Sheng Silu, Jia Han, Topiol Sidney, Farinas Edgardo T
New Jersey Institute of Technology, Department of Chemistry and Environmental Science, University Heights, Newark, NJ 07102, USA.
Center for Healthcare Innovation, Stevens Institute of Technology, Hoboken, NJ 07030, USA.
J Microbiol Biotechnol. 2017 Mar 28;27(3):507-513. doi: 10.4014/jmb.1608.08026.
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-(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 () 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 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.
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保持活力，且基因型/表型联系保持完整。此外，天然的孢子形成过程消除了与目标蛋白穿过细胞膜相关的蛋白质折叠问题。而且，存在依赖ATP的伴侣蛋白来协助蛋白质折叠。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中具有优势。例如，蛋白质可轻松从反应中分离且更稳定。目的是利用孢子展示提高pH稳定性。对于底物ABTS（ABTS = 2,2'-叠氮基双（3-乙基苯并噻唑啉-6-磺酸）二铵），CotA的最大活性在pH 4至5之间。然而，在pH 4时活性急剧下降。在pH 5时活性没有显著变化。筛选了一个约3000个克隆的文库。鉴定出E498G变体在pH 4时的失活半衰期（）比野生型CotA大24.8倍。在先前的一项研究中，筛选了一个CotA文库以寻找有机溶剂抗性，并发现了一个T480A突变体。因此，构建了T480A/E498G-CotA，其比野生型CotA大62.1倍。最后，在42小时内将生物催化剂循环使用7次后，E498G-CotA和T480A/E498G-CotA产生的产物分别比野生型CotA多3.7倍和5.3倍。
