+ apparently is able to replace this cation to a certain extent. The kinetics of the isocitrate lyase-catalyzed cleavage reaction were investigated, and the amino acid composition of the maize enzyme was determined. Finally the occurrence of an association between maize isocitrate lyase and catalase was observed. Such a multienzyme complex may be postulated to play a protective role in vivo. PMID:1637186

  18. Effect of Oxygen Tension and Medium Components on Monomer Distribution of Alginate.

    PubMed

    Kıvılcımdan Moral, Çiğdem; Doğan, Özdemir; Sanin, Faika Dilek

    2015-06-01

    Alginate is a natural biopolymer composed of mannuronic and guluronic acid monomers. It is produced by algae and some species of Azotobacter and Pseudomonas. This study aims to investigate the effect of dissolved oxygen tension (DOT) and growth medium substrate and calcium concentrations on the monomeric composition of alginate produced by Azotobacter vinelandii ATCC® 9046 in a fermenter. Results showed that alginate production increased with increasing DOT from 1 to 5 %. The highest alginate production was obtained as 4.51 g/L under 20 g/L of sucrose and 50 mg/L of calcium at 5 % DOT. At these conditions, alginate was rich in mannuronic acid (up to 61 %) and it was particularly high at low calcium concentration. On the other hand, at extreme conditions such as high DOT level (10 % DOT) and low sucrose concentration (10 g/L), guluronic acid was dominant (ranging between 65 and 100 %).

  19. Cloning of Pseudomonas aeruginosa algG, which controls alginate structure.

    PubMed Central

    Chitnis, C E; Ohman, D E

    1990-01-01

    The biochemical mechanism by which alpha-L-guluronate (G) residues are incorporated into alginate by Pseudomonas aeruginosa is not understood. P. aeruginosa first synthesizes GDP-mannuronate, which is used to incorporate beta-D-mannuronate residues into the polymer. It is likely that the conversion of some beta-D-mannuronate residues to G occurs by the action of a C-5 epimerase at either the monomer (e.g., sugar-nucleotide) or the polymer level. This study describes the results of a molecular genetic approach to identify a gene involved in the formation or incorporation of G residues into alginate by P. aeruginosa. Mucoid P. aeruginosa FRD1 was chemically mutagenized, and mutants FRD462 and FRD465, which were incapable of incorporating G residues into alginate, were independently isolated. Assays using a G-specific alginate lyase from Klebsiella aerogenes and 1H-nuclear magnetic resonance analyses showed that G residues were absent in the alginates secreted by these mutants. 1H-nuclear magnetic resonance analyses also showed that alginate from wild-type P. aeruginosa contained no detectable blocks of G. The mutations responsible for defective incorporation of G residues into alginate in the mutants FRD462 and FRD465 were designated algG4 and algG7, respectively. Genetic mapping experiments revealed that algG was closely linked (greater than 90%) to argF, which lies at 34 min on the P. aeruginosa chromosome and is adjacent to a cluster of genes required for alginate biosynthesis. The clone pALG2, which contained 35 kilobases of P. aeruginosa DNA that included the algG and argF wild-type alleles, was identified from a P. aeruginosa gene bank by a screening method that involved gene replacement. A DNA fragment carrying algG was shown to complement algG4 and algG7 in trans. The algG gene was physically mapped on the alginate gene cluster by subcloning and Tn501 mutagenesis. Images PMID:2160929

  20. [Spatial structure and mechanism of tyrosine phenol-lyase and tryptophan indole-lyase].

    PubMed

    Demidkina, T V; Anston, A A; Faleev, N G; Phillips, R S; Zakomyrdina, L N

    2009-01-01

    The bacterial tyrosine phenol-lyase (EC 4.1.99.2) and tryptoptophan indole-lyase (EC 4.1.99.1) belong to pyridoxal-5'-phosphate dependent beta-eliminating lyases, catalysing the reversible decomposition of L-tyrosine and L-tryptophan to pyruvate, ammonia, and phenol or indole correspondingly. Data on the three dimentional structures of the holoenzymes of tyrosine phenol-lyase and tryptophan indole-lyase and several enzyme-inhibitor complexes, modeling distinct reaction stages of the beta-elimination of L-tyrosine are described in the paper and structural bases of monovalent cations influence of activity of the enzymes are discussed. The spectral and catalytic properties of the mutant enzymes were studied. The data thus obtained have allowed us to elucidate the catalytic functions of a number of amino acid residues and conclude that the acid-base properties of the catalytic groups of the enzymes under the optimal for the catalysis conditions in hydrophobic active sites of tyrosine phenol-lyase and tryptoptophan indol-lyase are different from those in water solutions. Study of the mechanisms of labilization of Calpha-proton of the bound amino acids and activation of the leaving groups of the substrates during the catalytic process has demonstrated that in certain cases concerted reaction pathways are realized instead of stepwise ones. PMID:19425498

  1. Induction of Phenylalanine Ammonia-lyase in Xanthium Leaf Disks. Photosynthetic Requirement and Effect of Daylength 1

    PubMed Central

    Zucker, Milton

    1969-01-01

    A cycloheximide-sensitive increase in the activity of phenylalanine ammonia-lyase (EC 4.3.1.5) occurs in Xanthium leaf disks exposed to light. Radioactive ammonia-lyase has been isolated by means of sucrose density gradient centrifugation and starch gel electrophoresis from disks fed l-isoleucine-U-14C or l-arginine-U-14C. The incorporation of radioactive amino acids into phenylalanine ammonia-lyase together with the inhibitory effects of cycloheximide indicate that the observed increase in enzyme activity involves the induction of lyase synthesis. The light-dependent synthesis of the ammonia-lyase is completely inhibited by 50 μm 3-(4-chlorophenyl)-1,1-dimethylurea (CMU) indicating that photosynthesis is involved. Only a trace quantity of some photosynthetic product must be needed because half light saturation occurs at very low intensity (ca. 30 ft-c). Exogenous carbohydrate is also required for continuing enzyme synthesis over a 72 hr period. But carbohydrate does not replace the photosynthetic requirement in darkness. Enzyme formed in light disappears rapidly from disks placed in the dark. The decay of ammonia-lyase activity follows first order kinetics. The half-life of the lyase ranged from 6 to 15 hr in leaf material used. Cyoloheximide inhibits the decay of lyase activity. Thus the maintenance of turnover in Xanthium leaf disks requires de novo synthesis of protein. That turnover, i.e., degradation as well as synthesis of lyase protein occurs is suggested by the apparent loss of radioactive ammonia-lyase from leaf disks placed in darkness. Light-induced synthesis coupled with rapid turnover can produce a diurnal fluctuation of ammonia-lyase activity in Xanthium leaf disks. Alternating periods of enzyme synthesis and degradation were observed in disks exposed to 24 hr cycles of light and dark. The average level of enzyme activity maintained in the tissue was directly related to the length of the light period. Induction of lyase synthesis was also observed

  2. 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

  3. 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.

  4. A strategic study using mutant-strain entrapment in calcium alginate for the production of Saccharomyces cerevisiae cells with high invertase activity.

    PubMed

    Rossi-Alva, Juan Carlos; Rocha-Leão, Maria Helena Miguez

    2003-08-01

    Entrapped cells and entrapped cells grown inside of a calcium alginate matrix as well as free cells have been investigated using Saccharomyces cerevisiae mutant strains with regard to their pattern of growth and invertase activity. The repression of invertase by glucose and glucose-consumption ability were considered in the selection process of the mutants. Efficient sucrose bioconversion due to high invertase activity was obtained when entrapped mutant strain Q6R2 cells were grown within calcium alginate gel beads using sucrose plus glucose as the carbon source. Under these conditions, 1 mg (dry weight) of entrapped cells is able to produce 20 micromol of inverted sugar in 3 min (the maximum activity obtained was 20 units x mg(-1)). The experiments were carried out for 6 months without appreciable loss of either bead integrity or invertase activity. The biocatalyst was also stored at 4 degrees C for 6 months without appreciable loss of the invertase activity. This work shows that entrapped yeast cells with a weak ability to consume sugar may be used to produce inverted sugar.

  5. Preparation of unnatural amino acids with ammonia-lyases and 2,3-aminomutases.

    PubMed

    Poppe, László; Paizs, Csaba; Kovács, Klaudia; Irimie, Florin-Dan; Vértessy, Beáta

    2012-01-01

    Ammonia-lyases catalyze a wide range of processes leading to α,β-unsaturated compounds by elimination of ammonia. In this chapter, ammonia-lyases are reviewed with major emphasis on their synthetic applications in stereoselective preparation of unnatural amino acids. Besides the synthesis of various unnatural α-amino acids with the aid of phenylalanine ammonia-lyases (PALs) utilizing the 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) prosthetic groups, the biotransformations leading to various unnatural β-amino acids with phenylalanine 2,3-aminomutases using the same catalytic MIO prosthetic group are discussed. Cloning, production, purification, and biotransformation protocols for PAL are described in detail.

  6. Healing and Preventive Effects of Calcium Alginate on Carbon Tetrachloride Induced Liver Injury in Rats

    PubMed Central

    Khotimchenko, Yuri S.; Khotimchenko, Maxim Y.

    2004-01-01

    The purpose of this study was to investigate the pharmacological effects of calcium alginate on carbon tetrachloride (CCL4)-induced hepatotoxicity in rats. The study included two experiments. In the first experiment the animals were given daily CCL4 through gavage for 7 days and then 10, 50, or 250 mg/kg b.w. of calcium alginate for 21 days. The increased bilirubin level, enhanced alanine and aspartate aminotransferase activity in plasma and reduced liver glycogen content induced by CCL4 were partly normalized by alginate administration in a dose-dependent manner. In addition, alginate significantly improved CCL4-induced alterations of pro-oxidant and antioxidant biochemical parameters in liver and plasma compared to those of rats administered CCL4. In the second experiment the animals were given daily 10, 50 or 250 mg/kg b.w. of calcium alginate for 21 days before 7-day administration of CCL4. Pretreatment with alginate before CCL4 administration resulted in significantly inhibited increase of the blood enzymatic activities of alanine and aspartate aminotransferases and bilirubin level in a dose-dependent manner. Also, preliminary administration of alginate prevented elevation of lipid peroxidation products and reduction of liver glutathione content in rats given CCL4. These results suggest that calcium alginate exerts healing and preventive effects on CCL4-induced hepatotoxicity in rats.

  7. Effects of ambroxol on alginate of mature Pseudomonas aeruginosa biofilms.

    PubMed

    Li, Fang; Yu, Jialin; Yang, Hua; Wan, Zhenyan; Bai, Dan

    2008-07-01

    Biofilm-forming bacteria Pseudomonas aeruginosa is a common pathogen in mechanically ventilated newborns, which can cause life-threatening infections. Alginate of mucoid Pseudomonas aeruginosa biofilms is considered an important virulence factor which contributes to the resistance to antibiotics. Traditionally, ambroxol is widely used in newborns with lung problems as a mucolytic agent and antioxidant agent as well. And there are few studies that demonstrated the anti-biofilm activity of ambroxol. In this study, we found that ambroxol can affect the structure of mucoid Pseudomonas aeruginosa biofilms. Further, we found that ambroxol reduces the production of alginate, the expression of the important genes and the activity of key enzyme guanosine diphospho-D-mannose dehydrogenase (GDP-mannose dehydrogenase; GMD) which were involved in alginate biosynthesis.

  8. 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...

  9. 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,...

  10. 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,...

  11. 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,...

  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. 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.

  14. (PCG) Protein Crystal Growth Isocitrate Lyase

    NASA Technical Reports Server (NTRS)

    1989-01-01

    (PCG) Protein Crystal Growth Isocitrate Lyase. Target enzyme for fungicides. A better understanding of this enzyme should lead to the discovery of more potent fungicides to treat serious crop diseases such as rice blast. It regulates the flow of metabolic intermediates required for cell growth. Principal Investigator for STS-26 was Charles Bugg.

  15. Use of alginate beads as carriers for lactic acid bacteria in a structured system and preliminary validation in a meat product.

    PubMed

    Corbo, Maria Rosaria; Bevilacqua, Antonio; Speranza, Barbara; Di Maggio, Barbara; Gallo, Mariangela; Sinigaglia, Milena

    2016-01-01

    This paper proposes the microencapsulation into alginate beads of 4 isolates of lactic acid bacteria (Lactobacillus spp.), previously isolated from pork meat. First, the beads were studied in relation to the encapsulation yield (EY), kinetic of cell release in a structured system, and survival throughout bead storage at 4 °C. EY was 93-96% and the survival of the encapsulated microorganisms was variable, with two isolates showing a bacterial population of 6.1-6.9 log cfu/g after 35 days under refrigerated conditions. Thereafter, the paper addressed a preliminary validation in a meat model system, containing salt, nitrites and nitrates, lactose, pepper, and then in a commercial preparation of pork meat. For the validation in pork meat, free cells were used as controls. Cell released from beads were able to achieve a significant acidification; in particular, after 7 days they showed the same results of free cells. PMID:26476507

  16. Use of alginate beads as carriers for lactic acid bacteria in a structured system and preliminary validation in a meat product.

    PubMed

    Corbo, Maria Rosaria; Bevilacqua, Antonio; Speranza, Barbara; Di Maggio, Barbara; Gallo, Mariangela; Sinigaglia, Milena

    2016-01-01

    This paper proposes the microencapsulation into alginate beads of 4 isolates of lactic acid bacteria (Lactobacillus spp.), previously isolated from pork meat. First, the beads were studied in relation to the encapsulation yield (EY), kinetic of cell release in a structured system, and survival throughout bead storage at 4 °C. EY was 93-96% and the survival of the encapsulated microorganisms was variable, with two isolates showing a bacterial population of 6.1-6.9 log cfu/g after 35 days under refrigerated conditions. Thereafter, the paper addressed a preliminary validation in a meat model system, containing salt, nitrites and nitrates, lactose, pepper, and then in a commercial preparation of pork meat. For the validation in pork meat, free cells were used as controls. Cell released from beads were able to achieve a significant acidification; in particular, after 7 days they showed the same results of free cells.

  17. 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.

  18. 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. PMID:26253667

  19. Perfluorinated alginate for cellular encapsulation.

    PubMed

    Gattás-Asfura, Kerim M; Fraker, Christopher A; Stabler, Cherie L

    2012-08-01

    Molecules of pentadecafluorooctanoyl chloride (PFC) were grafted onto alginate (Alg) using a linear poly(ethylene glycol) linker and amide bonds. The resulting Alg-PFC material was characterized by proton nuclear magnetic resonance and infrared spectroscopies. The degree of PFC functionalization significantly influenced the physical and chemical properties of Alg-PFC, particularly when the resulting polymer was ionically crosslinked into hydrogels. Alg-PFC hydrogel beads fabricated via Ba(2+) crosslinking were found to match the permeability properties of control alginate beads, except upon swelling over time in culture media. When used to encapsulate MIN6 cells, a beta cell line, Alg-PFC beads demonstrated enhanced cell proliferation over alginate control beads. These results indicate that Alg-PFC hydrogels retain some of the PFC's biological-relevant benefits, such as enhancement of mass transport and bioinertness, to enhance cellular viability within alginate three-dimensional hydrogel environments. We envision these functionalized hydrogels to be particularly useful in the encapsulation of cells with a high metabolic demand, such as pancreatic islets.

  20. Evidence for cysteine persulfide as reaction product of L-Cyst(e)ine C-S-lyase (C-DES) from Synechocystis. Analyses using cystine analogues and recombinant C-DES.

    PubMed

    Lang, T; Kessler, D

    1999-01-01

    The pyridoxal phosphate-dependent monomeric L-cysteine/cystine C-S-lyase (C-DES), previously isolated from Synechocystis PCC 6714 by its capacity to direct [2Fe-2S] cluster assembly of ferredoxin in vitro (Leibrecht, I., and Kessler, D. (1997) J. Biol. Chem. 272, 10442-10447), has now been cloned, sequenced, and overexpressed in Escherichia coli. The amino acid sequence of C-DES was found to be nearly identical (92% identity) to the open reading frame slr2143 of Synechocystis PCC 6803 and showed a more distant relationship to the NifS family of proteins (about 27% identity). Recombinant C-DES displayed activities equal to the isolate from Synechocystis in terms of the cyst(e)ine lyase reaction and holoferredoxin formation which recommended its use for functional and mechanistic studies. Investigation of the substrate spectrum for beta-elimination found L-cysteine to be a poor substrate (kcat approximately 0.15 s-1) in contrast to L-cystine (kcat = 36 s-1) and several related compounds. Of these compounds, desaminocystine (S-(carboxyethylthio)-L-cysteine) was used for C-DES-mediated persulfide generation. Stabilization of the linear persulfide 3-(disulfanyl)-propionic acid was achieved by cyclization as a novel intramolecular trapping reaction; this yielded 1,2-dithiolan-3-one which was isolated and identified by chemical analyses.

  1. Evidence of histidine phosphorylation in isocitrate lyase from Escherichia coli

    SciTech Connect

    Roberston, E.F.; Hoyt, J.C.; Reeves, H.C.

    1987-05-01

    Escherichia coli isocitrate lyase can be phosphorylated in vitro in an ATP-dependent reaction. Partially purified extracts were incubated with ..gamma..-/sup 32/P-ATP and analyzed by two-dimensional polyacrylamide gel electrophoresis followed by a Western blot and autoradiography. Radioactivity was associated with the lyase only when blotting was performed under alkaline conditions. This suggests that phosphate groups are attached to the lyase via an acid-labile P-N bond rather than a more stable P-O bond. Treatment of the lyase with diethyl pyrocarbonate, a histidine modifying agent, blocks incorporation of /sup 32/P-phosphate. Treatment with phosphoramidate, a histidine phosphorylating agent, alters the isoelectric point of the lyase suggesting that the enzyme can be phosphorylated at histidine residues. Loss of catalytic activity after treatment with potato acid phosphatase indicates that isocitrate lyase activity may be modulated by phosphorylation.

  2. Engineering tissues with a perfusable vessel-like network using endothelialized alginate hydrogel fiber and spheroid-enclosing microcapsules.

    PubMed

    Liu, Yang; Sakai, Shinji; Taya, Masahito

    2016-02-01

    Development of the technique for constructing an internal perfusable vascular network is a challenging issue in fabrication of dense three-dimensional tissues in vitro. Here, we report a method for realizing it. We assembled small tissue (about 200 μm in diameter)-enclosing hydrogel microcapsules and a single hydrogel fiber, both covered with human vascular endothelial cells in a collagen gel. The microcapsules and fiber were made from alginate and gelatin derivatives, and had cell adhesive surfaces. The endothelial cells on the hydrogel constructs sprouted and spontaneously formed a network connecting the hydrogel constructs with each other in the collagen gel. Perfusable vascular network-like structure formation after degrading the alginate-based hydrogel constructs by alginate lyase was confirmed by introducing solution containing tracer particles of about 3 μm in diameter into the lumen templated by the alginate hydrogel fiber. The introduced solution flowed into the spontaneously formed capillary branches and passed around the individual spherical tissues.

  3. A protective association between catalase and isocitrate lyase in peroxisomes.

    PubMed

    Yanik, Tulin; Donaldson, Robert Paul

    2005-03-15

    Glyoxysomes are specialized peroxisomes in germinating seeds, which catalyze many reactions that convert fatty acids into carbohydrates thus generating H(2)O(2). They are characterized by the presence of catalase (CAT, E.C. 1.11.1.6) in their matrix which protects cells from oxidative stress. Here, we investigated the possibility that a protein can be protected from oxidative damage by its association with CAT. We purified peroxisomal CAT from germinating castor beans by ion exchange, gel filtration, and hydroxylapatite chromatography. Gel filtration of the matrix proteins, cross-linking, and co-immunoprecipitation studies indicate that CAT associates with a glyoxysomal matrix protein, isocitrate lyase (ICL, E.C. 4.1.3.1). In addition, we found that H(2)O(2) inactivates ICL and degrades its product, glyoxylate, when CAT is inactive. ICL and its product appear to be sensitive to oxidative damage; thus, association of CAT with ICL would afford protection from H(2)O(2).

  4. Structural Basis for Alginate Secretion Across the Bacterial Outer Membrane

    SciTech Connect

    J Whitney; I Hay; C Li; P Eckford; H Robinson; M Amaya; L Wood; D Ohman; C Bear; et al.

    2011-12-31

    Pseudomonas aeruginosa is the predominant pathogen associated with chronic lung infection among cystic fibrosis patients. During colonization of the lung, P. aeruginosa converts to a mucoid phenotype characterized by the overproduction of the exopolysaccharide alginate. Secretion of newly synthesized alginate across the outer membrane is believed to occur through the outer membrane protein AlgE. Here we report the 2.3 {angstrom} crystal structure of AlgE, which reveals a monomeric 18-stranded {beta}-barrel characterized by a highly electropositive pore constriction formed by an arginine-rich conduit that likely acts as a selectivity filter for the negatively charged alginate polymer. Interestingly, the pore constriction is occluded on either side by extracellular loop L2 and an unusually long periplasmic loop, T8. In halide efflux assays, deletion of loop T8 ({Delta}T8-AlgE) resulted in a threefold increase in anion flux compared to the wild-type or {Delta}L2-AlgE supporting the idea that AlgE forms a transport pathway through the membrane and suggesting that transport is regulated by T8. This model is further supported by in vivo experiments showing that complementation of an algE deletion mutant with {Delta}T8-AlgE impairs alginate production. Taken together, these studies support a mechanism for exopolysaccharide export across the outer membrane that is distinct from the Wza-mediated translocation observed in canonical capsular polysaccharide export systems.

  5. Structural basis for alginate secretion across the bacterial outer membrane

    SciTech Connect

    Whitney, J.C.; Robinson, H.; Hay, I. D.; Li, C.; Eckford, P. D. W.; Amaya, M. F.; Wood, L. F.; Ohman, D. E.; Bear, C. E.; Rehm, B. H.; Howell, P. L.

    2011-08-09

    Pseudomonas aeruginosa is the predominant pathogen associated with chronic lung infection among cystic fibrosis patients. During colonization of the lung, P. aeruginosa converts to a mucoid phenotype characterized by the overproduction of the exopolysaccharide alginate. Secretion of newly synthesized alginate across the outer membrane is believed to occur through the outer membrane protein AlgE. Here we report the 2.3 {angstrom} crystal structure of AlgE, which reveals a monomeric 18-stranded {beta}-barrel characterized by a highly electropositive pore constriction formed by an arginine-rich conduit that likely acts as a selectivity filter for the negatively charged alginate polymer. Interestingly, the pore constriction is occluded on either side by extracellular loop L2 and an unusually long periplasmic loop, T8. In halide efflux assays, deletion of loop T8 ({Delta}T8-AlgE) resulted in a threefold increase in anion flux compared to the wild-type or {Delta}L2-AlgE supporting the idea that AlgE forms a transport pathway through the membrane and suggesting that transport is regulated by T8. This model is further supported by in vivo experiments showing that complementation of an algE deletion mutant with {Delta}T8-AlgE impairs alginate production. Taken together, these studies support a mechanism for exopolysaccharide export across the outer membrane that is distinct from the Wza-mediated translocation observed in canonical capsular polysaccharide export systems.

  6. Bioinspired preparation of alginate nanoparticles using microbubble bursting.

    PubMed

    Elsayed, Mohamed; Huang, Jie; Edirisinghe, Mohan

    2015-01-01

    Nanoparticles are considered to be one of the most advanced tools for drug delivery applications. In this research, alginate (a model hydrophilic polymer) nanoparticles 80 to 200 nm in diameter were obtained using microbubble bursting. The natural process of bubble bursting occurs through a number of stages, which consequently produce nano- and microsized droplets via two main production mechanisms, bubble shell disintegration and a jetting process. In this study, nano-sized droplets/particles were obtained by promoting the disintegrating mechanism and suppressing (limiting) the formation of larger microparticles resulting from the jetting mechanism. A T-junction microfluidic device was used to prepare alginate microbubbles with different sizes in a well-controlled manner. The size of the bubbles was varied by controlling two processing parameters, the solution flow rate and the bubbling pressure. Crucially, the bubble size was found to be the determining factor for inducing (or limiting) the bubble shell disintegration mechanism and the size needed to promote this process was influenced by the properties of the solution used for preparing the bubbles, particularly the viscosity. The size of alginate nanoparticles produced via the disintegration mechanism was found to be directly proportional to the viscosity of the alginate solution.

  7. Expression of alginases and alginate polymerase genes in response to oxygen, and their relationship with the alginate molecular weight in Azotobacter vinelandii.

    PubMed

    Flores, Celia; Moreno, Soledad; Espín, Guadalupe; Peña, Carlos; Galindo, Enrique

    2013-07-10

    The transcription of genes involved in alginate polymerization and depolymerization, as well as the alginase activity (extracellular and intracellular) under oxygen-limited and non oxygen-limited conditions in cultures of A. vinelandii, was studied. Two levels of dissolved oxygen tension (DOT) (1% and 5%, oxygen-limited and non-oxygen-limited, respectively) strictly controlled by gas blending, were evaluated in a wild type strain. In cultures at low DOT (1%), in which a high molecular weight alginate (1200 kDa) was synthesized, the transcription levels of alg8 and alg44 (genes encoding alginate polymerase complex), and algX (encoding a protein involved in polymer transport through periplasmic space) were considerably higher as compared to cultures conducted at 5% DOT, under which an alginate with a low MW (42 kDa) was produced. In the case of genes encoding for intracellular and extracellular alginases, the levels of these transcripts were higher at 1% DOT. However, intracellular and extracellular alginase activity were lower (0.017 and 0.01 U/mg protein, respectively) in cultures at 1% DOT, as compared with the activities measured at 5% DOT (0.027 and 0.052 U/mg protein for intracellular and extracellular maximum activity, respectively). The low alginase activity measured in cultures at 1% DOT and the high level of transcription of genes constituting alginate polymerase complex might be mechanisms by which oxygen regulates the production of alginates with a high MW.

  8. Cysteine-S-conjugate beta-lyase activity and pyridoxal phosphate binding site of onion alliin lyase.

    PubMed

    Kitamura, N; Shimomura, N; Iseki, J; Honma, M; Chiba, S; Tahara, S; Mizutani, J

    1997-08-01

    Purification of onion alliin lyase gave two fractions by cation exchange chromatography. Both fractions showed the comparable high catalytic activity of cysteine-S-conjugate beta-lyase with that of alliin lyase using S-(2-chloro-6-nitrophenyl)-L-cysteine and alliin, S-allyl-L-cysteine sulfoxide as substrates. All the active substrates tested with onion alliin lyase were also active to the cysteine-S-conjugate beta-lyase of Mucor javanicus, but the catalytic activity of the Mucor enzyme was lower for all the substrates. The pyridoxal phosphate binding site of the onion alliin lyase was identified as Lys 285 in the amino acid sequence deduced from cDNA which has been reported. This lysine was conserved in all the sequences from the alliin lyase cDNAs, while similarity was not found between the sequences around pyridoxal phosphate binding sites of both the onion alliin lyase and the Mucor cysteine-S-conjugate beta-lyase. PMID:9301115

  9. Effect of Alginate Concentration on Alginate-TiO2 Hydrogel for Lead Ion Removal

    NASA Astrophysics Data System (ADS)

    Teoh, W. T.; Saito, N.; Sato, K.

    2011-03-01

    Alginate-TiO2 hydrogel was investigated for lead ion (Pb(II)) removal. By immobilizing TiO2 powder onto an alginate biopolymer, it is possible to utilize the ion exchange properties of the alginate and the photoreducibility of TiO2 to recover Pb(II). However, these photocatalytic activities degrade the alginate biopolymer in addition to removing Pb(II). This study examines photolytic degradation of alginate-TiO2 hydrogels prepared with alginate concentrations of 1, 1.5, 2, and 2.5%w/v; the same amount (0.4%w/v) of TiO2 was added to each alginate solution. The alginate-TiO2 hydrogels were formed by dripping the alginate-TiO2 suspension into a 0.2 M calcium chloride solution. The samples were washed and dried and then photoirradiated. The samples with alginate concentrations of 1 and 1.5%w/v were depolymerized, whereas the surface morphology of the sample that prepared from the 2%w/v alginate solution remained unchanged. The samples prepared from 1.5, 2, and 2.5%w/v alginate solutions had Pb(II) uptakes of 24.0, 39.8, and 39.7 mg/g, respectively.

  10. 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. PMID:24998183

  11. 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

  12. Manipulating the molecular weight of alginate produced by Azotobacter vinelandii in continuous cultures.

    PubMed

    Díaz-Barrera, Alvaro; Silva, Paulina; Berrios, Julio; Acevedo, Fernando

    2010-12-01

    Alginate production by Azotobacter vinelandii in chemostat cultures was evaluated at different dilution rates (D) and inlet sucrose concentrations of 5 and 20 g l(-1). At the low inlet sucrose concentration, the molecular weight of alginate increased from 800 to 1800 kDa when D increased from 0.05 to 0.10 h(-1), whereas the opposite trend was observed with the high inlet sucrose concentration. This behaviour can be explained by changes in specific sucrose uptake rate. Thus, a decrease in alginate molecular weight was dependent on the specific sucrose uptake rate when this rate was higher than 0.42 g g(-1) h(-1). The manipulation of the D can be used to select the molecular weight of alginate in continuous culture.

  13. 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.

  14. 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.

  15. Microgravity Separation of Alginate Empty Capsules from Encapsulated Pancreatic Islets Using a Microfluidic System.

    PubMed

    Shin, Soojeong; Yoo, Young Je; Hong, Jong Wook

    2015-10-01

    Although microencapsulated pancreatic islets have merits, such as ease of transplantation, viability and functionality improvement, and immune protection in vivo, the co-production of alginate empty capsules during the encapsulation of islets with alginate makes them unusable for biomedical application. In previous research, the removal of empty alginate capsules with high yield was achieved using density-gradient centrifugation. Here, we report advanced microgravity-based separation techniques in a microfluidic format for alginate empty capsules. The optimal separation conditions were mathematically evaluated using Stokes' law and the separation of the encapsulation product was accomplished. A microfluidic chip was designed with two inlets and two outlets at different elevations to mimic the vertical percoll gradient in density-gradient centrifugation. The separation of alginate empty capsules using microgravitational force resulted in effective separation of encapsulated islets from alginate empty capsules with more than 70% efficiency. Moreover, no loss of encapsulated islets was expected because the process is a one-pot separation, unlike the previous method. This type of microgravitational particle separation could be used both for the fractionization of heterogeneous encapsulated cells and to remove empty capsules. PMID:26726432

  16. Microgravity Separation of Alginate Empty Capsules from Encapsulated Pancreatic Islets Using a Microfluidic System.

    PubMed

    Shin, Soojeong; Yoo, Young Je; Hong, Jong Wook

    2015-10-01

    Although microencapsulated pancreatic islets have merits, such as ease of transplantation, viability and functionality improvement, and immune protection in vivo, the co-production of alginate empty capsules during the encapsulation of islets with alginate makes them unusable for biomedical application. In previous research, the removal of empty alginate capsules with high yield was achieved using density-gradient centrifugation. Here, we report advanced microgravity-based separation techniques in a microfluidic format for alginate empty capsules. The optimal separation conditions were mathematically evaluated using Stokes' law and the separation of the encapsulation product was accomplished. A microfluidic chip was designed with two inlets and two outlets at different elevations to mimic the vertical percoll gradient in density-gradient centrifugation. The separation of alginate empty capsules using microgravitational force resulted in effective separation of encapsulated islets from alginate empty capsules with more than 70% efficiency. Moreover, no loss of encapsulated islets was expected because the process is a one-pot separation, unlike the previous method. This type of microgravitational particle separation could be used both for the fractionization of heterogeneous encapsulated cells and to remove empty capsules.

  17. Purification and Properties of a Glucuronan Lyase from Sinorhizobium meliloti M5N1CS (NCIMB 40472)

    PubMed Central

    Da Costa, Alexandre; Michaud, Philippe; Petit, Emmanuel; Heyraud, Alain; Colin-Morel, Philippe; Courtois, Bernard; Courtois, Josiane

    2001-01-01

    A glucuronan lyase extracted from Sinorhizobium meliloti strain M5N1CS was purified to homogeneity by anion-exchange chromatography. The purified enzyme corresponds to a monomer with a molecular mass of 20 kDa and a pI of 4.9. A specific activity was found only for polyglucuronates leading to the production of 4,5-unsaturated oligoglucuronates. The enzyme activity was optimal at pH 6.5 and 50°C. Zn2+, Cu2+, and Hg2+ (1 mM) inhibited the enzyme activity. No homology of the enzyme N-terminal amino acid sequence was found with any of the previously published protein sequences. This enzyme purified from S. meliloti strain M5N1CS corresponding to a new lyase was classified as an endopolyglucuronate lyase. PMID:11679345

  18. Drug release behavior of poly (lactic-glycolic acid) grafting from sodium alginate (ALG-g-PLGA) prepared by direct polycondensation.

    PubMed

    Shi, Gang; Ding, Yuanyuan; Zhang, Xin; Wu, Luyan; He, Fei; Ni, Caihua

    2015-01-01

    Hydrophobically modified sodium alginate, poly (lactic-glycolic acid) grafting from sodium alginate (ALG-g-PLGA), was successfully synthesized through direct one-step polymerization of sodium alginate, glycolic acid, and lactic acid. ALG-g-PLGA self-assembled to colloidal nanoparticles and subsequently hydrogel microspheres were obtained by crosslinking ALG-g-PLGA nanoparticles in the solution of calcium chloride. The modified hydrogel microspheres could be used as the drug delivery vehicles for a hydrophobic ibuprofen. Compared with sodium alginate, ALG-g-PLGA demonstrated an improved drug loading rate, encapsulation efficiency, and prolonged release speed. The products, as novel and highly promising biomaterials, have potential applications.

  19. The effect of alginate impressions on the surface of cast gypsum. II. The role of sodium sulphate in incompatibility.

    PubMed

    Jarvis, R G; Earnshaw, R

    1981-02-01

    Of the materials tested only two alginates, Palginex (D), and Echo (C), show good surface reproduction when Vel-Mix (1), Ainsworth Greenstone (4), and Investo Greenstone (5) are cast against them. The above two alginates were shown to have a potassium and sulphate rich exudate. The reasonable surface produced by CA-37 (A) can be attributed to a proportional decrease in the ratio of potassium to sulphate present in the exudate, compared with Echo (C) and Palginex (D). S.S. White (B), Kromopan (E), and Tissutex (G) produce poor cast surfaces when high strength gypsum products are set against them. The exudates from these alginates were found to consist almost entirely of sodium sulphate. This study suggests that one way to improve the surface compatibility of alginates with gypsum products would be to modify the chemistry of the impression material so that a reactor other than calcium sulphate is used, to eliminate the appearance of sulphate ions in the exudate. Moreover, sources of sodium ions should also be eliminated. Therefore soluble alginates other than sodium alginate should be preferred, and a retarder other than one or other of the forms of sodium phosphate should be used. These changes would ensure that sulphate, particularly sodium sulphate, would not be produced as a by-product of the setting reaction. This in turn would eliminate the effects of incompatibility at present encountered to a greater or lesser extent with most combinations of alginate impression materials and gypsum cast materials. PMID:7018476

  20. Mechanical properties of C-5 epimerized alginates.

    PubMed

    Mørch, Y A; Holtan, S; Donati, I; Strand, B L; Skjåk-Braek, G

    2008-09-01

    There is an increased need for alginate materials with both enhanced and controllable mechanical properties in the fields of food, pharmaceutical and specialty applications. In the present work, well-characterized algal polymers and mannuronan were enzymatically modified using C-5 epimerases converting mannuronic acid residues to guluronic acid in the polymer chain. Composition and sequential structure of controls and epimerized alginates were analyzed by (1)H NMR spectroscopy. Mechanical properties of Ca-alginate gels were further examined giving Young's modulus, syneresis, rupture strength, and elasticity of the gels. Both mechanical strength and elasticity of hydrogels could be improved and manipulated by epimerization. In particular, alternating sequences were found to play an important role for the final mechanical properties of alginate gels, and interestingly, a pure polyalternating sample resulted in gels with extremely high syneresis and rupture strength. In conclusion, enzymatic modification was shown to be a valuable tool in modifying the mechanical properties of alginates in a highly specific manner.

  1. In vitro fermentation of alginate and its derivatives by human gut microbiota.

    PubMed

    Li, Miaomiao; Li, Guangsheng; Shang, Qingsen; Chen, Xiuxia; Liu, Wei; Pi, Xiong'e; Zhu, Liying; Yin, Yeshi; Yu, Guangli; Wang, Xin

    2016-06-01

    Alginate (Alg) has a long history as a food ingredient in East Asia. However, the human gut microbes responsible for the degradation of alginate and its derivatives have not been fully understood yet. Here, we report that alginate and the low molecular polymer derivatives of mannuronic acid oligosaccharides (MO) and guluronic acid oligosaccharides (GO) can be completely degraded and utilized at various rates by fecal microbiota obtained from six Chinese individuals. However, the derivative of propylene glycol alginate sodium sulfate (PSS) was not hydrolyzed. The bacteria having a pronounced ability to degrade Alg, MO and GO were isolated from human fecal samples and were identified as Bacteroides ovatus, Bacteroides xylanisolvens, and Bacteroides thetaiotaomicron. Alg, MO and GO can increase the production level of short chain fatty acids (SCFA), but GO generates the highest level of SCFA. Our data suggest that alginate and its derivatives could be degraded by specific bacteria in the human gut, providing the basis for the impacts of alginate and its derivates as special food additives on human health. PMID:26891629

  2. One-step synthesis of platinum nanoparticles loaded in alginate bubbles.

    PubMed

    Yang, Chih-Hui; Wang, Wei-Ting; Grumezescu, Alexandru Mihai; Huang, Keng-Shiang; Lin, Yung-Sheng

    2014-01-01

    Composite particles with multifunctions have been extensively utilized for various applications. Bubble particles can be applied for ultrasound-mediated imaging, drug delivery, absorbers, cell culture, etc. This study proposes a one-step strategy to obtain Pt nanoparticles loaded in alginate bubbles. A needle-based droplet formation was used to generate uniform alginate particles about 2 mm in diameter. The hydrolysis reaction of NaBH4 was utilized to produce gaseous hydrogen and then trapped within alginate particles to form bubbles. The Pt(4+) mixed with alginate solution was dropped into the reservoir to react with reducing NaBH4 and hardening CaCl2 to form Pt nanoparticles-alginate composite bubbles. Results indicate that the size of bubbles decreases with the CaCl2 concentration (1% ~ 20%), and size of bubbles increases with the NaBH4 concentration (1 ~ 20 mM). The advantages for the present approach include low cost, easy operation, and effective production of Pt nanoparticles-alginate composite bubbles.

  3. In vitro fermentation of alginate and its derivatives by human gut microbiota.

    PubMed

    Li, Miaomiao; Li, Guangsheng; Shang, Qingsen; Chen, Xiuxia; Liu, Wei; Pi, Xiong'e; Zhu, Liying; Yin, Yeshi; Yu, Guangli; Wang, Xin

    2016-06-01

    Alginate (Alg) has a long history as a food ingredient in East Asia. However, the human gut microbes responsible for the degradation of alginate and its derivatives have not been fully understood yet. Here, we report that alginate and the low molecular polymer derivatives of mannuronic acid oligosaccharides (MO) and guluronic acid oligosaccharides (GO) can be completely degraded and utilized at various rates by fecal microbiota obtained from six Chinese individuals. However, the derivative of propylene glycol alginate sodium sulfate (PSS) was not hydrolyzed. The bacteria having a pronounced ability to degrade Alg, MO and GO were isolated from human fecal samples and were identified as Bacteroides ovatus, Bacteroides xylanisolvens, and Bacteroides thetaiotaomicron. Alg, MO and GO can increase the production level of short chain fatty acids (SCFA), but GO generates the highest level of SCFA. Our data suggest that alginate and its derivatives could be degraded by specific bacteria in the human gut, providing the basis for the impacts of alginate and its derivates as special food additives on human health.

  4. Proinflammatory activity of an alginate isolated from Sargassum vulgare.

    PubMed

    Lins, Kézia O A L; Vale, Mariana L; Ribeiro, Ronaldo A; Costa-Lotufo, Letícia V

    2013-01-30

    Alginates are unbranched polymers of polysaccharide presented as the structural components of marine brown algae. The proinflammatory activity of SVHV, an alginate isolated from Sargassum vulgare, was investigated using models of paw edema, mast cells degranulation and neutrophil migration in vivo. SVHV induced a dose dependent paw edema, with a peak at 2 h, associated with an increased myeloperoxidase activity and production of TNF-α and IL-1β. Pharmacological modulators, remarkably dexamethasone and indomethacin, inhibited the edema. SVHV (1.0 mg) also led to a significant induction of neutrophil migration in the peritoneal cavity of rats. This neutrophil migration was significantly reduced by peritoneal resident macrophages depletion, but was not affected by the depletion of mast cells. Our data suggest that SVHV has proinflammatory activity dependent of the activation of resident cells, being the macrophages the main cells involved. PMID:23218314

  5. Differential depolymerization mechanisms of pectate lyases secreted by Erwinia chrysanthemi EC16.

    PubMed Central

    Preston, J F; Rice, J D; Ingram, L O; Keen, N T

    1992-01-01

    The four pectate lyases (EC 4.2.2.2) secreted by Erwinia chrysanthemi EC16 have been individually produced as recombinant enzymes in Escherichia coli. Oligogalacturonates formed from polygalacturonic acid during reactions catalyzed by each enzyme have been determined by high-performance liquid chromatography analysis. PLa catalyzes the formation of a series of oligomers ranging from dimer to dodecamer through a random endolytic depolarization mechanism. PLb and PLc are trimer- and tetramer-generating enzymes with an identical combination of endolytic and exolytic mechanisms. PLe catalyzes a nonrandom endolytic depolymerization with the formation of dimer as the predominant product. The pectate lyases secreted by E. chrysanthemi EC16 represent a battery of enzymes with three distinct approaches to the depolymerization of plant cell walls. PMID:1548242

  6. Induction of L-phenylalanine ammonia-lyase during utilization of phenylalanine as a carbon or nitrogen source in Rhodotorula glutinis

    SciTech Connect

    Marusich, W.C.; Jensen, R.A.; Zamir, L.O.

    1981-06-01

    Rhodotorula glutinis is a convenient source of L-phenylalanine ammonia-lyase, an enzyme that is useful as a biochemical reagent in the assay of L-phenylalanine. There have been previous descriptions of induced lyase production in complex medium where induction occurs late in exponential growth, suggesting a role in secondary metabolism such as is the case in higher plants. A higher specific activity of L-phenylalanine ammonia-lyase (sixfold higher than in complex medium) can be obtained during midexponential growth in a defined medium containing L-phenylalanine as the sole source of carbon. L-phenylalanine will also induce lyase synthesis during exponential growth in minimal medium in which L-phenylalanine is the sole source of nitrogen. The appearance of lyase in complex medium supplemented with L-phenylalanine is probably triggered fortuitously by exhaustion late in growth of a prime source of nitrogen. In this study, R. glutinis appeared to express a single lyase enzyme, regardless of whether induction was nitrogen signaled or carbon signaled. Thin-layer chromatographic analysis of ether extracts prepared fom cultures induced with doubly labeled (U-/sup 14/C; ring-4-/sup 3/H) L-phenylalanine provided evidence of a catabolic sequence containing cinnamic acid, benzoic acid, and 4-hydroxybenzoic acid as degradative intermediates. 3,4-Dihydroxybenzoic acid was not identified as a catabolic intermediate.

  7. Pectate lyase PelI of Erwinia chrysanthemi 3937 belongs to a new family.

    PubMed

    Shevchik, V E; Robert-Baudouy, J; Hugouvieux-Cotte-Pattat, N

    1997-12-01

    Erwinia chrysanthemi 3937 secretes five major isoenzymes of pectate lyases encoded by the pel4, pelB, pelC, pelD, and pelE genes and a set of secondary pectate lyases, two of which, pelL and pelZ, have been already identified. We cloned the pelI gene, encoding a ninth pectate lyase of E. chrysanthemi 3937. The pelI reading frame is 1,035 bases long, corresponding to a protein of 344 amino acids including a typical amino-terminal signal sequence of 19 amino acids. The purified mature PelI protein has an isoelectric point of about 9 and an apparent molecular mass of 34 kDa. PelI has a preference for partially methyl esterified pectin and presents an endo-cleaving activity with an alkaline pH optimum and an absolute requirement for Ca2+ ions. PelI is an extracellular protein secreted by the Out secretory pathway of E. chrysanthemi. The PelI protein is very active in the maceration of plant tissues. A pelI mutant displayed reduced pathogenicity on chicory leaves, but its virulence did not appear to be affected on potato tubers or Saintpaulia ionantha plants. The pelI gene constitutes an independent transcriptional unit. As shown for the other pel genes, the transcription of pelI is dependent on various environmental conditions. It is induced by pectic catabolic products and affected by growth phase, oxygen limitation, temperature, nitrogen starvation, and catabolite repression. Regulation of pelI expression appeared to be dependent on the three repressors of pectinase synthesis, KdgR, PecS, and PecT, and on the global activator of sugar catabolism, cyclic AMP receptor protein. A functional KdgR binding site was identified close to the putative pelI promoter. Analysis of the amino acid sequence of PelI revealed high homology with a pectate lyase from Erwinia carotovora subsp. carotovora (65% identity) and low homology with pectate lyases of the phytopathogenic fungus Nectria haematococca (Fusarium solani). This finding indicates that PelI belongs to pectate lyase class

  8. Exploration of the action pattern of Streptomyces hyaluronate lyase using high-resolution capillary electrophoresis.

    PubMed

    Park, Y; Cho, S; Linhardt, R J

    1997-02-01

    Hyaluronic acid was treated exhaustively with a hyaluronate lyase (hyaluronidase, EC 4.2.2.1) from Streptomyces hyalurolyticus to obtain a tetrasaccharide and a hexasaccharide product in a molar ratio of 1 to 1.2. The tetrasaccharide product was fluorescently labeled at the reducing end by reductive amination with 7-amino 1,3-naphthalene disulfonic acid (AGA) and the structure of the conjugate was determined spectroscopically. Partial treatments of hyaluronic acid with hyaluronate lyase afforded complex mixtures of oligosaccharides that were similarly fluorescently labeled. These labeled oligosaccharide mixtures were analyzed using high-resolution capillary electrophoresis. The resulting electropherograms showed the content of each hyaluronic acid derived oligosaccharide, having a degree of polymerization (dp) from 4 to 50, throughout the enzymatic reaction. Computer simulation studies gave comparable kinetic profiles suggesting that hyaluronate lyase exhibits a random endolytic action pattern. Interestingly, oligosaccharides of certain size (dp) were under-represented in these oligosaccharide mixtures suggesting that linkages at spacings of 10 to 12 saccharide units are somewhat resistant to this enzyme. The cause of this resistance might be the result of secondary or higher order structural features present in the hyaluronic acid polymer.

  9. 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

  10. Purification and Characterization of a Unique Pectin Lyase from Aspergillus giganteus Able to Release Unsaturated Monogalacturonate during Pectin Degradation.

    PubMed

    Pedrolli, Danielle Biscaro; 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 Pb(2+) and was not significantly affected by Hg(2+). Like other pectin lyases, PLIII is stimulated by but is not dependent on Ca(2+). 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.

  11. Purification and Characterization of a Unique Pectin Lyase from Aspergillus giganteus Able to Release Unsaturated Monogalacturonate during Pectin Degradation.

    PubMed

    Pedrolli, Danielle Biscaro; 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 Pb(2+) and was not significantly affected by Hg(2+). Like other pectin lyases, PLIII is stimulated by but is not dependent on Ca(2+). 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

  12. Mycoparasitism studies of Trichoderma species against three phytopathogenic fungi: evaluation of antagonism and hydrolytic enzyme production.

    PubMed

    Qualhato, Thiago Fernandes; Lopes, Fabyano Alvares Cardoso; Steindorff, Andrei Stecca; Brandão, Renata Silva; Jesuino, Rosália Santos Amorim; Ulhoa, Cirano José

    2013-09-01

    Trichoderma spp. are used for biocontrol of several plant pathogens. However, their efficient interaction with the host needs to be accompanied by production of secondary metabolites and cell wall-degrading enzymes. Three parameters were evaluated after interaction between four Trichoderma species and plant-pathogenic fungi: Fusarium solani, Rhizoctonia solani and Sclerotinia sclerotiorum. Trichoderma harzianum and T. asperellum were the most effective antagonists against the pathogens. Most of the Trichoderma species produced toxic volatile metabolites, having significant effects on growth and development of the plant pathogens. When these species were grown in liquid cultures with cell walls from these plant pathogens, they produced and secreted β-1,3-glucanase, NAGAse, chitinase, acid phosphatase, acid proteases and alginate lyase.

  13. Fatty acid hydroperoxide lyase is a heme protein.

    PubMed

    Shibata, Y; Matsui, K; Kajiwara, T; Hatanaka, A

    1995-02-01

    Fatty acid hydroperoxide lyase (HPO lyase) is an enzyme that cleaves hydroperoxides of polyunsaturated fatty acids to form short chain aldehydes and omega-oxoacids. Spectrophotometric analyses of HPO lyase highly purified from green bell pepper fruits indicate that it is a heme protein. The heme species was revealed to be heme b (protoheme IX) from the absorption spectrum of the pyridine hemochromogen. Although the spectrum highly resembles that of a plant cytochrome P450, allene oxide synthase from flaxseed, CO treatment of the enzyme caused no appearance of a peak at 450 nm, which is an essential diagnostic feature of a cytochrome P450. Internal amino acid sequences determined with peptide fragments obtained from the lyase showed no homology with any reported sequences.

  14. Purification and characterization of Acinetobacter calcoaceticus isocitrate lyase.

    PubMed Central

    Hoyt, J C; Johnson, K E; Reeves, H C

    1991-01-01

    Acinetobacter calcoaceticus is capable of growing on acetate or compounds that are metabolized to acetate. During adaptation to growth on acetate, A. calcoaceticus B4 exhibits an increase in NADP(+)-isocitrate dehydrogenase and isocitrate lyase activities. In contrast, during adaptation to growth on acetate, Escherichia coli exhibits a decrease in NADP(+)-isocitrate dehydrogenase activity that is caused by reversible phosphorylation of specific serine residues on this enzyme. Also, in E. coli, isocitrate lyase is believed to be active only in the phosphorylated form. This phosphorylation of isocitrate lyase may regulate entry of isocitrate into the glyoxylate bypass. To understand the relationships between these two isocitrate-metabolizing enzymes and the metabolism of acetate in A. calcoaceticus B4 better, we have purified isocitrate lyase to homogeneity. Physical and kinetic characterization of the enzyme as well as the inhibitor specificity and divalent cation requirement have been examined. Images FIG. 1 PMID:1938889

  15. 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.

  16. Improvement of enantioselectivity of the B-type halohydrin hydrogen-halide-lyase from Corynebacterium sp. N-1074.

    PubMed

    Watanabe, Fumiaki; Yu, Fujio; Ohtaki, Akashi; Yamanaka, Yasuaki; Noguchi, Keiichi; Odaka, Masafumi; Yohda, Masafumi

    2016-09-01

    Halohydrin hydrogen-halide-lyase (H-Lyase) is a bacterial enzyme involved in the degradation of halohydrins. This enzyme catalyzes the intramolecular nucleophilic displacement of a halogen by a vicinal hydroxyl group in halohydrins, producing the corresponding epoxides. The H-Lyases have been classified into A, B and C subtypes based on amino acid sequence similarities. These enzymes have attracted much attention as industrial catalysts in the synthesis of chiral chemicals from prochiral halohydrins. In the present study, we constructed mutants of B-type H-Lyase from Corynebacterium sp. N-1074 (HheB) displaying higher enantioselectivity by structure-based site-directed mutagenesis and random mutagenesis. A triple mutant of HheB exhibited 98.5% enantioselectivity, the highest ever reported, toward (R)-4-chloro-3-hydroxy-butyronitrile production, with the yield reaching approximately two-fold that of the wild-type enzyme. We discuss the structural basis of the high enantioselectivity and productivity of the mutant by comparing the crystal structures of the mutant HheB and the wild-type enzyme in complex with or without the substrate analogue.

  17. Improvement of enantioselectivity of the B-type halohydrin hydrogen-halide-lyase from Corynebacterium sp. N-1074.

    PubMed

    Watanabe, Fumiaki; Yu, Fujio; Ohtaki, Akashi; Yamanaka, Yasuaki; Noguchi, Keiichi; Odaka, Masafumi; Yohda, Masafumi

    2016-09-01

    Halohydrin hydrogen-halide-lyase (H-Lyase) is a bacterial enzyme involved in the degradation of halohydrins. This enzyme catalyzes the intramolecular nucleophilic displacement of a halogen by a vicinal hydroxyl group in halohydrins, producing the corresponding epoxides. The H-Lyases have been classified into A, B and C subtypes based on amino acid sequence similarities. These enzymes have attracted much attention as industrial catalysts in the synthesis of chiral chemicals from prochiral halohydrins. In the present study, we constructed mutants of B-type H-Lyase from Corynebacterium sp. N-1074 (HheB) displaying higher enantioselectivity by structure-based site-directed mutagenesis and random mutagenesis. A triple mutant of HheB exhibited 98.5% enantioselectivity, the highest ever reported, toward (R)-4-chloro-3-hydroxy-butyronitrile production, with the yield reaching approximately two-fold that of the wild-type enzyme. We discuss the structural basis of the high enantioselectivity and productivity of the mutant by comparing the crystal structures of the mutant HheB and the wild-type enzyme in complex with or without the substrate analogue. PMID:27215832

  18. Improvement of aromatic thiol release through the selection of yeasts with increased β-lyase activity.

    PubMed

    Belda, Ignacio; Ruiz, Javier; Navascués, Eva; Marquina, Domingo; Santos, Antonio

    2016-05-16

    The development of a selective medium for the rapid differentiation of yeast species with increased aromatic thiol release activity has been achieved. The selective medium was based on the addition of S-methyl-l-cysteine (SMC) as β-lyase substrate. In this study, a panel of 245 strains of Saccharomyces cerevisiae strains was tested for their ability to grow on YCB-SMC medium. Yeast strains with an increased β-lyase activity grew rapidly because of their ability to release ammonium from SMC in comparison to others, and allowed for the easy isolation and differentiation of yeasts with promising properties in oenology, or another field, for aromatic thiol release. The selective medium was also helpful for the discrimination between those S. cerevisiae strains, which present a common 38-bp deletion in the IRC7 sequence (present in around 88% of the wild strains tested and are likely to be less functional for 4-mercapto-4-methylpentan-2-one (4MMP) production), and those S. cerevisiae strains homozygous for the full-length IRC7 allele. The medium was also helpful for the selection of non-Saccharomyces yeasts with increased β-lyase activity. Based on the same medium, a highly sensitive, reproducible and non-expensive GC-MS method for the evaluation of the potential volatile thiol release by different yeast isolates was developed. PMID:26971012

  19. A facile stable-isotope dilution method for determination of sphingosine phosphate lyase activity.

    PubMed

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

    2016-01-01

    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 50fmol, 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.

  20. Phosphoserine Lyase Deoxyribozymes: DNA-Catalyzed Formation of Dehydroalanine Residues in Peptides

    PubMed Central

    Chandrasekar, Jagadeeswaran; Wylder, Adam C.; Silverman, Scott K.

    2015-01-01

    Dehydroalanine (Dha) is a nonproteinogenic electrophilic amino acid that is a synthetic intermediate or product in the biosynthesis of several bioactive cyclic peptides such as lantibiotics, thiopeptides, and microcystins. Dha also enables labeling of proteins and synthesis of post-translationally modified proteins and their analogues. However, current chemical approaches to introducing Dha into peptides have substantial limitations. Using in vitro selection, here we show that DNA can catalyze Zn2+ or Zn2+/Mn2+-dependent formation of Dha from phosphoserine (pSer), i.e., exhibit pSer lyase activity, a fundamentally new DNA-catalyzed reaction. Two new pSer lyase deoxyribozymes, named Dha-forming deoxyribozymes 1 and 2 (DhaDz1 and DhaDz2), each function with multiple turnover on the model hexapeptide substrate that was used during selection. Using DhaDz1, we generated Dha from pSer within an unrelated linear 13-mer peptide. Subsequent base-promoted intramolecular cyclization of homocysteine into Dha formed a stable cystathionine (thioether) analogue of the complement inhibitor compstatin. These findings establish the fundamental catalytic ability of DNA to eliminate phosphate from pSer to form Dha and suggest that with further development, pSer lyase deoxyribozymes will have broad practical utility for site-specific enzymatic synthesis of Dha from pSer in peptide substrates. PMID:26200899

  1. Improvement of aromatic thiol release through the selection of yeasts with increased β-lyase activity.

    PubMed

    Belda, Ignacio; Ruiz, Javier; Navascués, Eva; Marquina, Domingo; Santos, Antonio

    2016-05-16

    The development of a selective medium for the rapid differentiation of yeast species with increased aromatic thiol release activity has been achieved. The selective medium was based on the addition of S-methyl-l-cysteine (SMC) as β-lyase substrate. In this study, a panel of 245 strains of Saccharomyces cerevisiae strains was tested for their ability to grow on YCB-SMC medium. Yeast strains with an increased β-lyase activity grew rapidly because of their ability to release ammonium from SMC in comparison to others, and allowed for the easy isolation and differentiation of yeasts with promising properties in oenology, or another field, for aromatic thiol release. The selective medium was also helpful for the discrimination between those S. cerevisiae strains, which present a common 38-bp deletion in the IRC7 sequence (present in around 88% of the wild strains tested and are likely to be less functional for 4-mercapto-4-methylpentan-2-one (4MMP) production), and those S. cerevisiae strains homozygous for the full-length IRC7 allele. The medium was also helpful for the selection of non-Saccharomyces yeasts with increased β-lyase activity. Based on the same medium, a highly sensitive, reproducible and non-expensive GC-MS method for the evaluation of the potential volatile thiol release by different yeast isolates was developed.

  2. Phosphoserine Lyase Deoxyribozymes: DNA-Catalyzed Formation of Dehydroalanine Residues in Peptides.

    PubMed

    Chandrasekar, Jagadeeswaran; Wylder, Adam C; Silverman, Scott K

    2015-08-01

    Dehydroalanine (Dha) is a nonproteinogenic electrophilic amino acid that is a synthetic intermediate or product in the biosynthesis of several bioactive cyclic peptides such as lantibiotics, thiopeptides, and microcystins. Dha also enables labeling of proteins and synthesis of post-translationally modified proteins and their analogues. However, current chemical approaches to introducing Dha into peptides have substantial limitations. Using in vitro selection, here we show that DNA can catalyze Zn(2+) or Zn(2+)/Mn(2+)-dependent formation of Dha from phosphoserine (pSer), i.e., exhibit pSer lyase activity, a fundamentally new DNA-catalyzed reaction. Two new pSer lyase deoxyribozymes, named Dha-forming deoxyribozymes 1 and 2 (DhaDz1 and DhaDz2), each function with multiple turnover on the model hexapeptide substrate that was used during selection. Using DhaDz1, we generated Dha from pSer within an unrelated linear 13-mer peptide. Subsequent base-promoted intramolecular cyclization of homocysteine into Dha formed a stable cystathionine (thioether) analogue of the complement inhibitor compstatin. These findings establish the fundamental catalytic ability of DNA to eliminate phosphate from pSer to form Dha and suggest that with further development, pSer lyase deoxyribozymes will have broad practical utility for site-specific enzymatic synthesis of Dha from pSer in peptide substrates.

  3. Mechanism of benzaldehyde lyase studied via thiamin diphosphate-bound intermediates and kinetic isotope effects.

    PubMed

    Chakraborty, Sumit; Nemeria, Natalia; Yep, Alejandra; McLeish, Michael J; Kenyon, George L; Jordan, Frank

    2008-03-25

    Direct spectroscopic observation of thiamin diphosphate-bound intermediates was achieved on the enzyme benzaldehyde lyase, which carries out reversible and highly enantiospecific conversion of ( R)-benzoin to benzaldehyde. The key enamine intermediate could be observed at lambda max 393 nm in the benzoin breakdown direction and in the decarboxylase reaction starting with benzoylformate. With benzaldehyde as substrate, no intermediates could be detected, only formation of benzoin at 314 nm. To probe the rate-limiting step in the direction of ( R)-benzoin synthesis, the (1)H/ (2)H kinetic isotope effect was determined for benzaldehyde labeled at the aldehyde position and found to be small (1.14 +/- 0.03), indicating that ionization of the C2alphaH from C2alpha-hydroxybenzylthiamin diphosphate is not rate limiting. Use of the alternate substrates benzoylformic and phenylpyruvic acids (motivated by the observation that while a carboligase, benzaldehyde lyase could also catalyze the slow decarboxylation of 2-oxo acids) enabled the observation of the substrate-thiamin covalent intermediate via the 1',4'-iminopyrimidine tautomer, characteristic of all intermediates with a tetrahedral C2 substituent on ThDP. The reaction of benzaldehyde lyase with the chromophoric substrate analogue ( E)-2-oxo-4(pyridin-3-yl)-3-butenoic acid and its decarboxylated product ( E)-3-(pyridine-3-yl)acrylaldehyde enabled the detection of covalent adducts with both. Neither adduct underwent further reaction. An important finding of the studies is that all thiamin-related intermediates are in a chiral environment on benzaldehyde lyase as reflected by their circular dichroism signatures.

  4. Differential gene regulation of steroidogenic transcripts and estradiol production following in vitro pig embryo elongation in alginate hydrogel three-dimensional matrix

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Between d 10 and 12 of gestation, the pig embryo elongates from a sphere to a long thin, filament. During this time, the embryo increases the production of estrogen via an increase in steroidogenic transcripts, which is critical for maternal recognition of pregnancy. To date, attempts to elongate po...

  5. Stability of alginate-immobilized algal cells

    SciTech Connect

    Dainty, A.L.; Goulding, K.H.; Robinson, P.K.; Simpkins, I; Trevan, M.D.

    1986-01-01

    Investigations were carried out using immobilized Chlorella cells to determine the diameter, compressibility, tolerance to phosphate chelation, and ability to retain algal cells during incubation of various alginate beads. These physical bead-characteristics were affected by a variety of interactive factors, including multivalent cation type (hardening agent) and cell, cation, and alginate concentration, the latter exhibiting a predominant influence. The susceptibility of alginate beads to phosphate chelation involved a complex interaction of cation type, concentration, and pH of phosphate solution. A scale of response ranging from gel swelling to gel shrinking was observed for a range of conditions. However, stable Ca alginate beads were maintained in incubation media with a pH of 5.5 and a phosphate concentration of 5 micro M. A preliminary investigation into cell leakage from the beads illustrated the importance of maintaining a stable gel structure and limiting cell growth to reduce leakage.

  6. Alginate dressing as a donor site haemostat.

    PubMed Central

    Groves, A. R.; Lawrence, J. C.

    1986-01-01

    An alginate fibre dressing has been used to reduce blood loss from skin graft donor sites. Significant haemostasis has been achieved in the immediate post surgery phase and no adverse reactions observed. Images Fig. 1 PMID:3511833

  7. 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. PMID:25062547

  8. Kynurenine aminotransferase III and glutamine transaminase L are identical enzymes that have cysteine S-conjugate β-lyase activity and can transaminate L-selenomethionine.

    PubMed

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

    2014-11-01

    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

  9. 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

  10. Kynurenine aminotransferase III and glutamine transaminase L are identical enzymes that have cysteine S-conjugate β-lyase activity and can transaminate L-selenomethionine.

    PubMed

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

    2014-11-01

    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.

  11. Freeze-thaw induced gelation of alginates.

    PubMed

    Zhao, Ying; Shen, Wei; Chen, Zhigang; Wu, Tao

    2016-09-01

    Adding divalent ions or lowering pH below the pKa values of alginate monomers are common ways in preparing alginate gels. Herein a new way of preparing alginate gels using freeze-thaw technique is described. Solvent crystallization during freezing drove the polymers to associate into certain structures that became the junction zones of hydrogels after thawing. It enabled the preparation of alginate gels at pH 4.0 and 3.5, two pH at which the gel could not be formed previously. At pH 3.0 where alginate gel could be formed initially, applying freeze-thaw treatment increased the gel storage modulus almost 100 times. The formation of hydrogels and the resulting gel properties, such as dynamic moduli and gel syneresis were influenced by the pH values, number of freeze-thaw cycles, alginate concentrations, and ionic strengths. The obtained hydrogels were soft and demonstrated a melting behavior upon storage, which may find novel applications in the biomedical industry.

  12. Stability of alginate microbead properties in vitro

    PubMed Central

    Moya, Monica L.; Morley, Michael; Khanna, Omaditya; Opara, Emmanuel C.

    2013-01-01

    Alginate microbeads have been investigated clinically for a number of therapeutic interventions, including drug delivery for treatment of ischemic tissues, cell delivery for tissue regeneration, and islet encapsulation as a therapy for type I diabetes. The physical properties of the microbeads play an important role in regulating cell behavior, protein release, and biological response following implantation. In this research alginate microbeads were synthesized, varying composition (mannuronic acid to guluronic acid ratio), concentration of alginate and needle gauge size. Following synthesis, the size, volume fraction, and morphometry of the beads were quantified. In addition, these properties were monitored over time in vitro in the presence of varying calcium levels in the microenvironment. The initial volume available for solute diffusion increased with alginate concentration and mannuronic (M) acid content, and bead diameter decreased with M content but increased with needle diameter. Interestingly, microbeads eroded completely in saline in less than 3 weeks regardless of synthesis conditions much faster than what has been observed in vivo. However, microbead stability was increased by the addition of calcium in the culture medium. Beads synthesized with low alginate concentration and high G content exhibited a more rapid change in physical properties even in the presence of calcium. These data suggest that temporal variations in the physical characteristics of alginate microbeads can occur in vitro depending on synthesis conditions and microbead environment. The results presented here will assist in optimizing the design of the materials for clinical application in drug delivery and cell therapy. PMID:22350778

  13. 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.

  14. 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 %.

  15. Adsorption of ochratoxin A from grape juice by yeast cells immobilised in calcium alginate beads.

    PubMed

    Farbo, Maria Grazia; Urgeghe, Pietro Paolo; Fiori, Stefano; Marceddu, Salvatore; Jaoua, Samir; Migheli, Quirico

    2016-01-18

    Grape juice can be easily contaminated with ochratoxin A (OTA), one of the known mycotoxins with the greatest public health significance. Among the different approaches to decontaminate juice from this mycotoxin, microbiological methods proved efficient, inexpensive and safe, particularly the use of yeast or yeast products. To ascertain whether immobilisation of the yeast biomass would lead to successful decontamination, alginate beads encapsulating Candida intermedia yeast cells were used in our experiments to evaluate their OTA-biosorption efficacy. Magnetic calcium alginate beads were also prepared by adding magnetite in the formulation to allow fast removal from the aqueous solution with a magnet. Calcium alginate beads were added to commercial grape juice spiked with 20 μg/kg OTA and after 48 h of incubation a significant reduction (>80%), of the total OTA content was achieved, while in the subsequent phases (72-120 h) OTA was slowly released into the grape juice by alginate beads. Biosorption properties of alginate-yeast beads were tested in a prototype bioreactor consisting in a glass chromatography column packed with beads, where juice amended with OTA was slowly flowed downstream. The adoption of an interconnected scaled-up bioreactor as an efficient and safe tool to remove traces of OTA from liquid matrices is discussed.

  16. Effects of Lactobacillus plantarum immobilization in alginate coated with chitosan and gelatin on antibacterial activity.

    PubMed

    Trabelsi, Imen; Ayadi, Dorra; Bejar, Wacim; Bejar, Samir; Chouayekh, Hichem; Ben Salah, Riadh

    2014-03-01

    The present study aimed to investigate and evaluate the efficiency of immobilizing the Lactobacillus plantarum TN9 strain in alginate using chitosan and gelatin as coating materials, in terms of viability and antibacterial activity. The results indicate that maximum concentrations of L. plantarum TN9 strain were produced with 2% sodium alginate, 10(8)UFC/ml, and 1M calcium chloride. The viability and antibacterial activity of the L. plantarum TN9 cultures before and after immobilization in alginate, chitosan-coated alginate, and gelatin-coated alginate, were studied. The findings revealed that the viability of encapsulated L. plantarum could be preserved more than 5.8 log CFU/ml after 35 day of incubation at 4 °C, and no effects were observed when gelatin was used. The antibacterial activity of encapsulated L. plantarum TN9 against Gram-positive and Gram-negative pathogenic bacteria was enhanced in the presence of chitosan coating materials, and no activity was observed in the presence of gelatin. The effects of catalase and proteolytic enzymes on the culture supernatant of L. plantarum TN9 were also investigated, and the results suggested that the antibacterial activity observed was due to the production of organic acids. Taken together, the findings indicated that immobilization in chitosan enhanced the antibacterial activity of L. plantarum TN9 against several pathogenic bacteria. This encapsulated strain could be considered as a potential strong candidate for future application as an additive in the food and animal feed industries.

  17. A novel direct homogeneous assay for ATP citrate lyase.

    PubMed

    Ma, Zhengping; Chu, Ching-Hsuen; Cheng, Dong

    2009-10-01

    ATP citrate lyase (ACL) is a cytosolic enzyme that catalyzes the synthesis of acetyl-CoA and oxaloacetate using citrate, CoA, and ATP as substrates and Mg(2+) as a necessary cofactor. The ACL-dependent synthesis of acetyl-CoA is thought to be an essential step for the de novo synthesis of fatty acids and cholesterol. For this reason, inhibition of ACL has been pursued as a strategy to treat dyslipidemia and obesity. Traditionally, ACL enzyme activity is measured indirectly by coupling to enzymes such as malate dehydrogenase or chloramphenicol acetyl transferase. In this report, however, we describe a novel procedure to directly measure ACL enzyme activity. We first identified a convenient method to specifically detect [(14)C]acetyl-CoA without detecting [(14)C]citrate by MicroScint-O. Using this detection system, we devised a simple, direct, and homogeneous ACL assay in 384-well plate format that is suitable for high-throughput screening. The current assay consists of 1) incubation of ACL enzyme with [(14)C]citrate and other substrates/cofactors CoA, ATP, and Mg(2+), 2) EDTA quench, 3) addition of MicroScint-O, the agent that specifically detects product [(14)C]acetyl-CoA, and 4) detection of signal by TopCount. This unique ACL assay may provide more efficient identification of new ACL inhibitors and allow detailed mechanistic characterization of ACL/inhibitor interactions.

  18. Fatty acid 9- and 13-hydroperoxide lyases from cucumber.

    PubMed

    Matsui, K; Ujita, C; Fujimoto, S; Wilkinson, J; Hiatt, B; Knauf, V; Kajiwara, T; Feussner, I

    2000-09-15

    Fatty acid hydroperoxide lyase (HPL) is a novel P-450 enzyme that cleaves fatty acid hydroperoxides to form short-chain aldehydes and oxo-acids. In cucumber seedlings, the activities of both fatty acid 9HPL and 13HPL could be detected. High 9HPL activity was especially evident in hypocotyls. Using a polymerase chain reaction-based cloning strategy, we isolated two HPL-related cDNAs from cucumber hypocotyls. One of them, C17, had a frameshift and it was apparently expressed from a pseudogene. After repairing the frameshift, the cDNA was successfully expressed in Escherichia coli as an active HPL with specificity for 13-hydroperoxides. The other clone, C15, showed higher sequence similarity to allene oxide synthase (AOS). This cDNA was also expressed in E. coli, and the recombinant enzyme was shown to act both on 9- and 13-hydroperoxides, with a preference for the former. By extensive product analyses, it was determined that the recombinant C15 enzyme has only HPL activity and no AOS activity, in spite of its higher sequence similarity to AOS.

  19. Characterization of the exopolygalacturonate lyase PelX of Erwinia chrysanthemi 3937.

    PubMed

    Shevchik, V E; Kester, H C; Benen, J A; Visser, J; Robert-Baudouy, J; Hugouvieux-Cotte-Pattat, N

    1999-03-01

    Erwinia chrysanthemi 3937 secretes several pectinolytic enzymes, among which eight isoenzymes of pectate lyases with an endo-cleaving mode (PelA, PelB, PelC, PelD, PelE, PelI, PelL, and PelZ) have been identified. Two exo-cleaving enzymes, the exopolygalacturonate lyase, PelX, and an exo-poly-alpha-D-galacturonosidase, PehX, have been previously identified in other E. chrysanthemi strains. Using a genomic bank of a 3937 mutant with the major pel genes deleted, we cloned a pectinase gene identified as pelX, encoding the exopolygalacturonate lyase. The deduced amino acid sequence of the 3937 PelX is very similar to the PelX of another E. chrysanthemi strain, EC16, except in the 43 C-terminal amino acids. PelX also has homology to the endo-pectate lyase PelL of E. chrysanthemi but has a N-terminal extension of 324 residues. The transcription of pelX, analyzed by gene fusions, is dependent on several environmental conditions. It is induced by pectic catabolic products and affected by growth phase, oxygen limitation, nitrogen starvation, and catabolite repression. Regulation of pelX expression is dependent on the KdgR repressor, which controls almost all the steps of pectin catabolism, and on the global activator of sugar catabolism, cyclic AMP receptor protein. In contrast, PecS and PecT, two repressors of the transcription of most pectate lyase genes, are not involved in pelX expression. The pelX mutant displayed reduced pathogenicity on chicory leaves, but its virulence on potato tubers or Saintpaulia ionantha plants did not appear to be affected. The purified PelX protein has no maceration activity on plant tissues. Tetragalacturonate is the best substrate of PelX, but PelX also has good activity on longer oligomers. Therefore, the estimated number of binding subsites for PelX is 4, extending from subsites -2 to +2. PelX and PehX were shown to be localized in the periplasm of E. chrysanthemi 3937. PelX catalyzed the formation of unsaturated digalacturonates by

  20. Characterization of the pelL gene encoding a novel pectate lyase of Erwinia chrysanthemi 3937.

    PubMed

    Lojkowska, E; Masclaux, C; Boccara, M; Robert-Baudouy, J; Hugouvieux-Cotte-Pattat, N

    1995-06-01

    Erwinia chrysanthemi 3937 secretes five major isoenzymes of pectate lyases encoded by the pelA, pelB, pelC, pelD and pelE genes. Recently, a new set of pectate lyases was identified in E. chrysanthemi mutants deleted of those pel genes. We cloned the pelL gene, encoding one of these secondary pectate lyases of E. chrysanthemi 3937, from a genomic bank of a strain deleted of the five major pel genes. The nucleotide sequence of the region containing the pelL gene was determined. The pelL reading frame is 1275 bases long, corresponding to a protein of 425 amino acids including a typical amino-terminal signal sequence of 25 amino acids. Comparison of the amino acid sequences of PelL and the exo-pectate lyase PelX of E. chrysanthemi EC16 revealed a low homology, limited to 220 residues of the central part of the proteins. No homology was detected with other bacterial pectinolytic enzymes. Regulation of pelL transcription was analysed using gene fusion. As shown for the other pel genes, the transcription of pelL is dependent on various environmental conditions. It is induced by pectic catabolic products and affected by growth phase, temperature, iron starvation, osmolarity, anaerobiosis, nitrogen starvation and catabolite repression. Regulation of pelL expression appeared to be independent of the KdgR repressor, which controls all the steps of pectin catabolism. In contrast, the pecS gene, which is involved in regulation of the synthesis of the major pectate lyases and of cellulase, also appeared to be involved in pelL expression. The PelL protein is able to macerate plant tissue. This enzyme has a basic isoelectric point, presents an endo-cleaving activity on polygalacturonate or partially methylated pectin, with a basic pH optimum and an absolute requirement for Ca2+. The pelL mutant displayed a reduced virulence on potato tubers and Saintpaulia ionantha plants, demonstrating the important role of this enzyme in soft-rot disease. PMID:8577252

  1. Gelling process for sodium alginate: New technical approach by using calcium rich micro-spheres.

    PubMed

    Vicini, Silvia; Castellano, Maila; Mauri, Marco; Marsano, Enrico

    2015-12-10

    Alginate based materials have become an important class of products in many fields from the pharmaceutical industry to tissue engineering, because of their ability to create stimuli responsive hydrogels. We present a new technical approach for obtaining a controlled gelling process, based on the quantities of Ca(2+) rich alginate micro-beads added as crosslinkers. The gels have been evaluated in light of the amount of Ca(2+) added to the alginate solution, and in light of the different dimensions of the micro-beads, using rheological measurements to assess the variation in the storage modulus (G'), loss modulus (G'') and complex viscosity (η(*)) as well as swelling and deswelling tests. The methodology was developed to obtain a material with specific characteristics for application in the field of conservation. The material had to be able to create a stable gel after being applied on the artwork surface and to confine the solvent action at the interface during cleaning operations.

  2. Radiation effects on agar, alginates and carrageenan to be used as food additives

    NASA Astrophysics Data System (ADS)

    Aliste, A. J. A. J.; Vieira, F. F. F. F.; Del Mastro, N. L. N. L.

    2000-03-01

    Agar, alginates and carrageenan are hydrocolloids that induce stabilization of physical properties of the food product during shelf life and prevention of undesirable changes such as moisture migration, gas cell coalescence or textural profile changes. In this work, agar, alginates and carrageenan was irradiated as powder with different doses (0-10 kGy) of Co-60 and the rheological functional performance of water solutions of these irradiated additives was studied. The results are analyzed taking in account the future applications of those additives in irradiated foods.

  3. Effect of immobilized cells in calcium alginate beads in alcoholic fermentation

    PubMed Central

    2013-01-01

    Saccharomyces cerevisiae cells were immobilized in calcium alginate and chitosan-covered calcium alginate beads and studied in the fermentation of glucose and sucrose for ethanol production. The batch fermentations were carried out in an orbital shaker and assessed by monitoring the concentration of substrate and product with HPLC. Cell immobilization in calcium alginate beads and chitosan-covered calcium alginate beads allowed reuse of the beads in eight sequential fermentation cycles of 10 h each. The final concentration of ethanol using free cells was 40 g L-1 and the yields using glucose and sucrose as carbon sources were 78% and 74.3%, respectively. For immobilized cells in calcium alginate beads, the final ethanol concentration from glucose was 32.9 ± 1.7 g L-1 with a 64.5 ± 3.4% yield, while the final ethanol concentration from sucrose was 33.5 ± 4.6 g L-1 with a 64.5 ± 8.6% yield. For immobilized cells in chitosan-covered calcium alginate beads, the ethanol concentration from glucose was 30.7 ± 1.4 g L-1 with a 61.1 ± 2.8% yield, while the final ethanol concentration from sucrose was 31.8 ± 6.9 g L-1 with a 62.1 ± 12.8% yield. The immobilized cells allowed eight 10 h sequential reuse cycles to be carried out with stable final ethanol concentrations. In addition, there was no need to use antibiotics and no contamination was observed. After the eighth cycle, there was a significant rupture of the beads making them inappropriate for reuse. PMID:23721664

  4. Effect of immobilized cells in calcium alginate beads in alcoholic fermentation.

    PubMed

    Duarte, Juliana C; Rodrigues, J Augusto R; Moran, Paulo J S; Valença, Gustavo P; Nunhez, José R

    2013-05-30

    Saccharomyces cerevisiae cells were immobilized in calcium alginate and chitosan-covered calcium alginate beads and studied in the fermentation of glucose and sucrose for ethanol production. The batch fermentations were carried out in an orbital shaker and assessed by monitoring the concentration of substrate and product with HPLC. Cell immobilization in calcium alginate beads and chitosan-covered calcium alginate beads allowed reuse of the beads in eight sequential fermentation cycles of 10 h each. The final concentration of ethanol using free cells was 40 g L-1 and the yields using glucose and sucrose as carbon sources were 78% and 74.3%, respectively. For immobilized cells in calcium alginate beads, the final ethanol concentration from glucose was 32.9 ± 1.7 g L-1 with a 64.5 ± 3.4% yield, while the final ethanol concentration from sucrose was 33.5 ± 4.6 g L-1 with a 64.5 ± 8.6% yield. For immobilized cells in chitosan-covered calcium alginate beads, the ethanol concentration from glucose was 30.7 ± 1.4 g L-1 with a 61.1 ± 2.8% yield, while the final ethanol concentration from sucrose was 31.8 ± 6.9 g L-1 with a 62.1 ± 12.8% yield. The immobilized cells allowed eight 10 h sequential reuse cycles to be carried out with stable final ethanol concentrations. In addition, there was no need to use antibiotics and no contamination was observed. After the eighth cycle, there was a significant rupture of the beads making them inappropriate for reuse.

  5. 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

    2007-09-01

    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.

  6. Gene acquisition, duplication and metabolic specification: the evolution of fungal methylisocitrate lyases.

    PubMed

    Müller, Sebastian; Fleck, Christian B; Wilson, Duncan; Hummert, Christian; Hube, Bernhard; Brock, Matthias

    2011-06-01

    Gene duplication represents an evolutionary mechanism for expanding metabolic potential. Here we analysed the evolutionary relatedness of isocitrate and methylisocitrate lyases, which are key enzymes of the glyoxylate and methylcitrate cycle respectively. Phylogenetic analyses imply that ancient eukaryotes acquired an isocitrate lyase gene from a prokaryotic source, but it was lost in some eukaryotic lineages. However, protists, oomycetes and most fungi maintained this gene and successfully integrated the corresponding enzyme into the glyoxylate cycle. A second gene, encoding a highly related enzyme, is present in fungi, but absent from other eukaryotes. This methylisocitrate lyase is specifically involved in propionyl-CoA degradation via the methylcitrate cycle. Although bacteria possess methylisocitrate lyases with a structural fold similar to that of isocitrate lyases, their sequence identity to fungal methylisocitrate lyases is low. Phylogenetic analyses imply that fungal methylisocitrate lyases arose from gene duplication of an ancient isocitrate lyase gene from the basidiomycete lineage. Mutagenesis of active-site residues of a bacterial and fungal isocitrate lyase, which have been predicted to direct the substrate specificity of iso- and methylisocitrate lyases, experimentally confirmed the possibility of direct evolution of methylisocitrate lyases from isocitrate lyases. Thus, gene duplication has increased the metabolic capacity of fungi.

  7. Sustained release of vascular endothelial growth factor from calcium-induced alginate hydrogels reinforced by heparin and chitosan.

    PubMed

    Lee, K W; Yoon, J J; Lee, J H; Kim, S Y; Jung, H J; Kim, S J; Joh, J W; Lee, H H; Lee, D S; Lee, S K

    2004-10-01

    A possible alternative for immunosuppression is a microencapsulation technique using hydrogels, which have been utilized for cell immobilization and drug delivery systems. Angiogenesis is crucial for delivery of the metabolic products to the host tissues as well as to supply oxygen and nutrients to cells. The local delivery of angiogenic growth factors, such as VEGF and basic FGF, has been recently studied to enhance angiogenesis on peripheral tissue of graft. In this study, we evaluated sustained VEGF release with a model using hydrogels coated with chitosan and heparin in vitro. We fabricated calcium alginate gels and chitosan-coated calcium alginate gels. Heparinized chitosan-coated calcium-induced alginate hydrogel beads were prepared by soaking chitosan-coated calcium alginate gels in heparin solution. We compared the stability and VEGF release manner between three kinds of hydrogels. To compare the stability, 5 mL of each hydrogel was incubated with 20 mL PBS under the rotational culture. Compression forces were measured using a rheometer. The amount of VEGF released from the gels was measured by ELISA. The heparin-coated chitosan alginate hydrogels showed the highest surface stability among the three hydrogels. VEGF from the heparinized gel was released in sustained manner up to 10 days in vitro. Chitosan-coated alginate gels released 90% of loaded VEGF within 5 days. These results suggest that local delivery of VEGF using a heparinized hydrogel may provide a long-term supply of angiogenic growth factor that might induce new vessel formation in vivo.

  8. 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

  9. Role of calcium alginate and mannitol in protecting Bifidobacterium.

    PubMed

    Dianawati, Dianawati; Mishra, Vijay; Shah, Nagendra P

    2012-10-01

    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 (a(w)) 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 CaCl(2) solution to obtain Ca-alginate beads and Ca-alginate-mannitol beads, respectively. The wet beads were then freeze-dried. The a(w) 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 a(w) adjustment. Mannitol in the Ca-alginate system interacted with cell envelopes during freeze-drying and during storage at low a(w)s. 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 a(w)s. 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

  10. Occurrence of Isocitrate Lyase in a Thermophilic Bacillus Species

    PubMed Central

    Daron, Harlow H.

    1967-01-01

    A thermophilic, sporeforming bacterium has been isolated from soil on a medium containing acetate as a carbon source. This organism is similar to Bacillus stearothermophilus in most respects but differs in its inability to hydrolyze starch. Isocitrate lyase is present in cell-free extracts of organisms grown in a medium with acetate as a carbon source. The specific activity was 400 times lower in extracts of organisms utilizing glucose as a carbon source. With crude extracts, enzyme activity was strongly stimulated by Mg++, but cysteine and ethylenediaminetetraacetate had little effect. It appeared to be more heat-stable than the pure isocitrate lyase from Pseudomonas indigofera. Images PMID:6020570

  11. Michael addition of dehydroalanine-containing MAPK peptides to catalytic lysine inhibits the activity of phosphothreonine lyase.

    PubMed

    Zhang, Yuan; Yang, Ru; Huang, Juan; Liang, Qiujin; Guo, Yanmin; Bian, Weixiang; Luo, Lingfei; Li, Hongtao

    2015-11-30

    The phosphothreonine lyases OspF and SpvC irreversibly inactivate host dual-phosphorylated mitogen-activated protein kinases (MAPKs) [pThr-X-pTyr motif] through β-elimination. We found that dual-phosphorylated (pSer-X-pTyr) MAPK substrate peptides and their resulting catalytic products cross-link to OspF and SpvC. Mass spectrometry results revealed that these linkages form between lysine, which acts as a general base, and dehydroalanine (Dha) on catalytic products. The nucleophilic addition efficiency is dependent on the K136 residue being in a deprotonated state. Peptide cross-linking inhibits the activity of SpvC and blocks the inactivation of MAPK signaling by SpvC. Small compounds mimicking these sequences may act as phosphothreonine lyase inhibitors. PMID:26519561

  12. The role of alginate in Pseudomonas aeruginosa EPS adherence, viscoelastic properties and cell attachment.

    PubMed

    Orgad, Oded; Oren, Yoram; Walker, Sharon L; Herzberg, Moshe

    2011-08-01

    Among various functions, extracellular polymeric substances (EPS) provide microbial biofilms with mechanical stability and affect initial cell attachment, the first stage in the biofilm formation process. The role of alginate, an abundant polysaccharide in Pseudomonas aeruginosa biofilms, in the viscoelastic properties and adhesion kinetics of EPS was analyzed using a quartz crystal microbalance with dissipation (QCM-D) monitoring technology. EPS was extracted from two P. aeruginosa biofilms, a wild type strain, PAO1, and a mucoid strain, PAOmucA22 that over-expresses alginate production. The higher alginate content in the EPS originating from the mucoid biofilms was clearly shown to increase both the rate and the extent of attachment of the EPS, as well as the layer's thickness. Also, the presence of calcium and elevated ionic strength increased the thickness of the EPS layer. Dynamic light scattering (DLS) showed that the presence of calcium and elevated ionic strength induced intermolecular attractive interactions in the mucoid EPS molecules. For the wild type EPS, in the presence of calcium, an elevated shift in the distribution of the diffusion coefficients was observed with DLS due to a more compacted conformation of the EPS molecules. Moreover, the alginate over-expression effect on EPS adherence was compared to the effect of alginate over-expression on P. aeruginosa cell attachment. In a parallel plate flow cell, under similar hydraulic and aquatic conditions as those applied for the EPS adsorption tests in the QCM-D flow cell, reduced adherence of the mucoid strain was clearly observed compared to the wild type isogenic bacteria. The results suggest that alginate contributes to steric hindrance and shielding of cell surface features and adhesins that are known to promote cell attachment.

  13. 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.

  14. 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.

  15. The alginate regulator AlgR and an associated sensor FimS are required for twitching motility in Pseudomonas aeruginosa.

    PubMed Central

    Whitchurch, C B; Alm, R A; Mattick, J S

    1996-01-01

    Mucoid strains of Pseudomonas aeruginosa isolated from the lungs of cystic fibrosis patients produce large amounts of the exopolysaccharide alginate. AlgR has long been considered a key regulator of alginate production, but its cognate sensor has not been identified. Here we show that AlgR is required for twitching motility, which is a form of bacterial surface translocation mediated by type 4 fimbriae. Adjacent to algR we have identified a sensor gene (fimS), which is also required for twitching motility. However, FimS does not appear to be required for alginate production in mucoid strains. FimS and AlgR are representative of a new subclass of two-component transmitter-receiver regulatory systems. The alternative sigma factor AlgU also affects both alginate production and twitching motility. Therefore, these two virulence determinants appear to be closely associated and coordinately regulated. Images Fig. 1 Fig. 2 Fig. 3 PMID:8790418

  16. 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

  17. The immobilization of microbial cells, subcellular organelles, and enzymes in calcium alginate gels. Reprinted from Biotechnology and Bioengineering, Vol. XIX, No. 3, Pages 387-397 (1977).

    PubMed

    Kierstan, M; Bucke, C

    2000-03-20

    Saccharomyces cerevisiae cells, Kluyveromyces marxianus cells, inulase, glucose oxidase, chloroplasts, and mitochondria were immobilized in calcium alginate gels. Ethanol production from glucose solutions by an immobilized preparation of S. cerevisiae was demonstrated over a total of twenty-three days, and the half-life of such a preparation was shown to be about ten days. Immobilized K. marxianus, inulase, and glucose oxidase preparations were used to demonstrate the porosity and retraining properties of calcium alginate gels. Calcium alginate-immobilized chloroplasts were shown to perform the Hill reaction. Some experiments with immobilized mitochondria are reported.

  18. 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

  19. Structural insights into catalysis by βC-S lyase from Streptococcus anginosus.

    PubMed

    Kezuka, Yuichiro; Yoshida, Yasuo; Nonaka, Takamasa

    2012-10-01

    Hydrogen sulfide (H(2)S) is a causative agent of oral malodor and may play an important role in the pathogenicity of oral bacteria such as Streptococcus anginosus. In this microorganism, H(2)S production is associated with βC-S lyase (Lcd) encoded by lcd gene, which is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the α,β-elimination of sulfur-containing amino acids. When Lcd acts on L-cysteine, H(2)S is produced along with pyruvate and ammonia. To understand the H(2)S-producing mechanism of Lcd in detail, we determined the crystal structures of substrate-free Lcd (internal aldimine form) and two reaction intermediate complexes (external aldimine and α-aminoacrylate forms). The formation of intermediates induced little changes in the overall structure of the enzyme and in the active site residues, with the exception of Lys234, a PLP-binding residue. Structural and mutational analyses highlighted the importance of the active site residues Tyr60, Tyr119, and Arg365. In particular, Tyr119 forms a hydrogen bond with the side chain oxygen atom of L-serine, a substrate analog, in the external aldimine form suggesting its role in the recognition of the sulfur atom of the true substrate (L-cysteine). Tyr119 also plays a role in fixing the PLP cofactor at the proper position during catalysis through binding with its side chain. Finally, we partly modified the catalytic mechanism known for cystalysin, a βC-S lyase from Treponema denticola, and proposed an improved mechanism, which seems to be common to the βC-S lyases from oral bacteria.

  20. 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, §...

  1. 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...

  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. Formation of C-C bonds by mandelonitrile lyase in organic solvents.

    PubMed

    Wehtje, E; Adlercreutz, P; Mattiasson, B

    1990-06-01

    Mandelonitrile lyase (EC 4.1.2.10) catalyzes the formation of D-mandelonitrile from HCN and benzaldehyde. Mandelonitrile lyase was immobilized by adsorption to support materials, for example, Celite. The enzyme preparations were used in diisopropyl ether for production of D-mandelonitrile. In order to obtain optically pure D-mandelonitrile it was necessary to use reaction conditions which favor the enzymatic reaction and suppress the competing spontaneous reaction, which yields a racemic mixture of D, L-mandelonitrile. The effects of substrate concentrations, water content, and support materials on both the spontaneous and enzymatic reactions were studied. The enzymatic reaction was carried out under conditions where the importance of the spontaneous reaction was negligible and high enantiomeric purity of D-mandelonitrile was achieved (at least 98% enantiomeric excess). The operational stability of the enzyme preparations was studied in batch as well as in continuous systems. It was vital to control the water content in the system to maintain an active preparation. In a packed bed reactor the enzyme preparations were shown to be active and stable. The reactors were run for 50 h with only a small decrease in product yield. PMID:18592607

  4. 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.

  5. Raman probing of molecular interactions of alginate biopolymers with cells

    NASA Astrophysics Data System (ADS)

    Chourpa, Igor; Carpentier, Philippe; Maingault, Philippe; Fetissoff, Franck; Dubois, Pierre

    2000-05-01

    The biological polymers extracted from brown algae, alginates, are novel materials in biotechnology and biomedicine. Their ability to form viscous gels is used to immobilize or encapsulate yeast, enzymes, living cells and drugs. Calcium-alginate fibers are extensively used in wound dressings since exhibit antihaemostatic and healing properties. The problem with alginate-made dressings in surgery is their slow biodegradability: if entrapped within tissues, they can induce a local cellular recruitment with an inflammatory response contemporaneous to the resorption phase. In part, this problem is a consequence of poor solubility of the calcium alginates in water. Although calcium alginate fibers can exchange calcium ions with sodium ions from the wound exudate to create a calcium/sodium alginate fibers, the residual alginates are thought to be not totally degradable in vivo. Rapid and non- destructive characterization of series of the crude alginates and calcium alginate fibers has been performed using Raman spectroscopy with near IR excitation. Study of structural organization of the polymeric chains within calcium alginate fibers have been previously reported as made by confocal Raman multispectral imaging (CRMSI) in visible. Here, the Raman approach has been used to monitor the ion exchange reactions for different types of alginates and their salts in vitro. For in vivo evaluation, histological sections of alginate-treated rat tissue have been analyzed by light microscopy and CRMSI. The in vitro Raman modeling and the histochemical mapping were a necessary precursor for application of the Raman microprobe to follow in a non-invasive way the alginate-cell molecular interactions in rat tissue.

  6. Survival of different cell lines in alginate-agarose microcapsules.

    PubMed

    Orive, G; Hernández, R M; Gascón, A R; Igartua, M; Pedraz, J L

    2003-01-01

    Cell microencapsulation has emerged as a promising therapeutic strategy to treat a wide range of diseases. The optimisation of this technology depends on several critical issues such as the careful selection of the cell line, the controlled manufacture of microcapsules and the suitable adaptation of the construct design to the selected cell line. In this work, we studied the behavior of hybridoma cells once enclosed in solid and liquefied core alginate-agarose beads. Results show that hybridoma cells presented a better growing pattern and improved their viability and antibody production within liquefied beads. However, when these beads were evaluated with a compression resistance study, they were found to be mechanically more fragile than solid ones. To address this problem, we entrapped non-autologous cells (BHK fibroblast and C2C12 myoblast) in solid alginate-agarose beads and observed that they showed an improved growing profile and prolonged their viability up to 70 days in comparison to the 15 days seen for the hybridoma cells.

  7. Development of a new antibacterial biomaterial by tetracycline immobilization on calcium-alginate beads.

    PubMed

    Ozseker, Emine Erdogan; Akkaya, Alper

    2016-10-20

    In recent years, increasing risk of infection, caused by resistant microorganism to antibiotics, has become the limelight discovery of new and natural antibacterial materials. Heavy metals, such as silver, copper, mercury and titanium, have antibacterial activity. Products, which improved these metals, do not have stable antibacterial property. Therefore, use of these products is restricted. The aim of this study was to immobilize tetracycline to alginate and improve an antibacterial biomaterial. For this purpose, calcium-alginate beads were formed by dropping to calcium-chloride solution and tetracycline was immobilized to beads using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at optimum conditions. After immobilization, actualization of immobilization was investigated by analyzing ATR-FTIR spectrum and SEM images. Also, antibacterial property of obtained product was tested. Improved product demonstrated antibacterial property. It has potential for open wound, surgical drapes, bed and pillow sheath in hospitals and it may also be used for increasing human comfort in daily life. PMID:27474587

  8. Development of a new antibacterial biomaterial by tetracycline immobilization on calcium-alginate beads.

    PubMed

    Ozseker, Emine Erdogan; Akkaya, Alper

    2016-10-20

    In recent years, increasing risk of infection, caused by resistant microorganism to antibiotics, has become the limelight discovery of new and natural antibacterial materials. Heavy metals, such as silver, copper, mercury and titanium, have antibacterial activity. Products, which improved these metals, do not have stable antibacterial property. Therefore, use of these products is restricted. The aim of this study was to immobilize tetracycline to alginate and improve an antibacterial biomaterial. For this purpose, calcium-alginate beads were formed by dropping to calcium-chloride solution and tetracycline was immobilized to beads using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at optimum conditions. After immobilization, actualization of immobilization was investigated by analyzing ATR-FTIR spectrum and SEM images. Also, antibacterial property of obtained product was tested. Improved product demonstrated antibacterial property. It has potential for open wound, surgical drapes, bed and pillow sheath in hospitals and it may also be used for increasing human comfort in daily life.

  9. Quantitative chemical mapping of sodium acrylate- and N-vinylpyrrolidone-enhanced alginate microcapsules.

    PubMed

    Araki, Tohru; Hitchcock, Adam P; Shen, Feng; Chang, Patricia L; Wang, Maggie; Childs, Ronald F

    2005-01-01

    Alginate microcapsules enclosing recombinant cells secreting therapeutic products have been used successfully to treat several murine models of human diseases. The mechanical and chemical properties of these alginate capsules can be improved by the addition and in situ photo-polymerization of sodium acrylate and N-vinylpyrrolidone in the alginate capsule. The purpose of this modification was to form additional covalent cross-links. In this work we have used scanning transmission X-ray microscopy (STXM) to probe the nature and location of the chemical modifications in the modified capsules by comparison with unmodified capsules. Analysis of X-ray image sequences and selected area spectra has been used to map the calcium gradient in capsules, to identify the presence of polyacrylate throughout the capsules and the localization of poly-N-vinylpyrrolidone in the outer regions of the alginate capsules. The differences in the spatial distributions of these species have led to better understanding of the chemical modifications that provide a mechanically more stable capsule structure.

  10. Use of chitosan-alginate as alternative pelletization aid to microcrystalline cellulose in extrusion/spheronization.

    PubMed

    Charoenthai, Nattawut; Kleinebudde, Peter; Puttipipatkhachorn, Satit

    2007-09-01

    Two types of different molecular weight chitosan were investigated as a pelletization aid in extrusion/spheronization using water as granulation liquid. Spherical pellets with a maximum fraction of 60% w/w chitosan could be produced when 1.25-2.5% w/w sodium alginate was included in the formulations with no microcrystalline cellulose (MCC). Chitosan with lower molecular weight of 190 kDa showed a better pellet forming property. The pellets obtained had acceptable physical characteristics and a fast drug release. The results from Fourier transform infrared spectroscopy, differential scanning calorimetry and (13)C CP-MAS nuclear magnetic resonance spectroscopy confirmed the formation of polyelectrolyte complex (PEC) between chitosan and sodium alginate, which might be a reason for successful pelletization by extrusion/spheronization. Moreover, the presence of PEC might influence the physical characteristics and dissolution behavior of chitosan-alginate pellets. The results indicated an achievement in production of pellets by extrusion/spheronization without using MCC. Moreover, chitosan combined with sodium alginate could be used as a promising alternative pelletization aid to MCC in extrusion/spheronization. PMID:17286294

  11. 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.

  12. Mobility of atrazine from alginate-bentonite controlled release formulations in layered soil.

    PubMed

    Fernández-Pérez, M; González-Pradas, E; Villafranca-Sánchez, M; Flores-Céspedes, F

    2001-04-01

    The mobility of atrazine [6-chloro-N2-ethyl-N4-isopropyl-1,3,5-triazine-2,4-diamine] from alginate-bentonite-based controlled release (CR) formulations was investigated by using soil columns. Two CR formulations based on sodium alginate (14.0 g kg(-1), atrazine (6.0 g kg(-1), natural or acid-treated bentonite (50 g kg(-1), and water (924 g kg(-1) were compared to technical grade product and commercial liquid (CL) formulation (Gesaprim 500FW). All herbicide treatments were applied to duplicate layered bed systems simulating the typical arrangement under a plastic greenhouse, which is composed of sand (10 cm), peat (2 cm), amended soil (20 cm) and native soil (20 cm). The columns were leached with 39 cm (1500 ml) and 156 cm (6000 ml) of 0.02 M CaCl2 solution to evaluate the effect of water volume applied on herbicide movement. When 39 cm of 0.02 M CaCl2 solution was applied, there was no presence of herbicide in the leachate for the alginate-bentonite CR treatments. However, 0.11% and 0.14% of atrazine appeared in the leachate when the treatment was carried out with technical grade and CL formulations, respectively. When 156 cm of 0.02 M CaCl2 solution was applied, the use of the alginate-acid treated bentonite CR formulation retards and reduces the presence of atrazine in the leachate as compared to technical product. Analysis of the soil columns showed the highest atrazine concentration in the peat layer. Alginate-bentonite CR formulations might be an efficient system for reducing atrazine leaching in layered soil and thus, it could reduce the risks of pollution of groundwater.

  13. Antibacterial Performance of Alginic Acid Coating on Polyethylene Film

    PubMed Central

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

    2014-01-01

    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. PMID:25196604

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

    PubMed

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

    2014-01-01

    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. PMID:25196604

  15. α-Glucosidases and α-1,4-glucan lyases: structures, functions, and physiological actions.

    PubMed

    Okuyama, Masayuki; Saburi, Wataru; Mori, Haruhide; Kimura, Atsuo

    2016-07-01

    α-Glucosidases (AGases) and α-1,4-glucan lyases (GLases) catalyze the degradation of α-glucosidic linkages at the non-reducing ends of substrates to release α-glucose and anhydrofructose, respectively. The AGases belong to glycoside hydrolase (GH) families 13 and 31, and the GLases belong to GH31 and share the same structural fold with GH31 AGases. GH13 and GH31 AGases show diverse functions upon the hydrolysis of substrates, having linkage specificities and size preferences, as well as upon transglucosylation, forming specific α-glucosidic linkages. The crystal structures of both enzymes were determined using free and ligand-bound forms, which enabled us to understand the important structural elements responsible for the diverse functions. A series of mutational approaches revealed features of the structural elements. In particular, amino-acid residues in plus subsites are of significance, because they regulate transglucosylation, which is used in the production of industrially valuable oligosaccharides. The recently solved three-dimensional structure of GLase from red seaweed revealed the amino-acid residues essential for lyase activity and the strict recognition of the α-(1 → 4)-glucosidic substrate linkage. The former was introduced to the GH31 AGase, and the resultant mutant displayed GLase activity. GH13 and GH31 AGases hydrate anhydrofructose to produce glucose, suggesting that AGases are involved in the catabolic pathway used to salvage unutilized anhydrofructose. PMID:27137181

  16. 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

  17. Mechanistic studies of a novel C-S lyase in ergothioneine biosynthesis: the involvement of a sulfenic acid intermediate.

    PubMed

    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.

  18. 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.

  19. Rapid reagentless quantification of alginate biosynthesis in Pseudomonas fluorescens bacteria mutants using FT-IR spectroscopy coupled to multivariate partial least squares regression.

    PubMed

    Correa, Elon; Sletta, Håvard; Ellis, David I; Hoel, Sunniva; Ertesvåg, Helga; Ellingsen, Trond E; Valla, Svein; Goodacre, Royston

    2012-07-01

    Alginate is an important medical and commercial product and currently is isolated from seaweeds. Certain microorganisms also produce alginate and these polymers have the potential to replace seaweed alginates in some applications, mainly because such production will allow much better and more reproducible control of critical qualitative polymer properties. The research conducted here presents the development of a new approach to this problem by analysing a transposon insertion mutant library constructed in an alginate-producing derivative of the Pseudomonas fluorescens strain SBW25. The procedure is based on the non-destructive and reagent-free method of Fourier transform infrared (FT-IR) spectroscopy which is used to generate a complex biochemical infrared fingerprint of the medium after bacterial growth. First, we investigate the potential differences caused by the growth media fructose and glycerol on the bacterial phenotype and alginate synthesis in 193 selected P. fluorescens mutants and show that clear phenotypic differences are observed in the infrared fingerprints. In order to quantify the level of the alginate we also report the construction and interpretation of multivariate partial least squares regression models which were able to quantify alginate levels successfully with typical normalized root-mean-square error in predictions of only approximately 14%. We have demonstrated that this high-throughput approach can be implemented in alginate screens and we believe that this FT-IR spectroscopic methodology, when combined with the most appropriate chemometrics, could easily be modified for the quantification of other valuable microbial products and play a valuable screening role for synthetic biology.

  20. 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.

  1. 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.

  2. Priming ammonia lyases and aminomutases for industrial and therapeutic applications.

    PubMed

    Heberling, Matthew M; Wu, Bian; Bartsch, Sebastian; Janssen, Dick B

    2013-04-01

    Ammonia lyases (AL) and aminomutases (AM) are emerging in green synthetic routes to chiral amines and an AL is being explored as an enzyme therapeutic for treating phenylketonuria and cancer. Although the restricted substrate range of the wild-type enzymes limits their widespread application, the non-reliance on external cofactors and direct functionalization of an olefinic bond make ammonia lyases attractive biocatalysts for use in the synthesis of natural and non-natural amino acids, including β-amino acids. The approach of combining structure-guided enzyme engineering with efficient mutant library screening has extended the synthetic scope of these enzymes in recent years and has resolved important mechanistic issues for AMs and ALs, including those containing the MIO (4-methylideneimidazole-5-one) internal cofactor.

  3. Regulation of the Aspergillus nidulans pectate lyase gene (pelA).

    PubMed Central

    Dean, R A; Timberlake, W E

    1989-01-01

    Aspergillus nidulans pectate lyase was purified from culture filtrates. The enzyme catalyzed a random eliminative cleavage reaction, had an apparent molecular weight of 40,000, and a pl of 4.2. Pectate lyase antisera were produced and used to identify pectate lyase clones in a cDNA expression library. Thirteen of 14 clones identified immunologically cross-hybridized. The identity of the single-copy pectate lyase gene, which we designated pelA, was confirmed in two ways. First, several cDNA clones expressed pectate lyase activity in Escherichia coli. Second, targeted mutation of the gene in A. nidulans resulted in complete loss of enzyme activity. pelA encodes a 1,300-nucleotide mRNA that was present in cells grown with polygalacturonic acid as carbon source but absent from cells grown with glucose or acetate as carbon source. Thus, pectate lyase expression is regulated at the level of mRNA accumulation. PMID:2535502

  4. 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.

  5. 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.

  6. 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. PMID:26613390

  7. Redox-responsive alginate microsphere containing cystamine.

    PubMed

    Kwon, Kyeongnan; Kim, Jin-Chul

    2016-10-01

    Redox-responsive microspheres were prepared by solidifying the alginate- and cystamine-containing water droplets of O/W emulsion using calcium ion. Emulsions were prepared using alginate/cystamine mixture solution whose the carboxylic group/the amino group molar ratio was 1:1, 1:2, and 1:3, and whose the total concentration was kept to 2% (w/v). The microspheres on Scanning electron microscopy photographs were almost spherical and they were less than 1 μm in diameter. According to the energy-dispersive X-ray spectroscopy, the sulfur content of the microspheres was found to be 6.1, 11.4, and 14.8% (w/w), respectively, not markedly different from the calculated content. The release degree of blue dextran loaded in the microspheres was higher as the cystamine content was higher. Microspheres released almost the same amount of dye regardless of dithiothreitol (DTT, a reducing agent) concentration when the cystamine content was relatively low (e.g. 14.5% (w/w)), whereas they released dye in DTT concentration-dependent manner when the cystamine content was relatively high (e.g. 27.0 and 35.1% (w/w)). The light scattering intensity of alginate/cystamine mixture solution was stronger at a larger amount of cystamine, indicating that cystamine could cross-link alginate chains. The light scattering intensity decreased with increasing DTT concentration, possibly because of the breakdown of the disulfide bond of cystamine. The breakdown of the disulfide bond could account for why the DTT concentration-dependent release of dye loaded in the microspheres was observed. PMID:27484719

  8. 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

  9. 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. PMID:25656542

  10. Biodegradation of crystal violet using Burkholderia vietnamiensis C09V immobilized on PVA-sodium alginate-kaolin gel beads.

    PubMed

    Cheng, Ying; Lin, HongYan; Chen, Zuliang; Megharaj, Mallavarapu; Naidu, Ravi

    2012-09-01

    The strain, Burkholderia vietnamiensis C09V was immobilized on PVA-alginate-kaolin gel beads as a biomaterial to improve the degradation of crystal violet from aqueous solution. The results show that 98.6% (30 mg L(-1)) crystal violet was removed from aqueous solution using immobilized cells on PVA-alginate-kaolin gel beads, while 94.0% crystal violet was removed by free cells after degradation at the pH 5 and 30°C for 30 h. Kinetics studies show that the pseudo-second-order kinetics well described the adsorption of crystal violet on the PVA-alginate-kaolin beads. Biodegradation of crystal violet on immobilized cells was fitted well by first-order reaction kinetics, indicating that CV was adsorbed onto kaolin and followed their degradation by immobilized cells onto the the PVA-alginate-kaolin beads. Characterization with SEM shows that cells attached well to the surface of PVA-alginate-kaolin beads, leading to improved crystal violet transfer from aqueous solution to immobilized cells. In addition, UV-vis show that the absorption peak at 588 nm was reduced by the degraded N-bond linkages, as well as the formation of degrading products were observed by Fourier transform infrared (FTIR). These results suggest that crystal violet was biodegraded to N,N-dimethylaminophenol and Michler's Ketone prior to these intermediates being further degraded. PMID:22789742

  11. Biodegradation of crystal violet using Burkholderia vietnamiensis C09V immobilized on PVA-sodium alginate-kaolin gel beads.

    PubMed

    Cheng, Ying; Lin, HongYan; Chen, Zuliang; Megharaj, Mallavarapu; Naidu, Ravi

    2012-09-01

    The strain, Burkholderia vietnamiensis C09V was immobilized on PVA-alginate-kaolin gel beads as a biomaterial to improve the degradation of crystal violet from aqueous solution. The results show that 98.6% (30 mg L(-1)) crystal violet was removed from aqueous solution using immobilized cells on PVA-alginate-kaolin gel beads, while 94.0% crystal violet was removed by free cells after degradation at the pH 5 and 30°C for 30 h. Kinetics studies show that the pseudo-second-order kinetics well described the adsorption of crystal violet on the PVA-alginate-kaolin beads. Biodegradation of crystal violet on immobilized cells was fitted well by first-order reaction kinetics, indicating that CV was adsorbed onto kaolin and followed their degradation by immobilized cells onto the the PVA-alginate-kaolin beads. Characterization with SEM shows that cells attached well to the surface of PVA-alginate-kaolin beads, leading to improved crystal violet transfer from aqueous solution to immobilized cells. In addition, UV-vis show that the absorption peak at 588 nm was reduced by the degraded N-bond linkages, as well as the formation of degrading products were observed by Fourier transform infrared (FTIR). These results suggest that crystal violet was biodegraded to N,N-dimethylaminophenol and Michler's Ketone prior to these intermediates being further degraded.

  12. Alginate beads as a carrier for omeprazole/SBA-15 inclusion compound: A step towards the development of personalized paediatric dosage forms.

    PubMed

    Del Gaudio, Pasquale; De Cicco, Felicetta; Sansone, Francesca; Aquino, Rita Patrizia; Adami, Renata; Ricci, Maurizio; Giovagnoli, Stefano

    2015-11-20

    The treatment of gastro-esophageal reflux disease (GERD) shows several issues among paediatric patients. This work aims to the formulation of enteric alginate beads loaded with omeprazole (OME) allowing age- and weight-related personalized dosages in children. OME was entrapped in SBA-15 mesoporous compound, characterized and loaded into alginate beads by prilling at different OME and alginate concentrations. The beads resulted of homogeneous size, spherical morphology and very consistent in drug loading and distribution. Formulations demonstrated limited swelling and release (about 10%) in simulated gastric fluid (SGF) after 2h and a prolonged release in simulated intestinal fluid (SIF), till 6h, due to a mixed diffusion-case II transport mechanism. The beads were superior to the market product, which showed lower release in SGF but immediate dissolution in SIF. The high alginate beads uniformity and release properties make them a potential novel tool for a personalized treatment of GERD in children. PMID:26344303

  13. Refeeding syndrome in a young woman with argininosuccinate lyase deficiency.

    PubMed

    Stuy, M; Chen, G-F; Masonek, J M; Scharschmidt, B F

    2015-09-01

    A severely chronically protein and calorie restricted young woman with argininosuccinate lyase deficiency developed transient refeeding syndrome (RFS) and hyperammonemia after modest diet liberalization following initiation of glycerol phenylbutyrate (GPB). The patient required IV supportive care and supplementation with potassium, magnesium and calcium. She is now doing well on GPB and an appropriate maintenance diet. Susceptibility to RFS should be considered in chronically nutritionally restricted patients with metabolic disorders after liberalization of diet.

  14. Refeeding syndrome in a young woman with argininosuccinate lyase deficiency☆

    PubMed Central

    Stuy, M.; Chen, G.-F.; Masonek, J.M.; Scharschmidt, B.F.

    2015-01-01

    A severely chronically protein and calorie restricted young woman with argininosuccinate lyase deficiency developed transient refeeding syndrome (RFS) and hyperammonemia after modest diet liberalization following initiation of glycerol phenylbutyrate (GPB). The patient required IV supportive care and supplementation with potassium, magnesium and calcium. She is now doing well on GPB and an appropriate maintenance diet. Susceptibility to RFS should be considered in chronically nutritionally restricted patients with metabolic disorders after liberalization of diet. PMID:26937403

  15. Toward a mechanism for biliprotein lyases: revisiting nucleophilic addition to phycocyanobilin.

    PubMed

    Tu, Jun-Ming; Zhou, Ming; Haessner, Rainer; Plöscher, Matthias; Eichacker, Lutz; Scheer, Hugo; Zhao, Kai-Hong

    2009-04-22

    Biliprotein lyases attach linear-tetrapyrrolic bilins covalently to apoproteins, which is a prerequisite for the assembly of phycobiliproteins into phycobilisomes, the light-harvesting complexes of cyanobacteria. On the basis of the addition of thiol and imidazole to phycocyanobilin, we propose a generalized lyase reaction mechanism. The adducts contain isomerized phycocyanobilin that can be transferred by the lyase to apoproteins by either back-isomerization, generating phycocyanobilin-containing proteins, or direct transfer, generating phycoviolobilin-containing proteins.

  16. Induction of chondroitin sulfate lyase activity in Bacteroides thetaiotaomicron.

    PubMed Central

    Salyers, A A; Kotarski, S F

    1980-01-01

    Chondroitin sulfate lyase (EC 4.2.2.4) was present constitutively at low levels (0.06 to 0.08 U/mg of protein) in cells of Bacteroides thetaiotaomicron which were growing on glucose or other monosaccharides. When these uninduced bacteria were incubated with chondroitin sulfate A (5 mg/ml), chondroitin sulfate lyase specific activity increased more than 10-fold within 90 min. Synthesis of ribonucleic acid and of protein was required for induction, and induction was sensitive to oxygen. The disaccharides which resulted from chondroitinase action did not act as inducers, nor did tetrasaccharides or hexasaccharides obtained by digestion of chondroitin sulfate with bovine testicular hyaluronidase. None of these substances was taken up by uninduced cells; they may not have been able to penetrate the outer membrane. The smallest oligomer capable of acting as an inducer was the outer membrane. The smallest oligomer capable of acting as an inducer was the octassacharide. Oligomers larger than the octassacharide induced chondroitin lyase activity nearly as well as intact chondroitin sulfate. PMID:6782077

  17. 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.

  18. Sphingosine 1-phosphate lyase enzyme assay using a BODIPY-labeled substrate.

    PubMed

    Bandhuvula, Padmavathi; Li, Zaiguo; Bittman, Robert; Saba, Julie D

    2009-03-01

    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(m) of 35 microM for BODIPY-sphingosine 1-phosphate.

  19. Regulation of pH attenuates toxicity of a byproduct produced by an ethanologenic strain of Sphingomonas sp. A1 during ethanol fermentation from alginate.

    PubMed

    Fujii, Mari; Yoshida, Shiori; Murata, Kousaku; Kawai, Shigeyuki

    2014-01-01

    Marine macroalgae is a promising carbon source that contains alginate and mannitol as major carbohydrates. A bioengineered ethanologenic strain of the bacterium Sphingomonas sp. A1 can produce ethanol from alginate, but not mannitol, whereas the yeast Saccharomyces paradoxus NBRC 0259-3 can produce ethanol from mannitol, but not alginate. Thus, one practical approach for converting both alginate and mannitol into ethanol would involve two-step fermentation, in which the ethanologenic bacterium initially converts alginate into ethanol, and then the yeast produces ethanol from mannitol. In this study, we found that, during fermentation from alginate, the ethanologenic bacterium lost viability and secreted toxic byproducts into the medium. These toxic byproducts inhibited bacterial growth and killed bacterial cells and also inhibited growth of S. paradoxus NBRC 0259-3. We discovered that adjusting the pH of the culture supernatant or the culture medium containing the toxic byproducts to 6.0 attenuated the toxicity toward both bacteria and yeast, and also extended the period of viability of the bacterium. Although continuous adjustment of pH to 6.0 failed to improve the ethanol productivity of this ethanologenic bacterium, this pH adjustment worked very well in the two-step fermentation due to the attenuation of toxicity toward S. paradoxus NBRC 0259-3. These findings provide information critical for establishment of a practical system for ethanol production from brown macroalgae.

  20. Genes required for and effects of alginate overproduction induced by growth of Pseudomonas aeruginosa on Pseudomonas isolation agar supplemented with ammonium metavanadate.

    PubMed

    Damron, F Heath; Barbier, Mariette; McKenney, Elizabeth S; Schurr, Michael J; Goldberg, Joanna B

    2013-09-01

    Pseudomonas aeruginosa is an opportunistic pathogen that can adapt to changing environments and can secrete an exopolysaccharide known as alginate as a protection response, resulting in a colony morphology and phenotype referred to as mucoid. However, how P. aeruginosa senses its environment and activates alginate overproduction is not fully understood. Previously, we showed that Pseudomonas isolation agar supplemented with ammonium metavanadate (PIAAMV) induces P. aeruginosa to overproduce alginate. Vanadate is a phosphate mimic and causes protein misfolding by disruption of disulfide bonds. Here we used PIAAMV to characterize the pathways involved in inducible alginate production and tested the global effects of P. aeruginosa growth on PIAAMV by a mutant library screen, by transcriptomics, and in a murine acute virulence model. The PA14 nonredundant mutant library was screened on PIAAMV to identify new genes that are required for the inducible alginate stress response. A functionally diverse set of genes encoding products involved in cell envelope biogenesis, peptidoglycan remodeling, uptake of phosphate and iron, phenazine biosynthesis, and other processes were identified as positive regulators of the mucoid phenotype on PIAAMV. Transcriptome analysis of P. aeruginosa cultures growing in the presence of vanadate showed differential expression of genes involved in virulence, envelope biogenesis, and cell stress pathways. In this study, it was observed that growth on PIAAMV attenuates P. aeruginosa in a mouse pneumonia model. Induction of alginate overproduction occurs as a stress response to protect P. aeruginosa, but it may be possible to modulate and inhibit these pathways based on the new genes identified in this study.

  1. 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.

  2. Imaging contrast effects in alginate microbeads containing trapped emulsion droplets.

    PubMed

    Hester-Reilly, Holly J; Shapley, Nina C

    2007-09-01

    This study focuses on spherical microparticles made of cross-linked alginate gel and microcapsules composed of an oil-in-water emulsion where the continuous aqueous phase is cross-linked into an alginate gel matrix. We have investigated the use of these easily manufactured microbeads as contrast agents for the study of the flow properties of fluids using nuclear magnetic resonance imaging. Results demonstrate that combined spin-spin (T(2)) relaxation and diffusion contrast in proton NMR imaging can be used to distinguish among rigid polymer particles, plain alginate beads, and alginate emulsion beads. Multi-echo CPMG spin-echo imaging indicates that the average spin-lattice (T(1)) and spin-spin (T(2)) relaxation times of the plain alginate and alginate emulsion beads are comparable. Meanwhile, diffusion-weighted imaging produces sharp contrast between the two types of alginate beads, due to restricted diffusion inside the embedded oil droplets of the alginate emulsion beads. While the signal obtained from most materials is severely attenuated under applied diffusion gradients, the alginate emulsion beads maintain signal strength. The alginate emulsion beads were added to a suspension and imaged in an abrupt, annular expansion flow. The emulsion beads could be clearly distinguished from the surrounding suspending fluid and rigid polystyrene particles, through either T(2) relaxation or diffusion contrast. Such a capability allows future use of the alginate emulsion beads as tracer particles and as one particle type among many in a multimodal suspension where detailed concentration profiles or particle size separation must be quantified during flow. PMID:17600742

  3. 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

  4. Cloning of the Trichoderma reesei cDNA Encoding a Glucuronan Lyase Belonging to a Novel Polysaccharide Lyase Family▿ †

    PubMed Central

    Konno, Naotake; Igarashi, Kiyohiko; Habu, Naoto; Samejima, Masahiro; Isogai, Akira

    2009-01-01

    The filamentous fungus Trichoderma reesei produces glucuronan lyase (TrGL) when it is grown on β-(1→4)-polyglucuronate (cellouronate) as a sole carbon source. The cDNA encoding TrGL was cloned, and the recombinant enzyme was heterologously expressed in Pichia pastoris. The cDNA of TrGL includes a 777-bp open reading frame encoding a 20-amino-acid signal peptide and the 238-amino-acid mature protein. The amino acid sequence showed no similarity to the amino acid sequences of previously described functional proteins, indicating that the enzyme should be classified in a novel polysaccharide lyase (PL) family. Recombinant TrGL catalyzed depolymerization of cellouronate endolytically by β-elimination and was highly specific for cellouronate. The enzyme was most active at pH 6.5 and 50°C, and its activity and thermostability increased in the presence of Ca2+, suggesting that its calcium dependence is similar to that of other PLs, such as pectate lyases. PMID:18978091

  5. ZnO nanoparticle fate in soil and zinc bioaccumulation in corn plants (Zea mays) influenced by alginate.

    PubMed

    Zhao, Lijuan; Hernandez-Viezcas, Jose Angel; Peralta-Videa, Jose R; Bandyopadhyay, Susmita; Peng, Bo; Munoz, Berenice; Keller, Arturo A; Gardea-Torresdey, Jorge L

    2013-01-01

    Nanoparticles (NPs) can interact with naturally occurring inorganic and organic substances in soils, which may change their transport behavior in soil and plants. This study was performed in two steps. In the first step, corn (Zea mays) plants were cultivated for one month in soil amended with 10 nm commercial spheroid ZnO NPs at 0–800 mg kg−1 and sodium alginate at 10 mg kg−1. In the second step, the plants were grown with ZnO NPs at 400 mg kg−1 and alginate at 0, 10, 50, and 100 mg kg−1. The dynamics of Zn concentrations in soil solution and Zn accumulation in plant tissues were determined by ICP-OES. Biomass accumulation, chlorophyll concentration, and the activity of antioxidant enzymes in leaves were also quantified. Results indicate that ZnO NPs coexisting with Zn dissolved species were continuously released to the soil solution to replenish the Zn ions or ZnO NPs scavenged by roots. At 400 and 800 mg kg−1, without alginate, ZnO NPs significantly reduced the root and shoot biomass production; however, plants treated with these NP concentrations, plus alginate, had significantly more Zn in tissues with no reduction in biomass production. Alginate significantly reduced the activity of stress enzymes catalase and peroxidase, which could indicate damage in the defense system. The effects of ZnO NPs in a food crop grown in alginate enriched soil, showing an excess of Zn in the aerial parts, are yet to be reported.

  6. A Controlled Drug-Delivery Experiment Using Alginate Beads

    ERIC Educational Resources Information Center

    Farrell, Stephanie; Vernengo, Jennifer

    2012-01-01

    This paper describes a simple, cost-effective experiment which introduces students to drug delivery and modeling using alginate beads. Students produce calcium alginate beads loaded with drug and measure the rate of release from the beads for systems having different stir rates, geometries, extents of cross-linking, and drug molecular weight.…

  7. Composite coating of alginate-olive oil enriched with antioxidants enhances postharvest quality and shelf life of Ber fruit (Ziziphus mauritiana Lamk. Var. Gola).

    PubMed

    Ramana Rao, T V; Baraiya, Nilanjana S; Vyas, Pinal B; Patel, Dhara M

    2016-01-01

    The present study has been carried out to evaluate the effect of a composite edible coating of 2 % Sodium alginate and 0.2 % Olive oil with combination of 1 % ascorbic acid and 1 % citric acid on the post harvest nutritional quality and shelf life of Ber fruit stored at 25 ± 2 °C and 65 % R.H. The coatings reduced the decay occurrence, weight loss, accumulation of total soluble solids (TSS) and total sugars in Ber fruit and enhanced the level of antioxidants. The delayed activity of polygalacturonase (PG), Pectate lyase (PL) and Pectin methyl esterase (PME) was noticed in coated fruits than that of the control fruit indicating the reduced softening and ripening process. These findings suggest that the composite edible coating tested under the current study has the potential to control decaying incidence of Ber fruit, extends its storage life and also improves its valuable nutritional characteristics.

  8. Preparation and characterization of alginate and alginate-resistant starch microparticles containing nisin.

    PubMed

    Hosseini, Seyede Marzieh; Hosseini, Hedayat; Mohammadifar, Mohammad Amin; German, J Bruce; Mortazavian, Amir Mohammad; Mohammadi, Abdorreza; Khosravi-Darani, Kianoosh; Shojaee-Aliabadi, Saeedeh; Khaksar, Ramin

    2014-03-15

    Delivery systems with sustained release of nisin have been proposed to improve stability and long-term effectiveness of this bacteriocin in foods. In this study, nisin was encapsulated in alginate (Alg) and alginate-resistant starch (Alg-RS) microparticles and its release was investigated. Studies found that the nisin concentration has significant influence on encapsulation efficiency (EE), loading capacity (LC) and size of both microparticles. Furthermore, encapsulation efficiency and loading capacity values were more increased by the addition of resistant starch to the alginate formulation. The highest encapsulation efficiency was obtained with Alg-RS microparticles prepared using initial nisin to alginate weight ratio of 25% w/w (59.77 ± 2.26%). Fourier transform-infrared (FT-IR) spectroscopy, X-ray diffraction (XRD) and differential scanning calorimetry (DSC) results confirmed the presence of nisin in the microparticles. The in vitro nisin release from these microparticles followed a controlled-release pattern consistent with a Fickian diffusion mechanism. The release rate from Alg-RS microparticles was less than that from the Alg microparticles.

  9. Ultra-structural changes and expression of chondrogenic and hypertrophic genes during chondrogenic differentiation of mesenchymal stromal cells in alginate beads

    PubMed Central

    Dashtdar, Havva; Selvaratnam, Lakshmi; Balaji Raghavendran, Hanumantharao; Suhaeb, Abdulrazzaq Mahmod; Ahmad, Tunku Sara

    2016-01-01

    Chondrogenic differentiation of mesenchymal stromal cells (MSCs) in the form of pellet culture and encapsulation in alginate beads has been widely used as conventional model for in vitro chondrogenesis. However, comparative characterization between differentiation, hypertrophic markers, cell adhesion molecule and ultrastructural changes during alginate and pellet culture has not been described. Hence, the present study was conducted comparing MSCs cultured in pellet and alginate beads with monolayer culture. qPCR was performed to assess the expression of chondrogenic, hypertrophic, and cell adhesion molecule genes, whereas transmission electron microscopy (TEM) was used to assess the ultrastructural changes. In addition, immunocytochemistry for Collagen type II and aggrecan and glycosaminoglycan (GAG) analysis were performed. Our results indicate that pellet and alginate bead cultures were necessary for chondrogenic differentiation of MSC. It also indicates that cultures using alginate bead demonstrated significantly higher (p < 0.05) chondrogenic but lower hypertrophic (p < 0.05) gene expressions as compared with pellet cultures. N-cadherin and N-CAM1 expression were up-regulated in second and third weeks of culture and were comparable between the alginate bead and pellet culture groups, respectively. TEM images demonstrated ultrastructural changes resembling cell death in pellet cultures. Our results indicate that using alginate beads, MSCs express higher chondrogenic but lower hypertrophic gene expression. Enhanced production of extracellular matrix and cell adhesion molecules was also observed in this group. These findings suggest that alginate bead culture may serve as a superior chondrogenic model, whereas pellet culture is more appropriate as a hypertrophic model of chondrogenesis. PMID:26966647

  10. Synthesis of D- and L-phenylalanine derivatives by phenylalanine ammonia lyases: a multienzymatic cascade process.

    PubMed

    Parmeggiani, Fabio; Lovelock, Sarah L; Weise, Nicholas J; Ahmed, Syed T; Turner, Nicholas J

    2015-04-01

    The synthesis of substituted D-phenylalanines in high yield and excellent optical purity, starting from inexpensive cinnamic acids, has been achieved with a novel one-pot approach by coupling phenylalanine ammonia lyase (PAL) amination with a chemoenzymatic deracemization (based on stereoselective oxidation and nonselective reduction). A simple high-throughput solid-phase screening method has also been developed to identify PALs with higher rates of formation of non-natural D-phenylalanines. The best variants were exploited in the chemoenzymatic cascade, thus increasing the yield and ee value of the D-configured product. Furthermore, the system was extended to the preparation of those L-phenylalanines which are obtained with a low ee value using PAL amination.

  11. 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. PMID:23795723

  12. Biotechnologically produced microbial alginate dressings show enhanced gel forming capacity compared to commercial alginate dressings of marine origin.

    PubMed

    Hoefer, Dirk; Schnepf, Julia K; Hammer, Timo R; Fischer, Melissa; Marquardt, Christoph

    2015-04-01

    Marine alginate fibre dressings are well established in wound management. Alginate fibres can absorb plenty of wound exudate due to their gel forming abilities and ion exchange. Alginates from bacteria have never been studied for medical applications so far, although the microbial polymer raises expectations for improved gelling capacity due to its unique O-acetylation. To prove the gelling capacity of bacterial alginate, we extracted the co-polymer from fermentation of the soil bacterium Azotobacter vinelandii ATCC 9046, cultivated on crude glycerol as an alternative carbon source. Bacterial alginate was isolated in high purity and extruded by a wet spinning method. Fibre structure and properties were characterised by infrared spectroscopy, NMR, GPC, scanning electron microscopy and tensile testing. The fibres could be processed into biocompatible needle web dressings, which showed more than twice the gel formation in saline compared to commercial dressings made of marine alginates. Gelled dressings of bacterial alginate formed stable hydrogels of sufficient shape and strength for wound healing applications. This work suggests that the increased gel formation of bacterial alginate from A. vinelandii may be optimal for the preparation of novel wound dressings.

  13. 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.

  14. Characterization of algG encoding C5-epimerase in the alginate biosynthetic gene cluster of Pseudomonas fluorescens.

    PubMed

    Morea, A; Mathee, K; Franklin, M J; Giacomini, A; O'Regan, M; Ohman, D E

    2001-10-31

    The organization of the alginate gene cluster in Pseudomonas fluorescens was characterized. A bank of genomic DNA from P. fluorescens was mobilized to a strain of Pseudomonas aeruginosa with a transposon insertion (algJ::Tn501) in the alginate biosynthetic operon that rendered it non-mucoid. Phenotypic complementation in this heterologous host was observed, and a complementing clone containing 32 kb of P. fluorescens DNA was obtained. Southern hybridization studies showed that genes involved in alginate biosynthesis (e.g. algD, algG, and algA) were approximately in the same order and position as in P. aeruginosa. When the clone was mobilized to a P. aeruginosa algG mutant that produced alginate as polymannuronate due to its C5-epimerase defect, complementation was observed and the alginate from the recombinant strain contained L-guluronate as determined by proton nuclear magnetic resonance spectroscopy. A sequence analysis of the P. fluorescens DNA containing algG revealed sequences similar to P. aeruginosa algG that were also flanked by algE- and algX-like sequences. The predicted AlgG amino acid sequence of P. fluorescens was 67% identical (80% similar) to P. aeruginosa AlgG and 60% identical (76% similar) to Azotobacter vinelandii AlgG. As in P. aeruginosa, AlgG from P. fluorescens appeared to have a signal sequence that would localize it to the periplasm where AlgG presumably acts as a C5-epimerase at the polymer level. Non-polar algG knockout mutants of P. fluorescens were defective in alginate production, suggesting a potential role for this protein in polymer formation.

  15. Possible role of cysteine-S-conjugate β-lyase in species differences in cisplatin nephrotoxicity.

    PubMed

    Katayama, Rieko; Nagata, Saori; Iida, Hiroko; Yamagishi, Norio; Yamashita, Tetsuro; Furuhama, Kazuhisa

    2011-09-01

    To better understand species differences in cisplatin nephrotoxicity, we focused on renal cysteine-S-conjugate β-lyase (C-S lyase), which may play a crucial role in the metabolism of platinum (Pt)-cysteine conjugates. Aminooxyacetic acid hemihydrochloride (AOAA), an inhibitor of C-S lyase, reduced renal injuries due to cisplatin in rats, suggesting involvement of C-S lyase. On day 5 following a bolus cisplatin injection, three species showed in vivo nephrotoxic potentials in the order of rats>mice=rabbits (the highest to lowest), based on body surface. The levels of renal Pt residue at the nephrotoxic dose were in order of rabbits>rats>mice. Meanwhile, the activity of endogenous (basal) mitochondrial aspartate aminotransferase (AST), one of the C-S lyases, in the renal cortex of naive animals was rats>mice=rabbits. In a qualitative Western blot analysis, expression of mitochondrial C-S lyase in the kidney was observed at approximately 37kDa in all five species used. In in vitro studies, the cytotoxicity of cisplatin was dependent on the expression level of C-S lyase mRNA in the respective renal cells. These results demonstrate that species differences in cisplatin nephrotoxicity are attributable to an interaction of renal Pt transition with C-S lyase activity.

  16. Composite ECM-alginate microfibers produced by microfluidics as scaffolds with biomineralization potential.

    PubMed

    Angelozzi, Marco; Miotto, Martina; Penolazzi, Letizia; Mazzitelli, Stefania; Keane, Timothy; Badylak, Stephen F; Piva, Roberta; Nastruzzi, Claudio

    2015-11-01

    A novel approach to produce artificial bone composites (microfibers) with distinctive features mimicking natural tissue was investigated. Currently proposed inorganic materials (e.g. apatite matrixes) lack self-assembly and thereby limit interactions between cells and the material. The present work investigates the feasibility of creating "bio-inspired materials" specifically designed to overcome certain limitations inherent to current biomaterials. We examined the dimensions, morphology, and constitutive features of a composite hydrogel which combined an alginate based microfiber with a gelatin solution or a particulate form of urinary bladder matrix (UBM). The effectiveness of the composite microfibers to induce and modulate osteoblastic differentiation in three-dimensional (3D) scaffolds without altering the viability and morphological characteristics of the cells was investigated. The present study describes a novel alginate microfiber production method with the use of microfluidics. The microfluidic procedure allowed for precise tuning of microfibers which resulted in enhanced viability and function of embedded cells.

  17. Enhanced stability of Bacillus licheniformis L-arabinose isomerase by immobilization with alginate.

    PubMed

    Zhang, Ye-Wang; Prabhu, Ponnandy; Lee, Jung-Kul; Kim, In-Won

    2010-01-01

    Recombinant Escherichia coli whole cells harboring Bacillus licheniformis L-arabinose isomerase (BLAI) were harvested to prepare alginate-immobilized biocatalysts. The operational conditions for immobilization were optimized according to relative activity and the cell leakage of the immobilized cell. The optimal conditions are as follows: alginate concentration, Ca(2+) concentration, cell mass loading, and curing time were 2% (w/v), 0.1 M, 50 g l(-1), and 4 hours, respectively. After immobilization, cross-linking with 0.1% glutaraldehyde significantly reduced cell leakage. The immobilized whole cells harboring BLAI were very stable with 89% residual activity remaining after 33 days of incubation at 50 degrees C and were much more stable than the free enzyme and cells. The results showed that immobilizing whole cells harboring BLAI is suitable for use as a biocatalyst in the production of L-ribulose, largely due to its high stability and low cost.

  18. Starch filler and osmoprotectants improve the survival of rhizobacteria in dried alginate beads.

    PubMed

    Schoebitz, Mauricio; Simonin, Hélène; Poncelet, Denis

    2012-01-01

    This work deals with optimising the cell survival of rhizobacteria encapsulated in alginate beads filled with starch. Immobilisation of rhizobacteria was done by dripping alginate-starch solution mixed with rhizobacteria into a calcium solution. Beads were analysed based on matrix formulation, bacteria growth phase, osmoprotectants and nature of calcium solution. Maximum cell recovery was obtained on Raoultella terrigena grown in medium supplemented with trehalose and calcium gluconate as gelling agent. Furthermore, dried beads containing Azospirillum brasilense presented 76% of viable cells after one year of storage. The survival of rhizobacteria during the bioencapsulation process can be improved by incorporating starch on beads composition, varying the growth phase of cells and using trehalose in growth culture medium. This work provides a selection of appropriate methods to improve the surviving rate of encapsulated cells during their production and long-term storage (∼1 year at 4°C).

  19. Characterization of dextransucrase immobilized on calcium alginate beads from Leuconostoc mesenteroides PCSIR-4.

    PubMed

    Ul Qader, Shah Ali; Aman, Afsheen; Syed, Noman; Bano, Saeeda; Azhar, Abid

    2007-06-01

    Immobilization of dextransucrase from Leuconostoc mesenteroides PCSIR-4 on alginate is optimized for application in the production of dextran from sucrose. Dextransucrase was partially purified by ethanol upto 2.5 fold. Properties of dextransucrase were less affected by immobilization on alginate beads from soluble enzyme. Highest activities of both soluble and immobilized dextransucrase found to be at 35 degrees C and optimum pH for activity remain 5.00. Substrate maxima for immobilized enzyme changed from 125 mg/ml to 200 mg/ml. Incubation time for enzyme-substrate reaction for maximum enzyme activity was increased from 15 minutes to 60 minutes in case of immobilized enzyme. Maximum stability of immobilized dextransucrase was achieved at 25 degrees C with respect to time.

  20. Preparation and activity of bubbling-immobilized cellobiase within chitosan-alginate composite.

    PubMed

    Wang, Fang; Su, Rong-Xin; Qi, Wei; Zhang, Ming-Jia; He, Zhi-Min

    2010-01-01

    Cellobiase can hydrolyze cellobiose into glucose; it plays a key role in the process of cellulose hydrolysis by reducing the product inhibition. To reuse the enzyme and improve the economic value of cellulosic ethanol, cellobiase was immobilized using sodium alginate and chitosan as carriers by the bubbling method. The immobilization conditions were optimized as follows: enzyme loading of 100 U cellobiase/g carrier, 30 min immobilization, 3.5 wt% sodium alginate, 0.25 wt% chitosan, and 2 wt% calcium chloride. Compared to free enzyme, the immobilized cellobiase had a decreased apparent K(m) and the maximum activity at a lower pH, indicating its higher acidic and thermal stability. The immobilized cellobiase was further tested in the hydrolysis of cellobiose and various cellulosic substrates (microcrystalline cellulose, filter paper, and ammonia-pretreated corn cobs). Together with cellulases, the immobilized cellobiase converted the cellulosic substrates into glucose with the rate and extent similar to the free enzyme.

  1. Structure and mechanism of the phycobiliprotein lyase CpcT.

    PubMed

    Zhou, Wei; Ding, Wen-Long; Zeng, Xiao-Li; Dong, Liang-Liang; Zhao, Bin; Zhou, Ming; Scheer, Hugo; Zhao, Kai-Hong; Yang, Xiaojing

    2014-09-26

    Pigmentation of light-harvesting phycobiliproteins of cyanobacteria requires covalent attachment of open-chain tetrapyrroles, bilins, to the apoproteins. Thioether formation via addition of a cysteine residue to the 3-ethylidene substituent of bilins is mediated by lyases. T-type lyases are responsible for attachment to Cys-155 of phycobiliprotein β-subunits. We present crystal structures of CpcT (All5339) from Nostoc (Anabaena) sp. PCC 7120 and its complex with phycocyanobilin at 1.95 and 2.50 Å resolution, respectively. CpcT forms a dimer and adopts a calyx-shaped β-barrel fold. Although the overall structure of CpcT is largely retained upon chromophore binding, arginine residues at the opening of the binding pocket undergo major rotameric rearrangements anchoring the propionate groups of phycocyanobilin. Based on the structure and mutational analysis, a reaction mechanism is proposed that accounts for chromophore stabilization and regio- and stereospecificity of the addition reaction. At the dimer interface, a loop extending from one subunit partially shields the opening of the phycocyanobilin binding pocket in the other subunit. Deletion of the loop or disruptions of the dimer interface significantly reduce CpcT lyase activity, suggesting functional relevance of the dimer. Dimerization is further enhanced by chromophore binding. The chromophore is largely buried in the dimer, but in the monomer, the 3-ethylidene group is accessible for the apophycobiliprotein, preferentially from the chromophore α-side. Asp-163 and Tyr-65 at the β- and α-face near the E-configured ethylidene group, respectively, support the acid-catalyzed nucleophilic Michael addition of cysteine 155 of the apoprotein to an N-acylimmonium intermediate proposed by Grubmayr and Wagner (Grubmayr, K., and Wagner, U. G. (1988) Monatsh. Chem. 119, 965-983). PMID:25074932

  2. Structure and mechanism of the phycobiliprotein lyase CpcT.

    PubMed

    Zhou, Wei; Ding, Wen-Long; Zeng, Xiao-Li; Dong, Liang-Liang; Zhao, Bin; Zhou, Ming; Scheer, Hugo; Zhao, Kai-Hong; Yang, Xiaojing

    2014-09-26

    Pigmentation of light-harvesting phycobiliproteins of cyanobacteria requires covalent attachment of open-chain tetrapyrroles, bilins, to the apoproteins. Thioether formation via addition of a cysteine residue to the 3-ethylidene substituent of bilins is mediated by lyases. T-type lyases are responsible for attachment to Cys-155 of phycobiliprotein β-subunits. We present crystal structures of CpcT (All5339) from Nostoc (Anabaena) sp. PCC 7120 and its complex with phycocyanobilin at 1.95 and 2.50 Å resolution, respectively. CpcT forms a dimer and adopts a calyx-shaped β-barrel fold. Although the overall structure of CpcT is largely retained upon chromophore binding, arginine residues at the opening of the binding pocket undergo major rotameric rearrangements anchoring the propionate groups of phycocyanobilin. Based on the structure and mutational analysis, a reaction mechanism is proposed that accounts for chromophore stabilization and regio- and stereospecificity of the addition reaction. At the dimer interface, a loop extending from one subunit partially shields the opening of the phycocyanobilin binding pocket in the other subunit. Deletion of the loop or disruptions of the dimer interface significantly reduce CpcT lyase activity, suggesting functional relevance of the dimer. Dimerization is further enhanced by chromophore binding. The chromophore is largely buried in the dimer, but in the monomer, the 3-ethylidene group is accessible for the apophycobiliprotein, preferentially from the chromophore α-side. Asp-163 and Tyr-65 at the β- and α-face near the E-configured ethylidene group, respectively, support the acid-catalyzed nucleophilic Michael addition of cysteine 155 of the apoprotein to an N-acylimmonium intermediate proposed by Grubmayr and Wagner (Grubmayr, K., and Wagner, U. G. (1988) Monatsh. Chem. 119, 965-983).

  3. Design and Fabrication of Anatomical Bioreactor Systems Containing Alginate Scaffolds for Cartilage Tissue Engineering

    PubMed Central

    Gharravi, Anneh Mohammad; Orazizadeh, Mahmoud; Ansari-Asl, Karim; Banoni, Salem; Izadi, Sina; Hashemitabar, Mahmoud

    2012-01-01

    The aim of the present study was to develop a tissue-engineering approach through alginate gel molding to mimic cartilage tissue in a three-dimensional culture system. The perfusion biomimetic bioreactor was designed to mimic natural joint. The shear stresses exerting on the bioreactor chamber were calculated by Computational Fluid Dynamic (CFD). Several alginate/bovine chondrocyte constructs were prepared, and were cultured in the bioreactor. Histochemical and immunohistochemical staining methods for the presence of glycosaminoglycan(GAG), overall matrix production and type II collagen protein were performed, respectively. The dynamic mechanical device applied a linear mechanical displacement of 2 mm to 10 mm. The CFD modeling indicated peak velocity and maximum wall shear stress were 1.706×10−3 m/s and 0.02407 dyne/cm 2, respectively. Histochemical and immunohistochemical analysis revealed evidence of cartilage-like tissue with lacunas similar to those of natural cartilage and the production of sulfated GAG of matrix by the chondrons, metachromatic territorial matrix-surrounded cells and accumulation of type II collagen around the cells. The present study indicated that when chondrocytes were seeded in alginate hydrogel and cultured in biomimetic cell culture system, cells survived well and secreted newly synthesized matrix led to improvement of chondrogenesis. PMID:23408660

  4. Alginate Particles as Platform for Drug Delivery by the Oral Route: State-of-the-Art

    PubMed Central

    2014-01-01

    Pharmaceutical research and development aims to design products with ensured safety, quality, and efficacy to treat disease. To make the process more rational, coherent, efficient, and cost-effective, the field of Pharmaceutical Materials Science has emerged as the systematic study of the physicochemical properties and behavior of materials of pharmaceutical interest in relation to product performance. The oral route is the most patient preferred for drug administration. The presence of a mucus layer that covers the entire gastrointestinal tract has been exploited to expand the use of the oral route by developing a mucoadhesive drug delivery system that showed a prolonged residence time. Alginic acid and sodium and potassium alginates have emerged as one of the most extensively explored mucoadhesive biomaterials owing to very good cytocompatibility and biocompatibility, biodegradation, sol-gel transition properties, and chemical versatility that make possible further modifications to tailor their properties. The present review overviews the most relevant applications of alginate microparticles and nanoparticles for drug administration by the oral route and discusses the perspectives of this biomaterial in the future. PMID:25101184

  5. Energy Landscape of Alginate-Epimerase Interactions Assessed by Optical Tweezers and Atomic Force Microscopy.

    PubMed

    Håti, Armend Gazmeno; Aachmann, Finn Lillelund; Stokke, Bjørn Torger; Skjåk-Bræk, Gudmund; Sletmoen, Marit

    2015-01-01

    Mannuronan C-5 epimerases are a family of enzymes that catalyze epimerization of alginates at the polymer level. This group of enzymes thus enables the tailor-making of various alginate residue sequences to attain various functional properties, e.g. viscosity, gelation and ion binding. Here, the interactions between epimerases AlgE4 and AlgE6 and alginate substrates as well as epimerization products were determined. The interactions of the various epimerase-polysaccharide pairs were determined over an extended range of force loading rates by the combined use of optical tweezers and atomic force microscopy. When studying systems that in nature are not subjected to external forces the access to observations obtained at low loading rates, as provided by optical tweezers, is a great advantage since the low loading rate region for these systems reflect the properties of the rate limiting energy barrier. The AlgE epimerases have a modular structure comprising both A and R modules, and the role of each of these modules in the epimerization process were examined through studies of the A- module of AlgE6, AlgE6A. Dynamic strength spectra obtained through combination of atomic force microscopy and the optical tweezers revealed the existence of two energy barriers in the alginate-epimerase complexes, of which one was not revealed in previous AFM based studies of these complexes. Furthermore, based on these spectra estimates of the locations of energy transition states (xβ), lifetimes in the absence of external perturbation (τ0) and free energies (ΔG#) were determined for the different epimerase-alginate complexes. This is the first determination of ΔG# for these complexes. The values determined were up to 8 kBT for the outer barrier, and smaller values for the inner barriers. The size of the free energies determined are consistent with the interpretation that the enzyme and substrate are thus not tightly locked at all times but are able to relocate. Together with the

  6. Energy Landscape of Alginate-Epimerase Interactions Assessed by Optical Tweezers and Atomic Force Microscopy

    PubMed Central

    Håti, Armend Gazmeno; Aachmann, Finn Lillelund; Stokke, Bjørn Torger; Skjåk-Bræk, Gudmund; Sletmoen, Marit

    2015-01-01

    Mannuronan C-5 epimerases are a family of enzymes that catalyze epimerization of alginates at the polymer level. This group of enzymes thus enables the tailor-making of various alginate residue sequences to attain various functional properties, e.g. viscosity, gelation and ion binding. Here, the interactions between epimerases AlgE4 and AlgE6 and alginate substrates as well as epimerization products were determined. The interactions of the various epimerase–polysaccharide pairs were determined over an extended range of force loading rates by the combined use of optical tweezers and atomic force microscopy. When studying systems that in nature are not subjected to external forces the access to observations obtained at low loading rates, as provided by optical tweezers, is a great advantage since the low loading rate region for these systems reflect the properties of the rate limiting energy barrier. The AlgE epimerases have a modular structure comprising both A and R modules, and the role of each of these modules in the epimerization process were examined through studies of the A- module of AlgE6, AlgE6A. Dynamic strength spectra obtained through combination of atomic force microscopy and the optical tweezers revealed the existence of two energy barriers in the alginate-epimerase complexes, of which one was not revealed in previous AFM based studies of these complexes. Furthermore, based on these spectra estimates of the locations of energy transition states (xβ), lifetimes in the absence of external perturbation (τ0) and free energies (ΔG#) were determined for the different epimerase–alginate complexes. This is the first determination of ΔG# for these complexes. The values determined were up to 8 kBT for the outer barrier, and smaller values for the inner barriers. The size of the free energies determined are consistent with the interpretation that the enzyme and substrate are thus not tightly locked at all times but are able to relocate. Together with the

  7. 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

  8. The reinforcement and healing of asphalt mastic mixtures by rejuvenator encapsulation in alginate compartmented fibres

    NASA Astrophysics Data System (ADS)

    Tabaković, A.; Post, W.; Cantero, D.; Copuroglu, O.; Garcia, S. J.; Schlangen, E.

    2016-08-01

    This paper explores the potential use of compartmented alginate fibres as a new method of incorporating rejuvenators into asphalt pavement mixtures. The compartmented fibres are employed to locally distribute the rejuvenator and to overcome the problems associated with spherical capsules and hollow fibres. The work presents proof of concept of the encapsulation process which involved embedding the fibres into the asphalt mastic mixture and the survival rate of fibres in the asphalt mixture. To prove the effectiveness of the alginate as a rejuvenator encapsulating material and to demonstrate its ability survive asphalt production process, the fibres containing the rejuvenator were prepared and subjected to thermogravimetric analysis and uniaxial tensile test. The test results demonstrated that fibres have suitable thermal and mechanical strength to survive the asphalt mixing and compaction process. The CT scan of an asphalt mortar mix containing fibres demonstrated that fibres are present in the mix in their full length, undamaged, providing confirmation that the fibres survived the asphalt production process. In order to investigate the fibres physiological properties and ability to release the rejuvenator into cracks in the asphalt mastic, the environmental scanning electron microscope and optical microscope analysis were employed. To prove its success as an asphalt healing system, compartmented alginate fibres containing rejuvenator were embedded in asphalt mastic mix. The three point bend tests were performed on the asphalt mastic test samples and the degree to which the samples began to self-heal in response was measured and quantified. The research findings indicate that alginate fibres present a promising new approach for the development of self-healing asphalt pavement systems.

  9. Improvement of 2,3-Butanediol Yield in Klebsiella pneumoniae by Deletion of the Pyruvate Formate-Lyase Gene

    PubMed Central

    Jung, Moo-Young; Mazumdar, Suman; Shin, Sang Heum; Yang, Kap-Seok; Lee, Jinwon

    2014-01-01

    Klebsiella pneumoniae is considered a good host strain for the production of 2,3-butanediol, which is a promising platform chemical with various industrial applications. In this study, three genes, including those encoding glucosyltransferase (wabG), lactate dehydrogenase (ldhA), and pyruvate formate-lyase (pflB), were disrupted in K. pneumoniae to reduce both its pathogenic characteristics and the production of several by-products. In flask cultivation with minimal medium, the yield of 2,3-butanediol from rationally engineered K. pneumoniae (ΔwabG ΔldhA ΔpflB) reached 0.461 g/g glucose, which was 92.2% of the theoretical maximum, with a significant reduction in by-product formation. However, the growth rate of the pflB mutant was slightly reduced compared to that of its parental strain. Comparison with similar mutants of Escherichia coli suggested that the growth defect of pflB-deficient K. pneumoniae was caused by redox imbalance rather than reduced level of intracellular acetyl coenzyme A (acetyl-CoA). From an analysis of the transcriptome, it was confirmed that the removal of pflB from K. pneumoniae significantly repressed the expression of genes involved in the formate hydrogen lyase (FHL) system. PMID:25085487

  10. 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.

  11. Evaluation of sodium alginate as drug release modifier in matrix tablets.

    PubMed

    Liew, Celine Valeria; Chan, Lai Wah; Ching, Ai Ling; Heng, Paul Wan Sia

    2006-02-17

    Alginates are useful natural polymers suitable for use in the design of pharmaceutical dosage forms. However, the effects of particle size, viscosity and chemical composition of alginates on drug release from alginate matrix tablets are not clearly understood. Hence, 17 grades of sodium alginate with different particle size distributions, viscosities and chemical compositions were used to prepare matrix tablets at various concentrations to screen the factors influencing drug release from such matrices. Particle size was found to have an influence on drug release from these matrices. Sodium alginate was subsequently classified into several size fractions and also cryogenically milled to produce smaller particle size samples. Cryogenic milling could be successfully applied to pulverize coarse alginate particles without changing the quality through degradation or segregation. This study showed the significance of each alginate property in modulating drug release: particle size is important in initial alginic acid gel barrier formation as it affected the extent of burst release; higher alginate viscosity slowed down drug release rate in the buffer phase but enhanced release rate in the acid phase; high M-alginate might be more advantageous than high-G-alginate in sustaining drug release; and, the effect of increasing alginate concentration was greater with larger alginate particles. This can serve as a framework for formulators working with alginates. Furthermore, the results showed that sodium alginate matrices can sustain drug release for at least 8 h, even for a highly water-soluble drug in the presence of a water-soluble excipient. PMID:16364576

  12. Chitosan-alginate membranes accelerate wound healing.

    PubMed

    Caetano, Guilherme Ferreira; Frade, Marco Andrey Cipriani; Andrade, Thiago Antônio Moretti; Leite, Marcel Nani; Bueno, Cecilia Zorzi; Moraes, Ângela Maria; Ribeiro-Paes, João Tadeu

    2015-07-01

    The purpose of this study was to evaluate the efficacy of chitosan-alginate membrane to accelerate wound healing in experimental cutaneous wounds. Two wounds were performed in Wistar rats by punching (1.5 cm diameter), treated with membranes moistened with saline solution (CAM group) or with saline only (SL group). After 2, 7, 14, and 21 days of surgery, five rats of each group were euthanized and reepithelialization was evaluated. The wounds/scars were harvested for histological, flow cytometry, neutrophil infiltrate, and hydroxyproline analysis. CAM group presented higher inflammatory cells recruitment as compared to SL group on 2(nd) day. On the 7(th) day, CAM group showed higher CD11b(+) level and lower of neutrophils than SL group. The CAM group presented higher CD4(+) cells influx than SL group on 2(nd) day, but it decreased during the follow up and became lower on 14(th) and 21(st) days. Higher fibroplasia was noticed on days 7 and 14 as well as higher collagenesis on 21(st) in the CAM group in comparison to SL group. CAM group showed faster reepithelialization on 7(th) day than SL group, although similar in other days. In conclusion, chitosan-alginate membrane modulated the inflammatory phase, stimulated fibroplasia and collagenesis, accelerating wound healing process in rats.

  13. 3-Methylglutaconyl-CoA hydratase, 3-methylcrotonyl-CoA carboxylase and 3-hydroxy-3-methylglutaryl-CoA lyase deficiencies: a coupled enzyme assay useful for their detection.

    PubMed

    Narisawa, K; Gibson, K M; Sweetman, L; Nyhan, W L

    1989-09-15

    A coupled assay has been developed using 3-methylcrotonyl-CoA and NaH14CO3 which permits the detection of deficiencies of 3-methylcrotonyl-CoA carboxylase, 3-methylglutaconyl-CoA hydratase and 3-hydroxy-3-methylglutaryl CoA-lyase. The products of the reaction were analyzed by high performance liquid chromatography. Using this method the site of the defect was documented in a patient with deficiency of 3-methylcrotonyl-CoA carboxylase, 2 patients with deficiency of 3-methyl-glutaconyl-CoA hydratase, and 2 patients with deficiency of 3-hydroxy-3-methyl-glutaryl-CoA lyase.

  14. 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.

  15. 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. PMID:27393563

  16. An anomalous behavior of trypsin immobilized in alginate network.

    PubMed

    Ganachaud, Chrystelle; Bernin, Diana; Isaksson, Dan; Holmberg, Krister

    2013-05-01

    Alginate is a biopolymer used in drug formulations and for surgical purposes. In the presence of divalent cations, it forms solid gels, and such gels are of interest for immobilization of cells and enzymes. In this work, we entrapped trypsin in an alginate gel together with a known substrate, N α-benzoyl-L-arginine-4-nitroanilide hydrochloride (L-BAPNA), and in the presence or absence of D-BAPNA, which is known to be a competitive inhibitor. Interactions between alginate and the substrate as well as the enzyme were characterized with transmission electron microscopy, rheology, and nuclear magnetic resonance spectroscopy. The biocatalysis was monitored by spectrophotometry at temperatures ranging from 10 to 42 °C. It was found that at 37 and 42 °C a strong acceleration of the reaction was obtained, whereas at 10 °C and at room temperature, the presence of D-BAPNA leads to a retardation of the reaction rate. The same effect was found when the reaction was performed in a non-cross-linked alginate solution. In alginate-free buffer solution, as well as in a solution of carboxymethylcellulose, a biopolymer that resembles alginate, the normal behavior was obtained; however, with D-BAPNA acting as an inhibitor at all temperatures. A more detailed investigation of the reaction kinetics showed that at higher temperature and in the presence of alginate, the curve of initial reaction rate versus L-BAPNA concentration had a sigmoidal shape, indicating an allosteric behavior. We believe that the anomalous behavior of trypsin in the presence of alginate is due to conformational changes caused by interactions between the positively charged trypsin and the strongly negatively charged alginate.

  17. Altered Fermentative Metabolism in Chlamydomonas reinhardtii Mutants Lacking Pyruvate Formate Lyase and Both Pyruvate Formate Lyase and Alcohol Dehydrogenase[W

    PubMed Central

    Catalanotti, Claudia; Dubini, Alexandra; Subramanian, Venkataramanan; Yang, Wenqiang; Magneschi, Leonardo; Mus, Florence; Seibert, Michael; Posewitz, Matthew C.; Grossman, Arthur R.

    2012-01-01

    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. PMID:22353371

  18. Chemical modification of alginic acid by ultrasonic irradiation

    NASA Astrophysics Data System (ADS)

    Murdzheva, Dilyana; Denev, Panteley

    2016-03-01

    Abstract: Chemical modification of alginic acid has been done by ultrasonic irradiation to obtain its methylated, ethylated and isopropylated derivatives. The influence of ultrasonic frequency and power on esterification process of alginic acid has been investigated. Alginate derivatives have been characterized by degree of esterification (DE) and IR-FT spectroscopy. It has been found that 45 kHz ultrasonic frequency accelerated modification process as reduced the reaction time from 16 hours to 2 hours. The obtained results showed that ultrasound irradiation increased the reaction efficiency in methanol and depended on the ratio of the M/G.

  19. Cloning of Escherichia coli and Pseudomonas aeruginosa phosphomannose isomerase genes and their expression in alginate-negative mutants of Pseudomonas aeruginosa.

    PubMed Central

    Darzins, A; Nixon, L L; Vanags, R I; Chakrabarty, A M

    1985-01-01

    The phosphomannose isomerase (pmi) gene of Escherichia coli was cloned on a broad-host-range cosmid vector and expressed in Pseudomonas aeruginosa at a low level. Plasmid pAD3, which harbors the E. coli pmi gene, contains a 6.2-kilobase-pair HindIII fragment derived from the chromosome of E. coli. Subcloning produced plasmids carrying the 1.5-kilobase-pair HindIII-HpaI subfragment of pAD3 that restored alginic acid production in a nonmucoid, alginate-negative mutant of P. aeruginosa. This fragment also complemented mannose-negative, phosphomannose isomerase-negative mutants of E. coli and showed no homology by DNA-DNA hybridization to P. aeruginosa chromosomal DNA. By using a BamHI constructed cosmid clone bank of the stable alginate producing strain 8830, we have been able to isolate a recombinant plasmid of P. aeruginosa origin that also restores alginate production in the alginate-negative mutant. This new recombinant plasmid, designated pAD4, contained a 9.9-kilobase-pair EcoRI-BamHI fragment with the ability to restore alginate synthesis in the alginate-negative P. aeruginosa. This fragment showed no homology to E. coli chromosomal DNA or to plasmid pAD3. Both mucoid and nonmucoid strains of P. aeruginosa had no detectable levels of phosphomannose isomerase activity as measured by mannose 6-phosphate-to-fructose 6-phosphate conversion. However, P. aeruginosa strains harboring the cloned pmi gene of E. coli contained measurable levels of phosphomannose isomerase activity as evidenced by examining the conversion of mannose 6-phosphate to fructose 6-phosphate. Images PMID:3918000

  20. Hydroxyapatite-doped alginate beads as scaffolds for the osteoblastic differentiation of mesenchymal stem cells.

    PubMed

    Wang, Martha O; Bracaglia, Laura; Thompson, Joshua A; Fisher, John P

    2016-09-01

    This work investigates the role of an osteoblastic matrix component, hydroxyapatite (HA), in modular alginate scaffolds to support osteoblastic differentiation of human mesenchymal stem cells for the purpose of tissue engineered bone constructs. This system is first evaluated in a tubular perfusion bioreactor, which has been shown to improve osteoblastic differentiation over static culture conditions. HMSCs in alginate scaffolds that contain HA show increased osteoblastic gene expression compared to cells in pure alginate scaffolds, as well as significantly more matrix production and mineralization. The differentiated hMSCs and cell-laid matrix are ultimately evaluated in an in vivo site specific model. Implantation of these scaffolds with preformed matrix into the rat femoral condyle defects results in abundant bone growth and significant incorporation of the scaffold into the surrounding tissue. The developed mineralized matrix, induced in part by the HA component in the scaffold, could lead to increased tissue development in critically sized defects, and should be included in future implant strategies. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2325-2333, 2016.

  1. Prosopis alba exudate gum as excipient for improving fish oil stability in alginate-chitosan beads.

    PubMed

    Vasile, Franco Emanuel; Romero, Ana María; Judis, María Alicia; Mazzobre, María Florencia

    2016-01-01

    The aim of the present work was to employ an exudate gum obtained from a South American wild tree (Prosopis alba), as wall material component to enhance the oxidative stability of fish oil encapsulated in alginate-chitosan beads. For this purpose, beads were vacuum-dried and stored under controlled conditions. Oxidation products, fatty acid profiles and lipid health indices were measured during storage. Alginate-chitosan interactions and the effect of gum were manifested in the FT-IR spectra. The inclusion of the gum in the gelation media allowed decreasing the oxidative damage during storage in comparison to the free oil and alginate-chitosan beads. The gum also improved wall material properties, providing higher oil retention during the drying step and subsequent storage. Fatty acids quality and lipid health indices were widely preserved in beads containing the gum. Present results showed a positive influence of the gum on oil encapsulation and stability, being the main mechanism attributed to a physical barrier effect.

  2. Alginate/chitosan nanoparticles for encapsulation and controlled release of vitamin B2.

    PubMed

    Azevedo, Maria A; Bourbon, Ana I; Vicente, António A; Cerqueira, Miguel A

    2014-11-01

    This work aims at evaluating encapsulation and controlled release of vitamin B2 from alginate/chitosan nanoparticles. Ionotropic polyelectrolyte pre-gelation was used as production method being chitosan and alginate used as main materials. Nanoparticles were characterized in terms of average size, polydispersity index (PDI), zeta potential and vitamin entrapment efficiency. The average size for alginate/chitosan nanoparticles was 119.5±49.9nm for samples without vitamin B2 and 104.0±67.2nm with the encapsulation of vitamin B2, presenting a PDI of 0.454±0.066 and 0.319±0.068, respectively. The nanoparticles showed encapsulation efficiency and loading capacity values of 55.9±5.6% and 2.2±0.6%, respectively. Release profiles were evaluated at different conditions showing that the polymeric relaxation was the most influent phenomenon in vitamin B2 release. In order to study their stability nanoparticles were stored at 4°C being particles sizes and PDI evaluated during 5 months showing the results that vitamin B2-loaded nanoparticles are more stable (in terms of size and PDI) than nanoparticles without vitamin B2.

  3. Psychrotrophic Streptomyces spp. cells immobilisation in alginate microspheres produced by emulsification-internal gelation.

    PubMed

    Cotârleţ, Mihaela; Dima, Stefan; Bahrim, Gabriela

    2014-01-01

    The objective of the investigations was the optimisation of the parameters for cold-adapted Streptomyces MIUG 4 Alga strain cells immobilisation using emulsification-internal gelation technique in calcium alginate microspheres and testing their ability to produce cold-active β-amylase. By Box-Behnken design and response surface methodology, the effects of independent variables were established, which included sodium alginate concentration (A), sodium alginate:living cell ratio (B) and the Span 80 concentration (C) upon microspheres formation and their functionality. Mean diameter of formed microspheres with immobilised biomass and cold-active β-amylase production were chosen as dependent variables in order to increase the yield of starch hydrolysis. Diameters of microspheres <25.5 μm provided large yield of cold-active β-amylase comparing with microspheres with bigger diameter. A 1.5-fold increase in the substrate hydrolysis yield was achieved using the immobilised biocatalyst compared with the crude enzyme extract, after 96 h of substrate bioconversion.

  4. Encapsulation of liquid smoke flavoring in ca-alginate and ca-alginate-chitosan beads.

    PubMed

    Petzold, Guillermo; Gianelli, María Pia; Bugueño, Graciela; Celan, Raymond; Pavez, Constanza; Orellana, Patricio

    2014-01-01

    Encapsulation is a technique used in foods that may protect some compounds with sensory impact, in particular flavoring as liquid smoke. We used the dripping method, obtaining two different layers for encapsulation of liquid smoke: calcium alginate and calcium alginate-chitosan. The results show that the load capacity of liquid smoke encapsulation reached values above 96 %. The beads exhibit syneresis at room temperature, but in opposite side, refrigeration temperature stabilizes the hydrogel of beads, allowing the samples loss weight less than 3 % after 72 h. Heated capsules with liquid smoke released several volatile compounds in the headspace and may identify 66 compounds. Among these volatile compounds, phenols derivatives can be considered sensory descriptors to contribute to the specific flavor of smoke. We conclude that the dripping method is highly efficient to encapsulate liquid smoke and released several volatile compounds, although it is necessary to minimize syneresis at room temperature.

  5. Genetics of Bacterial Alginate: Alginate Genes Distribution, Organization and Biosynthesis in Bacteria

    PubMed Central

    Muhammadi; Ahmed, Nuzhat

    2007-01-01

    Bacterial alginate genes are chromosomal and fairly widespread among rRNA homology group I Pseudomonads and Azotobacter. In both genera, the genetic pathway of alginate biosynthesis is mostly similar and the identified genes are identically organized into biosynthetic, regulatory and genetic switching clusters. In spite of these similarities,still there are transcriptional and functional variations between P. aeruginosa and A. vinelandii. In P. aeruginosa all biosynthetic genes except algC transcribe in polycistronic manner under the control of algD promoter while in A. vinelandii, these are organized into many transcriptional units. Of these, algA and algC are transcribed each from two different and algD from three different promoters. Unlike P. aeruginosa, the promoters of these transcriptional units except one of algC and algD are algT-independent. Both bacterial species carry homologous algG gene for Ca2+-independent epimerization. But besides algG, A. vinelandii also has algE1-7 genes which encode C-5-epimerases involved in the complex steps of Ca2+-dependent epimerization. A hierarchy of alginate genes expression under σ22(algT) control exists in P. aeruginosa where algT is required for transcription of the response regulators algB and algR, which in turn are necessary for expression of algD and its downstream biosynthetic genes. Although algTmucABCD genes cluster play similar regulatory roles in both P. aeruginosa and A. vinelandii but unlike, transcription of A. vinelandii, algR is independent of σ22. These differences could be due to the fact that in A. vinelandii alginate plays a role as an integrated part in desiccation-resistant cyst which is not found in P. aeruginosa. PMID:18645604

  6. Fabrication of cross-linked alginate beads using electrospraying for adenovirus delivery.

    PubMed

    Park, Hongkwan; Kim, Pyung-Hwan; Hwang, Taewon; Kwon, Oh-Joon; Park, Tae-Joon; Choi, Sung-Wook; Yun, Chae-Ok; Kim, Jung Hyun

    2012-05-10

    Cross-linked alginate beads containing adenovirus (Ad) were successfully fabricated using an electrospraying method to achieve the protection and release of Ad in a controlled manner. An aqueous alginate solution containing Ad was electrosprayed into an aqueous phase containing a cross-linking agent (calcium chloride) at different process variables (voltages, alginate concentrations, and flow rates). Alginate beads containing Ad were used for transduction of U343 glioma cells and the transduction efficiency of the alginate beads was measured by quantification of gene expression using a fluorescence-activated cell sorter at different time points. In vitro results of gene expression revealed that the Ad encapsulated in the alginate beads with 0.5 wt% of alginate concentration exhibited a high activity for a long period (over 7 days) and was released in a sustained manner from the alginate beads. The Ad-encapsulating alginate beads could be promising materials for local delivery of Ad at a high concentration into target sites.

  7. Preparation of novel silica-coated alginate gel beads for efficient encapsulation of yeast alcohol dehydrogenase.

    PubMed

    Xu, Song-Wei; Lu, Yang; Li, Jian; Zhang, Yu-Fei; Jiang, Zhong-Yi

    2007-01-01

    Biomimetic formation has undoubtedly inspired the preparation of novel organic-inorganic hybrid composites. In this study, silica-coated alginate gel beads were prepared by coating the surface of alginate gel beads with silica film derived from tetramethoxysilane (TMOS). The composition and structure of the silica film were characterized by FT-IR and SEM equipped with EDX. The swelling behavior of silica-coated alginate gel beads was studied to be more stable against swelling than that of alginate gel beads. The results showed that silica-coated alginate gel beads exhibited appropriate diffusion property. The effective diffusion coefficient (D(e)) of NADH in silica-coated alginate beads was 1.76 x 10(-10) m2/s, while the effective diffusion coefficient in alginate beads was 1.84 x 10(-10) m2/s. The model enzyme yeast alcohol dehydrogenase (YADH) was encapsulated in silica-coated alginate and pure alginate beads, respectively. Enzyme leakage of YADH in alginate gel beads was determined to be 32%, while the enzyme leakage in silica-coated alginate gel beads was as low as 11%. Furthermore, the relative activity of YADH in alginate gel beads decreased almost to zero after 10 recycles, while the relative activity of YADH in silica-coated alginate gel beads was 81.3%. The recycling stability of YADH in silica-coated alginate gel beads was found to be increased significantly mainly due to the effective inhibition of enzyme leakage by compact silica film.

  8. Development of functionalized multi-walled carbon-nanotube-based alginate hydrogels for enabling biomimetic technologies

    NASA Astrophysics Data System (ADS)

    Joddar, Binata; Garcia, Eduardo; Casas, Atzimba; Stewart, Calvin M.

    2016-08-01

    Alginate is a hydrogel commonly used for cell culture by ionically crosslinking in the presence of divalent Ca2+ ions. However these alginate gels are mechanically unstable, not permitting their use as scaffolds to engineer robust biological bone, breast, cardiac or tumor tissues. This issue can be addressed via encapsulation of multi-walled carbon nanotubes (MWCNT) serving as a reinforcing phase while being dispersed in a continuous phase of alginate. We hypothesized that adding functionalized MWCNT to alginate, would yield composite gels with distinctively different mechanical, physical and biological characteristics in comparison to alginate alone. Resultant MWCNT-alginate gels were porous, and showed significantly less degradation after 14 days compared to alginate alone. In vitro cell-studies showed enhanced HeLa cell adhesion and proliferation on the MWCNT-alginate compared to alginate. The extent of cell proliferation was greater when cultured atop 1 and 3 mg/ml MWCNT-alginate; although all MWCNT-alginates lead to enhanced cell cluster formation compared to alginate alone. Among all the MWCNT-alginates, the 1 mg/ml gels showed significantly greater stiffness compared to all other cases. These results provide an important basis for the development of the MWCNT-alginates as novel substrates for cell culture applications, cell therapy and tissue engineering.

  9. Development of functionalized multi-walled carbon-nanotube-based alginate hydrogels for enabling biomimetic technologies

    PubMed Central

    Joddar, Binata; Garcia, Eduardo; Casas, Atzimba; Stewart, Calvin M.

    2016-01-01

    Alginate is a hydrogel commonly used for cell culture by ionically crosslinking in the presence of divalent Ca2+ ions. However these alginate gels are mechanically unstable, not permitting their use as scaffolds to engineer robust biological bone, breast, cardiac or tumor tissues. This issue can be addressed via encapsulation of multi-walled carbon nanotubes (MWCNT) serving as a reinforcing phase while being dispersed in a continuous phase of alginate. We hypothesized that adding functionalized MWCNT to alginate, would yield composite gels with distinctively different mechanical, physical and biological characteristics in comparison to alginate alone. Resultant MWCNT-alginate gels were porous, and showed significantly less degradation after 14 days compared to alginate alone. In vitro cell-studies showed enhanced HeLa cell adhesion and proliferation on the MWCNT-alginate compared to alginate. The extent of cell proliferation was greater when cultured atop 1 and 3 mg/ml MWCNT-alginate; although all MWCNT-alginates lead to enhanced cell cluster formation compared to alginate alone. Among all the MWCNT-alginates, the 1 mg/ml gels showed significantly greater stiffness compared to all other cases. These results provide an important basis for the development of the MWCNT-alginates as novel substrates for cell culture applications, cell therapy and tissue engineering. PMID:27578567

  10. Development of functionalized multi-walled carbon-nanotube-based alginate hydrogels for enabling biomimetic technologies.

    PubMed

    Joddar, Binata; Garcia, Eduardo; Casas, Atzimba; Stewart, Calvin M

    2016-01-01

    Alginate is a hydrogel commonly used for cell culture by ionically crosslinking in the presence of divalent Ca(2+) ions. However these alginate gels are mechanically unstable, not permitting their use as scaffolds to engineer robust biological bone, breast, cardiac or tumor tissues. This issue can be addressed via encapsulation of multi-walled carbon nanotubes (MWCNT) serving as a reinforcing phase while being dispersed in a continuous phase of alginate. We hypothesized that adding functionalized MWCNT to alginate, would yield composite gels with distinctively different mechanical, physical and biological characteristics in comparison to alginate alone. Resultant MWCNT-alginate gels were porous, and showed significantly less degradation after 14 days compared to alginate alone. In vitro cell-studies showed enhanced HeLa cell adhesion and proliferation on the MWCNT-alginate compared to alginate. The extent of cell proliferation was greater when cultured atop 1 and 3 mg/ml MWCNT-alginate; although all MWCNT-alginates lead to enhanced cell cluster formation compared to alginate alone. Among all the MWCNT-alginates, the 1 mg/ml gels showed significantly greater stiffness compared to all other cases. These results provide an important basis for the development of the MWCNT-alginates as novel substrates for cell culture applications, cell therapy and tissue engineering. PMID:27578567

  11. 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

  12. The Alginate Demonstration: Polymers, Food Science, and Ion Exchange

    NASA Astrophysics Data System (ADS)

    Waldman, Amy Sue; Schechinger, Linda; Govindarajoo, Geeta; Nowick, James S.; Pignolet, Louis H.

    1998-11-01

    We have recently devised a polymer demonstration involving the crosslinking and decrosslinking of alginate, a polysaccharide isolated from seaweed. The polymer is composed of D-mannuronic acid and L-guluronic acid subunits and is a component of cell walls. It is commonly used as a thickener in foods such as ice cream and fruit-filled snacks. For the demonstration, a 2% solution of sodium alginate is poured into a 1% solution of calcium chloride. Nontoxic calcium alginate "worms" form due to crosslinking of the polymer. Alternatively, the commercially available antacid Gaviscon can be used as a source of sodium alginate. The crosslinks can then be broken by shaking the worms in brine. The demonstration is a fine addition to any chemical educator's repertoire of polymer experiments.

  13. 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

  14. Enzyme-entrapping behaviors in alginate fibers and their papers

    SciTech Connect

    Kobayashi, Y.; Matsuo, R.; Ohya, T.; Yokoi, N.

    1987-01-01

    Enzyme immobilization in the form of fiber and paper was easily achieved by wet spinning of aqueous admixture of sodium alginate and enzymes into divalent metallic ion solution as a coagulating bath, followed by paper making of resultant shortly cut fibers. Entrapment yields of enzymes used, e.g., glucoamylase, cyclodextrin glucanotransferase, endo-polygalacturonase, and protease, were always higher in calcium alginate fibers and their papers than those in corresponding beads. It was found that the yields increased with an increase of the discharge rate through the spinning nozzle because the higher discharge rate could provide more highly oriented metal-chelate linear polymer molecules along the fiber axis for preventing leakage of entrapped enzymes. Divalent metallic ions affected greatly the entrapment of glucoamylase in alginate fibers, the order of which followed rougly the ionotropic series of Thiele. Entrapment of glucoamylase in bicomponent systems comprising alginate and other water-soluble polymers was also investigated. (Refs. 41).

  15. Modified alginate and chitosan for lactic acid bacteria immobilization.

    PubMed

    Le-Tien, Canh; Millette, Mathieu; Mateescu, Mircea-Alexandru; Lacroix, Monique

    2004-06-01

    Beads with enhanced-stability acid media, which were based on alginate and chitosan functionalized by succinylation (increasing the anionic charges able to retain protons) or by acylation (improving matrix hydrophobicity), were developed for immobilization of bacterial cells. Beads (3 mm diameter) formed by ionotropic gelation with CaCl(2) presented good mechanical characteristics. After 30 min incubation of viable free Lactobacillus rhamnosus cells in simulated gastric fluid (pH 1.5), we noticed that the level of viable bacteria was undetectable. Bacterial immobilization in native-alginate-based beads generated a viable-cell count of 22-26%, whereas, when entrapped in succinylated alginate and chitosan beads, the percentage of viable cells was of 60 and 66%, respectively. Best viability (87%) was found for bacteria immobilized in N -palmitoylaminoethyl alginate, which affords a high protective effect, probably due to long alkyl pendants that improve the beads' hydrophobicity, limiting hydration in the acidic environment.

  16. Calcium phosphate-alginate microspheres as enzyme delivery matrices.

    PubMed

    Ribeiro, C C; Barrias, C C; Barbosa, M A

    2004-08-01

    The present study concerns the preparation and initial characterisation of novel calcium titanium phosphate-alginate (CTP-alginate) and hydroxyapatite-alginate (HAp-alginate) microspheres, which are intended to be used as enzyme delivery matrices and bone regeneration templates. Microspheres were prepared using different concentrations of polymer solution (1% and 3% w/v) and different ceramic-to-polymer solution ratios (0.1, 0.2 and 0.4 w/w). Ceramic powders were characterised using X-ray diffraction, laser granulometry, Brunauer, Emmel and Teller (BET) method for the determination of surface area, zeta potential and Fourier transform infrared spectroscopy (FT-IR). Alginate was characterised using high performance size exclusion chromatography. The methodology followed in this investigation enabled the preparation of homogeneous microspheres with a uniform size. Studies on the immobilisation and release of the therapeutic enzyme glucocerebrosidase, employed in the treatment of Gaucher disease, were also performed. The enzyme was incorporated into the ceramic-alginate matrix before gel formation in two different ways: pre-adsorbed onto the ceramic particles or dispersed in the polymeric matrix. The two strategies resulted in distinct release profiles. Slow release was obtained after adsorption of the enzyme to the ceramic powders, prior to preparation of the microspheres. An initial fast release was achieved when the enzyme and the ceramic particles were dispersed in the alginate solution before producing the microspheres. The latter profile is very similar to that of alginate microspheres. The different patterns of enzyme release increase the range of possible applications of the system investigated in this work.

  17. Nanocellulose-alginate hydrogel for cell encapsulation.

    PubMed

    Park, Minsung; Lee, Dajung; Hyun, Jinho

    2015-02-13

    TEMPO-oxidized bacterial cellulose (TOBC)-sodium alginate (SA) composites were prepared to improve the properties of hydrogel for cell encapsulation. TOBC fibers were obtained using a TEMPO/NaBr/NaClO system at pH 10 and room temperature. The fibrillated TOBCs mixed with SA were cross-linked in the presence of Ca(2+) solution to form hydrogel composites. The compression strength and chemical stability of the TOBC/SA composites were increased compared with the SA hydrogel, which indicated that TOBC performed an important function in enhancing the structural, mechanical and chemical stability of the composites. Cells were successfully encapsulated in the TOBC/SA composites, and the viability of cells was investigated. TOBC/SA composites can be a potential candidate for cell encapsulation engineering. PMID:25458293

  18. Viscoelastic properties of mineralized alginate hydrogel beads.

    PubMed

    Olderøy, Magnus O; Xie, Minli; Andreassen, Jens-Petter; Strand, Berit L; Zhang, Zhibing; Sikorski, Pawel

    2012-07-01

    Alginate hydrogels have applications in biomedicine, ranging from delivery of cells and growth factors to wound management aids. However, they are mechanically soft and have shown little potential for the use in bone tissue engineering. Here, the viscoelastic properties of alginate hydrogel beads mineralized with calcium phosphate, both by a counter-diffusion (CD) and an enzymatic approach, are characterized by a micro-manipulation technique and mathematical modeling. Fabricated hydrogel materials have low mineral content (below 3 % of the total hydrogel mass, which corresponds to mineral content of up to 60 % of the dry mass) and low dry mass content (<5 %). For all samples compression and hold (relaxation after compression) data was collected and analyzed. The apparent Young's modulus of the mineralized beads was estimated by the Hertz model (compression data) and was shown to increase up to threefold upon mineralization. The enzymatically mineralized beads showed higher apparent Young's modulus compared to the ones mineralized by CD, even though the mineral content of the former was lower. Full compression-relaxation force-time profiles were analyzed using viscoelastic model. From this analysis, infinite and instantaneous Young's moduli were determined. Similarly, enzymatic mineralized beads, showed higher instantaneous and infinite Young's modulus, even if the degree of mineralization is lower then that achieved for CD method. This leads to the conclusion that both the degree of mineralization and the spatial distribution of mineral are important for the mechanical performance of the composite beads, which is in analogy to highly structured mineralized tissues found in many organisms.

  19. Sodium alginate decreases the permeability of intestinal mucus

    PubMed Central

    Mackie, Alan R.; Macierzanka, Adam; Aarak, Kristi; Rigby, Neil M.; Parker, Roger; Channell, Guy A.; Harding, Stephen E.; Bajka, Balazs H.

    2016-01-01

    In the small intestine the nature of the environment leads to a highly heterogeneous mucus layer primarily composed of the MUC2 mucin. We set out to investigate whether the soluble dietary fibre sodium alginate could alter the permeability of the mucus layer. The alginate was shown to freely diffuse into the mucus and to have minimal effect on the bulk rheology when added at concentrations below 0.1%. Despite this lack of interaction between the mucin and alginate, the addition of alginate had a marked effect on the diffusion of 500 nm probe particles, which decreased as a function of increasing alginate concentration. Finally, we passed a protein stabilised emulsion through a simulation of oral, gastric and small intestinal digestion. We subsequently showed that the addition of 0.1% alginate to porcine intestinal mucus decreased the diffusion of fluorescently labelled lipid present in the emulsion digesta. This reduction may be sufficient to reduce problems associated with high rates of lipid absorption such as hyperlipidaemia. PMID:26726279

  20. Encapsulation of fibroblasts causes accelerated alginate hydrogel degradation.

    PubMed

    Hunt, N C; Smith, A M; Gbureck, U; Shelton, R M; Grover, L M

    2010-09-01

    Calcium-alginate hydrogel has been widely studied as a material for cell encapsulation for tissue engineering. At present, the effect that cells have on the degradation of alginate hydrogel is largely unknown. We have shown that fibroblasts encapsulated at a density of 7.5 x 10(5) cells ml(-1) in both 2% and 5% w/v alginate remain viable for at least 60 days. Rheological analysis was used to study how the mechanical properties exhibited by alginate hydrogel changed during 28 days in vitro culture. Alginate degradation was shown to occur throughout the study but was greatest within the first 7 days of culture for all samples, which correlated with a sharp release of calcium ions from the construct. Fibroblasts were shown to increase the rate of degradation during the first 7 days when compared with acellular samples in both 2% and 5% w/v gels, but after 28 days both acellular and cell-encapsulating samples retained disc-shaped morphologies and gel-like spectra. The results demonstrate that although at an early stage cells influence the mechanical properties of encapsulating alginate, over a longer period of culture, the hydrogels retain sufficient mechanical integrity to exhibit gel-like properties. This allows sustained immobilization of the cells at the desired location in vivo where they can produce extracellular matrix and growth factors to expedite the healing process.

  1. A Pseudomonas aeruginosa alginate-exotoxin A conjugate that elicits anti-alginate and exotoxin A-neutralizing antibodies.

    PubMed

    Coin, D; Vacheron, M J; Guinand, M; Michel, G

    1991-08-01

    Pseudomonas aeruginosa alginate was covalently coupled to exotoxin A by reductive amination using adipic acid dihydrazide as spacer. The conjugate was composed of 25% alginate and 75% exotoxin A and possessed an average molecular mass higher than 700 kDa as determined by polyacrylamide gel electrophoresis. The conjugate had virtually no ADP-ribosyltransferase activity and a reduced cytotoxicity for TSA8 murine cells, derived from Friend erythroleukemia cells, as indicated by a greater than 50-fold increased LD50. Anti-conjugate antibodies recognized exotoxin A and alginate. A booster injection resulted in markedly increased antibody ELISA titers to both exotoxin A and alginate. The antibodies neutralized the exotoxin A toxicity. PMID:1931130

  2. Study of the interpolyelectrolyte reaction between chitosan and alginate: influence of alginate composition and chitosan molecular weight.

    PubMed

    Becherán-Marón, L; Peniche, C; Argüelles-Monal, W

    2004-04-01

    The interpolyelectrolyte reaction between chitosan (CHI) and alginate (ALG) was followed by conductimetry and potentiometry. Five chitosan samples, all with almost the same degree of N-acetylation (DA approximately 0.20) and molecular weights ranging from 5 x 10(3) to 2.5 x 10(5) Da were used. The polyelectrolyte complex was formed using alginate samples with three different M/G values (0.44, 1.31 and 1.96). The composition of the complex, Z (Z = [CHI]/[ALG]) resulted 0.70 +/- 0.02, independently of the molecular weight of chitosan and the composition of the alginate used. The degree of complexation was 0.51 with no dependence on the alginate composition.

  3. Deoxyribophosphate lyase activity of mammalian endonuclease VIII-like proteins.

    PubMed

    Grin, Inga R; Khodyreva, Svetlana N; Nevinsky, Georgy A; Zharkov, Dmitry O

    2006-09-01

    Base excision repair (BER) protects cells from nucleobase DNA damage. In eukaryotic BER, DNA glycosylases generate abasic sites, which are then converted to deoxyribo-5'-phosphate (dRP) and excised by a dRP lyase (dRPase) activity of DNA polymerase beta (Polbeta). Here, we demonstrate that NEIL1 and NEIL2, mammalian homologs of bacterial endonuclease VIII, excise dRP by beta-elimination with the efficiency similar to Polbeta. DNA duplexes imitating BER intermediates after insertion of a single nucleotide were better substrates. NEIL1 and NEIL2 supplied dRPase activity in BER reconstituted with dRPase-null Polbeta. Our results suggest a role for NEILs as backup dRPases in mammalian cells.

  4. An investigation of agitation speed as a factor affecting the quantity and monomer distribution of alginate from Azotobacter vinelandii ATCC(®) 9046.

    PubMed

    Kıvılcımdan Moral, C; Sanin, F D

    2012-03-01

    Alginate is a copolymer of β-D: -mannuronic and α-L: -guluronic acids. Distribution of these monomers in the alginate structure is one of the important characteristics that affect the commercial value of the polymer. In the present work, the effect of agitation speed in the range of 200-700 rpm on alginate production by Azotobacter vinelandii ATCC(®) 9046 was investigated at a dissolved oxygen tension of 5% of air saturation. Experiments were conducted in a fermentor operated in batch mode for 72 h while the production of biomass and alginate, the consumption of substrate and the change in culture broth viscosity and monomer distribution of the polymer were monitored. Results showed that the growth rate of the bacteria increased from 0.165 to 0.239 h(-1) by the increase of mixing speed from 200 to 400 rpm. On the other hand, alginate production was found to be the most efficient at 400 rpm with the highest value of 4.51 g/l achieved at the end of fermentation. The viscosity of culture broth showed similar trends to alginate production. Viscosity was recorded as 24.61 cP at 400 rpm while it was only 4.26 cP at 700 rpm. The MM- and GG-block contents were almost equal in most of the culture times at 400 rpm. On the other hand, GG-blocks dominated at both low and high mixing speeds. Knowing that GG-blocks make rigid and protective gels with divalent cations, due to the higher GG-block content, the gel formation potential is higher at 200 rpm as well at 700 rpm, which might originate from the unfavorable environmental conditions that the bacteria were exposed to. PMID:22009058

  5. Removal of uranium and other metals from wastewaters using calcium alginate and bacteria immobilized in calcium alginate

    SciTech Connect

    Gardner, K.R.; Montgomery, J.R.; Adams, D.J.

    1995-12-31

    Calcium alginate beads, containing immobilized bacteria, removed turanium and other metals from mine wastewaters. Batch tests using 2% calcium alginate beads with and without immobilized bacteria showed both were effective at removing uranium. Beads containing live and dead cells removed essentially the same amount of uranium and removed significantly more uranium than beads without bacteria. The bacteria-alginate system was used to treat waters containing 24.5 mg/L uranium and produced effluents containing 1.0 mg/L uranium in 1 hr and <0.1 mg/L uranium in 24 hr. Calcium alginate beads, with and without immobilized bacteria, also removed arsenic, selenium, and other metals from wastewaters. Bacteria investigated for immobilization in the alginate beads include Bacillus and Pseudomonas sp. A spray technique using an air nozzle has been tested to disperse the alginate into mine wastewater eliminating the bead-making process. Ninety-nine percent of the uranium was removed in less than 15 min contact.

  6. 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. PMID:26952484

  7. Insulin-stimulated phosphorylation of ATP-citrate lyase in isolated hepatocytes. Stoichiometry and relation to the phosphoenzyme intermediate.

    PubMed

    Alexander, M C; Palmer, J L; Pointer, R H; Kowaloff, E M; Koumjian, L L; Avruch, J

    1982-02-25

    We have estimated the insulin-stimulated phosphorylation of ATP-citrate lyase by two methods. Isolated hepatocytes incorporate extracellular 32P into [gamma-35P] ATP and immunoprecipitated ATP-citrate lyase to steady state levels by 1 h. The content of acid-stable 32P in hepatocyte ATP-citrate lyase at steady state is 0.33 +/- 0.038 mol of P/mol (tetrameric) holoenzyme. Insulin (1 milliunit/ml) increases the 32P content of immunoprecipitated lyase 2- to 3-fold in 10 min. Over 90% of acid-stable 32P on lyase is 32P-serine in enzyme isolated from both control and insulin-treated cells. ATP-citrate lyase isolated from hepatocytes contains 0.95 +/- 0.1 mol of alkali-labile phosphate/mol of holoenzyme. Insulin treatment of hepatocytes (1 milliunit/ml for 10 min) increases the alkali-labile P content by 45%. Evidence is presented which indicates that the insulin-stimulated phosphorylation does not arise by intramolecular migration from the catalytic phosphoenzyme intermediate. These observations support the conclusion that insulin-stimulated phosphorylation of ATP-citrate lyase is mediated either by an insulin-induced increase in the activity of lyase kinase and/or decrease in a lyase phosphatase. The functional role of the substoichiometric phosphorylation of ATP-citrate lyase remains unknown.

  8. 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.

  9. A Possible Role of Divalent Manganese Ions in the Photoinduction of Phenylalanine Ammonia-Lyase

    PubMed Central

    Engelsma, G.

    1972-01-01

    Divalent Mn ions cause an increase in the level of phenylalanine ammonia-lyase in gherkin hypocotyls. With the exception of Mg ions, which had a small effect, no other metal ion has so far been found which could replace the Mn ion in this respect. Invertase and peroxidase were not significantly affected by the Mn treatment. The increase in phenylalanine ammonialyase activity is explained by the removal, under the influence of Mn ions, of hydroxycinnamic acids, which cause repression of phenylalanine ammonia-lyase synthesis and/or inactivation of phenylalanine ammonia-lyase. Arguments are advanced for the hypothesis that photochemical transformations of Mn complexes are involved in the photoinduction of phenylalanine ammonia-lyase in dark-grown gherkin seedlings. PMID:16658225

  10. The pir gene of Erwinia chrysanthemi EC16 regulates hyperinduction of pectate lyase virulence genes in response to plant signals

    PubMed Central

    Nomura, Kinya; Nasser, William; Kawagishi, Hirokazu; Tsuyumu, Shinji

    1998-01-01

    The plant pathogenic bacterium Erwinia chrysanthemi secretes pectate lyase proteins that are important virulence factors attacking the cell walls of plant hosts. Bacterial production of these enzymes is induced by the substrate polypectate-Na (NaPP) and further stimulated by the presence of plant extracts. The bacterial regulator responsible for induction by plant extracts was identified and purified by using a DNA-binding assay with the promoter region of pelE that encodes a major pectate lyase. A novel bacterial protein, called Pir, was isolated that produced a specific gel shift of the pelE promoter DNA, and the corresponding pir gene was cloned and sequenced. The Pir protein contains 272 amino acids with a molecular mass of 30 kDa and appears to function as a dimer. A homology search indicates that Pir belongs to the IclR family of transcriptional regulators. Pir bound to a 35-bp DNA sequence in the promoter region of pelE. This site overlaps that of a previously described negative regulator, KdgR. Gel shift experiments showed that the binding of either Pir or KdgR interfered with binding of the other protein. PMID:9826648

  11. 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

  12. Molecular cloning of acetone cyanohydrin lyase from flax (Linum usitatissimum). Definition of a novel class of hydroxynitrile lyases.

    PubMed

    Trummler, K; Wajant, H

    1997-02-21

    Acetone cyanohydrin lyase from Linum usitatissimum is a hydroxynitrile lyase (HNL) which is involved in the catabolism of cyanogenic glycosides in young seedlings of flax. We have isolated a full-length cDNA clone encoding L. usitatissimum HNL (LuHNL) from a cDNA expression library by immunoscreening. LuHNL cDNA was expressed in Escherichia coli and isolated from the respective soluble fraction in an active form which was biochemically indistinguishable from the natural enzyme. An open reading frame of 1266 base pairs encodes for a protein of 45,780 kDa. The derived amino acid sequence shows no overall homologies to the to date cloned HNLs, but has significant similarities to members of the alcohol dehydrogenase (ADH) family of enzymes. In particular, the cysteine and histidine residues responsible for coordination of an active site Zn2+ and a second structurally important Zn2+ in alcohol dehydrogenases are conserved. Nevertheless, we found neither alcohol dehydrogenase activity in LuHNL nor HNL activity in ADH. Moreover, well known inhibitors of ADHs, which interfere with the coordination of the active site Zn2+, fail to affect HNL activity of LuHNL, suggesting principally different mechanisms of cyanohydrin cleavage and alcohol oxidation. Interestingly, LuHNL like ADH and Prunus serotina (PsHNL) possesses an ADP-binding betaalphabeta unit motif, pointing to the possibility that the non-flavoprotein PsHNL and the flavoprotein LuHNL have developed from two independent lines of evolution of a common ancestor with an ADP-binding betaalphabeta unit. PMID:9030531

  13. Biosynthesis of cyanobacterial phycobiliproteins in Escherichia coli: chromophorylation efficiency and specificity of all bilin lyases from Synechococcus sp. strain PCC 7002.

    PubMed

    Biswas, Avijit; Vasquez, Yasmin M; Dragomani, Tierna M; Kronfel, Monica L; Williams, Shervonda R; Alvey, Richard M; Bryant, Donald A; Schluchter, Wendy M

    2010-05-01

    Phycobiliproteins are water-soluble, light-harvesting proteins that are highly fluorescent due to linear tetrapyrrole chromophores, which makes them valuable as probes. Enzymes called bilin lyases usually attach these bilin chromophores to specific cysteine residues within the alpha and beta subunits via thioether linkages. A multiplasmid coexpression system was used to recreate the biosynthetic pathway for phycobiliproteins from the cyanobacterium Synechococcus sp. strain PCC 7002 in Escherichia coli. This system efficiently produced chromophorylated allophycocyanin (ApcA/ApcB) and alpha-phycocyanin with holoprotein yields ranging from 3 to 12 mg liter(-1) of culture. This heterologous expression system was used to demonstrate that the CpcS-I and CpcU proteins are both required to attach phycocyanobilin (PCB) to allophycocyanin subunits ApcD (alpha(AP-B)) and ApcF (beta(18)). The N-terminal, allophycocyanin-like domain of ApcE (L(CM)(99)) was produced in soluble form and was shown to have intrinsic bilin lyase activity. Lastly, this in vivo system was used to evaluate the efficiency of the bilin lyases for production of beta-phycocyanin.

  14. Diversity of function in the isocitrate lyase enzyme superfamily: the Dianthus caryophyllus petal death protein cleaves alpha-keto and alpha-hydroxycarboxylic acids.

    PubMed

    Lu, Zhibing; Feng, Xiaohua; Song, Ling; Han, Ying; Kim, Alexander; Herzberg, Osnat; Woodson, William R; Martin, Brian M; Mariano, Patrick S; Dunaway-Mariano, Debra

    2005-12-20

    The work described in this paper was carried out to define the chemical function a new member of the isocitrate lyase enzyme family derived from the flowering plant Dianthus caryophyllus. This protein (Swiss-Prot entry Q05957) is synthesized in the senescent flower petals and is named the "petal death protein" or "PDP". On the basis of an analysis of the structural contexts of sequence markers common to the C-C bond lyases of the isocitrate lyase/phosphoenolpyruvate mutase superfamily, a substrate screen that employed a (2R)-malate core structure was designed. Accordingly, stereochemically defined C(2)- and C(3)-substituted malates were synthesized and tested as substrates for PDP-catalyzed cleavage of the C(2)-C(3) bond. The screen identified (2R)-ethyl, (3S)-methylmalate, and oxaloacetate [likely to bind as the hydrate, C(2)(OH)(2) gem-diol] as the most active substrates (for each, k(cat)/K(m) = 2 x 10(4) M(-)(1) s(-)(1)). In contrast to the stringent substrate specificities previously observed for the Escherichia coli isocitrate and 2-methylisocitrate lyases, the PDP tolerated hydrogen, methyl, and to a much lesser extent acetate substituents at the C(3) position (S configuration only) and hydoxyl, methyl, ethyl, propyl, and to a much lesser extent isobutyl substituents at C(2) (R configuration only). It is hypothesized that PDP functions in oxalate production in Ca(2+) sequestering and/or in carbon scavenging from alpha-hydroxycarboxylate catabolites during the biochemical transition accompanying petal senescence.

  15. Cellobiose hydrolysis using Pichia etchellsii cells immobilized in calcium alginate

    SciTech Connect

    Jain, D.; Ghose, T.K.

    1984-04-01

    Cellulose degradation rates can be increased by the hydrolysis of cellobiose using immobilized beta-glucosidase. Production of beta-glucosidase in four yeasts was studied and a maximum activity of 1.22 IU/mg cells was obtained in cells of Pichia etchellsii grown on 3% cellobiose. The immobilization of beta-glucosidase containing cells on various solid supports was studied and entrapment in calcium alginate gel beads was found to be the best method. After ten sequential batch uses of the preparation, 96.5% of the initial activity was retained. The pH and temperature optima for free and immobilized cells were pH 6.5 (0.05M Maleate buffer) and 50/sup 0/C however, the enzyme has a better thermal stability at 45/sup 0/C. Beads stored at 4/sup 0/C for six months retain 80% of their activity. Kinetic studies performed on free and immobilized cells show that glucose is a noncompetitive product inhibitor. The immobilized preparation was limited by pore diffusion but exhibited no film-diffusion resistance during packed bed reactor operation. Good plug flow characteristics were observed. A model for reaction with pore diffusion for a noncompetitive type of inhibited system was developed and applied to this system. The reation rate with diffusional limitations was determined by using the model and effectiveness factors were calculated for different particle sizes. The modified rate expression using the effectiveness factor represented batch and packed bed reactor operation satisfactorily. The productivity in the packed bed column fell rapidly with an increase in conversion rate indicating that the operating conditions of the column would have to balance high conversion rates with acceptable productivity. The half-life in the column was affected by temperature, increasing to over seventeen days at 40/sup 0/C and decreasing to less than two days at 50/sup 0/C.

  16. Aging and microwave effects on alginate/chitosan matrices.

    PubMed

    Wong, Tin Wui; Chan, Lai Wah; Kho, Shyan Bin; Heng, Paul Wan Sia

    2005-06-01

    The influence of microwave irradiation on the drug release properties of freshly prepared and aged alginate, alginate-chitosan and chitosan beads was investigated. The beads were prepared by extrusion method with sulphathiazole as a model drug. The dried beads were subjected to microwave irradiation at 80 W for 10 min, 20 min or three consecutive cycles of 10 and 20 min, respectively. The profiles of drug dissolution, drug content, drug stability, drug polymorphism, drug-polymer interaction, polymer crosslinkage and complexation were determined by dissolution testing, drug content assay, differential scanning calorimetry and Fourier transform infra-red spectroscopy. The chemical stability of drug embedded in beads was unaffected by microwave conditions and length of storage time. The release property of drug was mainly governed by the extent of polymer interaction in beads. The aged alginate beads required intermittent cycles of microwave irradiation to induce drug release retarding effect in contrast to their freshly prepared samples. Unlike the alginate beads, the level of polymer interaction was higher in aged alginate-chitosan beads than the corresponding fresh beads. The drug release retarding property of aged alginate-chitosan beads could be significantly enhanced through subjecting the beads to microwave irradiation for 10 min. No further change in drug release from these beads was observed beyond 30 min of microwave irradiation. Unlike beads containing alginate, the rate and extent of drug released from the aged chitosan beads were higher upon treatment by microwave in spite of the higher degree of polymer interaction shown by the latter on prolonged storage. The observation suggested that the response of polymer matrix to microwave irradiation in induction of drug release retarding property was largely affected by the molecular arrangement of the polymer chains.

  17. Extraction and physicochemical characterization of Sargassum vulgare alginate from Brazil.

    PubMed

    Torres, Marcia R; Sousa, Alessandra P A; Silva Filho, Eduardo A T; Melo, Dirce F; Feitosa, Judith P A; de Paula, Regina C M; Lima, Maria G S

    2007-10-15

    Alginate fractions from Sargassum vulgare brown seaweed were characterized by (1)H NMR and fluorescence spectroscopy and by rheological measurements. The alginate extraction conditions were investigated. In order to carry out the structural and physicochemical characterization, samples extracted for 1 and 5h at 60 degrees C were further purified by re-precipitation with ethanol and denoted as SVLV (S. vulgare low viscosity) and SVHV (S. vulgare high viscosity), respectively. The M/G ratio values for SVLV and SVHV were 1.56 and 1.27, respectively, higher than the ratio for most Sargassum spp. alginates (0.19-0.82). The homopolymeric blocks F(GG) and F(MM) of these fractions characterized by (1)H NMR spectroscopy were 0.43 and 0.55 for SVHV and 0.36 and 0.58 for SVLV samples, respectively, these values typically being within 0.28-0.77 and 0.07-0.41, respectively. Therefore, the alginate samples from S. vulgare are much richer in mannuronic block structures than those from other Sargassum species. Values of M(w) for alginate samples were also calculated using intrinsic viscosity data. The M(w) value for SVLV (1.94 x 10(5)g/mol) was lower than that for SVHV (3.3 x 10(5)g/mol). Newtonian behavior was observed for a solution concentration as high as 0.7% for SVLV, while for SVHV the solutions behaved as a Newtonian fluid up to 0.5%. The optimal conditions for obtaining the alginates from S. vulgare were 60 degrees C and 5h extraction. Under these conditions, a more viscous alginate in higher yield was extracted from the seaweed biomass. PMID:17585891

  18. Isolation and spectroscopic characterization of a recombinant bell pepper hydroperoxide lyase.

    PubMed

    Psylinakis, E; Davoras, E M; Ioannidis, N; Trikeriotis, M; Petrouleas, V; Ghanotakis, D F

    2001-09-28

    Fatty acid hydroperoxide (HPO) lyase is a component of the oxylipin pathway and holds a central role in elicited plant defense. HPO lyase from bell pepper has been identified as a heme protein which shares 40% homology with allene oxide synthase, a cytochrome P450 (CYP74A). HPO lyase of immature bell pepper fruits was expressed in Escherichia coli and the enzyme was purified and characterized by spectroscopic techniques. The electronic structure and ligand coordination properties of the heme were investigated by using a series of exogenous ligands. The various complexes were characterized by using UV-visible absorption and electron paramagnetic resonance spectroscopy. The spectroscopic data demonstrated that the isolated recombinant HPO lyase has a pentacoordinate, high-spin heme with thiolate ligation. Addition of the neutral ligand imidazole or the anionic ligand cyanide results in the formation of hexacoordinate adducts that retain thiolate ligation. The striking similarities between both the ferric and ferrous HPO lyase-NO complexes with the analogous P450 complexes, suggest that the active sites of HPO lyase and P450 share common structural features.

  19. Structural Basis for Glycyl Radical Formation By Pyruvate Formate-Lyase Activating Enzyme

    SciTech Connect

    Vey, J.L.; Yang, J.; Li, M.; Broderick, W.E.; Broderick, J.B.; Drennan, C.L.

    2009-05-26

    Pyruvate formate-lyase activating enzyme generates a stable and catalytically essential glycyl radical on G{sup 734} of pyruvate formate-lyase via the direct, stereospecific abstraction of a hydrogen atom from pyruvate formate-lyase. The activase performs this remarkable feat by using an iron-sulfur cluster and S-adenosylmethionine (AdoMet), thus placing it among the AdoMet radical superfamily of enzymes. We report here structures of the substrate-free and substrate-bound forms of pyruvate formate-lyase-activating enzyme, the first structures of an AdoMet radical activase. To obtain the substrate-bound structure, we have used a peptide substrate, the 7-mer RVSGYAV, which contains the sequence surrounding G{sup 734}. Our structures provide fundamental insights into the interactions between the activase and the G{sup 734} loop of pyruvate formate-lyase and provide a structural basis for direct and stereospecific H atom abstraction from the buried G{sup 734}4 of pyruvate formate-lyase.

  20. Immobilization of a Plant Lipase from Pachira aquatica in Alginate and Alginate/PVA Beads.

    PubMed

    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.

  1. An acid/alkaline stress and the addition of amino acids induce a prolonged viability of Lactobacillus plantarum loaded into alginate gel.

    PubMed

    Bevilacqua, Antonio; Sinigaglia, Milena; Corbo, Maria Rosaria

    2010-08-15

    This study reports on the investigation on the effects of the conditions used throughout the step of biomass production on the survival of Lactobacillus plantarum loaded into alginate gels. L. plantarum was grown under different conditions (MRS or a laboratory medium-LB(2)-at acidic or alkaline pHs, with NaCl, phenols, vitamins or amino acids) and immobilized in sodium alginate; cell number was evaluated throughout the storage and death (delta(stand)) and first-reduction times (delta) were calculated. The storage of alginate gels at 4 degrees C prolonged cell viability up to 60 days (ca. 20 days for cells produced in MRS and stored at 30 degrees C); however, a similar prolongation was achieved for cells produced in LB(2) adjusted to pH 5.0 and 9.0 or added with amino acids (death time>50-60 days).

  2. Mechanistic deductions from kinetic isotope effects and pH studies of pyridoxal phosphate dependent carbon-carbon lyases: Erwinia herbicola and Citrobacter freundii tyrosine phenol-lyase

    SciTech Connect

    Kiick, D.M.; Phillips, R.S.

    1988-09-20

    The pH dependence of the kinetic parameters and primary deuterium isotope effects have been determined for tyrosine phenol-lyase from both Erwinia herbicola and Citrobacter freundii. The primary deuterium isotope effects indicate that proton abstraction from the 2-position of the substrate is partially rate-limiting for both enzymes. The C. freundii enzyme primary deuterium isotope effects (DV = 3.5 and D(V/Ktyr) = 2.5) are pH independent, indicating that tyrosine is not sticky (i.e., does not dissociate slower than it reacts to give products). Since Vmax for both tyrosine and the alternate substrate S-methyl-L-cysteine is also pH independent, substrate binds only to the correctly protonated form of the enzyme. For the E. herbicola enzyme, both Vmax and V/K for tyrosine or S-methyl-L-cysteine are pH dependent, as well as both DV and D(V/Ktyr). Thus, while both the protonated and unprotonated enzyme can bind substrate, and may be interconverted directly, only the unprotonated Michaelis complex is catalytically competent. At pH 9.5, DV = 2.5 and D(V/Ktyr) = 1.5. However, at pH 6.4 the isotope effect on both parameters is equal to 4.1. From these data, the forward commitment factor (cf = 5.2) and catalytic ratio (cvf = 1.1) for tyrosine and S-methyl-L-cysteine (cf = 2.2, cvf = 24) are calculated. Also, the Michaelis complex partition ratio (cf/cvf) for substrate and products is calculated to be 4.7 for tyrosine and 0.1 for S-methyl-L-cysteine.

  3. Green synthesis and characterization of alginate nanoparticles and its role as a biosorbent for Cr(VI) ions

    NASA Astrophysics Data System (ADS)

    Geetha, P.; Latha, M. S.; Pillai, Saumya S.; Deepa, B.; Santhosh Kumar, K.; Koshy, Mathew

    2016-02-01

    Green synthesis of nanoparticles has attained considerable attention in recent years because of its myriad of applications including drug delivery, tissue engineering and water purification. In the present study, alginate nanoparticles stabilized by honey were prepared by cross-linking aqueous solution of alginate with calcium ions. Honey mediated synthesis has been reported earlier for the production of metal nanoparticles. However no literature is available on the use of this technique for polymeric nanoparticles. Highly stable nanoparticles of 10-100 nm size were generated by this technique. The synthesised nanoparticles were characterized by transmission electron microscopy, scanning electron microscopy, atomic force microscopy, dynamic light scattering and Fourier transform infrared spectroscopic techniques. Potential of using these nanoparticles for heavy metal removal was studied by using Cr(VI) from aqueous solution, where a maximum removal efficiency of 93.5% was obtained. This method was also successfully employed for the production of other polymeric nanoparticles like casein, chitosan and albumin.

  4. Regulation of cystathionine γ-lyase in mammalian cells by hypoxia.

    PubMed

    Wang, Maoxian; Guo, Zhanyun; Wang, Shilong

    2014-02-01

    Hydrogen sulfide (H2S), an endogenous signaling molecule in mammalian cells, shows a variety of biological effects. Cystathionine γ-lyase (CSE) is a key enzyme in the trans-sulfuration pathway responsible for the production of endogenous H2S. Whether CSE expression is regulated by hypoxia in mammalian cells remains largely unknown. This study revealed that these regulatory effects changed with time at transcriptional and post-transcriptional levels. Hypoxia regulated CSE expression in mammalian cells in a complex manner; CSE transcription went through a down-regulation and recovery period, while CSE mRNA and protein levels increased during hypoxia. Taken together, the results suggest that CSE can respond to hypoxia through transcriptional and post-transcriptional regulation, and CSE expression can be up-regulated by hypoxia to a certain extent. Therefore, the up-regulation of CSE expression during hypoxia may be useful for increasing the production and concentration of H2S in mammalian cells and indirectly protecting cells from hypoxia.

  5. Continuous synthesis of hexanal by immobilized hydroperoxide lyase in packed-bed reactor.

    PubMed

    Liu, Qingqing; Hua, Yufei

    2015-12-01

    This study aimed to develop an optimal continuous procedure of immobilized hydroperoxide lyase (HPL)-catalyzed synthesis of hexanal. A central composite design was used to study the combined effect of substrate concentration and the residence time of the reactant on hexanal concentration. The optimum conditions for hexanal synthesis included a 13-HPOD concentration of 43.54 mM and a residence time of 60.99 min. The maximum hexanal concentration was 3560 ± 130 mg/L when 16 U of immobilized HPLwas used. Furthermore, the stability of immobilized HPL was significantly improved in the packed-bed reactor, as evidenced by the slowed enzyme inactivation and prolonged operation time. The immobilized HPL remained activity until 40 mL substrate solution flowed past the packed-bed reactor. The catalyst productivity of hexanal in the packed-bed reactor was 5.35 ± 0.34 mg/U, much higher than that in the batch stirred reactor. This study was greatly meaningful for providing a green method to the large-scale production of hexanal.

  6. Chondrocyte Culture in Three Dimensional Alginate Sulfate Hydrogels Promotes Proliferation While Maintaining Expression of Chondrogenic Markers

    PubMed Central

    Mhanna, Rami; Kashyap, Aditya; Palazzolo, Gemma; Vallmajo-Martin, Queralt; Becher, Jana; Möller, Stephanie; Schnabelrauch, Matthias

    2014-01-01

    The loss of expression of chondrogenic markers during monolayer expansion remains a stumbling block for cell-based treatment of cartilage lesions. Here, we introduce sulfated alginate hydrogels as a cartilage biomimetic biomaterial that induces cell proliferation while maintaining the chondrogenic phenotype of encapsulated chondrocytes. Hydroxyl groups of alginate were converted to sulfates by incubation with sulfur trioxide–pyridine complex (SO3/pyridine), yielding a sulfated material cross-linkable with calcium chloride. Passage 3 bovine chondrocytes were encapsulated in alginate and alginate sulfate hydrogels for up to 35 days. Cell proliferation was five-fold higher in alginate sulfate compared with alginate (p=0.038). Blocking beta1 integrins in chondrocytes within alginate sulfate hydrogels significantly inhibited proliferation (p=0.002). Sulfated alginate increased the RhoA activity of chondrocytes compared with unmodified alginate, an increase that was blocked by β1 blocking antibodies (p=0.017). Expression and synthesis of type II collagen, type I collagen, and proteoglycan was not significantly affected by the encapsulation material evidenced by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry. Alginate sulfate constructs showed an opaque appearance in culture, whereas the unmodified alginate samples remained translucent. In conclusion, alginate sulfate provides a three dimensional microenvironment that promotes both chondrocyte proliferation and maintenance of the chondrogenic phenotype and represents an important advance for chondrocyte-based cartilage repair therapies providing a material in which cell expansion can be done in situ. PMID:24320935

  7. Electrodeposition of alginate gels for construction of vascular-like structures.

    PubMed

    Ozawa, Fumisato; Ino, Kosuke; Takahashi, Yasufumi; Shiku, Hitoshi; Matsue, Tomokazu

    2013-04-01

    In this study, tubular hydrogel structures were constructed via electrodeposition using alginate gels. Electrolysis of water in alginate solutions with calcium carbonate particles induced gel aggregation around Pt wire electrodes, forming tubular alginate gel structures. The simple method is a promising approach for construction of multi-layer tubular hydrogel structures for tissue engineering.

  8. Cellobiose hydrolysis using Pichia etchellsii cells immobilized in calcium alginate

    SciTech Connect

    Jain, D.; Ghose, T.K.

    1984-01-01

    The rate of cellulose degradation, limited by inhibition by cellobiose, can be increased by hydrolysis of cellobiose to glucose using immobilized ..beta..-glucosidase. Production of ..beta..-glucosidase in four yeasts was studied and a maximum activity of 1.22 IU/mg cells was obtained in cells of Pichia etchellsii when grown on 3% cellobiose as the sole carbon source. Immobilization of ..beta..-glucosidase containing cells of Pichia etchellsii on various solid supports was conducted and immobilization by entrapment in calcium alginate gel beads was found to be the most simple and efficient method. The immobilized preparation was found to be limited by pore diffusion but exhibited no film-diffusion resistance during packed bed reactor operation. Good plug flow characteristics were observed in the packed bed column indicated by a low dispersion number of 0.1348. A model for reaction with pore diffusion for a noncompetitive type of inhibited system was developed and applied to the cellobiose hydrolysis system. The rate of reaction with diffusional limitations was determined by using the model and effectiveness factors were calculated for different particle sizes. An effectiveness factor of 0.49 was obtained for a particle diameter of 2.5 mm. The modified rate expression using the effectiveness factor represented batch and packed bed reactor operation satisfactorily. The productivity in the packed bed column was found to fall rapidly with increase in conversion rate indicating that the operating conditions of the column would have to be a compromise between high conversion rates and reasonable productivity. A half-life of over seven days was obtained at the operating temperature of 45/sup 0/C in continuous operation of the packed bed reactor. However, the half-life in the column was found to be greatly affected by temperature, increasing to over seve

  9. Zinc cross-linked hydroxamated alginates for pulsed drug release

    PubMed Central

    Raut, Neha S; Deshmukh, Prasad R; Umekar, Milind J; Kotagale, Nandkishor R

    2013-01-01

    Introduction: Alginates can be tailored chemically to improve solubility, physicochemical, and biological properties and its complexation with metal ion is useful for controlling the drug release. Materials And Methods: Synthesized N,O-dimethyl, N-methyl, or N-Benzyl hydroxylamine derivatives of sodium alginate were subsequently complexed with zinc to form beads. Hydroxamation of sodium alginate was confirmed by Fourier transform infra-red spectroscopy (FTIR) and differential scanning calorimetry (DSC). Results: The synthesized polymeric material exhibited reduced aqueous, HCl and NaOH solubility. The hydroxamated derivatives demonstrated pulsed release where change in pH of the dissolution medium stimulated the atenolol release. Conclusion: Atenolol loaded Zn cross-linked polymeric beads demonstrated the sustained the plasma drug levels with increased half-life. Although the synthesized derivatives greatly altered the aqueous solubility of sodium alginate, no significant differences in in vitro and in vivo atenolol release behavior amongst the N,O-dimethyl, N-methyl, or N-Benzyl hydroxylamine derivatives of sodium alginate were observed. PMID:24350039

  10. Oxidized alginate hydrogels as niche environments for corneal epithelial cells

    PubMed Central

    Wright, Bernice; De Bank, Paul A; Luetchford, Kim A; Acosta, Fernando R; Connon, Che J

    2014-01-01

    Chemical and biochemical modification of hydrogels is one strategy to create physiological constructs that maintain cell function. The aim of this study was to apply oxidised alginate hydrogels as a basis for development of a biomimetic niche for limbal epithelial stem cells that may be applied to treating corneal dysfunction. The stem phenotype of bovine limbal epithelial cells (LEC) and the viability of corneal epithelial cells (CEC) were examined in oxidised alginate gels containing collagen IV over a 3-day culture period. Oxidation increased cell viability (P ≤ 0.05) and this improved further with addition of collagen IV (P ≤ 0.01). Oxidised gels presented larger internal pores (diameter: 0.2–0.8 µm) than unmodified gels (pore diameter: 0.05–0.1 µm) and were significantly less stiff (P ≤ 0.001), indicating that an increase in pore size and a decrease in stiffness contributed to improved cell viability. The diffusion of collagen IV from oxidised alginate gels was similar to that of unmodified gels suggesting that oxidation may not affect the retention of extracellular matrix proteins in alginate gels. These data demonstrate that oxidised alginate gels containing corneal extracellular matrix proteins can influence corneal epithelial cell function in a manner that may impact beneficially on corneal wound healing therapy. © 2013 The Authors. Journal of Biomedical Materials Research Part A Published byWiley Periodicals, Inc. Part A: 102A: 3393–3400, 2014. PMID:24142706

  11. 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

  12. Towards antimicrobial yet bioactive Cu-alginate hydrogels.

    PubMed

    Madzovska-Malagurski, I; Vukasinovic-Sekulic, M; Kostic, D; Levic, S

    2016-06-15

    The simplest approach to enhance alginate hydrogel characteristics and functional properties is to replace the calcium in the process of alginate gelation with other metallic ions which are essential for living systems. Gelling of alginate with other ions and using modern encapsulation techniques can provide new delivery systems with required properties. Hence, in this study Cu-alginate hydrogels in the form of microbeads were produced by electrostatic extrusion using gelling solutions with Cu(II) concentrations in the range 13.5-270 mM and comprehensively characterized in vitro. The variation of gelling solution concentration influenced the microbead Cu(II) content, size, biomechanical properties, Cu(II) release and subsequently potential biomedical application. The formulations chosen for biomedical evaluation showed potential for antimicrobial and tissue engineering applications. Microbeads with higher Cu(II) loading (~100 μmol g(-1)) induced immediate bactericidal effects against Escherichia coli and Staphylococcus aureus. Conversely, Cu(II) release from microbeads with the Cu(II) content of ~60 μmol g(-1) was slower and they were suitable for promoting and maintaining chondrogenic phenotype of bovine calf chondrocytes in 3D culture. Results of this study have shown possibilities for tuning Cu-alginate properties for potential biomedical applications such as antimicrobial wound dressings, tissue engineering scaffolds or articular cartilage implants.

  13. Preparation of alginate beads containing a prodrug of diethylenetriaminepentaacetic acid

    PubMed Central

    Yang, Yu-Tsai; Di Pasqua, Anthony J.; He, Weiling; Tsai, Tsuimin; Sueda, Katsuhiko; Zhang, Yong; Jay, Michael

    2012-01-01

    A penta-ethyl ester prodrug of the radionuclide decorporation agent diethylenetriaminepentaacetic acid (DTPA), which exists as an oily liquid, was encapsulated in alginate beads by the ionotropic gelation method. An optimal formulation was found by varying initial concentrations of DTPA pentaethyl ester, alginate polymer, Tween 80 surfactant and calcium chloride. All prepared alginate beads were ~1.6 mm in diameter, and the optimal formulation had loading and encapsulation efficiencies of 91.0 ± 1.1 and 72.6 ± 2.2%, respectively, and only 3.2 ± 0.8% water absorption after storage at room temperature in ~80% relative humidity. Moreover, Fourier transform infrared spectroscopy showed that DTPA penta-ethyl ester did not react with excipients during formation of the DTPA penta-ethyl ester-containing alginate beads. Release of prodrug from alginate beads was via anomalous transport, and its stability enhanced by encapsulation. Collectively, these data suggest that this solid dosage form may be suitable for oral administration after radionuclide contamination. PMID:23399237

  14. 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.

  15. Oxidized alginate hydrogels as niche environments for corneal epithelial cells.

    PubMed

    Wright, Bernice; De Bank, Paul A; Luetchford, Kim A; Acosta, Fernando R; Connon, Che J

    2014-10-01

    Chemical and biochemical modification of hydrogels is one strategy to create physiological constructs that maintain cell function. The aim of this study was to apply oxidised alginate hydrogels as a basis for development of a biomimetic niche for limbal epithelial stem cells that may be applied to treating corneal dysfunction. The stem phenotype of bovine limbal epithelial cells (LEC) and the viability of corneal epithelial cells (CEC) were examined in oxidised alginate gels containing collagen IV over a 3-day culture period. Oxidation increased cell viability (P ≤ 0.05) and this improved further with addition of collagen IV (P ≤ 0.01). Oxidised gels presented larger internal pores (diameter: 0.2-0.8 µm) than unmodified gels (pore diameter: 0.05-0.1 µm) and were significantly less stiff (P ≤ 0.001), indicating that an increase in pore size and a decrease in stiffness contributed to improved cell viability. The diffusion of collagen IV from oxidised alginate gels was similar to that of unmodified gels suggesting that oxidation may not affect the retention of extracellular matrix proteins in alginate gels. These data demonstrate that oxidised alginate gels containing corneal extracellular matrix proteins can influence corneal epithelial cell function in a manner that may impact beneficially on corneal wound healing therapy.

  16. 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. PMID:27540886

  17. Evaluation of sodium alginate for bone marrow cell tissue engineering.

    PubMed

    Wang, L; Shelton, R M; Cooper, P R; Lawson, M; Triffitt, J T; Barralet, J E

    2003-09-01

    Sodium alginate has applications as a material for the encapsulation and immobilisation of a variety of cell types for immunoisolatory and biochemical processing applications. It forms a biodegradable gel when crosslinked with calcium ions and it has been exploited in cartilage tissue engineering since chondrocytes do not dedifferentiate when immobilised in it. Despite its attractive properties of degradability, ease of processing and cell immobilisation, there is little work demonstrating the efficacy of alginate gel as a substrate for cell proliferation, except when RGD is modified. In this study we investigated the ability of rat bone marrow cells to proliferate and differentiate on alginates of differing composition and purity. The mechanical properties of the gels were investigated. It was found that high purity and high G-type alginate retained 27% of its initial strength after 12 days in culture and that comparable levels of proliferation were observed on this material and tissue culture plastic. Depending on composition, calcium crosslinked alginate can act as a substrate for rat marrow cell proliferation and has potential for use as 3D degradable scaffold.

  18. Oral delivery of probiotic expressing M cell homing peptide conjugated BmpB vaccine encapsulated into alginate/chitosan/alginate microcapsules.

    PubMed

    Jiang, Tao; Singh, Bijay; Maharjan, Sushila; Li, Hui-Shan; Kang, Sang-Kee; Bok, Jin-Duck; Cho, Chong-Su; Choi, Yun-Jaie

    2014-11-01

    Oral administration of live probiotics as antigen delivery vectors is a promising approach in vaccine development. However, the low survival of probiotics in the gastrointestinal tract limits this approach. Therefore, the aim of this study was the encapsulation of probiotic expressing vaccine into alginate/chitosan/alginate (ACA) microcapsules (MCs) for efficient oral vaccine delivery. Here, recombinant Lactobacillus plantarum 25 (LP25) expressing M cell homing peptide fused BmpB protein was used as a model probiotic. The viability of LP25 in ACA MCs was more than 65% in simulated gastric fluid (SGF, pH 2.0) and 75% in simulated small intestinal fluid (SIF, pH 7.2) up to 2h. Encapsulated LP25 was completely released from ACA MCs in SIF within 12h. When stored at room temperature (RT) or 4°C, the viability of LP25 in ACA MCs was higher than free LP25. Interestingly, the viability of LP25 in ACA MCs at 4°C for 5weeks was above 58%, whereas viability of free LP25 stored at RT up to 5weeks was zero. After 4weeks from the first immunization, LP25-M-BmpB-loaded ACA MCs induced a stronger BmpB-specific IgG and IgA production in mice. Collectively, these findings suggest that encapsulation of probiotic by ACA MCs is a promising delivery system for oral administration of probiotic expressing vaccine.

  19. 3 dimensional cell cultures: a comparison between manually and automatically produced alginate beads.

    PubMed

    Lehmann, R; Gallert, C; Roddelkopf, T; Junginger, S; Wree, A; Thurow, K

    2016-08-01

    Cancer diseases are a common problem of the population caused by age and increased harmful environmental influences. Herein, new therapeutic strategies and compound screenings are necessary. The regular 2D cultivation has to be replaced by three dimensional cell culturing (3D) for better simulation of in vivo conditions. The 3D cultivation with alginate matrix is an appropriate method for encapsulate cells to form cancer constructs. The automated manufacturing of alginate beads might be an ultimate method for large-scaled manufacturing constructs similar to cancer tissue. The aim of this study was the integration of full automated systems for the production, cultivation and screening of 3D cell cultures. We compared the automated methods with the regular manual processes. Furthermore, we investigated the influence of antibiotics on these 3D cell culture systems. The alginate beads were formed by automated and manual procedures. The automated steps were processes by the Biomek(®) Cell Workstation (celisca, Rostock, Germany). The proliferation and toxicity were manually and automatically evaluated at day 14 and 35 of cultivation. The results visualized an accumulation and expansion of cell aggregates over the period of incubation. However, the proliferation and toxicity were faintly and partly significantly decreased on day 35 compared to day 14. The comparison of the manual and automated methods displayed similar results. We conclude that the manual production process could be replaced by the automation. Using automation, 3D cell cultures can be produced in industrial scale and improve the drug development and screening to treat serious illnesses like cancer.

  20. 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

  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. Bacterial conversion of hydroxylamino aromatic compounds by both lyase and mutase enzymes involves intramolecular transfer of hydroxyl groups.

    PubMed

    Nadeau, Lloyd J; He, Zhongqi; Spain, Jim C

    2003-05-01

    Hydroxylamino aromatic compounds are converted to either the corresponding aminophenols or protocatechuate during the bacterial degradation of nitroaromatic compounds. The origin of the hydroxyl group of the products could be the substrate itself (intramolecular transfer mechanism) or the solvent water (intermolecular transfer mechanism). The conversion of hydroxylaminobenzene to 2-aminophenol catalyzed by a mutase from Pseudomonas pseudoalcaligenes JS45 proceeds by an intramolecular hydroxyl transfer. The conversions of hydroxylaminobenzene to 2- and 4-aminophenol by a mutase from Ralstonia eutropha JMP134 and to 4-hydroxylaminobenzoate to protocatechuate by a lyase from Comamonas acidovorans NBA-10 and Pseudomonas sp. strain 4NT were proposed, but not experimentally proved, to proceed by the intermolecular transfer mechanism. GC-MS analysis of the reaction products formed in H(2)(18)O did not indicate any (18)O-label incorporation during the conversion of hydroxylaminobenzene to 2- and 4-aminophenols catalyzed by the mutase from R. eutropha JMP134. During the conversion of 4-hydroxylaminobenzoate catalyzed by the hydroxylaminolyase from Pseudomonas sp. strain 4NT, only one of the two hydroxyl groups in the product, protocatechuate, was (18)O labeled. The other hydroxyl group in the product must have come from the substrate. The mutase in strain JS45 converted 4-hydroxylaminobenzoate to 4-amino-3-hydroxybenzoate, and the lyase in Pseudomonas strain 4NT converted hydroxylaminobenzene to aniline and 2-aminophenol but not to catechol. The results indicate that all three types of enzyme-catalyzed rearrangements of hydroxylamino aromatic compounds proceed via intramolecular transfer of hydroxyl groups.

  3. Continuous bioconversion of starch to ethanol by calcium-alginate immobilized enzymes and yeasts

    SciTech Connect

    McGhee, J.E.; Carr, M.E.; St. Julian, G.

    1984-01-01

    Continuous bioconversion of starch to EtOH by immobilized enzymes and yeasts was studied. Commercial corn starch (10%) was 1st batch-liquefied with bacterial alpha-amylase. In continuous-flow systems, liquefied starch was then converted to glucose with Ca alginate-entrapped fungal glucoamylase, and the resulting glucose was fermented to EtOH by Ca alginate-entrapped active dry yeast. The continuous-flow saccharification-fermentation processes were performed in either 2-stage (sequential) or single-stage (simultaneous) operations. In the single-stage operation, immobilized glucoamylase produced glucose from liquefied starch continuously for 11 days. In the simultaneous saccharification technique using immobilized glucoamylase and yeast mixture in a single-stage column, EtOH production was 69% of theoretical for 5 days. In the 2-stage operation, in which immobilized glucoamylase and yeast were contained in separate columns connected in tandem, EtOH production averaged 97% of theoretical for 5 days. The overall alcoholic production efficiency was significantly greater in the 2-stage system than in the single-stage system.

  4. Methylene blue adsorption on graphene oxide/calcium alginate composites.

    PubMed

    Li, Yanhui; Du, Qiuju; Liu, Tonghao; Sun, Jiankun; Wang, Yonghao; Wu, Shaoling; Wang, Zonghua; Xia, Yanzhi; Xia, Linhua

    2013-06-01

    Graphene oxide has been used as an adsorbent in wastewater treatment. However, the dispersibility in aqueous solution and the biotoxicity to human cells of graphene oxide limits its practical application in environmental protection. In this research, a novel environmental friendly adsorbent, calcium alginate immobilized graphene oxide composites was prepared. The effects of pH, contact time, temperature and dosage on the adsorption properties of methylene blue onto calcium alginate immobilized graphene oxide composites were investigated. The equilibrium adsorption data were described by the Langmuir and Freundlich isotherms. The maximum adsorption capacity obtained from Langmuir isotherm equation was 181.81 mg/g. The pseudo-first order, pseudo-second order, and intraparticle diffusion equation were used to evaluate the kinetic data. Thermodynamic analysis of equilibriums indicated that the adsorption reaction of methylene blue onto calcium alginate immobilized graphene oxide composites was exothermic and spontaneous in nature.

  5. Fundamental Characteristics of Bioprint on Calcium Alginate Gel

    NASA Astrophysics Data System (ADS)

    Umezu, Shinjiro; Hatta, Tatsuru; Ohmori, Hitoshi

    2013-05-01

    The goal of this study is to fabricate precision three-dimensional (3D) biodevices those are micro fluidics and artificial organs utilizing digital fabrication. Digital fabrication is fabrication method utilizing inkjet technologies. Electrostatic inkjet is one of the inkjet technologies. The electrostatic inkjet method has following two merits; those are high resolution to print and ability to eject highly viscous liquid. These characteristics are suitable to print biomaterials precisely. We are now applying for bioprint. In this paper, the electrostatic inkjet method is applied for fabrication of 3D biodevices that has cave like blood vessel. When aqueous solution of sodium alginate is printed to aqueous solution of calcium chloride, calcium alginate is produced. 3D biodevices are fabricated in case that calcium alginate is piled.

  6. Alginate based polyurethanes: A review of recent advances and perspective.

    PubMed

    Zia, Khalid Mahmood; Zia, Fatima; Zuber, Mohammad; Rehman, Saima; Ahmad, Mirza Nadeem

    2015-08-01

    The trend of using biopolymers in combination with synthetic polymers was increasing rapidly from last two or three decades. Polysaccharide based biopolymers especially starch, cellulose, chitin, chitosan, alginate, etc. found extensive applications for different industrial uses, as they are biocompatible, biodegradable, bio-renewable resources and chiefly environment friendly. Segment block copolymer character of polyurethanes that endows them a broad range of versatility in terms of tailoring their properties was employed in conjunction with various natural polymers resulted in modified biomaterials. Alginate is biodegradable, biocompatible, bioactive, less toxic and low cost anionic polysaccharide, as a part of structural component of bacteria and brown algae (sea weed) is quite abundant in nature. It is used in combination with polyurethanes to form elastomers, nano-composites, hydrogels, etc. that especially revolutionized the food and biomedical industries. The review summarized the development in alginate based polyurethanes with their potential applications.

  7. Mechanical properties of alginate hydrogels manufactured using external gelation.

    PubMed

    Kaklamani, Georgia; Cheneler, David; Grover, Liam M; Adams, Michael J; Bowen, James

    2014-08-01

    Alginate hydrogels are commonly used in biomedical applications such as scaffolds for tissue engineering, drug delivery, and as a medium for cell immobilisation. Multivalent cations are often employed to create physical crosslinks between carboxyl and hydroxyl moieties on neighbouring polysaccharide chains, creating hydrogels with a range of mechanical properties. This work describes the manufacture and characterisation of sodium alginate hydrogels using the divalent cations Mg(2+), Ca(2+) and Sr(2+) to promote gelation via non-covalent crosslinks. Gelation time and Young׳s modulus are characterised as a function of cation and alginate concentrations. The implications of this work towards the use of environmental elasticity to control stem cell differentiation are discussed.

  8. Optimization of alpha-amylase immobilization in calcium alginate beads.

    PubMed

    Ertan, Figen; Yagar, Hulya; Balkan, Bilal

    2007-01-01

    alpha-Amylase enzyme was produced by Aspergillus sclerotiorum under SSF conditions, and immobilized in calcium alginate beads. Effects of immobilization conditions, such as alginate concentration, CaCl(2) concentration, amount of loading enzyme, bead size, and amount of beads, on enzymatic activity were investigated. Optimum alginate and CaCl(2) concentration were found to be 3% (w/v). Using a loading enzyme concentration of 140 U mL(-1), and bead (diameter 3 mm) amount of 0.5 g, maximum enzyme activity was observed. Beads prepared at optimum immobilization conditions were suitable for up to 7 repeated uses, losing only 35% of their initial activity. Among the various starches tested, the highest enzyme activity (96.2%) was determined in soluble potato starch hydrolysis for 120 min at 40 degrees C.

  9. Efficient aspartic acid production by a psychrophile-based simple biocatalyst.

    PubMed

    Tajima, Takahisa; Hamada, Mai; Nakashimada, Yutaka; Kato, Junichi

    2015-10-01

    We previously constructed a Psychrophile-based Simple bioCatalyst (PSCat) reaction system, in which psychrophilic metabolic enzymes are inactivated by heat treatment, and used it here to study the conversion of aspartic acid from fumaric acid mediated by the activity of aspartate ammonia-lyase (aspartase). In Escherichia coli, the biosynthesis of aspartic acid competes with that of L-malic acid produced from fumaric acid by fumarase. In this study, E. coli aspartase was expressed in psychrophilic Shewanella livingstonensis Ac10 heat treated at 50 °C for 15 min. The resultant PSCat could convert fumaric acid to aspartic acid without the formation of L-malic acid because of heat inactivation of psychrophilic fumarase activity. Furthermore, alginate-immobilized PSCat produced high yields of aspartic acid and could be re-used nine times. The results of our study suggest that PSCat can be applied in biotechnological production as a new approach to increase the yield of target compounds. PMID:26254042

  10. Ocular hypotensive efficacy and safety of once daily carteolol alginate

    PubMed Central

    Demailly, P.; Allaire, C.; Trinquand, C.

    2001-01-01

    BACKGROUND/AIM—Carteolol is a β adrenoceptor antagonist used topically to reduce intraocular pressure, typically twice daily. In an effort to provide a once daily dosing regimen, carteolol was formulated with 1% alginic acid. The objective of this study was to evaluate the efficacy and safety of carteolol alginate solution in comparison with standard carteolol solution.
METHODS—This was a double masked, parallel group, multicentre study. Patients with ocular hypertension or open angle glaucoma (n=235) were randomly assigned to receive either carteolol alginate four times daily or standard carteolol solution, twice daily. The masking was maintained through the use of a vehicle in the evening for the alginate group. Patients were evaluated at baseline, 15, 60, and 120 days.
RESULTS—At 0900 (presumed trough) on day 60, mean reductions in intraocular pressure (IOP) from baseline were 6.09 (SD 2.97) and 6.09 (3.18) mm Hg for the standard carteolol and alginate, respectively. At 1100 (presumed peak), mean reductions were 6.51 (2.53) and 6.47 (2.76) mm Hg, respectively. Results were similar at other times (day 15 and day 120). The most common side effect was transient stinging on instillation of drops, which did not differ significantly between groups. There were no differences of note in other ocular or systemic signs or symptoms.
CONCLUSION—The new alginate formulation of carteolol 2% given once daily was as effective as standard carteolol 2% given twice daily with no meaningful differences regarding safety.

 PMID:11466245

  11. Injectable hydrogels derived from phosphorylated alginic acid calcium complexes.

    PubMed

    Kim, Han-Sem; Song, Minsoo; Lee, Eun-Jung; Shin, Ueon Sang

    2015-06-01

    Phosphorylation of sodium alginate salt (NaAlg) was carried out using H3PO4/P2O5/Et3PO4 followed by acid-base reaction with Ca(OAc)2 to give phosphorylated alginic acid calcium complexes (CaPAlg), as a water dispersible alginic acid derivative. The modified alginate derivatives including phosphorylated alginic acid (PAlg) and CaPAlg were characterized by nuclear magnetic resonance spectroscopy for (1)H, and (31)P nuclei, high resolution inductively coupled plasma optical emission spectroscopy, Fourier transform infrared spectroscopy, and thermogravimetric analysis. CaPAlg hydrogels were prepared simply by mixing CaPAlg solution (2w/v%) with NaAlg solution (2w/v%) in various ratios (2:8, 4:6, 6:4, 8:2) of volume. No additional calcium salts such as CaSO4 or CaCl2 were added externally. The gelation was completed within about 3-40min indicating a high potential of hydrogel delivery by injection in vivo. Their mechanical properties were tested to be ≤6.7kPa for compressive strength at break and about 8.4kPa/mm for elastic modulus. SEM analysis of the CaPAlg hydrogels showed highly porous morphology with interconnected pores of width in the range of 100-800μm. Cell culture results showed that the injectable hydrogels exhibited comparable properties to the pure alginate hydrogel in terms of cytotoxicity and 3D encapsulation of cells for a short time period. The developed injectable hydrogels showed suitable physicochemical and mechanical properties for injection in vivo, and could therefore be beneficial for the field of soft tissue engineering. PMID:25842118

  12. Argininosuccinate lyase in enterocytes protects from development of necrotizing enterocolitis

    PubMed Central

    Premkumar, M. H.; Sule, G.; Nagamani, S. C.; Chakkalakal, S.; Nordin, A.; Jain, M.; Ruan, M. Z.; Bertin, T.; Dawson, B.; Zhang, J.; Schady, D.; Bryan, N. S.; Campeau, P. M.; Erez, A.

    2014-01-01

    Necrotizing enterocolitis (NEC), the most common neonatal gastrointestinal emergency, results in significant mortality and morbidity, yet its pathogenesis remains unclear. Argininosuccinate lyase (ASL) is the only enzyme in mammals that is capable of synthesizing arginine. Arginine has several homeostatic roles in the gut and its deficiency has been associated with NEC. Because enterocytes are the primary sites of arginine synthesis in neonatal mammals, we evaluated the consequences of disruption of arginine synthesis in the enterocytes on the pathogenesis of NEC. We devised a novel approach to study the role of enterocyte-derived ASL in NEC by generating and characterizing a mouse model with enterocyte-specific deletion of Asl (Aslflox/flox; VillinCretg/+, or CKO). We hypothesized that the presence of ASL in a cell-specific manner in the enterocytes is protective in the pathogenesis of NEC. Loss of ASL in enterocytes resulted in an increased incidence of NEC that was associated with a proinflammatory state and increased enterocyte apoptosis. Knockdown of ASL in intestinal epithelial cell lines resulted in decreased migration in response to lipopolysaccharide. Our results show that enterocyte-derived ASL has a protective role in NEC. PMID:24904080

  13. Regulation by glutathionylation of isocitrate lyase from Chlamydomonas reinhardtii.

    PubMed

    Bedhomme, Mariette; Zaffagnini, Mirko; Marchand, Christophe H; Gao, Xing-Huang; Moslonka-Lefebvre, Mathieu; Michelet, Laure; Decottignies, Paulette; Lemaire, Stéphane D

    2009-12-25

    Post-translational modification of protein cysteine residues is emerging as an important regulatory and signaling mechanism. We have identified numerous putative targets of redox regulation in the unicellular green alga Chlamydomonas reinhardtii. One enzyme, isocitrate lyase (ICL), was identified both as a putative thioredoxin target and as an S-thiolated protein in vivo. ICL is a key enzyme of the glyoxylate cycle that allows growth on acetate as a sole source of carbon. The aim of the present study was to clarify the molecular mechanism of the redox regulation of Chlamydomonas ICL using a combination of biochemical and biophysical methods. The results clearly show that purified C. reinhardtii ICL can be inactivated by glutathionylation and reactivated by glutaredoxin, whereas thioredoxin does not appear to regulate ICL activity, and no inter- or intramolecular disulfide bond could be formed under any of the conditions tested. Glutathionylation of the protein was investigated by mass spectrometry analysis, Western blotting, and site-directed mutagenesis. The enzyme was found to be protected from irreversible oxidative inactivation by glutathionylation of its catalytic Cys(178), whereas a second residue, Cys(247), becomes artifactually glutathionylated after prolonged incubation with GSSG. The possible functional significance of this post-translational modification of ICL in Chlamydomonas and other organisms is discussed.

  14. Novel Proton MR Spectroscopy Findings in Adenylosuccinate Lyase Deficiency

    PubMed Central

    Zulfiqar, Maria; Lin, Doris D.M.; Van der Graaf, Marinette; Barker, Peter B.; Fahrner, Jill A.; Marie, Sandrine; Morava, Eva; De Boer, Lonneke; Willemsen, Michel A.A.P; Vining, Eileen; Horská, Alena; Engelke, Udo; Wevers, Ron A.; Maegawa, Gustavo H.B.

    2016-01-01

    Adenylosuccinate lyase (ADSL) deficiency is a rare inborn error of metabolism resulting in accumulation of metabolites including succinylaminoimidazole carboxamide riboside (SAICAr) and succinyladenosine (S-Ado) in the brain and other tissues. Patients with ADSL have progressive psychomotor retardation, neonatal seizures, global developmental delay, hypotonia, and autistic features, although variable clinical manifestations may make the initial diagnosis challenging. Two cases of the severe form of the disease are reported here: an 18-month-old boy with global developmental delay, intractable neonatal seizures, progressive cerebral atrophy, and marked hypomyelination, and a 3-month-old girl presenting with microcephaly, neonatal seizures, and marked psychomotor retardation. In both patients in vivo proton magnetic resonance spectroscopy (MRS) showed the presence of S-Ado signal at 8.3 ppm, consistent with a prior report. Interestingly, SAICAr signal was also detectable at 7.5 ppm in affected white matter, which has not been reported in vivo before. A novel splice-site mutation, c.IVS12 + 1/G > C, in the ADSL gene was identified in the second patient. Our findings confirm the utility of in vivo proton MRS in suggesting a specific diagnosis of ADSL deficiency, and also demonstrate an additional in vivo resonance (7.5 ppm) of SAICAr in the cases of severe disease. PMID:23055421

  15. Genomic Characterization of Phenylalanine Ammonia Lyase Gene in Buckwheat.

    PubMed

    Thiyagarajan, Karthikeyan; Vitali, Fabio; Tolaini, Valentina; Galeffi, Patrizia; Cantale, Cristina; Vikram, Prashant; Singh, Sukhwinder; De Rossi, Patrizia; Nobili, Chiara; Procacci, Silvia; Del Fiore, Antonella; Antonini, Alessandro; Presenti, Ombretta; Brunori, Andrea

    2016-01-01

    Phenylalanine Ammonia Lyase (PAL) gene which plays a key role in bio-synthesis of medicinally important compounds, Rutin/quercetin was sequence characterized for its efficient genomics application. These compounds possessing anti-diabetic and anti-cancer properties and are predominantly produced by Fagopyrum spp. In the present study, PAL gene was sequenced from three Fagopyrum spp. (F. tataricum, F. esculentum and F. dibotrys) and showed the presence of three SNPs and four insertion/deletions at intra and inter specific level. Among them, the potential SNP (position 949th bp G>C) with Parsimony Informative Site was selected and successfully utilised to individuate the zygosity/allelic variation of 16 F. tataricum varieties. Insertion mutations were identified in coding region, which resulted the change of a stretch of 39 amino acids on the putative protein. Our Study revealed that autogamous species (F. tataricum) has lower frequency of observed SNPs as compared to allogamous species (F. dibotrys and F. esculentum). The identified SNPs in F. tataricum didn't result to amino acid change, while in other two species it caused both conservative and non-conservative variations. Consistent pattern of SNPs across the species revealed their phylogenetic importance. We found two groups of F. tataricum and one of them was closely related with F. dibotrys. Sequence characterization information of PAL gene reported in present investigation can be utilized in genetic improvement of buckwheat in reference to its medicinal value. PMID:26990297

  16. Genomic Characterization of Phenylalanine Ammonia Lyase Gene in Buckwheat

    PubMed Central

    Thiyagarajan, Karthikeyan; Vitali, Fabio; Tolaini, Valentina; Galeffi, Patrizia; Cantale, Cristina; Vikram, Prashant; Singh, Sukhwinder; De Rossi, Patrizia; Nobili, Chiara; Procacci, Silvia; Del Fiore, Antonella; Antonini, Alessandro; Presenti, Ombretta; Brunori, Andrea

    2016-01-01

    Phenylalanine Ammonia Lyase (PAL) gene which plays a key role in bio-synthesis of medicinally important compounds, Rutin/quercetin was sequence characterized for its efficient genomics application. These compounds possessing anti-diabetic and anti-cancer properties and are predominantly produced by Fagopyrum spp. In the present study, PAL gene was sequenced from three Fagopyrum spp. (F. tataricum, F. esculentum and F. dibotrys) and showed the presence of three SNPs and four insertion/deletions at intra and inter specific level. Among them, the potential SNP (position 949th bp G>C) with Parsimony Informative Site was selected and successfully utilised to individuate the zygosity/allelic variation of 16 F. tataricum varieties. Insertion mutations were identified in coding region, which resulted the change of a stretch of 39 amino acids on the putative protein. Our Study revealed that autogamous species (F. tataricum) has lower frequency of observed SNPs as compared to allogamous species (F. dibotrys and F. esculentum). The identified SNPs in F. tataricum didn’t result to amino acid change, while in other two species it caused both conservative and non-conservative variations. Consistent pattern of SNPs across the species revealed their phylogenetic importance. We found two groups of F. tataricum and one of them was closely related with F. dibotrys. Sequence characterization information of PAL gene reported in present investigation can be utilized in genetic improvement of buckwheat in reference to its medicinal value. PMID:26990297

  17. 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.

  18. 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

    2015-09-01

    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.

  19. Genomic Characterization of Phenylalanine Ammonia Lyase Gene in Buckwheat.

    PubMed

    Thiyagarajan, Karthikeyan; Vitali, Fabio; Tolaini, Valentina; Galeffi, Patrizia; Cantale, Cristina; Vikram, Prashant; Singh, Sukhwinder; De Rossi, Patrizia; Nobili, Chiara; Procacci, Silvia; Del Fiore, Antonella; Antonini, Alessandro; Presenti, Ombretta; Brunori, Andrea

    2016-01-01

    Phenylalanine Ammonia Lyase (PAL) gene which plays a key role in bio-synthesis of medicinally important compounds, Rutin/quercetin was sequence characterized for its efficient genomics application. These compounds possessing anti-diabetic and anti-cancer properties and are predominantly produced by Fagopyrum spp. In the present study, PAL gene was sequenced from three Fagopyrum spp. (F. tataricum, F. esculentum and F. dibotrys) and showed the presence of three SNPs and four insertion/deletions at intra and inter specific level. Among them, the potential SNP (position 949th bp G>C) with Parsimony Informative Site was selected and successfully utilised to individuate the zygosity/allelic variation of 16 F. tataricum varieties. Insertion mutations were identified in coding region, which resulted the change of a stretch of 39 amino acids on the putative protein. Our Study revealed that autogamous species (F. tataricum) has lower frequency of observed SNPs as compared to allogamous species (F. dibotrys and F. esculentum). The identified SNPs in F. tataricum didn't result to amino acid change, while in other two species it caused both conservative and non-conservative variations. Consistent pattern of SNPs across the species revealed their phylogenetic importance. We found two groups of F. tataricum and one of them was closely related with F. dibotrys. Sequence characterization information of PAL gene reported in present investigation can be utilized in genetic improvement of buckwheat in reference to its medicinal value.

  20. A new family of β-helix proteins with similarities to the polysaccharide lyases

    DOE PAGES

    Close, Devin W.; D'Angelo, Sara; Bradbury, Andrew R. M.

    2014-09-27

    Microorganisms that degrade biomass produce diverse assortments of carbohydrate-active enzymes and binding modules. Despite tremendous advances in the genomic sequencing of these organisms, many genes do not have an ascribed function owing to low sequence identity to genes that have been annotated. Consequently, biochemical and structural characterization of genes with unknown function is required to complement the rapidly growing pool of genomic sequencing data. A protein with previously unknown function (Cthe_2159) was recently isolated in a genome-wide screen using phage display to identify cellulose-binding protein domains from the biomass-degrading bacterium Clostridium thermocellum. Here, the crystal structure of Cthe_2159 is presentedmore » and it is shown that it is a unique right-handed parallel β-helix protein. Despite very low sequence identity to known β-helix or carbohydrate-active proteins, Cthe_2159 displays structural features that are very similar to those of polysaccharide lyase (PL) families 1, 3, 6 and 9. Cthe_2159 is conserved across bacteria and some archaea and is a member of the domain of unknown function family DUF4353. This suggests that Cthe_2159 is the first representative of a previously unknown family of cellulose and/or acid-sugar binding β-helix proteins that share structural similarities with PLs. More importantly, these results demonstrate how functional annotation by biochemical and structural analysis remains a critical tool in the characterization of new gene products.« less

  1. Decreased Warburg effect induced by ATP citrate lyase suppression inhibits tumor growth in pancreatic cancer.

    PubMed

    Zong, Haifeng; Zhang, Yang; You, Yong; Cai, Tiantian; Wang, Yehuang

    2015-03-01

    ATP citrate lyase (ACLY) is responsible for the conversion of cytosolic citrate into acetyl-CoA and oxaloacetate, and the first rate-limiting enzyme involved in de novo lipogenesis. Recent studies have demonstrated that inhibition of elevated ACLY results in growth arrest and apoptosis in a subset of cancers; however, the expression pattern and underlying biological function of ACLY in pancreatic ductal adenocarcinoma (PDAC) remains unclear. In the current study, overexpressed ACLY was more commonly observed in PDAC compared to normal pancreatic tissues. Kaplan-Meier survival analysis showed that high expression level of ACLY resulted in a poor prognosis of PDAC patients. Silencing of endogenous ACLY expression by siRNA in PANC-1 cells led to reduced cell viability and increased cell apoptosis. Furthermore, significant decrease in glucose uptake and lactate production was observed after ACLY was knocked down, and this effect was blocked by 2-deoxy-D-glucose, indicating that ACLY functions in the Warburg effect affect PDAC cell growth. Collectively, this study reveals that suppression of ACLY plays an anti-tumor role through decreased Warburg effect, and ACLY-related inhibitors might be potential therapeutic approaches for PDAC. PMID:25701462

  2. Immobilization of Cross-Linked Phenylalanine Ammonia Lyase Aggregates in Microporous Silica Gel

    PubMed Central

    Cui, Jian Dong; Li, Lian Lian; Bian, Hong Jie

    2013-01-01

    A separable and highly-stable enzyme system was developed by adsorption of phenylalanine ammonia lyase (PAL) from Rhodotorula glutinis in amino-functionalized macroporous silica gel and subsequent enzyme crosslinking. This resulted in the formation of cross-linked enzyme aggregates (PAL-CLEAs) into macroporous silica gel (MSG-CLEAs). The effect of adsorptive conditions, type of aggregating agent, its concentration as well as that of cross-linking agent was studied. MSG-CLEAs production was most effective using ammonium sulfate (40%-saturation), followed by cross-linking for 1 h with 1.5% (v/v) glutaraldehyde. The resulting MSG-CLEAs extended the optimal temperature and pH range compared to free PAL and PAL-CLEAs. Moreover, MSG-CLEAs exhibited the excellent stability of the enzyme against various deactivating conditions such as temperature and denaturants, and showed higher storage stability compared to the free PAL and the conventional PAL-CLEAs. Such as, after 6 h incubation at 60°C, the MSG-CLEAs still retained more than 47% of the initial activity whereas PAL-CLEAs only retained 7% of the initial activity. Especially, the MSG-CLEAs exhibited good reusability due to its suitable size and active properties. These results indicated that PAL-CLEAs on MSG might be used as a feasible and efficient solution for improving properties of immobilized enzyme in industrial application. PMID:24260425

  3. A new family of β-helix proteins with similarities to the polysaccharide lyases.

    PubMed

    Close, Devin W; D'Angelo, Sara; Bradbury, Andrew R M

    2014-10-01

    Microorganisms that degrade biomass produce diverse assortments of carbohydrate-active enzymes and binding modules. Despite tremendous advances in the genomic sequencing of these organisms, many genes do not have an ascribed function owing to low sequence identity to genes that have been annotated. Consequently, biochemical and structural characterization of genes with unknown function is required to complement the rapidly growing pool of genomic sequencing data. A protein with previously unknown function (Cthe_2159) was recently isolated in a genome-wide screen using phage display to identify cellulose-binding protein domains from the biomass-degrading bacterium Clostridium thermocellum. Here, the crystal structure of Cthe_2159 is presented and it is shown that it is a unique right-handed parallel β-helix protein. Despite very low sequence identity to known β-helix or carbohydrate-active proteins, Cthe_2159 displays structural features that are very similar to those of polysaccharide lyase (PL) families 1, 3, 6 and 9. Cthe_2159 is conserved across bacteria and some archaea and is a member of the domain of unknown function family DUF4353. This suggests that Cthe_2159 is the first representative of a previously unknown family of cellulose and/or acid-sugar binding β-helix proteins that share structural similarities with PLs. Importantly, these results demonstrate how functional annotation by biochemical and structural analysis remains a critical tool in the characterization of new gene products.

  4. Molecular Cloning and Characterization of Hydroperoxide Lyase Gene in the Leaves of Tea Plant (Camellia sinensis).

    PubMed

    Deng, Wei-Wei; Wu, Yi-Lin; Li, Ye-Yun; Tan, Zhen; Wei, Chao-Ling

    2016-03-01

    Hydroperoxide lyase (HPL, E.C. 4.1.2.) is the major enzyme in the biosynthesis of natural volatile aldehydes and alcohols in plants, however, little was known about HPL in tea plants (Camellia sinensis). A unique cDNA fragment was isolated by suppressive subtractive hybridization (SSH) from a tea plant subjected to herbivory by tea geometrid Ectropis obliqua. This full length cDNA acquired by RACE was 1476 bp and encoded 491 amino acids. DNA and protein BLAST searches showed high homology to HPL sequences from other plants. The His-tag expression vector pET-32a(+)/CsHPL was constructed and transferred into Escherichia coli Rosetta (DE3). The expression product of recombinant CsHPL in E. coli was about 60 kDa. The enzyme activity of CsHPL was 0.20 μmol·min(-1)·mg(-1). Quantitative RT-PCR analysis indicated CsHPL was strongly up-regulated in tea plants after Ectropis obliqua attack, suggesting that it may be an important candidate for defense against insects in tea plants.

  5. Enhanced selenium tolerance and accumulation in transgenic Arabidopsis expressing a mouse selenocysteine lyase.

    PubMed

    Pilon, Marinus; Owen, Jennifer D; Garifullina, Gulnara F; Kurihara, Tatsuo; Mihara, Hisaaki; Esaki, Nobuyoshi; Pilon-Smits, Elizabeth A H

    2003-03-01

    Selenium (Se) toxicity is thought to be due to nonspecific incorporation of selenocysteine (Se-Cys) into proteins, replacing Cys. In an attempt to direct Se flow away from incorporation into proteins, a mouse (Mus musculus) Se-Cys lyase (SL) was expressed in the cytosol or chloroplasts of Arabidopsis. This enzyme specifically catalyzes the decomposition of Se-Cys into elemental Se and alanine. The resulting SL transgenics were shown to express the mouse enzyme in the expected intracellular location, and to have SL activities up to 2-fold (cytosolic lines) or 6-fold (chloroplastic lines) higher than wild-type plants. Se incorporation into proteins was reduced 2-fold in both types of SL transgenics, indicating that the approach successfully redirected Se flow in the plant. Both the cytosolic and chloroplastic SL plants showed enhanced shoot Se concentrations, up to 1.5-fold compared with wild type. The cytosolic SL plants showed enhanced tolerance to Se, presumably because of their reduced protein Se levels. Surprisingly, the chloroplastic SL transgenics were less tolerant to Se, indicating that (over) production of elemental Se in the chloroplast is toxic. Expression of SL in the cytosol may be a useful approach for the creation of plants with enhanced Se phytoremediation capacity. PMID:12644675

  6. A new family of β-helix proteins with similarities to the polysaccharide lyases

    SciTech Connect

    Close, Devin W.; D'Angelo, Sara; Bradbury, Andrew R. M.

    2014-09-27

    Microorganisms that degrade biomass produce diverse assortments of carbohydrate-active enzymes and binding modules. Despite tremendous advances in the genomic sequencing of these organisms, many genes do not have an ascribed function owing to low sequence identity to genes that have been annotated. Consequently, biochemical and structural characterization of genes with unknown function is required to complement the rapidly growing pool of genomic sequencing data. A protein with previously unknown function (Cthe_2159) was recently isolated in a genome-wide screen using phage display to identify cellulose-binding protein domains from the biomass-degrading bacterium Clostridium thermocellum. Here, the crystal structure of Cthe_2159 is presented and it is shown that it is a unique right-handed parallel β-helix protein. Despite very low sequence identity to known β-helix or carbohydrate-active proteins, Cthe_2159 displays structural features that are very similar to those of polysaccharide lyase (PL) families 1, 3, 6 and 9. Cthe_2159 is conserved across bacteria and some archaea and is a member of the domain of unknown function family DUF4353. This suggests that Cthe_2159 is the first representative of a previously unknown family of cellulose and/or acid-sugar binding β-helix proteins that share structural similarities with PLs. More importantly, these results demonstrate how functional annotation by biochemical and structural analysis remains a critical tool in the characterization of new gene products.

  7. Design of benzimidazole- and benzoxazole-2-thione derivatives as inhibitors of bacterial hyaluronan lyase.

    PubMed

    Braun, Stephan; Botzki, Alexander; Salmen, Sunnhild; Textor, Christian; Bernhardt, Günther; Dove, Stefan; Buschauer, Armin

    2011-09-01

    Bacterial hyaluronan lyases (Hyal) degrade hyaluronan, an important component of the extracellular matrix, and are involved in microbial spread. Hyal inhibitors may serve as tools to study the role of the enzyme, its substrates and products in the course of bacterial infections. Moreover, such enzyme inhibitors are potential candidates for antibacterial combination therapy. Based on crystal structures of Streptococcus pneumoniae Hyal in complex with a hexasaccharide substrate and with different inhibitors, 1-acylated benzimidazole-2-thiones and benzoxazole-2-thiones were derived as new leads for the inhibition of Streptococcus agalactiae strain 4755 Hyal. Structure-based optimization led to N-(3-phenylpropionyl)benzoxazole-2-thione, one of the most potent compounds known to date (IC(50) values: 24 μM at pH 7.4, 15 μM at pH 5). Among the 27 new derivatives, other N-acylated benzimidazoles and benzoxazoles are just as active at pH 7.4, but not at pH 5. The results support a binding mode characterized by interactions with residues in the catalytic site and with a hydrophobic patch.

  8. Synthesis and biocompatibility of porous nano-hydroxyapatite/collagen/alginate composite.

    PubMed

    Zhang, S M; Cui, F Z; Liao, S S; Zhu, Y; Han, L

    2003-07-01

    Porous nano-hydroxyapatite/collagen/alginate (nHAC/Alginate) composite containing nHAC and Ca-crosslinked alginate is synthesized biomimetically. This composite shows a significant improvement in mechanical properties over nHAC material. Mechanical test results show that the compressive modulus and yield strength of this composite are in direct proportion to the percentage of Ca-crosslinked alginate in the composite. Primary biocompatibility experiments in vitro including fibroblasts and osteoblasts co-culture with nHAC/alginate composite indicated the high biocompatibility of this composite. Therefore the composite can be a promising candidate of scaffold material for bone tissue engineering.

  9. Microencapsulation of probiotics in hydrogel particles: enhancing Lactococcus lactis subsp. cremoris LM0230 viability using calcium alginate beads.

    PubMed

    Yeung, Timothy W; Arroyo-Maya, Izlia J; McClements, David J; Sela, David A

    2016-04-01

    Probiotics are beneficial microbes often added to food products to enhance the health and wellness of consumers. A major limitation to producing efficacious functional foods containing probiotic cells is their tendency to lose viability during storage and gastrointestinal transit. In this study, the impact of encapsulating probiotics within food-grade hydrogel particles to mitigate sensitivity to environmental stresses was examined. Confocal fluorescence microscopy confirmed that Lactococcus lactis were trapped within calcium alginate beads formed by dripping a probiotic-alginate mixture into a calcium solution. Encapsulation improved the viability of the probiotics during aerobic storage: after seven days, less than a two-log reduction was observed in encapsulated cells stored at room temperature, demonstrating that a high concentration of cells survived relative to non-encapsulated bacteria. These hydrogel beads may have applications for improving the stability and efficacy of probiotics in functional foods.

  10. 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

    2014-11-01

    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. PMID:25129727

  11. 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

    2014-11-01

    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.

  12. Design and application of an in vivo reporter assay for phenylalanine ammonia-lyase.

    PubMed

    Wang, Siyuan; Zhang, Shuwei; Zhou, Tong; Zeng, Jia; Zhan, Jixun

    2013-09-01

    Phenylalanine ammonia-lyase (PAL) is an important enzyme that links primary metabolism to secondary metabolism. Its efficiency is often a critical factor that affects the overall flux of a related metabolic pathway, the titer of the final products, and the efficacy of PAL-based therapies. Thus, PAL is a common target for metabolic engineering, and it is of significant interest to screen efficient PALs for industrial and medical applications. In this study, a novel and efficient visible reporter assay for screening of PAL efficiency in Escherichia coli was established based on a plant type III polyketide biosynthetic pathway. The candidate PALs were co-expressed with a 4-coumarate:CoA ligase 4CL1 from Arabidopsis thaliana and curcuminoid synthase (CUS) from Oryza sativa in E. coli BL21(DE3) to form a dicinnamoylmethane biosynthetic pathway. Taking advantage of the yellow color of the product, a microplate-based assay was designed to measure the titer of dicinnamoylmethane, which was validated by HPLC analysis. The different titers of the product reflect the overall performance (expression level and enzymatic activity) of the individual PALs in E. coli. Using this system, we have screened three PALs (PAL1, PAL3, and PAL4) from Trifolium pratense, among which PAL1 showed the best performance in E. coli. The engineered E. coli strain containing PAL1, 4CL1, and CUS led to the production of dicinnamoylmethane at a high level of 0.36 g/l. Supplement of 2-fluoro-phenylalanine yielded two fluorinated dicinnamoylmethane derivatives, 6,6'-difluoro-dicinnamoylmethane and 6-fluoro-dicinnamoylmethane, of which the latter is a new curcuminoid.

  13. On the specificity of lipid hydroperoxide fragmentation by fatty acid hydroperoxide lyase from Arabidopsis thaliana.

    PubMed

    Kandzia, Romy; Stumpe, Michael; Berndt, Ekkehardt; Szalata, Marlena; Matsui, Kenji; Feussner, Ivo

    2003-07-01

    Fatty acid hydroperoxide lyase (HPL) is a membrane associated P450 enzyme that cleaves fatty acid hydroperoxides into aldehydes and omega-oxo fatty acids. One of the major products of this reaction is (3Z)-hexenal. It is a constituent of many fresh smelling fruit aromas. For its biotechnological production and because of the lack of structural data on the HPL enzyme family, we investigated the mechanistic reasons for the substrate specificity of HPL by using various structural analogues of HPL substrates. To approach this 13-HPL from Arabidopsis thaliana was cloned and expressed in E. coli utilising a His-Tag expression vector. The fusion protein was purified by affinity chromatography from the E. coli membrane fractions and its pH optimum was detected to be pH 7.2. Then, HPL activity against the respective (9S)- and (13S)-hydroperoxides derived either from linoleic, alpha-linolenic or gamma-linolenic acid, respectively, as well as that against the corresponding methyl esters was analysed. Highest enzyme activity was observed with the (13S)-hydroperoxide of alpha-linolenic acid (13alpha-HPOT) followed by that with its methyl ester. Most interestingly, when the hydroperoxy isomers of gamma-linolenic acid were tested as substrates, 9gamma-HPOT and not 13gamma-HPOT was found to be a better substrate of the enzyme. Taken together from these studies on the substrate specificity it is concluded that At13HPL may not recognise the absolute position of the hydroperoxy group within the substrate, but shows highest activities against substrates with a (1Z4S,5E,7Z)-4-hydroperoxy-1,5,7-triene motif. Thus, At13HPL may not only be used for the production of C6-derived volatiles, but depending on the substrate may be further used for the production of Cg-derived volatiles as well. PMID:12940547

  14. Chitosan and alginate biopolymer membranes for remediation of contaminated water with herbicides.

    PubMed

    Agostini de Moraes, Mariana; Cocenza, Daniela Sgarbi; da Cruz Vasconcellos, Fernando; Fraceto, Leonardo Fernandes; Beppu, Marisa Masumi

    2013-12-15

    This study investigated the adsorption behavior of the herbicides diquat, difenzoquat and clomazone on biopolymer membranes prepared with alginate and chitosan (pristine and multi-layer model) for contaminated water remediation applications. Herbicides, at concentrations ranging from 5 μM to 200 μM, were adsorbed in either pure alginate, pure chitosan or a bilayer membrane composed of chitosan/alginate. No adsorption of clomazone was observed on any of the membranes, probably due to lack of electrostatic interactions between the herbicide and the membranes. Diquat and difenzoquat were only adsorbed on the alginate and chitosan/alginate membranes, indicating that this adsorption takes place in the alginate layer. At a concentration of 50 μM, diquat adsorption reaches ca. 95% after 120 min on both the alginate and chitosan/alginate membranes. The adsorption of difenzoquat, at the same concentration, reaches ca. 62% after 120 min on pure alginate membranes and ca. 12% on chitosan/alginate bilayer membranes. The adsorption isotherms for diquat and difenzoquat were further evaluated using the isotherm models proposed by Langmuir and by Freundlich, where the latter represented the best-fit model. Results indicate that adsorption occurs via coulombic interactions between the herbicides and alginate and is strongly related to the electrostatic charge, partition coefficients and dissociation constants of the herbicides. Biopolymer based membranes present novel systems for the removal of herbicides from contaminated water sources and hold great promise in the field of environmental science and engineering.

  15. Effect of chitosan coating on a bacteria-based alginate microrobot.

    PubMed

    Park, Sung Jun; Lee, Yu Kyung; Cho, Sunghoon; Uthaman, Saji; Park, In-Kyu; Min, Jung-Joon; Ko, Seong Young; Park, Jong-Oh; Park, Sukho

    2015-04-01

    To develop an efficient bacteria-based microrobot, first, therapeutic bacteria should be encapsulated into microbeads using biodegradable and biocompatible materials; second, the releasing rate of the encapsulated bacteria for theragnostic function should be regulated; and finally, flagellated bacteria should be attached on the microbeads to ensure the motility of the microrobot. For the therapeutic bacteria encapsulation, an alginate can be a promising candidate as a biodegradable and biocompatible material. Owing to the non-regulated releasing rate of the encapsulated bacteria in alginate microbeads and the weak attachment of flagellated bacteria on the surface of alginate microbeads, however, the alginate microbeads cannot be used as effective cargo for a bacteria-based microrobot. In this paper, to enhance the stability of the bacteria encapsulation and the adhesion of flagellated bacteria in alginate microbeads, we performed a surface modification of alginate microbeads using chitosan coating. The bacteria-encapsulated alginate microbeads with 1% chitosan coating maintained their structural integrity up to 72 h, whereas the control alginate microbead group without chitosan coating showed severe degradations after 24 h. The chitosan coating in alginate microbeads shows the enhanced attachment of flagellated bacteria on the surface of alginate microbeads. The bacteria-actuated microrobot with the enhanced flagellated bacteria attachment could show approximately 4.2 times higher average velocities than the control bacteria-actuated microrobot without chitosan coating. Consequently, the surface modification using chitosan coating enhanced the structural stability and the motility of the bacteria-based alginate microrobots.

  16. Effects of composition and structure of alginates on adsorption of divalent metals

    NASA Astrophysics Data System (ADS)

    Nai-Yu, Zheng; Yan-Xia, Zhang; Xiao, Fan; Li-Jun, Han

    1994-03-01

    Results of a series of experiments (on the adsorption of divalent metal ions by dried alginic acid, Na and Ca alginates of different composition and block structure) conducted in this systematic study of the effects of the composition and structure of alginates on the static adsorption equilibrium of divalent metal ions indicate that the properties of alginate adsorption to divalent metal ions are highly different, depending not only on the cations used, but also on the form and structure of the alginates. There is close correlation between the adsorption properties and the structure of the alginates. The selectivity coefficient of Na alginate for Cd-Sr ion exchange tends to increase with the increase of the M/G ratio in alginate, whereas the adsorption capacity of Ca alginate for Cu2+ ion decrease with the increase of the G-block or the average length of the G-block(bar N_G ) and the total adsorption capacity of alginic acid is found to vary in the same order as the F MM(diad frequency) in alginate in the mixed solution of Sr2+, Ba2+ and Cd2+.

  17. Antimicrobial activity of lactoperoxidase system incorporated into cross-linked alginate films.

    PubMed

    Yener, Fatih Y G; Korel, Figen; Yemenicioğlu, Ahmet

    2009-03-01

    In this study, the antimicrobial effect of lactoperoxidase (LPS) incorporated alginate films was investigated on Escherichia coli (NRRL B-3008), Listeria innocua (NRRL B-33314), and Pseudomonas fluorescens (NRRL B-253) in presence of different concentrations of H(2)O(2) (0.2, 0.4, and 0.8 mM) and KSCN (1, 2, and 4 mM). The incorporation of 70 nmol ABTS/min/cm(2) LPS into alginate films gave 0.66 to 0.85 nmol ABTS/min/cm(2) enzyme activity at 0.2 to 0.8 mM H(2)O(2) concentration range. The antimicrobial activity of LPS system on target bacteria changed according to the concentrations of KSCN and H(2)O(2). The growth of all tested bacteria was prevented for a 6-h period by applying LPS system in presence of 0.4 or 0.8 mM H(2)O(2) and 4 mM KSCN. At 0.8 mM H(2)O(2) and 4 mM KSCN, the LPS system also inhibited growth of L. innocua and P. fluorescens for a 24-h incubation period, whereas E. coli growth could not be inhibited for 24 h under these conditions. At 0.2 mM H(2)O(2) and 1 to 4 mM KSCN, a considerable inhibitory effect was obtained only on P. fluorescens. The decreasing order of the resistance of studied bacteria to LPS system is as follows: E. coli, L. innocua, and P. fluorescens. The developed antimicrobial system has a good potential for use in meat, poultry, and seafood since alginate coatings are already used in these products. Further studies are needed to test the LPS incorporated edible films in real food systems.

  18. Effects of alginate oligosaccharide mixtures on the growth and fatty acid composition of the green alga Chlamydomonas reinhardtii.

    PubMed

    Yamasaki, Yasuhiro; Yokose, Takeshi; Nishikawa, Toru; Kim, Daekyung; Jiang, Zedong; Yamaguchi, Kenichi; Oda, Tatsuya

    2012-01-01

    Alginate is a natural acidic linear polysaccharide that is produced by brown seaweeds. It is currently used in a broad range of commercial enterprises, such as the food and medical products industries. Recent evidence has demonstrated that alginate oligosaccharides may function as growth promoting agents for certain plant cells, including those of some green algae. Chlamydomonas reinhardtii is a green alga that is used as a model organism in fundamental molecular biology studies; it is also a producer of biohydrogen. In the present study, we examined effects of two types of alginate oligosaccharide mixtures (AOMs), which were prepared by either enzymatic degradation (ED) or acid hydrolysis (AH), on the growth of C. reinhardtii. Growth was significantly promoted by AOM (ED) in a concentration-dependent manner. The maximum effect was observed on day 4 of treatment. The fatty acid composition of C. reinhardtii was also influenced by AOM (ED); the levels of C16:0, C18:2 cis and C18:3 n-3 increased in treated cells. AOM (AH) and the other saccharides that we tested did not affect the growth of C. reinhardtii. The effects that we identified could promote efficient biomass production by reducing culture times and by changing cellular fatty acid levels.

  19. The Effect of Chondroitin Sulphate and Hyaluronic Acid on Chondrocytes Cultured within a Fibrin-Alginate Hydrogel.

    PubMed

    Little, Christopher J; Kulyk, William M; Chen, Xiongbiao

    2014-01-01

    Osteoarthritis is a painful degenerative joint disease that could be better managed if tissue engineers can develop methods to create long-term engineered articular cartilage tissue substitutes. Many of the tissue engineered cartilage constructs currently available lack the chemical stimuli and cell-friendly environment that promote the matrix accumulation and cell proliferation needed for use in joint cartilage repair. The goal of this research was to test the efficacy of using a fibrin-alginate hydrogel containing hyaluronic acid (HA) and/or chondroitin sulphate (CS) supplements for chondrocyte culture. Neonatal porcine chondrocytes cultured in fibrin-alginate hydrogels retained their phenotype better than chondrocytes cultured in monolayer, as evidenced by analysis of their relative expression of type II versus type I collagen mRNA transcripts. HA or CS supplementation of the hydrogels increased matrix glycosaminoglycan (GAG) production during the first week of culture. However, the effects of these supplements on matrix accumulation were not additive and were no longer observed after two weeks of culture. Supplementation of the hydrogels with CS or a combination of both CS and HA increased the chondrocyte cell population after two weeks of culture. Statistical analysis indicated that the HA and CS treatment effects on chondrocyte numbers may be additive. This research suggests that supplementation with CS and/or HA has positive effects on cartilage matrix production and chondrocyte proliferation in three-dimensional (3D) fibrin-alginate hydrogels. PMID:25238548

  20. Alginate gel microwell arrays using electrodeposition for three-dimensional cell culture.

    PubMed

    Ozawa, Fumisato; Ino, Kosuke; Arai, Toshiharu; Ramón-Azcón, Javier; Takahashi, Yasufumi; Shiku, Hitoshi; Matsue, Tomokazu

    2013-08-01

    In this study, we developed a novel method for fabricating microwell arrays constructed from alginate gels, and the alginate gel microwells were used for three-dimensional (3D) cell culture. The alginate gel microwells were fabricated on a patterned ITO electrode using alginate gel electrodeposition. Embryonic stem (ES) cells or hepatocellular carcinoma cells (HepG2) were cultured in the alginate gel microwells containing 3T3 cells. During the culture, embryoid bodies (EBs) or HepG2 spheroids were successfully fabricated in the alginate gel microwells. The oxygen consumption of the EBs indicated that they were successfully cultured. Liver-specific gene expressions of the HepG2 spheroids apparently increased by performing 3D co-culture in the microwell arrays with 3T3 cells. These results show that the alginate gel microwells are a useful 3D culture system.

  1. 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

  2. Ironing out their differences: dissecting the structural determinants of a phenylalanine aminomutase and ammonia lyase.

    PubMed

    Heberling, Matthew M; Masman, Marcelo F; Bartsch, Sebastian; Wybenga, Gjalt G; Dijkstra, Bauke W; Marrink, Siewert J; Janssen, Dick B

    2015-04-17

    Deciphering the structural features that functionally separate ammonia lyases from aminomutases is of interest because it may allow for the engineering of more efficient aminomutases for the synthesis of unnatural amino acids (e.g., β-amino acids). However, this has proved to be a major challenge that involves understanding the factors that influence their activity and regioselectivity differences. Herein, we report evidence of a structural determinant that dictates the activity differences between a phenylalanine ammonia lyase (PAL) and aminomutase (PAM). An inner loop region that closes the active sites of both PAM and PAL was mutated within PAM (PAM residues 77-97) in a stepwise approach to study the effects when the equivalent residue(s) found in the PAL loop were introduced into the PAM loop. Almost all of the single loop mutations triggered a lyase phenotype in PAM. Experimental and computational evidence suggest that the induced lyase features result from inner loop mobility enhancements, which are possibly caused by a 310-helix cluster, flanking α-helices, and hydrophobic interactions. These findings pinpoint the inner loop as a structural determinant of the lyase and mutase activities of PAM.

  3. 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

  4. KdgF, the missing link in the microbial metabolism of uronate sugars from pectin and alginate.

    PubMed

    Hobbs, Joanne K; Lee, Seunghyae M; Robb, Melissa; Hof, Fraser; Barr, Christopher; Abe, Kento T; Hehemann, Jan-Hendrik; McLean, Richard; Abbott, D Wade; Boraston, Alisdair B

    2016-05-31

    Uronates are charged sugars that form the basis of two abundant sources of biomass-pectin and alginate-found in the cell walls of terrestrial plants and marine algae, respectively. These polysaccharides represent an important source of carbon to those organisms with the machinery to degrade them. The microbial pathways of pectin and alginate metabolism are well studied and essentially parallel; in both cases, unsaturated monouronates are produced and processed into the key metabolite 2-keto-3-deoxygluconate (KDG). The enzymes required to catalyze each step have been identified within pectinolytic and alginolytic microbes; yet the function of a small ORF, kdgF, which cooccurs with the genes for these enzymes, is unknown. Here we show that KdgF catalyzes the conversion of pectin- and alginate-derived 4,5-unsaturated monouronates to linear ketonized forms, a step in uronate metabolism that was previously thought to occur spontaneously. Using enzyme assays, NMR, mutagenesis, and deletion of kdgF, we show that KdgF proteins from both pectinolytic and alginolytic bacteria catalyze the ketonization of unsaturated monouronates and contribute to efficient production of KDG. We also report the X-ray crystal structures of two KdgF proteins and propose a mechanism for catalysis. The discovery of the function of KdgF fills a 50-y-old gap in the knowledge of uronate metabolism. Our findings have implications not only for the understanding of an important metabolic pathway, but also the role of pectinolysis in plant-pathogen virulence and the growing interest in the use of pectin and alginate as feedstocks for biofuel production. PMID:27185956

  5. Enzymatic cellulose hydrolysis: enzyme reusability and visualization of β-glucosidase immobilized in calcium alginate.

    PubMed

    Tsai, Chien-Tai; Meyer, Anne S

    2014-01-01

    The high cellulase enzyme dosages required for hydrolysis of cellulose is a major cost challenge in lignocellulosic ethanol production. One method to decrease the enzyme dosage and increase biocatalytic productivity is to re-use β-glucosidase (BG) via immobilization. In the present research, glutaraldehyde cross-linked BG was entrapped in calcium alginate gel particles. More than 60% of the enzyme activity could be recovered under optimized conditions, and glutaraldehyde cross-linking decreased leakage of BG from the calcium alginate particles. The immobilized BG aggregates were visualized by confocal laser scanning microscopy (CLSM). The CLSM images, which we believe are the first to be published, corroborate that more BG aggregates were entrapped in the matrix when the enzymes were cross-linked by glutaraldehyde as opposed to when they are not cross-linked. The particles with the immobilized BG were recycled for cellulase catalyzed hydrolysis of Avicel. No significant loss in BG activity was observed for up to 20 rounds of reaction recycle steps of the BG particles of 48 h each, verifying a significant stabilization of the BG by immobilization. Similar high glucose yields were obtained by one round of enzymatic hydrolysis of hydrothermally pretreated barley straw during a 72 h reaction with immobilized BG and free BG. PMID:25429563

  6. Chemical oxidation of a malodorous compound, indole, using iron entrapped in calcium alginate beads.

    PubMed

    Ben Hammouda, Samia; Adhoum, Nafaâ; Monser, Lotfi

    2016-01-15

    Iron-alginate beads (Fe-ABs) were successfully prepared by the ion-gelation method, and applied as heterogeneous Fenton catalysts for the removal of a malodorous compound 'indole'. Similarly, copper-enriched alginate beads (Cu-ABs) were synthesized and tested as like-Fenton catalyst, however, their application proved not to be effective for this purpose. Fe-ABs catalysts were characterized by FTIR, SEM, EDS and AAS spectroscopy. Results pointed out that the parameters affecting Fenton catalysis must be carefully chosen to avoid excessive iron release. Under optimal conditions, complete indole removal and considerably high reduction of TOC, without significant leaching was achieved. Indole decay followed a pseudo-first-order kinetics. The absolute rate constant for indole hydroxylation was 3.59×10(9) M(-1) s(-1), as determined by the competition kinetics method. Four reaction intermediates (Isatin, Dioxindole, Oxindole and Anthralinic acid) were identified by ULC/MS/MS analysis. Short-chain aliphatic carboxylic acids like formic, acetic, oxalic, maleic, oxamic and pyruvic acids were identified by ion exclusion chromatography and as end-products. Based on the identified by-products, a plausible mineralization pathway was proposed. Moreover, the catalyst was recovered quantitatively by simple filtration and reused for several times without significant loss of activity. PMID:26384996

  7. Enzymatic cellulose hydrolysis: enzyme reusability and visualization of β-glucosidase immobilized in calcium alginate.

    PubMed

    Tsai, Chien-Tai; Meyer, Anne S

    2014-11-25

    The high cellulase enzyme dosages required for hydrolysis of cellulose is a major cost challenge in lignocellulosic ethanol production. One method to decrease the enzyme dosage and increase biocatalytic productivity is to re-use β-glucosidase (BG) via immobilization. In the present research, glutaraldehyde cross-linked BG was entrapped in calcium alginate gel particles. More than 60% of the enzyme activity could be recovered under optimized conditions, and glutaraldehyde cross-linking decreased leakage of BG from the calcium alginate particles. The immobilized BG aggregates were visualized by confocal laser scanning microscopy (CLSM). The CLSM images, which we believe are the first to be published, corroborate that more BG aggregates were entrapped in the matrix when the enzymes were cross-linked by glutaraldehyde as opposed to when they are not cross-linked. The particles with the immobilized BG were recycled for cellulase catalyzed hydrolysis of Avicel. No significant loss in BG activity was observed for up to 20 rounds of reaction recycle steps of the BG particles of 48 h each, verifying a significant stabilization of the BG by immobilization. Similar high glucose yields were obtained by one round of enzymatic hydrolysis of hydrothermally pretreated barley straw during a 72 h reaction with immobilized BG and free BG.

  8. Simultaneous removal of phenol, Cu and Cd from water with corn cob silica-alginate beads.

    PubMed

    Shim, Jaehong; Lim, Jeong-Muk; Shea, Patrick J; Oh, Byung-Taek

    2014-05-15

    Phenol and heavy metals in petroleum waste are environmental and human health concerns, but physicochemical removal is often cost-prohibitive and can produce toxic secondary products and treatment residues. An environmentally benign alternative combines corn cob silica with alginate and immobilized bacteria into beads for treating contaminated water. The concentration of phenol was decreased >92% by Pseudomonas putida YNS1 on aliginate-silica beads (2%, w/v) after equilibrating for 96h with water containing 214mg phenol/L. GC-MS analysis indicated formation of benzoquinone and other polar products. Beads containing corn cob silica decreased Cu concentrations by 84-88% and Cd by 83-87% within 24h. In a mixture of 114mg phenol, 43mg Cu and 51mg Cd/L, phenol removal (93% within 96h) only occurred with beads containing the silica and bacterial strain. Beads containing corn cob silica removed >97% of the Cu and >99% of the Cd, critical for reducing toxicity to the bacteria. Beads with the immobilized strain removed phenol when zeolite was used instead of corn cob silica, but beads with silica were more effective for Cu and Cd removal. Results show the potential of corn cob silica combined with alginate and immobilized bacteria for removing phenol and heavy metals from contaminated water. PMID:24685529

  9. Establishment of chondroitin B lyase-based analytical methods for sensitive and quantitative detection of dermatan sulfate in heparin.

    PubMed

    Wu, Jingjun; Ji, Yang; Su, Nan; Li, Ye; Liu, Xinxin; Mei, Xiang; Zhou, Qianqian; Zhang, Chong; Xing, Xin-hui

    2016-06-25

    Dermatan sulfate (DS) is one of the hardest impurities to remove from heparin products due to their high structural similarity. The development of a sensitive and feasible method for quantitative detection of DS in heparin is essential to ensure the clinical safety of heparin pharmaceuticals. In the current study, based on the substrate specificity of chondroitin B lyase, ultraviolet spectrophotometric and strong anion-exchange high-performance liquid chromatographic methods were established for detection of DS in heparin. The former method facilitated analysis in heparin with DS concentrations greater than 0.1mgmL(-1) at 232nm, with good linearity, precision and recovery. The latter method allowed sensitive and accurate detection of DS at concentrations lower than 0.1mgmL(-1), exhibiting good linearity, precision and recovery. The linear range of DS detection using the latter method was between 0.01 and 0.5mgmL(-1).

  10. Effects of CO/sub 2/ on total phenolics, phenylalanine ammonia lyase, and polyphenol oxidase in lettuce tissue

    SciTech Connect

    Siriphanich, J.; Kader, A.A.

    1985-01-01

    An atmosphere of air + 15% CO/sub 2/ caused CO/sub 2/ injury in lettuce (Lactuca sativa L.) in about 10 days at 0/sup 0/C. However, subsequent removal of CO/sub 2/ was necessary for the brown stain symptoms to develop. Under CO/sub 2/ treatment, phenylalanine ammonia lyase (PAL) was induced and its activity correlated well with the development of the injury. Nevertheless, PAL activity did not seem responsible for the differences in susceptibility to CO/sub 2/ injury among the 3 lettuce cultivars included in this study. Prevention of the development of brown stain symptoms by CO/sub 2/ probably was due to its inhibition of phenolics production and the inhibition of polyphenol oxidase activity. 27 references, 10 figures.

  11. 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

  12. Strains for the production of flavonoids from glucose

    DOEpatents

    Stephanopoulos, Gregory; Santos, Christine; Koffas, Mattheos

    2015-11-13

    The invention relates to the production of flavonoids and flavonoid precursors in cells through recombinant expression of tyrosine ammonia lyase (TAL), 4-coumarate:CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI).

  13. A carbon-nitrogen lyase from Leucaena leucocephala catalyzes the first step of mimosine degradation.

    PubMed

    Negi, Vishal Singh; Bingham, Jon-Paul; Li, Qing X; Borthakur, Dulal

    2014-02-01

    The tree legume Leucaena leucocephala contains a large amount of a toxic nonprotein aromatic amino acid, mimosine, and also an enzyme, mimosinase, for mimosine degradation. In this study, we isolated a 1,520-bp complementary DNA (cDNA) for mimosinase from L. leucocephala and characterized the encoded enzyme for mimosine-degrading activity. The deduced amino acid sequence of the coding region of the cDNA was predicted to have a chloroplast transit peptide. The nucleotide sequence, excluding the sequence for the chloroplast transit peptide, was codon optimized and expressed in Escherichia coli. The purified recombinant enzyme was used in mimosine degradation assays, and the chromatogram of the major product was found to be identical to that of 3-hydroxy-4-pyridone (3H4P), which was further verified by electrospray ionization-tandem mass spectrometry. The enzyme activity requires pyridoxal 5'-phosphate but not α-keto acid; therefore, the enzyme is not an aminotransferase. In addition to 3H4P, we also identified pyruvate and ammonia as other degradation products. The dependence of the enzyme on pyridoxal 5'-phosphate and the production of 3H4P with the release of ammonia indicate that it is a carbon-nitrogen lyase. It was found to be highly efficient and specific in catalyzing mimosine degradation, with apparent Km and Vmax values of 1.16×10(-4) m and 5.05×10(-5) mol s(-1) mg(-1), respectively. The presence of other aromatic amino acids, including l-tyrosine, l-phenylalanine, and l-tryptophan, in the reaction did not show any competitive inhibition. The isolation of the mimosinase cDNA and the biochemical characterization of the recombinant enzyme will be useful in developing transgenic L. leucocephala with reduced mimosine content in the future. PMID:24351687

  14. Endogenous carbon monoxide downregulates hepatic cystathionine-γ-lyase in rats with liver cirrhosis

    PubMed Central

    GUO, SHI-BIN; DUAN, ZHI-JUN; WANG, QIU-MING; ZHOU, QIN; LI, QING; SUN, XIAO-YU

    2015-01-01

    The aim of the present study was to investigate the effect of endogenous carbon monoxide (CO) on the hydrogen sulfide/cystathionine-γ-lyase (H2S/CSE) pathway in cirrhotic rat livers. The rats were allocated at random into four groups: Sham, cirrhosis, cobalt protoporphyrin (CoPP) and zinc protoporphyrin IX (ZnPP). The expression of hepatic CSE mRNA was evaluated using a quantitative polymerase chain reaction, while CSE protein expression was determined using immunohistochemical analysis. Hematoxylin and eosin staining was performed for the histological evaluation of liver fibrosis. The levels of H2S, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL) and carboxyhemoglobin (COHb) in the arterial blood were determined, in addition to the portal vein pressure. The mRNA and protein expression levels of hepatic CSE and the serum levels of H2S were significantly decreased in the cirrhosis group compared with those in the sham group (P<0.05). Compared with the cirrhosis group, rats in the ZnPP group had significantly lower levels of serum ALT, AST and TBIL, arterial COHb and hepatic fibrosis, while hepatic CSE expression and the production of H2S were significantly increased (P<0.05). The CoPP group exhibited decreased hepatic CSE expression and H2S production, but aggravated hepatic function and fibrosis (P<0.05). In conclusion, the H2S/CSE pathway is involved in the formation of liver cirrhosis and serves a crucial function in protecting liver cells against the progression of liver fibrosis. Endogenous CO downregulates hepatic CSE mRNA and protein expression and the production of H2S in rats with liver cirrhosis. PMID:26668593

  15. Effects of Perfusion and Dynamic Loading on Human Neocartilage Formation in Alginate Hydrogels

    PubMed Central

    Grogan, Shawn P.; Sovani, Sujata; Pauli, Chantal; Chen, Jianfen; Hartmann, Andreas; Colwell, Clifford W.; Lotz, Martin K.

    2012-01-01

    Dynamic loading and perfusion culture environments alone are known to enhance cartilage extracellular matrix (ECM) production in dedifferentiated articular chondrocytes. In this study, we explored whether a combination of these factors would enhance these processes over a free-swelling (FS) condition using adult human articular chondrocytes embedded in 2% alginate. The alginate constructs were placed into a bioreactor for perfusion (P) only (100 μL/per minute) or perfusion and dynamic compressive loading (PL) culture (20% for 1 h, at 0.5 Hz), each day. Control FS alginate gels were maintained in six-well static culture. Gene expression analysis was conducted on days 7 and 14, while cell viability, immunostaining, and mechanical property testing were performed on day 14 only. Total glycosaminoglycan (GAG) content and GAG synthesis were assessed after 14 days. Col2a1 mRNA expression levels were significantly higher (at least threefold; p<0.05) in both bioreactor conditions compared with FS by days 7 and 14. For all gene studies, no significant differences were seen between P and PL treatments. Aggrecan mRNA levels were not significantly altered in any condition although both GAG/DNA and 35S GAG incorporation studies indicated higher GAG retention and synthesis in the FS treatment. Collagen type II protein deposition was low in all samples, link protein distribution was more diffuse in FS condition, and aggrecan deposition was located in the outer regions of the alginate constructs in both bioreactor conditions, yet more uniformly in the FS condition. Catabolic gene expression (matrix metalloproteinase 3 [MMP3] and inducible nitric oxide synthase [iNOS]) was higher in bioreactor conditions compared with FS, although iNOS expression levels decreased to approximately fourfold less than the FS condition by day 14. Our data indicate that conditions created in the bioreactor enhanced both anabolic and catabolic responses, similar to other loading studies. Perfusion

  16. Argininosuccinate lyase: a new autoantigen in liver disease.

    PubMed

    Pelli, N; Fensom, A H; Slade, C; Boa, F; Mieli-Vergani, G; Vergani, D

    1998-12-01

    Anti-liver cytosol 1 autoantibody (LC1) characterizes a severe form of autoimmune hepatitis (AIH), staining the cytoplasm of periportal hepatocytes and targeting an unidentified 60-kD liver cytosolic antigen. To identify its target, we used high-titre anti-LCI+ sera from two patients with AIH to screen 18 cytoplasm enzymes with periportal location by double immunodiffusion (DDI). Both sera gave a broad precipitin line against human liver cytosol, suggesting that they may recognize two distinct antigens, a possibility confirmed by the appearance of two precipitin lines when DDI conditions were optimized (0.8% agarose and 3% polyethylene glycol (PEG)). Experiments by DDI and Western blot (WB) identified a liver cytosolic autoantigen of 50 kD, different from LC1, giving a line of identity with argininosuccinate lyase (ASL). Reactivity to ASL was then investigated by DDI and WB in 57 patients with AIH, 17 with primary biliary cirrhosis (PBC), 15 with chronic hepatitis B virus (HBV) infection, 13 with alphal-antitrypsin deficiency, 17 with Wilson's disease, 18 with extrahepatic autoimmune disorders, and in 48 healthy controls. Anti-ASL was found in 16% of AIH and 23% of PBC patients by DDI and in 14% of AIH, 23% of PBC and 20% of HBV patients by WB. No argininosuccinate was present in the urine of four anti-ASL+ patients tested, excluding an inhibition of enzymatic activity by anti-ASL. The addition of anti-ASL+ serum to human fibroblast cultures induced a significant increase in ASL activity. ASL is a new autoantigen in liver disease and its clinical relevance warrants further investigation.

  17. Cystathionine γ-lyase: clinical, metabolic, genetic, and structural studies

    PubMed Central

    Kraus, Jan P.; Hašek, Jindrich; Kožich, Viktor; Collard, Renata; Venezia, Sarah; Janošíková, Bohumila; Wang, Jian; Stabler, Sally P.; Allen, Robert H.; Jakobs, Cornelis; Finn, Christine T.; Chien, Yin-Hsiu; Hwu, Wuh-Liang; Hegele, Robert A.; Mudd, S. Harvey

    2009-01-01

    We report studies of six individuals with marked elevations of cystathionine in plasma and/or urine. Studies of CTH, the gene that encodes cystathionine γ-lyase, revealed the presence among these individuals of either homozygous or compound heterozygous forms of a novel large deletion, p.Gly57_Gln196del, two novel missense mutations, c.589C>T (p.Arg197Cys) and c.932C>T (p.Thr311Ile), and one previously reported alteration, c.200C>T (p.Thr67Ile). Another novel missense mutation, c.185G>T (p.Arg62His), was found in heterozygous form in three mildly hypercystathioninemic members of a Taiwanese family. In one severely hypercystathioninemic individual no CTH mutation was found. Brief clinical histories of the cystathioninemic/cystathioninuric patients are presented. Most of the novel mutations were expressed and the CTH activities of the mutant proteins determined. The crystal structure of the human enzyme, hCTH, and the evidence available as to the effects of the mutations in question, as well as those of the previously reported p.Gln240Glu, on protein structure, enzymatic activity, and responsiveness to vitamin B6 administration are discussed. Among healthy Czech controls, 9.3% were homozygous for CTH c.1208G>T (p.Ser403Ile), previously found homozygously in 7.5% of Canadians for whom plasma total homocysteine (tHcy) had been measured. Compared to wild-type homozygotes, among the 55 Czech c.1208G>T (p.Ser403Ile) homozygotes a greater level of plasma cystathionine was found only after methionine loading. Three of the four individuals homozygous or compound heterozygous for inactivating CTH mutations had mild plasma tHcy elevations, perhaps indicating a cause-and-effect relationship. The experience with the present patients provides no evidence that severe loss of CTH activity is accompanied by adverse clinical effects. PMID:19428278

  18. A Radical Transfer Pathway in Spore Photoproduct Lyase

    PubMed Central

    Yang, Linlin; Nelson, Renae S.; Benjdia, Alhosna; Lin, Gengjie; Telser, Joshua; Stoll, Stefan; Schlichting, Ilme; Li, Lei

    2013-01-01

    Spore photoproduct lyase (SPL) repairs a covalent UV-induced thymine dimer, spore photoproduct (SP), in germinating endospores and is responsible for endospores’ strong UV resistance. SPL is a radical SAM enzyme, which uses a [4Fe-4S]1+ cluster to reduce the S-adenosyl-L-methionine (SAM), generating a catalytic 5′-deoxyadenosyl radical (5′-dA•). This in turn abstracts an H atom from SP, generating an SP radical that undergoes β scission to form a repaired 5′-thymine and a 3′-thymine allylic radical. Recent biochemical and structural data suggest that a conserved cysteine donates an H atom to the thymine radical, resulting in a putative thiyl radical. Here we present structural and biochemical data which suggest that two conserved tyrosines are also critical in enzyme catalysis. One (Y99(Bs) in Bacillus subtilis SPL) is downstream of the cysteine, suggesting that SPL uses a novel hydrogen atom transfer (HAT) pathway with a pair of cysteine-tyrosine residues to regenerate SAM. The other tyrosine (Y97(Bs)) has a structural role to facilitate SAM binding; it may also contribute to the SAM regeneration process by interacting with the putative •Y99(Bs) and/or 5′-dA• intermediates to lower the energy barrier for the second H-abstraction step. Our results indicate that SPL is the first member of the radical SAM superfamily (comprising more than 44,000 members) to bear a catalytically operating HAT chain. PMID:23607538

  19. Inhibition of tobramycin diffusion by binding to alginate.

    PubMed Central

    Nichols, W W; Dorrington, S M; Slack, M P; Walmsley, H L

    1988-01-01

    [3H]tobramycin bound to sodium alginate and to exopolysaccharide prepared from two mucoid strains of Pseudomonas aeruginosa. Binding to sodium alginate was similar to binding to exopolysaccharide, both in the dependence on tobramycin concentration and in the maximum binding observed at saturation. Incorporation of sodium alginate into agar plates reduced the zone sizes of growth inhibition caused by tobramycin. The reductions in zone sizes were quantitatively accounted for by the binding of tobramycin to sodium alginate during diffusion of the antibiotic away from the well in which it had been placed at the start of the experiment. However, the binding of tobramycin to the exopolysaccharide of P. aeruginosa, and the resulting inhibition of diffusion of the antibiotic, did not significantly increase the penetration time of a spherical microcolony with a radius of 125 micron, such as might be found in the respiratory tract of a patient with cystic fibrosis (from a 90% penetration time of 12 s in the absence of exopolysaccharide to one of 35 s with an exopolysaccharide concentration of 1.0% [wt/vol]). PMID:3132093

  20. 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.