Procedimiento para la purificación de dos isoenzimas lipasa de Candida rugosa

Referencia OEPM: P9201378.-- Fecha de solicitud: 03/07/1992.-- Titular: Consejo Superior de Investigaciones Científicas (CSIC).Procedimiento para la purificación de dos isoenzimas lipasa de Candida rugosa. Procedimiento para la purificación de dos lipasas extracelulares presentes en lipasa bruta comercial de la levadura Candida rugosa. El método consta de un sólo paso de cromatografía hidrofóbica en matriz de agarosa. Condiciones de elución muy diferentes permiten la completa separación y purificación de dos enzimas con actividad lipásica (lipasas A y B) presentes en el extracto comercial de partida. Para ello se utiliza un tampón concentrado de pH 6 a 8, con el que se eluyen de la columna gran parte de los contaminantes presentes en el extracto; luego con la misma solución tampón más diluida se arrastra la lipasa B. Posteriormente, con un di o polialcohol disuelto en el tampón diluido se eluye la lipasa A, de cuya solución, mediante concentración por ultrafiltración y cromatografía de exclusión molecular con dextrano, se elimina el alcohol. Se aplica industrialmente en la purificación de enzimas.Peer reviewe

Galactooligosaccharides formation during enzymatic hydrolysis of lactose: towards a prebiotic enriched milk

The formation of galacto-oligosaccharides (GOS) in skim milk during the treatment with several commercial β-galactosidases (Bacillus circulans, Kluyveromyces lactis and Aspergillus oryzae) was analyzed in detail, at 4°C and 40°C. The maximum GOS concentration was obtained at a lactose conversion of approximately 40-50% with B. circulans and A. oryzae -galactosidases, and at 95% lactose depletion for K. lactis -galactosidase. Using an enzyme dosage of 0.1% (v/v), the maximum GOS concentration with K. lactis -galactosidase was achieved in 1 h and 5 h at 40°C and 4°C, respectively. With this enzyme, it was possible to obtain a treated milk with 7.0 g/L GOS −the human milk oligosaccharides (HMOs) concentration is between 5 and 15 g/L−, and with a low content of residual lactose (2.1 g/L, compared with 44-46 g/L in the initial milk sample). The major GOS synthesized by this enzyme were 6-galactobiose [Gal-β(1→6)-Gal], allolactose [Gal-β(1→6)-Glc] and 6´-O-β-galactosyl-lactose [Gal-β(1→6)-Gal-β(1→4)-Glc].The project BIO2010-20508-C04-01 from the Spanish Ministry of Science and Innovation supported this research. B.R.C was supported by a fellowship (FPI program)Peer reviewe

Combinatorial Saturation Mutagenesis by in vivo Overlap Extension for the Engineering of Fungal Laccases

Combinatorial saturation mutagenesis -CSM- is a valuable tool for improving enzymatic properties from hot-spot residues discovered by directed enzyme evolution or performing semi-rational studies. CSM coupled to a reliable high-throughput screening assay -CV below 10 %- has been used to enhance turnover rates in the fungal laccase variant T2 from Myceliophthora thermophila. The influence of the highly conserved pentapeptide 509-513 on the redox potential of blue-copper containing enzymes is well described. We focused combinatorial saturation mutagenesis in residues Ser510 and Leu513. Libraries were constructed in Saccharomyces cerevisiae by in vivo overlap extension -IVOE- of the PCR products. This methodology provides a simple manner to build CSM libraries avoiding extra PCR reactions, by-products formation and in vitro ligation steps. After exploring more than 1,700 clones, mutant (7E1) with 3-fold better kinetics than parent type was found. 7E1 showed one synonymous mutation (L513L, CGT/TTG) and beneficial mutation S510G (TCG/GGG) which can not be achieved by conventional error-prone PCR techniques. Mutation S510G seems to affect the C-terminal plug, which modulates the transit of water and oxygen to the trinuclear copper cluster.This material is based upon work founded by Spanish Ministry of Education and Science (project VEM2004-08559), Comunidad de Madrid/CSIC (project 20058M121) and Ramón y Cajal Research Program. MZ thanks Gobierno Vasco for fellowship.Peer reviewe

Immobilization of Pycnoporus coccineus laccase on Eupergit C: stabilization and treatment of olive oil mill wastewaters

The use of olive oil mill wastewaters (OMW) as organic fertilizer is limited by its phytotoxic effect, due to the high concentration of phenolic compounds. As an alternative to physico-chemical methods for OMW detoxification, the laccase from Pycnoporus coccineus, a white-rot fungus that is able to decrease the chemical oxygen demand (COD) and colour of the industrial effluent, is being studied. In this work, the P. coccineus laccase was immobilized on two acrylic epoxy-activated resins, Eupergit C and Eupergit C 250L. The highest activity was obtained with the macroporous Eupergit C 250L, reaching 110 U g-1 biocatalyst. A substantial stabilization effect against pH and temperature was obtained upon immobilization. The soluble enzyme maintained ≥80% of its initial activity after 24 h at pH 7.0-10.0, whereas the immobilized laccase kept the activity in the pH range 3.0-10.0. The free enzyme was quickly inactivated at temperatures above 50 oC, whereas the immobilized enzyme was very stable up to 70 oC. Gel filtration profiles of the OMW treated with the immobilized enzyme (for 8 h at room temperature) showed both degradation and polymerization of the phenolic compounds.We thank Dr. J. Martinez and T. de la Rubia (University of Granada, Spain) for giving us lyophilized OMW. We thank Thomas Boller (Degussa, Darmstadt, Germany) for supplying Eupergit C samples and for technical help. The authors thank the financial support received from the Spanish Projects BIO2003-00621, VEM2004-08559 and CAM S-0505/AMB0100.Peer reviewe

Procedimiento para la acilación específica enzimática de un hidroxilo secundario de la sacarosa

Referencia OEPM: P9702602.-- Fecha de solicitud: 15/12/1997.-- Titular: Consejo Superior de Investigaciones Científicas (CSIC).Procedimiento para la acilación específica enzimática de un hidroxilo secundario de la sacarosa. Es un procedimiento enzimático para la acilación regioespecífica de un hidroxilo secundario de la sacarosa. En esencia, consiste en hacer reaccionar la sacarosa con el éster vinílico de un ácido graso en un disolvente orgánico, en presencia de una lipasa de un hongo filamentoso, preferiblemente inmovilizada. Permite obtener tensioactivos con una estructura química distinta a los obtenidos industrialmente (químicamente los hidroxilos más reactivos son los primarios). Además, se trata de un proceso notablemente más sencillo para la acilación específica de un hidroxilo secundario de la sacarosa. Aplicaciones en farmacia, cosmética, alimentación y detergentes.Peer reviewe

Continuous Packed Bed Reactor with Immobilized β-Galactosidase for Production of Galactooligosaccharides (GOS)

The β-galactosidase from Bacillus circulans was covalently attached to aldehyde-activated (glyoxal) agarose beads and assayed for the continuous production of galactooligosaccharides (GOS) in a packed-bed reactor (PBR). The immobilization was fast (1 h) and the activity of the resulting biocatalyst was 97.4 U/g measured with o-nitrophenyl-β-d-galactopyranoside (ONPG). The biocatalyst showed excellent operational stability in 14 successive 20 min reaction cycles at 45 °C in a batch reactor. A continuous process for GOS synthesis was operated for 213 h at 0.2 mL/min and 45 °C using 100 g/L of lactose as a feed solution. The efficiency of the PBR slightly decreased with time; however, the maximum GOS concentration (24.2 g/L) was obtained after 48 h of operation, which corresponded to 48.6% lactose conversion and thus to maximum transgalactosylation activity. HPAEC-PAD analysis showed that the two major GOS were the trisaccharide Gal-β(1→4)-Gal-β(1→4)-Glc and the tetrasaccharide Gal-β(1→4)-Gal-β(1→4)-Gal-β(1→4)-Glc. The PBR was also assessed in the production of GOS from milk as a feed solution. The stability of the bioreactor was satisfactory during the first 8 h of operation; after that, a decrease in the flow rate was observed, probably due to partial clogging of the column. This work represents a step forward in the continuous production of GOS employing fixed-bed reactors with immobilized β-galactosidases.This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (BIO2013-48779-C4-1-R and BIO2016-76601-C3-1-R). We thank the support of COST-Action CM1303 on Systems Biocatalysis. P.S.-M. thanks the Spanish Ministry of Education for FPU Grant (FPU13/01185). We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)

Synthesis of methyl Alpha-D-Glucoolicosaccharides by Entrapped Dextransucrase from Leuconostoc mesenteroides B-1299

The synthesis of methyl alpha-D-glucooligosaccharides, using sucrose as glucosyl donor and methyl alpha-D-glucopyranoside as acceptor, was studied with dextransucrase from Leuconostoc mesenteroides NRRL B-1299. The enzyme was immobilized by entrapment in alginate. By NMR and mass spectrometry we identified three homologous series (S1-S3) of methyl alpha-D-glucooligosaccharides. Series S2 and S3 were characterized by the presence of alpha(1-2) linkages, in combination with alpha(1-6) bonds. Two parameters, sucrose to acceptor concentration ratio (S/A) and the total sugar concentration (TSC) determined the yield of methyl alpha-D-glucooligosaccharides. The maximum concentration achieved of the first acceptor product, methyl alpha-D-isomaltoside, was 65 mM using a S/A 1:4 and a TSC of 336 g l-1. When increasing temperature, a shift of selectivity towards compounds containing alpha(1-2) bonds was observed. The formation of leucrose as a side process was very significant (reaching values of 32 g l-1) at high sucrose concentrations.We are very grateful to Profs. P. Monsan and M. Remaud-Simeon (INSA, Toulouse) for providing us with dextransucrase. We thank Prof. A. Cortés (ICP, CSIC) for helpful comments. We thank Gobierno Vasco and Instituto Danone for research fellowships. This work was supported by the Spanish Education and Science Ministry (Project BIO2004-03773-C04-01).Peer reviewe

Biocatalizador inmovilizado basado en alginato para la biotransformación de carbohidratos

Biocatalizador inmovilizado basado en alginato para la biotransformación de carbohidratos. Procedimiento de obtención de un biocatalizador, que comprende: inmovilizar una enzima fúngica por inclusión en un gel de alginato cálcico; y el secado posterior el biocatalizador inmovilizado obtenido en el paso (a). La invención también se refiere al biocatalizador obtenido por el procedimiento de la invención y que comprende enzimas fúngicas, preferiblemente fructosiltransferasa o - fructofuranosidasa, inmovilizadas en alginato. Además la invención se refiere al uso de dicho biocatalizador para la biotransformación en las que el sustrato es una disolución concentrada de un carbohidrato y se puede llevar a cabo en un reactor continuo.Peer reviewedConsejo Superior de Investigaciones Científicas (España), Universidad Autónoma de MadridA1 Solicitud de patente con informe sobre el estado de la técnic

Selective Synthesis of Galactooligosaccharides Containing β(1→3) Linkages with β-Galactosidase from Bifidobacterium bifidum (Saphera)

[EN] The transglycosylation activity of a novel commercial β-galactosidase from Bifidobacterium bifidum (Saphera) was evaluated. The optimal conditions for the operation of this enzyme, measured with o-nitrophenyl-β-d-galactopyranoside, were 40 °C and pH around 6.0. Although at low lactose concentrations the property of this enzyme was basically hydrolytic, an increase of lactose concentration to 400 g/L resulted in a significant formation (107.2 g/L, 27% yield) of prebiotic galactooligosaccharides (GOS). The maximum amount of GOS was obtained at a lactose conversion of approximately 90%, which contrasts with other β-galactosidases, for which the highest GOS yield is achieved at 40-50% lactose conversion. Using high-performance anion-exchange chromatography with pulsed amperometric detection, semipreparative high-performance liquid chromatography-hydrophilic interaction liquid chromatography, mass spectrometry, and 1D and 2D NMR, we determined the structure of most of the GOS synthesized by this enzyme. The main identified products were Gal-β(1→3)-Gal-β(1→4)-Glc (3′-O-β-galactosyl-lactose), Gal-β(1→6)-Glc (allolactose), Gal-β(1→3)-Glc (3-galactosyl-glucose), Gal-β(1→3)-Gal (3-galactobiose), and the tetrasaccharide Gal-β(1→3)-Gal-β(1→3)-Gal-β(1→4)-Glc. In general, B. bifidum β-galactosidase showed a tendency to form β(1→3) linkages followed by β(1→6) and more scarcely β(1→4).This work was supported by a grant from the Spanish Ministry of Economy and Competitiveness (BIO2016-76601-C3-1). The group at CIC bioGUNE thanks Agencia Estatal de Investigación (Spain) for the Severo Ochoa Excellence Accreditation (SEV-2016-0644)

Regioselective synthesis of neo-erlose by the beta-fructofuranosidase from Xanthophyllomyces dendrorhous

The beta-fructofuranosidase from the yeast Xanthophyllomyces dendrorhous (Xd-INV) catalyzes the synthesis of neo-fructooligosaccharides (neo-FOS of the 6G-series), which contain a beta(2-6) linkage between a fructose and the glucosyl moiety of sucrose. In this work we demonstrate that the enzyme is also able to fructosylate other carbohydrates that contain glucose, in particular disaccharides (maltose, isomaltulose, isomaltose, trehalose) and higher oligosaccharides (maltotriose, raffinose, maltotetraose), but not monosaccharides (glucose, fructose, galactose). With maltose as acceptor, the reaction in the presence of Xd-INV proceeded with high regioselectivity; the product was purified and chemically characterized, and turned out to be 6’-O--fructosylmaltose (neo-erlose). Using 100 g/L sucrose as fructosyl donor and 300 g/L maltose as acceptor, the maximum concentration of neo-erlose was 38.3 g/L. Thus, novel hetero-fructooligosaccharides with potential applications in the functional food and pharmaceutical industries can be obtained with Xd-INV.Projects BIO2010-20508-C04-01 and BIO2010-20508-C04-04 from Spanish Ministry of Science and Innovation supported this research. We thank Fundación Ramon Areces for an institutional grant to the Centro de Biología Molecular Severo Ochoa.Peer reviewe