摘要
从天蓝色链霉菌Streptomyces coelicolor克隆得到海藻糖合酶基因(ScTreS),在大肠杆菌Escherichia coli BL21(DE3)中进行了异源表达,通过Ni-NTA亲和柱对表达产物进行分离纯化得到纯酶,经SDS-PAGE测定其分子量约为62.3 kDa。研究其酶学性质发现该酶最适温度35℃;最适pH7.0,对酸性条件比较敏感。通过同源建模和序列比对分析,对该基因进行定点突变。突变酶K246A比酶活比野生酶提高了1.43倍,突变酶A165T相对提高了1.39倍,海藻糖转化率分别提高了14%和10%。利用突变体重组菌K246A进行全细胞转化优化海藻糖的合成条件并放大进行5L罐发酵,结果表明:在麦芽糖浓度300g/L、初始反应温度和pH分别为35℃和7.0的条件下,转化率最高达到71.3%,产量为213.93 g/L;当底物浓度增加到700g/L时,海藻糖产量仍可达到465.98g/L。
The trehalose synthase(ScTreS)gene from Streptomyces coelicolor was successfully cloned and heterologously expressed in Escherichia coli BL21(DE3).The protein purified by Ni-NTA affinity column showed an apparent molecular weight(MW)of 62.3 kDa analyzed by SDS-PAGE.The optimum temperature of the enzyme was 35℃and the optimum pH was 7.0;the enzyme was sensitive to acidic conditions.By homologous modeling and sequence alignment,the enzyme was modified by site-directed mutagenesis.The relative activities of the mutant enzymes K246A and A165T were 1.43 and 1.39 times that of the wild type,an increased conversion rate of 14%and 10%respectively.To optimize the synthesis conditions of trehalose,the mutant strain K246A was cultivated in a 5-L fermentor and used for whole-cell transformation.The results showed that with the substrate maltose concentration of 300 g/L at 35℃and pH 7.0,the highest conversion rate reached 71.3%,and the yield of trehalose was 213.93 g/L.However,when maltose concentration was increased to 700 g/L,the yield of trehalose can reach 465.98 g/L with a conversion rate of 66%.
作者
吴傲
张显
徐美娟
杨套伟
李华钟
饶志明
Ao Wu;Xian Zhang;Meijuan Xu;Taowei Yang;Huazhong Li;Zhiming Rao Key(Laboratory of Industrial Biotechnology of Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi 214122,Jiangsu,China)
出处
《生物工程学报》
CAS
CSCD
北大核心
2019年第7期1348-1358,共11页
Chinese Journal of Biotechnology
基金
中央高校基本科研业务费专项资金(No.JUSRP51708A)
江苏高校优势学科建设工程,江苏高校品牌专业建设工程资助项目资助~~
关键词
海藻糖合酶
克隆表达
定点突变
全细胞转化
trehalose synthase
clone and expression
site-directed mutagenesis
whole-cell conversion
