A tailor-made, self-sufficient and recyclable monooxygenase catalyst based on coimmobilized cytochrome P450 BM3 and glucose dehydrogenase

Abstract

Cytochrome P450 monooxygenases (P450s) promote hydroxylations in a broad variety of substrates. Their prowess in C-H bond functionalization renders P450s promising catalysts for organic synthesis. However, operating P450 reactions involve complex management of the main substrates, O₂ and nicotinamide adenine dinucleotide phosphate (NAD(P)H) reducing equivalents against an overall background of low operational stability. Whole-cell biocatalysis, although often used, offers no general solution to these problems. Herein, we present the design of a tailor-made, self-sufficient, operationally stabilized and recyclable P450 catalyst on porous solid support. Using enzymes as fusion proteins with the polycationic binding module Z_basic2 , the P450s BM3 (from Bacillus megaterium) was coimmobilized with glucose dehydrogenase (type IV; from B. megaterium) on anionic sulfopropyl-activated carrier (ReliSorb SP). Immobilization via Z_basic2 enabled each enzyme to be loaded in controllable amount, thus maximizing the relative portion of the rate limiting P450 BM3 (up to 19.5 U/g_carrier ) in total enzyme immobilized. Using lauric acid as a representative P450 substrate that is poorly accessible to whole-cell catalysts, we demonstrate complete hydroxylation at low catalyst loading (≤0.1 mol%) and efficient electron coupling (74%), inside of the catalyst particle, to the regeneration of NADPH from glucose (27 cycles) was achieved. The immobilized P450 BM3 showed a total turnover number of ∼18,000, thus allowing active catalyst to be recycled up to 20 times. This study therefore supports the idea of practical heterogeneous catalysis by P450s systems immobilized on solid support.

Keywords: cytochrome P450; fatty acids; glucose dehydrogenase; heterogeneous biocatalysis; immobilization.

Figure 1

Immobilization of individual enzymes on ReliSorb SP carrier. For each step, immobilization is shown in terms of immobilized enzyme and immobilization yield (%) for Z_P450 BM3 (a) and Z_GDH (b). Error bars show standard deviations from four independent experiments

Figure 2

Characterization of immobilized Z_P450 BM3. (a) Shows the time course of anisole hydroxylation from the initial reaction phase using 0.2 mM NADPH. (b) Shows the dependency of immobilized Z_P450 BM3 activity on the NADPH concentration. The reaction mixture contained 20 mM anisole and 6 mg carrier (containing 0.6 nmol enzyme) in 1 ml of 50 mM potassium phosphate; 25°C, pH 7.5.NADPH: nicotinamide adenine dinucleotide phosphate reduced form

Figure 3

Characterization of immobilized Z_GDH. The dependency of Z_GDH activity on the NADP⁺ concentration is shown in (a) for soluble Z_GDH and in (b) for immobilized Z_GDH. The reaction mixture contained 200 mM glucose and 0.03 U (0.014 nmol) of soluble or immobilized enzyme (0.02 mg carrier) in 1 ml of 50 mM potassium phosphate; 25°C, pH 7.5. NADP⁺: nicotinamide adenine dinucleotide phosphate

Figure 4

Coimmobilization of Z_P450 BM3 and Z_GDH. The immobilization of the individual enzymes is shown

Figure 5

Time courses of anisole hydroxylation by the enzyme coimmobilizate. The reaction was performed in the presence (•) and in the absence (○) of catalase. The reaction mixture contained 0.39 U/ml Z_P450 BM3 (1.8 µM), 14 U/ml Z_GDH, 1000 U/ml catalase, 20 mM anisole, 0.8 mM NADP⁺, and 200 mM glucose in 50 mM potassium phosphate, 25°C, pH 7.5

Figure 6

Time courses of lauric acid hydroxylation for the soluble enzymes and the enzyme coimmobilizate. The symbols show soluble enzymes (○) and the coimmobilizate (•). The reaction mixture contained 0.24 U/ml Z_P450 BM3 (0.36 µM), 2.8 U/ml Z_GDH, 1000 U/ml catalase, 2 mM lauric acid, 0.1 mM NADP⁺, and 200 mM glucose in 50 mM potassium phosphate, 25°C, pH 7.5

Figure 7

Reuse of the enzyme coimmobilizate. The reaction mixture contained 1.2 U/ml Z_P450 BM3 (1.8 µM), 14 U/ml Z_GDH, 1000 U/ml catalase, 2 mM lauric acid, 0.8 mM NADP⁺, and 200 mM glucose in 50 mM potassium phosphate, 25°C, pH 7.5. For each cycle, the carrier was separated by centrifugation from the reaction mixture, washed with 4 ml loading buffer and added to a new reaction mixture