Purification and partial characterization of L-asparaginase enzyme produced by newly isolated Bacillus sp
A new bacterial producing L-asparaginase was successfully isolated from Sungai Klah Hot Spring, Perak, Malaysia and identified as Bacillus sp. It was the best L-asparaginase producer as compared to other isolates. Production of L-asparaginase from the microbial strain was carried out under liquid fe...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English English |
| Published: |
Kulliyah of Engineering, International Islamic University Malaysia (IIUM)
2017
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| Subjects: | |
| Online Access: | http://irep.iium.edu.my/59636/ http://irep.iium.edu.my/59636/ http://irep.iium.edu.my/59636/1/PURIFICATION%20AND%20PARTIAL%20CHARACTERIZATION%20OF%20L-ASPARAGINASE%20ENZYME%20PRODUCED%20BY%20NEWLY%20ISOLATED%20BACILLUS%20sp.pdf http://irep.iium.edu.my/59636/7/Purification%20and%20partial%20characterization%20of%20L-asparaginase%20enzyme%20produced%20by%20newly%20isolated%20Bacillus%20Sp..pdf |
| Summary: | A new bacterial producing L-asparaginase was successfully isolated from Sungai Klah Hot Spring, Perak, Malaysia and identified as Bacillus sp. It was the best L-asparaginase producer as compared to other isolates. Production of L-asparaginase from the microbial strain was carried out under liquid fermentation. The crude enzyme was then centrifuged and precipitated with ammonium sulfate before being further purified by chromatographic method. HiTrap DEAE-Sepharose Fast Flow ion exchange chromatography followed by separation on Superose 12 gel filtration were used to obtain pure enzyme. The purified enzyme showed 10.11 U/mg of specific activity, 50.07% yield with 2.21 fold purification. The purified enzyme was found to be dimer in form, with a molecular weight of 65 kDa as estimated by SDS-PAGE. The maximum activity of the purified L-asparaginase was observed at pH 9 and temperature of 60 oC.
Kajian penyelidikan ini telah berjaya menghasilkan enzim L-aspraginase
daripada bakteria-bakteria baru yang diambil dari Kolam Air Panas, Sungai Klah, Perak,
Malaysia dan ia dikenal pasti sebagai Bacillus sp. Teknik isolasi bakteria ini adalah teknik
terbaik dalam menghasilkan enzim L-asparaginase berbanding teknik-teknik lain.
Penghasilan enzim ini dibuat menerusi proses fermentasi. Kultur bakteria yang diperolehi
diempar dan diikuti presipitasi menggunakan ammonium sulfat sebelum proses penulenan
seterusnya dilakukan menggunakan kaedah kromatografi. Kolum penukaran ion jenis
HiTrap DEAE-Sepharose Fast Flow diikuti dengan pemisahan oleh gel Superose 12 telah
digunakan untuk mendapatkan enzim L-asparaginase yang tulen. Hasil kajian mendapati
enzim tulen yang diperolehi mempunyai aktiviti spesifik sebanyak 10.11 U/mg daripada
50.07% enzim yang dihasilkan dan berjaya mencapai 2.21 kali ganda tulen dari enzim
tanpa proses penulenan. Enzim tulen yang diperolehi didapati dalam bentuk “dimer“
dengan berat molekular sebanyak 65 kDa yang ditentukan menerusi SDS-PAGE. Enzim
ini juga menunjukkan aktiviti yang tinggi pada pH 9 dan suhu 60 oC. |
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