重组超耐热酸性α-淀粉酶的分离纯化及其性质研究  

Purification and properties of recombinant extremely thermostable and acid-stable amylase

作　　者：李辉[1] 郭建强 岳丽丽 李运敏 矫庆华[1] 

机构地区：[1]中国科学院微生物研究所 [2]三元基因有限公司 [3]VA Medical Centre 111 C5,Department of Medicine University of California at San Francisco 4150 Clement Street,San Francisco,CA94121,USA

出　　处：《微生物学报》2005年第4期547-550,共4页Acta Microbiologica Sinica

基　　金：国家科技攻关计划(2001BA708B0303)~~

摘　　要：基因工程菌所产生的重组超耐热酸性α-淀粉酶,通过超滤浓缩、脱盐和聚丙烯酰胺垂直板凝胶电泳进行纯化,得到电泳纯的超耐热酸性α-淀粉酶,纯化倍数为11.7,活力回收率为29.8%。用SDSPAGE测得该酶的分子量为55kD,酶的等电点pI(室温)为5.0,以可溶性淀粉为底物的Km值为1.12gL,用硫酸酚法测得其含糖量为15.4%。该酶的最适反应温度为95℃,最适反应pH值为4.5。在pH4.0～7.0室温放置48h酶活没有变化,110℃保温1h残留60%活力。Cr3+、Fe2+、Cu2+抑制酶的活性,Ca2+对酶活无影响。EDTA和DTT对酶的活性无影响。Extremely thermostable and acid-stable α-amylase produced by Pichia pastoris GS115/pPIC9K-Amy-228 was purified to electrophoretic homogeneity by the steps of ultrafiltration and PAGE. Purification of about 11.7 fold was achieved with an overall yield of 29.8%. Its molecular weight was estimated to be about 55kD by SDS-PAGE . The isoelectric point was 5.0 (room temperature). Michaelis constant of the enzyme for soluble starch was 1.12g/L.The carbohydrate content was 15.4% by the phenol-sulfuric acid method. The optimum temperature and pH of the enzyme activity were 95℃and 4.5 respectively. The enzyme activity was stable under room temperature in the pH rang of 4.0～7.0 for 48 hours.About 60% of the initial enzyme activity was measured after 1h of incubation at 110℃. The activity was strongly inhibited by Fe 2+ ,Cr 2+ and Cu 2+ ,While Ca 2+ had no effect on it. DTT and EDTA had no effect on the activity.

关 键 词：超耐热酸性α-淀粉酶 纯化 性质 

分 类 号：Q814[生物学—生物工程]