有关真菌α-淀粉酶的研究主要集中在酶的底物催化形式等生化性质方面，该类酶中的大量氨基酸残基组成与酶功能性质之间的关系尚不明确。本文利用分子对接和同源比对等生物信息学分析方法发现米根霉α-淀粉酶中有3个特殊的组氨酸残基（H120，H200和H286），并利用定点突变的方法构建了系列突变体。性质研究表明，286位氨基酸残基结构与该酶的最适反应温度、最适反应pH、酸耐受性以及麦芽糖生成能力均有关联；突变体H286L的酸耐受性和水解淀粉的麦芽糖生成能力得到显著提高；120位和200位氨基酸残基的变化不改变该酶的最适反应温度和最适反应pH，而两者的组合突变可显著提升该酶的酸耐受性。此外，数据表明该酶的高麦芽糖生成能力与酶对麦芽三糖的亲和力不直接相关。研究结果可为真菌α-淀粉酶高麦芽糖生成能力的原因及其定向进化提供一定理论参考。

The research on fungal α-amylase mainly focused on the biochemical properties such as the substrate catalytic mechanism, and the relationship between the composition of amino acids residues and the functional properties of this enzyme is unclear. In this paper, three special conserved histidine residues (H120, H200 and H286) that existed in the catalytic domain of Rhizopus oryzae α-amylase were identified by bioinformatics analysis methods of molecular docking and comparative analysis. A series of mutants were constructed base on the above three identified sites using site-directed mutagenesis. The results showed that the structure of the amino acid residue at position 286 has been proved to be related with the optimum temperature, optimum pH, acid resistance and maltose-forming ability of the enzyme. The acid resistance and maltose-forming ability on soluble starch of the mutant H286L were significantly improved. Changes in 120 and 200 amino acid residues exhibited no effects on the optimal temperature, optimum pH and maltose-forming ability, but the combined mutations at both sites of 120 and 200 could improve its acid resistance. The resulted data indicated that the high maltose-forming ability of the enzyme was not directly related to its affinity on maltotriose, which may provide a theoretical reference for the mechanism of high maltose-forming ability and its directed evolution for fungal α-amylase.