Institute Ciencias Of La Alimentacion

Madrid, Spain

Institute Ciencias Of La Alimentacion

Madrid, Spain
Time filter
Source Type

De Lima Damasio A.R.,University of Ribeirão Preto | Pessela B.C.,Institute Ciencias Of La Alimentacion | Mateo C.,CSIC - Institute of Catalysis | Segato F.,CTBE - Brazilian Bioethanol Science and Technology Laboratory | And 3 more authors.
Journal of Molecular Catalysis B: Enzymatic | Year: 2012

An endo-1,5-arabinanase (abnA) encoding gene from Aspergillus niveus was identified, cloned and successfully expressed in Aspergillus nidulans strain A773. Based on amino acid sequence comparison, the 34-kDa enzyme could be assigned to CAZy GH family 43. Characterization of purified recombinant endo-1,5-arabinanase (AbnA) revealed that it is active at a wide pH range (pH 4.0-7.0) and an optimum temperature at 70 °C. The immobilization of the AbnA was performed via covalent binding onto agarose-modified supports: glyoxyl iminodiacetic acid-Ni 2+, glyoxyl amine, glyoxyl (4% and 10%) and cyanogen bromide activated sepharose. The yield of immobilization was similar on glyoxyl amine and glyoxyl (96%), and higher than glyoxyl iminodiacetic acid-Ni 2+ (43%) support. The thermal inactivation of these immobilized preparations showed that the stability of the AbnA immobilized on glyoxyl 4 and 10% was improved by 4.0 and 10.3-fold factor at 70 °C. The half-life of glyoxyl 4% derivative at 60 °C was >48 h (pH 5), 9 h (pH 7) and 88 min (pH 9). The major hydrolysis product of debranched arabinan or arabinopentaose by glyoxyl agarose-immobilized AbnA was arabinobiose. © 2012 Elsevier B.V. All rights reserved.

Damasio A.R.D.L.,University of Sao Paulo | Pessela B.C.,Institute Ciencias Of La Alimentacion | Segato F.,Oklahoma State University | Prade R.A.,Oklahoma State University | And 2 more authors.
Process Biochemistry | Year: 2012

A gene encoding α-l-arabinofuranosidase (abfA) from Aspergillus niveus was identified, cloned, and successfully expressed in Aspergillus nidulans. Based on amino acid sequence comparison, the 88.6 kDa enzyme could be assigned to the GH family 51. The characterization of the purified recombinant AbfA revealed that the enzyme was active at a limited pH range (pH 4.0-5.0) and an optimum temperature of 70 °C. The AbfA was able to hydrolyze arabinoxylan, xylan from birchwood, debranched arabinan, and 4-nitrophenyl arabinofuranoside. Synergistic reactions using both AbfA and endoxylanase were also assessed. The highest degree of synergy was obtained after the sequential treatment of the substrate with endoxylanase, followed by AbfA, which was observed to release noticeably more reducing sugars than that of either enzyme acting individually. The immobilization of AbfA was performed via ionic adsorption onto various supports: agarose activated by polyethyleneimine polymers, cyanogen bromide activated Sepharose, DEAE-Sepharose, and Sepharose-Q. The Sepharose-Q derivative remained fully active at pH 5 after 360 min at 60 °C, whereas the free AbfA was inactivated after 60 min. A synergistic effect of arabinoxylan hydrolysis by AbfA immobilized in Sepharose-Q and endoxylanase immobilized in glyoxyl agarose was also observed. The stabilization of arabinofuranosidases using immobilization tools is a novel and interesting topic. © 2012 Elsevier Ltd.

Benassi V.M.,University of Sao Paulo | Silva T.M.D.,University of Sao Paulo | Pessela B.C.,Institute Ciencias Of La Alimentacion | Guisan J.M.,CSIC - Institute of Catalysis | And 4 more authors.
Journal of Molecular Catalysis B: Enzymatic | Year: 2013

β-Xylosidases have important applications in many biotechnological processes. In this context, the aim of this work was the purification, immobilization and characterization of a β-xylosidase produced by a new isolate of Aspergillus niger USP-67. β-Xylosidase was produced on static conditions in liquid Benassi medium supplemented with xylan birchwood, initial pH 3.0, for 6 days, at 30 °C. The enzyme was purified on DEAE-Sepharose followed of Superdex™ 200, and the molecular mass of the β-xylosidase was estimated to be 100 kDa, with 90% similarly to the β-xylosidase xlnD from A. niger (gi 146230215 accession), using MS sequencing. The enzyme was immobilized on DEAE-Sepharose, Polyethyleneimine (PEI)-Sepharose, Q-Sepharose, CM-Sepharose, Sulphopropil-Sepharose and MANAE-agarose, but the best result was obtained with PEI-Sepharose, which presented 94% of immobilization yield. Moreover, this derivative was more thermal stable than the soluble enzyme and other supports, which presented a half-life of about 50 min, at 65 °C. The enzyme immobilized on PEI-Sepharose had an optimum pH more acidic (around 4.5) than the purified enzyme (pH 5.5). Metal ions inhibited the soluble enzyme activity more than the immobilized form; however, Zn2+ increased the activity of the immobilized enzyme in 29%. The specific activity of the immobilized enzyme corresponded to 98.15 U/mg, but the soluble enzyme was 77.96 U/mg. Furthermore, the KM and Kcat values for the purified enzyme with p-nitrophenyl-xylopyranoside as substrate were 0.654 mM and 58.87 s-1 and for the immobilized enzyme the values were 0.587 mM and 88.95 s-1, respectively. The purified enzyme efficiently hydrolyzed xylooligosaccharides until xylose, but other xylooligosaccharides (X2-X6) were formed, suggesting transxylosylation action. The immobilized β-xylosidase of A. niger was not inhibited by xylose (100 mM) and glucose (200 mM), what confers to this enzyme a potential application in biotechnological processes. © 2012 Elsevier B.V. All rights reserved.

Pereira M.G.,University of Sao Paulo | Facchini F.D.A.,University of Sao Paulo | Polizeli A.M.,University of Sao Paulo | Vici A.C.,University of Sao Paulo | And 5 more authors.
Journal of Molecular Catalysis B: Enzymatic | Year: 2015

Lipase from Hypocrea pseudokoningii was purified using the support Octyl-Sepharose. This adsorption resulted in a 3-fold increase in activity of the immobilized enzyme. Following, still on this support, the lipase was enriched in surface amino groups (by reaction of carboxy groups with ethylendiamine). After amination, the lipase was desorbed from Octyl-Sepharose, while the 2-fold hyper-activation was maintained. The aminated lipase was also successfully immobilized on Glyoxyl-Agarose. The derivative was 45-fold more stable than was the free enzyme at 50 and 60 °C. The derivative was also stable in 50% of organic solvents such as methanol, ethanol, propanol and cyclohexane. The multipoint immobilization also increased the enzyme stability in relation to the free enzyme in the presence of ethanol, methanol and cyclohexane for up to 72 h. For example, the stabilized derivative was 9-fold more stable than the free enzyme in presence of methanol. The derivatives hydrolyzed fish, cupuaçu (Theobroma grandiflorum), bacuri (Latonia insignis) and murumuru (Astrocaryum murumuru) oils. The multipoint immobilization process increased the hydrolysis of oils up to 15-fold compared with the control, what makes these derivatives attractive for industrial application. © 2015 Elsevier B.V.

De Lima Damasio A.R.,University of Sao Paulo | Pessela B.C.,Institute Ciencias Of La Alimentacion | Da Silva T.M.,University of Sao Paulo | Guimaraes L.H.S.,University of Sao Paulo | And 3 more authors.
Journal of Biochemistry | Year: 2013

Plant cell-wall arabinoxylans have a complex structure that requires the action of a pool of debranching (arabinofuranosidases) and depolymerizing enzymes (endo-xylanase). Two Aspergillus nidulans strains over-secreting endo-xylanase and arabinofuranosidase were inoculated in defined 2% maltose-minimum medium resulting in the simultaneously production of these enzymes. To study the synergistic hydrolysis was used arabinoxylan with 41% of arabinose and 59% of xylose residues. Thus, it was adopted different approaches to arabinoxylan hydrolysis using immobilized arabinofuranosidase and endo-xylanase: (i) endo-xylanase immobilized on glyoxyl agarose; (ii) arabinofuranosidase immobilized on glyoxyl agarose; (T1) hydrolysis of arabinoxylan with arabinofuranosidase immobilized on glyoxyl agarose for debranching, followed by a second hydrolysis with endo-xylanase immobilized on glyoxyl agarose; (T2) hydrolysis using (i) and (ii) simultaneously; and (T3) hydrolysis of arabinoxylan with endo-xylanase and arabinofuranosidase co-immobilized on glyoxyl agarose. It was concluded that arabinoxylan hydrolysis using two derivatives simultaneously (T2) showed greater hydrolytic efficiency and consequently a higher products yield. However, the hydrolysis with multi-enzymatic derivative (T3) results in direct release of xylose and arabinose from a complex substrate as arabinoxylan, which is a great advantage as biotechnological application of this derivative, especially regarding the application of biofuels, since these monosaccharides are readily assimilable for fermentation and ethanol production. © The Authors 2013. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Pereira M.G.,University of Ribeirão Preto | Facchini F.D.A.,University of Ribeirão Preto | Filo L.E.C.,University of Ribeirão Preto | Polizeli A.M.,University of Ribeirão Preto | And 6 more authors.
Process Biochemistry | Year: 2015

Hypocrea pseudokoningii purified lipase was immobilized on hydrophobic supports (phenyl-sepharose, butyl-sepharose, octyl-sepharose, Hexyl Toyopearl, Lewatit, Purolite, Decaoctyl sepabeads) and ionic supports (Duolite, DEAE-agarose, PEI-agarose, MANAE-agarose, and Q-sepharose). The immobilization processes resulted in derivatives with excellent thermal stabilities, increasing the half-life up to 500-fold. The derivatives had excellent stability to organic solvents compared to the crude lipase. In the presence of 50% ethanol, hexyl and Decaoctyl derivatives increased by about 6- and 3.5-fold their stability to organic solvents, respectively. When tested for methanol, phenyl-sepharose derivative also increased their stability to organic solvents in approximately 2-fold. Octyl-sepharose derivative was fully stable for 48. h in the presence of propanol, which showed a half-life of about 7.5. h. The greater activation of the derivatives occurred using 50% cyclohexane, in which the hexyl derivative obtained an increase in the activity of 9-fold and phenyl and octyl derivatives had their activity increased by 6-fold. The lipase showed activity on different oils. Therefore, the adsorption of lipases in low ionic strength and highly hydrophobic supports is shown to be a simple and rapid tool for the immobilization of H. pseudokoningii lipase. These derivatives strongly increase the chances of this biocatalyst for industrial application. © 2015.

Loading Institute Ciencias Of La Alimentacion collaborators
Loading Institute Ciencias Of La Alimentacion collaborators