title: Production of recombinant Rhizopus oryzae lipase by the yeast Yarrowia lipolytica results in increased enzymatic thermostability.
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.
authors.affiliation: Russian State Collection of Industrial Microorganisms (VKPM), State Research Institute of Genetics and Selection of Industrial Microorganisms, Moscow 117545, Russia. t_yuzbashev@genetika.ru
authors.forename: Tigran V
authors.initials: TV
authors.lastname: Yuzbashev
authors.forename: Evgeniya Y
authors.initials: EY
authors.lastname: Yuzbasheva
authors.forename: Tatiana V
authors.initials: TV
authors.lastname: Vibornaya
authors.forename: Tatiana I
authors.initials: TI
authors.lastname: Sobolevskaya
authors.forename: Ivan A
authors.initials: IA
authors.lastname: Laptev
authors.forename: Alexey V
authors.initials: AV
authors.lastname: Gavrikov
authors.forename: Sergey P
authors.initials: SP
authors.lastname: Sineoky
mesh_terms: D003001:Cloning, Molecular; D004795:Enzyme Stability; D015870:Gene Expression; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012233:Rhizopus / Q000201:enzymology* / Q000235:genetics; D013696:Temperature; D025062:Yarrowia / Q000235:genetics*
chemical_list: D011994:Recombinant Proteins; D008049:Lipase