Supplementary Information
An isolated Amycolatopsis sp. GDS for cellulase and xylanase production using
agricultural waste biomass and fermentative ethanol production
Siddheshwar D. Kshirsagar1§, Ganesh D. Saratale2§, Rijuta G. Saratale3, Sanjay P.
Govindwar4 and Min-Kyu Oh2*
1Department
of Biotechnology, Shivaji University, Kolhapur 416004, Maharashtra, India
2Department
of Chemical and Biological Engineering, Korea University, Seongbuk-gu, Seoul
136-713, South Korea
3Department
of Environmental Science and Engineering, Ewha Womans University, Seoul
120-750, South Korea
4Department
of Biochemistry, Shivaji University, Kolhapur 416004, Maharashtra, India
*Corresponding author:
Min-Kyu Oh
Department of Chemical & Biological Engineering
Korea University, Seoul, 136–713
Republic of Korea.
Phone: +822–3290–3308
Fax:
+822–926-6102
Email: mkoh@korea.ac.kr
§ Both
authors contributed equally to this manuscript
1
Figure legends:
Figure S1 Neighbor-joining showing phylogenetic positions of Amycolatopsis sp. GDS and
Amycolatopsis species based on 16S rRNA gene sequence comparisons.
Figure S2 Volumetric activity of cellulase and xylanase of Amycolatopsis sp. GDS using
wheat straw (10 g l-1) as a cellulosic substrate at different incubation period (at
30°C, initial pH of Dubos media; 6.5) under static condition [exoglucanase (●);
cellobiase (▲); FPase (○); xylanase (□) and endoglucanase (■)]
Figure S3 HPTLC profile of sugars produced during enzymatic hydrolysis of wheat straw
by crude enzymes produced byAmycolatopsis sp. GDS after 48 hours of enzymatic
hydrolysis.(Standard Glu-glucose, Xyl- Xylose, Cel-cellobiose, and EH-enzymatic
hydrolysate of WS.
Table S1 Biochemical characterization of isolated cellulose degrading bacteria.
Table S2 Effects of incubation temperature, initial pH of Dubos media and under static (no
aeration and agitation) and shaking (100 rpm) condition on endoglucanase, FPase
and xylanase production by Amycolatopsis sp. GDS in Dubos medium containing
CMC (10 g l−1) as a carbon source
2
21
74
Amycolatopsis sp. ACT-0101
Amycolatopsis sp. ACT-0100
Amycolatopsis sp. PENDO-1789
7
Amycolatopsis tolypomycina type strain:DSM 4454
3
Amycolatopsis sp. GY125 GY125
27
2
Amycolatopsis rifamycinica DSM 46095
Amycolatopsis sp. GY122
1
Amycolatopsis tolypomycina IMSNU 20061
Amycolatopsis sp. 4-36
37
32
Amycolatopsis sp. 2-15
47
Amycolatopsis sp. 308201
52
Amycolatopsis sp. OS3-28
Amycolatopsis sp. 232065
9
Amycolatopsis sp. SF27
2
99
Amycolatopsis balhimycina DSM 44591
Amycolatopsis sp. AR1438
55
Amycolatopsis lexingtonensis NRRL B-24131
Amycolatopsis tolypomycina type strain:DSM 44544
Amycolatopsis mediterranei NRRL B-3240
26
Amycolatopsis pretoriensis NRRL B-24133
78
89
Amycolatopsis sp. GDS
1
Figure S1
3
Enzyme activity (U ml-1)
10
8
6
4
2
Enzyme activity (U ml-1)
0
600
500
400
300
200
100
0
0
2
4
5
6
7
8
10
Time (days)
Figure S2
4
Figure S3
5
Table S1 Biochemical characterization of isolated cellulose degrading bacteria.
Biochemical Test
Amycolatopsis sp. GDS
Gram staining
Gram positive
Color
White
Motility
Non-motile
Inulin
- ve
Sodium gluconate
- ve
Glycerol
- ve
Salicin
- ve
Alpha Methyl D-Glucoside
- ve
Alpha Methyl D-Monoside
- ve
ONPG
- ve
Esculin hydrolysis
+ ve
D-Arabinose
+ ve
Citrate Utilization
+ ve
Malonate
+ ve
Lysine
+ ve
Ornithine
+ ve
H2S Production
- ve
Utilization of the following compounds as a carbon source
Lactose
- ve
Xylose
+ ve
Maltose
+ ve
Fructose
+ ve
Dextrose
+ ve
Galactose
+ ve
Raffinose
- ve
Trehalose
+ ve
Melibiose
+ ve
Sucrose
+ ve
L-Arabinose
+ ve
Glucose
+ ve
Mannose
+ ve
Arabinose
+ ve
Sorbose
- ve
Xylitol
- ve
Erythritol
- ve
Rhamnose
- ve
Cellobiose
+ ve
Arabitol
- ve
Adonitol
- ve
Mannitol
- ve
Sorbitol
- ve
Inositol
- ve
Dulcitol
- ve
Melezitose
- ve
6
Table S2 Effects of incubation temperature, initial pH of Dubos media and under static (no aeration and agitation) and shaking (100 rpm)
condition on endoglucanase, FPase and xylanase production by Amycolatopsis sp. GDS in Dubos medium containing CMC (10 g l−1) as a carbon
source.
Incubation temperature (0C)
Enzyme
25
Endoglucanasea
FPasea
Xylanasea
a
30
5.5
6.0
6.5
7.0
7.5
8.0
Static
Shaking
30.24 44.20 42.45 35.65
15.45
30.24
38.54
44.20
35.65
18.45
8.45
31.25
44.20
±0.55 ±0.75 ±0.65 ±0.75
±0.35
±0.55
±0.55
±0.75
±0.75
±0.35
±0.35
±0.75
±0.75
0.52
0.32
0.41
0.54
0.72
0.65
0.32
0.15
0.72
0.35
±0.02 ±0.02 ±0.02 ±0.02
±0.01
±0.01
±0.015 ±0.02
±0.01
±0.01
±0.01
±0.02
±0.01
42.00 69.52 69.52 52.32
32.54
32.52
48.54
69.52
61.54
42.12
12.12
69.52
41.25
±0.98 ±1.16 ±1.25 ±1.15
±1.00
±0.98
±1.00
±1.16
±1.15
±1.00
±0.85
±1.16
±1.15
0.70
37
Agitation
40
0.72
35
Initial pH of Dubos media
0.55
Enzyme activity (U ml-1).
Values are mean of three experiments; (±) standard error (SE) by one-way ANOVA with Tukey–Kramer Multiple Comparisons Test.
7