Cross-linked crystals of chloroperoxidase Marcela Ayala, * Eduardo Horjales, Michael A. Pickard, and Rafael Vazquez-Duhalt Institute of Biotechnology, UNAM. Apartado Postal 510-3, Cuernavaca, Morelos 62250, Mexico Received 14 June 2002 Abstract Chloroperoxidase from Caldariomyces fumago was crystallized. The crystals were modified with several cross-linkers, but only glurataldehyde was able to produce catalytically active and insoluble crystals. Unlike other immobilized chloroperoxidase prepa- rations, these catalytic crystals are more thermostable than the unmodified soluble enzyme. The enhanced stability is probably due to the structure conservation in the crystalline matrix. In addition, non-cross-linked chloroperoxidase crystals retained more activity than the soluble enzyme after incubation in an organic solvent with low water content. Although the cross-linked crystals were catalytically active, they showed lower specific activity than the soluble enzyme. This low activity may be due to non-specific re- actions between the cross-linker and essential residues for catalysis. Alternative cross-linking strategies are discussed. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Biocatalysis; Chloroperoxidase; CLEC; Cross-linked crystals; Stability Protein stability is one of the major challenges for large-scale use of enzymes. Cross-linked enzyme crystals are a suitable preparation that confers structural resis- tance to proteins by stabilization of the crystalline matrix [1,2]. In crystals, protein molecules are symmetrically arranged and their native conformation is stabilized [3]. Thesecrystallinebiocatalystsaremorestablethansoluble enzymes when exposed to organic solvents and high temperatures, conditions normally found in many in- dustrial processes. Moreover, cross-linked enzyme crys- tals are mechanically resistant and can be recycled and reused. Although there are several cross-linked crystals commercially available, most of them are prepared with hydrolytic enzymes, but few are available for other reac- tions such as redox processes. Chloroperoxidase from Caldariomyces fumago is the most versatile and unusual heme-peroxidase. In vitro, chloroperoxidase shows halogenase-, peroxidase-, cata- lase-, and cytochrome P450-like activities [4]. This en- zyme has wide potential applications, ranging from synthesis of optically pure compounds [5] to environ- mentally related processes [6]. In spite of this, few re- ports dealing with chloroperoxidase immobilization are available [7–9]. Immobilized forms of chloroperoxidase have shown some advantages over the soluble form [10– 12], nevertheless no improved stability was conferred by the immobilization procedure. Chloroperoxidase in both soluble and immobilized preparations is readily inacti- vated above 50 °C [13], limiting its use in many fields. This is the first report of a cross-linked crystal of a peroxidase. The chloroperoxidase cross-linked crystals showed lower activity but increased thermostability when compared to the soluble enzyme. Materials and methods Chemicals. 1,6-Hexandiamine, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, glutaraldehyde, cacodylic acid, thioani- sole, and thianthrene were purchased from Sigma–Aldrich (St. Louis, MO). Zinc acetate was obtained from Fluka Chemie GmbH (Buchs, Switzerland). Polyethylene glycol 8000 was purchased from Hampton Research (Laguna Niguel, CA). Buffer salts and organic solvents were obtained from J.T. Baker (Phillipsburg, NJ). Chloroperoxidase from C. fumago CMI 89362 was obtained and purified as previously re- ported [14]. The Rz ðA 403 =A 280 Þ of the enzyme was 1.42. Crystallization of chloroperoxidase. The enzyme was crystallized in sitting drops using the vapor diffusion method. Thirty microliters of a 7 mg/mL chloroperoxidase solution in 10 mM phosphate buffer, pH 5 was mixed with 30 lL of the crystallization solution. The crystalliza- tion solution contained 14% polyethylene glycol 8000, 0.1 M zinc ac- etate, and 0.1 M sodium cacodylate, pH 5.5. The reservoir contained Biochemical and Biophysical Research Communications 295 (2002) 828–831 www.academicpress.com BBRC * Corresponding author. Fax: +52-777-317-2388. E-mail address: maa@ibt.unam.mx (M. Ayala). 0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII:S0006-291X(02)00766-0