High Stabilization of Enzymes Immobilized on Rigid Hydrophobic Glyoxyl-Supports: Generation of Hydrophilic Environments on Support Surfaces

© 2020 by the authors.Very rigid supports are useful for enzyme immobilization to design continuous flow reactors and/or to work in non-conventional media. Among them, epoxy-methacrylic supports are easily functionalized with glyoxyl groups, which makes them ideal candidates for enzyme stabilization via multipoint covalent immobilization. However, these supports present highly hydrophobic surfaces, which might promote very undesirable effects on enzyme activity and/or stability. The hydrophilization of the support surface after multipoint enzyme immobilization is proposed here as an alternative to reduce these undesirable effects. The remaining aldehyde groups on the support are modified with aminated hydrophilic small molecules (glycine, lysine or aspartic acid) in the presence of 2-picoline borane. The penicillin G acylase from Escherichia coli (PGA) and alcohol dehydrogenase from Thermus thermophilus HB27 (ADH2) were immobilized on glyoxyl-functionalized agarose, Relizyme and Relisorb. Despite the similar density of aldehyde groups displayed by functionalized supports, their stabilization effects on immobilized enzymes were quite different: up to 300-fold lower by hydrophobic supports than by highly hydrophilic glyoxyl-agarose. A dramatic increase in the protein stabilities was shown when a hydrophilization treatment of the hydrophobic support surface was done. The PGA immobilized on the glyoxyl-Relisorb hydrophilized with aspartic acid becomes 280-fold more stable than without any treatment, and it is even more stable than the PGA immobilized on the glyoxyl agarose.This research was funded by the Spanish Ministry of Economy, Industry and Competitiveness (projects BIO2012-36861 and CTQ2015-70348) and the EU FP7 project SuSy (Sucrose Synthase as Cost-Effective Mediator of Glycosylation Reactions, C-KBBE/3293). 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.Peer reviewe

Stabilization of multimeric sucrose synthase from Acidithiobacillus caldus via immobilization and post-immobilization techniques for synthesis of UDP-glucose

Sucrose synthases (SuSys) have been attracting great interest in recent years in industrial biocatalysis. They can be used for the cost-effective production of uridine 5′-diphosphate glucose (UDP-glucose) or its in situ recycling if coupled to glycosyltransferases on the production of glycosides in the food, pharmaceutical, nutraceutical, and cosmetic industry. In this study, the homotetrameric SuSy from Acidithiobacillus caldus (SuSyAc) was immobilized-stabilized on agarose beads activated with either (i) glyoxyl groups, (ii) cyanogen bromide groups, or (iii) heterogeneously activated with both glyoxyl and positively charged amino groups. The multipoint covalent immobilization of SuSyAc on glyoxyl agarose at pH 10.0 under optimized conditions provided a significant stabilization factor at reaction conditions (pH 5.0 and 45 °C). However, this strategy did not stabilize the enzyme quaternary structure. Thus, a post-immobilization technique using functionalized polymers, such as polyethyleneimine (PEI) and dextran-aldehyde (dexCHO), was applied to cross-link all enzyme subunits. The coating of the optimal SuSyAc immobilized glyoxyl agarose with a bilayer of 25 kDa PEI and 25 kDa dexCHO completely stabilized the quaternary structure of the enzyme. Accordingly, the combination of immobilization and post-immobilization techniques led to a biocatalyst 340-fold more stable than the non-cross-linked biocatalyst, preserving 60% of its initial activity. This biocatalyst produced 256 mM of UDP-glucose in a single batch, accumulating 1 M after five reaction cycles. Therefore, this immobilized enzyme can be of great interest as a biocatalyst to synthesize UDP-glucose.EU FP7 project SuSy (Sucrose Synthase as Cost-Effective Mediator of Glycosylation Reactions, CKBBE/ 3293(IJCI-2014-19260) Spanish Ministry of Economy, Industry and Competitiveness.3.670 JCR (2018) Q2, 41/162 Biotechnology & Applied Microbiology1.127 SJR (2018) Q1, 53/342 Biotechnology, 22/114 Applied Microbiology and Biotechnology, 451/2844 Medicine (miscellaneous)No data IDR 2018UE

Turn-on Fluorescent Biosensors for Imaging Hypoxia-like Conditions in Living Cells

We present the synthesis, photophysical properties, and biological application of nontoxic 3-azo-conjugated BODIPY dyes as masked fluorescent biosensors of hypoxia-like conditions. The synthetic methodology is based on an operationally simple N=N bond-forming protocol, followed by a Suzuki coupling, that allows for a direct access to simple and underexplored 3-azo-substituted BODIPY. These dyes can turn on their emission properties under both chemical and biological reductive conditions, including bacterial and human azoreductases, which trigger the azo bond cleavage, leading to fluorescent 3-amino-BODIPY. We have also developed a practical enzymatic protocol, using an immobilized bacterial azoreductase that allows for the evaluation of these azo-based probes and can be used as a model for the less accessible and expensive human reductase NQO1. Quantum mechanical calculations uncover the restructuration of the topography of the S1 potential energy surface following the reduction of the azo moiety and rationalize the fluorescent quenching event through the mapping of an unprecedented pathway. Fluorescent microscopy experiments show that these azos can be used to visualize hypoxia-like conditions within living cellsThis article is dedicated to Professor M. Carmen Carreño on the occasion of her retirement. We thank MINECO (grant CTQ2017-85454-C2-2-P), MICINN (grant PID2020- 113059GB-C22), MCIU (grant PGC2018-094644-B-C21), the Ramón y Cajal Program (grant RYC-2016-20489), the Fundación La Caixa (grant no. LCF/BQ/DR19/11740024), and the Comunidad Autónoma de Madrid (B2017/BMD-3867 RENIMCM) and co-financed by the European Structural and investment fund for financial support. I.C. and F.R.-G. also acknowledge the Red Española de Supercomputación, the MareNostrum Supercomputer Center, and the Centro de Computación Científica of the UAM (CCC-UAM) for the generous allocation of computer time and for their technical support. We thank the “Servicio de Microscopía óptica y confocal CBMSO” facility for their assistance. We also thank Prof. M. C. Carreño for her advice and helpful discussions during the wor

Stabilization of Immobilized Lipases by Intense Intramolecular Cross-Linking of Their Surfaces by Using Aldehyde-Dextran Polymers

Immobilized enzymes have a very large region that is not in contact with the support surface and this region could be the target of new stabilization strategies. The chemical amination of these regions plus further cross-linking with aldehyde-dextran polymers is proposed here as a strategy to increase the stability of immobilized enzymes. Aldehyde-dextran is not able to react with single amino groups but it reacts very rapidly with polyaminated surfaces. Three lipases—from Thermomyces lanuginosus (TLL), Rhizomucor miehiei (RML), and Candida antarctica B (CALB)—were immobilized using interfacial adsorption on the hydrophobic octyl-Sepharose support, chemically aminated, and cross-linked. Catalytic activities remained higher than 70% with regard to unmodified conjugates. The increase in the amination degree of the lipases together with the increase in the density of aldehyde groups in the dextran-aldehyde polymer promoted a higher number of cross-links. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of those conjugates demonstrates the major role of the intramolecular cross-linking on the stabilization of the enzymes. The highest stabilization was achieved by the modified RML immobilized on octyl-Sepharose, which was 250-fold more stable than the unmodified conjugate. The TLL and the CALB were 40-fold and 4-fold more stable than the unmodified conjugate.Sin financiación4.183 JCR (2018) Q2, 78/299 Biochemistry & Molecular Biology, 48/177 Chemistry, Multidisciplinary1.312 SJR (2018) Q1, 13/60 Catalysis, 11/72 Inorganic Chemistry, 22/185 Organic Chemistry, 26/166 Physical and Theoretical Chemistry, 9/80 Spectroscopy, 71/1875 Computer Science Applications, 335/2844 Medicine (miscellaneous); Q2, 151/421 Molecular BiologyNo data IDR 2018UE

Stabilization of Enzymes by Multipoint Covalent Attachment on Aldehyde-Supports: 2-Picoline Borane as an Alternative Reducing Agent

Enzyme immobilization by multipoint covalent attachment on supports activated with aliphatic aldehyde groups (e.g., glyoxyl agarose) has proven to be an excellent immobilization technique for enzyme stabilization. Borohydride reduction of immobilized enzymes is necessary to convert enzyme–support linkages into stable secondary amino groups and to convert the remaining aldehyde groups on the support into hydroxy groups. However, the use of borohydride can adversely affect the structure–activity of some immobilized enzymes. For this reason, 2-picoline borane is proposed here as an alternative milder reducing agent, especially, for those enzymes sensitive to borohydride reduction. The immobilization-stabilization parameters of five enzymes from different sources and nature (from monomeric to multimeric enzymes) were compared with those obtained by conventional methodology. The most interesting results were obtained for bacterial (R)-mandelate dehydrogenase (ManDH). Immobilized ManDH reduced with borohydride almost completely lost its catalytic activity (1.5% of expressed activity). In contrast, using 2-picoline borane and blocking the remaining aldehyde groups on the support with glycine allowed for a conjugate with a significant activity of 19.5%. This improved biocatalyst was 357-fold more stable than the soluble enzyme at 50 °C and pH 7. The results show that this alternative methodology can lead to more stable and active biocatalysts

Stabilization of glycosylated β-glucosidase by intramolecular crosslinking between oxidized glycosidic chains and lysine residues

Many industrial enzymes can be highly glycosylated, including the β-glucosidase enzymes. Although glycosylation plays an important role in many biological processes, such chains can cause problems in the multipoint immobilization techniques of the enzymes, since the glycosylated chains can cover the reactive groups of the protein (e.g., Lys) and do not allow those groups to react with reactive groups of the support (e.g., aldehyde and epoxy groups). Nevertheless, the activated glycosylated chains can be used as excellent crosslinking agents. The glycosylated chains when oxidized with periodate can generate aldehyde groups capable of reacting with the amino groups of the protein itself. Such intramolecular crosslinks may have significant stabilizing effects. In this study, we investigated if the intramolecular crosslinking occurs in the oxidized β-glucosidase and its effect on the stability of the enzyme. For this, the oxidation of glycosidic chains of β-glucosidase was carried out, allowing to demonstrate the formation of aldehyde groups and subsequent interaction with the amine groups and to verify the stability of the different forms of free enzyme (glycosylated and oxidized). Furthermore, we verified the influence of the glycosidic chains on the immobilization of β-glucosidase from Aspergillus niger and on the consequent stabilization. The results suggest that intramolecular crosslinking occurred and consequently the oxidized enzyme showed a much greater stabilization than the native enzyme (glycosylated). When the multipoint immobilization was performed in amino-epoxy-agarose supports, the stabilization of the oxidized enzyme increases by a 6-fold factor. The overall stabilization strategy was capable to promote an enzyme stabilization of 120-fold regarding to the soluble unmodified enzyme.The authors thank the support from Research Institute of Food Science (CIAL), Embrapa Instrumentation of São Carlos, Foundation for Research Support of Espírito Santo (FAPES), and Federal University of Espírito Santo (UFES).Peer reviewe

Immobilization and stabilization of beta-xylosidases from Penicillium janczewskii

β-Xylosidases are critical for complete degradation of xylan, the second main constituent of plant cell walls. A minor β-xylosidase (BXYL II) from Penicillium janczewskii was purified by ammonium sulfate precipitation (30% saturation) followed by DEAE-Sephadex chromatography in pH 6.5 and elution with KCl. The enzyme presented molecular weight (MW) of 301 kDa estimated by size exclusion chromatography. Optimal activity was observed in pH 3.0 and 70-75 °C, with higher stability in pH 3.0-4.5 and half-lives of 11, 5, and 2 min at 65, 70, and 75 °C, respectively. Inhibition was moderate with Pb+2 and citrate and total with Cu+2, Hg+2, and Co+2. Partially purified BXYL II and BXYL I (the main β-xylosidase from this fungus) were individually immobilized and stabilized in glyoxyl agarose gels. At 65 °C, immobilized BXYL I and BXYL II presented half-lives of 4.9 and 23.1 h, respectively, therefore being 12.3-fold and 33-fold more stable than their unipuntual CNBr derivatives (reference mimicking soluble enzyme behaviors). During long-term incubation in pH 5.0 at 50 °C, BXYL I and BXYL II glyoxyl derivatives preserved 85 and 35% activity after 25 and 7 days, respectively. Immobilized BXYL I retained 70% activity after 10 reuse cycles of p-nitrophenyl-β-D-xylopyranoside hydrolysis.This work was partially sponsored by FAPESP (Project 2010/16582-0) and the Spanish Ministry of Science and Innovation (Project BIO-2012-36861). C.R.F.T. gratefully acknowledges PROPe/UNESP and CAPES/Ministry of Education, Brazil, through the Program Science Without Borders for the postdoctoral scholarship (3134-13-0).Peer reviewe

Designing continuous flow reaction of xylan hydrolysis for xylooligosaccharides production in packed-bed reactors using xylanase immobilized on methacrylic polymer-based supports

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.Sin financiación6.669 JCR (2018) Q1, 1/13 Agricultural Engineering, 13/162 Biotechnology & Applied Microbiology, 13/103 Energy & Fuels2.157 SJR (2018) Q1, 13/188 Bioengineering, 14/352 Renewable Energy, Sustainability and the Environment, 2/200 Environmental Engineering, 2/129 Waste Management and Disposal, 134/2844 Medicine (miscellaneous)No data IDR 2018UE

Designing continuous flow reaction of xylan hydrolysis for xylooligosaccharides production in packed-bed reactors using xylanase immobilized on methacrylic polymer-based supports

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.Peer reviewe

Functionalization of porous cellulose with glyoxyl groups as a carrier for enzyme immobilization and stabilization

The functionalization of the internal surface of macroporous carriers with glyoxyl groups has proven to highly stabilize a large variety of enzymes through multipoint covalent immobilization. In this work, we have translated the surface chemistry developed for the fabrication of glyoxyl-agarose carriers to macroporous cellulose (CEL). To that aim, CEL-based microbeads were functionalized with glyoxyl groups through a stepwise alkoxylation (or alkylation)/oxidation synthetic scheme. This functionalization sequence was analyzed by solid-state NMR, while the scanning electron miscroscopy of CEL microbeads reveals that the mild oxidation conditions negligibly affect the morphological properties of the material. Through the optimal functionalization protocol using rac-glycidol, we introduce up to 200 μmols of aldehyde groups per gram of wet CEL, a similar density to the one obtained for the benchmarked agarose-glyoxyl carrier. This novel CEL-based carrier succeeds to immobilize and stabilize industrially relevant enzymes such as d-amino acid oxidase from Trigonopsis variabilis and xylanases from Trichoderma reseei. Remarkably, the xylanases immobilized on the optimal CEL-based materials present a half-life time of 51 h at 60 °C and convert up to 90% of the xylan after four operation cycles for the synthesis of xylooligosaccharides.The authors acknowledge funding from the National Council for Scientific and Technological Development (CNPq) for financial support for the PhD scholarships of S.M.O (Process CsF 201683/2014–2008). J.M.G thanks the funding from the EU FP7 project Lignofood (Ingredients for Food and Beverage Industry from a Lignocellulosic Source, grant agreement no 606073). F.L-G acknowledges the funding of IKERBASQUE. This work was performed under the Maria de Maeztu Units of Excellence Program from the Spanish State Research Agency—grant no. MDM-2017-0720. J.M.F., thanks to the funding from the Spanish Ministerio de Ciencia e Innovación (grant RTI2018-093431-BI00).Peer reviewe