L-asparaginase release from Escherichia coli cells with K2HPO4 and Triton X100.
A method to release L-asparaginase (EC 3.5.1.1) from ATCC Escherichia coli 11303 cells by chemical permeabilization was studied. It was found that a combination of K2HPO4 and Triton X100 was effective. The influences of K2HPO4 concentration, Triton concentration, E. coli cell concentration and pH on the release of enzyme and proteins were investigated in detail. Experimental results showed that 12.5% (w/v) K2HPO4, 2% (w/v) Triton X100 and 3 x 10(8) cells/mL made the amount of enzyme released over 70%. L-Asparaginase in K2HPO4 and Triton solution could remain stable at least for 24 h. The release effect of K2HPO4 and Triton X100 used simultaneously was better than that of K2HPO4 and Triton X100 used separately in succession. Electron microscopy indicated that the chemical treatment altered the surface structure of E. coli cells but did not break them. As the method does not produce a large amount of cell fragments and the amount of enzyme released is relatively high, it can be thought to be an valuable and economic method to release intracellular enzyme.
Zhao
F
F
Department of Biological Science and Technology, Shanghai Jiao Tong University, Shanghai 200030, P.R. China. fszhao@mail.sjtu.edu.cn
Yu
J
J
D001215:Asparaginase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D001671:Biochemistry / Q000379:methods*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D008854:Microscopy, Electron; D017830:Octoxynol / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry*; D017680:Potassium Compounds / Q000737:chemistry*; D011489:Protein Denaturation; D012492:Salts / Q000737:chemistry; D013501:Surface-Active Agents / Q000737:chemistry
D010710:Phosphates; D017680:Potassium Compounds; D012492:Salts; D013501:Surface-Active Agents; D017830:Octoxynol; C013216:potassium phosphate; D001215:Asparaginase