Biomolecular Frontiers Research Centre, Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, Australia.
Appl Biochem Biotechnol. 2010 May;161(1-8):301-12. doi: 10.1007/s12010-009-8794-6. Epub 2009 Oct 16.
Characteristics that would make enzymes more desirable for industrial applications can be improved using directed evolution. We developed a directed evolution technique called random drift mutagenesis (RNDM). Mutant populations are screened and all functional mutants are collected and put forward into the next round of mutagenesis and screening. The goal of this technique is to evolve enzymes by rapidly accumulating mutations and exploring a greater sequence space by providing minimal selection pressure and high-throughput screening. The target enzyme was a beta-glucosidase isolated from the thermophilic bacterium, Caldicellulosiruptor saccharolyticus that cleaves cellobiose resulting from endoglucanase hydrolysis of cellulose. Our screening method was fluorescence-activated cell sorting (FACS), an attractive method for assaying mutant enzyme libraries because individual cells can be screened, sorted into distinct populations and collected very rapidly. However, FACS screening poses several challenges, in particular, maintaining the link between genotype and phenotype because most enzyme substrates do not remain associated with the cells. We employed a technique where whole cells were encapsulated in cell-like structures along with the enzyme substrate. We used RNDM, in combination with whole cell encapsulation, to create and screen mutant beta-glucosidase libraries. A mutant was isolated that, compared to the wild type, had higher specific and catalytic efficiencies (k(cat)/K(M)) with p-nitrophenol-glucopyranoside and -galactopyranoside, an increased catalytic turnover rate (k(cat)) with cellobiose, an improvement in catalytic efficiency with lactose and reduced inhibition (K(i)) with galactose and lactose. This mutant had three amino acid substitutions and one was located near the active site.
可以通过定向进化来改善使酶更适合工业应用的特性。我们开发了一种称为随机漂移诱变（RNDM）的定向进化技术。筛选突变体群体，并收集所有功能突变体并将其提交到下一轮诱变和筛选。该技术的目标是通过快速积累突变并通过提供最小的选择压力和高通量筛选来探索更大的序列空间来进化酶。靶酶是从嗜热细菌Caldicellulosiruptor saccharolyticus中分离出的β-葡萄糖苷酶，可切割纤维素内切葡聚糖水解产生的纤维二糖。我们的筛选方法是荧光激活细胞分选（FACS），这是一种用于测定突变酶文库的有吸引力的方法，因为可以对单个细胞进行筛选，将其分类为不同的群体并非常快速地收集。但是，FACS 筛选带来了一些挑战，特别是要保持基因型和表型之间的联系，因为大多数酶底物都不会与细胞保持关联。我们采用了一种技术，即将整个细胞与酶底物一起封装在类似细胞的结构中。我们使用 RNDM 与全细胞封装相结合，创建和筛选突变β-葡萄糖苷酶文库。分离出一种突变体，与野生型相比，该突变体对 p-硝基苯酚-β-D-吡喃葡萄糖苷和β-D-半乳糖吡喃糖苷具有更高的比活性和催化效率（kcat/KM），对纤维二糖具有更高的催化周转率（kcat），对乳糖具有更高的催化效率，对半乳糖和乳糖的抑制作用降低（Ki）。该突变体有三个氨基酸取代，其中一个位于活性部位附近。
