Anti-leukemic enzyme L-asparaginase despite having significant applicability in medicine, holds side effects attributed to glutaminase activity and endotoxin content. Glutaminase activity proves to be toxic to non-tumor cells as glutamine is an essential amino acid. Endotoxin illicit the production of vasoactive amines and induce septic shock. Hence there is a need for glutaminase free L-asparaginase with minimum endotoxin level. The report aims at the development of a downstream process for purification of glutaminase free L-asparaginase and subsequent endotoxin removal. Producing bacteria were isolated from various soil samples and screened initially for asparaginase and glutaminase activity. The glutaminase free L-asparaginase producing bacteria were identified as Bacillus altitudinis. Production of L-asparaginase was optimized. The optimum medium comprised of comprising Lactose (1.5 g/L), NaCl (1.2 g/L), Yeast extract (5 g/L), L-asparagine (20 g/L) with pH 7.0 and incubation time of 18 h. Kinetic parameters Km and Vmax were computed to be 9.09x10-2M and 0.09 M/S. L-asparaginase Purification was achieved with a specific activity of 800 U/mg of enzyme. Molecular weight of the purified L-asparaginase was determined to be around 35 KDa using SDS-PAGE. The developed process also brought down the endotoxin content below the FDA recommended level. The endotoxin content of the purified enzyme was determined to be 0.015EU/mL.

中文翻译：

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新型土壤分离物中低内毒素水平的无谷氨酰胺酶抗白血病L-天冬酰胺酶的生产，纯化和动力学表征。

抗白血病酶L-天冬酰胺酶尽管在医学上具有显着的适用性，但仍具有归因于谷氨酰胺酶活性和内毒素含量的副作用。谷氨酰胺酶活性被证明对非肿瘤细胞有毒，因为谷氨酰胺是必需氨基酸。内毒素非法产生血管活性胺并引起败血性休克。因此，需要具有最小内毒素水平的无谷氨酰胺酶的L-天冬酰胺酶。该报告的目的是开发无谷氨酰胺酶的L-天冬酰胺酶的下游工艺，并随后去除内毒素。从各种土壤样品中分离出生产细菌，并初步筛选天冬酰胺酶和谷氨酰胺酶活性。不含谷氨酰胺酶的产生L-天冬酰胺酶的细菌被鉴定为枯草芽孢杆菌。优化了L-天冬酰胺酶的生产。最佳培养基包括乳糖（1.5 g / L），氯化钠（1.2 g / L），酵母提取物（5 g / L），pH 7.0的L-天冬酰胺（20 g / L），孵育时间为18 h。动力学参数Km和Vmax经计算为9.09×10-2M和0.09M / S。L-天冬酰胺酶的纯化具有800 U / mg酶的比活性。使用SDS-PAGE确定纯化的L-天冬酰胺酶的分子量为约35KDa。开发的工艺还使内毒素含量降低到了FDA推荐的水平以下。纯化的酶的内毒素含量测定为0.015EU / mL。L-天冬酰胺酶的纯化具有800 U / mg酶的比活性。使用SDS-PAGE测定纯化的L-天冬酰胺酶的分子量为约35KDa。开发的工艺还使内毒素含量降低到了FDA推荐的水平以下。纯化的酶的内毒素含量测定为0.015EU / mL。L-天冬酰胺酶的纯化具有800 U / mg酶的比活性。使用SDS-PAGE确定纯化的L-天冬酰胺酶的分子量为约35KDa。开发的工艺还使内毒素含量降低到了FDA推荐的水平以下。纯化的酶的内毒素含量测定为0.015EU / mL。