Unique N-glycosylation of a recombinant exo-inulinase from Kluyveromyces cicerisporus and its effect on enzymatic activity and thermostability
文献类型：期刊论文
| 作者 | Ma,Junyan1,2; Li,Qian1; Tan,Haidong2; Jiang,Hao3; Li,Kuikui2; Zhang,Lihua3; Shi,Quan3; Yin,Heng2 |
| 刊名 | Journal of Biological Engineering
 |
| 出版日期 | 2019-10-29 |
| 卷号 | 13期号:1 |
| ISSN号 | 1754-1611 |
| 关键词 | N-glycosylation Exo-inulinase β-Sandwich domain Enzyme activity Thermostability |
| DOI | 10.1186/s13036-019-0215-y |
| 通讯作者 | Yin,Heng(yinheng@dicp.ac.cn) |
| 英文摘要 | AbstractBackgroundInulinase can hydrolyze polyfructan into high-fructose syrups and fructoligosaccharides, which are widely used in food, the medical industry and the biorefinery of Jerusalem artichoke. In the present study, a recombinant exo-inulinase (rKcINU1), derived from Kluyveromyces cicerisporus CBS4857, was proven as an N-linked glycoprotein, and the removal of N-linked glycan chains led to reduced activity.ResultsFive N-glycosylation sites with variable high mannose-type oligosaccharides (Man3–9GlcNAc2) were confirmed in the rKcINU1. The structural modeling showed that all five glycosylation sites (Asn-362, Asn-370, Asn-399, Asn-467 and Asn-526) were located at the C-terminus β-sandwich domain, which has been proven to be more conducive to the occurrence of glycosylation modification than the N-terminus domain. Single-site N-glycosylation mutants with Asn substituted by Gln were obtained, and the Mut with all five N-glycosylation sites removed was constructed, which resulted in the loss of all enzyme activity. Interestingly, the N362Q led to an 18% increase in the specific activity against inulin, while a significant decrease in thermostability (2.91?°C decrease in Tm) occurred, and other single mutations resulted in the decrease in the specific activity to various extents, among which N467Q demonstrated the lowest enzyme activity.ConclusionThe increased enzyme activity in N362Q, combined with thermostability testing, 3D modeling, kinetics data and secondary structure analysis, implied that the N-linked glycan chains at the Asn-362 position functioned negatively, mainly as a type of steric hindrance toward its adjacent N-glycans to bring rigidity. Meanwhile, the N-glycosylation at the other four sites positively regulated enzyme activity caused by altered substrate affinity by means of fine-tuning the β-sandwich domain configuration. This may have facilitated the capture and transfer of substrates to the enzyme active cavity, in a manner quite similar to that of carbohydrate binding modules (CBMs), i.e. the chains endowed the β-sandwich domain with the functions of CBM. This study discovered a unique C-terminal sequence which is more favorable to glycosylation, thereby casting a novel view for glycoengineering of enzymes from fungi via redesigning the amino acid sequence at the C-terminal domain, so as to optimize the enzymatic properties. |
| 语种 | 英语 |
| 出版者 | BioMed Central |
| WOS记录号 | BMC:10.1186/S13036-019-0215-Y |
| 源URL | [http://cas-ir.dicp.ac.cn/handle/321008/172608]  |
| 专题 | 大连化学物理研究所_中国科学院大连化学物理研究所 |
| 通讯作者 | Yin,Heng |
| 作者单位 | 1. 2. 3. |
| 推荐引用方式 GB/T 7714 | Ma,Junyan,Li,Qian,Tan,Haidong,et al. Unique N-glycosylation of a recombinant exo-inulinase from Kluyveromyces cicerisporus and its effect on enzymatic activity and thermostability[J]. Journal of Biological Engineering,2019,13(1). |
| APA | Ma,Junyan.,Li,Qian.,Tan,Haidong.,Jiang,Hao.,Li,Kuikui.,...&Yin,Heng.(2019).Unique N-glycosylation of a recombinant exo-inulinase from Kluyveromyces cicerisporus and its effect on enzymatic activity and thermostability.Journal of Biological Engineering,13(1). |
| MLA | Ma,Junyan,et al."Unique N-glycosylation of a recombinant exo-inulinase from Kluyveromyces cicerisporus and its effect on enzymatic activity and thermostability".Journal of Biological Engineering 13.1(2019). |
入库方式： OAI收割
来源：大连化学物理研究所
浏览0
下载0
收藏0
其他版本
除非特别说明，本系统中所有内容都受版权保护，并保留所有权利。