本研究以甘油三酯脂肪酶TLL为研究对象，将偏甘油酯脂肪酶AOL的C末端(Y246-H266)与其C末端(I241-P256)替换，获得突变体TLL_CC，并在毕赤酵母X-33中成功地进行了重组表达。乳化橄榄油的水解实验显示，C末端的置换不影响TLL脂肪酶的pH特性，但是TLL_CC的最适反应温度较TLL脂肪酶下降了15 ℃。这可能意味着长的C末端可以增加酶分子整体的柔性，进而降低其最适反应温度。三油酸甘油酯的水解实验发现，TLL脂肪酶和突变体TLL_CC反应体系中残留的三酯分别为28.31%和48.12%。而酯化合成甘油酯实验显示，TLL_CC催化的反应体系中TAG生成量仅为野生型的56.67%，但DAG的生成量却相同。上述研究表明，TLLC末端的这种置换明显减弱了TLL脂肪酶水解和合成TAG的能力，但并不影响其合成DAG的能力。这说明C末端的氨基酸替换可能对脂肪酶TLL的底物选择性起到调节作用。

In this study, the C-terminus (I241-P256) of Thermomyces lanuginosus lipase (TLL) was replaced by the corresponding region of Aspergillus oryzae lipase (Y246-H266) through site-directed substitution, and then the mutant of TLL, named TLL_CC, was successfully expressed in P. pastoris strain X-33. The hydrolysis experiments of emulsified olive oil indicated that the substitution for C-terminus of TLL led to a reduction of 15 ℃ in optimum reaction temperature but didn’t change the pH-depedent profile of the activity, implying that long C-terminus may increase the overall flexibility of enzymes and reduce the optimum reaction temperature. Hydrolysis experiments of triolein showed that the TAG substrate remained 28.31% and 48.12% in reaction systems of TLL lipase and mutant TLL_CC, respectively. On the other hand, esterification reactions showed that the yield of TAG produced by TLL_CC was 56.67% of TLL, while the same amount of DAG was obtained. The results indicated that the substitution of C-terminus of TLL distinctly decreased its ability to hydrolyze or produce TAG, but did not affect its ability to synthesize DAG.. Thus, the substitution of amino acids on C-terminus may play important role in regulating substrate selectivity of TLL.

广东省科技计划项目（2017A050503001）