[关键词]
[摘要]
基于单分子层技术研究了禾谷镰孢菌脂肪酶GZEL对不同磷脂单分子膜的吸附动力学。同时,采用截断突变方法分析C-端肽段(编号为269-319的氨基酸)缺失后对脂肪酶GZEL界面吸附动力学参数的影响。研究发现,脂肪酶GZEL对不同磷脂单分子层的吸附动力学参数 (吸附常数ka,解离常数kd,吸附平衡常数KAds)与磷脂单分子膜的种类及初始表面压力密切相关。尽管如此,相比于野生型GZEL,269-319肽段缺失后导致酶蛋白对不同磷脂单分子膜的吸附常数ka均显著降低,而解离常数kd则显著升高,两者共同导致酶蛋白对于磷脂单分子膜的亲和力KAds显著降低。野生型GZEL对不同磷脂单分子膜的选择性顺序为磷脂酰丝氨酸>磷脂酰胆碱>磷脂酰乙醇胺。而269-319肽段缺失后,酶蛋白对于这三种磷脂单分子膜的亲和力则表现为无显著差异。以上结果表明269-319肽段在脂肪酶GZEL的界面吸附及对磷脂选择性方面均发挥有重要作用。
[Key word]
[Abstract]
The adsorption kinetics of Gibberellazeae lipase (GZEL) onto different phospholipid monolayers was investigated based on monolayer technology. Meanwhile, was constructed to analyze the effect of deletion of C-terminal peptide (269-319 amino acids) on the adsorption kinetic parameters of GZEL onto various phospholipid monolayers was analyzed by the truncated mutation method. Results revealed that the adsorption kinetic parameters (adsorption constant ka, dissociation constant kd, adsorption equilibrium constant KAds) of GZEL onto different phospholipid monolayers were closely related to the type of phospholipid monolayer and initial surface pressure. Nonetheless, compared to the wild-type GZEL, a significant decrease of the adsorption constant ka and a significant increase of the dissociation constant kd towards different phospholipid monolayers were found after 269-319 peptide was deleted from GZEL. Both changes in adsorption and dissociation constants caused a significant reduction in the adsorption affinity (KAds) of the enzyme for various phospholipid monolayers. The order of preference for the wild-type GZEL for different phospholipid monolayers was: Phosphatidylserine > phosphatidylcholine > phosphatidylethanolamine. However, after the deletion of the 269-319 peptide, no significant difference was found in the affinity of the enzyme protein for the three phospholipid monolayers. The above results indicated that the 269-319 peptide played an important role in both the interfacial adsorption and selectivity of lipase GZEL to phospholipid monolayers.
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[基金项目]
国家自然科学基金项目(31671791);广东省科技计划项目(2016B090920082);广州市珠江科技新星专项(201610010074);中央高校基本科研业务费(2017ZD090)资助