Improvement of the Activity and Stability of Starch-Debranching Pullulanase from Bacillus naganoensis via Tailoring of the Active Sites Lining the Catalytic Pocket.
Improvement of the Activity and Stability of Starch-Debranching Pullulanase from Bacillus naganoensis via Tailoring of the Active Sites Lining the Catalytic Pocket.
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DOI：
10.1021/acs.jafc.8b06002
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发表时间：
2018-11
影响因子：
6.1
通讯作者：
Xinye Wang;Y. Nie;Yan Xu
中科院分区：
文献类型：
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作者：
Xinye Wang;Y. Nie;Yan Xu
支链淀粉酶是众所周知的脱支酶，可水解淀粉和寡糖中的 α-1,6-糖苷键。然而，大多数普鲁兰酶在实际应用中表现出有限的活性。在这里，长野芽孢杆菌支链淀粉酶催化口袋内的两个位点（787 和 621）被三重密码饱和诱变鉴定为对酶活性至关重要。随后，对两个位点进行基于NNK的饱和诱变以获得阳性变异。在活性增强的变体中，D787C的酶活性和比活性比野生型（WT）高1.5倍。 D787C 还显示 kcat 增加了 1.8 倍，kcat/Km 增加了 1.7 倍。此外，D787C在60℃以上的温度下仍保持比WT更高的活性。所有阳性变体均表现出较高的耐酸性，其中 D787C 在 pH 4.0 下保持 90% 的残留活性。因此，通过活性位点的饱和诱变获得了具有改进特性的酶。
Pullulanases are well-known debranching enzymes that hydrolyze α-1,6-glycosidic linkages in starch and oligosaccharides. However, most of the pullulanases exhibit limited activity for practical applications. Here, two sites (787 and 621) lining the catalytic pocket of Bacillus naganoensis pullulanase were identified as being critical for enzymatic activity by triple-code saturation mutagenesis. Subsequently, both sites were subjected to NNK-based saturation mutagenesis to obtain positive variants. Among the variants showing enhanced activity, the enzymatic activity and specific activity of D787C were 1.5-fold higher than those of the wild-type (WT). D787C also showed a 1.8-fold increase in kcat and a 1.7-fold increase in kcat/ Km. In addition, D787C maintained higher activity compared with that of WT at temperatures over 60 °C. All the positive variants showed higher acid resistance, with D787C maintaining 90% residual activity at pH 4.0. Thus, enzymes with improved properties were obtained by saturation mutagenesis at the active site.