双水相萃取法从风干香肠中分离提取蛋白酶  

Purification of Protease from Dry Fermented Sausages by Aqueous Two-phase System

作　　者：张兰威[1] 陈一[2] 韩雪[1] 杜明[1] 易华西[1] 

机构地区：[1]哈尔滨工业大学食品科学与工程学院,哈尔滨150090 [2]东北农业大学食品学院,哈尔滨150030

出　　处：《分析化学》2008年第7期900-904,共5页Chinese Journal of Analytical Chemistry

基　　金：黑龙江省教育厅科技攻关项目(No10511Z003);黑龙江省十五科技攻关项目(NoGB02B4031-1)资助

摘　　要：双水相萃取(ATPS)是近年来发展起来的蛋白纯化方法。为了扩展该方法适应领域,同时为风干香肠形成过程酶系的研究提供具体方法。本实验研究了运用双水相技术分离提取风干香肠中蛋白酶,对构成双水相体系中的PEG分子量、浓度和类型以及盐浓度的影响进行了分析。确定了双水相组成体系为20%PEG1000(m/m)和25%MgSO4(m/m),在此体系中风干香肠的蛋白酶主要分布在上相,最高酶活12.37U/μg;纯化倍数为4.61;回收率为85%。通过分子筛层析对比,表明风干香肠经过双水相分离提取杂蛋白峰被除去,而蛋白酶峰几乎未受到影响,说明该双水相体系萃取香肠中蛋白酶具有良好的专一性。调解双水相pH值对蛋白酶的萃取没有影响,而添加电解质NaCl反而产生不利影响。Aqueous two-phase system（ATPS） was used in the purification of proteins in recent years.It was widely used for extracting the bioactive substances.In order to extend the application for analyzing the forming of enzymes in the dry fermented sausages,the protease was extracted from dry fermented sausages by an ATPS.The phase compositions including polyethylene glyool（PEG） molecular mass,concentration as well as types and concentration of salts affected protein partitioning were studied.ATPS comprising PEG1000（20%,m/m） and magnesium sulfate（25%,m/m） provided the best condition for the maximum partitioning of the protease into the top phase and gave a highest specific activity（12.37 U/μg protein） and purification fold（4.61）.The yield of 85% was obtained.When Sephadex G-75 was used,it revealed that the band intensity of contaminating proteins in ATPS fraction almost disappeared and the band intensity of major protease slightly decreased.Therefore,ATPS was an effective method for partitioning and recovery of proteases from dry fermented sausages.Adjusted the pH in ATPS,there was no effect on protease extraction,while adding electrolyte has disadvantage effect.

关 键 词：双水相 蛋白酶 风干香肠 分离提取 

分 类 号：TS251.1[轻工技术与工程—农产品加工及贮藏工程]