State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, People's Republic of China.
J Ind Microbiol Biotechnol. 2014 Apr;41(4):693-700. doi: 10.1007/s10295-014-1406-7. Epub 2014 Feb 4.
β-Mannanases (EC 3.2.1.78) can catalyze the cleavage of internal β-1,4-D-mannosidic linkages of mannan backbones, and they have found applications in food, feed, pulp and paper, oil, pharmaceutical and textile industries. Suitable amino acid substitution can promote access to the substrate-binding groove and maintain the substrate therein, which probably improves the substrate affinity and, thus, increases catalytic efficiency of the enzyme. In this study, to improve the substrate affinity of AuMan5A, a glycoside hydrolase (GH) family 5 β-mannanase from Aspergillus usamii, had its directed modification conducted by in silico design, and followed by site-directed mutagenesis. The mutant genes, Auman5A (Y111F) and Auman5A (Y115F), were constructed by megaprimer PCR, respectively. Then, Auman5A and its mutant genes were expressed in Pichia pastoris GS115 successfully. The specific activities of purified recombinant β-mannanases (reAuMan5A, reAuMan5A(Y111F) and reAuMan5A(Y115F)) towards locust bean gum were 152.5, 199.6 and 218.9 U mg(-1), respectively. The two mutants were found to be similar to reAuMan5A regarding temperature and pH characteristics. Nevertheless, the K m values of reAuMan5A(Y111F) and reAuMan5A(Y115F), towards guar gum, decreased to 2.95 ± 0.22 and 2.39 ± 0.33 mg ml(-1) from 4.49 ± 0.07 mg ml(-1) of reAuMan5A, which would make reAuMan5A(Y111F) and reAuMan5A(Y115F) promising candidates for industrial processes. Structural analysis showed that the two mutants increased their affinity by decreasing the steric conflicts with those more complicated substrates. The results suggested that subtle conformational modification in the substrate-binding groove could substantially alter the substrate affinity of AuMan5A. This study laid a solid foundation for the directed modification of substrate affinities of β-mannanases and other enzymes.
β-甘露聚糖酶（EC 3.2.1.78）能够催化甘露聚糖骨架内部β-1,4-D-甘露糖苷键的裂解，已在食品、饲料、纸浆和造纸、石油、制药和纺织工业中得到应用。合适的氨基酸取代可以促进进入底物结合槽并保持其中的底物，这可能会提高底物亲和力，从而提高酶的催化效率。在这项研究中，为了提高来自土曲霉的糖苷水解酶（GH）家族 5β-甘露聚糖酶 AuMan5A 的底物亲和力，通过计算机设计进行了定向修饰，并随后进行了定点突变。通过 megaprimer PCR 分别构建了突变基因 Auman5A(Y111F)和 Auman5A(Y115F)。然后，成功在巴斯德毕赤酵母 GS115 中表达了 Auman5A 和其突变基因。纯化的重组β-甘露聚糖酶（reAuMan5A、reAuMan5A(Y111F)和 reAuMan5A(Y115F)）对罗望子豆胶的比活性分别为 152.5、199.6 和 218.9 U mg(-1)。这两个突变体在温度和 pH 特性方面与 reAuMan5A 相似。然而，reAuMan5A(Y111F)和 reAuMan5A(Y115F)对瓜尔豆胶的 K m 值分别降低至 2.95±0.22 和 2.39±0.33 mg ml(-1)，而 reAuMan5A 的 K m 值为 4.49±0.07 mg ml(-1)，这将使 reAuMan5A(Y111F)和 reAuMan5A(Y115F)成为工业过程有前途的候选者。结构分析表明，这两个突变体通过减少与更复杂底物的空间位阻，增加了它们的亲和力。结果表明，在底物结合槽中进行细微的构象修饰可以显著改变 AuMan5A 的底物亲和力。本研究为β-甘露聚糖酶和其他酶的定向修饰底物亲和力奠定了坚实的基础。
