Yuzbashev Tigran V, Yuzbasheva Evgeniya Y, Vibornaya Tatiana V, Sobolevskaya Tatiana I, Laptev Ivan A, Gavrikov Alexey V, Sineoky Sergey P
Russian State Collection of Industrial Microorganisms (VKPM), State Research Institute of Genetics and Selection of Industrial Microorganisms, Moscow 117545, Russia.
Protein Expr Purif. 2012 Mar;82(1):83-9. doi: 10.1016/j.pep.2011.11.014. Epub 2011 Dec 3.
The gene encoding Rhizopus oryzae lipase (ROL) was expressed in the non-conventional yeast Yarrowia lipolytica under the control of the strong inducible XPR2 gene promoter. The effects of three different preprosequence variants were examined: a preprosequence of the Y. lipolytica alkaline extracellular protease (AEP) encoded by XPR2, the native preprosequence of ROL, and a hybrid variant of the presequence of AEP and the prosequence of ROL. Lipase production was highest (7.6 U/mL) with the hybrid prepropeptide. The recombinant protein was purified by ion-exchange chromatography. The ROL included 28 amino acids of the C-terminal region of the prosequence, indicating that proteolytic cleavage occurred below the KR site through the activity of the Kex2-like endoprotease. The optimum temperature for recombinant lipase activity was between 30 and 40 °C, and the optimum pH was 7.5. The enzyme was shown not to be glycosylated. Furthermore, recombinant ROL exhibited greater thermostability than previously reported, with the enzyme retaining 64% of its hydrolytic activity after 30 min of incubation at 55 °C.
编码米根霉脂肪酶（ROL）的基因在强诱导型XPR2基因启动子的控制下，在非常规酵母解脂耶氏酵母中表达。研究了三种不同前原序列变体的效果：由XPR2编码的解脂耶氏酵母碱性细胞外蛋白酶（AEP）的前原序列、ROL的天然前原序列，以及AEP前序列和ROL前序列的杂交变体。使用杂交前原肽时脂肪酶产量最高（7.6 U/mL）。重组蛋白通过离子交换色谱法纯化。ROL包含前序列C末端区域的28个氨基酸，这表明通过类似Kex2的内切蛋白酶的活性，蛋白水解切割发生在KR位点下方。重组脂肪酶活性的最适温度在30至40°C之间，最适pH为7.5。该酶未被糖基化。此外，重组ROL表现出比先前报道更高的热稳定性，在55°C孵育30分钟后，该酶保留了64%的水解活性。
