Optimization, purification, and characterization of L-asparaginase fromActinomycetales bacteriumBkSoiiA

Abstract

Actinobacteria are promising source of a wide range of important enzymes, some of which are produced in industrial scale, with others yet to be harnessed. L-Asparaginase is used as an antineoplastic agent. The present work deals with the production and optimization of L-asparaginase from Actinomycetales bacterium BkSoiiA using submerged fermentation in M9 medium. Production optimization resulted in a modified M9 medium with yeast extract and fructose as carbon and nitrogen sources, respectively, at pH 8.0, incubated for 120 hr at 30 ± 2 °C. The crude enzyme was purified to near homogeneity by ammonium sulfate precipitation following dialysis, ion-exchange column chromatography, and finally gel filtration. The sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) revealed an apparent molecular weight of 57 kD. The enzyme was purified 95.06-fold and showed a final specific activity of 204.37 U/mg with 3.49% yield. The purified enzyme showed maximum activity at a pH 10.0 and was stable at pH 7.0 to 9.0. The enzyme was activated by Mn(2+) and strongly inhibited by Ba(2+). All these preliminary characterization suggests that the L-asparaginase from the source may be a tool useful to pharmaceutical industries after further research.

Experimental and bioinformatics study for production of l-asparaginase from Bacillus licheniformis: a promising enzyme for medical application

“…Halophilic enzymes have high negative charges so can be easily dissociated and become more flexible in the presence of sodium chloride (Han et al 2014). The current results agree with that of some researches (Elshafei et al 2012; Dash et al 2016; Shechtman 2013; Han et al 2014). A gradual increase in the enzyme activity was reported by increasing asparagine concentration followed by slight decrease in the activity at higher concentration.…”

Production, characterization and bioinformatics analysis of l-asparaginase from a new Stenotrophomonas maltophilia EMCC2297 soil isolate

“…The same authors reported that negative charges are associated with halophilic enzymes and the presence of sodium chloride renders these enzymes more flexible with an increase in enzyme activity. The results reported in this study agreed those obtained by many researchers (Dash et al 2016;Elshafei et al 2012;Han et al 2014;Shechtman 2013) who reported that sodium chloride increases enzyme activity. Regarding l-asparaginase activity produced by Stenotrophomonas maltophilia test isolate in relation to asparagine concentration, it was found that this relationship obeys the commonly exhibited by various enzymes.…”

Characterization of a Novel L-Asparaginase from Mycobacterium gordonae with Acrylamide Mitigation Potential

“…The Mg 2+ co-factors can activate L-asparaginases by first activating the substrate and then combining it with the complex enzyme substrate to complete the enzyme-catalyzed hydrolysis reaction [21]. In our study, Mg 2+ , Mn 2+ , and Ni 2+ played active roles in activating GmASNase, which were identical to those of the L-asparaginases from A. bacterium [49], B. amyloliquefaciens [31], T. zilligii [12], Cobetia amphilecti [51], and P. fluorescens [40]. However, divalent metals do not affect the enzymatic activity of L-asparaginase from T. kodakarensis [47].…”

“…GmASNase was quite stable at 4 to 35 • C, approximately 50% of its residual enzyme activity could be retained after 30 min at 40 • C, and a significant loss of stability was recorded with the increasing of the temperature. However, L-asparaginase from A. bacterium [49] is only stable from 4 to 10 • C. In addition, L-asparaginase from Acinetobacter soli shows poor thermostability with a half-life at 40 • C of 9.63 min [10]. These results suggest that GmASNase is thermally stable.…”

“…Surprisingly, GmASNase showed an outstanding stability over a wide pH range of 5.0-11.0 and even showed more than 80% residual activity at pH 6.0-11.0 after 24 h of incubation. Stud-ies show that L-asparaginases from actinomycete A. bacterium [49], S. gulbargensis [26], and S. spPKD2 [48] are only stable under alkaline conditions, and L-asparaginases from A. oryzae and A. niger L-asparaginases are only stable in the pH range of pH 6.0-9.0 and pH 5.0-9.0. In our study, GmASNase was stable in both acidic and alkaline conditions, which suggests that it would be suitable for high-pH baked foodstuffs such as corn flakes, crackers, and soda crackers.…”