Supplementary Data
O
173.3
-H
-O
OH
120
173.2
Relative absorbance
100
80
295.3
60
313.3
40
20
0
100
150
200
250
300
m/z
Supplementary Fig. 1 LC-MS/MS spectrum of 8-HPOME obtained from conversion of αlinolenic acid using diol synthase from A. nidulans double-site (H1004A-C1006S) variant.
The inset shows the major fragments formed.
120
Relative activity (%)
100
80
60
40
20
0
25
30
35
40
45
50
55
60
o
Temperature ( C)
Supplementary Fig. 2
Effect of temperature on the production of 5,8-diHOME from oleic
acid using whole recombinant cells expressing diol synthase from A. nidulans. The reactions
were performed by varying the temperature from 30 to 55 °C for 10 min in 50 mM HEPES
buffer (pH 7.5) containing 2 g cells l–1 and 0.14 g oleic acid l–1. Data represent the means of
three separate experiments and error bars represent the standard deviation.
120
Relative activity (%)
100
80
60
40
20
0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
pH
Supplementary Fig. 3 Effect of pH on the production of 5,8-diHOME from oleic acid using
whole recombinant cells expressing diol synthase from A. nidulans. The reactions were
performed by varying the pH from 6.0 to 8.0 at 40 °C for 10 min in 50 mM HEPES buffer
(filled circle, pH 6.0−8.0) and 50 mM EPPS buffer (empty circle, pH 8.0−8.5) containing 2 g
cells l−1 and 0.14 g oleic acid l−1. Data represent the means of three separate experiments and
error bars represent the standard deviation.
120
Residual activity (%)
100
80
60
40
20
0
25
30
35
40
45
50
55
o
Temperature ( C)
Supplementary Fig. 4 Effect of enzyme stability on the production of 5,8-diHOME from
oleic acid using whole recombinant cells expressing diol synthase from A. nidulans. The
reactions were performed in 50 mM HEPES buffer (pH 7.5) containing 2 g cells l−1 and 0.14
g oleic acid l−1 after incubation for 1 h at temperatures from 25 to 50 °C. Initial activity was
set at 100 %, and the residual activity after incubation is presented. Data represent the means
of three separate experiments and error bars represent the standard deviation.
140
Relative activity (%)
120
100
80
60
40
20
0
ol ol ol te ol ne de ol r ne ol
ntr an an eta an ta xi an the xa tan
Co EthoMethyl ac Prop o-ocsulfo Hexhyl e He Bu
Is yl
h
et
Et
im
eth
D
m
Di
Supplementary Fig. 5
Effect of solvents on the production of 5,8-diHOME from oleic acid
using whole recombinant cells expressing diol synthase from A. nidulans. The reactions were
performed at 40 °C for 10 min in 50 mM HEPES buffer (pH 7.5) containing 2 g cells l−1, 0.14
g oleic acid l−1, and 5 % (filled bar) or 10 % (empty bar) (v/v) solvent. Data represent the
means of three separate experiments and error bars represent the standard deviation.
140
Relative activity (%)
120
100
80
60
40
20
0
0
2
4
6
8
10
12
14
16
18
Dimethyl sulfoxide concentration (%, v/v)
Supplementary Fig. 6 Effect of dimethyl sulfoxide concentration on the production of 5,8diHOME from oleic acid using whole recombinant cells expressing diol synthase from A.
nidulans. The reactions were performed at 40 °C for 10 min in 50 mM HEPES buffer (pH
7.5) containing 2 g cells l−1, 0.14 g oleic acid l−1, and DMSO. Data represent the means of
three separate experiments and error bars represent the standard deviation.