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食品与发酵工业  2021, Vol. 47 Issue (20): 1-7    DOI: 10.13995/j.cnki.11-1802/ts.026871
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福氏志贺菌来源L-鼠李树胶糖激酶的克隆表达及酶学性质分析
冯林雪1, 陈洲1, 王亚森1, 冯康1, 许向阳2, 李子杰1*, 中西秀树1*, 高晓冬1
1(江南大学 生物工程学院,江苏 无锡,214122)
2(枣庄市杰诺生物酶有限公司,山东 枣庄,277100)
Cloning, expression and characterization of L-rhamnulose kinase from Shigella flexneri
FENG Linxue1, CHEN Zhou1, WANG Yasen1, FENG Kang1, XU Xiangyang2, LI Zijie1*, NAKANISHI Hideki1*, GAO Xiaodong1
1(School of Bioengineering, Jiangnan University, Wuxi 214122, China)
2(Zaozhuang Jienuo Biological Enzyme Co.Ltd., Zaozhuang 277100, China)
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摘要 D-阿洛酮糖是一种具有保健功能的稀有糖,目前其生产主要利用D-阿洛酮糖3-差向异构酶(D-psicose 3-epimerase,DPE)将D-果糖转化为D-阿洛酮糖,但该反应转化率低,仅能达到30%左右。基于L-鼠李树胶糖激酶(L-rhamnulose kinase,RhaB)的“磷酸化-脱磷酸”级联反应可提高反应转化率,然而目前有关RhaB的研究较少。该文研究了一种新型来源福氏志贺菌(Shigella flexneri 2a str.301)来源的RhaB,从福氏志贺菌(S.flexneri 2a str.301)的基因组DNA中克隆得到RhaB基因,将其与质粒载体pET28a连接,在 Escherichia coli BL21(DE3)中诱导表达。构建突变菌株RhaBE437Q,将酶活性提高了10倍,且大部分包涵体蛋白变为可溶性蛋白。利用载体上组氨酸标签对重组酶分离纯化,对其酶学性质进行了一系列研究。结果表明,重组蛋白RhaBE437Q为单体蛋白,分子质量为54 kDa;最适反应条件为40 ℃,pH 8.5,Mn2+;其只对C-3构型为R构型的糖有催化活性;将其与底物D-阿洛酮糖进行分子对接,对其催化机制进行了初步研究。
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冯林雪
陈洲
王亚森
冯康
许向阳
李子杰
中西秀树
高晓冬
关键词:  L-鼠李树胶糖激酶  福氏志贺菌  克隆与表达  酶学性质  D-阿洛酮糖  催化机制    
Abstract: D-Allulose is a kind of rare sugars with health care function and its production is mainly catalyzed by DPE (D-Psicose 3-epimerase) which can convert D-fructose to D-allulose. However, the conversion rate of this reaction is only about 30%. One method to improve the conversion rate is a phosphorylation-dephosphorylation cascade reaction based on L-rhamnulose kinase (RhaB). Up to now, the research about RhaB has been rarely reported. In this study, a gene encoding RhaB from Shigella flexneri 2a str. 301 was cloned into pET28a and expressed in Escherichia coli BL21(DE3). To enhance the activity of RhaB, we constructed a mutant strain RhaBE437Q. As a consequence, the activity was increased by 10 times and most of the inclusion body proteins became soluble. The purified recombinant RhaBE437Q had the maximum activity at 40 ℃, pH 8.5 and Mn2+, and was identified as a monomer with a molecular weight of 54 kDa. It could only catalyse sugars with the structure of R configuration of C-3. At last, we docked D-allulose into RhaBE437Q and performed a preliminary research on its catalytic mechanism.
Key words:  L-rhamnulose kinase    Shigella flexneri 2a str.301    cloning and expressing    enzymatic properties    D-allulose    catalytic mechanism
收稿日期:  2021-01-21      修回日期:  2021-03-01           出版日期:  2021-10-25      发布日期:  2021-11-18      期的出版日期:  2021-10-25
基金资助: 国家自然科学基金(32071467);山东省重点研发计划重大科技创新工程(2019JZZY011006);枣庄英才集聚工程
作者简介:  硕士研究生(李子杰副教授和中西秀树教授为共同通讯作者,E-mail:lizijie@jiangnan.edu.cn;hideki@jiangnan.edu.cn)
引用本文:    
冯林雪,陈洲,王亚森,等. 福氏志贺菌来源L-鼠李树胶糖激酶的克隆表达及酶学性质分析[J]. 食品与发酵工业, 2021, 47(20): 1-7.
FENG Linxue,CHEN Zhou,WANG Yasen,et al. Cloning, expression and characterization of L-rhamnulose kinase from Shigella flexneri[J]. Food and Fermentation Industries, 2021, 47(20): 1-7.
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http://sf1970.cnif.cn/CN/10.13995/j.cnki.11-1802/ts.026871  或          http://sf1970.cnif.cn/CN/Y2021/V47/I20/1
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