Structure/Function Relationships of Several Biopolymers as Related to Invertase Stability in Dehydrated Systems Patricio R. Santagapita, † Leissy Gómez Brizuela, ‡ M. Florencia Mazzobre, † Héctor L. Ramirez, ‡ Horacio R. Corti, §,4 Reynaldo Villalonga Santana, ‡ and M. Pilar Buera* ,† Departamentos de Industrias y de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria 1428, Buenos Aires, Argentina, Centro de Estudios de Tecnología Enzimática, Facultad de Agronomía, Universidad de Matanzas “Camilo Cienfuegos”, Autopista a Varadero km 3½, 44740, Matanzas, Cuba, Comisión Nacional de Energía Atómica, Centro Atómico Constituyentes, Av. Constituyentes, San Martín, Buenos Aires, Argentina, and Departamento de Química Inorgánica, Analítica y Química Física, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria 1428, Buenos Aires, Argentina Received November 2, 2007; Revised Manuscript Received December 3, 2007 Structure/function relationships of different biopolymers (alginate, dextran, or -cyclodextrin) were analyzed as single excipients or combined with trehalose in relation to their efficiency as enzyme stabilizers in freeze-dried formulations and compared to trehalose. Particularly, a novel synthesized polymer -cyclodextrin-branched alginate (-CD-A) was employed as excipient. During freeze-drying, the polymers or their mixtures did not confer better protection to invertase compared to trehalose. -CD-A (with or without trehalose), -cyclodextrin (-CD), or dextran with trehalose were the best protective agents during thermal treatment, while -CD and alginate showed a negative effect on invertase activity preservation. The -CD linked alginate combined the physical stability provided by alginate with the stabilization of hydrophobic regions of the enzyme provided by cyclodextrin. -CD-A was effective even at conditions at which trehalose lost its protective effect. A relatively simple covalent combination of two biopolymers significantly affected their functionalities and, consequently, their interactions with proteins, modifying enzyme stability patterns. 1. Introduction The use of enzymes at the industrial level is often limited by the low resistance of these proteins to technological conditions, especially at high temperatures. Important efforts based on site- direct mutagenesis, 1 immobilization on solid supports, 2 use of water-soluble additives, 3 and chemical, 4–7 enzymatic, 8 and chemoenzymatic 9 modifications of the protein surface have been reported to improve enzyme thermostability. From the industrial point of view, the use of hydrosoluble additives appears as one of the most promising approaches, considering the ease and low cost of this procedure. 10 Alginates and dextrans are employed as ingredients, excipi- ents, or protein stabilizers in food and pharmaceutical industries or for biotechnological purposes (immobilization media for enzyme or bacteria/preparation of hydrogels). 11–15 Alginate (A) is a binary copolymer of (1f4) linked -D-mannuronic and its C5 epimer (R-L-guluronic acid) residues, negatively charged at pH values higher than 3.65. 16 Dextran (D) is a D-glucose linear polymer composed of 95% R(1f6) linkage, is mostly uncharged in a wide pH range, and behaves as a very flexible and extended polymer. Although they are widely used, it has been demon- strated that they are not as good as trehalose (T) to protect enzymes after freeze-drying or thermal treatment. 17,18 It is well- known that the water replacement mechanism is one of the keys of the success of trehalose as a cryo- and dehydroprotectant of labile systems. 19 However, undesired trehalose crystallization processes have become a main issue in pharmaceutical and food ingredients industries because crystallization of the solid phase signicantly affects the mechanisms by which amorphous sugars manifest protective effects on biomolecules and, consequently, the shelf life of a product. 20–23 Several authors have demon- strated that the presence of a polymer delays sugar crystallization processes. 20,21,24–27 Another approach for increasing protein stability in dehy- drated formulations is the use of cyclodextrins. -Cyclodextrin (-CD) is a cyclic nonreducing oligosaccharide composed of seven glucopyranose units bonded together via R(1–4) glycoside linkages, with a hydrophobic central cavity and a hydrophilic outer surface. 28 CDs are capable of including a variety of hydrophobic guest compounds such as aromatic amino acids located at the proteins surface. 4,5,8 Thermal stability of enzymes could thus be improved by chemical conjugation of the enzyme with several CD derivatives that induced multipoint supramo- lecular advantageous interactions at the surface of the enzyme. 4,5,8 On the other side, covalently modified biopolymers (polysucrose or dextran) with cyclodextrins were used as excipients for improving enzyme protection in very diluted aqueous media. 10,29 Also, in diluted systems, a modification of alginate with R- cyclodextrin generated through the addition of the CD moieties * Author to whom correspondence should be addressed. E-mail: pilar@ di.fcen.uba.ar. Telephone/Fax: +(54) 114576-3366. † Departamentos de Industrias y de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires. Telephone/ Fax: +(54) 114576-3366. ‡ Centro de Estudios de Tecnología Enzimática, Facultad de Agronomía, Universidad de Matanzas “Camilo Cienfuegos”. Telephone: 5345 262251. Fax: 5345 253101. § Comisión Nacional de Energía Atómica, Centro Atómico Constituyentes. 4 Departamento de Química Inorgánica, Analítica y Química Física, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires. Biomacromolecules 2008, 9, 741–747 741 10.1021/bm7012108 CCC: $40.75  2008 American Chemical Society Published on Web 01/12/2008