Biofuels in Perspective

Specific methanolysis activity (U/mg)
0.16
0.12
0.08
0.04
a
b
c
Enzymatic Production of Biodiesel 143
0.00
0.2 1.0 2.0
Intensity of membrane-bound lipase (ROL31) band
Figure 8.14 Correlation between specific methanolysis activity and amount of membrane-bound lipase
(ROL31). Letters in plots represent immobilized cells cultivated for 4 days (a) without oils and fatty acids, or
with (b) stearic acid, (c) palmitic acid, (d) oleic acid, and (e) olive oil at 30 g/l each. The horizontal axis shows
the sum of the intensities of the pixels inside the bands corresponding to each membrane-bound lipase
detected by Western blot analysis. The specific methanolysis activity of the cells shows a linear relationship
with the intensity of the membrane-bound lipase (ROL31) band in a semilogarithmic plot. (From Ref. 56,
with permission of The Society for Biotechnology, Japan.)
It is interesting that the N-terminal 28-amino-acid residue of ROL34 is critical for determination
of the lipase localization. Generally, in eukaryotic cells, the topology of proteins
in the membrane is determined by the translocation across the endoplasmic reticulum (ER)
membrane after the cleavage of a signal peptide. 59 If once the secretory proteins are transported
into the ER, they are located in the cell wall and finally secreted extracellularly.
On the other hand, the proteins integrated into the ER membrane are finally accumulated
in the cell membrane as it does to the ER membrane. Thus, the fact that ROL34 is localized
mostly in the cell wall and very little in the membrane led to the hypothesis that the
N-terminal 28-amino-acid residue of ROL34 plays an important role in the translocation
of ROL across the ER membrane.
In the cell wall, where large amounts of ROL34 are localized, there was no significant
difference according to the different substrate-related compounds added to the culture
medium. In the membrane, however, cells cultivated with olive oil or oleic acid retained
larger amounts of ROL31. As can be seen in Figure 8.14, there was significant correlation
between the intracellular methanolysis activity and the amount of ROL31 localized in the
membrane (56). These findings suggest that ROL31 localized in the membrane plays a
crucial role in the methanolysis activity of R. oryzae cells.
8.5 Use of Cell-Surface Displaying Cells as Whole-Cell Biocatalyst
Cell-surface display of heterologous proteins using microorganisms has been widely utilized
in various areas. 61,62 For instance, cell-surface display of enzymes such as glucoamylase
and cellulase on bacteria 63–65 , and yeast 66–68 has been found effective for the
d
e