分别利用强碱性大孔树脂、氨基树脂、环氧树脂作为葡萄糖氧化酶（GOD）和过氧化氢酶（CAT）固定化载体制备固定化酶，并研究其在低pH值葡萄糖酸生产工艺中的应用效果。耐酸性试验表明固定化酶较游离酶具有更强的耐酸性能。分别利用游离酶和3种固定化酶在低pH值（pH值3.5）条件下制备葡萄糖酸，强碱性大孔树脂、氨基树脂和环氧树脂固定化酶所需反应时间分别为40 h、24 h和27 h，酶活损失率分别为46.44%、3.42%和21.84%，而游离酶在反应过程中完全失活。3种固定化酶反应液的澄清度及色度均显著优于游离酶反应液。正交试验进一步优化后氨基树脂固定化酶的制备工艺为混合酶液浓度10%、GOD:CAT=1.5:1、固定温度25 ℃。在该条件下，氨基树脂固定化酶的酶活回收率可达93.15%，转化100 g/L葡萄糖溶液所需时间为23 h，反应结束时葡萄糖酸:葡萄糖酸钠可达0.899:1（m/m）。并且氨基树脂固定化酶稳定性良好。因此，利用氨基树脂固定化酶可以建立低pH值固定化酶葡萄糖酸生产工艺，实现高品质葡萄糖酸产物的生产。

Immobilized enzymes were prepared by using strongly alkaline macroporous resin, amino resin, and epoxy resin as immobilized carriers of glucose oxidase and catalase, respectively, and their effect on low-pH gluconic acid production was assessed. The results of the acid resistance test showed that all the three immobilized enzymes had stronger acid resistance than did the free enzyme. The free enzyme and the three immobilized enzymes were used to prepare gluconic acid under low pH conditions (pH value 3.5). The reaction times required for the strongly alkaline macroporous resin-immobilized enzyme, amino resin-immobilized enzyme, and epoxy resin-immobilized enzyme were 40, 24, and 27 h respectively. The enzymatic activity loss rates were 46.44%, 3.42%, and 21.84%, respectively. The free enzyme was completely inactivated during the reaction. The clarity and chromaticity of the three immobilized enzyme reaction solutions were significantly better than those of the free enzyme reaction solutions. The preparation process of amino resin-immobilized enzyme-after further optimization through orthogonal experiments - was as follows: the concentration of mixed enzyme solution was 10%, the ratio of glucose oxidase to catalase was 1.5:1, and the fixation temperature was 25 ℃. Under these conditions, the recovery rate of amino resin-immobilized enzyme was as high as 93.15%, and the conversion time of the 100 g/L glucose solution was 23 h. At the end of the reaction, the ratio of gluconic acid to sodium gluconate had reached 0.899:1 (m/m), and the amino resin-immobilized enzyme was stable. Based on these results, we conclude that the amino resin immobilized enzyme can be used to establish a low-pH immobilized enzyme gluconic acid production process, with the goal of producing high-quality gluconic acid products.

山东省科学院-商河县产学研协同创新基金项目（2019-CXY15）；国家自然科学基金项目（32102454）；山东省重点研发计划（重大科技创新工程）项目（2020CXGC010602）；齐鲁工业大学生物及生物化学ESI培育学科开放课题（ESIBBC202012）；齐鲁工业大学（山东省科学院）科教产融合创新试点工程项目（2020KJC-ZD15）