钝齿棒杆菌精氨酸琥珀酸酶编码基因argH的克隆表达及其重组菌发酵产精氨酸研究

doi:Q78

中国生物工程杂志

2010, Vol. 30

Issue (09): 49-55 DOI: Q78

研究报告

钝齿棒杆菌精氨酸琥珀酸酶编码基因argH的克隆表达及其重组菌发酵产精氨酸研究

饶志明^1**,徐美娟¹,陆元修¹,周晨¹,蓝春燕¹,窦文芳²,张晓梅²,许泓瑜²,许正宏^1,2**

1.江南大学工业生物技术教育部重点实验室无锡 214122
2.江南大学医药学院制药工程实验室无锡 214122

Cloning, Expression and Analysis of the argH Gene Encoding Argininosuccinate Lyase from Corynebacterium crenatum

RAO Zhi-ming¹,XU Mei-juan¹,LU Yuan-xiu¹,ZHOU Chen¹,LAN Chun-yan¹,DOU Wen-fang²,ZHANG Xiao-mei²,XU Hong-yu²,XU Zheng-hong^1,2

1.Key Laboratory of Industrial Biotechnology, Ministry of Education,Wuxi 214122,China
2.Lab of Pharmaceutical Engineering, School of Medicine and Pharmaceutics, Jiangnan University, Wuxi 214122,China

全文: PDF(682 KB) HTML

摘要：

钝齿棒杆菌（Corynebacterium crenatum）SYPA是本实验室筛选获得的一株高产精氨酸生产菌株。精氨酸琥珀酸酶（AL）是精氨酸合成过程中的最后一个酶，催化底物精氨酸琥珀酸生成产物精氨酸。为进一步提高精氨酸产量，本文以钝齿棒杆菌基因组为模板，扩增得到其编码基因argH，全长为1434 bp，编码476个氨基酸，理论蛋白分子量大小为50.8 kDa，其与C. glutamicum ATCC 13032比对其同源性为99.4%，相差10 bp，3个氨基酸。将其在E.coli BL21(DE3)及C. crenatum SYPA中成功表达。利用载体pET-28a上的6×His?Tag选用Ni柱亲和层析纯化AL，纯化后获得的重组蛋白的比酶活达156.9mU/mg蛋白，总回收率为72.3%，对该酶的部分酶学性质进行了初步研究，并发现产物精氨酸对其具有反馈抑制作用。成功构建钝齿棒杆菌重组穿梭表达质粒pJC1-tac-argH并将其通过电击转化法转入C.crenatum SYPA中，加强其代谢途径中AL蛋白表达量，并对其发酵产精氨酸做了初步分析。结果表明与出发菌株相比，转化子在精氨酸琥珀酸酶酶活增强了66.8%的基础上精氨酸产量达40.9 g/L，比出发菌株产量的35.8 g/L提高了约14.2%。

关键词： L-精氨酸; 钝齿棒杆菌; 精氨酸琥珀酸酶; 酶学性质; 发酵

Abstract:

Argininosuccinate lyase (EC 4.3.2.1; AL) genes from L-arginine producing mutant Corynebacterium crenatum SYPA were cloned and sequenced. Analysis of argH sequences revealed that only one ORF existed , which used ATG as the initiation codon and coded a peptide of 476 amino acids with acalculated molecular weight of 50.8 kDa. Only 10 nucleotide difference was found in the structure gene and the difference caused 3 change of amino acid by comparision of the gene sequences between C. crenatum SYPA and the Corynebacterium glutamicum ATCC 13032. The ORF sequence of argHs showed homologies of 99.4%. The argH gene from C. crenatum was expressed both in E.coli and C. crenatum SYPA. Then AL was purified by Ni-NTA affinity chromatography and the enzymatic characterization of it was determines. The expression vector pJC1-tac-argH was transducted to C. crenatum SYPA. The AL was expressed well and the activity was improved by 66.8%. The fermentive character of CCH1(pJC1-tac-argH) was also primary analysed. The result shows that the acid producing ability of recombinant strain is improved by 14.2%.

Key words: L-arginine Corynebacterium crenatum Arginieosuccinate Lyase Characterization of enzyme Fermentation

收稿日期: 2010-04-09 出版日期: 2010-08-25

基金资助:

国家自然科学基金(20676053, 30970056)、国家“863”计划(2006AA020103, 2007AA02Z207)、国家“973”计划（2007CB707804）、霍英东青年基金(121020)资助项目

通讯作者: 饶志明;许正宏 E-mail: raozm@yahoo.com.cn;zhenghongxu@jiangnan.edu.cn

	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
	饶志明
	徐美娟
	陆元修
	周晨
	蓝春燕
	窦文芳
	张晓梅许
	泓瑜
	许正宏

引用本文:

饶志明徐美娟陆元修周晨蓝春燕窦文芳张晓梅许泓瑜许正宏. 钝齿棒杆菌精氨酸琥珀酸酶编码基因argH的克隆表达及其重组菌发酵产精氨酸研究[J]. 中国生物工程杂志, 2010, 30(09): 49-55.

RAO Zhi-Meng, XU Mei-Juan, LIU Yuan-Xiu, ZHOU Chen, LA Chun-Yan, DOU Wen-Fang, ZHANG Xiao-Mei-Hu, HONG Yu, HU Zheng-Hong. Cloning, Expression and Analysis of the argH Gene Encoding Argininosuccinate Lyase from Corynebacterium crenatum. China Biotechnology, 2010, 30(09): 49-55.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/Q78 或 https://manu60.magtech.com.cn/biotech/CN/Y2010/V30/I09/49

［1］ Ikeda M. Amino acid production processes. Adv Biochem Eng Biotechnol, 2003, 79: 135.
［2］ Rainer H B, Stefanie M B, Jiirgen C F. The Largininenitric oxide pathway: role in atherosclerosis and therapeutic implications. Atherosclerosis, 1996, 127(1):111.
［3］ Utagawa T. Production of arginine by fermentation. J Nutr, 2004, 134 (10): 28542867.
［4］熊筱晶, 窦文芳, 许正宏. L精氨酸高产菌的诱变育种及其摇瓶产酸条件. 无锡轻工大学学报, 2003, 22(2): 1013. Xiong Y J, Dou W F, Xu Z H.Journal of Wuxi University of Light Industry, 2003, 22(2): 1013.
［5］ Hirose Y, Shibai H. Amino acid fermentation. Biotechnol Bioeng, 1980, 22: 111-125.
［6］许正宏, 窦文芳, 王霞. 氮源及其添加模式对钝齿棒杆菌JDN2875合成L精氨酸的影响. 应用与环境生物学报, 2006, 12(3):381385. Xu Z H, Dou W F, Wang X.Chinese Journal of Applied & Environmental Biology, 2006, 12(3):381385.
［7］ Joseph S, David W R. Molecular Cloning. 3rd ed. New York: Cold Spring Harbor Laboratory Press, 2001.15231574.
［8］徐美娟, 杨套伟, 饶志明,等. 克雷伯氏菌甘油脱氢酶dhaD的克隆表达、纯化及酶学性质研究. 中国生物工程杂志, 2008, 28(2): 3035. Xu M J, Yang T W, Rao Z M et al.China Biotechnology, 2008, 28(2): 3035.
［9］ Bhaumik P, Koski M K , Bergmann U,et al. Structure determination and refinement at 2.44  resolution of argininosuccinate lyase from Escherichia coli. Acta Cryst, 2004, 60(11): 19641970.
［10］ Troshina O, Hansel A, Lindblad P. Cloning, characterization, and functional expression in Escherichia coli of argH encoding argininosuccinate lyase in the Cyanobacterium Nostoc sp. strain PCC 73102. Curr Microbiol, 2001, 43(4): 260264.
［11］ Bradford M M. A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem, 1976, 72: 248254.
［12］王镜岩，沈同.生物化学.第三版. 北京:高等教育出版社,2001. 78278. Wang J Y, Shen T. Biochemistry. 3 rd. Beijing: Hign Education Press, 2001.78278.
［13］郝宁,赵智,王宇,等.钝齿棒杆菌N乙酰谷氨酸激酶基因的克隆、序列分析及表达. 微生物学报, 2006, 46(1):9094. Hao N, Zhao Z, Wang Y.Acta Microbiologica Sinica, 2006, 46(1):9094.
［14］ Xu H, Dou W.F, Xu H Y, et al. A twostage oxygen supply strategy for enhanced Larginine production by Corynebacterium crenatum based on metabolic fluxes analysis. Biochem Eng J, 2009, 43(1): 4151.
［15］宁正祥. 食品成分分析手册. 北京：中国轻工业出版社, 2001.5055. Ning Z X. Handbook of Food Composition Analysis. Beijing: China Light Industry Press, 2001.5055.

[1]	高寅岭,张凤娇,赵贵众,张宏森,王风芹,宋安东. 衣康酸发酵研究进展[J]. 中国生物工程杂志, 2021, 41(5): 105-113.
[2]	梁爱玲,刘文婷,武攀,李倩,高健,张洁,刘卫东,贾士儒,郑迎迎. 来源于Exophiala aquamarina的新型玉米赤霉烯酮水解酶的性质及底物结合中心关键氨基酸的功能研究^*[J]. 中国生物工程杂志, 2021, 41(10): 19-27.
[3]	杨娜,吴群,徐岩. 解淀粉芽孢杆菌合成surfactin的发酵策略优化 ^*[J]. 中国生物工程杂志, 2020, 40(7): 51-58.
[4]	王泽建,栗波,王萍,张琴,杭海峰,梁剑光,庄英萍. 葡萄糖和麦芽糖碳源底物对粪产碱杆菌合成凝胶多糖的胞内代谢流影响^*[J]. 中国生物工程杂志, 2020, 40(5): 30-39.
[5]	朱衡,张继福,张云,胡云峰. 环氧交联剂和氨基载体固定化海洋假丝酵母脂肪酶^*[J]. 中国生物工程杂志, 2020, 40(5): 57-68.
[6]	王蒙,张全,高慧鹏,关浩,曹长海. 生物发酵法制备木糖醇的研究进展 ^*[J]. 中国生物工程杂志, 2020, 40(3): 144-153.
[7]	马翠萍,刘朵朵,潘炳菊,申会涛,宋亚囝. 来源于嗜碱芽孢杆菌N16-5甘露聚糖利用基因簇的乙酰酯酶AesA的克隆及性质分析^*[J]. 中国生物工程杂志, 2020, 40(3): 65-71.
[8]	朱衡,张继福,张云,孙爱君,胡云峰. 聚乙二醇二缩水甘油醚交联氨基载体LX-1000EA固定化脂肪酶 ^*[J]. 中国生物工程杂志, 2020, 40(1-2): 124-132.
[9]	王宝石,谭凤玲,李林波,李志刚,孟丽,邱立友,张明霞. 生物处理策略改善麸皮酚类化合物的生物可及性^*[J]. 中国生物工程杂志, 2020, 40(12): 88-94.
[10]	王菲,胡春辉,于浩. 6-羟基烟酸3-单加氧酶(NicC)催化反应机理研究 ^*[J]. 中国生物工程杂志, 2019, 39(7): 15-23.
[11]	彭强强,刘启,徐名强,张元兴,蔡孟浩. 新型重组毕赤酵母产人胰岛素前体的表达工艺研究 ^*[J]. 中国生物工程杂志, 2019, 39(7): 48-55.
[12]	王鑫淼,张康,陈晟,吴敬. 嗜热网球菌纤维二糖差向异构酶在枯草芽孢杆菌中的表达及发酵优化 ^*[J]. 中国生物工程杂志, 2019, 39(7): 24-31.
[13]	谢玉锋,韩雪梅,路福平. 副干酪乳杆菌β-葡糖苷酶的表达、纯化及酶学性质研究 ^*[J]. 中国生物工程杂志, 2019, 39(5): 72-79.
[14]	朱梦露,王雪雨,刘鑫,路福平,孙登岳,秦慧民. 一种新型亮氨酸5-羟化酶NmLEH的异源表达、纯化及酶学性质分析 ^*[J]. 中国生物工程杂志, 2019, 39(12): 24-34.
[15]	陈子晗,周海胜,尹新坚,吴坚平,杨立荣. **Amphibacillus xylanus谷氨酸脱氢酶基因工程菌培养条件优化 ^***[J]. 中国生物工程杂志, 2019, 39(10): 58-66.

Viewed

Full text

Abstract

Cited

Shared

Discussed