Preparation of cytochrome P450 enzyme-cobalt phosphate hybrid nano-flowers for oxidative coupling of benzylamine
Introduction
Cytochrome P450 s (CYP450 s) pervasively exist in animals, plants, fungi, protists, bacteria, archaea, and even in viruses [1], and attract extensive attention in many disciplines, mainly due to their unique catalytic properties in a wide scope of reactions, including hydrocarbon hydroxylation, olefin epoxidation/olefin oxygenation, alcohol/aldehyde oxidation, organic nitrogen/sulfide oxidation, etc [2,3]. However, the direct usage of CYP450 enzymes as catalysts usually suffers from high cost, easy inactivation and poor recyclability, which obviously hinder their industrial applications.
To overcome these disadvantages, various methods of enzyme immobilization have been developed [[4], [5], [6], [7]]. Among them, an early breakthrough reported by Ge et al. [8] prepared bovine serum albumin-copper phosphate hybrid nano-flowers with a low cost and facile coprecipitation method. Surprisingly, the hybrid materials exhibited enhanced activity and stability compared with the corresponding free enzymes, on account of their large surface-to-volume ratio and unique structure. Since then, various enzyme-inorganic hybrid materials, e.g. laccase–Cu3(PO4)2 [9], α-chymotrypsin–Ca3(PO4)2 [10], immunoglobulin G–Mn3(PO4)2 [11], papain–Zn3(PO4)2 [12] and bovine serum albumin–Co3(PO4)2 [13], were fabricated with the similar methods and most of them showed excellent catalytic properties, as reviewed thoroughly by Cui et al. [14] and Lei et al. [15]. Among the phosphates mentioned above, cobalt phosphate was frequently used as inorganic component in the hybrid materials, mainly because cobalt species were very active in the decomposition of peroxides, e.g. t-BuOOH and H2O2, to induce the radical chain reactions [[16], [17], [18]].
Imines are a kind of important chemical intermediates widely used in the synthesis of various biologically active compounds [11], and the oxidative coupling of amines is one of the most promising synthetic routes [12]. Therefore we select CYP450 2C9 and cobalt phosphate as organic component and inorganic one, respectively, to build the hybrid nano-flowers while the oxidation of benzylamine was selected as the model reaction to evaluate their catalytic performance. The hybrid nano-flowers exhibited superior catalytic performance that 99.9% benzylamine conversion and 97.9% selectivity to N-benzylidenebenzylamine were obtained under mild reaction conditions and the catalytic properties kept stable for at least 5 catalytic runs. The catalytic mechanisms were also investigated that the surface enzyme was ascribed to be the key active species, and radical intermediates were indispensable in the catalytic conversion process.
Section snippets
Experimental
A simple co-precipitation method [8] was adopted in the preparation of cytochrome CYP450-cobalt phosphate hybrid nano-flowers. In a typical synthesis, a certain amount of CYP450 2C9 (Sigma-Aldrich) was added to 20 mL of PBS buffer (Biosharp). Then 1 mL of 0.05 mol·L−1 cobalt nitrate (Aladdin) was introduced into the solution. After incubation for 72 h, the precipitations were obtained by centrifugation, washed with deionized water for three times and then dried in vacuum at 298 K.
The morphology
Results and discussion
The morphology of the as-obtained precipitates was shown in Fig. 1(a). The hybrid materials possessed a uniform and flower-like morphology with ca. 25 μm average diameter, similar to other protein - inorganic nano-composits in previous reports [8,19,20]. Hundreds of triangular nano-petals with ca. 1 μm average thickness were linked to each other tightly. The XRD pattern for CYP450 - Co3(PO4)2 hybrid nano-flowers was presented in Fig. 1(b), and the weak characteristic peaks at 26.8°, 30.31° and
Conclusions
In summary, a facile copercipitation method has been employed for the synthesis of CYP450 enzyme-cobalt phosphate hybrid nano-flowers. And such hybrid nano-flowers displayed superior catalytic performance in the oxidative coupling of benzylamine with high conversion, satisfactory selectivity and excellent recyclability. Furthermore, the hybrid nano-flowers with CYP450 and cobalt phosphate exhibited higher conversion than those with CYP450 and other phosphate salts, mainly ascribed to the unique
Declaration of Competing Interest
The authors declare no conflicts of interest.
Acknowledgments
This work was supported by the National Science for Distinguished Young Scholars of China (No. 21425617), the National Natural Science Foundation of China (Nos. 21606260, 21576302, 21376278, 21808247), the NSF of Guangdong Province (2015A030313104), the Fundamental Research Funds for the Central Universities of Sun Yat-sen University (15lgjc33).
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