碳源及阻遏蛋白CreA对黑曲霉产α-L-鼠李糖苷酶的影响

Effect of Carbon Sources and Repressor Protein CreA on α-L-Rhamnosidase Production of Aspergillus niger

【目的】研究碳源对黑曲霉JMU-TS528发酵α-L-鼠李糖苷酶的影响,并探讨葡萄糖阻遏黑曲霉合成α-L-鼠李糖苷酶的分子机制。【方法】分别用5种单一碳源进行摇瓶发酵,高效液相色谱法测定经黑曲霉发酵后产物中的α-L-鼠李糖苷酶活性;通过在培养基中添加葡萄糖进行液态发酵,研究葡萄糖对黑曲霉JMU-TS528产α-L-鼠李糖苷酶的影响,并用实时定量PCR技术检测α-L-鼠李糖苷酶基因rha和葡萄糖抑制基因Cre A的相对表达量。【结果】黑曲霉JMU-TS528在以柚皮苷、橘皮苷、芸香苷和鼠李糖为唯一碳源时可分泌α-L-鼠李糖苷酶,以葡萄糖为唯一碳源时不能合成α-L-鼠李糖苷酶;培养基中添加葡萄糖后,黑曲霉发酵产物中α-L-鼠李糖苷酶活性降低,黑曲霉α-L-鼠李糖苷酶基因rha表达量降低,而葡萄糖抑制基因Cre A表达量上升。【结论】黑曲霉JMU-TS528菌株液态发酵条件下合成α-L-鼠李糖苷酶需要诱导物作为碳源,该菌株合成α-L-鼠李糖苷酶的能力受到葡萄糖的调节作用,此调控过程可能通过一个依赖阻遏蛋白Cre A的机制实现。

[Objective] The aim of this work was to investigate the effects of different carbon sources on the production of α-L-rhamnosidase by Aspergillus niger, and to explore the molecular mechanism of glucose repression on the synthesis of α-L-rhamnosidase. [Methods] The A. niger JMU-TS528 strain was cultured in the medium contained naringin, hesperidin, rutin, rhamnose and glucose as sole carbon sources in shake flask, respectively. Then the α-L-rhamnosidase activity was measured by High Performance Liquid Chromatography（HPLC）. Further to study the effect of glucose on α-L-rhamnosidase production, the relative expression of the α-L-rhamnosidase gene rha and glucose repression gene CreA was examined by Real Time PCR（RT-PCR）to compare the different expression level with or without adding glucose during culture. [Results] α-L-rhamnosidase can be induced when naringin, hesperidin, rutin and rhamnose were used as the sole carbon source; while no activity of α-L-rhamnosidase was detected when glucose was used as the sole carbon source. Furthermore, a lower enzymatic activity was observed by adding of glucose, and the expression of α-L-rhamnosidase gene（rha） was decreased also. In the same time, it was found that the expression level of glucose repression gene CreA was increased greatly. [Conclusion] L-rhamnose or L-rhamnose-containing flavonoid glycosides（rutin, hesperidin, and naringin） can be used as specific inducers for α-L-rhamnosidase production. L-Rhamnose induction of A. niger JMU-TS528 α-L-rhamnosidase genes is glucose repressed and may function via a Cre A-dependent mechanism.