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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2019, Vol. 39 Issue (7): 24-31    DOI: 10.13523/j.cb.20190704
研究报告     
嗜热网球菌纤维二糖差向异构酶在枯草芽孢杆菌中的表达及发酵优化 *
王鑫淼,张康,陈晟,吴敬()
1 江南大学食品科学与技术国家重点实验室 江南大学生物工程学院工业生物技术教育部重点实验室教育部食品安全国际合作联合实验室 无锡 214122
Recombinant Expression and Fermentation Optimization of Dictyoglomus thermophilum Cellobiose 2-Epimerase in Bacillus subtilis
Xin-miao WANG,Kang ZHANG,Sheng CHEN,Jing WU()
1 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
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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%。
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    
Abstract:

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 7.5U/ml after cultivated in TB culture for 48h. 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 120min. In order to reduce the cost of fermentation, the fermentation medium was optimized.When 35g/L soybean meal was used as nitrogen source and 5g/L glycerin was used as carbon source, the intracellular activity reached 12.3U/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 56U/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 400g/L, the reaction temperature was 85℃, the initial pH was 8.5, the enzyme amount was 20U/ml, the enzyme converted lactose to 51% lactulose.
Key words: Dictyoglomus thermophilum    Cellobiose 2-epimerase    Bacillus subtilis    Enzymatic properties    Fermentation optimization
收稿日期: 2018-12-29 出版日期: 2019-08-05
ZTFLH:  Q819  
基金资助: * 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)项目资助项目(BE2015751)
通讯作者: 吴敬     E-mail: jingwu@jiangnan.edu.cn
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引用本文:

王鑫淼,张康,陈晟,吴敬. 嗜热网球菌纤维二糖差向异构酶在枯草芽孢杆菌中的表达及发酵优化 *[J]. 中国生物工程杂志, 2019, 39(7): 24-31.

Xin-miao WANG,Kang ZHANG,Sheng CHEN,Jing WU. Recombinant Expression and Fermentation Optimization of Dictyoglomus thermophilum Cellobiose 2-Epimerase in Bacillus subtilis. China Biotechnology, 2019, 39(7): 24-31.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20190704    或    https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I7/24

图1  纤维二糖差向异构酶核酸电泳分析
图2  纤维二糖差向异构酶SDS-PAGE分析
图3  纯化后的纤维二糖差向异构酶的SDS-PAGE分析
图4  重组CEase最适pH
图5  重组CEase最适温度
图6  重组CE的温度稳定性
图7  HPLC分析酶转化产物
图8  氮源种类对重组菌生长和发酵产酶的影响
氮源 浓度 OD600 酶活(U/ml)
15 9.02±0.2 6.77±0.2
豆粕粉 25 14.60±0.2 10.46±0.2
35 16.20±0.2 12.13±0.2
15 7.30±0.2 3.17±0.2
玉米浆 25 10.30±0.2 7.88±0.2
35 8.20±0.2 4.45±0.2
15 7.84±0.2 7.71±0.2
大豆蛋白胨 25 11.28±0.2 9.24±0.2
35 10.56±0.2 6.86±0.2
表1  氮源浓度对重组菌生长和发酵产酶的影响
图9  玉米浆和豆粕粉浓度对重组菌发酵生长(a)和产酶(b)的影响
大豆蛋白胨浓度
(g/L)		 豆粕粉浓度
(g/L)		 OD600 酶活(U/ml)
15 25 13.50±0.2 8.38±0.2
15 35 11.80±0.2 9.16±0.2
15 45 10.46±0.2 6.72±0.2
25 25 12.03±0.2 6.21±0.2
25 35 13.70±0.2 7.61±0.2
25 45 14.20±0.2 3.14±0.2
35 25 12.10±0.2 4.31±0.2
35 35 10.10±0.2 4.03±0.2
35 45 15.20±0.2 4.18±0.2
表2  氮源组合对重组菌生长和发酵产酶的影响
图10  碳源种类对重组菌生长和发酵产酶的影响
图11  碳源浓度对重组菌和产酶的影响
图12  3L发酵罐菌体生长产酶情况
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