利用3D生物列印固定化碳酸酐酶以提升固碳效率

Abstract

碳酸酐酶是一種催化CO2水合反應之含鋅金屬酶，透過兩步驟生化反應進行，包含鋅金屬上氫氧根離子對CO2之親核攻擊，產生碳酸氫根基團，以及水分子對鋅位點之再生。此類型酵素常見於生物體內，能調節酸鹼環境，並具有高轉換效率與CO2專一性，在碳捕捉技術上具開發潛力。然而，游離態酵素容易因環境變化而影響催化活性，導致酵素失活，故需透過固定化程序提高實用性。而使用包埋法進行酵素固定化能有效保持酵素活性且具備良好接枝穩定性，且結合3D生物列印技術能進一步打造載體形狀之多樣性，適用於碳捕捉材料之開發。 本研究試著利用3D生物列印技術進行酵素固定化，比較不同負載情形下之活性表現，並將載體置於兩種曝氣類型之反應槽內，試著分析氣、液相中無機碳，以及碳酸鈣沉澱量，綜合評估載體固碳效率。實驗結果顯示，酵素分布於載體之交聯結構孔洞中，顯示此類型三維結構能良好的負載酵素並表現活性。低酵素負載時，載體表現出高催化效率，比活性達最高207.49 WAU/mg CA，在經過14天乾式保存後仍保有71.47%活性，並在6次循環使用後保留84.98%酵素活性，展現良好酵素穩定性。另外，酵素負載增加能有效提升熱穩定性，負載最高之CA-3D-4 (0.171 mg/g)於50℃時表現最佳相對活性。此外，通過比較直接注入式及奈米曝氣式反應槽，可知若引入奈米氣泡能有效增強酵素催化CO2水合反應。藉由碳酸鈣沉澱結果得知進一步驗證低負載酵素之高催化效率，CA-3D-1 (0.028 mg/g)載體在直接注入式反應槽中表現出2724.4 mg CaCO3/mg CA之沉澱量，而在奈米氣泡式反應槽中更達到3016.0 mg CaCO3/mg CA之沉澱量。

Carbonic anhydrase is a zinc-containing metalloenzyme that catalyzes the hydration of CO2 through a two-step biochemical reaction. This process involves a nucleophilic attack of the hydroxide ion coordinated on the zinc metal center, producing bicarbonate ions, followed by the regeneration of the zinc-bound water molecule. This type of enzyme is commonly found in living organisms, where it regulates acid-base balance and exhibits high catalytic efficiency and CO2 specificity. It also holds great potential for development in carbon capture technologies. However, free enzymes are prone to activity loss due to environmental changes, resulting in enzyme deactivation. Therefore, immobilization techniques are necessary to enhance their practical applicability. Among these, enzyme immobilization by entrapment can effectively maintain enzyme activity and provide good grafting stability. When combined with 3D bioprinting technique, it allows for greater diversity in carrier shapes, making it suitable for the development of CO2 capture materials. This study attempts to immobilize the enzyme using 3D bioprinting technique, comparing the activity performance under different enzyme loading. The carriers were placed in two types of reactors, inorganic carbon analysis was conducted in the gas and liquid phase system, combined with calcium carbonate precipitation test to evaluate the carbon fixation efficiency of the carriers. Experimental results show that the enzyme is distributed within the crosslinked pores of the carrier, indicating that this type of three-dimensional structure can effectively load the enzyme and maintain its activity. At low enzyme loading, the carrier exhibited high catalytic efficiency, with a maximum specific activity of 207.49 WAU/mg CA. After 14 days of dry storage, it retained 71.47% of its activity. In reusability test, after six cycles, it preserved 84.98% of enzyme activity, demonstrating good enzyme stability. Additionally, increasing enzyme loading effectively improved thermal stability, with the highest enzyme loaded CA-3D-4 carrier (0.171 mg/g) showing the best relative activity at 50°C. Furthermore, by comparing direct injection and nano-bubble aeration reactors, it was found that the nano-bubbles can effectively enhance the enzyme-catalyzed CO2 hydration reaction. The calcium carbonate precipitation results further verified the high catalytic efficiency of the low-loaded enzyme. It shows that the CA-3D-1 carrier (0.028 mg/g) produced 2724.4 mg CaCO3/mg CA in the direct injection reactor and reached 3016.0 mg CaCO3/mg CA in the nano-bubble reactor.