59 results on '"Pastor FI"'
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2. Oxidized Product Profiles of AA9 Lytic Polysaccharide Monooxygenases Depend on the Type of Cellulose.
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Sun P, Valenzuela SV, Chunkrua P, Javier Pastor FI, Laurent CVFP, Ludwig R, van Berkel WJH, and Kabel MA
- Abstract
Lytic polysaccharide monooxygenases (LPMOs) are essential for enzymatic conversion of lignocellulose-rich biomass in the context of biofuels and platform chemicals production. Considerable insight into the mode of action of LPMOs has been obtained, but research on the cellulose specificity of these enzymes is still limited. Hence, we studied the product profiles of four fungal Auxiliary Activity family 9 (AA9) LPMOs during their oxidative cleavage of three types of cellulose: bacterial cellulose (BC), Avicel PH-101 (AVI), and regenerated amorphous cellulose (RAC). We observed that attachment of a carbohydrate-binding module 1 (CBM1) did not change the substrate specificity of LPMO9B from Myceliophthora thermophila C1 ( Mt LPMO9B) but stimulated the degradation of all three types of cellulose. A detailed quantification of oxidized ends in both soluble and insoluble fractions, as well as characterization of oxidized cello-oligosaccharide patterns, suggested that Mt LPMO9B generates mainly oxidized cellobiose from BC, while producing oxidized cello-oligosaccharides from AVI and RAC ranged more randomly from DP2-8. Comparable product profiles, resulting from BC, AVI, and RAC oxidation, were found for three other AA9 LPMOs. These distinct cleavage profiles highlight cellulose specificity rather than an LPMO-dependent mechanism and may further reflect that the product profiles of AA9 LPMOs are modulated by different cellulose types., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)
- Published
- 2021
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3. A bacterial GH6 cellobiohydrolase with a novel modular structure.
- Author
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Cerda-Mejía L, Valenzuela SV, Frías C, Diaz P, and Pastor FI
- Subjects
- Amino Acid Sequence, Bacterial Proteins chemistry, Bacterial Proteins genetics, Bacterial Proteins isolation & purification, Bacterial Proteins metabolism, Catalytic Domain, Cellulose metabolism, Cellulose 1,4-beta-Cellobiosidase genetics, Cellulose 1,4-beta-Cellobiosidase isolation & purification, Escherichia coli genetics, Hydrolysis, Kinetics, Protein Binding, Protein Structure, Secondary, Recombinant Proteins chemistry, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Sequence Deletion, Substrate Specificity, Cellulose 1,4-beta-Cellobiosidase chemistry, Cellulose 1,4-beta-Cellobiosidase metabolism, Paenibacillus enzymology
- Abstract
Cel6D from Paenibacillus barcinonensis is a modular cellobiohydrolase with a novel molecular architecture among glycosyl hydrolases of family 6. It contains an N-terminal catalytic domain (family 6 of glycosyl hydrolases (GH6)), followed by a fibronectin III-like domain repeat (Fn3
1,2 ) and a C-terminal family 3b cellulose-binding domain (CBM3b). The enzyme has been identified and purified showing catalytic activity on cellulosic substrates and cellodextrins, with a marked preference for phosphoric acid swollen cellulose (PASC). Analysis of mode of action of Cel6D shows that it releases cellobiose as the only hydrolysis product from cellulose. Kinetic parameters were determined on PASC showing a Km of 68.73 mg/ml and a Vmax of 1.73 U/mg. A series of truncated derivatives of Cel6D have been constructed and characterized. Deletion of CBM3b caused a notable reduction in hydrolytic activity, while deletion of the Fn3 domain abolished activity, as the isolated GH6 domain was not active on any of the substrates tested. Mutant enzymes Cel6D-D146A and Cel6D-D97A were constructed in the residues corresponding to the putative acid catalyst and to the network for the nucleophilic attack. The lack of activity of the mutant enzymes indicates the important role of these residues in catalysis. Analysis of cooperative activity of Cel6D with cellulases from the same producing P. barcinonensis strain reveals high synergistic activity with processive endoglucanase Cel9B on hydrolysis of crystalline substrates. The characterized cellobiohydrolase can be a good contribution for depolymerization of cellulosic substrates and for the deconstruction of native cellulose.- Published
- 2017
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4. The Glycoside Hydrolase Family 8 Reducing-End Xylose-Releasing Exo-oligoxylanase Rex8A from Paenibacillus barcinonensis BP-23 Is Active on Branched Xylooligosaccharides.
- Author
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Valenzuela SV, Lopez S, Biely P, Sanz-Aparicio J, and Pastor FI
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- Bacterial Proteins chemistry, Bacterial Proteins genetics, Catalytic Domain, Glucuronates chemistry, Glycoside Hydrolases chemistry, Glycoside Hydrolases genetics, Multigene Family, Oligosaccharides chemistry, Paenibacillus chemistry, Paenibacillus genetics, Substrate Specificity, Xylosidases genetics, Bacterial Proteins metabolism, Glucuronates metabolism, Glycoside Hydrolases metabolism, Oligosaccharides metabolism, Paenibacillus enzymology, Xylose metabolism, Xylosidases metabolism
- Abstract
Unlabelled: A GH8 family enzyme involved in xylan depolymerization has been characterized. The enzyme, Rex8A, is a reducing-end xylose-releasing exo-oligoxylanase (Rex) that efficiently hydrolyzes xylooligosaccharides and shows minor activity on polymeric xylan. Rex8A hydrolyzes xylooligomers of 3 to 6 xylose units to xylose and xylobiose in long-term incubations. Kinetic constants of Rex8A were determined on xylotriose, showing a Km of 1.64 ± 0.03 mM and a kcat value of 118.8 s(-1) Besides linear xylooligosaccharides, the enzyme hydrolyzed decorated xylooligomers. The catalytic activity on branched xylooligosaccharides, i.e., the release of xylose from the reducing end, is a newly described trait of xylose-releasing exo-oligoxylanases, as the exo-activity on these substrates has not been reported for the few of these enzymes characterized to date. Modeling of the three-dimensional (3D) structure of Rex8A shows an (α/α)6 barrel fold where the loops connecting the α-helices contour the active site. These loops, which show high sequence diversity among GH8 enzymes, shape a catalytic cleft with a -2 subsite that can accommodate methyl-glucuronic acid decorations. The hydrolytic ability of Rex8A on branched oligomers can be crucial for the complete depolymerization of highly substituted xylans, which is indispensable to accomplish biomass deconstruction and to generate efficient catalysts., Importance: A GH8 family enzyme involved in xylan depolymerization has been characterized. The Rex8A enzyme from Paenibacillus barcinonensis is involved in depolymerization of glucuronoxylan, a major component of the lignocellulosic substrates. The study shows that Rex8A is a reducing-end xylose-releasing exo-oligoxylanase that efficiently hydrolyzes xylose from neutral and acidic xylooligosaccharides generated by the action of other xylanases also secreted by the strain. The activity of a Rex enzyme on branched xylooligosaccharides has not been described to date. This report provides original and useful information on the properties of a new example of the rarely studied Rex enzymes. Depolymerization of highly substituted xylans is crucial for biomass valorization as a platform for generation of biofuels, chemicals, and solvents., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)
- Published
- 2016
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5. Recombinant expression of a GH12 β-glucanase carrying its own signal peptide from Stachybotrys atra in yeast and filamentous fungi.
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Picart P, Orejas M, and Pastor FI
- Subjects
- Amino Acid Sequence, Aspergillus nidulans genetics, Cloning, Molecular, Genetic Engineering, Molecular Weight, Recombinant Proteins metabolism, Saccharomyces cerevisiae genetics, Stachybotrys genetics, Glycoside Hydrolases genetics, Glycoside Hydrolases metabolism, Protein Sorting Signals, Stachybotrys enzymology
- Abstract
The β-glucanase Cel12A gene from Stachybotrys atra has been cloned and heterologously expressed in Aspergillus nidulans and Saccharomyces cerevisiae. The recombinant strains constructed, contained the exonic sequence of cel12A including its own signal peptide coding sequence. SDS-PAGE and zymography revealed that recombinant Cel12A has a molecular mass of 24 kDa which agrees with that deduced from its amino acid sequence, indicating that it is expressed in the non-glycosylated active form. Recombinant A. nidulans showed about eightfold greater activity yield than S. cerevisiae recombinant strain, namely 0.71 and 0.09 β-glucanase Units/ml of culture, respectively. In both host strains most of the activity was secreted to the extracellular media, evidencing the functionality of Cel12A signal peptide in yeast and fungi. This novel signal peptide might facilitate the expression and efficient secretion of other recombinant proteins difficult to secrete.
- Published
- 2016
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6. New GH16 β-glucanase from Paenibacillus barcinonensis BP-23 releases a complex pattern of mixed-linkage oligomers from barley glucan.
- Author
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Cerda LA, Valenzuela SV, Diaz P, and Pastor FI
- Subjects
- Cloning, Molecular, Glucans chemistry, Glycoside Hydrolases chemistry, Glycoside Hydrolases genetics, Hordeum chemistry, Hydrolysis, Paenibacillus chemistry, Paenibacillus genetics, Paenibacillus metabolism, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Glucans metabolism, Glycoside Hydrolases metabolism, Hordeum metabolism, Paenibacillus enzymology
- Abstract
The gene coding for a lichenase from Paenibacillus barcinonensis BP-23, a powerful carbohydrate-degrading strain, was obtained using a genome walking strategy and expressed in Escherichia coli for further characterization. The amino acid sequence deduced from lic16A revealed that the lichenase is a single-domain enzyme belonging to the GH16 family. Purified recombinant Lic16A showed exclusive activity on β-1,3-1,4-glucans, showing a Km of 16.88 mg/mL and a Vmax of 266.09 U/mg using lichenan as a substrate. Lic16A was stable at 55 °C for at least 3 H in moderate pH conditions. Thin-layer chromatography analysis showed that the enzyme released a complex mixture of hydrolysis products, which consisted of different length oligosaccharides of intermediate mobility among cellooligomers. The health benefits of β-glucans's consumption and the increased interest for the use of their oligomers as prebiotics add interest to the study of Lic16A for the production of β-glucan-derived oligosaccharides and the evaluation of their biotechnological potential. This is the first report on β-1,3-1,4-glucanase produced by P. barcinonensis., (© 2015 International Union of Biochemistry and Molecular Biology, Inc.)
- Published
- 2016
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7. Exploring Multimodularity in Plant Cell Wall Deconstruction: STRUCTURAL AND FUNCTIONAL ANALYSIS OF Xyn10C CONTAINING THE CBM22-1-CBM22-2 TANDEM.
- Author
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Sainz-Polo MA, González B, Menéndez M, Pastor FI, and Sanz-Aparicio J
- Subjects
- Amino Acid Sequence, Bacterial Proteins genetics, Base Sequence, Catalytic Domain, Crystallography, X-Ray, DNA, Bacterial genetics, Endo-1,4-beta Xylanases genetics, Ligands, Models, Molecular, Molecular Sequence Data, Paenibacillus genetics, Protein Conformation, Protein Interaction Domains and Motifs, Protein Structure, Quaternary, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Xylans metabolism, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Cell Wall metabolism, Endo-1,4-beta Xylanases chemistry, Endo-1,4-beta Xylanases metabolism, Paenibacillus enzymology, Plants metabolism
- Abstract
Elucidating the molecular mechanisms regulating multimodularity is a challenging task. Paenibacillus barcinonensis Xyn10C is a 120-kDa modular enzyme that presents the CBM22/GH10/CBM9 architecture found in a subset of large xylanases. We report here the three-dimensional structure of the Xyn10C N-terminal region, containing the xylan-binding CBM22-1-CBM22-2 tandem (Xyn10C-XBD), which represents the first solved crystal structure of two contiguous CBM22 modules. Xyn10C-XBD is folded into two separate CBM22 modules linked by a flexible segment that endows the tandem with extraordinary plasticity. Each isolated domain has been expressed and crystallized, and their binding abilities have been investigated. Both domains contain the R(W/Y)YYE motif required for xylan binding. However, crystallographic analysis of CBM22-2 complexes shows Trp-308 as an additional binding determinant. The long loop containing Trp-308 creates a platform that possibly contributes to the recognition of precise decorations at subsite S2. CBM22-2 may thus define a subset of xylan-binding CBM22 modules directed to particular regions of the polysaccharide. Affinity electrophoresis reveals that Xyn10C-XBD binds arabinoxylans more tightly, which is more apparent when CBM22-2 is tested against highly substituted xylan. The crystal structure of the catalytic domain, also reported, shows the capacity of the active site to accommodate xylan substitutions at almost all subsites. The structural differences found at both Xyn10C-XBD domains are consistent with the isothermal titration calorimetry experiments showing two sites with different affinities in the tandem. On the basis of the distinct characteristics of CBM22, a delivery strategy of Xyn10C mediated by Xyn10C-XBD is proposed., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2015
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8. Crystallization and preliminary X-ray diffraction analysis of the N-terminal domain of Paenibacillus barcinonensis xylanase 10C containing the CBM22-1-CBM22-2 tandem.
- Author
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Sainz-Polo MÁ, González B, Pastor FI, and Sanz-Aparicio J
- Subjects
- Amino Acid Sequence, Crystallization, Electrophoresis, Polyacrylamide Gel, Molecular Sequence Data, Protein Structure, Tertiary, Bacterial Proteins chemistry, Endo-1,4-beta Xylanases chemistry, Paenibacillus chemistry, X-Ray Diffraction
- Abstract
A construct containing the CBM22-1-CBM22-2 tandem forming the N-terminal domain of Paenibacillus barcinonensis xylanase 10C (Xyn10C) has been purified and crystallized. A xylan-binding function and an affinity for mixed β-1,3/β-1,4 glucans have previously been demonstrated for some members of the CBM22 family. The sequence of the tandem is homologous to the N-terminal domains found in several thermophilic enzymes. Crystals of this tandem were grown by the streak-seeding method after a long optimization strategy. The structure has been determined by molecular replacement to a resolution of 2.43 Å and refinement is under way. This study represents the first structure containing two contiguous CBM22 modules, which will contribute to a better understanding of the role that this multiplicity plays in fine-tuning substrate affinity.
- Published
- 2015
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9. Structural analysis of glucuronoxylan-specific Xyn30D and its attached CBM35 domain gives insights into the role of modularity in specificity.
- Author
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Sainz-Polo MA, Valenzuela SV, González B, Pastor FI, and Sanz-Aparicio J
- Subjects
- Amino Acid Sequence, Bacillus subtilis enzymology, Binding Sites, Biomass, Catalytic Domain, Cell Wall enzymology, Crystallography, X-Ray, Edetic Acid chemistry, Escherichia coli metabolism, Molecular Sequence Data, Polysaccharides chemistry, Protein Binding, Secale chemistry, Sequence Homology, Amino Acid, Substrate Specificity, Wood, Bacterial Proteins chemistry, Paenibacillus enzymology, Xylans chemistry, Xylosidases chemistry
- Abstract
Glucuronoxylanase Xyn30D is a modular enzyme containing a family 30 glycoside hydrolase catalytic domain and an attached carbohydrate binding module of the CBM35 family. We present here the three-dimensional structure of the full-length Xyn30D at 2.4 Å resolution. The catalytic domain folds into an (α/β)8 barrel with an associated β-structure, whereas the attached CBM35 displays a jellyroll β-sandwich including two calcium ions. Although both domains fold in an independent manner, the linker region makes polar interactions with the catalytic domain, allowing a moderate flexibility. The ancillary Xyn30D-CBM35 domain has been expressed and crystallized, and its binding abilities have been investigated by soaking experiments. Only glucuronic acid-containing ligands produced complexes, and their structures have been solved. A calcium-dependent glucuronic acid binding site shows distinctive structural features as compared with other uronic acid-specific CBM35s, because the presence of two aromatic residues delineates a wider pocket. The nonconserved Glu(129) makes a bidentate link to calcium and defines region E, previously identified as specificity hot spot. The molecular surface of Xyn30D-CBM35 shows a unique stretch of negative charge distribution extending from its binding pocket that might indicate some oriented interaction with its target substrate. The binding ability of Xyn30D-CBM35 to different xylans was analyzed by affinity gel electrophoresis. Some binding was observed with rye glucuronoarabinoxylan in presence of calcium chelating EDTA, which would indicate that Xyn30D-CBM35 might establish interaction to other components of xylan, such as arabinose decorations of glucuronoarabinoxylan. A role in depolymerization of highly substituted chemically complex xylans is proposed., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2014
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10. A glucuronoxylan-specific xylanase from a new Paenibacillus favisporus strain isolated from tropical soil of Brazil.
- Author
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Padilha IQ, Valenzuela SV, Grisi TC, Díaz P, de Araújo DA, and Pastor FI
- Subjects
- Bacterial Proteins chemistry, Bacterial Proteins genetics, Brazil, Cloning, Molecular, Enzyme Stability, Kinetics, Molecular Sequence Data, Paenibacillus chemistry, Paenibacillus genetics, Paenibacillus isolation & purification, Substrate Specificity, Xylosidases chemistry, Xylosidases genetics, Bacterial Proteins metabolism, Paenibacillus enzymology, Soil Microbiology, Xylosidases metabolism
- Abstract
A new xylanolytic strain, Paenibacillus favisporus CC02-N2, was isolated from sugarcane plantation fields in Brazil. The strain had a xylan-degrading system with multiple enzymes, one of which, xylanase Xyn30A, was identified and characterized. The enzyme is a single-domain xylanase belonging to family 30 of the glycosyl hydrolases (GH30). Xyn30A shows high activity on glucuronoxylans, with a Vmax of 267.2 U mg⁻¹, a Km of 4.0 mg/ml, and a kcat of 13,333 min⁻¹ on beechwood xylan, but it does not hydrolyze arabinoxylans. The three-dimensional structure of Xyn30A consists of a common (β/α)8 barrel linked to a side-chain-associated β-structure, similar to previously characterized GH30 xylanases. The hydrolysis products from glucuronoxylan were methylglucuronic-acid-substituted xylooligomers (acidic xylooligosaccharides). The enzyme bound to insoluble xylan but not to crystalline cellulose. Our results suggest a specific role for Xyn30A in xylan biodegradation in natural habitats. The enzyme is a good candidate for the production of tailored xylooligosaccharides for use in the food industry and in the biotechnological transformation of biomass., (Copyright© by the Spanish Society for Microbiology and Institute for Catalan Studies.)
- Published
- 2014
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11. Unusual carboxylesterase bearing a GGG(A)X-type oxyanion hole discovered in Paenibacillus barcinonensis BP-23.
- Author
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Infanzón B, Valenzuela SV, Fillat A, Pastor FI, and Diaz P
- Subjects
- Alcohols metabolism, Amino Acid Motifs, Carboxylesterase genetics, Cloning, Molecular, Conserved Sequence, Peptide Hydrolases metabolism, Phylogeny, Carboxylesterase chemistry, Carboxylesterase metabolism, Paenibacillus enzymology
- Abstract
Strain Paenibacillus barcinonensis BP-23, previously isolated from Ebro's river delta (Spain), bears a complex hydrolytic system showing the presence of at least two enzymes with activity on lipidic substrates. EstA, a cell-bound B-type carboxylesterase from the strain was previously isolated and characterized. The gene coding for a second putative lipase, located upstream cellulase Cel5A, was obtained using a genome walking strategy and cloned in Escherichia coli for further characterization. The recombinant clone obtained displayed high activity on medium/short-chain fatty acid-derivative substrates. The enzyme, named Est23, was purified and characterized, showing maximum activity on pNP-caprylate (C8:0) or MUF-heptanoate (C7:0) under conditions of moderate temperature and pH. Although Est23 displays a GGG(A)X-type oxyanion hole, described as an important motif for tertiary alcohol ester resolution, neither conversion nor enantiomeric resolution of tertiary alcohols could be detected. Amino acid sequence alignment of Est23 with those of known bacterial lipase families and with closely related proteins suggests that the cloned enzyme does not belong to any of the described bacterial lipase families. A phylogenetic tree including Est23 and similar amino acid sequences showed that the enzyme belongs to a differentiated sequence cluster which probably constitutes a new family of bacterial lipolytic enzymes., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)
- Published
- 2014
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12. Xyn11E from Paenibacillus barcinonensis BP-23: a LppX-chaperone-dependent xylanase with potential for upgrading paper pulps.
- Author
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Valenzuela SV, Diaz P, and Pastor FI
- Subjects
- Cloning, Molecular, DNA, Bacterial chemistry, DNA, Bacterial genetics, Escherichia coli genetics, Escherichia coli metabolism, Gene Expression, Hydrogen-Ion Concentration, Isoelectric Point, Kinetics, Molecular Sequence Data, Molecular Weight, Paenibacillus genetics, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Sequence Analysis, DNA, Substrate Specificity, Temperature, Xylosidases chemistry, Xylosidases isolation & purification, Paenibacillus enzymology, Xylosidases genetics, Xylosidases metabolism
- Abstract
A new xylanase from Paenibacillus barcinonensis BP-23, Xyn11E, has been identified and characterized. Xyn11E has been cloned and heterologously expressed in Escherichia coli. It is a single-domain xylanase belonging to the family 11 of glycosyl hydrolases (GH11) with a predicted molecular weight of 20.652 kDa and an isoelectric point (pI) of 8.7. Substrate specificity, kinetic properties, and mode of action of the purified xylanase were characterized. Xyn11E exhibited high activity toward branched xylans, being beechwood xylan the preferred substrate. The optimum pH and temperature of the purified enzyme were 6.5 and 50 °C, respectively. Catalytic constants were determined on beechwood xylan, on which Xyn11E showed a Km of 12.98 mg/ml and a Vmax of 3,023 U/mg. The enzyme hydrolyzed long xylooligosaccharides, while oligomers shorter than xylotetraose were not degraded. Products released from glucuronoxylans were shorter than those liberated from cereal arabinoxylans. The xylanase was dependent on P. barcinonensis BP-23 LppX for its expression in an active form. Coexpression of Xyn11E with E. coli chaperones could not replace the need of LppX, which seems to act as a specific chaperone for Xyn11E correct folding. Activity of the enzyme on bleached pulps was evaluated. Xyn11E liberated reducing sugars from ECF and TCF pulps from eucalyptus, sisal, and flax, which makes it a good candidate for the enzymatic-assisted production of high-cellulose-content pulps from paper-grade pulps.
- Published
- 2014
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13. Improving enantioselectivity towards tertiary alcohols using mutants of Bacillus sp. BP-7 esterase EstBP7 holding a rare GGG(X)-oxyanion hole.
- Author
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Fillat A, Romea P, Urpí F, Pastor FI, and Diaz P
- Subjects
- Amino Acid Motifs, Amino Acid Substitution, Bacillus genetics, Cold Temperature, Esterases genetics, Protein Engineering, Substrate Specificity, Bacillus enzymology, Esterases metabolism, Esters metabolism, tert-Butyl Alcohol metabolism
- Abstract
Lipases and esterases are important biocatalysts for synthetic organic fine chemistry. An esterase from Bacillus sp. BP-7 (EstBP7) bears in its amino acid sequence a rare GGG(A)X oxyanion hole motif, where an uncommon threonine (T) is found at the third position. Detection of this pattern motivated evaluation of the ability of EstBP7 for conversion of tertiary alcohols. The enzyme was engineered in order to optimize its performance to provide important chiral building blocks: five variants with mutations in the oxyanion hole motif were created to investigate the influence on activity and enantioselectivity in the kinetic resolution of eight acetates of tertiary alcohols. Wild-type enzyme converted all esters of tertiary alcohols assayed with low enantioselectivity, whereas some of the mutants displayed significantly increased E-values. One of the mutants (EstBP7-AGA; Mut 5) showed an E >100 towards a complex tertiary alcohol acetate (2-(4-pyridyl)but-3-yn-2-yl acetate) at low reaction temperature (4 °C). Therefore, the catalytic toolbox was expanded for biocatalysis of optically pure tertiary alcohols valuable for the pharmaceutical industry.
- Published
- 2014
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14. Fast and economic immobilization methods described for non-commercial Pseudomonas lipases.
- Author
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Cesarini S, Infanzón B, Pastor FI, and Diaz P
- Subjects
- Bacterial Proteins chemistry, Biotechnology methods, Enzymes, Immobilized chemistry, Lipase chemistry, Pseudomonas enzymology
- Abstract
Background: There is an increasing interest to seek new enzyme preparations for the development of new products derived from bioprocesses to obtain alternative bio-based materials. In this context, four non-commercial lipases from Pseudomonas species were prepared, immobilized on different low-cost supports, and examined for potential biotechnological applications., Results: To reduce costs of eventual scaling-up, the new lipases were obtained directly from crude cell extracts or from growth culture supernatants, and immobilized by simple adsorption on Accurel EP100, Accurel MP1000 and Celite®545. The enzymes evaluated were LipA and LipC from Pseudomonas sp. 42A2, a thermostable mutant of LipC, and LipI.3 from Pseudomonas CR611, which were produced in either homologous or heterologous hosts. Best immobilization results were obtained on Accurel EP100 for LipA and on Accurel MP1000 for LipC and its thermostable variant. Lip I.3, requiring a refolding step, was poorly immobilized on all supports tested (best results for Accurel MP1000). To test the behavior of immobilized lipases, they were assayed in triolein transesterification, where the best results were observed for lipases immobilized on Accurel MP1000., Conclusions: The suggested protocol does not require protein purification and uses crude enzymes immobilized by a fast adsorption technique on low-cost supports, which makes the method suitable for an eventual scaling up aimed at biotechnological applications. Therefore, a fast, simple and economic method for lipase preparation and immobilization has been set up. The low price of the supports tested and the simplicity of the procedure, skipping the tedious and expensive purification steps, will contribute to cost reduction in biotechnological lipase-catalyzed processes.
- Published
- 2014
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15. The influence of supramolecular structure of cellulose allomorphs on the interactions with cellulose-binding domain, CBD3b from Paenibacillus barcinonensis.
- Author
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Ciolacu D, Chiriac AI, Pastor FI, and Kokol V
- Subjects
- Adsorption, Amino Acid Sequence, Crystallization, Hydrogen Bonding, Molecular Sequence Data, Protein Binding, Protein Structure, Tertiary, Regression Analysis, Spectroscopy, Fourier Transform Infrared, Steam, Temperature, X-Ray Diffraction, Cellulase chemistry, Cellulase metabolism, Cellulose chemistry, Cellulose metabolism, Paenibacillus enzymology
- Abstract
The interaction of recombinant cellulose-binding domains (CBDs) of endoglucanase Cel9B from Paenibacillus barcinonensis with different cotton cellulose allomorphs (I, II and III) has been investigated, in order to bring new insights regarding the CBD adsorption and desorption processes. The highest CBD adsorption capacity was recorded for cellulose I, confirming the affinity of proteins to the most crystalline substrate. The weakening and splitting of the hydrogen bonds within cellulose structure after CBD adsorption, as well as a decrease of the crystallinity degree were identified by ATR-FTIR spectroscopy and XRD. The CBD's adsorption kinetic was shown to be rendered by properties as, specific surface area and porosity, being confirmed by dynamic vapor sorption measurements. An important influence of temperature (25, 37 and 50°C) and/or pH medium (4, 5.5, 7 and 10) on the CBD desorption capacity was confirmed, being related to the hydrophobic interactions formed between the CBD and the cellulose allomorphs., (Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.)
- Published
- 2014
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16. Special Rhodococcus sp. CR-53 esterase Est4 contains a GGG(A)X-oxyanion hole conferring activity for the kinetic resolution of tertiary alcohols.
- Author
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Bassegoda A, Fillat A, Pastor FI, and Diaz P
- Subjects
- Amino Acid Sequence, Cloning, Molecular, DNA, Bacterial chemistry, DNA, Bacterial genetics, Enzyme Inhibitors metabolism, Enzyme Stability, Esterases chemistry, Esterases genetics, Esterases isolation & purification, Hydrogen-Ion Concentration, Models, Molecular, Molecular Sequence Data, Protein Conformation, Rhodococcus genetics, Sequence Alignment, Sequence Analysis, DNA, Substrate Specificity, Temperature, tert-Butyl Alcohol, Alcohols metabolism, Esterases metabolism, Rhodococcus enzymology
- Abstract
Rhodococci are highly adaptable bacteria, capable to degrade or transform a large number of organic compounds, including recalcitrant or toxic products. However, little information is available on the lipases of the genus Rhodococcus, except for LipR, the first lipase isolated and described from strain Rhodococcus CR-53. Taking into consideration the interest raised by the enzymes produced by actinomycetes, a search for new putative lipases was performed in strain Rhodococcus CR-53. We describe here the isolation, cloning, and characterization of intracellular esterase Est4, a mesophilic enzyme showing preference for short-chain-length acyl groups, without interfacial activation. Est4 displays moderate thermal and pH stability and low tolerance to most tested ions, being inhibited by detergents like sodium dodecyl sulfate and Triton X-100®. Nevertheless, the enzyme shows good long-term stability when stored at 4-20 °C and neutral pH. Amino acid sequence analysis of Est4 revealed a protein of 313 amino acids without a signal peptide, bearing most of the conserved blocks that define bacterial lipase family IV, thus being assigned to this family. Detection of a GGG(A)X oxyanion hole in the enzyme motivated the evaluation of Est4 ability to convert tertiary alcohol esters. The newly discovered esterase Est4 from Rhodococcus CR-53 successfully hydrolyzed the tertiary alcohol esters linalyl acetate, terpinyl acetate, and 1,1,1-trifluoro-2-phenylbut-3-yn-2-yl acetate.
- Published
- 2013
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17. Acidic lipase Lip I.3 from a Pseudomonas fluorescens-like strain displays unusual properties and shows activity on secondary alcohols.
- Author
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Panizza P, Syfantou N, Pastor FI, Rodríguez S, and Díaz P
- Subjects
- Amino Acid Sequence, Binding Sites, Cloning, Molecular, DNA, Bacterial genetics, Enzyme Stability, Escherichia coli genetics, Escherichia coli metabolism, Hydrogen-Ion Concentration, Hydrolysis, Lipase genetics, Protein Structure, Secondary, Pseudomonas genetics, Sequence Analysis, DNA, Substrate Specificity, Alcohols metabolism, Lipase chemistry, Pseudomonas enzymology
- Abstract
Aims: Identification, cloning, expression and characterization of a novel lipase--Lip I.3--from strain Pseudomonas CR-611., Methods and Results: The corresponding gene was identified and isolated by PCR-amplification, cloned and expressed in Escherichia coli, and purified by refolding from inclusion bodies. Analysis of the deduced amino acid sequence revealed high homology with members of the bacterial lipase family I.3, showing 97% identity to a putative lipase from Pseudomonas fluorescens Pf0-1, and 93% identity to a crystallized extracellular lipase from Pseudomonas sp. MIS38. A typical C-terminal type I secretion signal and several putative Ca(2+) binding sites were also identified. Experimental data confirmed that Lip I.3 requires Ca(2+) ions for correct folding and activity. The enzyme differs from the previously reported family I.3 lipases in optimal pH, being the first acidophilic lipase reported in this family. Furthermore, Lip I.3 shows a strong preference for medium chain fatty acid esters and does not display interfacial activation. When tested for activity on secondary alcohol hydrolysis, Lip I.3 displayed higher efficiency on aromatic alcohols rather than on alkyl alcohols., Conclusions: A new family I.3 lipase with unusual properties has been isolated, cloned and described. This will contribute to a better knowledge of family I.3 lipases, a family that has been scarcely explored, and that might provide a novel source of biocatalysts., Significance and Impact of the Study: The unusual properties shown by Lip I.3 and the finding of activity and enantioselectivity on secondary alcohol esters may contribute to the development of new enzymatic tools for applied biocatalysis., (© 2013 The Society for Applied Microbiology.)
- Published
- 2013
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18. Functional characterization of ExFadLO, an outer membrane protein required for exporting oxygenated long-chain fatty acids in Pseudomonas aeruginosa.
- Author
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Martínez E, Estupiñán M, Pastor FI, Busquets M, Díaz P, and Manresa A
- Subjects
- Bacterial Outer Membrane Proteins genetics, Biological Transport, Escherichia coli genetics, Escherichia coli metabolism, Extracellular Space metabolism, Fatty Acid Transport Proteins genetics, Gene Expression, Genetic Complementation Test, Mutation, Oxidation-Reduction, Oxygen metabolism, Periplasm metabolism, Phylogeny, Pseudomonas aeruginosa genetics, Bacterial Outer Membrane Proteins metabolism, Fatty Acid Transport Proteins metabolism, Hydroxy Acids metabolism, Oleic Acids metabolism, Pseudomonas aeruginosa metabolism
- Abstract
Bacterial proteins of the FadL family have frequently been associated to the uptake of exogenous hydrophobic substrates. However, their outer membrane location and involvement in substrate uptake have been inferred mainly from sequence similarity to Escherichia coli FadL, the first well-characterized outer membrane transporters of Long-Chain Fatty Acids (LCFAs) in bacteria. Here we report the functional characterization of a Pseudomonas aeruginosa outer membrane protein (ORF PA1288) showing similarities to the members of the FadL family, for which we propose the name ExFadLO. We demonstrate herein that this protein is required to export LCFAs 10-HOME and 7,10-DiHOME, derived from a diol synthase oxygenation activity on oleic acid, from the periplasm to the extracellular medium. Accumulation of 10-HOME and 7,10-DiHOME in the extracellular medium of P. aeruginosa was abolished by a transposon insertion mutation in exFadLO (ExFadLO¯ mutant). However, intact periplasm diol synthase activity was found in this mutant, indicating that ExFadLO participates in the export of these oxygenated LCFAs across the outer membrane. The capacity of ExFadLO¯ mutant to export 10-HOME and 7,10-DiHOME was recovered after complementation with a wild-type, plasmid-expressed ExFadLO protein. A western blot assay with a variant of ExFadLO tagged with a V5 epitope confirmed the location of ExFadLO in the bacterial outer membrane under the experimental conditions tested. Our results provide the first evidence that FadL family proteins, known to be involved in the uptake of hydrophobic substrates from the extracellular environment, also function as secretion elements for metabolites of biological relevance., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)
- Published
- 2013
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19. Modular glucuronoxylan-specific xylanase with a family CBM35 carbohydrate-binding module.
- Author
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Valenzuela SV, Diaz P, and Pastor FI
- Subjects
- Binding Sites, Catalytic Domain, Escherichia coli enzymology, Escherichia coli genetics, Kinetics, Molecular Sequence Data, Paenibacillus genetics, Recombinant Proteins genetics, Recombinant Proteins metabolism, Sequence Analysis, DNA, Substrate Specificity, Xylans chemistry, Xylosidases genetics, Paenibacillus enzymology, Polysaccharides metabolism, Xylans metabolism, Xylosidases chemistry, Xylosidases metabolism
- Abstract
Xyn30D from the xylanolytic strain Paenibacillus barcinonensis has been identified and characterized. The enzyme shows a modular structure comprising a catalytic module family 30 (GH30) and a carbohydrate-binding module family 35 (CBM35). Like GH30 xylanases, recombinant Xyn30D efficiently hydrolyzed glucuronoxylans and methyl-glucuronic acid branched xylooligosaccharides but showed no catalytic activity on arabinose-substituted xylans. Kinetic parameters of Xyn30D were determined on beechwood xylan, showing a K(m) of 14.72 mg/ml and a k(cat) value of 1,510 min(-1). The multidomain structure of Xyn30D clearly distinguishes it from the GH30 xylanases characterized to date, which are single-domain enzymes. The modules of the enzyme were individually expressed in a recombinant host and characterized. The isolated GH30 catalytic module showed specific activity, mode of action on xylan, and kinetic parameters that were similar to those of the full-length enzyme. Computer modeling of the three-dimensional structure of Xyn30D showed that the catalytic module is comprised of a common (β/α)(8) barrel linked to a side-associated β-structure. Several derivatives of the catalytic module with decreasing deletions of this associated structure were constructed. None of them showed catalytic activity, indicating the importance of the side β-structure in the catalysis of Xyn30D. Binding properties of the isolated carbohydrate-binding module were analyzed by affinity gel electrophoresis, which showed that the CBM35 of the enzyme binds to soluble glucuronoxylans and arabinoxylans. Analysis by isothermal titration calorimetry showed that CBM35 binds to glucuronic acid and requires calcium ions for binding. Occurrence of a CBM35 in a glucuronoxylan-specific xylanase is a differential trait of the enzyme characterized.
- Published
- 2012
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20. Expression of novel β-glucanase Cel12A from Stachybotrys atra in bacterial and fungal hosts.
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Picart P, Goedegebuur F, Díaz P, and Javier Pastor FI
- Subjects
- Aspergillus niger genetics, Cloning, Molecular, DNA, Fungal chemistry, DNA, Fungal genetics, Electrophoresis, Polyacrylamide Gel, Enzyme Stability, Gene Expression, Glucans metabolism, Glycoside Hydrolases chemistry, Glycoside Hydrolases isolation & purification, Hydrogen-Ion Concentration, Isoelectric Focusing, Molecular Sequence Data, Molecular Weight, Recombinant Proteins genetics, Recombinant Proteins metabolism, Sequence Analysis, DNA, Substrate Specificity, Temperature, Glycoside Hydrolases genetics, Glycoside Hydrolases metabolism, Stachybotrys enzymology, Stachybotrys genetics
- Abstract
β-glucanase Cel12A from Stachybotrys atra has been cloned and expressed in Aspergillus niger. The purified enzyme showed high activity of β-1,3-1,4-mixed glucans, was also active on carboxymethylcellulose (CMC), while it did not hydrolyze crystalline cellulose or β-1,3 glucans as laminarin. Cel12A showed a marked substrate preference for β-1,3-1,4 glucans, showing maximum activity on barley β-glucans (27.69 U mg(-1)) while the activity on CMC was much lower (0.51 U mg(-1)). Analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focussing (IEF), and zymography showed the recombinant enzyme has apparent molecular weight of 24 kDa and a pI of 8.2. Optimal temperature and pH for enzyme activity were 50°C and pH 6.5. Thin layer chromatography analysis showed that major hydrolysis products from barley β-glucan and lichean were 3-O-β-cellotriosyl-D-glucose and 3-O-β-cellobiosyl-D-glucose, while glucose and cellobiose were released in smaller amounts. The amino acid sequence deduced from cel12A revealed that it is a single domain enzyme belonging to the GH12 family, a family that contains several endoglucanases with substrate preference for β-1,3-1,4 glucans. We believe that S. atra Cel12A should be considered as a lichenase-like or nontypical endoglucanase., (Copyright © 2012 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)
- Published
- 2012
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21. Rhodococcus sp. strain CR-53 LipR, the first member of a new bacterial lipase family (family X) displaying an unusual Y-type oxyanion hole, similar to the Candida antarctica lipase clan.
- Author
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Bassegoda A, Pastor FI, and Diaz P
- Subjects
- Burkholderia cenocepacia enzymology, Burkholderia cenocepacia genetics, Burkholderia cenocepacia metabolism, Candida enzymology, Candida genetics, Cloning, Molecular, Cluster Analysis, Conserved Sequence, Enzyme Stability, Hydrogen-Ion Concentration, Lipase chemistry, Lipase genetics, Models, Molecular, Phylogeny, Protein Conformation, Protein Structure, Tertiary, Rhodococcus genetics, Sequence Analysis, DNA, Sequence Homology, Amino Acid, Substrate Specificity, Temperature, Lipase isolation & purification, Lipase metabolism, Rhodococcus enzymology
- Abstract
Bacterial lipases constitute the most important group of biocatalysts for synthetic organic chemistry. Accordingly, there is substantial interest in developing new valuable lipases. Considering the lack of information concerning the lipases of the genus Rhodococcus and taking into account the interest raised by the enzymes produced by actinomycetes, a search for putative lipase-encoding genes from Rhodococcus sp. strain CR-53 was performed. We isolated, cloned, purified, and characterized LipR, the first lipase described from the genus Rhodococcus. LipR is a mesophilic enzyme showing preference for medium-chain-length acyl groups without showing interfacial activation. It displays good long-term stability and high tolerance for the presence of ions and chemical agents in the reaction mixture. Amino acid sequence analysis of LipR revealed that it displays four unique amino acid sequence motifs that clearly separate it from any other previously described family of bacterial lipases. Using bioinformatics tools, LipR could be related only to several uncharacterized putative lipases from different bacterial origins, all of which display the four blocks of consensus amino acid sequence motifs that contribute to define a new family of bacterial lipases, namely, family X. Therefore, LipR is the first characterized member of the new bacterial lipase family X. Further confirmation of this new family of lipases was performed after cloning Burkholderia cenocepacia putative lipase, bearing the same conserved motifs and clustering in family X. Interestingly, all lipases grouping in the new bacterial lipase family X display a Y-type oxyanion hole, a motif conserved in the Candida antarctica lipase clan but never found among bacterial lipases. This observation contributes to confirm that LipR and its homologs belong to a new family of bacterial lipases.
- Published
- 2012
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22. New xylanases to obtain modified eucalypt fibres with high-cellulose content.
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Valls C, Gallardo O, Vidal T, Pastor FI, Díaz P, and Roncero MB
- Subjects
- Color, Hexuronic Acids analysis, Lignin analysis, Oxygen analysis, Polysaccharides analysis, Polysaccharides chemistry, Spectrum Analysis, Viscosity, Waste Disposal, Fluid, Cellulose analysis, Endo-1,4-beta Xylanases metabolism, Eucalyptus chemistry, Paper
- Abstract
Modified fibres with high-cellulose content were obtained with two new bacterial xylanases from families 11 and 5. These xylanases were applied separately or simultaneously in a complete ECF (Elemental Chlorine Free) bleaching sequence. Both xylanases improved delignification and bleaching during the sequence and a synergistic effect of the enzymes was observed on several pulp and paper properties. The xylanases boosted the release of xylooligosaccharides branched with hexenuronic acids (HexA), giving rise to fibres with a reduced HexA and xylose content. However, these effects depended on the xylanase used, being the family 11 enzyme more efficient than the family 5 xylanase. Effluent properties such as absorbance spectra UV/Vis, COD and colour were affected by the enzymatic sequences as a consequence of the dissolution of lignin and xylooligosaccharides. Some changes in the fibre morphology were also produced without affecting the final paper strength properties., (Copyright 2010 Elsevier Ltd. All rights reserved.)
- Published
- 2010
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23. Characterization of a family GH5 xylanase with activity on neutral oligosaccharides and evaluation as a pulp bleaching aid.
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Gallardo O, Fernández-Fernández M, Valls C, Valenzuela SV, Roncero MB, Vidal T, Díaz P, and Pastor FI
- Subjects
- Amino Acid Sequence, Base Sequence, Chromatography, Gel, Chromatography, Ion Exchange, Chromatography, Liquid, Chromatography, Thin Layer, Cloning, Molecular, Eucalyptus chemistry, Hydrogen-Ion Concentration, Lignin metabolism, Molecular Sequence Data, Sequence Analysis, DNA, Temperature, Xylosidases metabolism, Bacillus enzymology, Bleaching Agents metabolism, Oligosaccharides metabolism, Xylosidases genetics
- Abstract
A new bacterial xylanase belonging to family 5 of glycosyl hydrolases was identified and characterized. The xylanase, Xyn5B from Bacillus sp. strain BP-7, was active on neutral, nonsubstituted xylooligosaccharides, showing a clear difference from other GH5 xylanases characterized to date that show a requirement for methyl-glucuronic acid side chains for catalysis. The enzyme was evaluated on Eucalyptus kraft pulp, showing its effectiveness as a bleaching aid.
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- 2010
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24. Use of cellulases and recombinant cellulose binding domains for refining TCF kraft pulp.
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Cadena EM, Chriac AI, Pastor FI, Diaz P, Vidal T, and Torres AL
- Subjects
- Cellulases genetics, Paenibacillus enzymology, Protein Binding, Protein Structure, Tertiary genetics, Protein Structure, Tertiary physiology, Recombinant Proteins genetics, Cellulases metabolism, Cellulose metabolism
- Abstract
The modular endoglucanase Cel9B from Paenibacillus barcinonensis is a highly efficient biocatalyst, which expedites pulp refining and reduces the associated energy costs as a result. In this work, we set out to identify the specific structural domain or domains responsible for the action of this enzyme on cellulose fibre surfaces with a view to facilitating the development of new cellulases for optimum biorefining. Using the recombinant enzymes GH9-CBD3c, Fn3-CBD3b, and CBD3b, which are truncated forms of Cel9B, allowed us to assess the individual effects of the catalytic, cellulose binding, and fibronectin-like domains of the enzyme on the refining of TCF kraft pulp from Eucalyptus globulus. Based on the physico-mechanical properties obtained, the truncated form containing the catalytic domain (GH9-CBD3c) has a strong effect on fibre morphology. Comparing its effect with that of the whole cellulase (Cel9B) revealed that the truncated enzyme contributes to increasing paper strength through improved tensile strength and burst strength and also that the truncated form is more effective than the whole enzyme in improving tear resistance. Therefore, the catalytic domain of Cel9B has biorefining action on pulp. Although cellulose binding domains (CBDs) are less efficient toward pulp refining, evidence obtained in this work suggests that CBD3b alters fibre surfaces and influences paper properties as a result., ((c) 2010 American Institute of Chemical Engineers)
- Published
- 2010
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25. Recombinant expression of an alkali stable GH10 xylanase from Paenibacillus barcinonensis.
- Author
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Valenzuela SV, Díaz P, and Javier Pastor FI
- Subjects
- Alkalies, Base Sequence, Cloning, Molecular, DNA Primers, Endo-1,4-beta Xylanases genetics, Paenibacillus genetics, Recombinant Proteins genetics, Recombinant Proteins metabolism, Endo-1,4-beta Xylanases metabolism, Paenibacillus enzymology
- Abstract
Xylanase A from Paenibacillus barcinonensis, a new species isolated from a rice field, has been cloned and expressed in Escherichia coli. Purified recombinant xylanase showed high activity on xylans from hardwoods and cereals, and exhibited K(m) and V(max) of 2.93 mg/mL and 50.67 U/mg on birchwood xylan. Xylanase A was highly active at 60 degrees C in alkaline pH values up to 9.5 and remained stable for at least 3 h in alkaline conditions. The amino acid sequence deduced from xynA revealed that it is a single domain xylanase belonging to the GH10 family. Thin layer chromatography analysis showed that the enzyme released a mixture of hydrolysis products including substituted xylooligomers from cereal arabinoxylans, while xylose, xylobiose, and aldotetraouronic acid were the main products released from glucuronoxylan from birchwood. The enzyme released a complex mixture of xylooligomers for acetylated xylan from eucalyptus, revealing its potential to depolymerize this widely used resource in the pulp and paper industry.
- Published
- 2010
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26. Engineering a family 9 processive endoglucanase from Paenibacillus barcinonensis displaying a novel architecture.
- Author
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Chiriac AI, Cadena EM, Vidal T, Torres AL, Diaz P, and Pastor FI
- Subjects
- Amino Acid Sequence, Bacterial Proteins chemistry, Cellulase chemistry, Cellulose metabolism, Molecular Sequence Data, Protein Binding, Protein Engineering, Protein Structure, Tertiary, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Sequence Alignment, Sequence Deletion, Bacterial Proteins genetics, Bacterial Proteins metabolism, Cellulase genetics, Cellulase metabolism, Paenibacillus enzymology, Paenibacillus genetics
- Abstract
Cel9B from Paenibacillus barcinonensis is a modular endoglucanase with a novel molecular architecture among family 9 enzymes that comprises a catalytic domain (GH9), a family 3c cellulose-binding domain (CBM3c), a fibronectin III-like domain repeat (Fn3(1,2)), and a C-terminal family 3b cellulose-binding domain (CBM3b). A series of truncated derivatives of endoglucanase Cel9B have been constructed and characterized. Deletion of CBM3c produced a notable reduction in hydrolytic activity, while it did not affect the cellulose-binding properties as CBM3c did not show the ability to bind to cellulose. On the contrary, CBM3b exhibited binding to cellulose. The truncated forms devoid of CBM3b lost cellulose-binding ability and showed a reduced activity on crystalline cellulose, although activity on amorphous celluloses was not affected. Endoglucanase Cel9B produced only a small ratio of insoluble products from filter paper, while most of the reducing ends produced by the enzyme were released as soluble sugars (91%), indicating that it is a processive enzyme. Processivity of Cel9B resides in traits contained in the tandem of domains GH9-CBM3c, although the slightly reduced processivity of truncated form GH9-CBM3c suggests a minor contribution of domains Fn3(1,2) or CBM3b, not contained in it, on processivity of endoglucanase Cel9B.
- Published
- 2010
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27. Differential behaviour of Pseudomonas sp. 42A2 LipC, a lipase showing greater versatility than its counterpart LipA.
- Author
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Bofill C, Prim N, Mormeneo M, Manresa A, Pastor FI, and Diaz P
- Subjects
- Bacterial Proteins genetics, Enzyme Stability, Isoenzymes genetics, Lipase genetics, Substrate Specificity, Bacterial Proteins metabolism, Isoenzymes metabolism, Lipase metabolism, Pseudomonas enzymology
- Abstract
Growth of Pseudomonas sp. 42A2 on oleic acid releases polymerized hydroxy-fatty acids as a result of several enzymatic conversions that could involve one or more lipases. To test this hypothesis, the lipolytic system of strain Pseudomonas sp. 42A2 was analyzed, revealing the presence of at least an intracellular carboxylesterase and a secreted lipase. Consensus primers derived from a conserved region of bacterial lipase subfamilies I.1 and I.2 allowed isolation of two secreted lipase genes, lipA and lipC, highly homologous to those of Pseudomonas aeruginosa PAO1. Homologous cloning of the isolated lipA and lipC genes was performed in Pseudomonas sp. 42A2 for LipA and LipC over-expression. The overproduced lipases were further purified and characterized, both showing preference for medium fatty acid chain-length substrates. However, significant differences could be detected between LipA and LipC in terms of enzyme kinetics and behaviour pattern. Accordingly, LipA showed maximum activity at moderate temperatures, and displayed a typical Michaelis-Menten kinetics. On the contrary, LipC was more active at low temperatures and displayed partial interfacial activation, showing a shift in substrate specificity when assayed at different temperatures, and displaying increased activity in the presence of certain heavy metal ions. The versatile properties shown by LipC suggest that this lipase could be expressed in response to variable environmental conditions., (Copyright (c) 2009 Elsevier Masson SAS. All rights reserved.)
- Published
- 2010
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28. Structural insights into the specificity of Xyn10B from Paenibacillus barcinonensis and its improved stability by forced protein evolution.
- Author
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Gallardo O, Pastor FI, Polaina J, Diaz P, Łysek R, Vogel P, Isorna P, González B, and Sanz-Aparicio J
- Subjects
- Catalysis, Catalytic Domain, Crystallography, X-Ray, Endo-1,4-beta Xylanases genetics, Mutagenesis, Site-Directed, Paenibacillus genetics, Protein Folding, Protein Structure, Secondary, Protein Structure, Tertiary, Soil Microbiology, Substrate Specificity, Teprotide pharmacology, Endo-1,4-beta Xylanases chemistry, Endo-1,4-beta Xylanases metabolism, Evolution, Molecular, Paenibacillus enzymology, Xylans metabolism
- Abstract
Paenibacillus barcinonensis is a soil bacterium bearing a complex set of enzymes for xylan degradation, including several secreted enzymes and Xyn10B, one of the few intracellular xylanases reported to date. The crystal structure of Xyn10B has been determined by x-ray analysis. The enzyme folds into the typical (beta/alpha)(8) barrel of family 10 glycosyl hydrolases (GH10), with additional secondary structure elements within the beta/alpha motifs. One of these loops -L7- located at the beta7 C terminus, was essential for xylanase activity as its partial deletion yielded an inactive enzyme. The loop contains residues His(249)-Glu(250), which shape a pocket opened to solvent in close proximity to the +2 subsite, which has not been described in other GH10 enzymes. This wide cavity at the +2 subsite, where methyl-2,4-pentanediol from the crystallization medium was found, is a noteworthy feature of Xyn10B, as compared with the narrow crevice described for other GH10 xylanases. Docking analysis showed that this open cavity can accommodate glucuronic acid decorations of xylo-oligosaccharides. Co-crystallization experiments with conduramine derivative inhibitors supported the importance of this open cavity at the +2 subsite for Xyn10B activity. Several mutant derivatives of Xyn10B with improved thermal stability were obtained by forced evolution. Among them, mutant xylanases S15L and M93V showed increased half-life, whereas the double mutant S15L/M93V exhibited a further increase in stability, showing a 20-fold higher heat resistance than the wild type xylanase. All the mutations obtained were located on the surface of Xyn10B. Replacement of a Ser by a Leu residue in mutant xylanase S15L can increase hydrophobic packing efficiency and fill a superficial indentation of the protein, giving rise to a more compact structure of the enzyme.
- Published
- 2010
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29. Stachybotrys atra BP-A produces alkali-resistant and thermostable cellulases.
- Author
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Picart P, Diaz P, and Pastor FI
- Subjects
- Alkalies analysis, Cellulases genetics, Cellulases metabolism, Enzyme Stability, Fungal Proteins genetics, Fungal Proteins metabolism, Hot Temperature, Isoelectric Focusing, Molecular Sequence Data, Phylogeny, Stachybotrys classification, Stachybotrys genetics, Substrate Specificity, Cellulases chemistry, Cellulose metabolism, Fungal Proteins chemistry, Stachybotrys enzymology, Stachybotrys isolation & purification
- Abstract
A cellulose-degrading fungal strain has been isolated from a rotten rag. Morphological characterization and ITS1, 5.8S and ITS2 rDNA sequencing showed that the strain is a new isolate of Stachybotrys atra. The strain secreted high cellulase activity in media supplemented with rice straw. However, cellulases were not produced in glucose-supplemented media. The crude cellulase showed the highest activity on amorphous celluloses such as carboxymethyl cellulose, while activity on crystalline celluloses such as Avicel was lower. The optimal temperature and pH for CMCase activity were 70 degrees C and pH 5 respectively, although a second peak of activity was found at pH 8. Activity was strongly inhibited by Cu(2+), Mn(2+) and Hg(2+). Analysis by SDS-PAGE, isoelectric focusing and zymography showed that the strain secretes a complex cellulase system comprising several enzymes. Most of these enzymes are alkali-resistant CMCases that remained stable at pH 9 and 65 degrees C for at least 1 h. Cellulose binding assays showed notable differences among the CMCases. While some CMCase bands did not bind Avicel, other bands bound to this polymer and were eluted either with NaCl or by boiling with SDS. Analysis by two-dimensional electrophoresis showed that the band eluted by SDS boiling contained at least 4 different polypeptides. The complex set of cellulases produced by the strain, and their activity and stability at alkaline pH and a high temperature indicate that both the isolated strain and the cellulases identified are good candidates for biotechnological applications involving cellulose modification.
- Published
- 2008
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30. Cellulases from two Penicillium sp. strains isolated from subtropical forest soil: production and characterization.
- Author
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Picart P, Diaz P, and Pastor FI
- Subjects
- Biotechnology methods, Cellulases classification, Cellulose metabolism, Electrophoresis, Polyacrylamide Gel, Penicillium growth & development, Penicillium isolation & purification, Trees growth & development, Cellulases biosynthesis, Penicillium enzymology, Soil Microbiology, Tropical Climate
- Abstract
Aims: To isolate new fungal strains from subtropical soils and to identify those that produce high cellulase activity. To select microbial strains producing thermostable cellulases with potential application in industry., Methods and Results: The new strains Penicillium sp. CR-316 and Penicillium sp. CR-313 have been identified and selected because they secreted a high level of cellulase in media supplemented with rice straw. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis, isoelectric focussing and zymography showed that the studied strains secreted multiple enzymes that hydrolyse cellulose. Cellulase activity of Penicillium sp. CR-316, the strain showing higher production, was analysed. Optimum temperature and pH of carboxymethyl cellulase activity were 65 degrees C and pH 4.5, respectively. Activity remained stable after incubation at 60 degrees C and pH 4.5 for 3 h., Conclusions: Fungal strains that secrete high levels of cellulase activity have been characterized and selected from soil. The isolated strains have complex sets of enzymes for cellulose degradation. Crude cellulase produced by Penicillium sp. CR-316 showed activity and stability at high temperature., Significance and Impact of the Study: Two fungal strains with biotechnological potential have been isolated. The strains secrete high levels of cellulase, and one of them, Penicillium sp. CR-316, produces a thermostable cellulase, that makes it a good candidate for industrial applications.
- Published
- 2007
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31. Helicobacter pylori EstV: identification, cloning, and characterization of the first lipase isolated from an epsilon-proteobacterium.
- Author
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Ruiz C, Falcocchio S, Pastor FI, Saso L, and Diaz P
- Subjects
- Carboxylesterase antagonists & inhibitors, Carboxylesterase genetics, Carboxylesterase isolation & purification, Carboxylesterase metabolism, Cloning, Molecular, Coenzymes pharmacology, Computational Biology, DNA, Bacterial genetics, Enzyme Inhibitors pharmacology, Enzyme Stability, Escin pharmacology, Genome, Bacterial genetics, Glycyrrhizic Acid pharmacology, Helicobacter pylori genetics, Hydrogen-Ion Concentration, Lipase antagonists & inhibitors, Lipase classification, Lipase isolation & purification, Polymerase Chain Reaction, Sequence Homology, Substrate Specificity, Temperature, Helicobacter pylori enzymology, Lipase genetics, Lipase metabolism
- Abstract
Bacterial lipases are attracting an enormous amount of attention due to their wide biotechnological applications and due to their roles as virulence factors in some bacteria. Helicobacter pylori is a significant and widespread pathogen which produces a lipase(s) and phospholipases that seem to play a role in mucus degradation and the release of proinflammatory and cytotoxic compounds. However, no H. pylori lipase(s) has been isolated and described previously. Therefore, a search for putative lipase-encoding genes was performed by comparing the amino acid sequences of 53 known lipolytic enzymes with the deduced proteome of H. pylori. As a result, we isolated, cloned, purified, and characterized EstV, a novel lipolytic enzyme encoded by open reading frame HP0739 of H. pylori 26695, and classified it in family V of the bacterial lipases. This enzyme has the properties of a small, cell-bound carboxylesterase (EC 3.1.1.1) that is active mostly with short-chain substrates and does not exhibit interfacial activation. EstV is stable and does not require additional cofactors, and the maximum activity occurs at 50 degrees C and pH 10. This unique enzyme is the first lipase isolated from H. pylori that has been described, and it might contribute to ulcer development, as inhibition by two antiulcer substances (beta-aescin and glycyrrhizic acid) suggests. EstV is also the first lipase from an epsilon-proteobacterium to be described. Furthermore, this enzyme is a new member of family V, probably the least-known family of bacterial lipases, and the first lipase of this family for which kinetic behavior, inhibition by natural substances, and other key biochemical features are reported.
- Published
- 2007
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32. Esterase EstA6 from Pseudomonas sp. CR-611 is a novel member in the utmost conserved cluster of family VI bacterial lipolytic enzymes.
- Author
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Prim N, Bofill C, Pastor FI, and Diaz P
- Subjects
- Amino Acid Sequence, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Carboxylic Ester Hydrolases chemistry, Carboxylic Ester Hydrolases metabolism, Cloning, Molecular, DNA, Bacterial chemistry, DNA, Bacterial genetics, DNA, Bacterial isolation & purification, Electrophoresis, Polyacrylamide Gel methods, Hydrogen-Ion Concentration, Isoelectric Focusing methods, Lipolysis, Molecular Sequence Data, Multigene Family genetics, Phylogeny, Pseudomonas genetics, Sequence Alignment, Sequence Analysis, DNA, Substrate Specificity, Temperature, Bacterial Proteins genetics, Carboxylic Ester Hydrolases genetics, Pseudomonas enzymology
- Abstract
Strain Pseudomonas sp. CR-611, previously isolated from a subtropical forest soil on tributyrine-supplemented plates, displays phenotypic and physiological properties consistent with those described for Pseudomonas fluorescens. However, no complete match to this species could be found after 16S rDNA comparison. Zymographic analysis of the strain revealed a complex lipolytic system, showing the presence of at least two enzymes with activity on MUF-butyrate. Alignment of Pseudomonas fluorescens lipase/esterase-coding sequences allowed the design of specific primers for family VI lipases, and the isolation and cloning of the resulting gene estA6. The recombinant clone obtained displayed high activity on fatty acid-derivative substrates, indicating that one of the lipolytic enzymes of the strain had been cloned. The enzyme, named EstA6, was then purified and characterized, showing maximum activity on short chain-length substrates under conditions of high temperature and neutral pH. Amino acid sequence alignment of EstA6 with other family VI esterases allowed identification of a highly conserved beta-/gamma-protobacterial cluster in family VI lipases, to which EstA6 belongs.
- Published
- 2006
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33. Identification of a carboxylesterase-producing Rhodococcus soil isolate.
- Author
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Falcocchio S, Ruiz C, Pastor FI, Saso L, and Diaz P
- Subjects
- Bacterial Typing Techniques, Butyrates metabolism, Carboxylesterase metabolism, DNA, Bacterial analysis, DNA, Ribosomal, Hymecromone analogs & derivatives, Hymecromone metabolism, Molecular Sequence Data, Phylogeny, RNA, Ribosomal, 16S genetics, Rhodococcus genetics, Sequence Analysis, DNA, Carboxylesterase biosynthesis, Rhodococcus classification, Rhodococcus enzymology, Soil Microbiology
- Abstract
Subtropical soil microbial isolates were screened for carbohydrate, tributyrin, or olive oil hydrolysis using agar plates supplemented with the corresponding substrates. A heterotrophic, aerobic, Gram-positive strain displaying activity on tributyrin was selected and further characterized. Analysis of the morphological and physiological traits of the strain placed it as a member of the genus Rhodococcus. Further 16S rDNA sequencing revealed a 99% identity to Rhodococcus erythropolis. The strain displayed lipolytic activity on fatty-acid-derivative substrates of short chain length, with cell extract fractions having highest activity, as confirmed by the presence, after zymogram analysis, of a ca. 60-kDa intracellular protein band with activity on 4-methylumbelliferone-butyrate substrate. The presence of such a lipolytic enzyme, similar to those found in other Gram-positive bacteria, indicates that the strain could be of interest for certain biotechnological applications, like the synthesis of pharmaceuticals or biocide detoxification.
- Published
- 2005
- Full Text
- View/download PDF
34. Use of the cell wall protein Pir4 as a fusion partner for the expression of Bacillus sp. BP-7 xylanase A in Saccharomyces cerevisiae.
- Author
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Andrés I, Gallardo O, Parascandola P, Javier Pastor FI, and Zueco J
- Subjects
- Bacillus chemistry, Bacillus genetics, Cell Wall chemistry, Cell Wall genetics, Endo-1,4-beta Xylanases genetics, Fungal Proteins chemistry, Fungal Proteins genetics, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins metabolism, Saccharomyces cerevisiae chemistry, Saccharomyces cerevisiae genetics, Bacillus enzymology, Cell Wall metabolism, Endo-1,4-beta Xylanases metabolism, Fungal Proteins metabolism, Saccharomyces cerevisiae metabolism
- Abstract
Xylanase A from Bacillus sp. BP7, an enzyme with potential applications in biotechnology, was used to test Pir4, a disulfide bound cell wall protein, as a fusion partner for the expression of recombinant proteins in standard or glycosylation-deficient mnn9 strains of Saccharomyces cerevisiae. Five different constructions were carried out, inserting in-frame the coding sequence of xynA gene in that of PIR4, with or without the loss of specific regions of PIR4. Targeting of the xylanase fusion protein to the cell wall was achieved in two of the five constructions, while secretion to the growth medium was the fate of the gene product of one of the constructions. In all three cases localization of the xylanase fusion proteins was confirmed both by Western blot and detection with Pir-specific antibodies and by xylanase activity determination. The cell wall-targeted fusion proteins could be extracted by reducing agents, showing that the inclusion of a recombinant protein of moderate size does not affect the way Pir4 is attached to the cell wall. Also, the construction that leads to the secretion of the fusion protein permitted us to identify a region of Pir4 responsible for cell wall retention. In summary, we have developed a Pir4-based system that allows selective targeting of an active recombinant enzyme to the cell wall or the growth medium. This system may be of general application for the expression of heterologous proteins in S. cerevisiae for surface display and secretion.
- Published
- 2005
- Full Text
- View/download PDF
35. Pectinolytic systems of two aerobic sporogenous bacterial strains with high activity on pectin.
- Author
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Soriano M, Diaz P, and Pastor FI
- Subjects
- Bacillus isolation & purification, Gram-Positive Endospore-Forming Rods isolation & purification, Hydrogen-Ion Concentration, Kinetics, Polysaccharide-Lyases isolation & purification, Polysaccharide-Lyases metabolism, Soil Microbiology, Species Specificity, Temperature, Bacillus metabolism, Gram-Positive Endospore-Forming Rods metabolism, Pectins metabolism
- Abstract
Strains Paenibacillus sp. BP-23 and Bacillus sp. BP-7, previously isolated from soil from a rice field, secreted high levels of pectinase activity in media supplemented with pectin. Production of pectinases in strain Paenibacillus sp. BP-23 showed catabolite repression, while in Bacillus sp. BP-7 production of pectin degrading enzymes was not negatively affected by glucose. The two strains showed lyase activities as the predominant pectinases, while hydrolase activity was very low. Analysis of Paenibacillus sp. BP-23 in SDS-polyacrylamide gels and zymograms showed five pectinase activity bands. The strict requirement of Ca(2+) for lyase activity of the strain indicates that correspond to pectate lyases. For Bacillus sp. BP-7, zymograms showed four bands of different size. The strain showed a Ca(2+) requirement for lyase activity on pectate but not on pectin, indicating that the pectinolytic system of Bacillus sp. BP-7 is comprised of pectate lyases and pectin lyases. The results show differences in pectin degrading systems between the two aerobic sporogenous bacterial strains studied.
- Published
- 2005
- Full Text
- View/download PDF
36. Isolation of lipid- and polysaccharide-degrading micro-organisms from subtropical forest soil, and analysis of lipolytic strain Bacillus sp. CR-179.
- Author
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Ruiz C, Pastor FI, and Diaz P
- Subjects
- Bacillus genetics, Biodegradation, Environmental, Genes, Bacterial genetics, Genes, Bacterial physiology, Hydrolysis, Lipase physiology, Lipolysis, Oils metabolism, Phylogeny, RNA, Ribosomal, 16S genetics, Bacillus enzymology, Bacillus isolation & purification, Lipase genetics, Lipid Metabolism, Polysaccharides metabolism, Soil Microbiology
- Abstract
Aims: To isolate the micro-organisms from three soil samples obtained from a subtropical forest of Puerto Iguazu (Argentina), to analyse them for detection of the biotechnologically interesting enzymatic activities lipase, esterase, cellulase, xylanase and pectinase, and to identify the most active strain., Methods and Results: A total of 724 strains were isolated using different culture media and temperatures, and 449 of them showed at least one of the hydrolytic activities pursued. Lipolytic activity of the lipid-degrading strains was further determined using MUF-butyrate and MUF-oleate as substrates. The alkalophilic strain CR-179, one of the most active for all the enzymatic activities assayed, was characterized and preliminarily identified by morphological, physiological and 16S rDNA tests, as a Bacillus sp. closely related to Bacillus subtilis., Conclusions: Highly hydrolytic strains were isolated from all soil samples, suggesting the existence of a microbial community well-adapted to nutrient recycling. Strain CR-179, one of the most active, has been preliminarily identified as a Bacillus sp., Significance and Impact of the Study: A collection of hydrolytic strains with high biotechnological potential was obtained. Presence of sequences codifying for a lipolytic system related to the B. subtilis group lipases was revealed by PCR for the best lipolytic strain.
- Published
- 2005
- Full Text
- View/download PDF
37. Synergistic activity of Paenibacillus sp. BP-23 cellobiohydrolase Cel48C in association with the contiguous endoglucanase Cel9B and with endo- or exo-acting glucanases from Thermobifida fusca.
- Author
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Sánchez MM, Irwin DC, Pastor FI, Wilson DB, and Diaz P
- Subjects
- Bacillus growth & development, Carboxymethylcellulose Sodium metabolism, Cellobiose analysis, Cellobiose metabolism, Cellulase chemistry, Cellulase genetics, Cellulose 1,4-beta-Cellobiosidase chemistry, Cellulose 1,4-beta-Cellobiosidase genetics, Chromatography, Thin Layer, Electrophoresis, Polyacrylamide Gel, Escherichia coli genetics, Glucose metabolism, Glycoside Hydrolases chemistry, Glycoside Hydrolases genetics, Glycoside Hydrolases metabolism, Isoelectric Point, Kinetics, Oligosaccharides analysis, Recombinant Proteins metabolism, Substrate Specificity, Actinomycetales enzymology, Bacillus enzymology, Cellulase metabolism, Cellulose metabolism, Cellulose 1,4-beta-Cellobiosidase metabolism
- Abstract
Cellobiohydrolase Cel48C from Paenibacillus sp. BP-23, an enzyme displaying limited activity on most cellulosic substrates, was assayed for activity in the presence of other bacterial endo- or exocellulases. Significant enhanced activity was observed when Cel48C was incubated in the presence of Paenibacillus sp. BP-23 endoglucanase Cel9B or Thermobifida fusca cellulases Cel6A and Cel6B, indicating that Cel48C acts synergistically with them. Maximum synergism rates on bacterial microcrystalline cellulose or filter paper were obtained with a mixture of Paenibacillus cellulases Cel9B and Cel48C, accompanied by T. fusca exocellulase Cel6B. Synergism was also observed in cell extracts from recombinant clone E. coli pUCel9-Cel48 expressing the two contiguous Paenibacillus cellulases Cel9B and Cel48C. The enhanced cellulolytic activity displayed by the cellulase mixtures assayed could be used as an efficient tool for biotechnological applications like pulp and paper manufacturing., (Copyright 2004 Wiley Periodicals, Inc.)
- Published
- 2004
- Full Text
- View/download PDF
38. Activation and inhibition of Candida rugosa and Bacillus-related lipases by saturated fatty acids, evaluated by a new colorimetric microassay.
- Author
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Ruiz C, Falcocchio S, Xoxi E, Pastor FI, Diaz P, and Saso L
- Subjects
- Chromatography, High Pressure Liquid, Microchemistry, Bacillus enzymology, Candida enzymology, Colorimetry methods, Fatty Acids pharmacology, Lipase antagonists & inhibitors, Lipase metabolism
- Abstract
Research on lipase inhibitors could help in the therapy of diseases caused by lipase-producing microorganisms and in the design of novel lipase substrate specificities for biotechnology. Here we report a fast and sensitive colorimetric microassay that is low-cost and suitable for high-throughput experiments for the evaluation of lipase activity and inhibition. Comparison of Candida rugosa activity and inhibition with previous HPLC results validated the method, and revealed the importance of the reaction mixture composition. The assay was used to evaluate the effect of saturated fatty acids on Bacillus-related lipases. Cell-bound esterases were strongly inhibited by fatty acids, suggesting a negative feedback regulation by product, and a role of these enzymes in cell membrane turnover. Bacillus subtilis LipA was moderately activated by low concentrations of fatty acids and was inhibited at greater concentrations. LipB-like esterases were highly activated by myristic and lauric acids and were only slightly inhibited by high capric acid concentrations. Such an activation, reported here for the first time in bacterial lipases, seems to be part of a regulatory system evolved to ensure a high use of carbon sources, and could be related to the successful adaptation of Bacillus strains to nutrient-rich environments with strong microbial competition.
- Published
- 2004
- Full Text
- View/download PDF
39. Cloning and characterization of xylanase A from the strain Bacillus sp. BP-7: comparison with alkaline pI-low molecular weight xylanases of family 11.
- Author
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Gallardo O, Diaz P, and Pastor FI
- Subjects
- Base Sequence, Cloning, Molecular, Codon, Endo-1,4-beta Xylanases chemistry, Isoelectric Point, Molecular Sequence Data, Molecular Weight, Bacillus enzymology, Endo-1,4-beta Xylanases genetics
- Abstract
The xynA gene encoding a xylanase from the recently isolated Bacillus sp. strain BP-7 has been cloned and expressed in Escherichia coli. Recombinant xylanase A showed high activity on xylans from hardwoods and cereals, and exhibited maximum activity at pH 6 and 60 degrees C. The enzyme remained stable after incubation at 50 degrees C and pH 7 for 3 h, and it was strongly inhibited by Mn(2+), Fe(3+), Pb(2+), and Hg(2+). Analysis of xylanase A in zymograms showed an apparent molecular size of 24 kDa and a pI of above 9. The amino acid sequence of xylanase A, as deduced from xynA gene, shows homology to alkaline pI-low molecular weight xylanases of family 11 such as XynA from Bacillus subtilis. Analysis of codon usage in xynA from Bacillus sp. BP-7 shows that the G+C content at the first and second codon positions is notably different from the mean values found for glycosyl hydrolase genes from Bacillus subtilis.
- Published
- 2004
- Full Text
- View/download PDF
40. Biochemical studies on cloned Bacillus sp. BP-7 phenolic acid decarboxylase PadA.
- Author
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Prim N, Pastor FI, and Diaz P
- Subjects
- Bacillus genetics, Base Sequence, Caffeic Acids metabolism, Carboxy-Lyases metabolism, Cloning, Molecular, Coumaric Acids metabolism, DNA, Bacterial chemistry, DNA, Bacterial genetics, Electrophoresis, Polyacrylamide Gel, Molecular Sequence Data, Propionates, Sequence Alignment, Sequence Analysis, DNA, Bacillus enzymology, Carboxy-Lyases genetics
- Abstract
Sequence analysis of a Bacillus sp. BP-7 recombinant clone coding for a previously described carboxylesterase revealed the presence of an additional ORF with homology to bacterial hydroxycinnamic acid decarboxylases. Analysis of the amino acid sequence of the encoded enzyme revealed the presence of a single, highly conserved domain of 161 amino acids, with a predicted molecular mass of 19,143 Da and a pI of 5.5. Crude cell extracts from the recombinant clone displayed activity on ferulic, p-coumaric and caffeic acids, with no need for added cofactors. The cloned enzyme, named PadA, displayed maximum activity at 40 degrees C and pH 5.5, being stable over a broad range of pH and up to 45 degrees C. HPLC analysis of the products of catalysis revealed the conversion of phenolic acids to their aromatic 4-vinyl derivatives, with no accumulation of other by-products. PadA was found as a homodimer in the parental Bacillus sp. BP-7 strain and its expression was induced by both hydroxycinnamic acids and their corresponding derivative products. The results obtained suggest that the enzyme could be involved in a stress response for conversion of toxic hydroxycinnamic acids released after plant cell wall degradation.
- Published
- 2003
- Full Text
- View/download PDF
41. Exo-mode of action of cellobiohydrolase Cel48C from Paenibacillus sp. BP-23. A unique type of cellulase among Bacillales.
- Author
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Sánchez MM, Pastor FI, and Diaz P
- Subjects
- Bacillus genetics, Bacterial Proteins genetics, Cellulase genetics, Cellulose metabolism, Cellulose 1,4-beta-Cellobiosidase, Open Reading Frames, Substrate Specificity, Bacillus enzymology, Bacterial Proteins metabolism, Cellulase metabolism
- Abstract
Sequence analysis of a Paenibacillus sp. BP-23 recombinant clone coding for a previously described endoglucanase revealed the presence of an additional truncated ORF with homology to family 48 glycosyl hydrolases. The corresponding 3509-bp DNA fragment was isolated after gene walking and cloned in Escherichia coli Xl1-Blue for expression and purification. The encoded enzyme, a cellulase of 1091 amino acids with a deduced molecular mass of 118 kDa and a pI of 4.85, displayed a multidomain organization bearing a canonical family 48 catalytic domain, a bacterial type 3a cellulose-binding module, and a putative fibronectin-III domain. The cloned cellulase, unique among Bacillales and designated Cel48C, was purified through affinity chromatography using its ability to bind Avicel. Maximum activity was achieved at 45 degrees C and pH 6.0 on acid-swollen cellulose, bacterial microcrystalline cellulose, Avicel and cellodextrins, whereas no activity was found on carboxy methyl cellulose, cellobiose, cellotriose, pNP-glycosides or 4-methylumbeliferyl alpha-d-glucoside. Cellobiose was the major product of cellulose hydrolysis, identifying Cel48C as a processive cellobiohydrolase. Although no chromogenic activity was detected from pNP-glycosides, TLC analysis revealed the release of p-nitrophenyl-glycosides and cellodextrins from these substrates, suggesting that Cel48C acts from the reducing ends of the sugar chain. Presence of such a cellobiohydrolase in Paenibacillus sp. BP-23 would contribute to widen up its range of action on natural cellulosic substrates.
- Published
- 2003
- Full Text
- View/download PDF
42. Characterization of a Paenibacillus cell-associated xylanase with high activity on aryl-xylosides: a new subclass of family 10 xylanases.
- Author
-
Gallardo O, Diaz P, and Pastor FI
- Subjects
- Amino Acid Sequence, Bacillus subtilis genetics, Base Sequence, Cloning, Molecular, Extracellular Space, Molecular Sequence Data, Phylogeny, Sequence Alignment, Sequence Homology, Amino Acid, Substrate Specificity, Xylan Endo-1,3-beta-Xylosidase, Xylosidases chemistry, Xylosidases genetics, Xylosidases isolation & purification, Bacillaceae cytology, Bacillaceae enzymology, Xylosidases metabolism
- Abstract
The sequence of gene xynB encoding xylanase B from Paenibacillus sp. BP-23 was determined. It revealed an open reading frame of 999 nucleotides encoding a protein of 38,561 Da. The deduced amino acid sequence of xylanase B shows that the N-terminal region of the enzyme lacks the features of a signal peptide. When the xylan-degrading system of Paenibacillus sp. BP-23 was analysed in zymograms, it revealed that xylanase B was not secreted to the extracellular medium but instead remained cell-associated, even in late stationary-phase cultures. When xynB was expressed in a Bacillus subtilis secreting host, it also remained associated with the cells. Sequence homology analysis showed that xylanase B from Paenibacillus sp. BP-23 belongs to family 10 glycosyl hydrolases, exhibiting a distinctive high homology to six xylanases of this family. The homologous enzymes were also found to be devoid of a signal peptide and seem to constitute, together with xylanase B, a separate group of enzymes. They all have two conserved amino acid regions not found in the other family 10 xylanases, and cluster in a separate group after dendrogram analysis. We propose that these enzymes constitute a new subclass of family 10 xylanases, that are cell-associated, and that hydrolyse the xylooligosaccharides resulting from extracellular xylan hydrolysis. Xylanase B shows similar specific activity on aryl-xylosides and xylans. This can be correlated to some, not yet identified, trait of catalytic activity of the enzyme on plant xylan.
- Published
- 2003
- Full Text
- View/download PDF
43. Isolation and characterization of Bacillus sp. BP-6 LipA, a ubiquitous lipase among mesophilic Bacillus species.
- Author
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Ruiz C, Javier Pastor FI, and Diaz P
- Subjects
- Bacillus genetics, Biotechnology methods, Cloning, Molecular, Escherichia coli enzymology, Escherichia coli genetics, Lipase chemistry, Lipase genetics, Molecular Sequence Data, Sequence Analysis, DNA, Substrate Specificity, Bacillus enzymology, Lipase isolation & purification, Lipase metabolism
- Abstract
Aims: The aim of this study was to perform the isolation, cloning and characterization of a lipase from Bacillus sp. BP-6 bearing the features of a biotechnologically important group of enzymes., Methods and Results: Strain Bacillus sp. BP-6, showing activity on tributyrin plates, was used for isolation of lipase-coding gene lipA by means of inverse and direct PCR. The complete 633 nucleotide ORF isolated was cloned in Escherichia coli for further characterization. The amino acid sequence of the cloned protein was 98% identical to B. subtilis and B. megaterium lipases, the enzyme also showing similar molecular and biochemical features., Conclusions: The gene coding for Bacillus sp. BP-6 LipA was found in all mesophilic Bacillus species assayed, indicating its ubiquity in the genus. The cloned enzyme displayed the same properties as those of homologous lipases., Significance and Impact of the Study: The overall profile of Bacillus sp. BP-6 LipA was found to be that of a ubiquitous and highly conserved subfamily I.4 bacterial lipase. Previously described lipases within this family have shown to be well suited for biotechnological applications, suggesting that the cloned enzyme could be used accordingly.
- Published
- 2003
- Full Text
- View/download PDF
44. Analysis of Bacillus megaterium lipolytic system and cloning of LipA, a novel subfamily I.4 bacterial lipase.
- Author
-
Ruiz C, Blanco A, Pastor FI, and Diaz P
- Subjects
- Base Sequence, Cloning, Molecular, Electrophoresis, Polyacrylamide Gel, Hot Temperature, Hydrogen-Ion Concentration, Lipolysis, Bacillus megaterium enzymology, Bacillus megaterium metabolism, Bacterial Proteins genetics, Genes, Bacterial, Lipase genetics
- Abstract
The lipolytic system of Bacillus megaterium 370 was investigated, showing the existence of at least two secreted lipases and a cell-bound esterase. A gene coding for an extracellular lipase was isolated and cloned in Escherichia coli. The cloned enzyme displayed high activity on short to medium chain length (C(4)-C(8)) substrates, and poor activity on C(18) substrates. On the basis of amino acid sequence homology, the cloned lipase was classified into subfamily I.4 of bacterial lipases., (Copyright 2002 Federation of European Microbiological Societies)
- Published
- 2002
- Full Text
- View/download PDF
45. Zymographic detection of cinnamic acid decarboxylase activity.
- Author
-
Prim N, Pastor FI, and Diaz P
- Subjects
- Bacteriological Techniques, Bromcresol Purple metabolism, Cinnamates metabolism, Culture Media, Electrophoresis, Polyacrylamide Gel methods, Hydrogen-Ion Concentration, Bacillus enzymology, Carboxy-Lyases chemistry, Carboxy-Lyases metabolism, Escherichia coli enzymology
- Abstract
The manuscript includes a concise description of a new, fast and simple method for detection of cinnamic acid decarboxylase activity. The method is based on a color shift caused a by pH change and may be an excellent procedure for large screenings of samples from natural sources, as it involves no complex sample processing or purification. The method developed can be used in preliminary approaches to biotransformation processes involving detection of hydroxycinnamic acid decarboxylase activity., (Copyright 2002 Elsevier Science B.V.)
- Published
- 2002
- Full Text
- View/download PDF
46. Engineering of baker's yeasts, E. coli and Bacillus hosts for the production of Bacillus subtilis Lipase A.
- Author
-
Sánchez M, Prim N, Rández-Gil F, Pastor FI, and Diaz P
- Subjects
- Bacillus subtilis enzymology, Escherichia coli metabolism, Gene Expression, Gene Transfer Techniques, Lipase genetics, Saccharomyces cerevisiae metabolism, Bacillus subtilis genetics, Escherichia coli genetics, Genetic Engineering methods, Lipase metabolism, Saccharomyces cerevisiae genetics
- Abstract
Lipases are versatile biocatalists showing multiple applications in a wide range of biotechnological processes. The gene lipA coding for Lipase A from Bacillus subtilis was isolated by PCR amplification, cloned and expressed in Escherichia coli, Saccharomyces cerevisiae and Bacillus subtilis strains, using pBR322, YEplac112 and pUB110-derived vectors, respectively. Lipase activity analysis of the recombinant strains showed that the gene can be properly expressed in all hosts assayed, this being the first time a lipase from bacterial origin can be expressed in baker's S. cerevisiae strains. An important increase of lipase production was obtained in heterologous hosts with respect to that of parental strains, indicating that the described systems can represent a useful tool to enhance productivity of the enzyme for biotechnological applications, including the use of the lipase in bread making, or as a technological additive., (Copyright 2002 Wiley Periodicals, Inc.)
- Published
- 2002
- Full Text
- View/download PDF
47. Cloning and characterization of a bacterial cell-bound type B carboxylesterase from Bacillus sp. BP-7.
- Author
-
Prim N, Pastor FI, and Diaz P
- Subjects
- Amino Acid Sequence, Bacterial Proteins chemistry, Bacterial Proteins genetics, Bacterial Proteins metabolism, Base Sequence, Carboxylesterase, Carboxylic Ester Hydrolases chemistry, Cloning, Molecular, Electrophoresis, Polyacrylamide Gel, Enzyme Stability, Escherichia coli enzymology, Escherichia coli genetics, Genome, Bacterial, Genomic Library, Hymecromone metabolism, Isoelectric Focusing, Lipolysis, Molecular Sequence Data, Nitrophenols metabolism, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Sequence Analysis, DNA, Substrate Specificity, Bacillus enzymology, Bacillus genetics, Carboxylic Ester Hydrolases genetics, Carboxylic Ester Hydrolases metabolism
- Abstract
A clone producing halos on tributyrin plates was isolated from a genomic library of Bacillus sp. BP-7. The insert contained an open reading frame that coded for a protein of 487 amino acids with homology to carboxylesterases. The cloned enzyme showed clear preference for esters of short-chain fatty acids, being classified as an esterase. Maximum activity was found at 45 degrees C and pH 7.5. The enzyme displayed stability in the pH range from 6 to 9.5, and at temperatures from 4 degrees to 45 degrees C. Zymogram analysis of the protein revealed a molecular mass of 53 kDa and a pI of 5.1. The enzyme showed homology to members of the bacterial subclass of type B carboxylesterases, a set of proteins potentially useful for biotechnological applications.
- Published
- 2001
- Full Text
- View/download PDF
48. Molecular cloning and characterization of a multidomain endoglucanase from Paenibacillus sp BP-23: evaluation of its performance in pulp refining.
- Author
-
Pastor FI, Pujol X, Blanco A, Vidal T, Torres AL, and Díaz P
- Subjects
- Bacillus genetics, Cellulase chemistry, Cloning, Molecular, Escherichia coli genetics, Molecular Sequence Data, Sequence Analysis, DNA, Bacillus enzymology, Biotechnology methods, Cellulase genetics, Cellulase metabolism, Escherichia coli enzymology, Paper
- Abstract
The gene celB encoding an endoglucanase from Paenibacillus sp. BP-23 was cloned and expressed in Escherichia coli. The nucleotide sequence of a 4161 bp DNA fragment containing the celB gene was determined, revealing an open reading frame of 2991 nucleotides that encodes a protein of 106,927 Da. Comparison of the deduced amino acid sequence of endoglucanase B with known beta-glycanase sequences showed that the encoded enzyme is a modular protein and exhibits high homology to enzymes belonging to family 9 cellulases. The celB gene product synthesized in E. coli showed high activity on carboxymethyl cellulose and lichenan while low activity was found on Avicel. Activity was enhanced in the presence of 10 mM Ca2+ and showed its maximum at 53 degrees C and pH 5.5. The effect of the cloned enzyme in modifying the physical properties of pulp and paper from Eucalyptus was tested (CelB treatment). An increase in mechanical strength of paper and a decrease in pulp dewatering properties were found, indicating that CelB treatment can be considered as a biorefining. Treatment with CelB gave rise to an improvement in paper strength similar to that obtained with 1,000 revolutions increase in mechanical refining. Comparison with the performances of recently developed endoglucanase A from the same strain and with a commercial cellulase showed that CelB produced the highest refining effect.
- Published
- 2001
- Full Text
- View/download PDF
49. Direct fluorescence-based lipase activity assay.
- Author
-
Diaz P, Prim N, and Javier Pastor FI
- Subjects
- Bacillus enzymology, Carboxylic Ester Hydrolases analysis, Carboxylic Ester Hydrolases genetics, Carboxylic Ester Hydrolases metabolism, Electrophoresis, Polyacrylamide Gel, Escherichia coli enzymology, Escherichia coli genetics, Gene Expression, Hymecromone metabolism, Indicators and Reagents, Isoelectric Focusing, Isoelectric Point, Lipase genetics, Lipase metabolism, Lipolysis, Recombinant Proteins, Sensitivity and Specificity, Ultraviolet Rays, Bacterial Proteins, Fluorescence, Lipase analysis
- Published
- 1999
- Full Text
- View/download PDF
50. Cloning of a new endoglucanase gene from Bacillus sp. BP-23 and characterisation of the enzyme. Performance in paper manufacture from cereal straw.
- Author
-
Blanco A, Díaz P, Martínez J, Vidal T, Torres AL, and Pastor FI
- Subjects
- Amino Acid Sequence, Bacillus genetics, Base Sequence, Carboxymethylcellulose Sodium chemistry, Cellulase genetics, Cellulose chemistry, Cloning, Molecular, Electrophoresis, Agar Gel, Electrophoresis, Polyacrylamide Gel, Escherichia coli chemistry, Isoelectric Focusing, Molecular Sequence Data, Phosphoenolpyruvate Sugar Phosphotransferase System genetics, Sequence Alignment, Sequence Analysis, DNA, Sequence Homology, Amino Acid, Substrate Specificity, Triticum ultrastructure, Bacillus enzymology, Cellulase chemistry, DNA, Bacterial chemistry, Paper, Phosphoenolpyruvate Sugar Phosphotransferase System chemistry, Triticum chemistry
- Abstract
The gene ce1A, encoding an endoglucanase from the strain Bacillus sp. BP-23, was cloned and expressed in Escherichia coli. The nucleotide sequence of a 1867-bp DNA fragment containing the ce1A gene was determined, revealing an open reading frame of 1200 nucleotides that encodes a protein of 44,803 Da. The deduced amino acid sequence of the encoded enzyme shows high homology to those of enzymes belonging to subtype 4 of the family-A cellulases. The ce1A gene product synthesized in E. coli showed activity on carboxymethylcellulose and lichenan but no activity was found on Avicel. Activity was enhanced in the presence of 10 mM Mg2+ and Ca2+ and showed its maximum at 40 degrees C and pH 4.0. Study of the performance of Ce1A on paper manufacture from agricultural fibres showed that treatment with the enzyme improved the properties of the pulp and the quality of paper. Ce1A treatment enhanced the physical properties (stretch and tensile index) of paper from wheat straw, while dewatering properties were slightly diminished. Electron-microscope analysis showed that the surface of straw fibres was modified by Ce1A.
- Published
- 1998
- Full Text
- View/download PDF
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