Diverse Activities of Carotenoid Cleavage Oxygenases - Carotenoids: Physical, Chemical, and Biological Functions and Properties - page 409

Aroma compounds generated by enzymatic activities or fermentation (including recombinant
production systems) are considered “natural” according to the U.S. Food and Drug Administration
Code of Federal Regulations (CFR 1999) and European EC 1998 legislation. Biotechnological pro-
duction of aroma compounds has therefore enormous economic implications because the price of
“natural” compounds (as the result of consumer demand) far exceeds that of chemically synthesized
compounds. For example “natural” vanillin costs $1000-4000 kg −1 while the price of chemically
synthesized vanillin is two orders of magnitude less ($10-15 kg −1 ). A promising route to natural
vanillin would be the application of stilbene degrading oxygenases in engineered microbial cells
that produce stilbene compounds (Watts et al. 2006).
As the signaling mechanisms of apocarotenoids in plants are elucidated, the opportunity for the
development of genetically modii ed plants with improved drought resistance or stress tolerance can
be developed. ABA mutant plants exhibited increased transpiration efi ciency and root conductiv-
ity (Thompson et al. 2007). The i nding of strigolactones as branching factors and the mycorrhizin
and cyclohexenones as AM signals inl uences crop production strategies (Akiyama 2007). Clearer
understanding of their interactions with plants may also improve plant growth and development.
19.6.2 I MPROVING CCO I N V ITRO A CTIVITY
The poor solubility of the CCO enzymes is one impediment to their use in biotransformation reac-
tions. CCOs have a tendency to form insoluble aggregates in recombinant hosts. Another challenge
lies in delivering the extremely hydrophobic substrates to the enzymes in vitro . Because detergent-
based aqueous micellar systems make enzymology difi cult, CCOs are only superi cially character-
ized in vitro . Comprehensive kinetic studies with purii ed CCO enzymes have not been published
to date. Recently, several studies have investigated assay methods to address these impediments
with some success (see below). However, although some improvements in in vitro activity have been
achieved, the catalytic activities of CCOs in vitro are still extremely low suggesting that perhaps a
critical cofactor (small molecule and/or protein) may still be missing. As discussed in Section 19.4,
these enzymes represent a novel class of oxygenases with a yet to be clearly dei ned mechanism.
It is possible that cofactors (i.e., those involved in electron transfer) are required for this class of
enzymes to function properly; although commonly with oxygenases associated cofactors have no or
little effect on the catalytic activity of this class of enzymes (Marasco et al. 2006).
Two distinct approaches for improved solubility of the CCO enzymes have been taken. The i rst
relies on coexpression of molecula r chaperones with CCO enzymes; expression of GroEL and GroES
with microbial CCOs resulted in improved solubility in E. coli (Marasco et al. 2006). Other com-
mon chaperones such as tig , dnaK , dnaJ , and grpE did not improve solubility (Marasco et al. 2006).
The second approach utilizes solubility-enhancing fusion proteins such as glutathione-S-transferase
(GST) and the E. coli transcription-termination anti-termination factor NusA (Schilling et al. 2007).
The specii c activity of GST-AtCCD1 in cellular extracts increased throughout the stationary phase
and there was a twofold increase in maximum specii c activity for the GST-AtCCD1 compared to the
His 6X -tag version ( K m = 1.81 compared to 0.90 mU mg −1 of total protein when a unit is described as
cleavage of 1 mmol of substrate per minute). NusA fusions resulted in reduced activity (0.35-0.90 mU
mg −1 ). Turnover numbers of the purii ed fusion proteins (GST and NusA) were reduced compared
to the His 6X -tagged version (0.040, 0.051-0.132). Schilling et al. (2007) suggest that weak promot-
ers may be benei cial for CCO expression (Schilling et al. 2007). Findings by Mathieu et al. (2007)
echo these reports by describing the benei ts of slow, low temperature growth, harvesting cells at the
end of the stationary phase, and the use of a detergent in the lysis buffer (Mathieu et al. 2007). The
addition of detergents to lysis buffers aided in the extraction of soluble protein (0.08%-0.2% Triton
X-100). An earlier examination of detergents in assays with BCOI found octylglucosylpyranoside to
be the most benei cial detergent (Lindqvist and Andersson 2002).
CCO activation by organic solvents is another aspect of in vitro activity to be optimized. It was
observed that the addition of a small amount of organic solvent 1%-15% improved the activity of