对三羟甲基磷交联Thermus oshimai葡萄糖异构酶重组菌的固定化工艺进行研究。确定所用硅藻土载体粒径36.8 μm、聚凝剂聚乙烯亚胺分子质量为70 000 Da、交联剂合成前体为四羟甲基硫酸磷；运用最优的固定化条件：0.3 g硅藻土、5 mmol/L Mn2+、0.12%(体积分数)聚乙烯亚胺絮凝1 h、1.5%(体积分数)三羟甲基硫酸磷交联1.5 h，所制备的固定化细胞酶活可达138.4 U/g。应用该催化剂于85 ℃和1 100 mmol/L D-葡萄糖底物条件下连续催化10批次，催化剂仍保留91%以上酶活，D-果糖转化率始终维持在51.7%以上。该工艺可以连续生产高果糖浓度高果糖浆，有效简化后续分离提取工艺，为进一步使用符合食品工业要求的表达系统连续制备高果糖浆奠定基础。

Glucose isomerase is capable for catalyzing D-glucose to form D-fructose. The obtained high fructose corn syrup (HFCS) is widely served as sweetener in various food fields. This study addressed the immobilization conditions optimization of recombinant Escherichia coli BL21 (DE3)/pET28b/ToGI using tetrakis(hydroxylmethyl)phosphonium as crosslinker. It was confirmed that the optimum particle diameter of diatomite carrier was 36.8 μm, the optimum molecular weight of polyethylene imine was 70000, and the optimum forming precursor of crosslinker was tetrakis(hydroxylmethyl)phosphonium sulfate. Furthermore, the optimum immobilized process was determined at the condition of 0.3 g carrier, 5 mmol/L Mn2+, 0.12% (v/v) polyethylene imine flocculation for 1.0 h, and 1.5% (v/v) tetrakis(hydroxylmethyl)phosphonium sulfate crosslinking for 1.5 h. The activity of the resulting immobilized cells reached 138.4 U/g. After 10 batches of bioconversion at 85 ℃ and 1 100 mmol/L D-glucose, the biocatalyst still exhibited at least 90.3% of initial activity and all the yield of D-fructose was above 51.7%. Successive production of HFCS with higher D-fructose using this craft has the advantage of simplifying the subsequent isolation and extraction step, and it has an important reference value for industrial application in HFCS production using food safety expression system.