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作 者:冯林雪 陈洲 王亚森 冯康 许向阳 李子杰[1] 中西秀树[1] 高晓冬[1] FENG Linxue;CHEN Zhou;WANG Yasen;FENG Kang;XU Xiangyang;LI Zijie;NAKANISHI Hideki;GAO Xiaodong(School of Bioengineering,Jiangnan University,Wuxi 214122,China;Zaozhuang Jienuo Biological Enzyme Co.Ltd.,Zaozhuang 277100,China)
机构地区:[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
出 处:《食品与发酵工业》2021年第20期1-7,共7页Food and Fermentation Industries
基 金: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。
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RhaB^(E437Q),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RhaB^(E437Q)为单体蛋白,分子质量为54 kDa;最适反应条件为40℃,pH 8.5,Mn^(2+);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。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 RhaB^(E437Q).As a consequence,the activity was increased by 10 times and most of the inclusion body proteins became soluble.The purified recombinant RhaB^(E437Q) had the maximum activity at 40℃,pH 8.5 and Mn^(2+),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 RhaB^(E437Q) and performed a preliminary research on its catalytic mechanism.
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