Optimization, purification, and characterization of L-asparaginase from Actinomycetales bacterium BkSoiiA
Optimization, purification, and characterization of L-asparaginase from Actinomycetales bacterium BkSoiiA
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DOI：
10.1080/10826068.2014.969437
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发表时间：
2016-01-02
影响因子：
2.9
通讯作者：
Maiti, Prasanta Kumar
中科院分区：
文献类型：
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作者：
Dash, Chitrangada;Mohapatra, Sukanti Bala;Maiti, Prasanta Kumar
放线菌是一种很有前途的重要酶的来源，其中一些是工业化生产的，另一些还有待利用。L-天门冬酰胺酶被用作抗肿瘤药物。研究了放线菌BkSoiA在M9液体发酵条件下生产L天冬酰胺酶的工艺条件及优化条件。优化后的M9发酵条件为：以酵母膏和果糖为碳源，以果糖为氮源，pH 8.0，30+/-2℃培养120小时。粗酶经透析、离子交换柱层析、凝胶过滤纯化至接近均一的水平。十二烷基硫酸钠(SDS)聚丙烯酰胺凝胶电泳法(PAGE)显示其表观分子量为57kD。该酶纯化倍数为95.06，最终比活力为204.37U/mg，得率为3.49%。纯化后的酶在pH为10.0时活性最高，在pH为7.0~9.0范围内稳定性较好。该酶被Mn2+激活，而被Ba2+强烈抑制。这些初步的表征表明，通过进一步的研究，L天冬酰胺酶有可能成为医药工业中有用的工具。
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 120hr at 30 +/- 2 degrees 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 204.37U/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 Mn2+ and strongly inhibited by Ba2+. All these preliminary characterization suggests that the L-asparaginase from the source may be a tool useful to pharmaceutical industries after further research.