摘要
研究微生物转谷氨酰胺酶(MTGase)反胶束纯化的工艺和条件,调节MTGase离心上清液等电点,除去部分杂蛋白,MTGase活力升高7.5倍;用截留分子量为10000的超滤膜除去小分子杂蛋白,MTGase活力升高1.33倍;用0.05mol/L的AOT/异辛烷反胶束进一步纯化MTGase,其最适萃取条件是粗MTGase蛋白质浓度20mg/mL,[Na+]0.12mol/L,水相pH4.80～5.20,相比1:1(v/v);荷载MTGase的AOT反胶束用2.0mol/LKCl进行反萃取,MTGase活力为14.2U/g,纯化8.875倍;冷冻干燥脱盐反萃取液,获得MTGase冻干粉,其活力为110.3U/g,与粗酶液相比较,纯化689.4倍。经过AOT/异辛烷反胶束萃取纯化的MTGase,其SDS-聚丙烯酰胺凝胶电泳为一条带。Ca2+与表面活性剂非极性尾上丁二酰羰基氧、极性头磺酸基硫氧基氧及MTGase分子表面具有孤电子对的基团的配位结合放大了AOT反胶束的另一种萃取作用——配位萃取,致使其对MTGase的萃取率高于K+而接近Na+。
Technics and conditions of purifying MTGase by AOT reversed micelles have been carried out. After adjusting pI of the raw protein solution and removing some impure proteins, activity of MTGase increases by 7.5 times. Some small molecule substances are removed by ultrafihration membrane which cuts off the substances of the molecular weight more than 10000, in this way, activity of MTGase increases by 1.33 times. Then , MTGase was extracted by 0.05 mol/L AOT/ isooctane reversed micelles,the optimum conditions are that the raw protein concentration is 20 mg/mL, [ Na^＋ ] 0. 12 mol/L ,aqueous phase pH 4.80-5.20, and the ratio of reversed micelles to extracted solution is 1：1 （v/v）. Reversed micelles dissolved MTGase is re-extracted by 2.0 mol/L KC1. Activity of MTGase is 14. 2 U/g, increasing by 8. 875 times, compared with the raw protein solution. The freeze drying powder of MTGase is attained by freezing and desalted, and its activity is 110.3 U/g, increasing by 689.4 times. Only one trace is shown in SDS-PAGE gel electrophoresis test. Coordination extraction of AOT reversed micelles is magnified by combining Ca^2＋ with succinyl carbonyl oxygen in the nonpolar end group of surfactant ,sulfonic and sulfoxide oxygen in the polar head group and the group with lone electron pair on the surface of MTGase molecule. Therefore, its extraction efficiency is higher than K^＋ and is close to Na^＋.
出处
《天然产物研究与开发》
CAS
CSCD
2007年第3期396-400,共5页
Natural Product Research and Development
作者简介
通讯作者 Tel：86-23-65111179；E-mail：wanglp118@yahoo.com.cn.
