Evaluation of a new system for developing particulate enzymes based on the surface (S)-layer protein (RsaA) of Caulobacter crescentus -: Fusion with the β-1,4-glycanase (Cex) from the cellulolytic bacterium cellulomonas fimi yields a robust, catalytically active product
Evaluation of a new system for developing particulate enzymes based on the surface (S)-layer protein (RsaA) of Caulobacter crescentus -: Fusion with the β-1,4-glycanase (Cex) from the cellulolytic bacterium cellulomonas fimi yields a robust, catalytically active product
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DOI：
10.1385/abab:127:2:095
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发表时间：
2005-11-01
影响因子：
3
通讯作者：
Smit, J
中科院分区：
文献类型：
--
作者：
Duncan, G;Tarling, CA;Smit, J
固定化生物催化剂，包括颗粒酶，代表了研究和工业应用的有吸引力的工具，因为它们将天然酶的特异性与它们可以容易地从最终产物分离并重复使用的优点联合收割机结合。我们证明了使用新月柄杆菌表面（S）层蛋白（RsaA）分泌装置的颗粒酶的产生。具体地，评价了先前通过来自纤维素分解细菌纤维单胞菌（Cellulomonasfimi）的β-1，4-聚糖酶（Cex）与RsaA的C-末端融合制备的候选蛋白。Cex/RsaA切割人工底物对硝基苯基-β-D-纤维二糖苷中的糖苷键，K-m与天然Cex相似（Cex/RsaA为1.1 mM，Cex为0.60 mM），表明颗粒状Cex酶能够以野生型亲和力结合底物。相比之下，k（cat）值显著降低（Cex/RsaA为0.08 s（-1），Cex为15.8 s（-1）），这可能是由于RsaA C末端诱导重组蛋白自发非结构化聚集的事实。在这里，我们证明了RsaA融合蛋白不仅可以廉价地生产并纯化至高产率（对于Cex/RsaA为76 mg/L干重），而且还可以有效地回收利用。因此，柄杆菌S层分泌系统提供了一个有吸引力的新的模型系统的颗粒生物催化剂的生产。
Immobilized biocatalysts, including particulate enzymes, represent an attractive tool for research and industrial applications because they combine the specificity of native enzymes with the advantage that they can be readily separated from end product and reused. We demonstrated the use of the Caulobacter crescentus surface (S)-layer protein (RsaA) secretion apparatus for the generation of particulate enzymes. Specifically, a candidate protein made previously by fusion of the beta-1, 4-glycanase (Cex) from the cellulolytic bacterium Cellulomonas fimi with the C-terminus of RsaA was evaluated. Cex/RsaA cleaved the glycosidic linkage in the artificial substrate p-nitrophenyl-beta-D-cellobioside with a K-m similar to that of native Cex (1.1 mM for Cex/RsaA vs 0.60 mM for Cex), indicating that the particulate Cex enzyme was able to bind substrate with wild-type affinity. By contrast, the k(cat) value was significantly reduced (0.08 s(-1) for Cex/RsaA vs 15.8 s(-1) for Cex), likely owing to the fact that the RsaA C-terminus induces spontaneous unstructured aggregation of the recombinant protein. Here, we demonstrated that not only can an RsaA fusion protein be cheaply produced and purified to a high yield (76 mg/L of dry wt for Cex/RsaA), but it can also be efficiently recycled. The Caulobacter S-layer secretion system therefore offers an attractive new model system for the production of particulate biocatalysts.