Department of Biosciences, Faculty of Agriculture, Yamagata University, Tsuruoka 997-8555, Japan.
Department of Biosciences, Faculty of Agriculture, Yamagata University, Tsuruoka 997-8555, Japan.
Enzyme Microb Technol. 2022 Nov;161:110112. doi: 10.1016/j.enzmictec.2022.110112. Epub 2022 Aug 8.
Purified recombinant rutinosidase from Aspergillus oryzae expressed in Pichia pastoris (rAoRutM) exhibits increase in thermal stability after treatment with endo-β-N-acetylglucosaminidase H (endo-H). In this study, the role of N-glycosylation in the activity and thermal stability of rAoRutM was analyzed via site-directed mutagenesis. Based on the crystal structure of AoRutM, five N-glycosylation sites (N32, N128, N176, N288, and N359) were identified in the AoRut protein. Among five single variants constructed for these sites, the N128D, N176D, and N359D variants exhibited similar mobility bands compared to that of the wild-type enzyme based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas the N32D and N288D variants exhibited slightly and considerably increasing mobility bands, respectively. The N128D and N288D variants showed increasing and decreasing rutinosidase activity, respectively, compared to the case for the wild-type, without and with endo-H treatments. While the N128D and N176D variants had lower K values, the N288D and N359D variants had higher K values, compared to the wild-type, without and with endo-H treatments. Surprisingly, the N32D and N176D variants exhibited considerably greater thermal stability than the wild-type, without or with the endo-H treatments, whereas the N128D and N359D variants exhibited drastically decreased thermal stability. Circular dichroism (CD) spectra of the N128D and N359D variants showed a similar CD profile to that of the wild-type treated with endo-H; however, the molar ellipticity values of the peaks at 208 nm and 212 nm in the above variants varied from those of the intact wild-type and other variants.
米曲霉来源的重组菊糖酶（rAoRutM）经内切-β-N-乙酰氨基葡萄糖苷酶 H（endo-H）处理后热稳定性增加。在本研究中，通过定点突变分析了 N-糖基化对 rAoRutM 活性和热稳定性的作用。基于 AoRutM 的晶体结构，鉴定了 AoRut 蛋白中的 5 个 N-糖基化位点（N32、N128、N176、N288 和 N359）。在构建的这 5 个单点突变体中，与野生型酶相比，N128D、N176D 和 N359D 突变体的 SDS-PAGE 迁移率带相似，而 N32D 和 N288D 突变体的迁移率带分别略有增加和显著增加。与野生型相比，N128D 和 N288D 突变体的菊糖酶活性分别增加和降低，而未经和经 endo-H 处理时均如此。与野生型相比，N128D 和 N176D 突变体的 K 值较低，而 N288D 和 N359D 突变体的 K 值较高，未经和经 endo-H 处理时均如此。令人惊讶的是，未经和经 endo-H 处理时，N32D 和 N176D 突变体的热稳定性明显高于野生型，而 N128D 和 N359D 突变体的热稳定性则急剧降低。N128D 和 N359D 突变体的圆二色性（CD）光谱与经 endo-H 处理的野生型具有相似的 CD 谱；然而，上述突变体中 208nm 和 212nm 峰的摩尔椭圆率值与完整的野生型和其他变体有所不同。
