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N-糖基化影响耐热纤维素酶热稳定性的机制研究
发布时间：2019
| 学术论文附件 | ||||
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论文题目 | Improvement of the catalytic activity and thermostability of a hyperthermostable endoglucanase by optimizing N-glycosylation sites; Improving the thermostability of a thermostable endoglucanase Chaetomium thermophilum by engineering the conserved noncatalytic residue and N-glycosylation site | ||
| 作者姓名 | 韩超 | |||
| 是否发表 | 是 | 刊 号 | ||
| 期刊名称 | Biotechnol Biofuels | 期刊期次 | (2020) 13:30 | |
| 项目名称 | N-糖基化影响耐热纤维素酶热稳定性的机制研究 | |||
| 项目类型 | 创新训练项目 | |||
| 项目编号 | 201910434007 | 学科类别 | 农学——植物生产类 | |
| 立项年份 | 2019 | 联系QQ号 | ||
| 项目组成员 | 姓 名 | 年 级 | 专 业 | 是否主持人 |
| 指导老师 | 姓 名 | 职称 | 研究方向 | |
| 韩超 | 讲师 | |||
| 论文摘要 | Abstract Background: Endoglucanase has been extensively employed in industrial processes as a key biocatalyst for ligno- cellulosic biomass degradation. Thermostable endoglucanases with high catalytic activity at elevated temperatures are preferred in industrial use. To improve the activity and thermostability, site-directed mutagenesis was con- ducted to modify the N-glycosylation sites of the thermostable β-1,4-endoglucanase CTendo45 from Chaetomium thermophilum. Results: In this study, structure-based rational design was performed based on the modification of N-glycosylation sites in CTendo45. Eight single mutants and one double mutant were constructed and successfully expressed in Pichia pastoris. When the unique N-glycosylation site of N88 was eliminated, a T90A variant was active, and its specific activity towards CMC-Na and β- d -glucan was increased 1.85- and 1.64-fold, respectively. The mutant R67S with an additional N-glycosylation site of N65 showed a distinct enhancement in catalytic efficiency. Moreover, T90A and R67S were endowed with extraordinary heat endurance after 200 min of incubation at different temperatures ranging from 30 to 90 °C. Likewise, the half-lives (t 1/2 ) indicated that T90A and R67S exhibited improved enzyme thermostability at 80 °C and 90 °C. Notably, the double-mutant T90A/R67S possessed better hydrolysis activity and thermal stability than its single-mutant counterparts and the wild type. Conclusions: This study provides initial insight into the biochemical function of N-glycosylation in thermostable endoglucanases. Moreover, the design approach to the optimization of N-glycosylation sites presents an effective and feasible strategy to improve enzymatic activity and thermostability.
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