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Cyclodextrin Polymers on Invertase Activity in FreezeDried Systems

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Cyclodextrin Polymers on Invertase Activity in Freeze�Dried Systems Formulation and Engineering of Biomaterials Biotechnology Progress DOI 10.1002/btpr.2067 Impact of supramolecular interactions of dextran-β-cyclodextrin polymers on invertase activity in freeze-dried systems Patricio R. Santagapita1, M. Florencia Mazzobre1, Héctor L. Ramirez2, Leissy Gómez Brizuela2, Horacio R. Corti3, Reynaldo Villalonga4 and M. Pilar Buera1,* 1 Industry Department and Organic Chemistry Department, Faculty of Exact and Natural Sciences, University of Buenos Aires, Intendente Güiraldes 2160 - Ciudad Universitaria - C1428EGA (FCEyN-UBA) & National Council of Scientific and Technical Research (CONICET), Ciudad Autónoma de Buenos Aires, Argentina. E- mails: [email protected]; [email protected]; [email protected] 2 Center for Enzyme Technology, University of Matanzas, Matanzas, C.P. 44740, Cuba. E-mails: [email protected]; [email protected] 3 Departamento de Física de la Materia Condensada, Comisión Nacional de Energía Atómica, Centro Atómico Constituyentes, Avda. General Paz 1499, San Martín, 1650, Buenos Aires, and Instituto de Química Física de los Materiales, Ambiente y Energía (INQUIMAE). Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Ciudad Universitaria. 1428, Buenos Aires, Argentina. E-mail: [email protected] 4 Department of Analytical Chemistry, Faculty of Chemistry, Complutense University of Madrid, Madrid, Av de Séneca, 2, 28040, Madrid, Spain. E-mail: [email protected] * Phone: +54 11 4576 3366; e-mail: [email protected] This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/btpr.2067 © 2015 American Institute of Chemical Engineers Biotechnol Prog Received: Aug 26, 2014; Revised: Nov 04, 2014; Accepted: Feb 24, 2015 This article is protected by copyright. All rights reserved. Biotechnology Progress Page 2 of 26 Abstract β-cyclodextrin-grafted dextrans with spacer arms of different length were employed to evaluate the impact of supramolecular interactions on invertase activity. The modified dextrans were used as single additives or combined with trehalose in freeze-dried formulations containing invertase. Enzyme activity conservation was analyzed after freeze-drying and thermal treatment. The change of glass transition temperatures (Tg) was also evaluated and related to effective interactions. Outstanding differences on enzyme stability were mainly related to the effect of the spacer arm length on polymer-enzyme interactions, since both the degree of substitution and the molecular weight were similar for the two polymers. This change of effective interactions was also manifested in the pronounced reduction of Tg values, and were related to the chemical modification of the backbone during oxidation, and to the attachment of the β-cyclodextrin units with spacer arms of different length on dextran. Keywords: supramolecular interactions; dextran; β-cyclodextrin; glass transition temperature (Tg); enzyme stability. John Wiley & Sons This article is protected by copyright. All rights reserved. Page 3 of 26 Biotechnology Progress 1. Introduction Protection of labile compounds during dehydration is based on the formation of an amorphous glassy matrix and its maintenance over storage, and by the presence of adequate molecular and supramolecular interactions between the excipients and the labile compound.1-4 Previous reports showed that the formation of an amorphous matrix does not guarantee the stabilization of immobilized labile materials, which requires the development of adequate protein-excipient interactions.5,6 In this way, biomolecules are kinetically stabilized even though they are not under a thermodynamically stable condition. However, once the glassy systems are above their glass transition temperature, they become supercooled and protein denaturation may be accelerated.7 Dehydration and freezing of proteins in the presence of polyols is frequently used for increasing protein stability, where the disaccharide trehalose is one of the most used 2,4 2,8 excipients. Besides trehalose, previous works showed that dextran (of Mw 70,000), a D-glucose linear polymer composed of 95% α(1 6) linkage, is a very good enzyme stabilizer in freeze-dried formulations. After freeze-drying, more than 80% of invertase,2 75% of α-chymotrypsin,8 and 50% of catalase activities8 were recovered. A less frequent approach for increasing protein stability in dehydrated formulations is the use of cyclodextrins (CDs). CDs have a hydrophobic central cavity and a hydrophilic outer surface,9 and are capable to include a variety of hydrophobic guest compounds such as aromatic amino acids located at the protein’s surface.10 The effect of dextran modification with several mono-6-alkylenediamino- 6-deoxy-β- cyclodextrin on trypsin thermo-resistance in diluted aqueous media was related to changes on supramolecular interactions.11,12 Then, the combination of the physical stability provided by dextran with the protective effect on hydrophobic regions of the enzyme provided by cyclodextrin (i.e., the combination of both molecular and supramolecular interactions) could offer improved protection to enzymes subJected to dehydration. Invertase from Saccharomyces cerevisiae was selected as a model enzyme to analyze its stability at considerably high temperatures, it is a well-known enzyme, and was previously used as a model in similar works.2,3 Besides, invertase has extensive industrial applications in food industry (such as in confectionary) and biotecnology (fermentation of cane molasses for ethanol production).13 Several of its hydrobobic aminoacids (for instance, those having a key role in membrane translocation)14 could be a target for CDs complexation. John Wiley & Sons This article is protected by copyright. All rights reserved. Biotechnology Progress Page 4 of 26 In present work two types of supramolecular interactions were considered in relation to invertase stability: on one side, the induction of multipoint interactions at the surface of enzymes by adding CD-modified polysaccharides to protein solutions has been considered as an interesting supramolecular alternative for stabilizing enzymes.10,12 On the other side, the glass transition and crystallization of the matrices in which the invertase is immersed are cooperative phenomena involving the concerted movements of several molecules.15 The purpose of this work was to evaluate the impact of enzyme-polymer supramolecular interactions by analyzing enzyme activity conservation and glass transitions of β-CD-modified dextrans with alkyl spacer arms of different lengths. The results may contribute to foster the knowledge on the structure/function relationships involved in the dehydro-protective effect of polymers on the activity of freeze-dried enzymes. 2. Materials and Methods 2.1. Materials Dextran 70, a D-glucose linear polymer composed of 95% α(1 6) linkage, of -1 Mw = 70,000 g mol , (Sigma-Aldrich, St. Louis, M.O., USA) was employed. β- cyclodextrin (β-CD), a cyclic non-reducing oligosaccharide composed of seven glucopyranose units bonded together via α(14) glycoside linkages, was from Amaizo, Hammond, IN, USA and α-α-trehalose dihydrate (Tr) from Hayashibara Co, Ltd., Shimoishii, Okayama, Japan/Cargill Inc., Minneapolis, Minnesota, USA. The enzyme invertase from Saccharomyces cerevisiae (β-fructofuranosidase, E.C. 3.2.1.26, 1840 U/mg, molecular mass 270 kDa) was from Fluka, Buchs, Switzerland. One unit of activity was defined as the amount of enzyme required to hydrolyze 1.0 µmol of sucrose per minute at pH 4.6, at 37 °C. 2.2. Synthesis of dextran-ethane-1,2-diamine-β-cyclodextrin and dextran- hexane-1,6-diamine-β-cyclodextrin polymers A two-step synthesis procedure was employed by following Ramírez et al.,16 as showed in Scheme 1: i) dextran was oxidized; ii) β-CD derivatives (previously prepared) were introduced into the polysaccharide chains after reduction. Briefly, the polysaccharide was oxidized by dissolving 500 mg of polymers in 15 ml H2O and treated with 500 mg mNaIO4 under continuous stirring at 0°C in the dark John Wiley & Sons This article is protected by copyright. All rights reserved. Page 5 of 26 Biotechnology Progress for 1 h. The oxidation reactions were stopped by adding 0.5 mL of ethylene glycol and stirred for another 1 h, and further dialyzed in dark and cold conditions against distilled H2O in a semi-permeable membrane of 10 kDa. The degree of dextran degradation by oxidation was negligible, since the oxidation conditions were very soft (1 hour, in darkness). β-CD was purified by cation exchange chromatography on CM-Shephadex C-25 + (NH4 form) and characterized by conventional NMR techniques. Mono-6-(ethane-1,2- diamine)-6-deoxy-β-CD and mono-6-(hexane-1,6-diamine)-6-deoxy-β-CD were previously obtained by treating mono-6-O-tosyl-β-CD17 with ethane-1,2-diamine and hexane-1,6-diamine, respectively, in dimethylformamide,18-20 and the mono-substitution was confirmed by NMR (Figures S1- S2, and Tables S1- S2). For introducing the β-CD derivatives into the polysaccharide chains, 350 mg of mono-6-(ethane-1,2-diamine)-6- deoxy-β-CD or mono-6-(hexane-1,6-diamine)-6-deoxy-β-CD was added to 5 mL of the activated polymer solutions and then treated with 20 mg NaBH4 for 4 h under continuous stirring (Scheme 1). The modified polymers solutions were further dialyzed against distilled H2O and finally freeze-dried (frozen at -26 °C for 24 h and finally freeze-dried in a Heto Holten A/S, cooling trap model CT 110 freeze-dryer (Heto Lab Equipment, Denmark) operating at a condenser plate temperature of -111 °C and a chamber pressure of 4.10-4 mbar); the secondary drying was performed at 25 °C). The final products, dextran-ethane-1,2-diamine-β-cyclodextrin (DECD) and dextran-hexane-1,6-diamine-β-cyclodextrin (DHCD) were characterized by 1H-NMR and its average molecular weight was obtained by viscosimetry. O O O O O O HO HO HO O OH OH NaIO4 O 1) aminated CD derivative O O (oxidation) O 2) NaBH4 (reduction) HO O O O O HO HO ( ) OH O n NH O O HN O O HO O O O HO HO OH O OH O O O Scheme 1.
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