Exploring the Biocatalytic Scope of Alditol Oxidase from Streptomyces coelicolor
FULL PAPERS
1
,3-propanediol, 2-aminoethanol, 1-amino-2-propanol, 2-
(100 mM), 1,2-pentanediol (50 mM), and 1-phenyl-1,2-etha-
nediol (50 mM) were performed in a closed 500-mL Erlen-
meyer flask containing 10 mL of 50 mM KPi buffer, pH 7.5,
10 mM AldO and 6800 U catalase. Reactions were per-
formed at 308C in a shaker-incubator at 100 rpm. Control
reactions without AldO were performed in parallel. Samples
were taken regularly for the determination of the oxidation
product(s) and analysis of the enantiomeric excess of the re-
amino-1-propanol, 4-amino-1-butanol, 1,2-butanediol, meth-
anol, ethanol, 1-propanol, 2-propanol, 1-butanol, 1,4-diami-
nobutane, 2-butanol, 1-pentanol, 2-pentanol, 1-octanol,
benzyl alcohol, 2-phenylethanol, isoamylalcohol, a-methyl-
benzyl alcohol, a-methylbenzyl amine, 1-phenyl-1,2-ethane-
diol, 1,2-propanediol, 2-amino-1-phenylethanol, d-2-amino-
2
-phenylethanol, l-2-amino-2-phenylethanol, 1,3-butanediol,
benzyl methylether, 1,4-butanediol, cinnamyl alcohol, d-
meso-2,3-butanediol, 2-methoxy-4-methylphenol, vanillyl al-
cohol, 1,2-butanediol, 1,2-pentanediol, 1,2-hexanediol, cis-
maining substrate (ee ).
s
Oxidation of (R)-1-Phenyl-1,2-ethanediol in Presence
1,2-cyclohexanediol. For substrate profiling, every well
18
of H O
2
(
100 mL) contained 50 mM potassium phosphate (KPi)
À1
buffer pH 7.5, 100 mM ABTS and 50 UmL HRP. The con-
centrations of AldO and the putative substrates were varied
resulting in four different screening conditions: a) 10 mM of
putative substrate, 12 nM AldO, b) 50 mM of putative sub-
strate, 12 nM of AldO, c) 10 mM of putative substrate and
To elucidate the mechanism of the AldO-catalyzed oxida-
tion of 1,2-diols to the corresponding a-hydroxy acids, the
oxidation of (R)-1-phenyl-1,2-ethanediol was performed
1
2
8
with and without H
in 2-mL Eppendorf tubes, in a total volume of 300 mL
O. Oxidation experiments were done
18
2
1
1
.2 mM of AldO and d) 50 mM of putative substrate and
.2 mM of AldO. For the putative substrate cholesterol the
50 mM KPi buffer, pH 7.5, containing 33% (v/v) H O. The
reaction mixture also contained 50 mM (R)-1-phenyl-1,2-
À1
concentrations were 2 mM cholesterol, also containing 0.2%
DMSO for conditions a) and c) and 10 mM, also containing
ethanediol, 7 UmL catalase and 20 mM AldO. Two control
1
2
8
reactions with H O were performed: one without enzyme
1
% DMSO for the conditions b) and d). The 96-well plates
and one without substrate, containing 50 mM (R)-mandelic
acid. The reaction mixtures were incubated overnight at
308C and then analyzed by MS. MS analysis of oxidation
products of 1,2-phenylethanediol was performed on a
Thermo LCQ Fleet mass spectrometer. Measurements were
performed by direct spray, operating in the negative ion
were incubated at room temperature for 24 h and regularly
checked by visual inspection for green colour formation as a
result of H O /HRP-mediated ABTS oxidation.
2
2
Enzyme Activity and Determination of Kinetic
Parameters
mode using 0.1% formic acid in H O:methanol (70:30).
2
Unless stated otherwise enzyme activity was measured at
Product Identification
258C in 50 mm KPi buffer pH 7.5 using the H O coupled
2 2
[9]
assay as described before containing 0.1 mM 4-aminoanti-
pyrine (AAP), 1.0 mM 3,5-dichloro-2-hydroxybenzenesul-
fonic acid (DCHBS) and 4 U of HRP. Steady-state kinetic
parameters were determined by measuring initial rates with
different substrate concentrations. Data were fitted with
Origin 7.0 using the Michaelis–Menten equation. Three dif-
ferent buffers were used to determine the effect of pH on
the enzyme activity: 50 mM citrate buffer (pH 2.5 to 6),
Product identification for the AldO-catalyzed conversion of
xylitol was performed by HPLC using a Shodex SUGAR
SP0810 column (8.0 mm IDꢁ300 mm) at 808C using H
an eluent with a flow rate of 0.7 mLmin . For detection an
RI detector was used. For product identification of the
AldO-catalyzed conversion of racemic diols samples were
analyzed by GC-MS (EI). All analyses were performed on a
O as
2
À1
CP-SIL 8CB column (30 mꢁ0.25 mm, d 0.5 mm) with the
f
50 mM sodium phosphate buffer (pH 6 to 8) and 50 mM gly-
following temperature program: from 408C to 3008C at 58C
À1
cine/NaOH buffer (pH 8.6 to 10.6). Initial activity was mea-
sured at 258C using the assay described above with 0.1 mM
ABTS instead of AAP/DCHBS as the HRP substrate. In
this case 0.1 mM ABTS, 5 U of HRP and 0.012 mM of AldO
were used and the initial activity was calculated from the in-
min . Helium was used as the carrier gas with a constant
À1
flow of 1 mLmin and split ratio of 25:1. The injector tem-
perature was 2508C. The mass spectrum was scanned from
m/z=15 to 550 with an acquisition rate of 1.8 scanss .
Products formed upon oxidation of 1,2-pentanediol and 1-
phenyl-1,2-ethanediol were analyzed by NMR. 1 mL of
sample was lyophilized and redissolved into 0.5 mL of D O.
Structures were elucidated by means of H, COSY, TOCSY,
HSQC and HMBC spectra on a Bruker Avance spectrome-
ter at 600 MHz using standard Bruker pulse programs. The
NMR data were also used to monitor the degree of conver-
sion.
À1
À1
À1
crease in A420 (e420 =36 mm cm ). At nine different tem-
peratures between 10 and 808C the initial activity was mea-
sured using a temperature-controlled spectrophotometer.
The effect of temperature on enzyme stability was deter-
mined by incubation of 250 mL samples of AldO (107 mM)
at 37 and 508C. Samples were taken and the residual activity
was determined with regular time intervals using the AAP/
DCHBS activity assay.
2
1
Chiral GC Analysis
AldO-Catalyzed Oxidation Reactions
To determine the enantioselectivity of AldO for chiral diols,
samples were analyzed by chiral GC. All samples were dilut-
ed 1:1 with acetonitrile before GC analysis, except for 1,3-
butanediol, which was further diluted as follows: 100 mL
sample+400 mL pyridine+400 mL [N,O-bis(trimetylsilyl)tri-
The oxidation of xylitol was performed in a 100-mL Erlen-
meyer flask with 20 mL of 50 mM KPi buffer, pH 7.5, con-
taining 10 mM xylitol, 0.24 mM AldO and 324 U catalase at
room temperature. Samples were taken regularly and ana-
lyzed by HPLC for substrate depletion and product forma-
tion. Oxidation reactions with racemic 1,3-butanediol
fluoroacetamide
(BSTFA)+1%
trimethylchlorosilane
(TMCS)]. The analyses were performed on a chiral Betadex
Adv. Synth. Catal. 2009, 351, 1523 – 1530
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1529
Van Hellemond, Erik W.
