Characterization and applications of glutaminase free L-asparaginase from indigenous Bacillus halotolerans ASN9.
L-asparaginase (L-ASNase) is a versatile anticancer and acrylamide reduction enzyme predominantly used in medical and food industries. However, the high specificity of L-asparaginase formulations for glutamine, low thermostability, and blood clearance are the major disadvantages. Present study descr...
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2023-01-01
|
| Series: | PLoS ONE |
| Online Access: | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0288620&type=printable |
| _version_ | 1797394621750312960 |
|---|---|
| author | Ifrah Shafqat Shaheen Shahzad Azra Yasmin Muhammad Tausif Chaudhry Safia Ahmed Aneela Javed Imran Afzal Monaza Bibi |
| author_facet | Ifrah Shafqat Shaheen Shahzad Azra Yasmin Muhammad Tausif Chaudhry Safia Ahmed Aneela Javed Imran Afzal Monaza Bibi |
| author_sort | Ifrah Shafqat |
| collection | DOAJ |
| description | L-asparaginase (L-ASNase) is a versatile anticancer and acrylamide reduction enzyme predominantly used in medical and food industries. However, the high specificity of L-asparaginase formulations for glutamine, low thermostability, and blood clearance are the major disadvantages. Present study describes production, characterization, and applications of glutaminase free extracellular L-asparaginase from indigenous Bacillus halotolerans ASN9 isolated from soil sample. L-asparaginase production was optimized in M9 medium (containing 0.2% sucrose and 1% L-asparagine) that yielded maximum L-ASNase with a specific activity of 256 U mg-1 at pH 6 and 37°C. L-asparaginase was purified through acetone precipitation and Sephadex G-100 column, yielding 48.9 and 24% recovery, respectively. Enzyme kinetics revealed a Vmax of 466 mM min-1 and Km of 0.097 mM. Purified L-ASNase showed no activity against glutamine. The purified glutaminase free L-ASNase has a molecular mass of 60 kDa and an optimum specific activity of 3083 U mg-1 at pH 7 and 37°C. The enzyme retains its activity and stability over a wide range of pH and temperature, in the presence of selected protein inhibitors (SDS, β-mercaptoethanol), CoCl2, KCl, and NaCl. The enzyme also exhibited antioxidant activity against DPPH radical (IC50 value 70.7 μg mL-1) and anticancer activity against U87 human malignant glioma (IC50 55 μg mL-1) and Huh7 human hepatocellular carcinoma (IC50 37 μg mL-1) cell lines. Normal human embryonic kidney cells (HEK293) had greater than 80% cell viability with purified L-ASNase indicating its least cytotoxicity against normal cells. The present work identified potent glutaminase free L-ASNase from B. halotolerans ASN9 that performs well in a wide range of environmental conditions indicating its suitability for various commercial applications. |
| first_indexed | 2024-03-09T00:22:25Z |
| format | Article |
| id | doaj.art-7c64e7d368c74a4ba06a5fdf42884d9a |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-03-09T00:22:25Z |
| publishDate | 2023-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-7c64e7d368c74a4ba06a5fdf42884d9a2023-12-12T05:33:23ZengPublic Library of Science (PLoS)PLoS ONE1932-62032023-01-011811e028862010.1371/journal.pone.0288620Characterization and applications of glutaminase free L-asparaginase from indigenous Bacillus halotolerans ASN9.Ifrah ShafqatShaheen ShahzadAzra YasminMuhammad Tausif ChaudhrySafia AhmedAneela JavedImran AfzalMonaza BibiL-asparaginase (L-ASNase) is a versatile anticancer and acrylamide reduction enzyme predominantly used in medical and food industries. However, the high specificity of L-asparaginase formulations for glutamine, low thermostability, and blood clearance are the major disadvantages. Present study describes production, characterization, and applications of glutaminase free extracellular L-asparaginase from indigenous Bacillus halotolerans ASN9 isolated from soil sample. L-asparaginase production was optimized in M9 medium (containing 0.2% sucrose and 1% L-asparagine) that yielded maximum L-ASNase with a specific activity of 256 U mg-1 at pH 6 and 37°C. L-asparaginase was purified through acetone precipitation and Sephadex G-100 column, yielding 48.9 and 24% recovery, respectively. Enzyme kinetics revealed a Vmax of 466 mM min-1 and Km of 0.097 mM. Purified L-ASNase showed no activity against glutamine. The purified glutaminase free L-ASNase has a molecular mass of 60 kDa and an optimum specific activity of 3083 U mg-1 at pH 7 and 37°C. The enzyme retains its activity and stability over a wide range of pH and temperature, in the presence of selected protein inhibitors (SDS, β-mercaptoethanol), CoCl2, KCl, and NaCl. The enzyme also exhibited antioxidant activity against DPPH radical (IC50 value 70.7 μg mL-1) and anticancer activity against U87 human malignant glioma (IC50 55 μg mL-1) and Huh7 human hepatocellular carcinoma (IC50 37 μg mL-1) cell lines. Normal human embryonic kidney cells (HEK293) had greater than 80% cell viability with purified L-ASNase indicating its least cytotoxicity against normal cells. The present work identified potent glutaminase free L-ASNase from B. halotolerans ASN9 that performs well in a wide range of environmental conditions indicating its suitability for various commercial applications.https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0288620&type=printable |
| spellingShingle | Ifrah Shafqat Shaheen Shahzad Azra Yasmin Muhammad Tausif Chaudhry Safia Ahmed Aneela Javed Imran Afzal Monaza Bibi Characterization and applications of glutaminase free L-asparaginase from indigenous Bacillus halotolerans ASN9. PLoS ONE |
| title | Characterization and applications of glutaminase free L-asparaginase from indigenous Bacillus halotolerans ASN9. |
| title_full | Characterization and applications of glutaminase free L-asparaginase from indigenous Bacillus halotolerans ASN9. |
| title_fullStr | Characterization and applications of glutaminase free L-asparaginase from indigenous Bacillus halotolerans ASN9. |
| title_full_unstemmed | Characterization and applications of glutaminase free L-asparaginase from indigenous Bacillus halotolerans ASN9. |
| title_short | Characterization and applications of glutaminase free L-asparaginase from indigenous Bacillus halotolerans ASN9. |
| title_sort | characterization and applications of glutaminase free l asparaginase from indigenous bacillus halotolerans asn9 |
| url | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0288620&type=printable |
| work_keys_str_mv | AT ifrahshafqat characterizationandapplicationsofglutaminasefreelasparaginasefromindigenousbacillushalotoleransasn9 AT shaheenshahzad characterizationandapplicationsofglutaminasefreelasparaginasefromindigenousbacillushalotoleransasn9 AT azrayasmin characterizationandapplicationsofglutaminasefreelasparaginasefromindigenousbacillushalotoleransasn9 AT muhammadtausifchaudhry characterizationandapplicationsofglutaminasefreelasparaginasefromindigenousbacillushalotoleransasn9 AT safiaahmed characterizationandapplicationsofglutaminasefreelasparaginasefromindigenousbacillushalotoleransasn9 AT aneelajaved characterizationandapplicationsofglutaminasefreelasparaginasefromindigenousbacillushalotoleransasn9 AT imranafzal characterizationandapplicationsofglutaminasefreelasparaginasefromindigenousbacillushalotoleransasn9 AT monazabibi characterizationandapplicationsofglutaminasefreelasparaginasefromindigenousbacillushalotoleransasn9 |