Multiple approaches of loop region modification for thermostability improvement of 4,6-α-glucanotransferase from Limosilactobacillus fermentum NCC 3057.
Multiple approaches of loop region modification for thermostability improvement of 4,6-α-glucanotransferase from Limosilactobacillus fermentum NCC 3057.
复制标题
DOI：
10.1016/j.ijbiomac.2023.123536
复制
发表时间：
2023-02
影响因子：
8.2
通讯作者：
D. Rao;Runtian Huo;Zheng-Fei Yan;Zhiyong Guo;Weiqiong Liu;Mengwei Lu;Hui-Bo Luo;Xiumei Tao;Weikang Yang;Lingqia Su;Sheng Chen;Lei Wang;Jing Wu
中科院分区：
文献类型：
--
作者：
D. Rao;Runtian Huo;Zheng-Fei Yan;Zhiyong Guo;Weiqiong Liu;Mengwei Lu;Hui-Bo Luo;Xiumei Tao;Weikang Yang;Lingqia Su;Sheng Chen;Lei Wang;Jing Wu
4，6-α-葡聚糖基转移酶（4，6-α-GT）是糖苷水解酶70（GH 70）家族的成员之一，能将淀粉/麦芽寡糖转化为含α，1 -6键的α-葡聚糖，在食品、医药等行业具有潜在的应用价值，但由于其热稳定性差，不能满足高温要求。本研究以硅乳木聚糖酶NCC 3057中的4，6-α-GT（ΔGtfB）为模型酶，研究其热稳定性。利用定向进化、序列比对和计算机辅助设计对作为靶区域的ΔGtfB的环进行优化。获得总共11个阳性突变体，并迭代组合以获得具有高耐温性（50 °C）的组合突变体CM 9。突变体CM 9的活性是野生型活性的2.08倍，伴随着高5 °C的最适温度、高5.76 °C的熔点（Tm，59.46 °C）和长11.95倍的半衰期（t1/2）。结果表明，ΔGtfB环区大部分极性残基突变为刚性脯氨酸残基。分子动力学模拟表明，CM 9的均方根波动显着降低的“呼吸”运动减少的环区。本研究为通过合理的环区修饰提高4，6-α-GT的热稳定性提供了一种新的策略。
4,6-α-glucanotransferase (4,6-α-GT), as a member of the glycoside hydrolase 70 (GH70) family, converts starch/maltooligosaccharides into α,1–6 bond-containing α-glucan and possesses potential applications in food, medical and related industries but does not satisfy the high-temperature requirement due to its poor thermostability. In this study, a 4,6-α-GT (ΔGtfB) fromLimosilactobacillus fermentumNCC 3057 was used as a model enzyme to improve its thermostability. The loops of ΔGtfB as the target region were optimized using directed evolution, sequence alignment, and computer-aided design. A total of 11 positive mutants were obtained and iteratively combined to obtain a combined mutant CM9, with high resistance to temperature (50 °C). The activity of mutant CM9 was 2.08-fold the activity of the wild type, accompanied by a 5 °C higher optimal temperature, a 5.76 °C higher melting point (Tm, 59.46 °C), and an 11.95-fold longer half-life time (t1/2). The results showed that most of the polar residues in the loop region of ΔGtfB are mutated into rigid proline residues. Molecular dynamics simulation demonstrated that the root mean square fluctuation of CM9 significantly decreased by “Breathing” movement reduction of the loop region. This study provides a new strategy for improving the thermostability of 4,6-α-GT through rational loop region modification.