School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, China.
School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China.
Int J Mol Sci. 2017 Jul 18;18(7):1535. doi: 10.3390/ijms18071535.
Using the classical emulsified system and the monomolecular film technique, the substrate specificity of recombinant lipase (rGZEL) that originates from was characterized in detail. Under the emulsified reaction system, both phospholipase and glycolipid hydrolytic activities were observed, except for the predominant lipase activity. The optimum conditions for different activity exhibition were also determined. Compared with its lipase activity, a little higher ratio of glycolipid hydrolytic activity (0.06) than phospholipase activity (0.02) was found. rGZEL preferred medium chain-length triglycerides, while lower activity was found for the longer-chain triglyceride. Using the monomolecular film technique, we found that the preference order of rGZEL to different phospholipids was 1,2-diacyl--glycero-3-phospho-l-serine (PS) > 1,2-dioleoyl--glycero-3-phospho-rac-(1-glycerol) sodium salt (PG) > 1,2-dioleoyl--glycero-3-phosphocholine (DOPC) > l-α-phosphatidylinositol (PI) > cardiolipin (CL) > 3--phosphatidic acid sodium salt (PA) > l-α-phosphatidylethanolamine (PE), while no hydrolytic activity was detected for sphingomyelin (SM). Moreover, rGZEL showed higher galactolipase activity on 1,2-distearoyimonoglactosylglyceride (MGDG). A kinetic study on the stereo- and regioselectivity of rGZEL was also performed by using three pairs of pseudodiglyceride enantiomers (DDGs). rGZEL presented higher preference for distal DDG enantiomers than adjacent ester groups, however, no hydrolytic activity to the -2 position of diglyceride analogs was found. Furthermore, rGZEL preferred the R configuration of DDG enantiomers. Molecular docking results were in concordance with in vitro tests.
利用经典乳化体系和单分子膜技术，详细研究了来源于的重组脂肪酶（rGZEL）的底物特异性。在乳化反应体系中，观察到除了主要的脂肪酶活性外，还存在磷脂酶和糖脂水解活性。还确定了不同活性表现的最佳条件。与脂肪酶活性相比，发现糖脂水解活性（0.06）的比值略高于磷脂酶活性（0.02）。rGZEL 更喜欢中链长三酰基甘油，而长链三酰基甘油的活性较低。使用单分子膜技术，我们发现 rGZEL 对不同磷脂的偏好顺序为 1,2-二酰基 -甘油-3-磷酸-l-丝氨酸（PS）>1,2-二油酰基 -甘油-3-磷酸-rac-（1-甘油）钠盐（PG）>1,2-二油酰基 -甘油-3-磷酸胆碱（DOPC）>l-α-磷脂酰肌醇（PI）>心磷脂（CL）>3-磷酸酰基钠盐（PA）>l-α-磷脂酰乙醇胺（PE），而神经鞘磷脂（SM）则没有水解活性。此外，rGZEL 在 1,2-二硬脂酰单甘油脂（MGDG）上表现出更高的半乳糖脂酶活性。还通过使用三对假二甘油对映体（DDG）对 rGZEL 的立体和区域选择性进行了动力学研究。rGZEL 对远端 DDG 对映体的偏好高于相邻酯基，但对二甘油类似物的-2 位没有水解活性。此外，rGZEL 更喜欢 DDG 对映体的 R 构型。分子对接结果与体外测试结果一致。
