高热稳定性己糖激酶在大肠杆菌中的表征和表达优化

Characterization and expression optimization of a highly thermostable hexokinase in Escherichia coli

摘要:

己糖激酶是血糖检测中的重要诊断试剂，因此对其酶活和热稳定性要求较高。目前国内己糖激酶主要依赖进口，大多是酵母来源酶，其价格昂贵且存在热稳定性较差等问题，限制了国内血糖诊断试剂的开发。因此，当前亟待实现高活性、高热稳定性己糖激酶的高效表达。本研究在大肠杆菌(Escherichia coli) BL21(DE3)中异源表达了一种来源于嗜热菌的ATP依赖型己糖激酶(glucokinase, Glk)，发现其对葡萄糖特异性高、依赖Mg²⁺，最适pH和温度分别为8.5和80 ℃，在30-37 ℃下保存7 d后酶活保留90%以上，属于热稳定的偏碱性葡萄糖激酶。随后系统优化了Glk表达的培养基、诱导时机、诱导剂终浓度、诱导温度、诱导时长等因素，优化后Glk表达量相比于优化前提高了4.71倍。Glk纯化后，比酶活达到(43.05±2.00) U/mg，纯度达到95%以上。本研究开发的高热稳定己糖激酶的表达和纯化方法，为突破血糖诊断试剂制备中的短板提供了更多可能性和发展空间。

Abstract:

Hexokinase is a crucial diagnostic reagent in blood glucose testing, which has high requirements for the enzyme activity and thermal stability. The hexokinases in China mainly rely on imports and are primarily sourced from yeast, with high costs and poor thermal stability, which limit the development of blood glucose diagnostic reagents. Therefore, there is an urgent need for the efficient expression of highly active and thermally stable hexokinases. In this study, an ATP-dependent hexokinase (glucokinase, Glk) from a thermophilic bacterium Glk was heterologously expressed in Escherichia coli BL21(DE3). Glk exhibited high specificity for glucose, dependence on Mg²⁺, and the highest activity at pH 8.5 and 80 ℃. It retained over 90% activity after storage at 30–37 ℃ for 7 days, demonstrating thermal stability as an alkaline glucose kinase. Subsequently, the factors influencing Glk expression, including culture medium, OD₆₀₀, final concentration of the inducer, induction temperature, and induction duration, were systematically optimized. The optimization increased the Glk expression by 4.71 folds Glk compared with non-optimized conditions. After purification, Glk exhibited a specific activity of (43.05±2.00) U/mg and the purity ≥98%. In conclusion, the developed expression and purification method for the highly thermostable hexokinase provides more possibilities for overcoming the shortcomings in the preparation of blood glucose diagnostic reagents in China.