Production and Characterization of Novel Glutaminase Free Recombinant L-asparaginase II of Erwinia carotovora subsp. atroseptica SCRI 1043 in E. coli BL21 (DE3)
Journal Title: Microbiology Research Journal International - Year 2015, Vol 6, Issue 2
Abstract
Aims: To clone and express, the gene encoding L-asparaginase II (ansB2) from Erwinia carotovora subsp. atroseptica SCRI 1043 in E. coli BL21 (DE3). Further, the work is also comprised of purification and detailed biochemical characterization of L-asparaginase II. Place and Duration of Study: Biochemical Engineering laboratory, Department of Biotechnology, Indian Institute of Technology Guwahati, Assam India. Experiments conducted as a part of project and a PhD thesis from December 2010 to January 2014. Methodology: The gene encoding L-asparaginase II (ansB2) from Erwinia carotovora subsp. atroseptica SCRI 1043 was cloned and expressed in E. coli BL21 (DE3). Affinity chromatography was employed to purify the enzyme to homogeneity. Detailed biochemical characterization, such as substrate specificity, operational stability in various effect or molecules, effect of pH and temperature, kinetic parameters were investigated. Results: The enzyme was found to be highly specific towards its natural substrate, L-asparagine. The activity of recombinant L-asparaginase II was activated by various effect or compounds, such as mono cations, L-cystine, L-histidine, 2-mercaptoethanol and glutathione. However, considerable inhibitory effect was observed with divalent cations and iodoactamide. Kinetic parameters (Vmax, Km, kcat and Kcat/Km) of purified recombinant L-asparaginase II were found to be 0.656 mM, 312.50 IU mg-1, 1.38×102 s-1 and 2.11×105 M-1s-1, respectively. Optimum range of pH and temperature for the hydrolysis of L-asparagine by purified recombinant L-asparaginase II were found to be 6.5-9.5 and 47-52ºC, respectively. Under optimal levels of medium components and physical process parameters (pH, inoculum size, agitation), the production of recombinant L-asparaginase II was increased by 1.95 fold. The purified recombinant L-asparaginase II has shown no glutaminase activity. Conclusion: The present characterization experiments of the L-asparaginase II from Erwinia carotovora subsp. atroseptica SCRI 1043 showed very high specificity for its natural substrate, L-asparagine and shown no glutaminase activity which makes it a better alternative in therapeutic applications like an anticancer drug.
Authors and Affiliations
Rachna Goswami, Krishnamoorthy Hegde, Venkata Dasu Veeranki
Keywords
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- EP ID EP354891
- DOI 10.9734/BMRJ/2015/13867
- Views 81
- Downloads 0
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