College of Life Science, Jiangxi Normal University, Nanchang, 330022, China.
Biotechnol Lett. 2020 Nov;42(11):2239-2250. doi: 10.1007/s10529-020-02935-9. Epub 2020 Jun 24.
β-Glucosidase (BGL) plays a key role in cellulose hydrolysis. However, it is still a great challenge to enhance product tolerance and enzyme activity of BGL simultaneously. Here, we utilized one round error-prone PCR to engineer the Penicillium oxalicum 16 BGL (16BGL) for improving the cellulosic ethanol yield. We identified a new variant (L-6C), a triple mutant (M280T/V484L/D589E), with enhanced catalytic efficiency ([Formula: see text]) for hydrolyzing pNPG and reduced strength of inhibition ([Formula: see text]) by glucose. To be specific, L-6C achieved a [Formula: see text] of 0.35 at a glucose concentration of 20 mM, which was 3.63 times lower than that attained by 16BGL. The catalytic efficiency for L-6C to hydrolyze pNPG was determined to be 983.68 mM s, which was 22% higher than that for 16BGL. However, experiments showed that L-6C had reduced binding affinity (2.88 mM) to pNGP compared with 16BGL (1.69 mM). L-6C produced 6.15 g/L ethanol whose yield increased by about 10% than 16BGL. We performed molecular docking and molecular dynamics (MD) simulation, and binding free energy calculation using the Molecular Mechanics/Poisson Boltzmann surface area (MM/PBSA) method. MD simulation together with the MM/PBSA calculation suggested that L-6C had reduced binding free energy to pNPG, which was consistent with the experimental data.
β-葡萄糖苷酶（BGL）在纤维素水解中起着关键作用。然而，同时提高 BGL 的产物耐受性和酶活性仍然是一个巨大的挑战。在这里，我们利用一轮易错 PCR 来工程化青霉 16BGL（16BGL），以提高纤维乙醇的产量。我们鉴定出一个新变体（L-6C），一个三重突变体（M280T/V484L/D589E），对 pNPG 的水解具有更高的催化效率（[公式：见正文]）和降低的葡萄糖抑制强度（[公式：见正文]）。具体来说，L-6C 在 20mM 葡萄糖浓度下达到[公式：见正文]为 0.35，比 16BGL 低 3.63 倍。L-6C 水解 pNPG 的催化效率被确定为 983.68mM·s，比 16BGL 高 22%。然而，实验表明，与 16BGL（1.69mM）相比，L-6C 对 pNGP 的结合亲和力降低（2.88mM）。L-6C 产生了 6.15g/L 的乙醇，其产量比 16BGL 提高了约 10%。我们进行了分子对接和分子动力学（MD）模拟，并使用分子力学/泊松-玻尔兹曼表面面积（MM/PBSA）方法进行了结合自由能计算。MD 模拟结合 MM/PBSA 计算表明，L-6C 对 pNPG 的结合自由能降低，这与实验数据一致。
