Structure and Function of Xanthomonas campestris alpha-amylase

本研究旨在进一步了解油菜黄单胞菌α -淀粉酶的结构/功能关系，得到了以下三个结果：1。产生了（α / β） 8桶中第6环缩短4、8或12个氨基酸残基的突变酶。在4个和8个残基缺失突变酶的情况下，酶对糖原的选择性分别提高了5倍和20倍。这表明，长6环的campestris α -淀粉酶引起酶与糖原之间的位阻。可以说，通过α -淀粉酶环的部分缺失可以产生新的酶，2。采用挂滴法结晶葡萄球菌α -淀粉酶。情况如下：酶浓度，10 mg / ml；pH 4.5；温度：20℃；2 .饱和硫酸铵在10 mM醋酸钠（pH 4.5）中，沉淀剂含量分别为15%和10%。甲硫氨酸82的自氧化被怀疑是葡萄球菌α -淀粉酶失活的原因，因此，残基被丙氨酸取代。该突变体酶在高浓度氧化剂的作用下具有相当的稳定性。

This study aimed to get further structure / function relationship of Xanthomonas campestris alpha-amylase and three results as below were obtained.1. Mutant enzymes of which the 6th loop in (alpha / beta) 8 barrel was shortened by 4,8, or 12 amino acid residues were created. The selectivity of enzymes toward glycogen increased fivefold and twentyfold in the case of 4 and 8 residues-deleted mutant enzyme, respectively. This suggests that the long 6th loop of X.campestris alpha-amylase caused the steric hindrance between enzyme and glycogen. It is possible to say that a new enzyme can be created by partial deletion of loop in alpha-amylase,2. X.campestris alpha-amylase was crystallized by the hanging drop method. Conditions were as follows ; enzyme concentration, 10 mg / ml ; pH 4.5 ; temperature, 20ﾟC ; precipitant in reservoir and drop, 15% and 10%, respectively, saturated ammonium sulfate in 10 mM sodium acetate (pH 4.5).3. Auto-oxidation of methionine 82 was suspected to be a reason for the inactivation of X.campestris alpha-amylase, thus, the residue was replaced by alanine. The mutant enzyme was found to be quite stable in the presence of oxidizer of high concentration.