摘要
L-谷氨酸氧化酶(L-glutamate oxidase,LGOX)能够专一性氧化L-谷氨酸生成α-酮戊二酸(α-ketoglutaric acid,α-KG),在食品领域以及临床生化检验中具有重要的价值。为了提高LGOX的活性,本文结合生物信息学方法和显色法,筛选得到一种新型的含有LGOX基因的茂源链霉菌株(Streptomyces mobaraensis CGMCC 4.1719),以其基因组序列为模板,通过PCR技术扩增LGOX基因,与pET21b质粒连接,构建表达载体pET21b-SmLGOX,化转至大肠杆菌E.coli BL21(DE3)中进行诱导表达,并对诱导条件加以优选。通过电泳分析结果可知,SmLGOX基因在大肠杆菌中成功表达。确定了最佳诱导条件为:IPTG终浓度0.4 mmol/L,30℃下诱导6 h。纯化后在不同pH条件下测定酶活,该重组菌最适pH为6.0,且在pH=4.0~6.0时相对酶活保持在80%以上,稳定性好,适于LGOX的工业化应用。
L-glutamate oxidase(LGOX)can specifically oxidize L-glutamate to produceα-ketoglutaric acid(αKG),which has important value in food field and clinical biochemistry.In order to improve the activity of LGOX,a novel strain of Streptomyces mobaraensis CGMCC 4.1719 containing LGOX gene was screened by combining bioinformatics method and chromogenic method.The LGOX gene was amplified by PCR using its genome sequence as a template,and was linked with pET21b plasmid to construct the expression vector pET21b-SmLGOX,which was transferred into E.coli BL21(DE3)for induced expression and the induced conditions were optimized.According to the results of electrophoresis,the SmLGOX gene was successfully expressed in E.coli.The optimal induction conditions were determined as follows:IPTG final concentration 0.4 mmol/L,induction at 30℃for 6h.After purification,the enzyme activity was determined under different pH conditions.The optimal pH was 6.0,and the relative enzyme activity remained above 80%at pH=4.0~6.0,showing good stability and was suitable for industrial application of LGOX.
作者
林怡颖
刘凯文
马小倩
连惠勇
李莹
王芳
张泽生
刘清岱
LIN Yiying;LIU Kaiwen;MA Xiaoqian;LIAN Huiyong;LI Ying;WANG Fang;ZHANG Zesheng;LIU Qingdai(College of Food Science and Engineering,Tianjin University of Science and Technology,Tianjin 300457;College of Food Science and Engineering,Hainan Tropical Ocean University,Sanya 572022)
出处
《中国食品添加剂》
CAS
北大核心
2023年第4期52-59,共8页
China Food Additives
基金
国家自然科学基金(51967006)。
关键词
茂源链霉菌
L-谷氨酸氧化酶
诱导表达
条件优化
酶活性
Streptomyces mobaraensis
L-glutamate oxidase
induced expression
conditions optimization
enzymatic activity
