Supplementary Table S1. Effect of Different Nutritional Components on Xylanase Production*
Dhiman et al. 2012
SupplementaryInformation
Supplementary Table S1. Effect of different nutritional components on xylanase production*
Carbon source(20 g l-1) / Xylanase activity(U ml-1) / Total protein(mg) / pHCellulose / 1125±88 / 6.82±0.56 / 3.98
Glucose / 1428±112 / 3.13±0.29 / 2.66
Lactose / 1372±119 / 5.27±0.48 / 3.13
Maltose / 1249±98 / 2.89±0.23 / 3.29
Cellobiose / 1088±86 / 3.29±0.34 / 2.59
CMC / 1179±112 / 6.12±0.59 / 4.24
Birchwood xylan / 1869±132 / 9.64±0.88 / 4.52
Rice straw / 1506±122 / 6.85±0.59 / 4.82
Wheat bran / 1662±109 / 5.03+0.45 / 4.88
Avicel / 926±69 / 4.62±0.39 / 4.51
Sucrose / 1129±119 / 7.59±0.64 / 4.38
Softwood powder / 826±89 / 5.19±0.48 / 6.53
Hardwood powder / 772±66 / 4.66±0.46 / 4.48
Nitrogen source(20 g l-1) / Xylanase activity(U ml-1) / Total protein(mg) / pH
Yeast extract + peptone / 1619±132 / 6.63±0.59 / 4.46
Yeast extract / 1429±122 / 5.29±0.49 / 4.52
Peptone / 1544±113 / 5.46±0.46 / 4.12
Corn steep powder / 1327±98 / 3.34±0.38 / 4.62
Urea / 1456±116 / 3.79±0.32 / 7.88
(NH4)2SO4 / 1182±129 / 4.12±0.39 / 5.26
KNO3 / 1383±120 / 4.29±0.44 / 5.15
NaNO3 / 1029±110 / 3.27±0.39 / 4.29
*Experiments were carried out at 30 ºC and pH 5.0 at 150 RPM with incubation of 8 days. Wheat bran preferred over birchwood xylan as carbon source due to cost economics
Supplementary Table S2. Different levels of independent variables
LevelsParameter / Independent Variable / –α / –1 / 0 / +1 / + α
for xylanase production
Temperature (°C) / A / 20 / 25 / 30 / 35 / 40
pH / B / 4.0 / 4.5 / 5.5 / 6.5 / 8.0
RPM / C / 150 / 200 / 250 / 300 / 350
for crude xylanase immobilization
Temperature (°C) / D / 20 / 25 / 30 / 35 / 40
pH / E / 3.0 / 4.0 / 5.0 / 6.0 / 7.0
RPM / F / 35 / 75 / 115 / 150 / 190
Incubation time (h) / G / 6 / 12 / 30 / 48 / 60
Supplementary Table S3. Regression equation analysis for xylanase production from Pholiota adiposa using 7liter jar fermenter
Source / Sum ofsquares / Degree of freedom / MeanSquare / F-value / p-value
Prob. > F
Model / 4819049 / 9 / 535449.9 / 10.47956 / 0.0005 / significant
A-Temp / 199395.9 / 1 / 199395.9 / 3.902475 / 0.0064
B-pH / 29183.49 / 1 / 29183.49 / 0.571165 / 0.0162
C-RPM / 293617.9 / 1 / 293617.9 / 5.746541 / 0.0037
AB / 30184.25 / 1 / 30184.25ta / 0.590751 / 0.4599
AC / 98879.05 / 1 / 98879.05 / 1.935211 / 0.1944
BC / 87236.65 / 1 / 87236.65 / 1.707351 / 0.2206
A2 / 2501142 / 1 / 2501142 / 48.95108 / < 0.0001
B2 / 1909887 / 1 / 1909887 / 37.37935 / 0.0001
C2 / 268637.5 / 1 / 268637.5 / 5.257638 / 0.0448
Residual / 510947.2 / 10 / 51094.72
Lack of Fit / 510947.2 / 5 / 102189.4
Pure Error / 0.0001 / 5 / 0.0001
Cor Total / 5329997 / 19
Supplementary Table S4.Effect of different combinations of reaction parameters (un-coded value) on xylanase production in 7-liter jar fermenters. P. adiposa cultures were incubated for 8 days*
Run / Temp. (°C) / pH / RPM / Xylanase activity (U/ ml)Actual / Predicted
1 / 30 / 5.5 / 250 / 1237±88 / 1245±54
2 / 30 / 5.5 / 250 / 1237±88 / 1245±54
3 / 25 / 6.5 / 200 / 350±23 / 339±21
4 / 30 / 5.5 / 250 / 1237±88 / 1245±54
5 / 30 / 8 / 250 / 665±41 / 638±44
6 / 35 / 6.5 / 300 / 868±51 / 826±56
7 / 30 / 5.5 / 250 / 1237±88 / 1245±54
8 / 25 / 4.5 / 200 / 526±43 / 584±45
9 / 40 / 5.5 / 250 / 368±29 / 270±19
10 / 30 / 5.5 / 350 / 1680±48 / 1662±77
11 / 30 / 5.5 / 250 / 1237±88 / 1245±54
12 / 30 / 5.5 / 150 / 652±52 / 613±40
13 / 25 / 6.5 / 300 / 377±34 / 340±24
14 / 35 / 6.5 / 200 / 380±33 / 402±29
15 / 35 / 4.5 / 200 / 227±19 / 280±27
16 / 22 / 5.5 / 250 / 371±31 / 335±24
17 / 30 / 5.5 / 250 / 1237±88 / 1245±54
18 / 30 / 4 / 250 / 672±57 / 293±19
19 / 35 / 4.5 / 300 / 281±22 / 587±47
20 / 25 / 4.5 / 300 / 452±33 / 446±39
*Composition of production medium (g/l) peptone (5.0), yeast extract (5.0), KH2PO4 (5.0), K2HPO4 (5.0), MgSO4.7H2O (3.0), thiamin-HCl (0.02), KNO3 (2.0), and wheat bran (20)
Supplementary Table S5. Substrate specificities of purified xylanase from Pholiota adiposa
Substrate (1%) / Specific activity (U/mg-protein) / Relative activity (%)Birchwood xylan / 418±27 / 100
Oat spelt xylan / 401 ±32 / 95.8
Avicel / 29.7±1.2 / 7.10
Carboxymethyl cellulose / 1.2 ±0.3 / 0.28
p-Nitrophenyl xylopyranoside / 0.6 ±0.1 / 0.14
p-Nitrophenyl glucopyranoside / ND* / ND*
p-Nitrophenyl cellobioside / ND* / ND*
*ND: Not Detectable
Supplementary Table S6.CCD-RSM design of different independent variables (un-coded) affecting the immobilization of crude xylanase from Pholiota adiposa on gluteraldehyde-activated SiO2 nanoparticles
Run / Temp. (°C) / pH / RPM / IT (h) / Yield (%) / Efficiency (%)Actual / Predicted
1 / 35 / 4 / 75 / 12 / 77.2±6.6 / 102±8.5 / 94.7±7.5
2 / 25 / 4 / 150 / 12 / 80.2±7.4 / 88.8±4.7 / 69.6±3.8
3 / 25 / 4 / 75 / 12 / 84.0±6.8 / 75.4±6.4 / 80.1±4.5
4 / 25 / 6 / 150 / 12 / 61.7±5.2 / 85.8±5.6 / 82.7±6.6
5 / 20 / 5 / 115 / 30 / 70.4±6.4 / 79.7±6.5 / 90.5±8.4
6 / 35 / 6 / 75 / 48 / 66.7±5.9 / 109±10.6 / 127±10.2
7 / 35 / 4 / 75 / 48 / 72.8±6.8 / 105±3.8 / 104±6.9
8 / 30 / 5 / 35 / 30 / 66.0±5.6 / 144±9.8 / 134±9.3
9 / 25 / 4 / 75 / 48 / 47.5±3.4 / 89.4±7.3 / 90.9±5.7
10 / 30 / 5 / 115 / 30 / 75.9±5.9 / 122±10.6 / 123±9.8
11 / 30 / 5 / 115 / 30 / 75.9±5.7 / 122±11.6 / 123±9.8
12 / 25 / 6 / 150 / 48 / 59.9±6.1 / 118±11.4 / 125±10.9
13 / 30 / 3 / 115 / 30 / 49.4±3.9 / 65.5±5.5 / 79.2±6.2
14 / 35 / 4 / 150 / 48 / 65.4±4.2 / 128±11.3 / 122±9.8
15 / 25 / 4 / 150 / 48 / 66.7±6.2 / 110±11.2 / 99.6±5.8
16 / 35 / 4 / 150 / 12 / 64.8±3.8 / 87.0±4.5 / 93.4±6.3
17 / 30 / 5 / 115 / 30 / 75.9±3.4 / 122±10.9 / 123±10.5
18 / 25 / 6 / 75 / 48 / 79.0±3.6 / 122±10.2 / 120±9.9
19 / 30 / 5 / 115 / 30 / 75.9±5.4 / 122±9.8 / 123±9.8
20 / 35 / 6 / 75 / 12 / 61.1±5.4 / 98.7±7.9 / 105±10.4
21 / 35 / 6 / 150 / 12 / 65.4±5.4 / 102±9.8 / 99.7±7.2
22 / 30 / 7 / 115 / 30 / 69.8±5.6 / 123±5.9 / 115±10.6
23 / 30 / 5 / 115 / 6 / 66.0±2.9 / 45.9±2.4 / 49.0±3.9
24 / 30 / 5 / 115 / 30 / 75.9±5.2 / 122±5.9 / 123±9.7
25 / 35 / 6 / 150 / 48 / 64.8±2.8 / 149±6.8 / 141±9.4
26 / 25 / 6 / 75 / 12 / 52.5±3.4 / 92.9±5.9 / 97.5±6.9
27 / 40 / 5 / 115 / 30 / 76.5±5.6 / 126±9.6 / 121±9.8
28 / 30 / 5 / 190 / 30 / 69.1±3.9 / 121±9.7 / 137±10.4
29 / 30 / 5 / 115 / 66 / 67.3±4.2 / 99.2±6.8 / 101±10.2
30 / 30 / 5 / 115 / 30 / 75.9±4.6 / 122±9.8 / 123±10.2
Supplementary Table S7. Regression equation analysis for immobilization of crude xylanase on gluteraldehyde activated SiO2 nanoparticle
Source / Sum ofsquares / Degree of freedom / MeanSquare / F-value / p-value
Prob. > F
Model / 13667.57 / 14 / 976.2551 / 7.316471 / 0.0002 / significant
A-Temp / 1404.54 / 1 / 1404.54 / 10.52622 / 0.0054
B-pH / 1958.427 / 1 / 1958.427 / 14.67728 / 0.0016
C-RPM / 15.68167 / 1 / 15.68167 / 0.117525 / 0.7365
D-IT / 4118.64 / 1 / 4118.64 / 30.86684 / < 0.0001
AB / 47.61 / 1 / 47.61 / 0.35681 / 0.5592
AC / 85.5625 / 1 / 85.5625 / 0.641242 / 0.4358
AD / 0.81 / 1 / 0.81 / 0.00607 / 0.9389
BC / 18.0625 / 1 / 18.0625 / 0.135368 / 0.7181
BD / 156.25 / 1 / 156.25 / 1.171004 / 0.2963
CD / 370.5625 / 1 / 370.5625 / 2.777153 / 0.1164
A2 / 477.1433 / 1 / 477.1433 / 3.575915 / 0.0781
B2 / 1095.852 / 1 / 1095.852 / 8.212781 / 0.0118
C2 / 294.9376 / 1 / 294.9376 / 2.210388 / 0.1578
D2 / 3832.652 / 1 / 3832.652 / 28.72353 / < 0.0001
Residual / 2001.488 / 15 / 133.4325
Lack of Fit / 2001.488 / 10 / 200.1488
Pure Error / 0.001 / 5 / 0.001
Cor Total / 15669.06 / 29
Supplementary Table S8. Comparison of immobilization yield and immobilization efficiency for different supporting matrix
Resin Type / Immobilization Yield (%) / Immobilization Efficiency (%)Amberlite XAD-16 / 14.8±1.6 / 17.5±1.9
XAD-4 / 22.2±1.8 / 19.7±1.8
Amberlite IRA-400 / 14.2±1.1 / 29.0±2.5
XAD-2 / 17.2±1.4 / 18.3±1.6
XAD-7HP / 19.7±1.6 / 14.6±1.3
DUO-A7 / 27.1±2.1 / 37.4±2.9
Supplementary Figure S1.The phylogenetic dendrogram for P. adiposa and related strains based on the IT rDNA sequence. Numbers following the names of the strains are accession numbers of published sequences.
Supplementary Figure S2.Effect of different temperature and pH values on xylanase activity.Xylanase activity was determined at various temperature () and pH () values to indentify the optimal conditions for purified PaXyl
Supplementary Figure S3. Effect of birchwood xylan concentration on the activity of xylanase. Xylanase activity was measured in the presence of the indicated concentrations of birchwood xylan at pH 5.0. The inset shows the Lineweaver-Burk plot of initial velocity versus various fixed substrate concentrations. Each value represents the mean of triplicate measurements and varied from the mean by not more than 10%.
Supplementary Figure S4.Thermal stability profile of purified xylanase from Pholiota adiposa. Relative xylanase activity (%)at 4°C (); at 25°C (); at 40°C (); at 50°C (); at 60°C (); at 70°C (); at 80°C ()
Supplementary Figure S5.Effect of immobilization on temperature and pH stability of crude xylanase from Pholiota adiposa. Relative activity (%) of free () and immobilized () crude xylanase at different temperatures; relative activity (%) of free () and immobilized () crude xylanase at different pHs
Supplementary Figure S6. Effect of unoptimized (low protein loading) and optimized (high protein loading) enzyme dose on the relative activity of immobilized PaXyl at different temperature and pH values.Relative activity (%) of the immobilized crude xylanase with low enzyme dose () and high enzyme dose () at different temperatures; relative activity (%) of the immobilized crude xylanase with low enzyme dose () and high enzyme dose () at different pHs
Supplementary Figure S7. Thermal stability profile of crude un-immobilized and immobilized PaXyl. Relative xylanase activity (%) at 50°C; un-immobilized enzyme (); immobilized enzyme ()
Supplementary Figure S8. Reusability of immobilized crude PaXylfor XOS synthesis. One cycle is defined as the time taken to hydrolyze all of the substrates present in the reaction mixture under the standard assay condition.
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Supplementary Figure S9.High-pressure liquid chromatography (HPLC) chromatogram showing synthesis of xylooligosaccharide (XOS). a. by crude unimmobilized xylanase; b. by crude immobilized xylanase
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