Preparation And Application Of Cross-linked Enzyme Aggregates Of Nitrile Hydratase

Nitrile substrates can be readily converted to the corresponding high value amides under mild conditions through the catalysis of nitrile hydratase(NHase, EC 4.2.1.84). For example, acrylonitrile could be translated into acrylamide and 3-cyanopyridine could be translated into vitamin nicotinamide through the catalysis of microbial cells or free isolated NHase. Although the whole-cell catalyzed processes have been widely used in biotransformation of nitriles and amides, the transformations that catalyzed by isolated enzymes are usually preferred. Compared with the whole cells, isolated enzymes can exhibit stringent chemo-, regio- and enantio-selectivity and broad substrate specificity. However, the use of free isolated NHase is often hampered by several limitations such as high costs, low pH, thermal and operational stability, and difficulties in recovery and reuse.Immobilization provides one of the most attractive concepts to overcome these drawbacks and to simplify the biocatalyst recycling and downstream processing. Cross-linked enzyme aggregates(CLEAs) technique is a carrier-free immobilization method and can be widely used because of simple operation and low cost. In order to improve the activity recovery of NHase, macromolecular cross-linker(dextran polyaldehyde) was employed to prepare CLEAs in this study. Spherezymes were also formed with cross-linked enzyme aggregates technique and spheroidizing enzyme technology. Compared with CLEAs, the controllability and dispersion of spherezyme were improved.The main researches in this study were summarized as follows:(1) CLEAs of NHase were prepared by using dextran polyaldehyde as cross-linker. The optimal conditions of the immobilization process were determined, NHase CLEAs with 49.63% of activity recovery were obtained. The NHase CLEAs exhibited increased stability at varied p H, temperature and storage conditions compared to its free counterpart. When exposed to high concentrations of acrylamide, NHase CLEAs also exhibited effective catalytic activity. After recycling eight times, 47.67% of its original activity was retained. The michaelis constants of free NHase and NHase CLEAs were 1.596 mmol/L and 2.068 mmol/L, respectively.(2)Spherezymes lost most of the activity when prepared with spheroidizing enzyme technology. The egg-white protein was spheroidized firstly, NHase was cross-linked outside of the balls with dextran polyaldehyde. After optimizing the preparation conditions, Spherezymes with over 50% of activity recovery were obtained. The individual spherical particles had a diameter of between 8–20 μm, the stabilities of spherezymes were better than free NHase.(3) CLEAs and Spherezymes were employed to catalyze 3-cyanopyridine converted to nicotinamide. High catalysis efficiency was showed when 50 mmol/L of 3-cyanopyridine catalyzed by 1.6 mg/mL of enzyme. The reaction was stable under pH 7 and 30 oC. After recycling 10 times, CLEAs and Spherezymes retained 73.45% and 61.26% of their original activity, respectively.