利用内源元件在谷氨酸棒杆菌中分泌表达木聚糖酶

Secretory production of xylanase in Corynebacterium glutamicum using its endogenous elements

摘要:

以来自于谷氨酸棒杆菌内源AH6启动子和5′ UTR及其前38 bp结合合适的Shine-Dalgarno (SD) 序列，构建双顺反子表达载体对木聚糖酶进行表达。为了能够实现分泌表达，选取了来自谷氨酸棒杆菌的两种分泌途径的信号肽，分别为Tat型的CgR0949及Sec型的CspB信号肽。在实现分泌表达之后，对其进行5 L发酵罐的扩大培养以提高分泌量。并对纯化的木聚糖酶进行了部分酶学性质的研究，包括最适催化pH及酸碱耐受性；最适催化温度及热稳定性。结果表明：在上述表达体系中，以CgR0949为信号肽木聚糖酶不能分泌到胞外；木聚糖酶能在CspB信号肽的引导下分泌到胞外，分泌表达量为486.2 U/mL。木聚糖酶的分泌量在5 L发酵罐水平上达到1 648.7 U/mL，是摇瓶培养的3.4倍。该木聚糖酶的最适反应pH 为4.5，最适温度为45 ℃；在pH 4–11范围内4 ℃处理24 h酶活保持在80%以上；在50 ℃前处理15 min酶活保持在95%以上，超过60 ℃则酶活迅速下降至20%及其以下。上述结果表明，谷氨酸棒杆菌内源元件能有效用于木聚糖酶的分泌表达，扩大培养能进一步提升木聚糖酶的分泌量。该双顺反子表达体系能为外源蛋白在谷氨酸棒杆菌中的分泌表达提供一种可用的工具。此外，通过酶学性质的研究可进一步提高木聚糖酶的催化效率。

Abstract:

We constructed bicistronic expression system containing AH6 promoter, 5′ UTR and its fore 38 bp sequence from Corynebacterium glutamicum, followed by a conserved Shine-Dalgarno (SD) sequence for xylanase expression. The two major secretory pathways signal peptide in C. glutamicum, Tat (CgR0949) and Sec (CspB) dependent signal peptide were added before xylanase for its secretion. Fed-batch cultivation was done in a 5 L jar for high-level xylanase secretion. The enzyme properties of the purified xylanase were then studied, including the effect of temperature and pH on its activity. The xylanase could be secreted into the culture supernatant when the Sec-dependent signal peptide CspB was used, but none was detected when CgR0949 was used. The secretory production level of xylanase in a flask was 486.2 U/mL and become 1 648.7 U/mL when in a 5 L jar, which was 3.4 fold as in the flask. The optimal pH and temperature of xylanase were pH 4.5 and 45 °C, respectively. Its activity was 80% of initial activity after pretreatment at 4 °C for 24 h at pH 4–11, 95% after incubation below 50 °C for 15 min, and 20% when the temperature above 60 °C. The xylanase could be efficiently secreted into the culture medium by C. glutamicum using its own genetic elements, and the secretion level could be improved through large-scale fed-batch cultivation. This bicistronic expression system can provide a useful tool for heterologous proteins secretion in C. glutamicum. In addition, the catalyze activity of xylanase could be further improved by enzyme properties study.