Background

Sortase A (SrtA) is a transpeptidase found in Staphylococcus aureus, which is widely used in site-specific protein modification. However, SrtA was expressed in Escherichia coli (E. coli) in rather low level (ranging from several milligrams to 76.9 mg/L at most). The present study aims to optimize fermentation conditions for improving SrtA expression in E. coli.

Results

Under the optimized media (0.48 g/L glycerol, 1.37 g/L tryptone, 0.51 g/L yeast extract, MOPS 0.5 g/L, PBS buffer 180 mL/L) and condition (30 °C for 8 h) in a 7-L fermentor, the enzyme activity and the yield of SrtA reached 2458.4 ± 115.9 U/mg DCW and 232.4 ± 21.1 mg/L, respectively, which were higher by 5.8- and 4.5-folds compared with initial conditions, respectively. The yield of SrtA also represented threefold increase than the previously reported maximal level. In addition, the enzymatic characterizations of SrtA (optimal temperature, optimal pH, the influence of metal irons, and tolerance to water-soluble organic solvents) were determined.

Conclusions

Enhanced expression of SrtA was achieved by optimization of medium and condition. This result will have potential application for production levels of SrtA on an industry scale. Moreover, the detailed enzymatic characterizations of SrtA were examined, which will provide a useful guide for its future application.

中文翻译：

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金黄色葡萄球菌分选酶A在大肠杆菌中的高效表达及其酶学特性。

背景

Sortase A（SrtA）是一种在金黄色葡萄球菌中发现的转肽酶，广泛用于位点特异性蛋白质修饰。但是，SrtA在大肠杆菌（E. coli）中的表达水平很低（范围从几毫克到最高76.9 mg / L）。本研究旨在优化发酵条件，以改善SrtA在大肠杆菌中的表达。

结果

在优化的培养基（0.48 g / L甘油，1.37 g / L胰蛋白,、 0.51 g / L酵母提取物，MOPS 0.5 g / L，PBS缓冲液180 mL / L）和条件下（30°C 8 h） -L发酵罐中，SrtA的酶活性和产量分别达到DCW的2458.4±115.9 U / mg和232.4±21.1 mg / L，分别比初始条件高5.8倍和4.5倍。SrtA的产量也比以前报道的最高水平提高了三倍。此外，测定了SrtA的酶学特性（最佳温度，最佳pH值，金属铁的影响以及对水溶性有机溶剂的耐受性）。

结论

通过优化培养基和条件可以实现SrtA的增强表达。该结果将有可能在工业规模上应用于SrtA的生产水平。此外，对SrtA的详细酶学表征进行了研究，这将为SrtA的未来应用提供有用的指导。