Kellock Miriam, Rahikainen Jenni, Borisova Anna S, Voutilainen Sanni, Koivula Anu, Kruus Kristiina, Marjamaa Kaisa
VTT Technical Research Centre of Finland Ltd, P.O. Box 1000, 02044 VTT, Espoo, Finland.
Aalto University, P.O. Box 16100, 00076 Aalto, Espoo, Finland.
Biotechnol Biofuels Bioprod. 2022 May 14;15(1):49. doi: 10.1186/s13068-022-02148-4.
Enzymatic hydrolysis of lignocellulosic biomass into platform sugars can be enhanced by the addition of accessory enzymes, such as xylanases. Lignin from steam pretreated biomasses is known to inhibit enzymes by non-productively binding enzymes and limiting access to cellulose. The effect of enzymatically isolated lignin on the hydrolysis of xylan by four glycoside hydrolase (GH) family 11 xylanases was studied. Two xylanases from the mesophilic Trichoderma reesei, TrXyn1, TrXyn2, and two forms of a thermostable metagenomic xylanase Xyl40 were compared.
Lignin isolated from steam pretreated spruce decreased the hydrolysis yields of xylan for all the xylanases at 40 and 50 °C. At elevated hydrolysis temperature of 50 °C, the least thermostable xylanase TrXyn1 was most inhibited by lignin and the most thermostable xylanase, the catalytic domain (CD) of Xyl40, was least inhibited by lignin. Enzyme activity and binding to lignin were studied after incubation of the xylanases with lignin for up to 24 h at 40 °C. All the studied xylanases bound to lignin, but the thermostable xylanases retained 22-39% of activity on the lignin surface for 24 h, whereas the mesophilic T. reesei xylanases become inactive. Removing of N-glycans from the catalytic domain of Xyl40 increased lignin inhibition in hydrolysis of xylan when compared to the glycosylated form. By comparing the 3D structures of these xylanases, features contributing to the increased thermal stability of Xyl40 were identified.
High thermal stability of xylanases Xyl40 and Xyl40-CD enabled the enzymes to remain partially active on the lignin surface. N-glycosylation of the catalytic domain of Xyl40 increased the lignin tolerance of the enzyme. Thermostability of Xyl40 was most likely contributed by a disulphide bond and salt bridge in the N-terminal and α-helix regions.
通过添加辅助酶（如木聚糖酶），可提高木质纤维素生物质酶解生成平台糖的效率。已知蒸汽预处理生物质中的木质素会通过非生产性结合酶并限制其与纤维素的接触来抑制酶的活性。研究了酶法分离的木质素对四种糖苷水解酶（GH）家族11木聚糖酶水解木聚糖的影响。比较了来自嗜温里氏木霉的两种木聚糖酶TrXyn1、TrXyn2，以及两种形式的热稳定宏基因组木聚糖酶Xyl40。
从蒸汽预处理云杉中分离得到的木质素在40℃和50℃时降低了所有木聚糖酶对木聚糖的水解产率。在50℃的较高水解温度下，热稳定性最差的木聚糖酶TrXyn1受木质素的抑制作用最强，而热稳定性最好的木聚糖酶Xyl40的催化结构域（CD）受木质素的抑制作用最弱。在40℃下将木聚糖酶与木质素孵育长达24小时后，研究了酶活性和与木质素的结合情况。所有研究的木聚糖酶都与木质素结合，但热稳定木聚糖酶在木质素表面24小时内保留了22%-39%的活性，而嗜温里氏木霉木聚糖酶则失活。与糖基化形式相比，去除Xyl40催化结构域的N-聚糖会增加木质素对木聚糖水解的抑制作用。通过比较这些木聚糖酶的三维结构，确定了有助于提高Xyl40热稳定性的特征。
木聚糖酶Xyl40和Xyl40-CD的高热稳定性使酶能够在木质素表面保持部分活性。Xyl40催化结构域的N-糖基化增加了酶对木质素的耐受性。Xyl40的热稳定性很可能是由N端和α-螺旋区域的二硫键和盐桥所致。
