Zhu Jian-Hang, Yan Xi-Luan, Chen Hong-Jun, Wang Zhi-Hui
Key Laboratory of Poyang Lake Ecology and Bio-Resource Utilization of Ministry of Education, School of Environmental Science and Engineering, Nanchang University, Nanchang, Jiangxi 330031, China.
J Chromatogr A. 2007 Apr 13;1147(1):127-34. doi: 10.1016/j.chroma.2007.02.035. Epub 2007 Feb 16.
The feasibility and generic applicability of directly integrating conventional discrete operations of cell disruption by high pressure homogenizer and the product capture by aqueous two-phase extraction (ATPE) system have been demonstrated for the extraction of intracellular L-asparaginase from E. coli. In a side-by-side comparison with the conventional ATPE process, including cell disruption, centrifugal clarification and following ATPE, purification of L-asparaginase via this novel in situ ATPE process yielded a product of L-asparaginase with a higher specific activity of 94.8 U/(mg protein) and a higher yield of 73.3%, both of which in the conventional ATPE process were 78.6 U/(mg protein) and 52.1%, respectively. In the purification of L-asparaginase (pI=4.9), product-debris interactions commonly diminish its recovery. It was demonstrated that immediate extraction of L-asparaginase in ATPE systems when it is released at pH 5.0 during cell disruption effectively increased its recovery in the top phase due to the reduced interaction between L-asparaginase and cell debris and the reduced degradation by contaminated protease. In addition, no clarification step and/or disruptate storage are required in this in situ ATPE, which reduced the number of unit operations and thus shortened the overall process time. This novel process has a good potential for the separation of other intracellular biological products.
通过高压匀浆器进行细胞破碎和水相两相萃取（ATPE）系统进行产物捕获的常规离散操作直接整合，已被证明可用于从大肠杆菌中提取细胞内L-天冬酰胺酶，具有可行性和普遍适用性。与包括细胞破碎、离心澄清和后续ATPE的传统ATPE工艺进行并行比较，通过这种新型原位ATPE工艺纯化L-天冬酰胺酶得到的L-天冬酰胺酶产物，比活性更高，为94.8 U/（mg蛋白），产率更高，为73.3%，而在传统ATPE工艺中，这两个指标分别为78.6 U/（mg蛋白）和52.1%。在纯化L-天冬酰胺酶（pI = 4.9）时，产物与碎片的相互作用通常会降低其回收率。结果表明，在细胞破碎过程中L-天冬酰胺酶在pH 5.0时释放后立即在ATPE系统中进行萃取，由于L-天冬酰胺酶与细胞碎片之间的相互作用减少以及受污染蛋白酶的降解减少，有效地提高了其在上相中回收率。此外，这种原位ATPE不需要澄清步骤和/或破碎物储存，减少了单元操作数量，从而缩短了整个工艺时间。这种新型工艺在分离其他细胞内生物产物方面具有良好的潜力。
