Please wait a minute...
 
 
食品与发酵工业  2021, Vol. 47 Issue (5): 7-11    DOI: 10.13995/j.cnki.11-1802/ts.025690
  研究报告 本期目录 | 过刊浏览 | 高级检索 |
普鲁兰酶N467G突变体的酶学性质分析
张亚楠1, 申培立2, 牛丹丹1, 田康明1, KUGEN PERMAUL3, SUREN SINGH3, 王正祥1*
1(天津科技大学 化工与材料学院, 天津, 300457)
2(天津科技大学 生物工程学院, 天津, 300457)
3(Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, Durban, 4001, South Africa)
Enzymatic characterization of the pullulanase mutant N467G
ZHANG Yanan1, SHEN Peili2, NIU Dandan1, TIAN Kangming1, KUGEN PERMAUL3, SUREN SINGH3, WANG Zhengxiang1*
1(College of Chemical Engineering and Materials Science, Tianjin University of Science and Technology, Tianjin 300457, China)
2(College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China)
3(Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, Durban, 4001, South Africa
下载:  HTML   PDF (2114KB) 
输出:  BibTeX | EndNote (RIS)      
摘要 在淀粉制糖工业中, 普鲁兰酶通常与糖化酶配合使用, 其在酸性pH和较高温度下的催化活力是影响淀粉脱支效率的主要因素。该研究通过对长野芽胞杆菌(Bacillus naganoensis)普鲁兰酶蛋白质解折叠自由能的差值(ΔΔG)的计算预测和突变位点稳定性分析, 选择突变位点并进行定点突变, 获得突变体N467G。与野生型普鲁兰酶分子相比, 突变体N467G的最适作用温度提高至60 ℃, 最适作用pH降低至4.5;其在60 ℃条件下孵育2.5 h或在pH 3.5~5.5孵育1 h残留酶活力均保持在80%以上, 稳定性明显提高。此突变体的获得为其后续高效表达与工业化应用奠定了重要基础。
服务
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章
张亚楠
申培立
牛丹丹
田康明
KUGEN PERMAUL
SUREN SINGH
王正祥
关键词:  普鲁兰酶  定向进化  最适作用pH  最适作用温度    
Abstract: Pullulanase is extensively used in preparation of glucose syrup from starch, combined with glucoamylase.However, its catalytic activity at low pH and high temperature is the main factor affecting its efficiency during starch debranching.In this study, the difference of the protein unfolding free energy (ΔΔG) of Bacillus naganoensis pullulanase (PulA) mutants was calculated and the stability of each mutation site was analyzed.A mutant, N467G, was obtained through site-directed mutagenesis of the Asn467 residue of PulA and its biochemical properties were examined.Compared to the wild-type PulA, the optimal temperature of mutant N467G was increased by 5 ℃ to 60 ℃ and the optimal pH was reduced by 0.5 pH value to 4.5.Its relative activity was over 80% after incubating the enzyme at 60 ℃ for 2.5 h or at pH 4.0-5.0 for 1 h, which represented a significantly higher stability.The result lays a foundation for the subsequent high-efficiency protein production and industrial application of this mutant.
Key words:  pullulanase    directed evolution    pH optima    temperature optima
收稿日期:  2020-09-17      修回日期:  2020-11-30                发布日期:  2021-03-31      期的出版日期:  2021-03-15
基金资助: 国家重点研发计划政府间国际科技创新合作重点专项项目(2018YFE0100400);天津市高等学校创新团队建设规划项目(TD13-5009)
作者简介:  硕士研究生(王正祥教授为通讯作者, E-mail: zxwang0519@tust.edu.cn)
引用本文:    
张亚楠,申培立,牛丹丹,等. 普鲁兰酶N467G突变体的酶学性质分析[J]. 食品与发酵工业, 2021, 47(5): 7-11.
ZHANG Yanan,SHEN Peili,NIU Dandan,et al. Enzymatic characterization of the pullulanase mutant N467G[J]. Food and Fermentation Industries, 2021, 47(5): 7-11.
链接本文:  
http://sf1970.cnif.cn/CN/10.13995/j.cnki.11-1802/ts.025690  或          http://sf1970.cnif.cn/CN/Y2021/V47/I5/7
[1] SHI J L, SWEEDMAN M C, SHI Y C.Structural changes and digestibility of waxy maize starch debranched by different levels of pullulanase[J].Carbohydrate Polymers, 2018, 194:350-356.
[2] VAN DER MAAREL M J, VAN DER VEEN B, UITDEHAAG J C, et al.Properties and applications of starch-converting enzymes of the α-amylase family[J].Journal of Biotechnology, 2002, 94(2):137-155.
[3] ROY A, MESSAOUD E B, BEJAR S.Isolation and purification of an acidic pullulanase type II from newly isolated Bacillus sp.US149[J].Enzyme and Microbial Technology, 2003, 33(5):720-724.
[4] ARA K, SAEKI K, IGARASHI K, et al.Purification and characterization of an alkaline amylopullulanase with both α-1, 4 and α-1, 6 hydrolytic activity from alkalophilic Bacillus sp.KSM-1378[J].Biochimica et Biophysica Acta, 1995, 1243(3):315-324.
[5] NIEHAUS F, PETERS A, GROUDIEVA T, et al.Cloning, expression and biochemical characterisation of a unique thermostable pullulan-hydrolysing enzyme from the hyperthermophilic archaeon Thermococcus aggregans[J].FEMS Microbiology Letters, 2000, 190(2):223-229.
[6] WANG X Y, NIE Y, XU Y.Industrially produced pullulanases with thermostability:Discovery, engineering, and heterologous expression[J].Bioresource Technology, 2019, 278:360-371.
[7] WANG Y P, LIU Y H, WANG Z X, et al.Infiuence of promoter and signal peptide on the expression of pullulanase in Bacillus subtilis[J].Biotechnology Letters, 2014, 36:1 783-1 789.
[8] DUFFNER F, BERTOLDO C, ANDERSEN J T, et al.A new thermoactive pullulanase from Desulfurococcus mucosus:Cloning, sequencing, purification, and characterization of the recombinant enzyme after expression in Bacillus subtilis[J].Journal of Bacteriology, 2000, 182(22):6 331-6 338.
[9] 王正祥, 刘吉泉, 诸葛健.微生物酶的分子改性和人工进化的研究进展[J].生物工程学报, 2000, 16(3):301-303.
WANG Z X, LIU J Q, ZHUGE J.Molecular modification and artificial evolution of microbial enzymes[J].Chinese Journal of Biotechnology, 2000, 16(3):301-303.
[10] CHAKRAVARTY S, VARADARAJAN R.Elucidation of determinants of protein stability through genome sequence analysis[J].FEBS Letters, 2000, 470 (1):65-69.
[11] DUAN X G, CHEN J, WU J.Improving the thermostability and catalytic efficiency of Bacillus deramificans pullulanase by site-directed mutagenesis[J].Applied and Environmental Microbiology, 2013, 79(13):4 072-4 077.
[12] YAMASHITA M.Random mutagenesis of pullulanase from Klebsiella aerogenes for studies of the structure and function of the enzyme[J].Journal of Biochemistry, 1994, 116(6):1 233-1 240.
[13] 王兵波, 沈微, 钱灵紫, 等.一种密码子优化的酸性普鲁兰酶基因在巴斯德毕赤酵母中的高效表达[J].食品与发酵工业, 2016, 42(7):9-15.
WANG B B, SHEN W, QIAN L Z, et al.High expression of a codon-optimized acid-resistant pullulanase-enconding gene in Pichia pastoris[J].Food and Fermentation Industries, 2016, 42(7):9-15.
[14] PANG B, ZHOU L, CUI W J, et al.Improvement of the thermostability and activity of pullulanase from Anoxybacillus sp.WB42[J].Applied Biochemistry and Biotechnology, 2020, 191 (3):942-954.
[15] 王正祥.微生物遗传育种[M].北京:高等教育出版社, 2020, 136-139.
WANG Z X.Genetic breeding of microorganisms[M].Beijing:Higher Education Press, 2020, 136-139.
[16] NIU D D, ZUO Z R, SHI G Y, et al.High yield recombinant thermostable α-amylase production using an improved Bacillus licheniformis system[J].Microbial Cell Factories, 2009, 8(1):58.
[17] NIU D D, WANG Z X.Development of a pair of bifunctional expression vectors for Escherichia coli and Bacillus licheniformis[J].Journal of Industrial Microbiology and Biotechnology, 2007, 34(5):357-362.
[18] 谢银珠, 沈微, 王正祥.酸性普鲁兰酶基因在地衣芽胞杆菌中的表达[J].食品与发酵工业, 2011, 37(2):7-10.
XIE Y Z, SHEN W, WANG Z X.Expression of the acid pullulanase in Bacillus licheniformis[J].Food and Fermentation Industries, 2011, 37(2):7-10.
[19] SAQIB A A N, SIDDIQUI K S.How to calculate thermostability of enzymes using a simple approach[J].Biochemistry and Molecular Biology Education, 2018, 46(4):398-402.
[20] WATERHOUSE A, BERTONI M, BIENERT S, et al.SWISS-MODEL:Homology modelling of protein structures and complexes[J].Nucleic Acids Research, 2018, 46(1):296-303.
[21] 诸葛健, 王正祥.工业微生物实验技术手册[M].北京:中国轻工业出版社, 1994.
ZHUGE J, WANG Z X.Handbook of industrial microbiology experiment[M].Beijing:China Light Industry Press, 1994.
[22] BRADFORD M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry, 1976, 72:248-254.
[23] 中华人民共和国国家质量监督检验检疫局, 中国国家标准化管理委员会.GB 1886.174—2016 食品安全国家标准 食品添加剂 食品工业用酶制剂[S].北京:中国标准出版社, 2016.
State Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China, China National Standardization Administration Committee.GB 1886.174—2016 National standard of food safety food additives enzyme preparations for food industries[S].Beijing:China Standards Press, 2016.
[24] SHAH P S, HOM G K, ROSS S A, et al.Full-sequence computational design and solution structure of a thermostable protein variant[J].Journal of Molecular Biology, 2007, 372(1):1-6.
[1] 王婕, 朱新文, 德青美朵, 姚雁, 沈微, 陈献忠, 樊游. 一种中性普鲁兰酶的高效表达及重组酶在粉丝制作中的应用[J]. 食品与发酵工业, 2019, 45(14): 122-127.
[2] 肖亚朋,沈微,李婷霖,陈献忠,樊游. 嗜热脂肪土芽孢杆菌普鲁兰酶基因的异源表达及重组酶性质[J]. 食品与发酵工业, 2017, 43(5): 30-.
[3] 谷海涛,李松,陈阿娜. 嵌合突变嗜酸普鲁兰芽孢杆菌普鲁兰酶结构域B对酶学性质及功能的影响[J]. 食品与发酵工业, 2017, 43(5): 43-.
[4] 王兵波,沈微,钱灵紫,李琛,罗枭,樊游,陈献忠. 一种密码子优化的酸性普鲁兰酶基因在巴斯德毕赤酵母中的高效表达[J]. 食品与发酵工业, 2016, 42(7): 9-.
[5] 申莹莹,郑宏臣,李树芳,付晓平,徐健勇,宋诙. 耐热普鲁兰酶CBM68结构域中关键位点对其酶学性质的影响[J]. 食品与发酵工业, 2016, 42(3): 12-.
[6] 严芬,杨光,连燕萍,王培松,吴晨烁. 产普鲁兰酶海单胞菌的分离鉴定及发酵条件优化[J]. 食品与发酵工业, 2015, 41(9): 40-.
[7] 王小兰,穆晓清,徐岩,聂尧. 重组大肠杆菌产普鲁兰酶发酵条件优化[J]. 食品与发酵工业, 2015, 41(7): 24-.
[8] 赵伟超,聂尧,穆晓清,张荣珍,徐岩. 采用复合保护剂提高重组普鲁兰酶稳定性[J]. 食品与发酵工业, 2015, 41(5): 48-.
[9] 许苗苗,焦国宝,王明道,孙利鹏,刘仲敏,邱立友. 启动子替代构建克雷伯氏菌普鲁兰酶高产菌株[J]. 食品与发酵工业, 2015, 41(10): 1-.
[10] 王苗苗,董海洲,张慧,侯汉学,尹训兰. 明胶/普鲁兰酶改性淀粉膜的制备与性能研究[J]. 食品与发酵工业, 2012, 38(09): 64-68.
[11] 王琳,武俊超,高群玉. 豌豆抗性淀粉的酶法制备及其性质研究[J]. 食品与发酵工业, 2012, 38(07): 108-113.
[12] 谢银珠,沈微,王正祥. 酸性普鲁兰酶基因在地衣芽孢杆菌中的表达[J]. 食品与发酵工业, 2011, 37(02): 7-10.
[13] 史苗苗,高群玉. 脱支蜡质玉米抗性淀粉的制备及性质[J]. 食品与发酵工业, 2010, 36(08): 31-35.
[14] 张永伟,徐学明,赵建伟,王金鹏,金征宇. 普鲁兰酶逆向合成麦芽糖基-α-环糊精[J]. 食品与发酵工业, 2009, 35(6): 46-.
[15] 夏子芳,王正祥. Thermotoga maritima普鲁兰酶的基因克隆与酶学性质研究[J]. 食品与发酵工业, 2007, 33(4): 19-.
No Suggested Reading articles found!
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
版权所有 © 《食品与发酵工业》编辑部
地址:北京朝阳区酒仙桥中路24号院6号楼111室
本系统由北京玛格泰克科技发展有限公司设计开发  技术支持:support@magtech.com.cn