Arnold F H, Moore J C
Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena 91125, USA.
Adv Biochem Eng Biotechnol. 1997;58:1-14. doi: 10.1007/BFb0103300.
Enzymes can be tailored for optimal performance in industrial applications by directing their evolution in vitro. This approach is particularly attractive for engineering industrial enzymes. We have created an efficient para-nitrobenzyl esterase over six generations of random point mutagenesis and recombination coupled with screening for improved variants. The best clones identified after four generations of sequential random mutagenesis and two generations of random recombination display more than 150 times the p-nitrobenzyl esterase activity of wild type towards loracarbef-p-nitrobenzyl ester in 15% dimethylformamide. Although the contributions of individual effective amino acid substitutions to enhanced activity are small (< 2-fold increases), the accumulation of multiple mutations by directed evolution allows significant improvement of the biocatalyst for reactions on substrates and under conditions not already optimized in nature. The positions of the effective amino acid substitutions have been identified in a pNB esterase structural model. None appear to interact directly with the antibiotic substrate, further underscoring the difficulty of predicting their effects in a 'rational' design effort.
通过在体外引导酶的进化，可以使其适应工业应用中的最佳性能。这种方法对于工程化工业酶特别有吸引力。我们通过六代随机点突变和重组，并结合对改良变体的筛选，创造了一种高效的对硝基苄酯酶。经过四代连续随机诱变和两代随机重组后鉴定出的最佳克隆，在15%二甲基甲酰胺中，对对氯卡比-对硝基苄酯的对硝基苄酯酶活性比野生型高出150倍以上。尽管单个有效氨基酸取代对活性增强的贡献很小（增加不到2倍），但通过定向进化积累多个突变可以显著改善生物催化剂在天然未优化的底物和条件下的反应性能。在对硝基苄酯酶结构模型中已经确定了有效氨基酸取代的位置。似乎没有一个与抗生素底物直接相互作用，这进一步强调了在“理性”设计中预测其效果的难度。
