渗压震扰法释放Aeromonas sp.2016菌株外周间质中几丁质酶的研究

气单胞菌Aeromonassp．2016菌株能产生多种几丁质酶,其中的胞外酶C可能聚集于细胞外周胞质。为了避免破碎菌体而产生过多的杂蛋白,探索了用渗压震扰法(osmoticshock)来释放这部分酶。主要步骤是:先将菌体悬浮在20％蔗糖-0．03mol／LTris-HCI(pH8．0)高渗透压的溶液中,再快速转移到纯水低渗透压溶液中,产生瞬间渗压震荡,释放细胞外周胞质中的酶。结果表明,通过渗压震扰法释放出的酶纯度最高,比活力达到142．79U／g,比培养液上清液的54．46U／g和菌体破碎样品的14．66U／g分别高1．6倍和8．7倍,可用于纯化目的蛋白。

The Release of Chitinase from Periplasm of Aeromonas sp. 2016 by Osmotic Shock

Aeromonas sp. strain 2016 produces several kinds of chitinolytic proteins, among which Enzyme C locates in periplasm. The traditional method for purifying enzymes not secreted into medium is to break the cell. But all of the proteins in the cytoplasm will also come out concomitantly, which makes it difficult to purify the targeting protein. To release Enzyme C, not breaking the cells, osmotic shock was tried. Cells were firstly suspended in high pressure solution, 20% sugar-0.03mol/L Tris-HCl (pH8.0) buffer, and then were transferred into water. Enzymes in periplasm were released by instant osmotic shock. The result shows that the specific activity of enzyme C released by osmotic shock is 142.79U/g, which is higher than that of supernatant by 1.6 folds, and than that of cell extraction by 8.7 folds. So it is reasonable to release enzyme C by osmotic shock.