(650h) Design of a Heterogeneous Biocatalyst for Cofactor Regeneration and Improved Catalytic Characteristics

Enzyme immobilization generally allows for improved lifetime and stability of catalytically active protein. Typically, immobilization systems involve adsorption to surfaces, cross-linked enzyme aggregates or entrapment of enzyme in a polymer network. However, these systems often can introduce unwanted limiting effects, such as high resistance to mass transfer or distortion in the enzyme structure due to adsorption or crosslinking. To overcome these shortcomings, it is possible to use more porous materials which are imbued with immobilization domains. Supraparticles formed by a hierarchically structured self-assembly of protein-inorganic nanoflowers provide a porous and stable support for enzyme capture with a high surface area and low mass transfer resistance. In this work, we used hybrid protein-inorganic supraparticles made of calcium phosphate and protein with leucine zipper binding domains for high-affinity immobilization of fusion proteins containing the complementary binding domain and enzymes. Particularly, we showed the modularity of the supraparticle system by immobilizing two distinct enzymes on the particle in tandem.

As a model system, we used the amine dehydrogenase, cFL1AmDH, which is coupled for cofactor regeneration with formate dehydrogenase, cbFDH. The cFL1AmDH oxidizes NADH to convert 4-fluorophenylacetone and ammonia to 4-fluoroamphetamine, an important chiral intermediate. However, the enzyme lacks desirable kinetic characteristics and reaction condition, given a current optimum at 5M ammonium chloride and a pH of 9.6. The cbFDH regenerates NADH from NAD+ by oxidizing formate to carbon dioxide, reducing the cost associated with replacing an expensive cofactor. By co-immobilizing these enzymes, we seek to improve the activity of cFL1AmDH by supplying a constant source of NADH. To account for the effects of alkalinity and ionic strength from the optimal cFL1AmDH reaction, the supraparticles were synthesized with several concentrations of ammonia and at neutral and alkaline pHs. The particles synthesized under the reaction conditions proved to be comparable to the neutral, ammonia-free synthesis in size and number. We then generated a set of fusion proteins with four different linker peptides connecting the catalytic domain to the immobilization domain and tested their activity. After selecting the enzymes with the highest specific activity, we incubated them with the particles and quantified the enzyme immobilization and specific activity. The particle porosity, effectiveness factor, and Thiele modulus were quantified to determine the effects of the particles on mass transfer. This work demonstrates our ability to create an industrially relevant heterogeneous biocatalyst which incorporates cofactor regeneration, is stable in enzymatically relevant conditions, and allows for enzyme recycling.

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