Preparation and Properties of Matri9-Supported Horseradish Persxidase Can. J. Biochem. Downloaded from www.nrcresearchpress.com by UNIVERSITY OF MICHIGAN on 11/09/14 For personal use only.

GREGJ. BARTLING,SWAWAJ K. CHATTQPADHYAY, CHARLES 851. BARKER, AND HARRY D. BROWN Cuj~cerResearch Ce~rter.,Columbia, hfissolwi 65201

Received December 18, 8974 Bastling, G . J., Chattopadhyay, S. K., Barker, C . W. & Brown, H. D. (1975) Preparation and Properties s f Matrix-Supported Horseradish Peroxidas?. Can. 9.Biochern. 53, 868-874 A new method of enzyme immobilization has been described using poly(4-rnethacryloxybenzoic acid) as the carrier. Activation of the polymer, prior to enzyme attachment, was achieved with hl-ethoxycarbonyl-2-eth0xy-1,2-dihy&oquinoliae. The enzyme coupling step proceeded through nucbophilic attack by the protein on a mixed carbonic anhydride. The degree of polymer activation was determined by analysis for quinoline, a by-product of the reaction. The polymer-enzyme complex was compared to the enzyme in solution in terms of pH optimum, substrate kinetics. send thermal denaturation. Potential uses of the polymerenzyme system in chemical synthesis of henzoquinone derivatives are discussed. Bartling, G. J., Chattopadhyshy. S . K., Barker, C . W. & Browam, H. D. (1975) Preparation and Properties of Matrix-Supported Horseradish Persxidase. Catz. J. Btochen~.53, 868-894 Nous dCc~vonsugae ~lslavellemkthode d'immrnobilisation des enzymes, wtilisant Be poly(neide 4m6thacry~oxybenz~ique) cornme support. Avant la fixation de l'enzyme, le support est active avec la. hl-Cthoxycarbony1-2-6thoxy-1,2-dihydr~uinsline. Lors du couplage de l'enzyme, il y a attaque nuclCophble par Hn protCime sur un anhydride carbonique mixte. ke degrC d'activation du poIym&reest dCterminC par analyw de la quinoline, un sous-produit de la rkaction. Chez le complexe pdymkre-enzyme et I'enayme en solution, nous comgarsns le pH optimum, la cindtique du substrat et Ha denaturation therrnique. Nous discutons de l'utilitk possible du systkme pslyrn&re-enzymedans la synthae chin~iqgledes derives & la benzoquinone. [Traduit gar Be journal]

Introduction Aside from providing a useful biological model for the study of complex intercellular relatisnships, matrix-supported enzymes have recently received a great deal of attention for their potential practical applications in the food, chemical, and pharmaceutical industries (1, 2). To attempt the preparation of an immobilized enzyme for use in a continuously operating system requires consideration of several parameters. First, the protein must be attacked to a carrier which is inert to the particular enviranrneirt with which it will come into contact. Second, sufficient protein must be attached per unit carrier to maintain the size of the reactor within acceptable limits, and simuHtaneously provide a useful level of catalytic activity. Finally, the enzyme complex must remain active over a reasonable period of use. Recently? much effort has been expended in devising chemical methods for the efficient immobilization of a variety of enzymes 43,4), but no one method sf insolubilization is completely general for all classes of enzymes. Thus, for a specific protein, a comparative evaluation of the available techniques is often necessary to determine the method of choice. Clearly, the need

exists for the development of a greater variety of iinrnobilization techniques both for synthesis in water 441, or under anhydrous conditions (5, 6). Four principle methods are currently used for enzyme iitasolubilkatisn: (I) adsorption s n a surface, (2) entrapprnent within a polymeric matrix, (4) inter- and intramolecular chemical cross-linking, and (4) covalent attachment to a carrier through protein functional groups not essential for maintenance of catalytic activity. The first two techniques frequently suffer the disadvantage of protein loss through Beaching during application. Covalent attachment avoids these difficulties but sometin~esresults in a degree of conformational distortion which causes a substantial loss in enzynne activity after attachment. However, in practice, the method of covalent attachment is better suited to the requirements of product characterization. Balleau and Malek reported 47) the compound (I), N-ethoxy-carbonyl-2-ethoxy-1,2-di8myd~noline, reacted efficiently with carboxylic acids to produce the mixed carbonic anhydride (Scheme 1). The latter species undergoes nucleophilic attack by, e.g,, aaslines to yield peptide bonds. A variety of peptides have been synthesized by this

Can. J. Biochem. Downloaded from www.nrcresearchpress.com by UNIVERSITY OF MICHIGAN on 11/09/14 For personal use only.

HARTLING ET At.: MATRIXSUPPOR7'ED HORSERADISH PERQXEDASE

CH3 0 1 II

@ H Z = C - @ - 6

Beazoy l CQOH

1

peroxide 80 Oc

Enzyme

- NR2

@- = Poly (methaeryl) NH 2

C H ' ~ ~ ~ C H+ , WZ4*

.

backbone

--,

Peroxidase ------

CH3

procedure (7) and, in general, the method is highly efficient. We recently adapted the technique to the preparation of ilnmobilized enzymes usihg a cross-linked version of the carbonyl containing polymer shown in Scheme 1 (8). Moreover, a brief study (8) revealed the procedure was equally general with respect to a variety of enzyme classes, being especially useful for the redox enzyme horseradish peroxidase. This enzyme was chosen for this study because

of its ability to catalyze the oxidation of a variety of substituted phenols and anilines to p-benzoquinone derivatives (9). For example, in the presence of hydrogen peroxide, 2,4,6-trimethylaniline is converted (Scheme 2) quantitatively to %,6-dimethylbenzoquinone-4-(2',4',6'-trimethyl)anil (compound 2). Under mild hydrolytic conditions the anil is smoothly converted to 2,6-dimethylbenzoquinone (Scheme 2). We are interested in evaluating the possibility s f using

The activated polymer was freed of THF by washing 8 X BO rnl with distilled water. A solution of 100 rng of peroxidase dissolved in 15 rnl of water (brown colored solution) was added to the support and the reaction mixture was stirred for 18 h at room temperature. The Methods and hXatesials product was centrifuged to yield a tan colored pellet. The Horseradish peroxidase (EC I. 1 1.1.S) (Donor: hydro- supernatant was decanted and the product was washed gen-peroxide oxidoreductase) was obtained from Sigma with water, then sodium chloride solution, and finally Chemical Co., St. Louis, Mo. and dried to constant again with water, until the washes exhibited no detectable weight over phosphorous pentoxide at 0.1 mm for 3 days. enzymic activity. The complex was finally transferred 4-Mydroxybenzoic acid, 2,4,6-trimethylaniline and meth- quantitatively to a 250 rnl volumetric flask and made up acryloyl chloride were purchased from Aldrich Chemical to the mark with distilled water. Two 20.0 ml aliquots of Co., Milwaukee,Wisc.The latter was distilled prior to use. the suspension were removed and filtered through tared The aniline derivative was converted to the hydrochloride Gooch crucibles. The crucibles were dried to constant by the standard method and twice recrystallized from weight at 105 'C to give 20.0 and 20.1 mg of dry complex, 6 N hydrcchloric acid. N-Ethoxycarbonyl-2-ethoxy-l,2- respectively. Zt was determined that 250 mg of complex dihydroquinoline (EEDQ) was prepared according to the was obtained, coincidentally yielding a concentration of method of Belleau as cited by Bruce and Walls (1 1) and 1.0 m g of complex per millilitre of suspension. Assuming complete precipitation of the activated polypurified by fractional distillation (b.p. 162-164 " C ,3 mm). Tetrahydrofuran (TMF), N,hT-dimethylformamide(DMF), mer from the above THF solution, it was calculated that benzoyl peroxide and hydrogen peroxide (30