摘要
将已构建好的表达载体pPIC3.5K/PAE转化毕赤酵母KM71,经甲醇诱导表达弹性蛋白酶,通过对发酵条件的初步优化,确定毕赤酵母重组菌株表达的最佳条件为:甲醇浓度1%,最佳诱导时间4d。通过DEAE-SepharoseFF弱阴离子交换柱一步纯化,获得良好的纯化效果。该重组蛋白酶最适pH值范围6.0～8.5,最适温度范围为25～40℃,催化反应条件温和。Ca2+和Mg2+对重组弹性蛋白酶活性有不同程度的促进作用,Zn2+、Cd2+、Mn2+、Fe2+、Co2+、Cu2+和EDTA对该酶有不同程度的抑制作用。
Elastase was expressed in Pichiapastoris KM7 with the transformation of a recombinant plasmid pPIC3.5K/PAE encoding a full-length lasB gene of PAE （Pseudomonas aeruginosa elastase） under the induction of methanol. The optimal expression conditions were 1% methanol and 4 d induction. The best purification efficiency was achieved through DEAE- Sepharose FF anion exchange chromatography. The optimal pH and temperature range for catalytic reaction of this elastase were 6.0-8.5 and 25-40 ℃, respectively. Its activity was revealed an enhancement effect from Ca^2＋ and Mg^2＋, and an inhibition effect from Zn^2＋, Cd^2＋, Mn^2＋, Fe^2＋, Co^2＋, Cu^2＋ and EDTA.
出处
《食品科学》
EI
CAS
CSCD
北大核心
2009年第19期227-231,共5页
Food Science
基金
国家"863"计划项目(2006AA10Z440)
农业部"948"项目(2007-Z8)
