Apendice-Biodegradation

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Supplementary information 
 
Biodegradation of acrylamide by a novel isolate, Cupriavidus oxalaticus 
ICTDB921: identification, and characterization of the acrylamidase produced 
 
Dattatray K. Bedade and Rekha S. Singhal* 
Food Engineering and Technology Department, Institute of Chemical Technology, 
Nathalal Parekh Marg, Matunga, Mumbai 400 019, India 
 
*Corresponding author: 
Prof. Rekha S. Singhal 
Professor and Dean (RCRM) 
Food Engineering and Technology Department, 
Institute of Chemical Technology, 
Nathalal Parekh Marg, Matunga, Mumbai 400 019, India 
Tel: +91 22 3361 2501, Fax: +91 22 3361 1020 
E-mail: rsinghal7@rediffmail.com 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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CH2=CH-CONH2 CH2=CH-COOH + NH3 
 Acrylamide Acrylamidase Acrylic acid Ammonia 
 
 Propionate 
 Nitrogen metabolism 
 
 Succinyl-CoA 
 Succinyl-CoA synthetase 
 Malate deshydogenase 
 Phosphoglycerate kinase 
 
 Acetyl-CoA ATP + CO2 + H2O 
 
 
Fig. S1 Proposed biochemical pathway for acrylamide biodegradation by C. oxalaticus 
ICTDB921 (Adapted from Charoenpanich and Tani, 2014) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Induces expression of 
genes relevant to 
acrylamide metabolism 
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Fig. S2 Phenol red pH indicator plate showing degradation of acrylamide. (Section I- 
control (without bacterium), Section II- Isolate (ICTDB921) with ability to degrade 
acrylamide, Section III- Isolate (ICTDB922) having no ability to degrade acrylamide) 
 
 
 
 
 
 
 
 
I III 
II 
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Fig. S3 Utilization of acrylamide along with acrylic acid, ammonia and biomass 
production profile by C. oxalaticus ICTDB921 under optimized conditions (4.2 g l-1 or 
60mM acrylamide, 30 °C and pH-7). (Total acrylic acid= acrylic acid detected in medium 
+ acrylic acid utilized for growth, Total ammonia= ammonia detected in medium+ 
ammonia utilized for growth). 
 
 
 
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Time (h)
Acrylamide Total acrylic acid Total ammonia DCW
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Table S1. Two enzymes from Cupriavidus oxalaticus and other reported strains 
showing identity based on protein BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). 
Sr. no Microbial culture/ amidase 
type 
Locus ID 
(NCBI) 
Amidase type from 
Cupriavidus 
oxalaticus 
Identity (%) 
1. Geobacillus 
thermoglucosidasius AUT-
01/acrylamidase 
 
ACL27228 
Formamidase 33 
N-carbamoyl-D-
amino-acid hydrolase 
24 
2. Voriovorax boronicumulans 
CGMCC 4969 /aliphatic 
amidase 
 
WP_095743110 
Formamidase 33 
N-carbamoyl-D-
amino-acid hydrolase 
26 
3. Geobacillus 
stearothermophilus/ Aliphatic 
amidase 
 
Q9RQ17.1 
Formamidase 33 
N-carbamoyl-D-
amino-acid hydrolase 
24 
4. Rhodococcus 
erythropolis/acylamide 
amidohydrolase 
 
WP_095973499 
Formamidase 31 
N-carbamoyl-D-
amino-acid hydrolase 
27 
5. Brevibacterium sp./amidase AAA22990 Formamidase 31 
N-carbamoyl-D-
amino-acid hydrolase 
27 
6. Geobacillus pallidus BTP-5X 
MTCC9225/amidase 
 
AF257487_1 
Formamidase 31 
N-carbamoyl-D-
amino-acid hydrolase 
25 
7. 
Bacillus sp. BR449/amidase 
 
AF257487_1 
Formamidase 31 
N-carbamoyl-D-
amino-acid hydrolase 
25 
8. 
Bacillus sp. RAPc8/ amidase 
 
AAO23013 
Formamidase 31 
N-carbamoyl-D-
amino-acid hydrolase 
25 
9. 
Aeribacillus pallidus/amidase 
 
CAX63366 
Formamidase 31 
N-carbamoyl-D-
amino-acid hydrolase 
25 
10. Bacillus stearothermophilus 
BR388/amidase 
 
AAF14257 
Formamidase 33 
N-carbamoyl-D-
amino-acid hydrolase 
24 
11. Rhodococcus 
erythropolis/amidase 
AAK11724 Amidase (locus ID: 
WP_084254745) 
42 
 
 
 
 
https://blast.ncbi.nlm.nih.gov/Blast.cgi
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Table S2. Maximum biomass and specific growth rate profiling of C. oxalaticus 
ICTDB921 under different concentrations of acrylamidea 
 
Acrylamide 
concentration 
(mM) 
Maximum biomass 
(O.D600nm) 
Specific growth 
rate, µ (h-1) 
10 1.16 ± 0.045 0.2427 ± 0.0013 
20 1.62 ± 0.061 0.2651 ± 0.0019 
30 2.35 ± 0.054 0.2755 ± 0.0017 
40 3.18 ± 0.043 0.3065 ± 0.0014 
50 4.90 ± 0.035 0.3316 ± 0.0017 
60 6.58 ± 0.028 0.3906 ± 0.0021 
70 3.05 ± 0.041 0.1442 ± 0.0024 
80 1.95 ± 0.051 0.0976 ± 0.0014 
90 1.05 ± 0.042 0.0910 ± 0.0017 
100 0.71 ± 0.036 0.0442 ± 0.0012 
 a Values are mean ± SD of 3 replicate determinations