Schmidt J, John M
Biochim Biophys Acta. 1979 Jan 12;566(1):88-99. doi: 10.1016/0005-2744(79)90252-3.
The extracellular maltotetraose-forming amylase of Pseudomonas stutzeri was purified to homogeneity by a combination of affinity and hydroxyapatite chromatography. Sodium dodecyl sulfate-gel electrophoresis indicated that the oligomeric enzyme contains two different subunits with molecular weights of 48 000 and 58 000. Cross-linking studies using dimethyl suberimidate have demonstrated that the native enzyme consists of dimers. Seven isozymes of the amylase have been identified after polyacrylamide gel electrophoresis and amylose-digestion zymograms. The amylase of Ps. stutzeri is known to produce maltotetraose from linear and branched alpha-glucans by an exomechanism. The relatively high conversion rate of starch (75% hydrolysis), and the hydrolysis of cross-linked blue starch by this amylase indicate that the enzyme can cleave its substrates also by an endomechanism. Further strong evidence for an endomechanism was obtained from the action of the amylase on maltotetraose units which are located within the pullulan molecule. Dextran, pullulan, and maltotetraose are compeititve inhibitors. EDTA caused reversible inactivation. Amylase activity could be restored by addition of Ca2+. Heavy metals are inhibitory.
通过亲和色谱和羟基磷灰石色谱相结合的方法，将施氏假单胞菌的细胞外形成麦芽四糖的淀粉酶纯化至同质。十二烷基硫酸钠-凝胶电泳表明，该寡聚酶含有两种不同的亚基，分子量分别为48000和58000。使用亚氨酯二甲酯进行的交联研究表明，天然酶由二聚体组成。在聚丙烯酰胺凝胶电泳和直链淀粉消化酶谱分析后，已鉴定出该淀粉酶的七种同工酶。已知施氏假单胞菌的淀粉酶通过外切机制从线性和支链α-葡聚糖产生麦芽四糖。淀粉的相对高转化率（75%水解）以及该淀粉酶对交联蓝色淀粉的水解表明，该酶也可以通过内切机制切割其底物。从淀粉酶对位于支链淀粉分子内的麦芽四糖单元的作用中获得了进一步支持内切机制的有力证据。葡聚糖、支链淀粉和麦芽四糖是竞争性抑制剂。EDTA导致可逆失活。添加Ca2+可恢复淀粉酶活性。重金属具有抑制作用。
