N-糖基化对植酸酶phy(Q<sub>F</sub>)酶学性质的影响

doi:10.7668/hbnxb.2015.01.030

华北农学报 ›› 2015, Vol. 30 ›› Issue (1): 177-181. doi: 10.7668/hbnxb.2015.01.030

N-糖基化对植酸酶phy(Q_F)酶学性质的影响

王茜¹, 宋玛丽¹, 杜军¹, 付月君¹, 胡风云², 梁爱华¹

1. 山西大学生物技术研究所, 化学生物学与分子工程教育部重点实验室, 山西太原 030006;
2. 山西省人民医院神经内科, 山西太原 030012

收稿日期:2014-12-02 出版日期:2015-02-28
通讯作者: 梁爱华(1957-),女,山西榆社人,教授,博士,主要从事分子生物学研究。
作者简介:王茜(1989-),女,山西临汾人,在读硕士,主要从事分子生物学研究。
基金资助:
国家自然科学基金项目(31372199;31272100);国家高技术研究发展计划"863"计划项目(2012AA020809);山西省回国留学人员科研资助项目(2012-重点1)

The Effects of N-glycosylation on Enzymological Properties of Phytase Phy(Q_F)

WANG Xi¹, SONG Ma-li¹, DU Jun¹, FU Yue-jun¹, HU Feng-yun², LIANG Ai-hua¹

1. Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan 030006, China;
2. Department of Neurology, Shanxi Provincial People's Hospital, Taiyuan 030012, China

Received:2014-12-02 Published:2015-02-28

摘要/Abstract

摘要： 为探讨N-糖基化对植酸酶phy(Q_F)酶学性质的影响,将植酸酶基因 phy(Q_F) 在毕赤酵母中进行了表达,利用脱糖基化酶Endo H_f去除重组植酸酶phy(Q_F)的糖链,对去除糖链前后的植酸酶phy(Q_F)进行了纯化和酶学性质研究与比较。结果表明,糖基化的植酸酶phy(Q_F)分子量约为53 kDa,酶活为3 201 U/mg,K_m=392 μmol/L,V_max=3 267 U/mg;糖基化的植酸酶phy(Q_F)最适pH值为4.5,最适反应温度为55 ℃,与去糖基化的植酸酶phy(Q_F)的最适pH值无明显区别,比去糖基化的植酸酶phy(Q_F)最适温度提高了5 ℃,T_m值提高约2 ℃。经70 ℃孵育10 min后,去糖基化的植酸酶phy(Q_F)完全失活,而糖基化的植酸酶phy(Q_F)仍残留13%的活性,说明N-糖基化作为一种重要的翻译后修饰,对植酸酶phy(Q_F)的稳定性有促进作用。

关键词: 植酸酶, N-糖基化, 毕赤酵母, 热稳定性, 圆二色谱

Abstract: In order to study the effects of N-glycosylation on enzymological properties of phytase phy(Q_F), the recombinant phytase gene phy(Q_F) was expressed in pichia pastoris GS115.Phytase phy(Q_F) was deglycosylated by endoglycosylase Endo H_f.Enzymology properties of glycosylated phy(Q_F) and deglycosylated phy(Q_F) were analyzed.The results showed that the glycosylated phy(Q_F) had a specific activity of 3 201 U/mg with an MW of approximately 53 kDa and the K_m and V_max of glycosylated phy(Q_F) were 392 μmol/L and 3 267 U/mg, respectively.The optimal pH value of glycosylated phy(Q_F) was 4.5 and was consistented with that of deglycosylated phy(Q_F).However, the optimal temperature of glycosylated phy(Q_F) was 55 ℃, 5 ℃ higher than that of deglycosylated phy(Q_F) and 2 ℃ increased in the melting temperature (T_m).The glycosylated phy(Q_F) exhibited 13% residual activity after treatment at 70 ℃ for 10 min and deglycosylated phy(Q_F)was completely deactivated under the condition.As one of posttranslational modifications, N-glycosylation may play an important role in improving the stability of phytase phy(Q_F).

Key words: Phytase, N-glycosylation, Pichia pastoris, Thermal stability, Circular dichroism

中图分类号:

王茜, 宋玛丽, 杜军, 付月君, 胡风云, 梁爱华. N-糖基化对植酸酶phy(Q_F)酶学性质的影响[J]. 华北农学报, 2015, 30(1): 177-181. doi: 10.7668/hbnxb.2015.01.030.

WANG Xi, SONG Ma-li, DU Jun, FU Yue-jun, HU Feng-yun, LIANG Ai-hua. The Effects of N-glycosylation on Enzymological Properties of Phytase Phy(Q_F)[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2015, 30(1): 177-181. doi: 10.7668/hbnxb.2015.01.030.

http://www.hbnxb.net/CN/Y2015/V30/I1/177

参考文献

[1] Khajal F,Slominsk B A.Factors that affect the nutritive value of canola mealfor poultry [J].Poultry Science 2012,91(10):2564-2575.
[2] Yao M Z,Zhang Y H,Lu W L,et al.Phytases:crystal structures,protein engineerinand potential biotechnological applications [J].J Appl Microbiol,2012,112(1):1-14.
[3] Tahir M,Shim M Y,Ward N E,et al.Phytate and other nutrient components of feed ingredients for poultry [J].2012,91(4):928-935.
[4] 王芳,张红岗,李燕萍.植酸酶在鹌鹑El粮中的应用[J].山西农业科学,2003,31(1):58-60.
[5] 王郡美,宋维平,张莉.宇佐美曲霉植酸酶phyA基因的克隆[J].华北农学报,2006,21(3):5-8.
[6] 柯晓静,郭润芳,于宏伟,等.植酸酶基因 PhyB₁的克隆及序列分析[J].华北农学报,2007,22(2):14-17.
[7] Han Y,Lei X G.Role of glycosylation in the functional expression of an Aspergillus niger phytase (phyA) in Pichia pastoris[J].Arch Biochem Biophys,1999,364(1):83-90.
[8] Kim Y O,Kim H W,Lee J H,et al.Molecular cloning of the phytase gene from Citrobacter braakii and its expression in Saccharomyces cerevisiae[J].Biotechnol Lett,2006,28(1):33-38.
[9] Zou L K,Wang H N,Pan X,et al.Design and expression of a synthetic phyC gene encoding the neutral phytase in Pichia pastoris[J].Acta Biochim Biophys Sin,2006,38(11):803-811.
[10] Rodriguez E,Wood Z A,Karplus P A,et al.Site-directed mutagenesis improves catalytic efficiency and thermostability of Escherichia coli pH 2.5 acid phosphatase/phytase expressed in Pichia pastoris[J].Arch Biochem Biophys,2000,382(1):105-112.
[11] 张耀华,姚明泽,卢文亮,等.大肠杆菌植酸酶在毕赤酵母中的表达与纯化[J].应用与环境生物学报,2012,18(4):678-681.
[12] 王茜,卢文亮,姚明泽,等.芽孢杆菌植酸酶phy(ycD)基因在大肠杆菌中的异源表达与纯化[J].应用与环境生物学报,2014,20(2):295-299.
[13] Pace C N,Hebert E J,Shaw K L,et al.Conformational stability and thermodynamics of folding of ribonucleases Sa,Sa2 and Sa3[J].J Mol Biol,1998,279(1):271-286.
[14] Correia A R,Adinolfi S,Pastore A,et al.Conformational stability of human frataxin and effect of Friedreich's ataxia-related mutations on protein folding[J].Biochem J,2006,398(3):605-611.
[15] Yao M Z,Wang X,Wang W,et al.Improving the thermostability of Escherichia coli phytase,AppA,by enhancement of glycosylation[J].Biotechnol Lett,2013,35(10):1669-1676.
[16] Kwon K S,Yu M H.Effect of glycosylation on the stability of alpha1-antitrypsin toward urea denaturation and thermal deactivation[J].Biochim Biophys Acta,1997,1335(3):265-272.
[17] Suzuki S,Furuhashi M,Additional N-glycosylation at Asn(13) rescues the human LH beta-subunit from disulfide-linked aggregation[J].Mol Cell Endocrinol,2000,160(1-2):157-163.
[18] 赵洪亮,刘志敏.蛋白质糖基化工程[J].中国生物工程杂志,2003,9(23):18-20.
[19] Panchal T,Wodzinski R J.Comparison of glycosylation pattens of phytase from Aspergillus niger (A.ficuum) NRRL 3135 and recombinant phytase[J].Prep Biochem Biotechnol,1998,28(3):201-217.
[20] 潘冬梅,李弘剑,李白桦,等.无花果曲霉植酸酶发酵及酶动力学[J].暨南大学学报,2001,22(5):107-112.
[21] 王玉琪,巫光宏,林先丰,等.多糖和糖蛋白聚丙烯酰胺凝胶电泳染色方法的改进[J].植物生理学通讯,2009,45(2):169-172.

选择文件类型/文献管理软件名称

选择包含的内容

N-糖基化对植酸酶phy(Q_F)酶学性质的影响

The Effects of N-glycosylation on Enzymological Properties of Phytase Phy(Q_F)

PDF

可视化

被引次数

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 11

编辑推荐

Metrics

本文评价

[1]	李琦, 成莉凤, 段盛文, 冯湘沅, 郑科, 杨琦, 刘志远, 彭源德. 一种新型木聚糖酶基因在毕赤酵母中的表达[J]. 华北农学报, 2018, 33(4): 126-130.
[2]	皇甫海燕, 燕孟娇, 宋培玲, 郝丽芬, 皇甫九茹, 贾晓清, 李子钦. *油菜pgip基因去信号肽片段在毕赤酵母中的分泌表达*[J]. 华北农学报, 2015, 30(6): 27-31.
[3]	张莉, 康雪燕, 章振华, 李永清, 盖丽丽, 姜世金, 张培君. 毕赤酵母表达的猪传染性胃肠炎病毒纤突糖蛋白及其免疫原性分析[J]. 华北农学报, 2015, 30(6): 84-90.
[4]	宋玛丽, 王茜, 杜军, 赵峰梅, 梁爱华. *重组植酸酶appA-2QN在毕赤酵母中的高效表达*[J]. 华北农学报, 2015, 30(2): 55-58.
[5]	张雅昆, 赵丹, 郭巍, 刘小民, 徐大庆, 孙伟明. 华北大黑鳃金龟丝氨酸羧肽酶在不同体系中的表达[J]. 华北农学报, 2013, 28(5): 44-47.
[6]	胡骁飞, 魏凤仙, 李青梅, 职爱民, 王方雨, 孙亚宁, 柴书军, 付晓鹤. 植酸酶单克隆抗体的制备及鉴定[J]. 华北农学报, 2012, 27(5): 44-48.
[7]	魏凤仙, 白献晓, 李绍钰, 舒畅, 王琳燚. 低磷加植酸酶日粮不同钙磷比对生长猪生产性能和血清学指标的影响[J]. 华北农学报, 2007, 22(5): 47-50.
[8]	马丽娜, 陈喜文, 陈德富. 糖化酶工业株的鉴定及糖化酶基因的克隆[J]. 华北农学报, 2007, 22(1): 103-107.
[9]	王郡美, 宋维平, 张莉. *宇佐美曲霉植酸酶phy* A基因的克隆**[J]. 华北农学报, 2006, 21(3): 5-8.
[10]	张梅申, 沈爱光, 熊晓辉, 沈昌. 植酸酶产生菌株的液体深层发酵条件研究[J]. 华北农学报, 1999, 14(4): 63-67.
[11]	周人纲, 樊志和, 李晓芝, 王占武, 韩炜. 高温锻炼对小麦细胞膜热稳定性的影响[J]. 华北农学报, 1993, 8(3): 33-37.

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

N-糖基化对植酸酶phy(QF)酶学性质的影响

The Effects of N-glycosylation on Enzymological Properties of Phytase Phy(QF)

PDF

可视化

被引次数

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 11

编辑推荐

Metrics

本文评价

N-糖基化对植酸酶phy(Q_F)酶学性质的影响

The Effects of N-glycosylation on Enzymological Properties of Phytase Phy(Q_F)