Download Supplementary materials Method 1: liquid chromatography for

Supplementary materials
Method 1: liquid chromatography for Enzyme purification
The dialyzed ammonium sulfate fraction (200 ml) was applied to a Fractogel DEAE-650M
column (2.5 × 30 cm). The enzyme was then eluted with buffer A (flow rate, 90 ml/h). Ammonium
sulfate was added to the pooled active fractions to a final 2-M concentration. The enzyme solution was
then applied to a Toyoperal Phenyl-650M column (2.5 × 15 cm) pre-equilibrated with buffer A
containing 2 M ammonium sulfate. After washed with 500 ml buffer A containing 2 M ammonium
sulfate, the column was eluted with a 1-L linear gradient of 0–2 M ammonium sulfate in buffer A (flow
rate, 85 ml/h). Ammonium sulfate was added to the pooled active fractions to a 3.5-M final
concentration. The enzyme solution was then applied to a Toyoperal MX-Trp-650M column (1.5 × 15
cm) pre-equilibrated with buffer A containing 3.5 M ammonium sulfate. After the column was washed
with 500-ml buffer A containing 3.5 M ammonium sulfate, it was eluted with a 1-L linear gradient of
3.5–1 M ammonium sulfate in buffer A (flow rate, 70 ml/h). To concentrate the pooled active fraction
(250 ml), ammonium sulfate was added to a 2-M final concentration, recharged to a small Toyoperal
Phenyl-650M column (1.5 × 15 cm) pre-equilibrated with buffer A containing 2 M ammonium sulfate,
and eluted directly within 20-ml buffer A. The concentrated enzyme solution was applied to a Fractogel
HW-50 column (2.5 × 115 cm). The column was eluted with 10 mM phosphate (pH 7.5) (flow rate, 15
ml/h). The pooled active fractions were further purified using an Ultragel-HA column (2.5 × 15 cm)
pre-equilibrated with 10 mM phosphate (pH 7.5). After washing with 10 mM phosphate buffer, the
column was eluted with a 500-ml linear gradient of 10–200 mM phosphate buffer (flow rate, 30 ml/h).
The active fractions were pooled and stored at −20°C.
1
2
75 KDa
73 KDa
sFig. 1 SDS-PAGE analysis of purified laccase.
purified laccase is shown in lane 2.
The 73-kDa band representing
Lane 1: protein marker.
SFig. 2 Cyclic voltammetry of borate–fructose complex (upper) and ABTS
(lower).
(A)
(B)
sFig. 3
Optimal temperature (A) and thermostability (B) of laccase. Relative
activities assay was performed by mixing ABTS, borate buffer, fructose, and
suitably diluted enzyme in 1-ml cuvettes as described in 1.4.
sFig. 4
pH stability of laccase. The four different pH regions using the following
buffers are examined (without substrate or mediator) for relative activities:
CH3COOH-CH3COONa (pH 4.5-5.5) (□), KH2PO4-NaHPO4 (pH 5.5-7.0) (○),
Tris-HCl (pH 7.0-9.0) (Δ), and Na2CO3-NaHCO3 (pH 9.0-10.0) ( ).
Relative
activities assay was performed by mixing ABTS, borate buffer, fructose, and suitably
diluted enzyme in 1-ml cuvettes as described in 1.4.
H
H
H
H
H
O
OH
O
H
+ H2O
OH
O
HO B
OH
H
H
O
+ H+
OH
OH
O
H
OH
O
H
HO BOH
sFigure 5 The structure and acid–base reaction of borate-fructose complex. The
complex resulting in a six-membered heterocyclic compound is similar to phenol, not
only structure but also size. It is suggest that the borate–fructose complex can be a
good substrate, serve as a mediator, and provided a hydrogen buffer on laccase
activity.
sTable 1 purification of laccase from strain Edenia sp. TS-76
Total
volume
(ml)
Total
protein
(mg)
Total
activity
(U)
Specific
activity
(U/mg)
Crude extract
800
1,268
2,576
2.03
1.0
100
Precipitation
80
659
1,555
2.36
1.2
60
180
359
2,267
6.31
3.1
88
525
311
1,748
5.62
2.8
68
325
218
1,749
8.02
4.0
68
104
55
1,311
23.84
11.7
51
120
12
818
68.2
33.6
32
Fractogel
DEAE-650M
Toyopearl
phenyl-650M
Toyoperal
MX-Trp-650M
Fractogel HW
50 gel filtration
Ultragel-HA
Purification Yield
fold
(%)