1
Supplementary data
2
Fig.S1 Multiple sequence alignment
3
4
Multiple sequence alignment of the consensus sequence and the catalytic residues of lipases
homologous to YLIP9.
5
6
Fig.S2 Kinetic parameters of ZZ-YLIP9
7
8
9
Assay was performed with different concentrations of p-NPdecanoate, p-NPlaurate and pNPpalmitate ranging from 0.6 mM to 1.6mM. The kinetic parameters were calculated using the
Lineweaver-Burk plot and Michaelis-Menten equation.
10
Fig. S3 Regioselectivity of ZZ-YLIP9
11
12
13
50 U of lyophilized ZZ-YLIP9 was incubated with 100 µM of triolein in 20 ml hexane for 24 h
at 60 °C/100 rpm. The filtered product was analyzed by (a) TLC and (b) Gas chromatography.
Retention time for oleic acid, diolein and triolein is 27, 32 and 40 min.
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15
Fig. S4 Thermal kinetics of ZZ-YLIP9
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17
18
The stability of ZZ-YLIP9 was determined at different temperatures (30-80 0C) by incubating the
enzyme at these temperatures for 2 h. The log of the percentage residual activity was plotted
against different time intervals and thermal kinetic parameters were calculated.
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20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
Figure S1
AFL1-1
CoPL
ANL
PDL
AOL
Lip18
Lip14
Lip7
Lip8
YLIP9
Lip11
LIP2
CdLIP1
122
130
140
130
143
148
123
133
130
158
194
128
144
.:
SYAALQWMSD--------HAAELGFNPRKLVVCGSSAGGNLTAGVTLLARDRSGPQIRGQ
GFM-TSWVNARAAILGPLSDTIAKYPDYQLVVTGHSLGGAVAAIASLELR---------GFM-NAWRLTRATILDTISAARDQYPDYALTLVGHSLGGAVAALAGTEMQ---------GFM-RSWLHTRTEILPAVTTLRQNYPDYAVTLVGHSLGGAVAALAGLEMR---------GFL-ASWLNTRPIILKHVSAARKQYRDYKVVLVGHSLGGAVAALAGLEMQ---------GFL-KGYLHSFHVVDSIVQRLTEKYPNYQLVITGHSLGGTAATLFGLNYR---------GWN-KAYNETMGIIGDKLADHVNSNPDYRLVVTGHSLGAAIAVLSATSLK---------GFS-KAFTETWHNIGDLLEQHLDSYPDYQLYVTGHSLGAAMALLGATSIK---------GFS-KAFTETWGNIGEDLQKHLDANPDYQLYVTGHSLGAAVALLGATSIK---------GFV-GIFNNSFKQIDSRLNLYKSMYPDYKLVVTGHSLGGAVALLYGVSLR---------GFL-QAYNLAYSEAKGAMDDTFAKYPDYQVIVTGHSLGGAATFLHGINLK---------GFI-QSYNNTYNQIGPKLDSVIEQYPDYQIAVTGHSLGGAAALLFGINLK---------GFI-ESYNNTFNQIGPKLDSVIAEHPDYEIAVTGHSLGGAAALLFGINLK---------:
: : * * *. :
:
Figure S2
(a)
1
173
178
188
178
191
196
170
181
178
106
242
176
192
2.5
1/V (µmol/ml/min)-1
2.0
1.5
1.0
0.5
0.0
-1.0
-0.5
0.0
1.0
1.5
2.0
-1
41
42
0.5
1/S (mM )
(b)
3.5
1/V (µmol/ml/min)-1
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-1.0
43
44
-0.5
0.0
0.5
1.0
1.5
2.0
-1
1/S (mM )
(c)
2
7
1/V (µmol/ml/min)-1
6
5
4
3
2
1
0
-1.0
45
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48
49
50
51
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53
54
55
56
57
58
59
60
61
62
63
64
65
-0.5
0.0
0.5
1.0
1.5
2.0
-1
1/S (mM )
Figure S3
(a)
(b)
3
66
67
68
Figure S4
2.05
Log (%) residual activity
2.00
1.95
1.90
1.85
1.80
1.75
1.70
1.65
0
20
40
60
80
100
120
140
Time (min)
0
69
70
Stability at 30-70 C
Stability at 80 0C
71
4