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中文摘要：
研究了毕赤酵母GS115中能够高效分泌表达的华根霉脂肪酶（r27RCLC）的糖基化情况。运用糖基化抑制剂衣霉素处理表达目的蛋白的毕赤酵母细胞,通过SDS-PAGE比较分析经衣霉素处理后的脂肪酶r27RCLC分泌表达量的变化,结果显示酶的分泌表达受到很大的抑制,说明脂肪酶r27RCLC在表达时候很可能发生了糖基化,且糖基化对其有重要影响。通过生物化学方法,利用内切糖苷酶PNGase F和Endo Hf酶切处理脂肪酶r27RCLC,SDS-PAGE和Western Blot,结果显示r27RCLC未发生糖基化。将样品r27RCLC通过胰蛋白酶酶解等步骤处理,进行MADLI-TOF-MS质谱分析,结果发现MADLI-TOF-MS鉴定结果与内切糖苷酶酶切处理结果一致,进一步证明毕赤酵母分泌表达的华根霉脂肪酶r27RCLC未发生糖基化。
英文摘要：
In this study,we identified the glycosylation situation of Rhizopus chinensis lipase CCTCC M201021 expressed in P. pastoris. At first,the yeast cells expressing lipase r27 RCLC were treated with inhibitor tunicamycin and SDS-PAGE was conducted to analyze the effect of tunicamycin on the expression level of lipase. Results showed that lipase r27 RCLC maybe occur glycosylation when formed in yeast cells. Then,lipase r27 RCLC was digested with endoglycosidase PNGase F and Endo Hf. SDS-PAGE and Western Blot analysis showed that r27 RCLC is not a glycoprotein. Finally,the sample of r27 RCLC was digested by trypsin and detected by MADLI-TOF mass spectrometry. The results of MADLI-TOF-MS identification were consistent with the results of endoglycosidase treatement,which further proved that lipase r27 RCLC expressed in P. pastoris did not occur glycosyaltion.
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