In the Laboratory
Biocatalytic Lactone Generation in Genetically Engineered
Escherichia coli and Identification of Products
by Gas Chromatography–Mass Spectroscopy
Chad Slawson
Department of Chemistry, University of South Florida, Tampa, FL 33620
Jon Stewart
Department of Chemistry, University of Florida, Gainesville, FL 32611
Robert Potter*
Department of Chemistry, University of South Florida, Tampa, FL 33620; potter@chuma1.cas.usf.edu
Using enzymes to perform organic synthesis is of growing
interest to chemists both in academe and in industry. Industrial
applications of catalytic enzymes and whole organisms as
bioreactors are increasing daily, since they efficiently catalyze
a number of reactions that provide unparalleled chiral and
positional selectivity (1).
In this project, we present a novel way to perform
Baeyer–Villiger oxidations using the enzyme cyclohexanone
monooxygenase (CHMO) genetically engineered into a
biocatalytic microorganism. The Baeyer–Villiger oxidation of
ketones to the corresponding lactone has been known since
the late 1800s (2). The reaction uses peroxy compounds that
are highly toxic, explosive, and costly to dispose of, and ra-
cemic products are produced (2). To avoid these problems, a
strain of nonpathogenic Escherichia coli has been engineered
to produce CHMO and thus to carry out Baeyer–Villiger
oxidations under mild conditions. These cells catalytically
produce specific lactones under conditions that are environ-
mentally less hazardous while producing their own cofactors
for the reaction and yielding an optically pure compound.
By expressing the necessary synthetic enzyme in a cellular
environment, the problems associated with enzyme stability
in extracellular conditions are circumvented along with the
problems of supplying the necessary cofactors (1). Most
importantly, the bacterial cell system, complete with the
necessary synthetic enzyme, is renewable through growth and
replication of the bacteria. A potential drawback of this tech-
nology is the need to separate products from the complex
cell mixture. In our case this is easily overcome through simple
extraction, which yields a relatively pure compound (2).
In this laboratory, students either work in teams to grow
the biocatalytic organism in the presence of ketone substrates
or, depending on time, use samples grown by the instructor.
Students extract the lactone produced by the organisms and
analyze their compound using thin layer chromatography, gas
chromatography, and mass spectroscopic techniques for prod-
uct identification. The experiment provides the flexibility to
utilize one or all three methods of identification. Extraction
of lactones from previously prepared bacterial cultures and
chromatographic and spectroscopic manipulations require
about 4 hours of class time. Data processing can be performed
outside of class or during another laboratory period.
Preparation of the bacterial cultures requires approximately
JChemEd.chem.wisc.edu • Vol. 78 No. 11 November 2001 • Journal of Chemical Education
W
30 minutes to an hour on each of the two days prior to the
actual experiment.W Students are asked to write a detailed
laboratory report discussing their results and answering sev-
eral questions requiring data analysis and calculations. The
project develops the student’s abilities in the areas of
biocatalysis, product identification, and decision-making.
Background
Cyclohexanone monooxygenase cloned from Acineto-
bacter sp. NCIB 9871 has a molecular weight of 53 kDa. It
consists of a single subunit with a tightly bound molecule of
FAD (flavin adenine dinucleotide). The enzyme catalyzes the
oxidation of a wide variety of cyclic ketones to the corre-
sponding lactones (2). Oxygen and NADPH (nicotinamide
adenine dinucleotide phosphate, reduced form) are consumed
in the reaction. The enzyme displays an absolute specificity for
O2 and NADPH but accepts a broad range of ketone substrates
(2). In most cases, the enzyme yields an optically pure compound
(3). In the case of racemic 3-substituted cyclohexanones, the
enzyme prefers the R isomer of the ketone owing to the shape
of the enzyme’s active site and the conformational state of the
substrates (4). In Acinetobacter cells, ε-caprolactone produced
from the oxidation of cyclohexanone is hydrolyzed to an α-
hydroxy acid that is ultimately degraded to form acetyl-CoA
and succinyl-CoA. Since Acinetobacter degrades most lactone
products and is a pathogen, the gene for cyclohexanone
monooxygenase has been transferred into a nonpathogenic
strain of E. coli. While some strains of E. coli can be dangerous
to humans, the majority are harmless. The latter are good
candidates for biocatalytic organisms because they are easy
and safe to grow, do not degrade lactones, and provide the
biocatalytic reactions with all the necessary cofactors.
The gene for CHMO was cloned into the BL21 (DE3)
strain of E. coli. In this plasmid construct, the CHMO gene is
genetically engineered to be under the control of the bacterio-
phage T7 transcription and translation signals, which in turn
are under control of the lac promotor (Fig. 4 of the supple-
mental materialW). This tightly regulated expression system
provides very high levels of inducible CHMO within the E.
coli cells, which in turn facilitates controllable Baeyer–Villiger
oxidations in the engineered strain. For more information
on the genetically engineered gene and its regulation see the
supplemental material.W
1533
 In the Laboratory
Experimental Procedure
The transformed bacteria can be obtained upon request to
Jon Stewart, Department of Chemistry at the University of
Florida (for the charge of shipping on dry ice). The bacteria
should be stored at ᎑70 °C in medium containing glycerol
or DMSO (20%) until they are used. Depending on time
and interest, students may participate in growing the
biocatalytic organism and seeding the reaction mixture. This
will require student involvement in the two days before prod-
uct isolation and identification. Alternatively, the instructor
can do the relatively simple microorganism preparation.W In
either case biocatalysis is initiated by mixing one of the pos-
sible substrates (cyclohexanone, 4-methylcyclohexanone, etc.
at 10 mM) with the E. coli 4–12 hours before use. The ex-
periment then proceeds as described below.
Teams of two to three students are given a flask with
40–60 mL of a liquid culture of the biocatalytic E. coli con-
taining the biosynthetic lactone produced during the previous
4–12 hours. We use several different substrates and give students
a biotechnology production problem that they must solve
through the isolation and identification of the product.
In a separatory funnel, the students begin the extraction
by adding a volume of methylene chloride one-fourth the
total volume of the liquid culture (10–15 mL of methylene
chloride). After mixing, the denser bottom layer consisting
of methylene chloride contains the majority of the lactone
product. The students perform the extraction at least three
times and combine the extracts. The lactone is concentrated
by rotary evaporation. The yield is calculated from the volume
and density of the lactone product and the initial amounts
of the cyclic ketone.
At this juncture, there are several choices of methodologies
for product identification. We use both thin layer chromatog-
raphy and gas chromatography–mass spectroscopy (GC–MS).
Teams or individual students first use 6 × 10-cm silica plates
and assorted controls to quickly examine the outcome of their
particular biocatalytic process. They spot the plates with 1 µL
of their unknown and the potential substrates and products.
The mobile phase is methylene chloride and the spots are
developed with phosphomolybdic acid in sulfuric acid (PMA
at 1% dissolved in 50% H2SO4), which leads to dark brown/
purple spots for the compounds. As an alternative to PMA,
the students can use an iodine chamber and develop for 10
minutes. The students examine their TLC plates and deter-
mine which of the lactone products they have isolated; they
also estimate the effectiveness of conversion from substrate
to product.
The purpose of the TLC evaluation is to make students
think critically about the reaction and experimental proce-
dure before they move on to more sophisticated methods of
analysis. Students further analyze and confirm their product
through GC–MS. If only a GC is available, the lactone product
can be identified by comparison to a standard elution profile
comprising the various lactones. Since this laboratory is de-
signed for an introductory biochemical class, the students are
not asked to interpret the fragmentation pattern of the mass
spectra. However, they are required to match the molecular
1534 Journal of Chemical Education • Vol. 78 No. 11 November 2001 • JChemEd.chem.wisc.edu
ion peak with the molecular weight of the unknown lactones
and use a spectrum library to identify the product. More
advanced classes can perform a more complete mass spectrum
analysis.
Hazards
Methylene chloride is toxic and should be handled with
care. The separation should be performed in the fume hood.
The TLC development solution contains concentrated
H2SO4, so extreme caution should be used.
Discussion and Conclusions
In two semesters (4 sections) of pilot testing, students
achieved their goal of identifying an unknown lactone product
isolated from a biocatalytic organism with a high degree of
success (75%). Their product yields can be as high as 84% for
cyclic ketones; yields are lower for substrates containing sub-
stituent groups, as expected from previous research findings (2).
It is important that the isolated compounds be diluted
with methylene chloride (typically 1:10) before use in TLC
and GC–MS, since the approximate sample size for the MS
instrument is 100 ng. If the isolated lactone spot on the TLC
appears large and amorphous, we suggest further diluting the
sample before separation and identification by GC–MS. A
concentrated sample could damage the mass spectrum de-
tector. The extracted lactones should be identified the day of
isolation because the compounds tend to degrade (hydrolyze)
during storage. As an interesting alternative project, this
degradation can be followed over time, since products of
hydrolysis are quite easily identified on TLC or GC–MS.
Since the lactones from the biocatalysis process are optically
pure, analysis by polarimetry offers students an additional
technique in their evaluation of biocatalytic products.
The project successfully introduced students to the use
of a biocatalytic organism to perform novel chemical functions.
It also gives the students experience in using several important
qualitative and quantitative identification tools. This labora-
tory is most effective late in the semester, as it encompasses
many of the concepts taught throughout the semester in a
typical biochemistry course from enzyme catalysis to genetic
cloning. Students enjoy the opportunity to apply skills gained
during the semester in carrying out this project.
W
Supplemental Material
A more detailed version of this article is available in this
issue of JCE Online.
Literature Cited
1. Schmid, A.; Dordick, J. S.; Hauer, B.; Kiener, A.; Wubbolts, M.;
Witholt, B. Nature 2001, 409, 258–268.
2. Stewart, J. Curr. Org. Chem. 1998, 2, 195–216.
3. Donoghue, N.; Norris, D.; Trudgill, P. Eur. J. Biochem. 1976,
63, 175–192.
4. Stewart, J.; Reed, K.; Martinez, C.; Zhu, J.; Chen, G.;
Kayser, M. J. Am. Chem. Soc. 1998, 15, 3541–3548.