Bovine enterokinase light chain (EK_L) is an industrially useful protease for accurate removal of affinity-purification tags from high-value biopharmaceuticals. However, recombinant expression in Escherichia coli produces insoluble inclusion bodies, requiring solubilisation, refolding, and autocatalytic activation to recover functional enzyme. Error-prone PCR and DNA shuffling of the EK_L gene, T7 promoter, lac operon, ribosome binding site, and pelB leader sequence, yielded 321 unique variants after screening ~ 6500 colonies. The best variants had > 11,000-fold increased total activity in lysates, producing soluble enzyme that no longer needed refolding. Further characterisation identified the factors that improved total activity from an inactive and insoluble starting point. Stability was a major factor, whereby melting temperatures > 48.4 °C enabled good expression at 37 °C. Variants generally did not alter catalytic efficiency as measured by k_cat/K_m, which improved for only one variant. Codon optimisation improved the total activity in lysates produced at 37 °C. However, non-optimised codons and expression at 30 °C gave the highest activity through improved protein quality, with increased k_cat and T_m values. The 321 variants were statistically analysed and mapped to protein structure. Mutations detrimental to total activity and stability clustered around the active site. By contrast, variants with increased total activity tended to combine stabilising mutations that did not disrupt the active site.

牛肠激酶轻链 (EK _L ) 是一种工业上有用的蛋白酶，可用于从高价值生物制药中准确去除亲和纯化标签。然而，大肠杆菌中的重组表达会产生不溶性包涵体，需要溶解、重折叠和自催化激活才能恢复功能性酶。_{EK L}基因、T7 启动子、lac的易错 PCR 和 DNA 改组操纵子、核糖体结合位点和 pelB 前导序列在筛选 ~ 6500 个菌落后产生了 321 个独特的变体。最好的变体在裂解物中的总活性增加了 > 11,000 倍，产生了不再需要重新折叠的可溶性酶。进一步的表征确定了从非活性和不溶性起点提高总活性的因素。稳定性是一个主要因素，熔解温度 > 48.4 °C 可在 37 °C 下实现良好表达。变体通常不会改变通过k _cat / K _{m测量的催化效率}，仅针对一个变体进行了改进。密码子优化提高了 37 °C 下产生的裂解物的总活性。然而，未优化的密码子和在 30 °C 下的表达通过提高蛋白质质量以及增加的k _cat和T _m值提供了最高的活性。对 321 个变体进行了统计分析并映射到蛋白质结构。对总活性和稳定性有害的突变聚集在活性位点周围。相比之下，总活性增加的变体倾向于结合不破坏活性位点的稳定突变。