Search

BACKGROUND: It has still to be ascertained whether severe acute respiratory syndrome coronavirus 2 infection in pregnancy is associated with worse maternal and fetal outcomes compared to low risk gestations. OBJECTIVE: This study aimed to evaluate maternal and perinatal outcomes in high- and low-risk pregnancies complicated by severe acute respiratory syndrome coronavirus 2 infection. STUDY DESIGN: This was a multinational retrospective cohort study involving women with laboratory-confirmed severe acute respiratory syndrome coronavirus 2 infection from 76 centers from 25 countries in Europe, the United States, South America, Asia, and Australia from April 4, 2020, to October 28, 2020. The primary outcome was a composite measure of maternal mortality and morbidity, including admission to the intensive care unit, use of mechanical ventilation, or death. The secondary outcome was a composite measure of adverse perinatal outcome, including miscarriage, fetal loss, neonatal and perinatal death, and admission to the neonatal intensive care unit. All outcomes were assessed in high- and low-risk pregnancies. Pregnancies were considered high risk in case of either preexisting chronic medical conditions in pregnancy or obstetrical disorders occurring in pregnancy. The Fisher exact test and logistic regression analysis were used to analyze the data. RESULTS: A total of 887 singleton pregnancies who tested positive for severe acute respiratory syndrome coronavirus 2 infection using reverse transcription-polymerase chain reaction of nasal and pharyngeal swab specimens were included in the study. The risk of composite adverse maternal outcomes was higher in high-risk pregnancies than in low-risk pregnancies (odds ratio, 1.52; 95% confidence interval, 1.03-2.24; P=.035). In addition, women carrying high-risk pregnancies were at higher risk of hospital admission (odds ratio, 1.48; 95% confidence interval, 1.07-2.04; P=.002), presence of severe respiratory symptoms (odds ratio, 2.13; 95% confidence interval, 0.41-3.21; P=.001), admission to the intensive care unit (odds ratio, 2.63; 95% confidence interval, 1.42-4.88), and invasive mechanical ventilation (odds ratio, 2.65; 95% confidence interval, 1.19-5.94; P=.002). When exploring perinatal outcomes, high-risk pregnancies were at high risk of adverse perinatal outcomes (odds ratio, 1.78; 95% confidence interval, 0.15-2.72; P=.009). However, such association was mainly because of the higher incidence of miscarriage in high-risk pregnancies compared with that in low-risk pregnancies (5.3% vs 1.6%, P=.008); furthermore, there was no difference in other explored outcomes between the 2 study groups. At logistic regression analysis, maternal age (odds ratio, 1.12; 95% confidence interval, 1.02-1.22; P=.023) and high-risk pregnancy (odds ratio, 4.21; 95% confidence interval, 3.90-5.11; P

In this paper, a novel procedure for the immobilization and stabilization of enzymes is proposed: the multipoint covalent attachment of bi-molecular enzyme aggregates. This immobilization protocol allows the “capture” and fixation of the enzyme aggregate on the support surface. In addition to stabilization by multipoint attachment, enzyme aggregation promotes very interesting stabilizing effects. In the presence of low concentrations of polyethylene glycol (30 %) the dimeric amine oxidase from Pisum sativum forms soluble bi-molecular aggregates. Enzyme aggregates were analyzed by Dynamic Light Scattering and by full chemical loading of a mesoporous support (10 % agarose gels activated with glyoxyl groups). The soluble aggregate was immobilized by multipoint attachment on glyoxyl- agarose at pH 8.5 though the four amino termini of the two dimeric molecules (Lys residues are not reactive at this pH). The immobilized aggregated structure cannot undergo any movement (translational or rotational) after multipoint attachment and the aggregate is “fixed” on the support surface even after the removal of PEG. The immobilized aggregate was further incubated at pH 10 in order to allow the Lys residues to react with the glyoxyl groups on the support. Enzyme aggregation has an important effect on enzyme stabilization: the aggregated derivative was 40 fold more stable than a similar derivative of the isolated enzyme and 200 fold more than native enzymes in experiments of thermal inactivation., This work was supported by the Ramon Areces Foundation by the project in the field of Sciences of Life and Matter titled “The detection and elimination of biogenic amines in foods: the design of new biosensors and new amino oxidase catalysts”. The authors thank the Ramon Areces Foundation for the pre-doctoral grant given to Paz Garcia-Garcia.

Stabilization of dimeric enzymes requires the stabilization of the quaternary structure as well as the 3D one. Both subunits may be easily immobilized on a highly activated support. Additional stabilization of the 3D structure may be achieved via multipoint covalent attachment (MCA) on highly activated supports. In the case of monomeric enzymes or thermophilic dimeric ones, the optimal stabilization is obtained via the most intense MCA and it is associated to a small loss of catalytic activity. However, in the case of mesophilic enzymes, a very intense MCA of both subunits may promote negative effects, e.g., associated to distortions of the assembly between subunits and a subsequent very important loss of catalytic activity. A dimeric mesophilic amine oxidase from P.sativum was stabilized by MCA on glyoxyl-agarose. Both subunits were covalently immobilized on the support through the region with the highest density in Lys residues. In addition to that, an interesting activity/stabilization binomial was obtained after only 3 h of enzyme-support multiinteraction (50 % of activity/350 fold stabilization). However, after 24 h of enzyme-support multi-interaction this binomial activity-stabilization decreased down to 30/150. A moderate multiinteraction seems to be the optimal strategy for immobilization-stabilization of mesophilic dimeric enzymes and it promotes moderate losses of activity and interesting stabilizations against the combined effect of heat, acid pH and ethanol. The control of the intensity of enzyme-support multi-interactions becomes now strictly necessary., This work was supported by Ramon Areces Foundation for the project in the field of Sciences of Life and Matter titled “The detection and elimination of biogenic amines in foods: the design of new biosensors and new amino oxidase catalysts”. The authors thank the Ramon Areces Foundation for the pre-doctoral grant awarded to Paz Garcia Garcia.

Immobilized enzymes have a very large surface region which is not in contact with the support surface and, thus, have potential as a target for novel stabilization strategies. In this paper, coating the surfaces of such enzymes with a highly hydrophilic and compact cross-linked poly-aminated polymer as a strategy to increase the thermal stability of the immobilized enzymes is proposed. In particular, Rhizomucor miehei lipase (RML) was immobilized by interfacial adsorption onto octyl-agarose and further coated with poly-allylamine (PAA), a polymer that is very rich in primary amino groups. Cross-linking of the PAA layer to coat the immobilized enzyme was carried out, in situ, by reaction with freshly oxidized dextran (aldehyde–dextran). The PAA layer only exerted moderate stabilizing effects (around 4-fold), but further cross-linking with aldehyde–dextran highly increased the stabilizing effects; the new derivative was 440-fold more stable than uncoated derivative at 55 °C and pH 7 and exhibited 6-fold more catalytic activity compared to the soluble enzyme used for immobilization. We hypothesize that the hydrophilicity of PAA reduces the exposure of internal hydrophobic pockets to the enzyme surface at high temperatures. Besides, the compactness of the polymer may reduce distortion of the enzyme surface during inactivation., This work was supported by the Spanish Ministry of Science and Innovation (grant numbers BIO2012-36861 and CTQ2015-70348-C2-1-R). We gratefully recognize the Spanish Ministry of Economy and Innovation for the “Juan de la Cierva” contract for Dr. Rocha-Martin (grant number IJCI-2014-19260)

Enzymes’ stabilization degree and protein structure modification have been associated with different immobilization techniques. Geotrichum candidum lipase (GCL) was immobilized using different supports and immobilization protocols: covalent bonding on Cyanogen bromide activated sepharose (CNBr) and amino-glyoxyl (AMG), and ionic bonding on anionic support diethylaminoethyl (DEAE) and cationic support carboxymethyl agarose. The lipase stability in medium with different pH values and thermal stability of different derivatives of GCL were evaluated for enantioselective hydrolysis of (R,S)-mandelic acid ethyl ester under different pH conditions at the same temperature. The properties of GCL can be modulated by direct immobilization, i.e., immobilization using different supports and the reaction pH conditions changes the catalytic properties leading to different selectivity, consequently improving the biocatalyst properties. Stereochemical preference to the R isomer was observed on carboxymethyl and AMG derivatives at pH 5.0 and 7.0, respectively, with enantiomeric excess higher than 99% and specificity above 200. On the other hand, the reaction has shown stereochemical preference to the S isomer when using CNBr and AMG derivatives at pH 5.0 and 9.0, respectively, achieving an enantiomeric excess higher than 99% and specificity above 200., The authors wish to thank CAPES (Brazil) for financial support through the doctorate scholarship provided by the post-graduate program and “Programa de Doutorado Sanduíche no Exterior” (PDSE) abroad by the project BEX 14174/13-8; and the “Instituto de Investigación en Ciencia de la Alimentación” (CIAL) for allowing them to accomplish an internship during the development of the experiments of this project.

The present study focuses on the development and optimization of a packed-bed reactor (PBR) for continuous production of xylooligosaccharides (XOS) from xylan. For this purpose, three different methacrylic polymer-based supports (Relizyme R403/S, Purolite P8204F and Purolite P8215F) activated with glyoxyl groups were morphologically characterized and screened for the multipoint covalent immobilization of a xylanase. Based on its physical and mechanical properties, maximum protein loading and thermal stability, Relizyme R403/S was selected to set up a PRB for continuous production of XOS from corncob xylan. The specific productivity for XOS at 10 mL/min flow rate was 3277 gXOS genzyme−1 h−1 with a PBR. This PBR conserved >90% of its initial activity after 120 h of continuous operation., This work was supported by the Spanish Ministry of Economy, Industry and Competitiveness (projects BIO2012-36861 and CTQ2015- 70348). The authors would like to thank Spanish Ministry of Economy, Industry and Competitiveness for financial support awarded the first author (BES-2013-065396). Javier Rocha-Martin is grateful for the “Juan de la Cierva” fellowship (IJCI-2014-19260) funded by the Spanish Ministry of Economy, Industry and Competitiveness.

Diamine oxidase (DAO) from Pisum sativum is an enzyme that catalyzes the degradation of biogenic amines (BA) present in wine, producing harmless aldehydes and hydrogen peroxide (HO). HO promotes a rapid inactivation of the immobilized enzyme. At first glance, co-immobilization of DAO and catalase (CAT) could improve the elimination of the released hydrogen peroxide. Two different co-immobilized derivatives were prepared: (a) both enzymes co-localized and homogeneously distributed across the whole structure of a porous support, and (b) both enzymes we de-localized inside the porous support: DAO immobilized on the outer part of the porous support and catalase immobilized in the inner part. Co-localized derivatives were seven-fold more effective than de-localized ones for the elimination of hydrogen peroxide inside the porous support. In addition to that, the degradation of putrescine by DAO was three-fold more rapid when using both co-localized enzymes. The optimal co-localized derivative (containing 1.25 mg of DAO plus 25 mg of CAT per g of support) promoted the instantaneous elimination of 91% HO released inside the porous support during putrescine oxidation. This optimal derivative preserves 92% of activity after three reaction cycles and DAO immobilized without catalase only preserves 41% of activity. Co-localization seems to be the key strategy to immobilize two sequential enzymes. When enzymes are immobilized in close proximity to each other in a co-localized pattern, the generation of byproducts as HO is strongly reduced., This work was sponsored by the Spanish Ministry of Economy, Industry and Competitiveness (projects BIO2012-36861 and CTQ2015-70348) and by Foundation Ramon Areces (Madrid. Spain). Javier Rocha-Martin is grateful for the Juan de la Cierva fellowship (IJCI-2014-19260) funded by the Spanish Ministry of Economy, Industry and Competitiveness.

Resumen: Introducción: El manejo del dolor y la sedación es una prioridad de los cuidados intensivos neonatales. Se diseñó un estudio con el objetivo de determinar la práctica clínica actual en relación con la sedación y la analgesia en unidades de cuidados intensivos neonatales en España e identificar factores asociados al uso de fármacos sedantes o analgésicos. Método: Estudio multicéntrico, observacional, longitudinal y prospectivo. Resultados: Participaron 30 unidades neonatales y se reclutó a 468 neonatos. De estos, 198 (42,3%) recibieron medicación sedante o analgésica. En total, se usaron durante el período de estudio 19 fármacos distintos, de los cuales el más utilizado fue el fentanilo. Solo fentanilo, midazolam, morfina y paracetamol se usaron al menos en un 20% de los neonatos que recibieron sedación y/o analgesia. Se usaron 14 pautas distintas de fármacos en perfusión, siendo las más frecuentes la infusión de fentanilo y la combinación de fentanilo y midazolam.Las variables asociadas a recibir sedación y/o analgesia fueron el haber precisado ventilación invasiva (p = < 0,001; OR = 23,79), un score de CRIB > 3 (p = 0,023; OR = 2,26), la existencia en la unidad de guías de evaluación del dolor (p 3 (P=.023; OR = 2.26), the existence of pain evaluation guides in the unit (P

An assemblage is a group of species co-occurring at one spatial location at one point in time. They are a central object of study for ecologists. There are a few important commonly measured properties of organisms (eg, body mass, energy flux, functional traits such as leaf mass per unit area) and properties of species (eg, abundance and range size). Exploring the means, full frequency distributions, and covariances (interrelationships) of these properties are concise ways to describe assemblages. One can also examine and try to predict how assemblages change across space and time. This approach of describing assemblages may provide a number of tools useful for understanding how humans are impacting assemblages.

This chapter describes the physicochemical coating of the surface of immobilized enzymes with a dense layer of polyethylene glycol (PEG) to improve enzyme stability. One hypothesis is that a dense, viscous, polar PEG layer around the enzyme would enhance enzyme thermal stability, while still providing access to the active site. PEG groups were attached by using aldehyde–dextran polymers, the dextran polymers are in turn attached to the enzyme surface that have been enriched with excess primary amino groups. The enzymes themselves were initially attached onto porous solids such that they may be separated easily from the reaction mixtures for easy downstream processing and that they may be recycled to reduce the cost of the biocatalyst. The hierarchical modification of enzyme surface with three different sublayers, under chemical design, provided a rational control at several structural levels. Few methods for increasing the number of amino groups on the surface of the enzyme are described: (a) chemical amination of carboxyl residues and (b) coating of the enzyme surface with cationic polymers containing a high percentage of primary amines. Reliable protocols for the PEGylation of four different enzymes are described here. For example, lipases from Thermomyces lanuginosa , Candida antarctica B, and Rhizomucor miehei attached to octyl sepharose and chemically modified via PEGylation are stabilized from 7- to 50-fold when compared to the stability of the corresponding unmodified enzyme. A derivative of endoxylanase from Trichoderma reesei , immobilized by multipoint covalent attachment on glyoxyl agarose, is stabilized by 50-fold. Very likely, the PEG layer generated a dense, high viscosity medium surrounding the enzyme surface and this increase in viscosity around the enzyme microenvironment resists distortion of enzyme structure by heat or other denaturing agents.

We report the synthesis of polyurethane (PU) with polyethylene glycol (PEG) by miniemulsion polymerization as technique to produce a support for enzyme immobilization and stabilization. In order to study the influence of molar weight and concentration of PEG in the enzyme-support interaction, different PEG (400, 4000 and 6000 Da) was used during the synthesis of PU-PEG support to immobilize Thermomyces lanuginosus lipase (TLL). The enzymatic derivatives were effectively used in the production of polyunsaturated fatty acids (PUFAs), particularly derivative TLL-PU-PEG6000. This derivative showed the best results in terms of Km (0.183 mM) and Vmax (45.79 mmol/min/mL), calculated by Lineweaver-Burk plot (double reciprocal plot) and catalytic efficient (250.22), 100% of yield immobilization and recovered activity, reaching the highest values to the ethyl esters production (260 mM ethyl esters. U-1) making this derivative a potential low cost catalyst for ethanolysis reactions in solvent free system, The authors thank the financial support from CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for the Programa de Doutorado Sanduíche no Exterior, Project BEX 2569/14-0; the direction of the Instituto de Investigación en Ciencias de la Alimentación, CIAL-CSIC for helping in the development of this research work, and Laboratório Central de Microscopia Eletrônica (LCME) at Federal University of Santa Catarina for the microscopy analyzes.

Soluble lipase from Thermomyces lanuginosa (TLL) was dramatically hyper-activated by a cationic surfactant, cetyltrimethylammonium bromide (CTAB). The greatest hyperactivation (above 340-fold) was observed with 0.005% CTAB. In addition to that, very high hyperactivation was also observed with much higher concentrations of surfactant (100-fold in the presence of 0.3% CTAB). Without surfactant or with very low surfactant concentrations, TLL was not adsorbed to the cationic exchangers. However, in the presence of high concentrations of surfactant (0.3%), the lipase was completely and strongly adsorbed on sulfopropyl-sepharose. The adsorbed enzyme remained hyperactivated (80-fold more active than the soluble enzyme) after elimination of the excess of surfactant. Complete desorption of the hyperactivated TLL from the cationic exchanger is only achieved at 2 M NaCl. The same level of hyperactivation was observed for the hydrolysis of a large substrate: fish oil. The release of EPA (eicosapentaenoic acid) was 80-fold more rapid with hyperactivated TLL derivatives than with TLL very mildly immobilized on CNBr activated agarose. Hyperactivated derivatives were very stable at 25 °C and 37 °C. Full activity was preserved after 1 week., This work was sponsored by the Spanish Ministry of Economy and Innovation (project AGL-2009-07526 and BIO-2012-36861). We gratefully recognize the Spanish Ministry of Science and Innovation for the Ramón y Cajal contract for Dr. Fernandez-Lorente and The Ministry of Science from Tunisia for supporting Nesrine Gattas.

Immobilized enzymes are preferred over their soluble counterparts due to their robustness in harsh industrial processes; the most stable enzyme derivatives are often produced through multipoint covalent attachment (MCA). However, most enzymes are unable to establish optimal MCA to electrophile-type supports given the heterogeneous distribution and/or low content of primary amino groups on their surfaces; this restricts both the diversity of areas prone to react and the number of attachments to the support. To overcome this we propose combining site-directed immobilization and protein engineering to increase the number of bonds between a specific enzyme surface and a tailor-made support. We applied this novel strategy to engineered mutants of the lipase 2 from Geobacillus thermocatenulatus with one Cys exposed residue, that after genetic amination and/or chemical amination, were immobilized on glyoxyl-disulfide support using a site-directed MCA protocol. Two highly stabilized derivatives of chemically aminated lipase variants, in which site-directed MCA implied the surrounding surface of residues Cys344 or Cys40, were produced: the first one was 2.4-fold more productive than the reference derivative (648 g of hydrolyzed ester); the second derivative was 40% more selective (EPA/DHA molar ratio) and as active (1 μmol g catalyst-1 min-1) as the reference in the production of PUFAs. © 2014 Elsevier Ltd., CICYT (project BIO-2005-6018576), Madrid Region council CAM (grants S0505/PPQ/0344) and CSIC-Spain.

Immobilization of enzymes on glutaraldehyde-activated supports has been largely used on supports previously activated with amine groups. Therefore, the supports are positively charged hence usually the immobilization is promoted through a two step mechanism: in a first step the enzyme is adsorbed on the support via an anionic exchange mechanism and then, the covalent immobilization occurs. In this paper a new glutaraldehyde activated support without a net charge is presented and characterized in immobilizations of trypsin, penicillin acylase G, lipase and E. coli BL21 cell extract. Immobilization mechanism was studied and this was produced without an adsorption step. This support promoted initially a reversible immobilization, converting into irreversible after incubation of the enzyme-support for several days or after a reduction step. In addition the stability of glutaraldehyde groups was studied retaining around 50 and 25% of its immobilization capacity for 24 h at pH 7 and 10 respectively. This fact allows the incubation of the enzyme with the support even at alkaline pH promoting an extra stabilization factor for trypsin on this support., D. Bezbradica had a postdoctoral fellowship of the Serbian Ministry of Science and Technological Development in research area related with project III 46010.

The soluble lipase from Pseudomonas fluorescens (PFL) forms bimolecular aggregates in which the hydrophobic active centers of the enzyme monomers are in close contact. This bimolecular aggregate could be immobilized by multipoint covalent linkages on glyoxyl supports at pH 8.5. The monomer of PFL obtained by incubation of the soluble enzyme in the presence of detergent (0.5% TRITON X-100) could not be immobilized under these conditions. The bimolecular aggregate has two amino terminal residues in the same plane. A further incubation of the immobilized derivative under more alkaline conditions (e.g., pH 10.5) allows a further multipoint attachment of lysine (Lys) residues located in the same plane as the amino terminal residues. Monomeric PFL was immobilized at pH 10.5 in the presence of 0.5% TRITON X-100. The properties of both PFL derivatives were compared. In general, the bimolecular derivatives were more active, more selective and more stable both in water and in organic solvents than the monomolecular ones. The bimolecular derivative showed twice the activity and a much higher selectivity (100 versus 20) for the hydrolysis of R,S-2-hydroxy-4-phenylbutyric acid ethyl ester (HPBEt) in aqueous media at pH 5.0 compared to the monomeric derivative. In experiments measuring thermal inactivation at 75 °C, the bimolecular derivative was 5-fold more stable than the monomeric derivative (and 50-fold more stable than a one-point covalently immobilized PFL derivative), and it had a half-life greater than 4 h. In organic solvents (cyclohexane and tert-amyl alcohol), the bimolecular derivative was much more stable and more active than the monomeric derivative in catalyzing the transesterification of olive oil with benzyl alcohol. © 2012 Elsevier Ltd. All rights reserved., This work was sponsored by the Spanish Ministry of Science and Innovation(project AGL-2009-07526), Consolider INGENIO 2010 CSD2007-00063 FUN-C-FOOD (CICYT) and ALIBIRD S2009/AGR-1469. We gratefully recognize the Spanish Ministry of Science and Innovation for the “Ramón y Cajal” contract for Dr. Fernandez-Lorente. We also thank the Brazilian agencies CAPES and CNPq for the scholarship of Lionete N. Lima.

The hydrolysis of phenolic compounds using an immobilized and highly active and stable derivative of laccase from Trametes versicolor is presented. The enzyme was immobilized on aldehyde supports. For this, the enzyme was enriched in amino groups by chemical modification of its carboxyl groups. The aminated enzyme was immobilized with a high recovered activity (over 60%). Aldehyde derivatives were more stable than soluble or aminated-soluble enzyme and the reference derivatives after incubation in different inactivating conditions (high temperatures, different pH values or presence of organic cosolvents). The most stable derivative was obtained immobilizing the chemically aminated enzyme at pH 10 on aldehyde supports with a stabilization factor approximately 280 fold after incubation at pH 7 and 55 °C. In addition, it was possible to prepare immobilized derivatives with a maximal enzyme loading of 60 mg g−1 of support. This derivative could be reused for 10 reaction cycles with negligible lost of activity., This work has been sponsored by the Spanish Ministry of Science intramural projects (2008801058).

β-galactosidase from Kluyveromyces lactis was covalently immobilised on a Glyoxyl Sepharose (GS) support by multi-point attachment. The enzyme immobilisation process was very efficient; the supports immobilised almost all the protein responsible for the catalytic activity in a short period of time, retaining approximately 82% of the activity in the case of the optimal immobilised preparations. Stability of the GS derivatives varied as a function of enzyme-support incubation time. The optimal immobilised preparation was produced after 2 h of incubation with the support at alkaline pH. This derivative, obtained by multi-point covalent attachment, was 100-fold more stable at pH 7 and 50 °C than the cyanogen bromide Sepharose derivative obtained by a one-point covalent immobilisation method. Stabilisation was also observed under a wide range of experimental conditions. This method allowed the immobilisation of 9000 IU enzyme g−1 of support, resulting in highly active and stable derivatives suitable for industrial processes., We thank COLCIENCIAS, Municipio de Rionegro and Universidad de Antioquia for financial support through the program “Becas doctorales 2007 en el convenio de cooperación # 030-2006”. We also thank the Spanish Ministry of Science for financial support through intramural projects (2008801058) and project No. (AGL-2009- 07625).

An endoxylanase from Streptomyces halstedii was stabilized by multipoint covalent immobilization on glyoxyl-agarose supports. The immobilized enzyme derivatives preserved 65% of the catalytic activity corresponding to the one of soluble enzyme that had been immobilized. These immobilized derivatives were 200 times more stable 200 times more stable than the one-point covalently immobilized derivative in experiments involving thermal inactivation at 60 °C. The activity and stability of the immobilized enzyme was higher at pH 5.0 than at pH 7.0. The optimal temperature for xylan hydrolysis was 10 °C higher for the stabilized derivative than for the non-stabilized derivative. On the other hand, the highest loading capacity of activated 10% agarose gels was 75 mg of enzyme per mL of support. To prevent diffusional limitations, low loaded derivatives (containing 0.2 mg of enzyme per mL of support) were used to study the hydrolysis of xylan at high concentration (close to 1% (w/v)). 80% of the reducing sugars were released after 3 h at 55 °C. After 80% of enzymatic hydrolysis, a mixture of small xylo-oligosaccharides was obtained (from xylobiose to xylohexose) with a high percentage of xylobiose and minimal amounts of xylose. The immobilized-stabilized derivatives were used for 10 reaction cycles with no loss of catalytic activity. © 2013 Elsevier Ltd. All rights reserved., This work has been supported by the Ministerio de Ciencia e Innovación, Spain (Grant No. EUI2008-03631 from ERA-IB to R. I. Santamaría and Grant AGL-2009-07625 to Jose M. Guisan). Gloria Fernández-Lorente is recipient of a Ramon y Cajal postdoctoral Contract. Caio Aragon thanks Brazilian agencies FAPESP (2008/09332-8) and CAPES (3756/10-6) for financial support.

The production of xylooligosaccharides (XOS) using a packed-bed enzymatic reactor was studied at lab-scale. For this, a xylanase from Aspergillus versicolor was immobilized on different supports. The optimal derivative was xylanase immobilized on glyoxyl-agarose supports. This derivative preserved 85% of its catalytic activity; it was around 700-fold more stable than the soluble enzyme after incubation at 60 C and was able to be reused for at least 10 1 h-cycles retaining full catalytic activity. About 18% of oligosaccharides with prebiotic interest (X2-X6) were produced by the glyoxyl derivative in batch hydrolysis. The production of xylobiose was 2.5-fold higher using the immobilized preparation than with soluble enzyme and small concentrations of xylose (, C.C. Aragon thanks Brazilian agencies FAPESP (2008/09332-8) and CNPq (245341/2012-6) for financial support.

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., This work was supported by grants from Fundaçâo de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho de Desenvolvimento Científico e Tecnológico (CNPq) and Banco SANTANDER S.A. This work is part of Doctor Thesis of André Ricardo de Lima Damásio (A.R.L. Damásio is recipient FAPESP Fellowship).RAP received funding from the Department of Energy, awards 06103-OKL and ZDJ-7-77608-01.

The influence of the intensity of enzyme–support attachment and enzyme load on the reactivation process has been studied considering three immobilized penicillin G acylase (PGA) biocatalysts: one immobilized by covalent attachment to cyanogen bromide Sepharose (CNBrSep-PGA) and two immobilized by multipoint covalent attachment to glyoxyl agarose with different intensity of attachment (Gx 1 -PGA and Gx 2 -PGA). The biocatalysts were inactivated in 70% (v/v) dioxane at 5 °C down to 25% residual activity. Biocatalysts were then recovered by filtration to remove the inactivation medium and re-incubated in aqueous medium to promote reactivation. The highest level of reactivation (73% recovery of activity with respect to the initial activity before inactivation) was obtained with Gx 1 -PGA, corresponding to the biocatalyst with stronger multipoint interactions. For Gx 2 -PGA and CNBrSep-PGA, only 33% and 31% of activity was recovered respectively. When the distortion caused by the organic solvent produces a inactive enzyme structure that cannot be significantly recovered by re-incubation in aqueous medium, complete unfolding of the enzyme molecule by chaotropic agents prior to its refolding by incubation in aqueous medium can be used to recover enzyme activity. When this strategy was used, 70% activity was recovered with Gx 1 -PGA and Gx 2 -PGA, but weakly linked CNBrSep-PGA was completely inactivated after unfolding and no activity was recovered by re-incubation in aqueous medium. With respect to enzyme load, full recovery of enzyme activity was obtained with Gx 1 -PGA by reactivation in aqueous medium up to 74 IU/g support . However, at higher enzyme loads recovery was significantly impaired because of intense protein–protein interaction during biocatalyst inactivation and reactivation.

Partial acylation of only one primary hydroxyl group of glycerol generates a chiral center at position 2. Rhizomucor miehei lipase (RML) catalyzes the kinetically controlled transesterification of different aromatic carboxylic acids methyl esters with glycerol. High synthetic yields of glyceryl esters (around 70-80%) were obtained even in the presence of significant concentrations of water (from 5% to 20%). After a long incubation of the reaction mixture in the presence of the biocatalyst only pure free acid was obtained. Other lipases (from Geobacillus thermocatenulatus and from Thermomyces lanuginose) also catalyzed similar kinetically controlled transesterifications although less efficiently. RML immobilized on Sepharose-Q showed a high activity and specificity, compared to the immobilization by other techniques, only producing monoglyceryl esters with all substrates. In particular, monoglyceryl-phenylmalonate product was synthesized in 82% overall yield and >99% diastereomeric excess at pH 7.0 and 37 °C and 90% glycerol. © 2010 Elsevier Ltd.

Commercial glucoamylase immobilizes very slowly on highly activated glyoxyl-agarose supports. The resulting derivatives were only 6-fold more stable than soluble enzyme. The unmodified glucoamylase, highly glycosylated, seems to have a low number of Lys able to react with glyoxyl groups on the support. Thus, the enzyme surface was highly enriched in amino groups by chemical modification of carboxyl groups (activated with carbodiimide) with ethylenediamine. The aminated enzyme preserves a good percentage of activity (80%) and it exhibits the same stability than unmodified enzyme. The aminated enzyme was immobilized very rapidly on highly activated glyoxyl-agarose support. The new resulting derivatives preserved 50% of activity and were more than 500-fold more stable than soluble enzyme in experiments of thermal inactivation. © 2010 Elsevier Ltd. All rights reserved.

The influence of additives on the activity of different covalently and site-specific chemically modified immobilized preparations of a lipase from Geobacillus thermocatenulatus (BTL2) was investigated with a view to obtain a very high active biocatalyst. Non-ionic surfactant and co-solvents at different concentration range were applied. The CNBr-BTL2 immobilized preparation, a very mild immobilized enzyme with similar properties to the soluble enzyme, exhibited an increase in activity of 3 fold in the presence of 20% (v/v) co-solvent (e.g., 1,4-dioxane) and 2.6 fold when Triton X-100 (v/v) was added in the hydrolysis of p-nitrophenylbutyrate. This immobilized preparation was hyper-activated in the presence of both additives although without a synergistic effect. The CNBr-BTL2 modified with polymers showed mild hyperactivation in the presence of each additives and even a synergy in the presence of both. In the best of cases, the HOOC-PEG1500-CNBr-BTL2 preparation showed up to 11 fold higher activity in the presence of additives combination than in absence of them. The glyoxyl-BTL2 preparation was hyper-activated in a similar way than CNBr-BTL2 in the presence of detergents but much less with co-solvents. However, the modified glyoxyl-BTL2 preparations were hyper-activated with solvent (2 fold) but not with detergent. An increase of 3 fold in activity for the modified glyoxyl-BTL2 preparations was observed in the presence of both additives. © 2011 Elsevier B.V. All rights reserved.

In this study the immobilization and stabilization of a lipase from Thermomyces lanuginosus (TLL) on aldehyde-Lewatit (Lew-TLL) are described. TLL immobilization was rapid and over 90% of lipase activity was recovered after the immobilization. Lew-TLL was 10-fold more thermo stable than the commercial TLL preparation, Lipozyme TL-IM. The stabilized Lew-TLL was used for the enzymatic transesterification of ethanol and soybean oil. The transesterification was carried out following different strategies. First, with 7.5:1 molar ratio of ethanol:soybean oil, 15% immobilized enzyme and 4% water at 30 °C. In the presence of n-hexane, the transesterification reached 100% conversion, while in solvent-free system the yield was 75%. Second, at stoichiometric molar ratio, the yield was 70% conversion after 10. h of reaction in both systems. After this, transesterification was carried out by three stepwise additions of ethanol with a yield of 80% conversion, while a two step ethanolysis produced 100% conversion after 10. h of reaction in both solvent and solvent-free systems. © 2010 Elsevier Ltd.

Polyethyleneimine (PEI) modification on immobilized lipases greatly enhanced their enantioselectivity in the kinetic resolution of (±)-2-hydroxy-phenylacetic acid methyl ester. The enantiomeric ratio (E) of CNBr-agarose-CRL rose from 8 (without coating) to 20 (ee = 90%) after PEI coating in the hydrolysis at pH 5. In the case of CAL-B, the coating highly improved the enantioselectivity of the immobilized lipase from E = 1.5 (without coating) to E > 100 (ee > 99%). Moreover, this coating strategy improved the stability of the biocatalyst at high temperatures and in the presence of high co-solvent concentrations.

A novel approach is proposed to prepare a set of immobilized derivatives of a enzyme covalently rigidified through different regions of its surface. Six different variants of penicillin G acylase (PGA) from Escherichia coli (which lacks Cys) were prepared by introducing a unique Cys residue via site-directed mutagenesis in six different enzyme regions which were rich in Lys residues. All variants exhibited a similar activity and stability compared to those of the native enzyme. Each variant was immobilized on supports having a low concentration of reactive disulfide moieties and a high concentration of poorly reactive epoxy groups. After immobilization at pH 7.0 by site-directed thiol-disulfide intermolecular exchange, derivatives were further incubated at pH 10.0 for 48 h to promote an additional intramolecular reaction between Lys residues of enzyme and epoxy groups of the support. The establishment of at least three covalent attachments per PGA molecule was determined for all immobilized enzyme variants. The different derivatives exhibited diverse stability against several distorting agents and different selectivity in two interesting reactions. The derivative of the PGA variant obtained by replacement of GlnB380 by Cys was the most stable against heat and organic cosolvents: it preserved 90% of the initial activity and was 30-fold more stable than soluble PGA. This derivative also exhibited an improved enantioselectivity in the hydrolysis of chiral esters (E was improved from 8 to 16) and in kinetically controlled synthesis of amides (synthetic yields were increased from 31 to 49%)., The authors gratefully recognize the support from the Spanish CICYT (project. BIO-2005-8576). We gratefully recognize MEC for a fellowship for López-Gallego and Montes. We thank Antibióticos SA for the kind donation of reagents. The help and comments from Dr. Angel Berenguer (Universidad de Alicante) are gratefully recognized.

A new anion exchanger support has been designed for the selective adsorption of small proteins. This has been achieved activating an aminated support with glutaraldehyde and further coating the support surface with bovine serum albumin (BSA). In this support, " wells" are generated by two neighborhoods BSA molecules, on the bottom of those " wells" glutaraldehyde groups are exposed out ready to react with small molecules that have a size small enough to be accommodated between two BSA molecules on the pre-existing support. However, the BSA surface was not inert enough adsorbing many proteins, thereby reducing the selectivity of the system. A further solution was coating the immobilized BSA molecules with dextran, reducing the adsorption of protein on the BSA surface. This new matrix has been evaluated in the selective adsorption of the very small beta-lactoglobulins and alpha-lactalbumin from dairy whey, achieving the selective adsorption of both small proteins while other larger proteins from dairy whey remained in the supernatant. Moreover, a protein crude extract has been offered to the new matrix, and only small proteins could be adsorbed on the support (as probed by gel filtration).Thus this amino-glutaraldehyde-BSA-dextran-Sepharose is a matrix that may be used to selectively ionically adsorb proteins that were smaller than BSA (62. kDa). This strategy may be used for any other kind of adsorbing groups (chelating agents, boronic acid, etc.), or using proteins with different sizes to coat the support, designing tailor-made supports that may permit the fractioning of proteins following their sizes and by adsorption/desorption on different matrices. © 2010 Elsevier B.V.

9 páginas, 3 figuras, 3 esquemas, 2 tablas.-- et al., Controlled and directed immobilization plus post-immobilization techniques are proposed to get full stabilization of the quaternary structure of most multimeric industrial enzymes. The sequential utilization of two stabilization approaches is proposed: (a) Multi-subunit immobilization: a very intense multi-subunit covalent immobilization has been achieved by performing very long immobilization processes between multimeric enzymes and porous supports composed by large internal surfaces and covered by a very dense layer of reactive groups secluded from the support surface through very short spacer arms. (b) Additional cross-linking with poly-functional macromolecules: additional chemical modification of multi-subunit immobilized derivatives with polyfunctional macromolecules promotes an additional cross-linking of all subunits of most of multimeric enzymes. A number of homo and hetero-dimeric enzymes has been stabilized by the simple application of multi-subunit immobilization but more complex multimeric enzymes (e.g., tetrameric ones) were only fully stabilized after the sequential application of both strategies. After such stabilization of the quaternary structure these three features were observed: no subunits were desorbed from derivatives after boiling them in SDS, thermal inactivation becomes independent from enzyme concentration and derivatives became much more stable than soluble enzymes as well as than non-stabilized derivatives. For example, thermal stability of -amino acid oxidase from Rhodotorula gracilis was increased 7.000 fold after stabilization of its quaternary structure., This work has been partially supported by grants from Resindion S.R.L. (Mitsubishi Chemical Corporation), from Comunidad Autonoma de Madrid and from Agencia Española de Cooperacion Internacional.

We have developed a new strategy to prepare very active and very stable derivatives of the enzyme ferredoxin-NADP + -reductase (FNR). This strategy is based on a very controlled process of multipoint covalent attachment between the enzyme, through its amino groups, and very dense monolayers of linear aldehyde groups on support surfaces. Because of the excellent properties of this enzyme and the ones of its derivatives, we have been able to regenerate a complex and interesting enzymatic cofactor, NADP + , by using molecular oxygen as oxidizing agent and under a very wide range of experimental conditions. In this way, by coupling our efficient oxidative system to a number of NADP-depending redox enzymes, we should be able to develop very specific and very selective oxidative processes (dehydrogenation, deaminations, hydroxylations…) under very mild oxidizing conditions (e.g. at room temperature and by using molecular oxygen as unique oxidizing agent).

Fully oxidized dextrans (poly-(glutaraldehyde-like) macromolecules) have been studied as intramolecular crosslinking reagents. The developed methodology has been applied to the stabilization of quaternary as well as tertiary structure of proteins. The following model proteins were chosen: Tetrameric: s-Galactosidase, proteins composed y several polypeptidic chains: Penicillin G Acylase and Chymotrypsin, and monomeric: Trypsin. In all cases the different polypeptide chains were linked, remarkably the four subunits of s-Galactosidase. Tertiary structure was also stabilized, and this was reflected in a significant increase of enzymes thermal stabilizations. Thus, crosslinked chymotrypsin was 1,000 fold more stable than the native one. The promising possibilities of these agents to develop general strategies of enzyme stabilization are discussed.