β-琼胶酶AgaB催化活性的定向进化

Directed evolution improves the activity of β-agarase AgaB

用体外定向进化技术提高了β-琼胶酶 AgaB 热稳定突变酶 M446的催化活性。采用易错 PCR 和化学诱变法相结合的方法,构建了 M446的突变体文库;利用琼胶酶降解琼胶在平板上产生水解圈的特性,建立了阳性克隆的高通量筛选体系。最终筛选得到酶活性得到提高且保持较高耐热性的突变酶 M117,其酶活提高为突变酶 M446的3倍。测序分析发现编码突变酶 M117的 DNA 序列中有4处点突变,其中2处为同义突变,另外2处引发氨基酸替换,分别为 R9K 和 I111V。对突变酶 M117与 M446的动力学常数进行了比较,发现 M117的 Km 值降低了93.4%,Kcat/Km 值提高了15倍,由此可以推断突变酶M117比活力提高的主要原因是酶与底物的亲和能力提高。

The activity of thermostable mutant M446 of β-agarase AgaB was improved using the external directed evolution. The gene of M446 was subjected to error-prone PCR and chemical mutation to construct the mutant library. For the screening of mutants, an efficient and reliable assay suitable for high throughput screening was developed based on the agarase characteristics of pits or clear zone formation on agar plate. A mutant, Mll7, was obtained through 2 successive rounds of mutations and screening. The thermosatbility of Ml17 was comparable with that of M446, and its catalytic activity was 3 folds higher than that of M446. Sequence analysis revealed that the mutant enzyme has two nonsense substitutions and two sense substitutions, R9K and III1V. In addition, the Km value of MII7 was 93.4% lower than that of M446, and the Kcat/Km value was 15 folds higher than that of M446. These results indicated that the increase of enzyme activity was due to the enhanced affinity between M117 and its substrates.