School of Chemistry and Biological Engineering, Department of Biological Science and Engineering, University of Science and Technology Beijing, Beijing, China.
Appl Biochem Biotechnol. 2024 Apr;196(4):1948-1965. doi: 10.1007/s12010-023-04626-3. Epub 2023 Jul 15.
A high ethanol usage of alcohol oxidase (AOX) was required in industry. In this study, a "expand substrate pocket" strategy achieved a high activity AOX from Hansenula polymorpha (H. polymorpha) by Phe to Val residue (F/V) site-directed mutation to enlarge ethanol channel. Although H. Polymorpha AOX (HpAOX) possessed respectively 71.3% and 76.1% similarity with AOX (PpAOX) from Pichia pastoris (P. pastoris) in DNA and protein sequences, their active site structures including catalytic site and substrate channel were similar according to computer-aided analysis. After 3D structure analysis, Phe99 residue of their substrate channels was the most important residue to impact enzyme activity because of its large aromatic side chains. F99V mutation of HpAOX (HpAOXF99V) was designed and executed based on the enzyme catalytic mechanism and molecular computation in order to allow more larger size ethanol into active site. The highest enzyme activity of the fourth strains of HpAOXF99V mutant strain exhibited 12.06-folds increase than that of the host GS115 strain. Furthermore, kinetic studies indicated that the HpAOXF99V significantly promoted catalytic efficiency of ethanol than HpAOX, including Km, Vmax, kcat and kcat/Km. We also provided a new insight that the cofactor FAD irritated both active AOX octamer biosynthesis production and enzyme-catalysed ability due to help enzyme assembly and redox potential.
工业上需要高乙醇使用率的醇氧化酶（AOX）。在这项研究中，通过苯丙氨酸到缬氨酸残基（F/V）的定点突变，采用“扩展底物口袋”策略，从汉逊酵母（Hansenula polymorpha，H. polymorpha）获得了高活性的 AOX，从而扩大了乙醇通道。尽管 H. polymorpha AOX（HpAOX）与毕赤酵母（Pichia pastoris，P. pastoris）的 AOX（PpAOX）在 DNA 和蛋白质序列上分别具有 71.3%和 76.1%的相似性，但根据计算机辅助分析，它们的活性位点结构包括催化位点和底物通道相似。经过 3D 结构分析，由于其较大的芳香侧链，底物通道中的苯丙氨酸 99 残基是影响酶活性的最重要残基。根据酶催化机制和分子计算，设计并执行了 HpAOX 的 F99V 突变（HpAOXF99V），以便让更大尺寸的乙醇进入活性位点。第四株 HpAOXF99V 突变株的最高酶活比宿主 GS115 株提高了 12.06 倍。此外，动力学研究表明，HpAOXF99V 显著提高了乙醇的催化效率，包括 Km、Vmax、kcat 和 kcat/Km。我们还提供了一个新的见解，即辅因子 FAD 由于有助于酶组装和氧化还原电位，同时刺激了活性 AOX 八聚体生物合成产物和酶催化能力。
