Supplementary table 1 Outer primers of xyl10 for dual asymmetrical PCR and overlap extention PCR
Fragments
Primers
Sequences (5′→3′)
GATCGGTAGATTAACCGCTCGAGAAAAGAGAACCTATCGA
X10-1F
GCCT
I
TAGCAGTTTCGAAAGCGATTCTAACGTAATCTTCACCAATA
X10-1R
ACTTTGTAGAA
TTAGAATCGCTTTCGAAACTGCTAGAGCTGCTGATCCAAAC
X10-2F
GCTA
II
X10-2R
GCTCCAAATAGTCTTTTGCTCTAGAT
GATCGGTAGATTAACCGCTCGAGAAAAGAGAACCTATCGA
X10-1F
GCCT
Full-length xyl10
X10-2R
GCTCCAAATAGTCTTTTGCTCTAGAT
Supplementary data Optimized xyl10 genge sequence
> The optimized endo-1,4-β-D-xylanase gene xyl10 sequence
ATGGTTCAAATCAAGGTTGCTGCTTTGGCTATGTTGTTCGCTTCTCAAGTTTTGTCTGAACCTATCGAGCCTAGACAAGCCTCTG
TTTCCATCGACACAAAATTCAAAGCACACGGTAAAAAGTATCTTGGTAATATCGGAGATCAATACACTTTGACTAAGAACTCTA
AGACTCCAGCTATTATTAAGGCTGATTTTGGTGCTTTGACTCCAGAAAATTCTATGAAATGGGATGCTACTGAACCATCCAGAG
GTCAATTTTCTTTTTCTGGTTCTGATTATTTGGTTAACTTCGCTCAATCTAACAATAAGTTGATCAGAGGTCATACTTTGGTTTGG
CACTCTCAATTGCCTTCTTGGGTTCAATCTATCACTGATAAGAACACTTTGATCGAAGTTATGAAGAACCATATCACTACTGTTA
TGCAACACTACAAGGGTAAAATCTACGCTTGGGATGTTGTTAACGAAATTTTTAATGAAGATGGTTCTTTGAGAGATTCTGTTT
TCTACAAAGTTATTGGTGAAGATTACGTTAGAATCGCTTTCGAAACTGCTAGAGCTGCTGATCCAAACGCTAAGTTGTACATCA
ACGATTACAATTTGGATTCTGCTTCTTACCCTAAATTGACTGGTATGGTTTCTCATGTTAAGAAATGGATTGCTGCTGGTATTCC
AATTGATGGTATTGGTTCTCAAACTCACTTGTCTGCTGGTGGTGGTGCTGGTATTTCTGGTGCTTTGAACGCTTTGGCTGGTGC
TGGTACTAAGGAAATTGCTGTTACTGAATTGGATATTGCTGGTGCTTCTTCTACTGATTATGTTGAAGTTGTTGAAGCTTGTTTG
AACCAACCTAAGTGTATCGGTATCACTGTTTGGGGTGTTGCTGATCCAGATTCTTGGAGATCGTCTTCCACTCCTCTTCTTTTTG
ACTCCAACTACAACCCTAAGCCAGCCTACACCGCAATCGCTAATGCCTTGTAA
Note: Signal peptide (underlined);
Start codon (highlighted in green);
Stop codon (highlighted in red).
Supplementary table 2 Comparison of Xyl10 properties with those of other endo-1,4-β-D-xylanase belonging to
GH family 10
Thermal
stability
pH stability (residual The specific
Km (mg Vmax (μmol
(residual
activity/%, T/°C, activity (U
ml-1) mg-1min-1)
activity/%, T/°C,
time/min)
mg-1)
time/min)
Origin
Expression
host
Molecular
mass (kDa)
Topta/°C
pHoptb
Acidothermus cellulolyticus
11B
E. coli
40-55
90
6
t1/2c, 90, 90
NRg
350d
0.53d
NRg
ABK52146.1
Alicyclobacillus sp. A4
E. coli
46.8
55
6.2
56%, 55, 60
>90%, 5.8-12.0, 37,
60
16.7d
5.2d
27.7d
ADK91076.1
3,174e
7.12e
5,391e
This study
56.0f
6.5f
435f
BAB69073.1
Aspergillus niger
P. pastoris
33
60
5
45.8%, 75, 5
More than 74%,
3.0-13.0, 120,
Room
temperature
Aspergillus oryzae KBN616
-h
32.44
58
5
Completele
stability under
60 oC
>90%, 6.0-8.0, 25,
720
Accession
number
Aspergillus terreus BCC129
P. pastoris
33
60
5
t1/2c, 60, 30
more than 80%, 410, 40, 240
533f
4.8f
757f
AAY86996.1
Bacteroides xylanisolvens
XB1A
E. coli
40
37
6
NRg
NRg
83.1d
1.6d
118d
CBH32823.1
Bispora sp.MEY-1
P. pastoris
44.9
85
>80%, 1.5-6.0, 37,
60
18,831d
0.98d
23,728d
ACS96449.1
Cellulosimicrobium sp. HY13
-h
36
55
6
t1/2c, 55, 15
NRg
1,092.0d
2.78d
4,067d
ACY75514.1
Geobacillus
thermoleovorans
E. coli
45
80
8.5
t1/2c, 80, 50-60
NRg
270f
2.1f
42.5f
AEW07375.1
Glaciecola mesophila
KMM241
E. coli
43
30
7
20%, 30, 60
>80%, 6.0-8.0, 15,
60
77e
1.22e
98.3e
ACN76857.1
Paenibacillus
curdlanolyticus strain B-6
E. coli
45
35
7.5
NRg
NRg
2.5d
2d
3.7d
BAJ41040.1
Paenibacillus sp. HPL-002
E. coli
38.4
50
8.0-9.0
About 40%, 50,
10
NRg
51.3f
0.061
μMf
55.3f
ADF42684.1
59.6d
0.51d
357.9d
ACY69972.1
About >90%, 2.07.0, 37, 60
100.7f
4.3f
195.4f
AEV41144.1
About 40%, 80, >70%, 3.0-9.0, 37,
5
60
207.2d
2.5d
373.6d
ADZ99358.1
3.0, 4.5–
>87%, 90, 10
5.0
Paenibacillus sp. Strain E18
E. coli
39
50
stable at 40 °C
but lost activity stable over a pH
7.5-9.0
rapidly above range of 6.0 to 10.0
50°C
Penicillium pinophilum C1
P. pastoris
38
50
4.0–5.5
Phialophora sp. G5
P. pastoris
40
70
4
Saccharopolyspora
pathumthaniensis S582
E. coli
36.5
70
6.5
t1/2c, 70, 120
More than 80%,
5.5-10.0, 50, 10
965e
3.92e
256e
ADL60499.1
Sorangium cellulosum
So9733-1
E. coli
45.8
30–35
7
t1/2c, 50, 5
more than 60%,
6.0-9.0, 30, 60
4.1e
25.8e
8.91e
AEB69780.1
Streptomyces megasporus
DSM41476
P. pastoris
47.6
70
5.5
About 50%, 70, >80%, 4.0-12.0, 37,
60
60
242.1d
1.7d
436.8d
ADE37527.1
Streptomyces sp. TN119
E. coli
38
60
6.5
Decreased
> 85%, 4.0-10.0, 37,
rapidly above
60
60 °C
57.9d
1.0d
74.8d
ACR61563.1
Streptomyces sp. S9
E. coli
46
55
6.5
>80%, 60, 60
>80%, 4.0-12.0, 37,
60
453.7d
2.85d
772.2d
ABX71815.1
Streptomyces sp. S27
E. coli
47
60
6.5
25%, 65, 60
About 80%, 2.212.0, 37, 60
790.7d
1.3d
1361.2d
ACF57946.1
50%, 70, 30
Streptomyces
thermocarboxydus HY-15
E. coli
43.96
55
6
t1/2c, 50, 40
NRg
87.3d
2.51d
127d
ACJ64840.1
Streptomyces sp.
SWU10
-h
44.00
60
6
>80%, 60, 60
>80%, 2-9, 4, 960
60d
NRg
NRg
BAK19338.1
Thermoanaerobacterium
saccharolyticum NTOU1
E. coli
50
63
6.4
t1/2c, 65, 55
70%, 5.5-8.0, 65, 30
91d
35d
1429d
ADQ57411.2
environmental DNA of
goat rumen
E. coli
52.4
30
6.5
537.0d
4.6d
884.7d
ADN44261.1
86.8%, 45, 60 50%, 4.0-8.0, 37, 60
a
Topt: the optimum temperature;
b
pHopt: the optimum pH;
c
t1/2: half time of xylanase activity;
d
the specific activity or kinetic parameters towards oat spelt xylan;
e
the specific activity or kinetic parameters towards beechwood xylan;
f
the specific activity or kinetic parameters towards birchwood xylan;
g
NR: not reported;
h
-: the original strain.
Supplementary table 3 Effects of metal ions and chemical reagents on Xyl10 activitya
Relative activity(%)b
Chemicals
a
1 Mm
10 mM
Li+
127.4±1.7
92.9 ±3.7
NH4+
115.2±1.1
112.6±1.3
K+
123.5±1.1
110.8±3.3
Cu2+
74.7±1.9
29.4±2.4
Ni2+
107.5±3.0
88.8±4.0
Co2+
96.9±3.0
58.8±2.4
Ba2+
119.3±0.4
103.9±3.6
Mg2+
118.0±2.7
102.3±2.6
Ca2+
104.5±0.6
87.8±1.9
Sr2+
105.8±3.4
110.0±5.1
Zn2+
101.1±4.6
100.9±1.4
Fe2+
100.7±0.5
92.6±2.6
Sn2+
106.1±4.3
37.6±3.5
Cd2+
102.5±1.4
98.7±2.1
Cr2+
118.2±0.7
117.6±2.7
Al3+
98.9±0.8
99.4±1.3
SDS
36.3±1.2
0.4±0.6
EDTA
94.0±2.7
91.0±3.5
Residual activities were determined at 60 °C in 0.1 M citric acid/Na2HPO4 buffer (pH 5.0) containing 1 or 10 mM
chemicals with 10 mg oat spelt xylan/ml; The reaction mixture without any additive was used as a control.
b
Values represent the mean ± standard deviation (SD, n = 3) relative to the control samples without
addition of any chemicals; 2.5×103 U mg-1 was taken as 100% of xylanase activity.
Supplementary figure 1 TLC analysis of the hydrolysis products of Xylan by purified Xyl10. (a) Hydrolysis of oat
spelt xylan at different time intervals. Lane S, a mixture xylooligosaccharides standards containing D-xylose (Xl),
xylobiose (X2), xylotriose (X3), xylotetraose (X4) and xylopentaose (X5); lane 1-5, hydrolysis products by purified
Xyl10 for 0 min, 10 min, 2 h, 6 h, and 12 h. (b) Hydrolysis of different xylans by purified Xyl10. Lane 1-3,
xylooligosaccharides produced after 12 h incubation of purified Xyl10 with 1% (w/v) oat spelt xylan, birchwood
xylan and beechwood xylan in 0.1 M citric acid/Na2HPO4 buffer (pH 5.0) at 60 °C, respectively. (c) Hydrolysis of
different xylans without enzymes. Lane 1-3, hydrolysates produced after 12 h incubation of 1% (w/v) oat spelt
xylan, birchwood xylan and beechwood xylan without purified Xyl10 in 0.1 M citric acid/Na2HPO4 buffer (pH 5.0)
at 60 °C, respectively.