Pcal_0768 gene encoding an amylomaltase, a 4-α-glucanatransferase belonging to family 77 of glycosyl hydrolases, from Pyrobaculum calidifontis was cloned and expressed in Escherichia coli. The recombinant protein was produced in E. coli in soluble and active form. However, the expression level was not very high. Analysis of the mRNA of initial seven codons at the 5′-end of the gene revealed the presence of a hair pin like secondary structure. This secondary structure was removed by site directed mutagenesis, without altering the amino acids, which resulted in enhanced expression of the cloned gene. Recombinant Pcal_0768 exhibited optimal amylomaltase activity at 80 °C and pH 6.9. Under these conditions, the specific activity was 690 U/ mg. Recombinant Pcal_0768 was highly thermostable with a half-life of 6 h at 100 °C. It exhibited the highest k_cat value among the characterized glucanotransferases. No metal ions were required for activity or stability of the enzyme. Recombinant Pcal_0768 was successfully employed in the synthesis of modified starch for producing thermoreversible gel. To the best of our knowledge, till now this is the most thermostable enzyme among the characterized amylomaltases. High thermostability and starch modification potential make it a novel and distinct amylomaltase.

克隆了来自拟南芥（Pyrobaculum calidifontis）的编码淀粉淀粉酶（属于糖基水解酶的家族77的4-α-葡聚糖转化酶）的Pcal_0768基因，并在大肠杆菌中表达。重组蛋白在大肠杆菌中产生呈可溶性和活性形式。但是，表达水平不是很高。对基因5'端最初七个密码子的mRNA的分析显示，存在发夹样二级结构。通过位点定向诱变去除了该二级结构，而没有改变氨基酸，这导致了克隆基因的表达增强。重组Pcal_0768在80°C和pH 6.9下表现出最佳的淀粉酶活性。在这些条件下，比活为690 U / mg。重组Pcal_0768具有很高的热稳定性，在100°C下的半衰期为6小时。它表现出最高ķ_猫在表征的葡聚糖转移酶中的价值。酶的活性或稳定性不需要金属离子。重组Pcal_0768被成功地用于合成变性淀粉以生产热可逆凝胶。据我们所知，到目前为止，这是特征淀粉淀粉酶中最热稳定的酶。高热稳定性和淀粉修饰潜力使其成为一种新颖独特的淀粉酶。