As the only glycoside hydrolase family 48 member in Clostridium thermocellum, the exoglucanase Cel48S plays a crucial role in the extremely high activity of the cellulosome against crystalline cellulose. Although the importance of Cel48S in the hydrolysis of crystalline cellulose has been widely accepted, an efficient production system has not yet been established because Cel48S is usually expressed in Escherichia coli within inactive inclusion bodies. For unstable proteins like Cel48S, translocation across the inner membrane can be more advantageous than cytoplasmic production due to the presence of folding modulators in the periplasm and the absence of cytoplasmic proteases. In this study, we evaluated whether the production of Cel48S in the periplasmic space of E. coli could enhance its functional expression. To do so, we attached the PelB signal peptide, which mediates post-translational secretion, to the N-terminal end of Cel48S (P-Cel48S). The PelB signal peptide allowed catalytically active Cel48S to be successfully produced in the culture medium. In addition, we investigated the role of an alternative co-translational pathway on the extracellular production of Cel48S, finding that co-translational secretion yielded a specific activity of recombinant Cel48S of 135.1 ± 10.0 U/mg cell in the culture medium, which was 2.2 times higher than that associated with P-Cel48S expression. Therefore, we believe that our approach has potential applications for the cost-effective conversion of lignocellulosic biomass and the industrial production of other unstable proteins.

作为热纤梭菌中唯一的糖苷水解酶家族 48 成员，外切葡聚糖酶 Cel48S 在纤维素体对结晶纤维素的极高活性中起着至关重要的作用。尽管 Cel48S 在结晶纤维素水解中的重要性已被广泛接受，但由于 Cel48S 通常在大肠杆菌中的非活性包涵体中表达，因此尚未建立有效的生产系统。对于像 Cel48S 这样的不稳定蛋白质，由于周质中存在折叠调节剂且不存在细胞质蛋白酶，因此跨内膜易位可能比细胞质生产更有利。在这项研究中，我们评估了 Cel48S 是否在大肠杆菌的周质空间中产生可以增强其功能表达。为此，我们将介导翻译后分泌的 PelB 信号肽连接到 Cel48S (P-Cel48S) 的 N 末端。PelB 信号肽允许在培养基中成功产生具有催化活性的 Cel48S。此外，我们研究了替代共翻译途径对 Cel48S 细胞外产生的作用，发现共翻译分泌在培养基中产生了 135.1 ± 10.0 U/mg 细胞的重组 Cel48S 的比活性，即 2.2比与 P-Cel48S 表达相关的高出数倍。因此，我们相信我们的方法在木质纤维素生物质的成本效益转化和其他不稳定蛋白质的工业生产方面具有潜在应用。