Kamoun Jannet, Schué Mathieu, Messaoud Wala, Baignol Justine, Point Vanessa, Mateos-Diaz Eduardo, Mansuelle Pascal, Gargouri Youssef, Parsiegla Goetz, Cavalier Jean-François, Carrière Frédéric, Aloulou Ahmed
University of Sfax, ENIS - Laboratory of Biochemistry and Enzymatic Engineering of Lipases, 3038 Sfax, Tunisia.
Proteabio Europe S.A.S., 290 Chemin de Saint-Dionysy, Bât A, 30980 Langlade, France.
Biochim Biophys Acta. 2015 Feb;1851(2):129-40. doi: 10.1016/j.bbalip.2014.10.012. Epub 2014 Nov 1.
Yarrowia lipolytica is a lipolytic yeast possessing 16 paralog genes coding for lipases. Little information on these lipases has been obtained and only the major secreted lipase, namely YLLIP2, had been biochemically and structurally characterized. Another secreted lipase, YLLIP8, was isolated from Y. lipolytica culture medium and compared with the recombinant enzyme produced in Pichia pastoris. N-terminal sequencing showed that YLLIP8 is produced in its active form after the cleavage of a signal peptide. Mass spectrometry analysis revealed that YLLIP8 recovered from culture medium lacks a C-terminal part of 33 amino acids which are present in the coding sequence. A 3D model of YLLIP8 built from the X-ray structure of the homologous YLLIP2 lipase shows that these truncated amino acids in YLLIP8 belong to an additional C-terminal region predicted to be mainly helical. Western blot analysis shows that YLLIP8 C-tail is rapidly cleaved upon enzyme secretion since both cell-bound and culture supernatant lipases lack this extension. Mature recombinant YLLIP8 displays a true lipase activity on short-, medium- and long-chain triacylglycerols (TAG), with an optimum activity at alkaline pH on medium chain TAG. It has no apparent regioselectivity in TAG hydrolysis, thus generating glycerol and FFAs as final lipolysis products. YLLIP8 properties are distinct from those of the 1,3-regioselective YLLIP2, acting optimally at acidic pH. These lipases are tailored for complementary roles in fatty acid uptake by Y. lipolytica.
解脂耶氏酵母是一种具有16个编码脂肪酶的旁系同源基因的解脂酵母。关于这些脂肪酶的信息很少，只有主要分泌的脂肪酶，即YLLIP2，进行了生化和结构表征。另一种分泌脂肪酶YLLIP8是从解脂耶氏酵母培养基中分离出来的，并与在毕赤酵母中产生的重组酶进行了比较。N端测序表明，YLLIP8在信号肽切割后以活性形式产生。质谱分析显示，从培养基中回收的YLLIP8缺少编码序列中存在的33个氨基酸的C端部分。根据同源YLLIP2脂肪酶的X射线结构构建的YLLIP8三维模型表明，YLLIP8中这些截短的氨基酸属于预计主要为螺旋结构的额外C端区域。蛋白质免疫印迹分析表明，由于细胞结合型和培养上清液中的脂肪酶都缺乏这种延伸，YLLIP8的C端尾巴在酶分泌时会迅速被切割。成熟的重组YLLIP8对短链、中链和长链三酰甘油（TAG）具有真正的脂肪酶活性，在碱性pH值下对中链TAG具有最佳活性。它在TAG水解中没有明显的区域选择性，因此产生甘油和游离脂肪酸作为最终的脂解产物。YLLIP8的特性与1,3-区域选择性的YLLIP2不同，YLLIP2在酸性pH值下活性最佳。这些脂肪酶为解脂耶氏酵母在脂肪酸摄取中的互补作用而量身定制。
