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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.

Caldicellulosiruptor bescii is the most thermophilic, cellulolytic bacterium known and has the native ability to utilize unpretreated plant biomass. Cellulase A (CelA) is the most abundant enzyme in the exoproteome of C. bescii and is primarily responsible for its cellulolytic ability. CelA contains a family 9 glycoside hydrolase and a family 48 glycoside hydrolase connected by linker regions and three carbohydrate-binding domains. A truncated version of the enzyme (TM1) containing only the endoglucanase domain is thermostable and actively degrades crystalline cellulose. A catalytically active TM1 was successfully produced via the attachment of the PelB signal peptide (P-TM1), which mediates post-translational secretion via the SecB-dependent translocation pathway. We sought to enhance the extracellular secretion of TM1 using an alternative pathway, the signal recognition particle (SRP)-dependent translocation pathway. The co-translational extracellular secretion of TM1 via the SRP pathway (D-TM1) resulted in a specific activity that was 4.9 times higher than that associated with P-TM1 overexpression. In batch fermentations, the recombinant Escherichia coli overexpressing D-TM1 produced 1.86 ± 0.06 U/ml of TM1 in the culture medium, showing a specific activity of 1.25 ± 0.05 U/mg cell, 2.7- and 3.7-fold higher than the corresponding values of the strain overexpressing P-TM1. We suggest that the TM1 secretion system developed in this study can be applied to enhance the capacity of E. coli as a microbial cell factory for the extracellular secretion of this as well as a variety proteins important for commercial production.

PURPOSE To evaluate the early-postoperative intraocular pressure (IOP) changes and frequency of IOP spike after cataract surgery in normal-tension glaucoma (NTG) eyes and to compare them with those of nonglaucomatous eyes. MATERIALS AND METHODS This was a case-control study. We reviewed the medical records of patients who had undergone cataract surgery. One-to-one (1:1) case matching was performed, each matched set consisting of 1 NTG and 1 nonglaucomatous eye as the control eye. Comparisons between those 2 groups were performed. Specifically, IOP was measured preoperatively and 1 day, 1, 2, and 4 weeks postoperatively. The incidence of IOP spike was evaluated according to the criteria of IOP higher than 21, 25, 30 mm Hg, higher than 5, 10 mm Hg over baseline IOP, and 50% above preoperative IOP. RESULTS A total of 298 eyes (149 NTG eyes and 149 control eyes) were enrolled. Preoperative IOP did not show any significant difference between the 2 groups (P=0.687). IOP significantly decreased in both the NTG and control groups [P