Engineering CotA Laccase for Acidic pH Stability Using Bacillus subtilis Spore Display
Engineering CotA Laccase for Acidic pH Stability Using Bacillus subtilis Spore Display
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DOI：
10.4014/jmb.1608.08026
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发表时间：
2017-03-01
影响因子：
2.8
通讯作者：
Farinas, Edgardo T.
中科院分区：
文献类型：
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作者：
Sheng, Silu;Jia, Han;Farinas, Edgardo T.
枯草芽孢杆菌孢子可用于蛋白质展示以改造蛋白质特性。该方法克服了与传统蛋白质展示方法相关的活力和蛋白质折叠问题。孢子在极端条件下仍能存活，并且基因型/表型连接保持完整。此外，自然孢子形成过程消除了与穿过细胞膜的目标蛋白耦合的蛋白折叠问题。此外，ATP 依赖性伴侣的存在有助于蛋白质折叠。 CotA 被优化为固定在孢子惰性基质中的全细胞生物催化剂。一般来说，固定化的蛋白质在生物催化方面具有优势。例如，蛋白质可以很容易地从反应中去除，并且更稳定。目的是利用孢子展示来提高 pH 稳定性。对于底物 ABTS（ABTS = 2,2'-连氮基双（3-乙基苯并噻唑啉-6-磺酸盐）二铵），CotA 的最大活性在 pH 4 和 5 之间。然而，在 pH 4 时活性急剧下降。在 pH 5 时活性没有显着改变。筛选了约 3,000 个克隆的文库。E498G 变体被鉴定为具有失活半衰期(t(1/2))在pH 4下比wt-CotA高24.8倍。在之前的研究中，筛选了CotA文库的有机溶剂抗性并发现了T480A突变体，因此构建了T480A/E498G-CotA，并且t(1/2)比wt-CotA高62.1倍。在 42 小时内回收生物催化剂 7 次后，T480A/E498G-CotA 的产量比 wt-CotA 多 3.7 倍和 5.3 倍。
Bacillus subtilis spores can be used for protein display to engineer protein properties. This method overcomes viability and protein-folding concerns associated with traditional protein display methods. Spores remain viable under extreme conditions and the genotype/phenotype connection remains intact. In addition, the natural sporulation process eliminates protein-folding concerns that are coupled to the target protein traveling through cell membranes. Furthermore, ATP-dependent chaperones are present to assist in protein folding. CotA was optimized as a whole-cell biocatalyst immobilized in an inert matrix of the spore. In general, proteins that are immobilized have advantages in biocatalysis. For example, the protein can be easily removed from the reaction and it is more stable. The aim is to improve the pH stability using spore display. The maximum activity of CotA is between pH 4 and 5 for the substrate ABTS (ABTS = diammonium 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate). However, the activity dramatically decreases at pH 4. The activity is not significantly altered at pH 5. A library of approximately 3,000 clones was screened. A E498G variant was identified to have a half-life of inactivation (t(1/2)) at pH 4 that was 24.8 times greater compared with wt-CotA. In a previous investigation, a CotA library was screened for organic solvent resistance and a T480A mutant was found. Consequently, T480A/E498G-CotA was constructed and the t(1/2) was 62.1 times greater than wt-CotA. Finally, E498G-CotA and T480A/E498G-CotA yielded 3.7-and 5.3-fold more product than did wt-CotA after recycling the biocatalyst seven times over 42 h.