摘要
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p H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 ofAspergillus oryzae lipase CY246-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 of15℃ 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 remained28.31%and48.12%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 was56.67%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.
作者
谭熙钰
赵泽鑫
杨博
王永华
TAN Xi-yu;ZHAO Ze-xin;YANG Bo;WANG Yong-hua(School of Food Science and Engineering,South China University of Technology,Guangzhou510640,China;School of Bioscience and Bioengineering,South China University of Technology,Guangzhou510006,China)
出处
《现代食品科技》
EI
CAS
北大核心
2018年第11期21-27,共7页
Modern Food Science and Technology
基金
广东省科技计划项目(2017A050503001)
关键词
甘油三酯脂肪酶
偏甘油酯脂肪酶
底物选择性
C末端
triacylglycerol lipase
mono-and diacylglycerol lipase
substrate selectivity
C-terminus
