biofuel Booster |
Engineered enzyme increases output of alkanes!!
Scientists studying an enzyme that naturally produces
alkanes—long carbon-chain molecules that could be a direct replacement for the
hydrocarbons in gasoline—have figured out why the natural reaction typically
stops after three to five cycles.
"Alkanes are very similar to the carbon-chain molecules
in gasoline.
They represent a potential renewable alternative to replace the
petrochemical component of gasoline," said Brookhaven biochemist John
Shanklin, who led the research, which was conducted in large part by former
Brookhaven postdoc Carl Andre, now working at BASF Plant Science in North
Carolina, and Xiaohong Yu of Brookhaven's Biosciences Department.
"Unlike
the process of breaking down plant biomass to sugars and fermenting them to
ethanol," Shanklin said, "biologically produced alkanes could be
extracted and used directly as fuel." Recent discovery of an enzyme known
as aldehyde-deformylating oxygenase (ADO), which naturally makes alkanes from
precursors in certain bacteria, stimulated interest in harnessing this enzyme's
action to make liquid biofuels. But early attempts to install ADO in
laboratory-based alkane "factories" produced disappointing results.
Likewise, the Brookhaven team's experiments in test tubes-using substrates
synthesized with the help of Sunny Kim in Brookhaven's Radiotracer and
Biological Imaging group-yielded the same result others had observed: The
enzyme mysteriously stopped working after three to five "turnovers"
and alkane production would cease.
Biochemical curiosity and ADO's remarkable
resemblance to a group of enzymes the Brookhaven scientists were familiar with
drew them deeper into the mystery of why the enzyme stopped working. "We
set to work to try to understand the biochemistry of ADO because it is so
similar to the desaturase enzymes that we study, but performs a very different
and interesting reaction," Shanklin said. The key discovery—that the
alkane-producing system creates a by-product that's toxic to the ADO enzyme—was
unexpected. It was also the key to solving the turnover problem.
To simplify the analysis of ADO, the scientists tested
whether they could substitute hydrogen peroxide for the electron transfer
proteins and oxygen normally required for the alkane-producing reaction-an
approach that had worked for a related enzyme.
But instead of stimulating
alkane production, no alkane at all was produced, and in control experiments
containing all the components plus hydrogen peroxide, alkane production was
also blocked. "It turns out one of the electron transport proteins was interacting
with oxygen to produce hydrogen peroxide, and the buildup of hydrogen peroxide
was 'poisoning' the ADO enzyme, completely inhibiting its activity.
To confirm that hydrogen peroxide buildup was the problem and to
simultaneously test whether its depletion might enhance alkane production,
Shanklin and his team tried adding another enzyme, catalase, which metabolizes
hydrogen peroxide to oxygen and water. "When we added both enzymes,
instead of the reaction turning over three times before stopping, it ran for
more than 225 cycles," Shanklin said. So the scientists decided to make a
"bi-functional" enzyme by linking the two together.
"We reasoned
that with the ADO and catalase enzymes linked, as the hydrogen peroxide concentration
near the enzyme increases, the catalase could convert it to oxygen, mitigating
the inhibition and thereby keeping the reaction going," he said. Living
cells often contain levels of hydrogen peroxide sufficient to cause ADO
inhibition. So there was a question about whether the dual enzyme would
increase alkane production under these natural conditions.
Results to date have
been encouraging: In experiments in test tubes and pilot studies in bacteria,
the bi-functional enzyme resulted in at least a five-fold increase in alkane
production compared with ADO alone. And, in addition to removing hydrogen
peroxide as an inhibitor of ADO, the combo enzyme actually helps drive the
alkane-producing reaction by producing oxygen, one of the key components
required for activity.
"This bi-functional enzyme simultaneously decreases
the concentration of the inhibitor and increases the concentration of a needed
reaction component by converting an inhibitor into a substrate," Shanklin
said. Now the scientists are working to install the combo enzyme in algae or
green plants.
"While ADO-containing bacteria convert sugar that we feed to
them into alkanes, it would be much more efficient to produce alkanes in
photosynthetic organisms using carbon dioxide and sunlight," Shanklin
said. The scientists also suggest that the general approach of strategically
designing fusion enzymes to break down small molecule inhibitors could be used
to improve the efficiency of a wide range of reactions. Defeating natural
inhibition, a process they describe as "protection via inhibitor
metabolism" (PIM), would allow such bifunctional enzymes to function more
efficiently than their natural counterparts.
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