脂肪酶TLL在毕赤酵母中表达及催化制备生物柴油初探

High-level expression of Thermomyces lanuginosus lipase in Pichia pastoris and its application in biodiesel preparation

[目的]拟实现疏棉状嗜热丝孢菌(Thermomyces lanuginosus)脂肪酶TLL在毕赤酵母菌中的高效表达;初步探索该脂肪酶催化生物柴油的可行性及催化条件,为大规模工业生产生物柴油提供一种可行方案。[方法]比较分析α-信号肽和脂肪酶tll基因自身的信号肽对其分泌表达量的影响;构建脂肪酶tll基因多拷贝表达框,提高该基因在宿主基因组中的剂量,从而实现高效表达;直接以液体脂肪酶TLL为催化剂,采用单因子方法初步探索了其制备生物柴油的条件。[结果]α-信号肽融合和多拷贝均能提升TLL脂肪酶的表达水平。在14 L发酵罐中培养144 h后,发酵上清液的酶活可达到769 U/m L;液体脂肪酶TLL可成功地催化制备生物柴油,且获得88.3%的转酯率。[结论]该研究显著地提高了脂肪酶TLL的表达水平,初步确定了液体脂肪酶TLL可以直接制备生物柴油。

[Objective] To trail a possibility for large-scale biodiesel preparation by realizing the high-level secretory expression of Thermomyces lanuginosus lipase TLL in Pichia pastoris,and optimizing the parameters for biodiesel preparation by using TLL lipase. [Methods]We comparatively analyzed the effect of native signal peptide and α-signal peptide on the secretive expression of TLL in P. pastoris. Meanwhile,multi-copy expression cassettes were constructed to obtain the high-level expression recombinants. The parameters were optimized for directly using the liquid lipase to catalyze the transesterification of plant oil to biodiesel. [Results] Both the α-signal peptide and the multi-copy of TLL constructs could improve the expression of TLL. After cultivated in a bioreactor for 144 h,the enzyme activity of TLL reached 769 U/m L. Under the optimized conditions,the liquid lipase could efficiently catalyze the transesterification,and the methyl ester yield reached 88. 3%. [Conclusion] We have successfully improved the expression level of TLL lipase,and acquired the optimized parameters for biodiesel preparation.