生姜蛋白酶提取及反胶束纯化工艺初步研究

PRIMARY RESEARCH OF PREPARATION AND ANTI-MICELLE EXTRACTING ABOUT GINGER PROTEASE(GP-Ⅱ)

本文研究了生姜中生姜蛋白酶的分布及贮藏中的活力变化 ,研究了从新鲜生姜中提取生姜粗蛋白酶及用AOT 异辛烷和CTAB庚烷 /辛醇反胶束萃取该酶的工艺和方法。实验结果指出 :在贮藏茎中生姜蛋白酶的活力为 2 .7μg/mL·min-1,在膨大茎中该酶活力为 0 .6 8μg/mL·min-1,而在幼嫩茎中活力最低 ,仅为0 .4 8μg/mL·min-1。新鲜生姜在 0℃下贮藏 2 4h即完全丧失活力 ,在室温下贮藏 3d后其活力损失达38 2 5 %。用 10倍 0 .2mol/L、pH =6 .0的磷酸缓冲液 (4℃ )三次提取生姜蛋白酶 ,其提取率分别为 6 4 .75 %、14 .2 8%和 5 .2 %。用 6 5 %饱和度的 (NH4) 2 SO4沉淀提取液中的生姜蛋白酶 ,再以 pH 6 .2、0 .1mol/L的柠檬酸缓冲液溶解 ,其比活力达到 4 .2 1(μgPro/ μgPro·min-1)。生姜蛋白酶的 pI =5 .4～ 5 .5 ,在pH 5 .4以上 ,用AOT 异辛烷反胶束不能萃取出生姜蛋白酶 ,但却可以萃取出 71.86 %的杂蛋白。用CTAB庚烷 /辛醇反胶束二次萃取AOT 异辛烷萃余液 ,其蛋白质萃取率为 6 0 .2 5 % ,萃取液中生姜蛋白酶理论比活力达到 4 9.77(μgPro/ μgPro·min-1)。

It was researched that existing place of Ginger Protease(GP-Ⅱ)and the activity change during storage and primary process about extracting and anti-micelle extracting of the enzyme by AOT-isooctane or CTAB-heptylane-capryl alcohol. The experiment result was as follows:activity of GP-Ⅱis 2.7 μg/mL·min -1 at storage stem, 0.68 μg/mL·min -1 at inflate stem, only 0.48 μg/mL·min -1 at tender stem respectively. The activity is dropped down zero at 0 ℃ during 24 h, and the activity loses is arrived to 38.25% at room temperature during stored 3 d. Using 0.2 mol/L phosphate buffer solution which pH is 6.0,and temperature at 4 ℃, extraction rate of three times is espectively 64.75%, 14.28% and 5.2%. GP-Ⅱ in extraction liquid be salted out by (NH 4) 2SO 4 while the last concentration is arrived to 65%,and after the settlings can be dissolved with 0.1 mol/L citric acid that pH is 6.2 the specific activity is increased to 4.21 μg Pro/μgPro·min -1. Isoelectric point of Ginger Protease(pI) is between pH 5.4 to 5.5, and while the solution's pH is above 5.4 the enzyme can't be extracted by AOT-isooctane anti-micelle, but other proteins are decreased 71.86%, and after using AOT-isooctane the solution is second extracted with CTAB-heptylane-capryl alcohol, and the percentage extraction of protein is 60.25%, in which the enzymatic specific activity in theory is arrived to 49.77 μg Pro/μg Pro·min -1.