Department of Biochemistry and Biophysics, Faculty of Biological Science, Tarbiat Modares University, PO Box: 14115-175, Tehran, Iran.
Appl Biochem Biotechnol. 2010 Sep;162(2):444-59. doi: 10.1007/s12010-009-8879-2. Epub 2010 Feb 23.
Successful industrial use of amylases requires that they are sufficiently stable and active at application conditions, e.g., at high temperature in starch-liquefaction process. In the present study, site-directed mutagenesis was used to enhance the thermal stability and calcium independency of a mesophilic alpha-amylase from Bacillus megaterium WHO. Mutations (A53S and H58I) were designed at the calcium-binding site based on the sequence alignment. Kinetic and thermostability parameters of the mutants were analyzed and compared with that of the wild type. In the presence of calcium, the affinity of the enzymes (wild type and mutants) toward starch was increased. In comparison to the wild type, calcium ion had more effect on the catalytic efficiency, k (cat)/K (m), and half-life (at 60 degrees C) of A53S mutant. In A53S, the dependence of half-life on calcium concentration showed that the enhanced calcium binding is likely to be responsible for the increased stability. In contrast, calcium-independent mutant (H58I) possessed high thermostability. In addition, thermodynamic parameters of amylolytic reaction exhibited an increase in the activation energy and the entropy of the system. Kinetics of irreversible thermal inactivation suggests that the activation energy increased by 1.4-fold in the most stable variant.
成功地将淀粉酶应用于工业生产需要它们在应用条件下具有足够的稳定性和活性，例如在淀粉液化过程中的高温条件下。在本研究中，采用定点突变技术来提高嗜温α-淀粉酶的热稳定性和钙离子非依赖性，该酶来自巨大芽孢杆菌 WHO。基于序列比对，在钙离子结合位点设计了突变（A53S 和 H58I）。分析了突变体的动力学和热稳定性参数，并与野生型进行了比较。在钙离子存在的情况下，酶（野生型和突变体）对淀粉的亲和力增加。与野生型相比，钙离子对 A53S 突变体的催化效率、k（cat）/K（m）和半衰期（在 60°C 时）的影响更大。在 A53S 中，半衰期对钙离子浓度的依赖性表明，增强的钙离子结合可能是导致稳定性提高的原因。相比之下，钙离子非依赖性突变体（H58I）具有较高的热稳定性。此外，淀粉水解反应的热力学参数显示出系统的活化能和熵增加。不可逆热失活动力学表明，最稳定的变体的活化能增加了 1.4 倍。
