Wang Fanzhi, Singh Suren, Permaul Kugen
Department of Biotechnology and Food Science, Durban University of Technology, Durban, 4001, South Africa.
Biotechnol Lett. 2025 Feb 26;47(2):30. doi: 10.1007/s10529-025-03566-8.
The hydrophilic microenvironment surrounding an enzyme's active site can influence its catalytic activity. This study examines the effect of enhancing this environment in the Aspergillus niger fructosyltransferase, SucC. Bioinformatics analysis identified a cysteine residue (C66) near the catalytic triad (D64, D194, E271) as vital for maintaining the active site's structure and facilitating substrate transport. Simulated mutagenesis suggested that mutating cysteine to serine (C66S) could increase hydrophilicity without altering the structure significantly. This mutation was predicted to enhance substrate affinity, with binding energy changing from -3.65 to -4.14 kcal mol. The C66S mutant, expressed in Pichia pastoris GS115, showed a 61.3% increase in specific activity, a 13.5% decrease in K (82.20/71.14 mM), and a 21.6% increase in k (112.23/136.48 min), resulting in a 40.1% increase in catalytic efficiency (1.37/1.92 min mM). For fructooligosaccharides (FOS) production, C66S demonstrated enhanced transfructosylation, particularly in the initial stages of the reaction, achieving higher overall FOS yields. These findings highlight that modifying the active site hydrophilicity, without causing major structural changes, is a promising strategy for improving an enzyme's catalytic efficiency.
酶活性位点周围的亲水性微环境会影响其催化活性。本研究考察了增强黑曲霉果糖基转移酶SucC中这种环境的效果。生物信息学分析确定，催化三联体（D64、D194、E271）附近的一个半胱氨酸残基（C66）对于维持活性位点的结构和促进底物转运至关重要。模拟诱变表明，将半胱氨酸突变为丝氨酸（C66S）可以增加亲水性，而不会显著改变结构。预计这种突变会增强底物亲和力，结合能从-3.65千卡/摩尔变为-4.14千卡/摩尔。在毕赤酵母GS115中表达的C66S突变体的比活性提高了61.3%，K值降低了13.5%（82.20/71.14毫摩尔），k值增加了21.6%（112.23/136.48分钟），催化效率提高了40.1%（1.37/1.92分钟/毫摩尔）。对于低聚果糖（FOS）的生产，C66S表现出增强的转果糖基化作用，尤其是在反应的初始阶段，实现了更高的FOS总产率。这些发现突出表明，在不引起重大结构变化的情况下改变活性位点亲水性是提高酶催化效率的一种有前景的策略。
