Independent production of two molecular forms of a recombinant Rhizopus oryzae lipase by KEX2-engineered strains of Saccharomyces cerevisiae.
A mixture of rProROL having the full-length prosequence (97 amino acids) for a recombinant lipase of Rhizopus oryzae (rROL) and r28ROL having 28 amino acids of the same prosequence has been produced as active forms by Saccharomyces cerevisiae [Takahashi et al. (1998) J Ferment Bioeng 86: 164-168]. However, the separation of rProROL and r28ROL has not been successful due to their identical behavior on column chromatographs, presumably because of the similarity of their surface properties. The independent production of two different molecular forms of rROL was carried out using KEX2-engineered strains of S. cerevisiae, since r28ROL was predicted to be a product from rProROL by a Kex2-like protease. rProROL was successfully obtained by expression of the ROL gene in the S. cerevisiae kex2 strain in which the KEX2 gene encoding Kex2p was disrupted, while r28ROL was obtained by co-expression of the gene (KEX2 delta 613) encoding the soluble form of the C-terminal truncated Kex2 protease (sKex2p). The specific lipase activities of rProROL and r28ROL were 92.9 U/mg and 140 U/mg, respectively. rProROL was stable at pH 2.2-8.0, and showed the optimal reaction temperature to be 30-35 degrees C with a T50 of 55 degrees C (T50 is the temperature resulting in 50% loss of activity). The values for r28ROL were pH 3.0-10.0, 25-30 degrees C, and 40 degrees C, respectively. rProROL was an N-linked glycosylated form, but r28ROL was not. The enhanced thermostability of rProROL did not seem to be due to the N-linked glycosylation, as judged by the results of the Endo H treatment. rProROL had the highest esterase activity toward p-nitrophenyl laurate (C12), whereas r28ROL had the highest esterase activity toward p-nitrophenyl caprylate (C8) and stearate (C18). These results suggest that the distinct properties of these two forms of lipase are caused by the different length of the ROL prosequence.
Takahashi
S
S
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Japan. atsuo@sbchem.kyoto-u.ac.jp
Ueda
M
M
Tanaka
A
A
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000737:chemistry; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000096:biosynthesis* / Q000737:chemistry; D043484:Proprotein Convertases; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012233:Rhizopus / Q000201:enzymology* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D013381:Subtilisins / Q000096:biosynthesis* / Q000235:genetics; D013696:Temperature
D001426:Bacterial Proteins; D005656:Fungal Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C075969:rol protein, Bacteria; D008049:Lipase; D043484:Proprotein Convertases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae