The Important Roles Played in Substrate Binding of Aromatic Amino Acids in Exo-Inulinase From Kluyveromyces cicerisporus CBS 4857.
The Important Roles Played in Substrate Binding of Aromatic Amino Acids in Exo-Inulinase From Kluyveromyces cicerisporus CBS 4857.
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克鲁维酵母 CicerisporusCBS 4857 外切菊粉酶中芳香族氨基酸底物结合的重要作用
DOI：
10.3389/fmolb.2020.569797
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发表时间：
2020
影响因子：
5
通讯作者：
Yin H
中科院分区：
文献类型：
--
作者：
Ma J;Li T;Tan H;Liu W;Yin H
菊粉酶是糖苷水解酶家族32 （GH32）的一员。它催化菊粉中2,1-β- d -果糖键的随机水解，并在高果糖糖浆的生产中发挥作用。本研究通过硅分析和定点诱变的方法，详细评价了克鲁维酵母（Kluyveromyces cicerisporus） CBS4857 （KcINU1）的外链菊粉酶的保守残基W79、F113、M117、R181、C239和W334在底物结合和稳定中的作用。这些残基属于GH32的保守的WG， FSGSMV， RDP， ECP和WQY区域，位于KcINU1的催化口袋周围。酶谱分析显示，与野生型相比，F113W对菊粉和蔗糖的酶谱带相对较弱，而M117A对菊粉和蔗糖的酶谱带相似，而其他所有变体在天然聚丙烯酰胺凝胶电泳上均未见明显的染色。用二硝基水杨酸比色法进一步证实了上述结果。结果表明，F113W对菊粉和蔗糖的残留活性分别为33.8±3.3%和96.2±5.5%，M117A对菊粉和蔗糖的残留活性分别为103.8±1.3%和166.5±12%。荧光光谱结果表明，与野生型酶相比，F113W发生了明显的构象变化，而M117A虽然淬灭作用增强，但影响有限。
Inulinase is a member of the glycoside hydrolase family 32 (GH32). It catalyzes the randomly hydrolyzation of 2,1-β-D-fructosidic linkages in inulin and plays a role in the production of high-fructose syrup. In this study, detailed roles of the conserved residues W79, F113, M117, R181, C239, and W334 of the exo-inulinase from Kluyveromyces cicerisporus CBS4857 (KcINU1) in substrate binding and stabilization were evaluated by in silico analysis and site-directed mutagenesis. These residues belong to the conserved WG, FSGSMV, RDP, ECP, and WQY regions of the GH32 and are located around the catalytic pocket of KcINU1. Zymogram assay showed relatively weaker band for F113W and similar band for M117A compared to the wild-type enzyme toward inulin and sucrose, whereas all other variants showed no observable stain on the native polyacrylamide gel electrophoresis. These results were further confirmed with the dinitrosalicylic acid colorimetric method. It showed that the residual activities of F113W toward inulin and sucrose were 33.8 ± 3.3% and 96.2 ± 5.5%, respectively, and that of M117A were 103.8 ± 1.3% and 166.5 ± 12%, respectively. Results from fluorescence spectra indicated that there is a significant conformational change that happened in F113W compared to the wild-type enzyme, while M117A exhibited limited impact although the quenching effect was increased.
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影响因子：
4.8
作者：
Alvaro-Benito, Miguel;Polo, Aitana;Sanz-Aparicio, Julia
通讯作者：
Sanz-Aparicio, Julia
影响因子：
5.4
作者：
Bujacz, Anna;Jedrzejczak-Krzepkowska, Marzena;Bujacz, Grzegorz
通讯作者：
Bujacz, Grzegorz
影响因子：
3.5
作者：
Wang, RX;Lai, LH;Wang, SM
通讯作者：
Wang, SM
影响因子：
7.4
作者：
Le Roy, Katrien;Lammens, Willem;Van den Ende, Wim
通讯作者：
Van den Ende, Wim
影响因子：
1.8
作者：
Goosen, C.;Van der Maarel, M. J. E. C.;Dijkhuizen, L.
通讯作者：
Dijkhuizen, L.