Han Nanyu, Miao Huabiao, Ding Junmei, Li Junjun, Mu Yuelin, Zhou Junpei, Huang Zunxi
School of Life Sciences, Yunnan Normal University, Kunming, 650500 China.
Key Laboratory of Enzyme Engineering, Yunnan Normal University, Kunming, 650500 China.
Biotechnol Biofuels. 2017 May 23;10:133. doi: 10.1186/s13068-017-0824-y. eCollection 2017.
Xylanases have been widely employed in many industrial processes, and thermophilic xylanases are in great demand for meeting the high-temperature requirements of biotechnological treatments. In this work, we aim to improve the thermostability of XynCDBFV, a glycoside hydrolase (GH) family 11 xylanase from the ruminal fungus , by site-directed mutagenesis. We report favorable mutations at the C-terminus from B-factor comparison and multiple sequence alignment.
C-terminal residues 207-NGGA-210 in XynCDBFV were discovered to exhibit pronounced flexibility based on comparison of normalized B-factors. Multiple sequence alignment revealed that beneficial residues 207-SSGS-210 are highly conserved in GH11 xylanases. Thus, a recombinant xylanase, Xyn-MUT, was constructed by substituting three residues (N207S, G208S, A210S) at the C-terminus of XynCDBFV. Xyn-MUT exhibited higher thermostability than XynCDBFV at ≥70 °C. Xyn-MUT showed promising improvement in residual activity with a thermal retention of 14% compared to that of XynCDBFV after 1 h incubation at 80 °C; Xyn-MUT maintained around 50% of the maximal activity after incubation at 95 °C for 1 h. Kinetic measurements showed that the recombinant Xyn-MUT had greater kinetic efficiency than XynCDBFV (, 0.22 and 0.59 µM, respectively). Catalytic efficiency values () of Xyn-MUT also increased (1.64-fold) compared to that of XynCDBFV. Molecular dynamics simulations were performed to explore the improved catalytic efficiency and thermostability: (1) the substrate-binding cleft of Xyn-MUT prefers to open to a larger extent to allow substrate access to the active site residues, and (2) hydrogen bond pairs S208-N205 and S210-A55 in Xyn-MUT contribute significantly to the improved thermostability. In addition, three xylanases with single point mutations were tested, and temperature assays verified that the substituted residues S208 and S210 give rise to the improved thermostability.
This is the first report for GH11 recombinant with improved thermostability based on C-terminus replacement. The resulting Xyn-MUT will be an attractive candidate for industrial applications.
木聚糖酶已广泛应用于许多工业过程,嗜热木聚糖酶对于满足生物技术处理的高温要求有很大需求。在本研究中,我们旨在通过定点突变提高来自瘤胃真菌的糖苷水解酶(GH)家族11木聚糖酶XynCDBFV的热稳定性。我们通过B因子比较和多序列比对报告了C端的有利突变。
基于标准化B因子的比较,发现XynCDBFV中C端残基207-NGGA-210表现出明显的灵活性。多序列比对显示,有益残基207-SSGS-210在GH11木聚糖酶中高度保守。因此,通过替换XynCDBFV C端的三个残基(N207S、G208S、A210S)构建了重组木聚糖酶Xyn-MUT。在≥70°C时,Xyn-MUT比XynCDBFV表现出更高的热稳定性。在80°C孵育1小时后,Xyn-MUT的残留活性有显著提高,热保留率为14%,相比之下XynCDBFV的热保留率较低;在95°C孵育1小时后,Xyn-MUT保持了约50%的最大活性。动力学测量表明,重组Xyn-MUT比XynCDBFV具有更高的动力学效率(分别为0.22和0.59μM)。与XynCDBFV相比,Xyn-MUT的催化效率值()也有所提高(1.64倍)。进行了分子动力学模拟以探索提高的催化效率和热稳定性:(1)Xyn-MUT的底物结合裂隙更倾向于更大程度地打开,以允许底物进入活性位点残基,(2)Xyn-MUT中的氢键对S208-N205和S210-A55对提高热稳定性有显著贡献。此外,测试了三种单点突变的木聚糖酶,温度测定验证了取代残基S208和S210提高了热稳定性。
这是首次基于C端替换提高GH11重组体热稳定性的报告。所得的Xyn-MUT将是工业应用中有吸引力的候选物。