R. J. Kazlauskas et al.
DNA polymerase kit (Invitrogen, Carlsbad, CA), was performed by ini-
Steady-state kinetics for the hydrolysis of ethyl acetate: Initial rates were
tially heating the mixture of pL29P (50 ng), dNTPs (0.2 mm), primers
measured by using a pH indicator assay; formation of acetic acid causes
[
21]
(
0.2 pmol), MgSO
4
(1.5 mm), and 1ꢂ buffer to 968C for 2 min, followed
a decreases in absorption of p-nitrophenoxide upon protonation to the
ꢀ
1
ꢀ1
by 29 cycles of 968C for 30 s, 558C for 30 s, 728C for 3 min and then a
final extension step for 10 min at 728C.
phenol (e404 =16600m cm ) at pH 7.2 at 238C. A typical reaction mix-
ture contained ethyl acetate (0.2–200 mm), p-nitrophenoxide (0.81 mm),
BES (5 mm), and enzyme (0.01–5 mg). The BES buffer stabilizes the pH
of the solution. The calculation of rated accounts for the protons that
The amplified PCR products were separately treated with DpnI (1 U for
1
h at 378C, Invitrogen, Carlsbad, CA), an endonuclease that cleaves
were buffered by BES: vtrue =vobs(1+ AHCUTNGRNENUG( 5 mm BES)/(0.81 mm pNP)).
template DNA, which is methylated, but not the amplified product
DNA. Next, the PCR product was purified to remove enzymes, primers,
buffers, and dNTPs by using MinElute (Stratagene, Santa Clara). The pu-
rified PCR products were mixed together in a 1:1 ratio for a second
round of PCR to join the two DNA strands. Reaction conditions were
similar to the first round of PCR except the template was products 1 and
Steady-state kinetic constants for perhydrolysis of acetic acid and ethyl
acetate: Kinetic constants for perhydrolysis were determined by using
[
20]
the monochlorodimedone (MCD) assay,
in which the amount of
enzyme added was adjusted to give a linear dependence of the reaction
rate to enzyme concentration. All reactions contained MCD (0.0472 mm)
and sodium bromide (149 mm). The concentrations of hydrogen peroxide
and acetic acid were varied to give evenly spaced data points above and
2
(200 ng for both). The 5.1 kbp product was purified by using MinElute,
then transformed into E. coli BL21-DE3 competent cells by the heat-
M
below K . When varying the concentration of hydrogen peroxide, the
shock method. Transformed cells were plated on LB (lysogeny broth)
ꢀ1
concentration of acetic acid was 2.00m; when varying the concentration
of acetic acid or ethyl acetate, the concentration of hydrogen peroxide
was 10 mm. For perhydrolysis of ethyl acetate, 50 mm citrate at pH 5.5
was substituted for acetate.
agar containing ampicillin (0.1 mgmL ) and incubated for 17 h at 378C.
Individual colonies were picked and inoculated into sterile 96-well plates
ꢀ
1
containing LB (1.2 mL with 0.1 mgmL ampicillin) and grown for 17 h
at 378C with shaking. The overnight culture was inoculated onto LB
ꢀ
1
Steady-state kinetic constants for hydrolysis of peracetic acid: Initial
rates were measured by using a pHstat to monitor the release of acetate
from hydrolysis of peracetic acid at pH 7.2 by titration with NaOH
agarose containing ampicillin (0.1 mgmL ) by using sterile 96 pins, incu-
bated, overnight, at 378C, then stored at 48C for up to two weeks. Plas-
mids were isolated from the 17 h culture by using a high-throughput
robot (Qiagen, Valencia). The mutations were confirmed by DNA se-
quencing by using sequencing primers in both the forward (64 bp up-
stream from the start codon, ATG) and reverse (15 bp downstream from
the stop codon, TGA) by the Biomedical Genomics Center (University
of Minnesota) with ABI BigDye Terminator version 3.1 chemistry.
(
0.01N). The amount of substrate was varied from 5 to 0.05 mm peracetic
acid in a 10 mL solution. A solution of 0.01N NaOH was added to in-
crease the pH to 7.2. The amount of enzyme added was 10 ng to insure
that the initial rates for the first few minutes were linear.
Steady-state kinetic constants for perhydrolysis of ethyl acetate: Initial
rates for perhydrolysis were determined by using the monochlorodime-
Protein expression and purification: Typically, LB medium (5 mL) con-
taining ampicillin (0.1 mgmL ) was inoculated from a single colony,
[
20]
ꢀ
1
done (MCD) assay, where the amount of enzyme added was adjusted
ꢀ
1
(
5 to 100 ngmL ) to give a linear dependence of the reaction rate to
then grown, overnight, at 378C. The overnight culture was diluted (1:100)
ꢀ
1
enzyme concentration. All reactions contained MCD (0.0472 mm) and
sodium bromide (149 mm) and 100 mm citrate buffer at pH 6.5. The con-
centration of ethyl acetate were varied to give a minimum of three data
with fresh LB media (100 mL containing 0.1 mgmL ampicillin) and in-
cubated at 378C until the absorbance reached 0.6 at 600 nm. Filter-steri-
lized rhamnose (20% w/v) was added to a concentration of 2% (w/v) to
induce protein expression and the culture was incubated for an additional
M
points above and below K , whereas hydrogen peroxide concentration
was kept constant at 14.7 mm. The kinetic constants were found by fitting
the initial rates to the Michaelis–Menten equation by using nonlinear re-
gression with Origin v.8.0 software (OriginLab, Northhampton, MA).
The single substrate Michaelis–Menten model is valid because the second
substrate, hydrogen peroxide is completely saturated.
3
h at 378C. The induced culture was centrifuged (4000g, 15 min) and the
cell paste was resuspended in buffer A (50 mm NaH PO , 300 mm NaCl,
0 mm imidazole) to a concentration of 20% (w/v). The resuspended cul-
ture was flash frozen in liquid nitrogen, thawed to room temperature, ly-
2
4
2
ꢀ
1
sozyme was added (final concentration of 1 mgmL ) and incubated on
ice for 30 min. The cell lysate was centrifuged (10000g, 60 min) and the
supernatant was poured onto a column of Ni-NTA agarose resin (5 mL,
Invitrogen) pre-equilibrated with buffer A (25 mL). The column was
Perhydrolysis to hydrolysis ratio: The perhydrolysis to hydrolysis ratio of
ethyl acetate was determined by measuring the concentration of perace-
tic and acetic acids by using HPLC. To generate peracetic acid, L29X
ꢀ
1
enzyme (0.1 mgmL
2 2
final concentration) was mixed with H O
washed with buffer B (50 mL, 50 mm NaH
dazole) and the protein was eluted with buffer C (10 mL, 50 mm
NaH PO , 300 mm NaCl, 250 mm imidazole). Typical yield was 10–15 mg
protein. Protein concentrations were measured by absorbance at 280 nm
2 4
PO , 300 mm NaCl, 40 mm imi-
(
250 mm), ethyl acetate (600 mm), and citrate (100 mm) at pH 6.5. The
mixture was allowed to react at 378C for 20 min with shaking (700 rpm)
before being quenched by diluting one part of the mixture in nine parts
of phosphoric acid (50 mm). The concentration of peracetic acid was
2
4
ꢀ1
ꢀ1 [32]
by using the calculated extinction coefficient of PFE (35410m cm ).
[4]
[33]
measured as described previously, which is a modified Karst assay.
Assay of L29X variants: Hydrolysis of p-nitrophenyl acetate, perhydroly-
The assay involves oxidation of thioanisole with peracetic acid and quan-
tifying the resulting sulfoxide by HPLC. To measure acetic acid, the
quenched solution was analyzed by HPLC by using ACE5 C18 column
(250ꢂ4.6 mm) eluted with H PO (0.1%) with H SO (20 mm) with a
sis of acetic acid, and perhydrolysis of ethyl acetate were assayed by
ꢀ
1
using purified PFE variants. Values are given in Umg protein, where
U=1 mmol product formed per min. Initial rates for hydrolysis of p-ni-
1
3
4
2
4
ꢀ
1
trophenyl acetate at pH 7.2 with N,N-bis(2-hydroxyethyl)-2-aminoethane-
sulfonic acid (BES, 5 mm) and acetonitrile (8%) were measured spectro-
photometrically by monitoring the increase in absorption of p-nitrophen-
flow rate of 0.5 mLmin and detected at 210 nm. The retention time for
acetic acid was 10.5 min. A standard curve was measured by using the
peak area of acetic acid versus concentration (Figure S2 in the Support-
ing Information).
ꢀ
1
ꢀ1
[16]
oxide (e404 =16600m cm ) at 404 nm.
Initial rates for perhydrolysis
of acetic acid were determined by using the monochlorodimeodone
Nucleophile competition between hydrogen peroxide and water: Initial
[
15]
assay. The assay solution contains 1.0m acetic acid/acetate at pH 5.5,
hydrogen peroxide (10 mm), sodium bromide (166 mm), and monochloro-
dimedone (0.22 mm). Peracetic acid reacts with sodium bromide to form
hypobromous acid, which then reacts with monochlorodimedone to form
a colorless product. The disappearance of monochlorodimedone was
monitored spectrophotometrically at 290 nm (e290 =19900m cm ). Ini-
tial rates for perhydrolysis of ethyl acetate (400 mm) was measured by
using the monochlorodimedone assay similar to that for acetic acid. In
one exception, 50 mm citrate buffer at pH 6.5 substituted for acetate
buffer.
rates for perhydrolysis and hydrolysis were determined by pHstat at
2
38C with NaOH (0.01N) as the titrant. The amount of hydrogen perox-
ide was varied from 14.7 to 147 mm while the concentration of methyl
acetate was held constant at 1.5m. The reaction solution was adjusted
with NaOH (0.01N) before the addition of enzyme to pH 7.2. The
ꢀ
1
ꢀ1
ꢀ1
amount of protein added was 1.2 mgmL for wild-type PFE and L29I
PFE. For perhydrolysis, NaBr (166 mm) was added to react with peracetic
acid to form BrOH. At pH 7.2, BrOH ionizes to release a proton. This
initial rate accounts for both hydrolysis and perhydrolysis. For hydrolysis
in the presence of hydrogen peroxide, the amount of acid released corre-
3044
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 3037 – 3046