Shad Mohsin, Akhtar Muhammad Waheed, Sajjad Muhammad
School of Biological Sciences, University of the Punjab, Quaid-e-Azam Campus, Lahore, P.O. 54590, Pakistan; Structural Biology, The Rosalind Franklin Institute, Harwell Science & Innovation Campus, Didcot OX11 0QS, United Kingdom.
School of Biological Sciences, University of the Punjab, Quaid-e-Azam Campus, Lahore, P.O. 54590, Pakistan.
Int J Biol Macromol. 2025 May;307(Pt 3):142243. doi: 10.1016/j.ijbiomac.2025.142243. Epub 2025 Mar 17.
Alpha-amylases are essential enzymes that cleave α-1,4-glycosidic bonds in starch, generating products such as glucose, maltose, dextrin's, and oligosaccharides, which play a key role in various industries. The structural and functional insights, along with biochemical dynamics, of Bacillus licheniformis α-amylase variants (BLAMWSP and BLAMCD) were investigated through N- and C-terminal truncations along with two previously reported mutations, Thr353Ile and His400Arg. MD simulation results demonstrated the stability of binding and catalytic residues as predicted through molecular docking. Analysis of the secondary structure and temperature ramping through CD spectroscopy revealed that both BLAMWSP and BLAMCD maintained structural stability at 90 °C. The specific activities of BLAMWSP and BLAMCD against wheat starch were determined to be 2343.09 ± 0.20 and 4237.88 ± 0.66 (μmol min/μmole protein), respectively at 90 °C in 100 mM phosphate buffer (pH 6.0). The BLAMCD variant exhibited a two-fold increase in enzymatic activity relative to BLAMWSP due to increased surface accessibility of its substrate-binding and catalytic residues. The enzymes' catalytic efficiencies (k /K) were 51.76 ± 1.76 and 114.10 ± 1.41, highlighting that BLAMCD exhibits significantly higher catalytic efficiency and substrate affinity than BLAMWSP. These modifications make BLAMCD a promising candidate for industrial starch liquefaction and scarification applications.
α-淀粉酶是一类重要的酶，可裂解淀粉中的α-1,4-糖苷键，生成葡萄糖、麦芽糖、糊精和寡糖等产物，这些产物在各个行业中发挥着关键作用。通过N端和C端截短以及两个先前报道的突变（Thr353Ile和His400Arg），研究了地衣芽孢杆菌α-淀粉酶变体（BLAMWSP和BLAMCD）的结构和功能见解以及生化动力学。分子动力学（MD）模拟结果证明了通过分子对接预测的结合和催化残基的稳定性。通过圆二色光谱（CD）对二级结构和温度变化的分析表明，BLAMWSP和BLAMCD在90°C时均保持结构稳定性。在90°C、100mM磷酸盐缓冲液（pH 6.0）中，BLAMWSP和BLAMCD对小麦淀粉的比活性分别测定为2343.09±0.20和4237.88±0.66（μmol·min/μmole蛋白质）。由于其底物结合和催化残基的表面可及性增加，BLAMCD变体的酶活性相对于BLAMWSP提高了两倍。这些酶的催化效率（kcat/Km）分别为51.76±1.76和114.10±1.41，突出表明BLAMCD比BLAMWSP表现出明显更高的催化效率和底物亲和力。这些修饰使BLAMCD成为工业淀粉液化和糖化应用的有前途的候选者。
