De Mot R, Verachtert H
Eur J Biochem. 1987 May 4;164(3):643-54. doi: 10.1111/j.1432-1033.1987.tb11175.x.
An alpha-amylase and a glucoamylase were purified to homogeneity from the culture fluid of beta-cyclodextrin-grown Candida antarctica CBS 6678 by protamine sulfate treatment, ammonium sulfate precipitation, gel filtration (Sephadex G-75 sf, Ultrogel AcA 54), DEAE-Sephacel chromatography, hydroxyapatite chromatography and affinity chromatography on acarbose--AH-Sepharose 4B. Both enzymes were monomeric glycoproteins with fairly different amino acid compositions. Their apparent relative molecular mass, sedimentation coefficient (Szero20,w), isoelectric point, absorption coefficient (280 nm), pH and temperature optima were estimated as 48,500, 4.7 S, 10.1, 1.74 cm2 mg-1, 4.2 and 57 degrees C, respectively, for glucoamylase and as 50,000, 4.9 S, 10.3, 1.53 cm2 mg-1, 4.2 and 62 degrees C, respectively, for alpha-amylase. Kinetic analyses indicated that both enzymes preferentially hydrolyzed high-molecular-mass substrates, including some raw starches. alpha-Amylase was active on cyclodextrins, whereas debranching activity was demonstrated for glucoamylase. Trestatins were potent inhibitors of both alpha-amylase (Ki less than 1 microM) and glucoamylase (Ki less than 0.1 microM), being more effective than Bay e 4609 (Ki less than 10 microM). Glucoamylase was selectivity and strongly inhibited by acarbose (Ki less than 0.1 microM). Activity of the latter enzyme was also affected by 1-deoxynojirimycin (Ki less than 1 mM), maltitol and amino alcohols (Ki less than 10 mM). Unlike alpha-amylase, glucoamylase adsorbed strongly onto raw starch, the adsorption site being non-identical with the active site.
通过硫酸鱼精蛋白处理、硫酸铵沉淀、凝胶过滤（Sephadex G - 75 sf、Ultrogel AcA 54）、DEAE - Sephacel层析、羟基磷灰石层析以及在阿卡波糖 - AH - Sepharose 4B上的亲和层析，从β - 环糊精培养的南极假丝酵母CBS 6678的培养液中纯化出了α - 淀粉酶和葡糖淀粉酶，使其达到均一状态。这两种酶都是单体糖蛋白，氨基酸组成差异较大。葡糖淀粉酶的表观相对分子质量、沉降系数（S₂₀,w⁰）、等电点、吸收系数（280 nm）、最适pH和最适温度分别估计为48,500、4.7 S、10.1、1.74 cm² mg⁻¹、4.2和57℃，而α - 淀粉酶的分别为50,000、4.9 S、10.3、1.53 cm² mg⁻¹、4.2和62℃。动力学分析表明，这两种酶都优先水解高分子质量底物，包括一些生淀粉。α - 淀粉酶对环糊精有活性，而葡糖淀粉酶表现出脱支活性。曲格列汀是α - 淀粉酶（Ki小于1 μM）和葡糖淀粉酶（Ki小于0.1 μM）的有效抑制剂，比拜耳e 4609（Ki小于10 μM）更有效。葡糖淀粉酶被阿卡波糖（Ki小于0.1 μM）选择性且强烈抑制。后一种酶的活性也受1 - 脱氧野尻霉素（Ki小于1 mM）、麦芽糖醇和氨基醇（Ki小于10 mM）影响。与α - 淀粉酶不同，葡糖淀粉酶强烈吸附在生淀粉上，吸附位点与活性位点不同。
