[1]李 飞,解静聪,李 琦,等.木聚糖酶XynB分子Ser/Thr 平面导入精氨酸对酶热稳定性的影响[J].南京林业大学学报(自然科学版),2014,38(04):107-112.[doi:10.3969/j.issn.1000-2006.2014.04.020]
 LI Fei,XIE Jingcong,LI Qi,et al.Thermostability of xylanase(XynB)by introducing Arg into its Ser/Thr surface[J].Journal of Nanjing Forestry University(Natural Science Edition),2014,38(04):107-112.[doi:10.3969/j.issn.1000-2006.2014.04.020]
点击复制

木聚糖酶XynB分子Ser/Thr 平面导入精氨酸对酶热稳定性的影响
分享到:

《南京林业大学学报(自然科学版)》[ISSN:1000-2006/CN:32-1161/S]

卷:
38
期数:
2014年04期
页码:
107-112
栏目:
研究论文
出版日期:
2014-07-01

文章信息/Info

Title:
Thermostability of xylanase(XynB)by introducing Arg into its Ser/Thr surface
文章编号:
1000-2006(2014)04-0107-06
作者:
李 飞12解静聪1李 琦1张雪松4赵林果1 3*
1.南京林业大学化学工程学院, 江苏 南京 210037;
2.南京师范大学泰州学院生物技术与化学工程学院, 江苏 泰州 225300;
3.江苏省生物质绿色燃料与化学品重点实验室,江苏 南京 210037;
4.江苏农林职业技术学院生物工程系,江苏 镇江 212400
Author(s):
LI Fei12 XIE Jingcong1LI Qi1ZHANG Xuesong4ZHAO Linguo13*
1.College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China;
2.College of Biochemical Engineering, Taizhou College, Nanjing Normal University, Taizhou 225300, China;
3.Jiangsu Key Lab of Biomass Based Green Fuels and Chemicals, Nanjing 210037, China;
4.Department of Biological Engineering, Jiangsu Polytechnic College of Agriculture and Forestry, Zhenjiang 212400, China
关键词:
定点突变 木聚糖酶 精氨酸 热稳定性
Keywords:
site-directed mutagenesis xylanase arginine thermostability
分类号:
TQ936
DOI:
10.3969/j.issn.1000-2006.2014.04.020
文献标志码:
A
摘要:
为提高木聚糖酶的热稳定性,通过定点突变的方法,在来源于黑曲霉Aspergillus niger nl-1的木聚糖酶XynB分子Ser/Thr 平面上引入5个精氨酸,实现了突变酶在毕赤酵母中的表达。通过酶学性质的研究表明,突变酶XynB-104和XynB-77的最适反应温度为50 ℃,且与原酶相比相对酶活力得到显著提高。在存在底物的情况下,50 ℃热处理2 h,突变酶XynB-104和XynB-77残余酶活力较原酶的相对转化效率由26%提高到80%左右。突变酶XynB-104的最适反应pH与原酶一致,而突变酶XynB-77最适pH由5.0提高到5.5。结果表明,在木聚糖酶分子Ser/Thr 平面不同折叠片引入精氨酸可以增强酶结构的稳定性,特别是能显著提高热稳定性。
Abstract:
Replacing several serine and threonine residues in the Ser/Thr surface of xylanase(XynB)with arginines effectively increased the thermostability of the enzyme by site-directed mutagenesis. The mutated enzymes XynB-104 and XynB-77 were expressed in Pichia pastoris and their enzymatic properties were determined. The optimal temperature of the mutations XynB-104 and XynB-77 were 50 ℃, and the relative activity of mutations were dramatically increased. Both mutated xylanases showed about 80% of maximal activity after incubating in xylan substrate for 2 h at 50 ℃ compared to only 26% activity of the native enzyme. The optimal pH of XynB-104 enzyme had no change, but the pH optimum of the XynB-77 enzyme was increased from 5.0 to 5.5. The results of the mutated enzymes indicated that properly arginines residues introduced into Ser/Thr surface of xylanase XynB might be effective to improve the enzyme thermostability and hydrolysis capacity.

参考文献/References:

[1] Liu M Q,Liu G F. Expression of recombinant Bacillus licheniformis xylanase A in Pichia pastoris and xylooligosaccharides released from xylans it[J]. Protein Expression and Purification, 2008, 57(2): 101-107.
[2] Qiu Z H, Shi P J, Luo H Y, et al. A xylanase with broad pH and temperature adaptability from Streptomyces megasporus DSM 41476, and its potential application in brewing industry[J]. Enzyme and Microbial Technology, 2010, 46(6): 506-512.
[3] Khandeparker R,Numan M T. Bifunctional xylanases and their potential use in biotechnology[J]. Journal of Industrial Microbiology & Biotechnology, 2008, 35(7): 635-644.
[4] Fred F, Matti L, Janne J, et al. A de novo designed N-terminal disulphide bridge stabilizes the Trichoderma reesei endo-1,4-xylanase II[J]. Journal of Biotechnology, 2004, 108(2): 137-143.
[5] 杨浩萌, 柏映国, 李江, 等. 木聚糖酶XYNB的N46D突变、表达及酶学性质变化[J]. 中国生物化学与分子生物学报, 2006, 22(3):204-211.Yang H M, Bai Y G, Li J, et al. N46D mutagenesis of xylanase XYNB and enzymatic changes of expression product[J]. Chinese Journal of Biochemistry and Molecular Biology, 2006, 22(3): 204-211.
[6] Tao X, Qin W. Directed evolution of Streptomyces lividans xylanase B toward enhanced thermal and alkaline pH stability[J]. World Journal of Microbiology and Biotechnology, 2009, 25(1): 93-100.
[7] Turunen O, Vuorio M, Fenel F, et al. Engineering of multiple arginines into the Ser/Thr surface of Trichoderma reesei Endo-1,4-beta-xylanase II increases the thermotolerance and shifts the pH optimum towards alkaline pH[J]. Protein Engineering, 2002, 15(2): 141-145.
[8] Chen X Z, Xu S Q, Zhu M S, et al. Site-directed mutagenesis of an Aspergillus niger xylanase B and its expression, purification and enzymatic characterization in Pichia pastoris[J]. Process Biochemistry, 2010, 45(1): 75-80.
[9] 周晨妍. 宇佐美曲霉木聚糖酶基因的克隆、表达及定向进化研究[D]. 无锡: 江南大学, 2008.Zhou C Y. Cloning, expression and directed mutagenesis of xylanase gene from Aspergillus usamii[D]. Wuxi: Jiangnan University, 2008.
[10] Li F, Yang S Y, Zhao L G, et al. Synonymous condon usage bias and overexpression of a synthetic xynB gene from Aspergillus niger NL-1 in Pichia pastoris[J]. BioResource, 2012, 7(2): 2330-2343.
[11] Li F, Zhao L G., Li G Q, et al. Cloning, expression and characterization of xylanase gene XYNB from Aspergillus niger in Pichia pastoris[C]// Sun Runcang,Fu Shiyu. 4th ISETPP. Guangzhou: South China University of Technology Press,2010:637-640.
[12] Miller G L. Use of dinitrosalicylic acid reagent for determination of reducing sugar[J]. Analytical Chemistry, 1959, 31(3): 426-428.
[13] Sriprang R, Asano K, Gobsuk J, et al. Improvement of thermostability of fungal xylanase by using site-directed mutagenesis[J]. Journal of Biotechnology, 2006, 126(4): 454-462.
[14] Amalia S, Johan W, Christophe L, et al. The endoxylanases from family 11: computer analysis of protein sequences reveals important structural and phylogenetic relationships[J]. Journal of Biotechnology, 2002, 95(2): 109-131.
[15] 杨浩萌, 姚斌, 罗会颖, 等. 木聚糖酶XYNB分子中折叠股B1和B2间的疏水作用对酶热稳定性的影响[J].生物工程学报, 2005, 21(3): 414-419.Yang H M, Yao B, Luo H Y, et al. Hydrophobic interaction between β-sheet B1 and B2 in xylanase XYNB influencing the enzyme thermostability[J]. Chinese Journal of Biotechnology, 2005, 21(3): 414-419.
[16] Kim S H, Pokhrel S, Yoo Y J. Mutation of non-conserved amino acids surrounding catalytic site to shift pH optimum of Bacillus circulans xylanase[J]. Journal Molecular Catalysis B: Enzymatic, 2008, 55(3): 130-136.

相似文献/References:

[1]董博,连之娜,勇强,等.碳源对里氏木霉β-聚糖酶合成的影响[J].南京林业大学学报(自然科学版),2005,29(06):088.[doi:10.3969/j.jssn.1000-2006.2005.06.022]
 DONG Bo,LIAN Zhi-na,YONG Qiang,et al.Effects of Carbon Sources on the Biosynthesis of β-glycanases by Trichoderma reesei[J].Journal of Nanjing Forestry University(Natural Science Edition),2005,29(04):088.[doi:10.3969/j.jssn.1000-2006.2005.06.022]
[2]侯光北,冯年捷,张 琳,等.麦草碱性亚硫酸盐制浆废液的木聚糖酶解特性[J].南京林业大学学报(自然科学版),2014,38(04):113.[doi:10.3969/j.issn.1000-2006.2014.04.021]
 HOU Guangbei,FENG Nianjie,ZHANG Lin,et al.Enzymatic hydrolysis characteristics of xylan in spent liquor of wheat straw alkaline sulfite[J].Journal of Nanjing Forestry University(Natural Science Edition),2014,38(04):113.[doi:10.3969/j.issn.1000-2006.2014.04.021]

备注/Memo

备注/Memo:
收稿日期:2013-01-10 修回日期:2013-10-21
基金项目:江苏省高校自然科学研究重大项目(13KJA220004); 江苏省普通高校研究生科研创新计划资助项目(CXLX12_0529); 江苏高校优势学科建设工程资助项目(PAPD)
第一作者:李飞,讲师。*通信作者:赵林果,教授。E-mail: lg.zhao@163.com。
引文格式:李飞,解静聪,李琦,等. 木聚糖酶XynB分子Ser/Thr 平面导入精氨酸对酶热稳定性的影响[J]. 南京林业大学学报:自然科学版,2014,38(4):107-112.
更新日期/Last Update: 2014-07-31