Protein Engineering of Novel Thermostable Pullulanase from Geobacillus stearothermophilus and Starch Saccharification Application

Starch Pub Date : 2024-04-05 DOI:10.1002/star.202300227

Lin Lin, Ruiqi Li, Ziyu Wang, Jing Yuan, Xiaojun Hu

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Abstract

A novel thermostable type I pullulanase gene (named pulGSM) is cloned from Geobacillus stearothermophilus DSMZ456. The purified PulGSM shows optimal activity at pH 6.0 and 65 °C. Protein engineering method (site‐directed mutagenesis and enzyme immobilization) is used to further improve the thermostability and catalytic efficiency of PulGSM. Through site‐directed mutagenesis, three mutants (carrying the mutations L211C, E306I and R471E named PulGSM‐M211, PulGSM‐M306 and PulGSM‐M471) are generated and characterized in detail. As showing the best enzymatic properties, PulGSM‐M471 is directly immobilized on epoxy‐functionalized supports to obtain the immobilized enzyme lx‐PulGSM‐M471. The temperature tolerance results show an enhanced T1/2 of 6.5 h (mutated PulGSM‐M471) and 8.5 h (mutated and immobilized lx‐PulGSM‐M471) at 70 °C in comparison to the wild type (4 h). Compared to the commercial pullulanase from Bacillus acidopullulyticus (PDB:2WAN), PulGSM or PulGSM‐M471 can be used directly in maize starch saccharification process without adjusting the pH, which reduces cost and improves efficiency.

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嗜热地衣芽孢杆菌新型可恒温普鲁兰酶的蛋白质工程及淀粉糖化应用

从嗜热地衣芽孢杆菌（Geobacillus stearothermophilus DSMZ456）中克隆出一种新型恒温 I 型拉鲁兰酶基因（命名为 PulGSM）。纯化的 PulGSM 在 pH 值为 6.0 和温度为 65 ℃ 时显示出最佳活性。蛋白质工程方法（定点突变和酶固定化）用于进一步提高 PulGSM 的耐热性和催化效率。通过定点突变，产生了三个突变体（携带 L211C、E306I 和 R471E 突变，分别命名为 PulGSM-M211、PulGSM-M306 和 PulGSM-M471），并对其进行了详细表征。由于 PulGSM-M471 显示出最佳的酶特性，因此将其直接固定在环氧树脂功能化的支持物上，得到了固定化酶 lx-PulGSM-M471。耐温结果表明，与野生型（4 小时）相比，在 70 °C 下，PulGSM-M471 的 T1/2 提高了 6.5 小时（突变的 PulGSM-M471）和 8.5 小时（突变和固定化的 lx-PulGSM-M471）。与来自酸性芽孢杆菌（PDB:2WAN）的商品化拉鲁糖酶相比，PulGSM 或 PulGSM-M471 可直接用于玉米淀粉糖化过程，无需调节 pH 值，从而降低了成本，提高了效率。

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Starch

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