Structure and Function of Xanthomonas campestris alpha-amylase

野油菜黄单胞菌α-淀粉酶的结构和功能

• 批准号: 07650964
• 负责人: ABE Jun-ichi
• 金额: $ 1.34万
• 依托单位: Kagoshima University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07650964/
• 关键词: Xanthomonas campestris; alpha-amylase; loop structure; crystallization; inactivation by oxidation; mutagenesis; substrate specificity; Resistance against oxidation; Xanthomonas Campestris; メチオニン残基; 部異指定突然変異; α-サイクロデキストリン; グリコーゲン; 変異酵素; 立体障害

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.

本研究旨在进一步研究野油菜黄单胞菌α-淀粉酶的结构与功能关系，主要结果如下：1.产生了（α/β）8桶中的第6环缩短了4、8或12个氨基酸残基的突变酶。在4和8个残基缺失的突变酶的情况下，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环较长，导致酶与糖原之间存在空间位阻。可以说，可以通过α-淀粉酶中的环的部分缺失来产生新的酶，2。采用悬滴法对野油菜黄单胞菌α-淀粉酶进行结晶。条件如下;酶浓度，10 mg / ml ; pH 4.5 ;温度，20 ℃;在储液器和液滴中的沉淀剂，分别为15%和10%，在10 mM乙酸钠中的饱和硫酸铵（pH 4.5）。甲硫氨酸82的自动氧化被怀疑是导致野油菜黄单胞菌α-淀粉酶失活的原因，因此，该残基被丙氨酸取代。突变酶在高浓度氧化剂存在下相当稳定。