N-糖基化对一种新型重组耐高温β-甘露聚糖酶(ReTMan26)稳定性的影响
Effect of N-glycosylation on the stability of a novel recombinant thermostable β-mannanase (ReTMan26)
  
DOI:  10.13344/j.microbiol.china.180192
中文关键词:N-糖基化,重组耐高温β-甘露聚糖酶,纯化,稳定性
英文关键词:N-glycosylation, Recombinant thermostable β-mannanase, Purification, Stability
基金项目:国家轻工技术与工程一流学科课题资助项目(LITE2018-11)
作者单位E-mail
罗长财 1 江南大学工业生物技术教育部重点实验室 江苏 无锡 214122  
缪静 2 鲁东大学生命科学学院 山东 烟台 264025  
李国莹 1 江南大学工业生物技术教育部重点实验室 江苏 无锡 214122  
杜瑶 1 江南大学工业生物技术教育部重点实验室 江苏 无锡 214122  
余晓斌 1 江南大学工业生物技术教育部重点实验室 江苏 无锡 214122 xbyu@jiangnan.edu.cn 
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中文摘要:
      【背景】对来源于嗜热枯草芽孢杆菌(TBS2)的一种新型重组耐高温β-甘露聚糖酶(ReTMan26)基因序列进行分析,该基因中含有3个N-糖基化位点(N8、N26与N255),经毕赤酵母表达时可进行N-糖基化修饰。【目的】确定N-糖基化对ReTMan26稳定性的影响。【方法】通过构建ReTMan26蛋白质三维结构模型,初步分析N-糖基化对该酶稳定性的影响。在此基础上,利用天然蛋白去糖基化试剂盒除去ReTMan26的N-多糖链,获得去除N-糖基化的耐高温β-甘露聚糖酶(ReTMan26-DG),并对纯化后的ReTMan26及ReTMan26-DG进行相应的稳定性对比检测。【结果】 ReTMan26与ReTMan26-DG的最适反应pH均为6.0,但在pH 1.5?9.0范围内,ReTMan26的稳定性比ReTMan26-DG有小幅提高。ReTMan26的最适反应温度为60 °C,比ReTMan26-DG高5 °C;ReTMan26经100 °C处理10 min,剩余酶活为58.6%,而ReTMan26-DG经93 °C处理10 min,剩余酶活为58.2%,100 °C处理10 min则完全失活。经胃蛋白酶及胰蛋白酶在37 °C处理2 h后,ReTMan26的剩余酶活分别为70.5%及91.2%,比ReTMan26-DG分别提高了23.7%及25.6%。【结论】N-糖基化可提高ReTMan26的pH稳定性、耐热稳定性及抗蛋白酶消化性能。
英文摘要:
      [Background] Based on the sequence analysis of a novel recombinant highly thermostable b-mannanase (ReTMan26) from a thermophilic Bacillus subtilis (TBS2), there are 3 N-glycosylation sites (N8, N26 and N255) in the encoding gene of ReTMan26, and ReTMan26 could be N-glycosylated when expressed by Pichia pastoris. [Objective] To determine the effects of N-glycosylation on the stability of ReTMan26. [Methods] Through constructing the three-dimensional structure models, the effects of N-glycosylation on the stability of ReTMan26 were analyzed. Then, the N-deglycosylated ReTMan26 (ReTMan26-DG) was obtained using Native Protein Deglycosylation Kit. After purification, the differences of enzymatic stability between ReTMan26 and ReTMan26-DG were determined. [Results] The optimum reaction pH of ReTMan26 was 6.0, identical with that of ReTMan26-DG, and pH stability of ReTMan26 was slightly higher than that of ReTMan26-DG in pH range between 1.5 and 9.0. The optimum temperature of ReTMan26 was 60 °C, 5 °C higher than that of ReTMan26-DG. ReTMan26 retained 58.6% of its maximum activity after treatment at 100 °C for 10 min. However, ReTMan26-DG retained 58.2% residual activity after treatment at 93 °C and was completely inactivated after treatment at 100 °C for 10 min. After treatment with trypsin or pepsin at 37 °C for 2 h, ReTMan26 retained 91.2% and 70.5% of its baseline activity, 23.7% and 25.6% higher than ReTMan26-DG, respectively. [Conclusion] N-glycosylation could improve the stability of ReTMan26 at different pH, high-temperature and the resistance to digestive proteases.
罗长财,缪静,李国莹,杜瑶,余晓斌.N-糖基化对一种新型重组耐高温β-甘露聚糖酶(ReTMan26)稳定性的影响[J].微生物学通报,2019,46(1):11~19
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