凝胶板植酸酶活力检测

DETECTION OF PHYTASE IN GELS

将１ｍｍ厚凝固于复印膜上的水琼脂（１５％～２．０％）凝胶板侵入含１２ｍｍｏｌ／Ｌ植酸钠的Ｇｌｙ—ＨＣｌ缓冲液中达１ｈ以上，取出干至胶表面无水迹，于上加Ａｓｐｅｒｇｉｌｌｕｓｓｐ．５９—２植酸酶或与电泳后的凝胶板紧贴１０～６０ｍｉｎ。然后水平置于恒温水浴锅中反应一定时间，取出浸入１ｍｏｌ／ＬＨ２ＳＯ４－２％（ＮＨ４）６Ｍｎ７Ｏ２４－１０％ＦｅＳＯ４溶液显色２～５ｍｉｎ。该法简便、快速、灵敏，反应１５ｍｉｎ即可检出每ｃｍ２低于０．００４５μ的植酸酶。此法适用于微生物的初筛、酶谱分析及酶比活的比较。

A sheet of agar gel(1.5%～2.0%,1mm thickness)on copy film was soaked in Gly-HCl buffer containing 1 .2m mol/L sodium phytate for over 1h and then taken out.After the moisture on thegel surface disappeared, phytase was spotted on or an electrophoresed polyacryamide gel contaillingphytase was placed in contact with it for 10～60min,then the agar gel was horizontally incubated inwater bath for 15 min at 37oC,finally the gel was immersed in lmol/L H2SO4-2%(NH4)6Mo7O24-10% FeSO4 for 2～5min.to develop its colour.This assay is simple, rapid andhighsensitive, as little as 0.0045 μ phytase on one cm2 of gel could be detected,which is suitable forscreening microorganism,zymogram analysis and comparing specific activities of phytases.