AOT反胶束萃取胰蛋白酶的STM及主要影响因素

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AOT反胶束萃取胰蛋白酶的STM及主要影响因素

周小华; 翁亚军

College of Chemical Engineering, Chongqing University, Chongqing 400044, China

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2006-06-28 发布日期:2006-06-28
通讯作者: 周小华

Factors Affecting Trypsin Extraction by AOT Reversed Micelles and Observation by STM

ZHOUXiaohua; WENGYajun

College of Chemical Engineering, Chongqing University, Chongqing 400044, China

Received:1900-01-01 Revised:1900-01-01 Online:2006-06-28 Published:2006-06-28
Contact: ZHOU Xiaohua

摘要/Abstract

摘要： In this article, the influence factors of trypsin extracted from crude pancreatin was investigated, and scanning tunneling microscope(STM) was used to observe the image of trypsin in butane-diacid-2-ethyl-hexyl-ester-sulfonic sodium (AOT)/ iso-octane reversed micelles. The STM image showed that trypsins bounded in reversed micelles was rigid, which weakened its conjugative effect and caused maximum ultraviolet absorption and fluorescence emissive absorption moving toward blue waves. AOT concentration, pH and cations were the main influence factors of ex-traction. Specifically, extraction percentage of trypsin decreased with the increase of AOT concentration from 0.01 to 0.1mol•L－1. When pH value is from 5.30 to 10.0, i.e. less than pI of trypsin, the extraction percentage is raised with the different increase of pI-pH, but when the pH value is less than 5.20, the extraction percentage is decreased with the acidity added. Besides, the extraction efficiency is negative, related with the concentrations of Ca2+, Na+, K+ which were in the range of 0.2—1.0mol•L－1, and influence of concentration of Ca2+ is greater than that of Na+, and K+ which has the minimum impact with the same concentration. Finally, optimum conditions to extract trypsin were: AOT reversed micelles 0.05mol•L－1, trypsin concentration in crude pancreatin solution 3mg•ml－1, pH 5.2—5.3, ratio (by volume）of extraction phase to strip-extraction phase 1︰1, and time of 5min. The corresponding per-centage of extraction was 22.7% and specific activity was 78.9 N-benzoyl-L-arginine ethyl ester (BAEE) U•mg－1 protein, three times than that in crude pancreatin. There was no lipase and amylopsin activity was decreased to 1/5 of crude pancreatin. Partly purifying solution was treated by condition mentioned above with 0.05mol•L－1 ceryl-trimethyl-ammonium bromide (CTAB), total extraction percentage of trypsin was 74.18% and specific activ-ity was 3148.3 BAEE U•mg－1, i.e. 48.16 times purer than that in crude pancreatin. Through sodium dodecyl sul-fate-polyacryl amide gel electrophoresis (SDS-PAGE) and image analysis of extracted product, there were only three bands in the trypsin, while seven in crude pancreatin, and electrophoresis location of main bend was almost identical with the standard enzyme.

关键词: AOT reversed micelles;extraction;trypsin;scanning;tunneling;microscope(STM)

Abstract: In this article, the influence factors of trypsin extracted from crude pancreatin was investigated, and scanning tunneling microscope(STM) was used to observe the image of trypsin in butane-diacid-2-ethyl-hexyl-ester-sulfonic sodium (AOT)/ iso-octane reversed micelles. The STM image showed that trypsins bounded in reversed micelles was rigid, which weakened its conjugative effect and caused maximum ultraviolet absorption and fluorescence emissive absorption moving toward blue waves. AOT concentration, pH and cations were the main influence factors of ex-traction. Specifically, extraction percentage of trypsin decreased with the increase of AOT concentration from 0.01 to 0.1mol•L－1. When pH value is from 5.30 to 10.0, i.e. less than pI of trypsin, the extraction percentage is raised with the different increase of pI-pH, but when the pH value is less than 5.20, the extraction percentage is decreased with the acidity added. Besides, the extraction efficiency is negative, related with the concentrations of Ca2+, Na+, K+ which were in the range of 0.2—1.0mol•L－1, and influence of concentration of Ca2+ is greater than that of Na+, and K+ which has the minimum impact with the same concentration. Finally, optimum conditions to extract trypsin were: AOT reversed micelles 0.05mol•L－1, trypsin concentration in crude pancreatin solution 3mg•ml－1, pH 5.2—5.3, ratio (by volume）of extraction phase to strip-extraction phase 1︰1, and time of 5min. The corresponding per-centage of extraction was 22.7% and specific activity was 78.9 N-benzoyl-L-arginine ethyl ester (BAEE) U•mg－1 protein, three times than that in crude pancreatin. There was no lipase and amylopsin activity was decreased to 1/5 of crude pancreatin. Partly purifying solution was treated by condition mentioned above with 0.05mol•L－1 ceryl-trimethyl-ammonium bromide (CTAB), total extraction percentage of trypsin was 74.18% and specific activ-ity was 3148.3 BAEE U•mg－1, i.e. 48.16 times purer than that in crude pancreatin. Through sodium dodecyl sul-fate-polyacryl amide gel electrophoresis (SDS-PAGE) and image analysis of extracted product, there were only three bands in the trypsin, while seven in crude pancreatin, and electrophoresis location of main bend was almost identical with the standard enzyme.

Key words: AOT reversed micelles, extraction, trypsin, scanning, tunneling, microscope(STM)

周小华, 翁亚军. AOT反胶束萃取胰蛋白酶的STM及主要影响因素[J]. CIESC Journal.

ZHOUXiaohua, WENGYajun. Factors Affecting Trypsin Extraction by AOT Reversed Micelles and Observation by STM[J]. .

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AOT反胶束萃取胰蛋白酶的STM及主要影响因素

Factors Affecting Trypsin Extraction by AOT Reversed Micelles and Observation by STM

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