海藻酸钙凝胶包埋乳酸氧化酶催化DL-乳酸生产丙酮酸

Preparation of Pyruvate from DL-Lactate by Immobilized Lactate Oxidase on Calcium Alginate

研究了用海藻酸钠包埋?戊二醛交联法固定耻垢分枝杆菌(Mycobacterium Sm egmatis)生成乳酸氧化酶的最佳条件,比较了原酶与固定化酶的酶学性质。将1 mL酶液和1 mL质量分数为3%的海藻酸钠溶液的混合液,用注射器滴加到20 mL 0.2 mol/L的CaC l2溶液中,25℃静置固化2 h后,过滤洗涤,将固体转移至20 mL质量分数为0.2%戊二醛溶液中37℃交联2 h后,过滤、洗涤和干燥得到球状固定化酶。固定化酶的活力回收率为39.8%。酶学性质研究表明,此固定化酶的热稳定性较好,游离酶在65℃保温1 h酶蛋白完全变性失活,而固定化酶在65℃保温1 h仍可保持86%的酶活力;其最适酶促反应温度可由37℃升至55℃,最适反应pH=7.4保持不变;在不加保护剂的条件下,4℃放置50 d后游离酶仅保持40%以上的酶活力,而固定化酶能保持80%以上的酶活力。该固定化乳酸氧化酶用于催化氧化DL-乳酸生产丙酮酸,3 h后丙酮酸产率可达75%,连续循环使用5次固定化酶活力仍保持85%。

The optimum conditions of immobilized Lactate Oxidase with sodium alginate and ross-linked with glutaraldehyde was studied. The Lactate Oxidase was compared with its immobilized enzyme in some properties. Enzyme of 1 mL and 1 mL of 3% sodium alginate were mixed and the mixture was added dropwise into 20 mL of a 0.2 mol/L CaCl2 solution to solidify for 2 h at 25 ℃. Then the precipitate was cross-linked in the solution of 20 mL of 0.2% glutaraldehyde for 2 h at 37 ℃. After filtration and drying, the immobilized enzyme beads were obtained with a activity recovery of 39. 8% for the total enzyme added. The thermal stability of the immobilized enzyme was tested. The free enzyme lost all its activity when heated at 65℃ for 1 h but the immobilized enzyme still kept 86% of the original activity. The optimal reaction temperature of the immobilized enzyme was 55 ℃ compared to 37 ℃ of free Lactate Oxidase, the optimal reaction pH of the immobilized enzyme was 7.4 as the same as that of the free enzyme. Stored at 4 ℃ for 50 days without any protection by reagent, the free enzyme only kept 40% of the original activity but the immobilized enzyme still retained 80% of the original activity. The calcified preparation of pyruvate from DL-Lactate by immobilized Lactate Oxidase, the yield could reach 75% after 3 h, and the immobilized enzyme maintained 85% of the activity after having been reused 5 times.