Studies On The Immobilization And Properties Of Carbonic Anhydrase

Carbonic anhydrase ( Carbonic anhydrase, CA,EC 4.2.1.1 ) is a zinc metalloenzyme,can effectively catalyze the reversible hydration reaction of CO 2. It is one of the highest catalytic rates of all known enzymes, and plays a significant role in biological processes such as photosynthesis, calcification, pH homoeostasis, and ion transport. Because the free enzyme is difficult to be used repeatedly in a batch reactor and can't be used in continues and automatic operations, it is necessary to immobilize it. This paper studied immobilization supports, immobilization methods and the properties of the immobilized enzyme.Using acidic alumina as adsorption carrier and glutaraldehyde as crosslinking agent, the optimal immobilization conditions were: adsorption temperature 25℃, adsorption time 6 h, pH 8.0, the load of enzyme 4 mg/g(support), the concentration of glutaraldehyde 0.2%, crosslinking time 1 h, crosslinking temperature 25℃. Under these conditions, the immobilized enzyme activity was 0.31 U/g and the activity recovery was 34.08%. The optimum reaction temperature of the immobilized enzyme was 30℃, which was 5℃higher than the free enzyme; the optimum reaction pH was 8.0, which was the same as the free enzyme. The thermal stability, pH stability, operational stability and storage stability of the immobilized enzyme were improved considerably.Carbonic anhydrase was immobilized on alkylated celite by means of covalent binding. The orthogonal experiment was designed to optimize reaction temperature, reaction time, pH, and the load of enzyme. The results showed that: the load of enzyme and reaction time had greater impacts on the activity of the immobilized enzyme. The optimal immobilization conditions were: the load of enzyme 6 mg/g(support), reaction time 16 h, reaction temperature 37℃, pH 7.0, the immobilized enzyme activity and the activity recovery were 0.46 U/g and 33.71%, respectively. The optimum reaction pH of the immobilized enzyme was 6.0, its optimum reaction temperature was 30℃, which was 5℃higher than the free enzyme. The pH stability of the immobilized enzyme increased; its thermal stability was also wider. At 60℃for 1 h, it still remained 54.19% of the relative activity. The operational stability had also significantly increased, the relative activity of the immobilized enzyme remained 45.58% after 6 time's uses.Sodium alginate-polyvinyl alcohol (PVA) was also used to immobilize carbonic anhydrase by the embedding-crosslinking method. The better immobilized enzyme was obtained under these conditions: sodium alginate concentration 2%, PVA concentration 6%, CaCl2 concentration 2%, glutaraldehyde concentration 1%, solidification time 40 min, the load of enzyme 10 mg/mL sodium alginate-PVA solution, crosslinking time 1 h. The immobilized enzyme activity was 0.28 U/g and the activity recovery was 10.20%. The optimum reaction pH of the immobilized enzyme was 7.0, the optimum reaction temperature was 30℃. The immobilized enzyme had good acid resistance, thermal stability and operational stability.