Please wait a minute...
 
 
食品与发酵工业  2018, Vol. 44 Issue (9): 9-14    DOI: 10.13995/j.cnki.11-1802/ts.017153
  研究报告 本期目录 | 过刊浏览 | 高级检索 |
分子改造提高谷氨酰胺转氨酶的催化活性
任蕊蕊1,2, 刘松1,2*, 李江华1,2, 堵国成1,2, 陈坚1,2
1(江南大学 生物工程学院,江苏 无锡,214122)
2(江南大学 工业生物技术教育部重点实验室,江苏 无锡,214122)
Improved catalytic activity of transglutaminase through molecular modification
REN Rui-rui1,2, LIU Song 1,2*, LI Jiang-hua 1,2, DU Guo-cheng 1,2, CHEN Jian 1,2
1(School of Biotechnology, Jiangnan University, Wuxi 214122, China)
2(Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China)
下载:  PDF (1985KB) 
输出:  BibTeX | EndNote (RIS)      
摘要 谷氨酰胺转氨酶(EC2.3.2.13, Transglutaminase, TGase)是一种重要的食品酶。为提高其催化活性,通过Discovery Studio 2017预测了Streptomyces mobaraense TGase中影响其与底物α-N-CBZ-GLN-GLY结合自由能的氨基酸位点,构建得到结合自由能下降的TGase突变体:Y24W、E300W和Y302R。与野生TGase相比,E300W的比酶活提高了31%;Kmkcat和kcat/Km值分别提高了10%、42%和29%,说明E300W比酶活的提高主要是由于酶转换数的增加。Y24W、E300W和Y302R的热稳定性均有不同程度下降。作用力分析发现,Y24W、E300W和Y302R主链-主链氢键分别减少1、2和4个。上述结果表明,基于蛋白质结合自由能分析的策略能迅速鉴定影响TGase催化活性关键氨基酸,进一步突变能有效提高其催化活性。
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
任蕊蕊
刘松
李江华
堵国成
陈坚
关键词:  谷氨酰胺转氨酶  解脂耶氏酵母  定点突变  高效表达  酶学性质    
Abstract: Transglutaminase (EC2.3.2.13, TGase) is an important food enzyme. In order to enhance its catalytic activity, the amino acid sites that could affect the binding energy of S. mobaraense TGase and its substrate α-N-CBZ-GLN-GLY were predicted through Discovery Studio 2017. Then mutants (Y24W, E300W and Y302R) with reduced binding energy were constructed. Compared with wild type of TGase, the specific activity of E300W was increased by 31%. Km, kcat and kcat/Km vaule of E300W were increased by 10%, 42% and 29% respectively. The results showed that the increase of specific enzyme activity was mainly due to the increase of enzymatic conversion number. While the thermal stability of all the mutants decreased in varying degrees. And the structure analysis indicated that the main-chain hydrogen bonds of Y24W, E300W and Y302R decreased by 1, 2 and 4, respectively, compared with original TGase. These results suggested that the strategy based on the analysis of binding free energy could rapidly identify the key amino acids that affect the catalytic activity of TGase, and further mutation might effectively improve its catalytic activity.
Key words:  transglutaminase    Yarrowia lipolytica    site-directed mutation    high level expression    enzymatic characteristics
收稿日期:  2018-03-02                出版日期:  2018-09-25      发布日期:  2018-10-30      期的出版日期:  2018-09-25
基金资助: 国家自然基金面上项目(31771913);江苏省重点研发计划社会发展项目(BE2016629)
作者简介:  硕士研究生(刘松副教授为通讯作者,E-mail: liusong@jiangnan.edu.cn)。
引用本文:    
任蕊蕊,刘松,李江华,等. 分子改造提高谷氨酰胺转氨酶的催化活性[J]. 食品与发酵工业, 2018, 44(9): 9-14.
REN Rui-rui,LIU Song,LI Jiang-hua,et al. Improved catalytic activity of transglutaminase through molecular modification[J]. Food and Fermentation Industries, 2018, 44(9): 9-14.
链接本文:  
http://sf1970.cnif.cn/CN/10.13995/j.cnki.11-1802/ts.017153  或          http://sf1970.cnif.cn/CN/Y2018/V44/I9/9
[1] UMEZAWA Y, OHTSUKA T, YOKOYAMA K, et al. Comparison of enzymatic properties of microbial transglutaminase from Streptomyces sp.[J]. Food Science and Technology Research, 2002, 8(2): 113-118.
[2] KIELISZEK M, MISIEWICZ A. Microbial transglutaminase and its application in the food industry. A review [J]. Folia Microbiologica, 2014, 59(3): 241-250.
[3] SANTHI D, KALAIKANNAN A, MALAIRAJ P, et al. Application of microbial transglutaminase in meat foods: A review [J]. Critical Reviews in Food Science and Nutrition, 2017, 57(10): 2 071-2 076.
[4] TATSUKAWA H, FURUTANI Y, HITOMI K, et al. Transglutaminase 2 has opposing roles in the regulation of cellular functions as well as cell growth and death [J]. Cell Death and Disease, 2016, 7(6): e2 244.
[5] MARINIELLO L, PORTA R, SORRENTINO A, et al. Transglutaminase-mediated macromolecular assembly: production of conjugates for food and pharmaceutical applications [J]. Amino Acids, 2014, 46(3): 767-776.
[6] ECKERT R L, KAARTINEN M T, NURMINSKAYA M, et al. Transglutaminase regulation of cell function [J]. Physiological Reviews, 2014, 94(2): 383-417.
[7] KUO T F, TATSUKAWA H, KOJIMA S. New insights into the functions and localization of nuclear transglutaminase 2 [J]. Febs Journal, 2011, 278(24): 4 756-4 767.
[8] FU Jia-jia, SU Jing, WANG Ping, et al. Enzymatic processing of protein-based fibers [J]. Applied Microbiology and Biotechnology, 2015, 99(24): 1 0387-1 097.
[9] GRIFFIN M, CASADIO R, BERGAMINI C M. Transglutaminases: Nature's biological glues [J]. Biochemical Journal, 2002, 368(Pt 2): 377-396.
[10] BOMMARIUS A S, BLUM J K, ABRAHAMSON M J. Status of protein engineering for biocatalysts: how to design an industrially useful biocatalyst [J]. Current Opinion in Chemical Biology, 2011, 15(2): 194-200.
[11] YOKOYAMA K, UTSUMI H, NAKAMURA T, et al. Screening for improved activity of a transglutaminase from Streptomyces mobaraensis created by a novel rational mutagenesis and random mutagenesis [J]. Applied Microbiology and Biotechnology, 2010, 87(6): 2 087-2 096.
[12] ZHANG Jun-hui, JIANG Yu-yan, LIN Ying, et al. Structure-guided modification of Rhizomucor miehei lipase for production of structured lipids [J]. PLoS One, 2013, 8(7): e67 892.
[13] LIPPOW S M, WITTRUP K D, TIDOR B. Computational design of antibody-affinity improvement beyond in vivo maturation [J]. Nature Biotechnology, 2007, 25(10): 1 171-1 176.
[14] LIU Song, WAN Dan, WANG Miao, et al. Overproduction of pro-transglutaminase from Streptomyces hygroscopicus in Yarrowia lipolytica and its biochemical characterization [J]. BMC Biotechnology, 2015, 15(1): 75.
[15] XUAN Jian-wu, FOURNIER P, GAILLARDIN C. Cloning of the LYS5 gene encoding saccharopine dehydrogenase from the yeast Yarrowia lipolytica by target integration [J]. Current Genetics, 1988, 14(1): 15-21.
[16] 刘松. Streptomyces hygroscopicus谷氨酰胺转胺酶的异源表达研究 [D]. 无锡: 江南大学, 2011.
[17] KASHIWAGI T, YOKOYAMA K, ISHIKAWA K, et al. Crystal structure of microbial transglutaminase from Streptoverticillium mobaraense [J]. The Journal of Biological Chemistry, 2002, 277(46): 44 252-44 260.
[18] LIU Song, ZHANG Dong-xu, WANG Miao, et al. The pro-region of Streptomyces hygroscopicus transglutaminase affects its secretion by Escherichia coli [J]. FEMS Microbiology Letters, 2011, 324(2): 98-105.
[19] CHEONG D E, KO K C, HAN Y, et al. Enhancing functional expression of heterologous proteins through random substitution of genetic codes in the 5′ coding region [J]. Biotechnology and Bioengineering, 2015, 112(4): 822-826.
[20] XIE Yuan, AN Jiao, YANG Guang-yu, et al. Enhanced enzyme kinetic stability by increasing rigidity within the active site [J]. Journal of Biological Chemistry, 2014, 289(11): 7 994-8 006.
[21] TAGAMI U, SHIMBA N, NAKAMURA M, et al. Substrate specificity of microbial transglutaminase as revealed by three-dimensional docking simulation and mutagenesis [J]. Protein Engineering Design and Selection, 2009, 22(12): 747-752.
[1] 陶大炜, 宁喜斌. 产α-环糊精葡萄糖基转移酶的菌株筛选、鉴定与酶学性质的初步研究[J]. 食品与发酵工业, 2021, 47(6): 145-151.
[2] 杨胜远, 林谦, 刘淑敏, 苏巧云, 黄慧玲. 屎肠球菌源谷氨酸脱羧酶的制备及其酶学性质研究[J]. 食品与发酵工业, 2021, 47(5): 28-34.
[3] 宋婷, 王帅静, 汪沉, 吕育财, 罗华军, 郭金玲, 龚大春. 近平滑假丝酵母ATCC 7330羰基还原酶CpCR的表达及酶学性质研究[J]. 食品与发酵工业, 2021, 47(3): 18-24.
[4] 于洁, 徐勤茜, 李子院, 刘红艳, 郝再彬, 李海云. 虎杖内生真菌Aspergillus aculeatus HZ001产β-葡萄糖苷酶的酶学特性[J]. 食品与发酵工业, 2021, 47(3): 31-35.
[5] 黄笛, 李翠云, 万敏惠, 程琴, 骆香远, 钟佶良, 叶劲松. 谷氨酰胺转氨酶添加量对蛋黄粉乳化性和凝胶性的影响[J]. 食品与发酵工业, 2021, 47(3): 101-106.
[6] 包怡, 胡友明, 朱林江, 陆跃乐, 陈小龙. 己糖氧化酶的研究进展[J]. 食品与发酵工业, 2021, 47(3): 218-223.
[7] 魏万涛, 李梦丽, 江波, 张涛. L-岩藻糖激酶/GDP-L-岩藻糖焦磷酸化酶的克隆表达及酶学性质研究[J]. 食品与发酵工业, 2020, 46(9): 18-24.
[8] 张庆芳, 王浚晨, 于爽, 刘春莹, 迟雪梅, 迟乃玉. 人体肠道中产尿酸氧化酶细菌的筛选、鉴定与酶学性质研究[J]. 食品与发酵工业, 2020, 46(8): 34-39.
[9] 宋丽丽, 闻格, 霍姗浩, 胡晓龙, 杨旭, 张志平. 白酒酒糟中产纤维素酶细菌的分离筛选和酶学性质研究[J]. 食品与发酵工业, 2020, 46(7): 43-49.
[10] 郑丹妮, 柏玉香, 纪杭燕, 李晓晓, 王禹, 蔣彤, 金征宇. γ-CGTase酶学性质及产物特异性影响因素[J]. 食品与发酵工业, 2020, 46(5): 38-45.
[11] 王乐, 包娜莎, 金征宇, 赵建伟, 周星, 田耀旗. 添加谷氨酰胺转氨酶对糙米蛋糕品质的影响[J]. 食品与发酵工业, 2020, 46(5): 174-180.
[12] 郑亚伦, 夏瑛, 李良, 董孝元, 方尚玲, 陈茂彬, 李琴. 源于解淀粉芽孢杆菌酸性木聚糖酶酶学性质的研究[J]. 食品与发酵工业, 2020, 46(24): 58-65.
[13] 周海岩, 周建宝, 易晓男, 李勉, 柳志强. 来源于Rhodohalobacter barkolensis的昆布多糖酶RbLam16的重组表达及生产条件优化[J]. 食品与发酵工业, 2020, 46(21): 9-15.
[14] 董艺凝, 陈卫, 陈海琴, 赵建新, 陈永泉, 张灏. 嗜热脂肪芽孢杆菌(Geobacillus stearothermophilus)来源耐热β-半乳糖苷酶BgaB转糖苷催化活性改造[J]. 食品与发酵工业, 2020, 46(2): 1-6.
[15] 庞翠萍, 刘松, 周景文, 张国强, 李江华. 细菌脂肪氧合酶酶学性质分析及表达优化[J]. 食品与发酵工业, 2020, 46(19): 1-8.
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