Fig 1.
Sequence alignment of Rhizopus oryzae lipase (ROL) and ROL-related lipases.
The full-length primary sequences of ROL, R. niveus lipase (RNL), R. stolonifer lipase (RSL), and R. chinensis lipase (RCL) are presented. Multiple-sequence alignments were generated using the ClustalW program (http://www.ebi.ac.uk/Tools/msa/clustalw2/). The underlined sequences in the propeptide of ROL (Ser20–Gly37 and Ser38–Glu57) indicate the regions that are essential for secretion and folding of mROL, respectively. The underlined sequences in the mature domain of ROL (Phe183–Asp189) indicate the lid domain. The shadowed region indicates residues that were replaced with hydrophilic amino acids (VDDDDK). In the original host, R. oryzae, the propeptide is also cleaved between the Ala97 and Ser98 residues [21]; however in P. pastoris and S. cerevisiae, the secondary cleavage has not been observed [14, 22]. Therefore, in this study, we defined the propeptide domain as the region between residues 1 and 69 and the mature domain as the region between residues 70 and 366 of ROL.
Fig 2.
Lipase activity assay with purified ROL.
(A) Measurement of the lipase activities of mROLWT and mROLimp using p-nitrophenyl esters with various acyl chain lengths (C2–C16) as substrates. The resultant p-nitrophenol was quantified to estimate the lipase activities. (B) Relative lipase activities normalized with the values obtained for mROLWT. The values are presented as mean ± standard error of the mean (SEM) based on at least three independent measurements. (C) Competitive lipase activity assay with purified mROLWT and mROLimp. Lipase activities were determined in the presence of the peptidase substrate, Suc-Ile-Ile-Trp-MCA, dissolved in dimethyl sulfoxide (DMSO). The P-values were determined using the Student’s t-test. * P < 0.05.
Table 1.
Kinetic parameters for mROL and mROLimp.
Fig 3.
Analysis of the structures and stabilities of mROLWT and mROLimp.
(A) Circular dichroism spectra of mROLWT and mROLimp. (B) The thermal stabilities of mROLWT and mROLimp. The negative ellipticities at 222 nm were measured to determine the fraction folded. The values are presented as mean ± SEM based on three independent measurements. The P-value was determined using a two-factor ANOVA with mROLs and temperatures as independent factors.
Fig 4.
Activity assay using yeast cell surface engineering.
(A) Peptidase activity assay of mROLWT, mROLimp, and mROLimp2 displayed on the yeast cell surface. (B) Lipase activity assay of mROLWT, mROLimp, and mROLimp2 displayed on the yeast cell surface. The resultant p-nitrophenol was quantified to estimate the lipase activities. The peptidase and lipase activities were corrected by the number of displayed enzymes (S3 Fig). The values are presented as mean ± SEM based on three independent measurements. The P-values were determined using one-way analysis of variance followed by Tukey’s test for multiple comparisons. ** P < 0.01.
Fig 5.
Structure of mROLWT, modeled using the SWISS-MODEL program.
The structure of mROLWT was modeled based on the open-lid structure of Rhizomucor miehei lipase (Protein Data Bank [PDB] ID: 4TGL), and visualized using PyMOL. The active site residues, S242, D301, and H354, are colored orange. The magenta-colored α-helix represents the lid domain. Green residues indicate hydrophilic amino acids and white residues indicate hydrophobic amino acids.