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
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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pH1.5-9.0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10min则完全失活。经胃蛋白酶及胰蛋白酶在37°C处理2h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。
[Background] Based on the sequence analysis of a novel recombinant highly thermostable ?-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.
[Background] Based on the sequence analysis of a novel recombinant highly thermostable ?-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.
引文
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[15]Yoon KH,Chung S,Lim BL.Characterization of the Bacillus subtilis WL-3 mannanase from a recombinant Escherichia coli[J].The Journal of Microbiology,2008,46(3):344-349
[16]Puchart V,Vr?anskáM,Svoboda P,et al.Purification and characterization of two forms of endo-β-1,4-mannanase from a thermotolerant fungus,Aspergillus fumigatus IMI 385708(formerly Thermomyces lanuginosus IMI 158749)[J].Biochimica et Biophysica Acta(BBA)-General Subjects,2004,1674(3):239-250
[17]Zou SP,Huang S,Kaleem I,et al.N-glycosylation enhances functional and structural stability of recombinantβ-glucuronidase expressed in Pichia pastoris[J].Journal of Biotechnology,2013,164(1):75-81
[18]Han MH,Wang XF,Ding HY,et al.The role of N-glycosylation sites in the activity,stability,and expression of the recombinant elastase expressed by Pichia pastoris[J].Enzyme and Microbial Technology,2014,54:32-37
[19]Kwon KS,Yu MH.Effect of glycosylation on the stability ofα1-antitrypsin toward urea denaturation and thermal deactivation[J].Biochimica et Biophysica Acta(BBA)-General Subjects,1997,1335(3):265-272
[2]Choct M,Annison G.The inhibition of nutrient digestion by wheat pentosans[J].British Journal of Nutrition,1992,67(1):123-132
[3]Turner P,Mamo G,Karlsson EN.Potential and utilization of thermophiles and thermostable enzymes in biorefining[J].Microbial Cell Factories,2007,6:9
[4]Viikari L,Alapuranen M,Puranen T,et al.Thermostable enzymes in lignocellulose hydrolysis[A]//Advances in Biochemical Engineering/Biotechnology[M].Vol.108.Berlin,Heidelberg:Springer,2007:121-145
[5]Luo ZC,Miao J,Li GY,et al.Screening and identification of a strain producing a highly thermostable?-mannanase[J].Letters in Biotechnology,2018,29(2):233-237(in Chinese)罗长财,缪静,李国莹,等.一株产耐高温β-甘露聚糖酶菌株的筛选鉴定[J].生物技术通讯,2018,29(2):233-237
[6]?elik E,?al?k P.Production of recombinant proteins by yeast cells[J].Biotechnology Advances,2012,30(5):1108-1118
[7]Macauley-Patrick S,Fazenda ML,McNeil B,et al.Heterologous protein production using the Pichia pastoris expression system[J].Yeast,2005,22(4):249-270
[8]Luo ZC,Miao J,Li GY,et al.A recombinant highly thermostableβ-mannanase(ReTMan26)from thermophilic Bacillus subtilis(TBS2)expressed in Pichia pastoris and its pHand temperature stability[J].Applied Biochemistry and Biotechnology,2017,182(4):1259-1275
[9]Gellissen G.Heterologous protein production in methylotrophic yeasts[J].Applied Microbiology and Biotechnology,2000,54(6):741-750
[10]Damasceno LM,Huang CJ,Batt CA.Protein secretion in Pichia pastoris and advances in protein production[J].Applied Microbiology and Biotechnology,2012,93(1):31-39
[11]Skropeta D.The effect of individual N-glycans on enzyme activity[J].Bioorganic&Medicinal Chemistry,2009,17(7):2645-2653
[12]Zhao YJ,Xue YF,Ma YH.Recent advances and prospect on structural biology ofβ-mannanase-a review[J].Acta Microbiologica Sinica,2009,49(9):1131-1137(in Chinese)赵月菊,薛燕芬,马延和.β-甘露聚糖酶的结构生物学研究现状和展望[J].微生物学报,2009,49(9):1131-1137
[13]Hilge M,Gloor SM,Rypniewski W,et al.High-resolution native and complex structures of thermostableβ-mannanase from Thermomonospora fusca-substrate specificity in glycosyl hydrolase family 5[J].Structure,1998,6(11):1433-1444
[14]Lv JN,Chen YQ,Pei HL,et al.Cloning,expression,and characterization ofβ-mannanase from Bacillus subtilis MAFIC-S11 in Pichia pastoris[J].Applied Biochemistry and Biotechnology,2013,169(8):2326-2340
[15]Yoon KH,Chung S,Lim BL.Characterization of the Bacillus subtilis WL-3 mannanase from a recombinant Escherichia coli[J].The Journal of Microbiology,2008,46(3):344-349
[16]Puchart V,Vr?anskáM,Svoboda P,et al.Purification and characterization of two forms of endo-β-1,4-mannanase from a thermotolerant fungus,Aspergillus fumigatus IMI 385708(formerly Thermomyces lanuginosus IMI 158749)[J].Biochimica et Biophysica Acta(BBA)-General Subjects,2004,1674(3):239-250
[17]Zou SP,Huang S,Kaleem I,et al.N-glycosylation enhances functional and structural stability of recombinantβ-glucuronidase expressed in Pichia pastoris[J].Journal of Biotechnology,2013,164(1):75-81
[18]Han MH,Wang XF,Ding HY,et al.The role of N-glycosylation sites in the activity,stability,and expression of the recombinant elastase expressed by Pichia pastoris[J].Enzyme and Microbial Technology,2014,54:32-37
[19]Kwon KS,Yu MH.Effect of glycosylation on the stability ofα1-antitrypsin toward urea denaturation and thermal deactivation[J].Biochimica et Biophysica Acta(BBA)-General Subjects,1997,1335(3):265-272