Biofuels in Perspective

ME content (wt.%)
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Reaction time (h)
Enzymatic Production of Biodiesel 141
Figure 8.11 Time course of methyl ester content during ten repeated batch cycles using cells cultivated
at various R f values. Symbols: (•) R f = 0.67; (◦) R f = 0.83; and (△) R f = 1.0. (From Ref. 50, with permission
of Elsevier.)
fatty acid composition in experiments using living cells 51 and lipid vesicle. 52–54 As for
tolerance to ethanol 51 and toluene, 55 cell membrane rigidity appears to prevent their
penetration. However, the effect of free fatty acids on the plasma membrane are still
uncertain in terms of the mechanism of action and their location within the cell.
The findings outlined above indicate that BSP-immobilized cells are significantly stabilized
by cultivation at optimal levels of Rf and could be used as whole-cell biocatalyst for
practical biodiesel fuel production.
8.4.4 Lipase Localization in Cells Immobilized within BSPs
To determine the lipase localization in R. oryzae cells, Hama et al. 56 performed Western
blot analysis. As can be seen in Figure 8.12, R. oryzae cells produced mainly two types of
lipase with different molecular mass values of 34 and 31 kDa. Inside the cells, the 34-kDa
lipase (ROL34) was bound to the cell wall and the 31-kDa lipase (ROL31) to the cell wall
or membrane. Lipase in the cytoplasmic fraction was difficult to detect because of the small
amount. It is notable that, in the suspension cells, the amount of membrane-bound lipase
decreased sharply with cultivation time (Figure 8.12A), while in the immobilized cells
large amounts of lipase remained even in the later period of cultivation (Figure 8.12B).
Although the relationship between cell morphology and enzyme secretion depends on the
fungal strain and the enzyme type, it was also found that cell immobilization strongly
inhibited the secretion of ROL31 into the culture medium.
The N-terminal amino acid sequences of ROL34 and ROL31 were ‘D-D-N-L-V’ and
‘S-D-G-G-K’, respectively, clearly indicating the processing site of the lipase precursor
(see Figure 8.13). 56 The R. oryzae lipase precursor consists of a signal sequence (26 amino
acids), a prosequence (97 amino acids), and a mature region (269 amino acids), as deduced
from the nucleotide sequence. 57 Of these regions, the prosequence contains a Lys–Arg