Palackal Nisha, Brennan Yali, Callen Walter N, Dupree Paul, Frey Gerhard, Goubet Florence, Hazlewood Geoffrey P, Healey Shaun, Kang Young E, Kretz Keith A, Lee Edd, Tan Xuqiu, Tomlinson Geoffery L, Verruto John, Wong Vicky W K, Mathur Eric J, Short Jay M, Robertson Dan E, Steer Brian A
Diversa Corp., 4955 Directors Place, San Diego, CA 92121, USA.
Protein Sci. 2004 Feb;13(2):494-503. doi: 10.1110/ps.03333504. Epub 2004 Jan 10.
Directed evolution technologies were used to selectively improve the stability of an enzyme without compromising its catalytic activity. In particular, this article describes the tandem use of two evolution strategies to evolve a xylanase, rendering it tolerant to temperatures in excess of 90 degrees C. A library of all possible 19 amino acid substitutions at each residue position was generated and screened for activity after a temperature challenge. Nine single amino acid residue changes were identified that enhanced thermostability. All 512 possible combinatorial variants of the nine mutations were then generated and screened for improved thermal tolerance under stringent conditions. The screen yielded eleven variants with substantially improved thermal tolerance. Denaturation temperature transition midpoints were increased from 61 degrees C to as high as 96 degrees C. The use of two evolution strategies in combination enabled the rapid discovery of the enzyme variant with the highest degree of fitness (greater thermal tolerance and activity relative to the wild-type parent).
定向进化技术被用于在不影响酶催化活性的前提下选择性地提高其稳定性。特别是，本文描述了两种进化策略的串联使用，以进化出一种木聚糖酶，使其能够耐受超过90摄氏度的温度。在每个残基位置生成了所有可能的19种氨基酸替换的文库，并在温度挑战后筛选活性。鉴定出九个增强热稳定性的单氨基酸残基变化。然后生成这九个突变的所有512种可能的组合变体，并在严格条件下筛选提高的耐热性。筛选产生了十一个具有显著提高的耐热性的变体。变性温度转变中点从61摄氏度提高到高达96摄氏度。两种进化策略的联合使用使得能够快速发现适应性最高的酶变体（相对于野生型亲本具有更高的耐热性和活性）。
