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.
出处
《微生物学报》
CAS
CSCD
北大核心
2005年第4期547-550,共4页
Acta Microbiologica Sinica
基金
国家科技攻关计划(2001BA708B0303)~~
关键词
超耐热酸性α-淀粉酶
纯化
性质
Extremely thermostable and acid-stable α-amylase, Purification, Properties
