Objective

To establish a temperature-induced chitosanase bacterial cell-surface display system to produce chitooligosaccharides (COSs) efficiently for industrial applications.

Results

Temperature-inducible chitosanase CSN46A bacterial surface display systems containing one or two copies of ice nucleation protein (InaQ-N) as anchoring motifs were successfully constructed on the basis of Escherichia coli and named as InaQ-N-CSN46A (1 copy) and 2InaQ-N-CSN46A (2 copies). The specific enzyme activity of 2InaQ-N-CSN46A reached 761.34 ± 0.78 U/g cell dry weight, which was 45.6% higher than that of InaQ-N-CSN46A. However, few proteins were detected in the 2InaQ-N-CSN46A hydrolysis system. Therefore, 2InaQ-N-CSN46A had higher hydrolysis efficiency and stability than InaQ-N-CSN46A. Gel permeation chromatography revealed that under the optimum enzymatic hydrolysis temperature, the final products were mainly chitobiose and chitotriose. Chitopentaose accumulated (77.62%) when the hydrolysis temperature reached 60 °C. FTIR and NMR analysis demonstrated that the structures of the two hydrolysis products were consistent with those of COSs.

Conclusions

In this study, chitosanase was expressed on the surfaces of E. coli by increasing the induction temperature, and chitosan was hydrolysed directly without enzyme purification steps. This study provides a novel strategy for industrial COS production.

中文翻译：

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一种用于高效生产壳寡糖的温度诱导壳聚糖酶细菌细胞表面展示系统

客观的

建立温度诱导的壳聚糖酶细菌细胞表面展示系统，以高效地生产用于工业应用的壳寡糖（COSs）。

结果

在大肠杆菌的基础上成功构建了含有一或两个冰核蛋白（InaQ-N）作为锚定基序的温度诱导壳聚糖酶CSN46A细菌表面展示系统并命名为InaQ-N-CSN46A（1份）和2InaQ-N-CSN46A（2份）。2InaQ-N-CSN46A的比酶活达到761.34±0.78 U/g细胞干重，比InaQ-N-CSN46A高45.6%。然而，在 2InaQ-N-CSN46A 水解系统中检测到的蛋白质很少。因此，2InaQ-N-CSN46A比InaQ-N-CSN46A具有更高的水解效率和稳定性。凝胶渗透色谱表明，在最佳酶解温度下，最终产物主要为壳二糖和壳三糖。当水解温度达到 60 °C 时，壳五糖积累 (77.62%)。FTIR和NMR分析表明两种水解产物的结构与COS的结构一致。

结论

在本研究中，壳聚糖酶通过提高诱导温度在大肠杆菌表面表达，壳聚糖直接水解，无需酶纯化步骤。本研究为工业 COS 生产提供了一种新策略。