重组L-天冬酰胺酶Ⅱ的中试工艺及其性质的研究

Studies on pilot-scale technology and properties of the recombinant L-asparaginase Ⅱ

目的 :通过重组L 天冬酰胺酶Ⅱ中试工艺及其性质的研究 ,旨在获得大量的高纯度产品。方法 :采用 5 0 0L发酵罐发酵 ,对所获得菌体进一步提取纯化 ,产品用还原SDS PAGE(十二烷基硫酸钠 聚丙酰胺凝胶电泳法 ) ,HPLC测定纯度 ,质谱和等电聚焦电泳分别测相对分子质量、等电点 ,并进行了紫外扫描。结果 :发酵液产酶活力达到 2 0 0U·mg-1,提取回收率为80 % ,纯化总回收率为 5 7% ,产品比活为 2 2 0U·ml-1,电泳 30 μg上样量显示一条带 ,HPLC测纯度大于 95 %。该酶Mr为13835 6 ,pI为 4.85 ,紫外最大吸收值在 2 78nm处。 结论 :本研究对实现重组L 天冬酰胺酶Ⅱ的工业化生产有重要意义。

OBJECTIVE:To obtain highly purified L asparaginase Ⅱ through the studies on pilot scale technology and product properties.METHODS:The engineering bacteria,which was cultivated in 500 L fermentor,were extracted and purified.The purity of the final products was detected by reductive SDS PAGE and HPLC.The molecular weight of enzyme was estimated by mass spectrometry.The pI was found by IEF and the maximum absorption of UV spectra was determined.RESULTS:The enzyme production in 500 L fermentor reached to 200 U·ml -1 .The recoveries of the extraction and purification were 80% and 57%,respectively.The specific activity of purified enzyme was 220 U·mg -1 .Only one band was found with SDS PAGE in reductive condition using 30 μg sample.Three kinds of HPLC analysis showed that the purity was greater than 95%.The Mr and pI of the enzyme were 138356 and 4.85,respectively.The maximum absorption was obtained at the wavelength of 278 nm.CONCLUSION:The industralized production of recombinant L asparaginase Ⅱ was promising.