Structure and Function of Xanthomonas campestris alpha-amylase

本研究旨在进一步研究野油菜黄单胞菌α-淀粉酶的结构/功能关系，获得以下三个结果。产生了突变酶，其中(α/β)8桶的第6环缩短了4，8或12个氨基酸残基。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增加了5倍和20倍。这表明，野油菜α-淀粉酶的第6个长环导致了酶和糖原之间的空间位阻。可以说，通过部分删除α-淀粉酶中的环可以产生一种新的酶。2.用悬滴法结晶野油菜α-淀粉酶。实验条件为：酶浓度10 mg/ml，pH值4.5，温度20ﾟC，沉淀剂中沉淀剂浓度分别为15%和10%，10 mM醋酸钠(pH 4.5)中加入饱和硫酸铵。甲硫氨酸82的自氧化可能是野油菜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存在下非常稳定。

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.