Genetika Articles Production of recombinant Rhizopus oryzae lipase by the yeast Yarrowia lipolytica results in increased enzymatic thermostability.

Production of recombinant Rhizopus oryzae lipase by the yeast Yarrowia lipolytica results in increased enzymatic thermostability.

Production of recombinant Rhizopus oryzae lipase by the yeast Yarrowia lipolytica results in increased enzymatic thermostability.

Tigran V Yuzbashev, Evgeniya Y Yuzbasheva, Tatiana V Vibornaya, Tatiana I Sobolevskaya, Ivan A Laptev, Alexey V Gavrikov, Sergey P Sineoky

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ABSTRACT: 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.

12.12.2011, 1547 просмотров.