Abstract: The present study entails the immobilization of carbonic anhydrase (CA) on geopolymer microspheres (GMS) and the enzymatic properties of free and immobilized enzyme. Carbonic anhydrase, a zinc-containing metalloenzyme which can catalyze the hydration of CO2 into bicarbonate. It is ubiquitous in animals and plants and has at least five distinct categories. Featured like other enzymes, it has the nature of high catalytic efficiency, excellent specificity and mild reaction conditions. However, the practical applications of CA are confronted with the challenge of the intrinsic fragile nature of enzyme in harsh environment, which makes them vulnerable to detrimental structural changes in industrial conditions, leading to inevitably denaturation, inactivation, poor stability, and laborious recycling. The enzyme immobilization technology overcomes the above problems. The immobilization technology is defined as the enzyme can be reused and recycled while still maintainlng the catalytic capability when it was confined or bound in a certain area by solid materials. The immobilized enzyme integrates the characteristics of mild reaction conditions, high efficiency and specificity, and makes up for the deficiency of free enzyme owing to its superiority in enhancing stability and overcoming the obstacle of enzyme recycling. In this research, geopolymer, a kind of green inorganic gel material, was used as carrier to immobilize carbonic anhydrase with NaOH as alkali activator and glutaraldehyde (GA) used as cross-linking agent by suspension dispersion solidification method. The immobilization efficiency of carbonic anhydrase was evaluated by enzyme activity recovery and relative enzyme activity. At the same time, the micro-morphology and structural changes of the carrier and the immobilized enzyme were compared and analyzed by different characterization methods. The optimal immobilization conditions were found out to be pH value, 8.0; temperature, 30 ℃; immobilization time, 2 h; GMS dose, 100 mg; GA concentration, 0.2wt%; cross-linking time, 1 h. Under these conditions, the highest recovery of activity of immobilized carbonic anhydrase can reach 55.9%.Compared with free CA, the optimum pH of the immobilized enzyme increased from 7.5 to 8.0 and the optimum temperature increased from 25 ℃ to 30 ℃. The storage stability of immobilized CA was better than its free counterpart at the circumstance of 4 ℃ and 25 ℃, respectively, and the relative activity of the immobilized enzyme remained 46.1% after 5 cycles of continuous operation. The Michaelis constant (Km) and maximum reaction rate in enzymatic reaction (Vmax) of free CA were determined to be 7.63 mmol·L-1 and 1.60 mmol·L-1·min-1, while that of the immobilized CA were 21.55 mmol·L-1 and 5.05 mmol·L-1·min-1, respectively. The overall activity (Kcat/Km) of them were 61.50 mol-1·L·s-1 and 12.36 mol-1·L·s-1, respectively.
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2023, Vol. 37 
