The common goal of various protein immobilization techniques has been to bypass the drawbacks of utilizing free enzymes as catalytic materials in industry. Crosslinked enzyme aggregates (CLEAs) have greatly improved the storage and operational stability of enzyme preparations as well as permitted their easy recovery. The general applicability of typical CLEA methods has occasionally been challenged by protein-specific anomalies, reflecting structural and functional traits, altering the effectiveness of aggregation and crosslinkability, as well as the resultant bioactivity of the material. In this work, the described limitations have been addressed using a CLEA-unfriendly protein starting material, namely, native urease. In this case, conventional CLEA methods led to low aggregation and crosslinking yields, and displayed insignificant catalytic activity in the immobilized product. Critical breakthrough was achieved by enforcing protein aggregation via lyophilization as opposed to routine precipitation. The subsequent crosslinking of the lyophilizate (yielding a crosslinked enzyme lyophilizate) in a suitable saturated salt solution/anti-solvent led to a much improved crosslinking yield and catalytic activity. Furthermore, urease CLELs have been shown to sustain activity and stability in organic solvents and temperatures nearing 100 oC. Conventional syntheses of industrially important reagents such as dimethyl carbonate, ethylene carbonate and carbodihydrazide are challenged by factors such as low efficiency due to side reactions, use of toxic starting materials, high energy input, and inconvenient reaction conditions. In view of the strategic importance of such compounds, alternative production methods boasting higher productivity and lower cost remain a subject of much interest. Urease CLELs were demonstrated to prompt formation of the above desired products by enforcing reaction between the inexpensive substrate urea, and a non-water nucleophile such as methanol, ethylene glycol or hydrazine. These results lead to assume the general utilization opportunity of similar heterogeneous catalyst systems in organic reactions to afford greener and more economic syntheses of many industrially important commodity compounds.

Urease Crosslinked Enzyme Lyophilizates (CLELs) streamlining conventional organic syntheses

253rd National Meeting (2017)

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