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 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

Statistical analysis of growth conditions of newly isolate bacillus sp producing l-asparaginase

The concentrations of nutrient elements together with several physical parameters were screened to find out the significant factors for the production of Lasparaginase from newly isolated strain, Bacillus sp. from Sg Klah, Hot Spring, Perak. Then, the significant factors were optimized for enhancing L-asparaginase production from the bacterium strain. Two statistical designs, Two Level Factorial Design and Face Centered Composite Design (FCCD), Design expert @version 8.0 were employed in screening and optimization of the process variables, respectively. The results for all experiment runs were analyzed by analysis of variance (ANOVA). Peptone (nitrogen source) concentration and temperature were found as significant factors, positively influenced the production of Lasparaginase. The two factors were then optimized to increase the desired enzyme production. The optimum peptone concentration and the temperature were found at 1.4 g/L and 30ºC, respectively. The L-asparaginase production under optimized conditions increased from 0.15±0.023 U/mL to 0.19 ± 0.03 U/mL. The kinetic studies showed that the biomass production dropped after 24 hours while L-asparaginase activity is active and positively increased until the fermentation period ended

PURIFICATION AND PARTIAL CHARACTERIZATION OF L-ASPARAGINASE ENZYME PRODUCED BY NEWLY ISOLATE BACILLUS SP.

A newly bacterial producing L-asparaginase was successful 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 further purified with chromatographic method. The ion exchange chromatography HiTrap DEAE-Sepharose Fast Flow column 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°C

Isolation and characterisation of thermophilic bacterial isolates producing L-asparaginase

This procedure is following Gulati, Saxena, and Gupta (1997) protocol which is known as semi-quantitative plate assay for screening L-asparaginase- producing bacterial from local hot spring. The plate agar medium containing asparagine (as sole nitrogen sourse) is prepared with addition of phenol red which later shows an indication of L-asparaginase based on changes of colour. Phenol red at acidic is yellow which might change colour to pink when it is at alkaline. Thus, it indicates the formation of pink zone around microbial colonies producing L-asparaginase. The inoculated agar plates are incubated at 370C in an incubator for 24 hours. Thus, the isolation and screening of bacterial producing L-asparaginase are based on phenol-red zone. After purification, the isolates are characterised using several biochemical tests