摘要
通过化学方法合成嗜热网球菌(Dictyoglomus thermophilum)来源的纤维二糖差向异构酶基因ce,将其引入到载体pBSuL3-ce,构建重组质粒pBSuL3-ce并转化进枯草芽孢杆菌,发酵48h后测定胞内酶活为7. 5U/ml。酶学性质结果表明:该酶的最适pH为8. 5;最适温度为85℃,85℃的半衰期为120min。为降低发酵成本,对发酵培养基进行优化:以35g/L豆粕粉为氮源、5g/L甘油为碳源时,酶活力最高可达12. 3U/ml。依据摇瓶优化的条件在3L发酵罐中扩大培养,胞内酶活达到56U/ml,比摇瓶培养酶活提高了8倍。利用发酵所得酶制备乳果糖,在乳糖浓度为400g/L、反应温度为85℃、初始pH 8. 5、加酶量为20U/ml的条件下,乳果糖转化率可达51%。
A cellobiose 2-epimerase gene ce derived from Dictyoglomus thermophilum was synthesized and introduced into the expression vector pBSuL3 to construct a recombinant plasmid pBSuL3-ce and then transformed it into Bacillus subtilis.The intracellular activity of cellobiose epimerase reached 7.5 U/ml after cultivated in TB culture for 48 h.The enzymatic properties showed that the optimum pH of the enzyme was 8.5;the optimum temperature was 85℃,and the half-life of 85℃was 120 min.In order to reduce the cost of fermentation,the fermentation medium was optimized.When 35 g/L soybean meal was used as nitrogen source and 5 g/L glycerin was used as carbon source,the intracellular activity reached 12.3 U/ml.Then,according to the optimized condition of the shake flask,the culture was expanded in the 3L fermenter,and the intracellular activity of cellobiose epimerase reached 56 U/ml,which was 8-fold higher than that of the shake flask culture.The lactulose was prepared by using the enzyme obtained by fermentation.When the lactose concentration was 400 g/L,the reaction temperature was 85℃,the initial pH was 8.5,the enzyme amount was 20 U/ml,the enzyme converted lactose to 51%lactulose.
作者
王鑫淼
张康
陈晟
吴敬
WANG Xin-miao;ZHANG Kang;CHEN Sheng;WU Jing(School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education,International Joint Laboratory on Food Safety,State Key Laboratory of Food Science and Technology,Jiangnan University,Wuxi 214122,China)
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2019年第7期24-31,共8页
China Biotechnology
基金
国家杰出青年基金(31425020)
国家自然科学基金(31571776)
中央高校基本科研业务费专项资(JUSRP51706A)
江苏省重点研发计划(社会发展)项目(BE2015751)资助项目
关键词
嗜热网球菌
纤维二糖差向异构酶
枯草芽孢杆菌
酶学性质
发酵优化
Dictyoglomus thermophilum
Cellobiose 2-epimerase
Bacillus subtilis
Enzymatic properties
Fermentation optimization
