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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  2018, Vol. 38 Issue (7): 83-88    DOI: 10.13523/j.cb.20180711
技术与方法     
D-阿洛酮糖 3-差向异构酶在枯草芽孢杆菌中的高效表达及固定化细胞研究 *
孙帆1,2,3,宿玲恰1,2,3,张康1,2,3,吴敬1,2,3,**()
1 江南大学食品科学与技术国家重点实验室 无锡 214122
2 江南大学生物工程学院 工业生物技术教育部重点实验室 无锡 214122
3 江南大学教育部食品安全国际合作联合实验室 无锡 214122
D-psicose 3-epimerase Gene Overexpression in Bacillus subtilis and Immobilization of Cells
Fan SUN1,2,3,Ling-qia SU1,2,3,Kang ZHANG1,2,3,Jing WU1,2,3,**()
1 State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China
2 School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, Wuxi 214122, China
3 International Joint Laboratory on Food Safety, Jiangnan University, Wuxi 214122, China
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摘要：

将来源于Clostridium cellulolyticum H10的DPEase基因在食品级表达系统Bacillus subtilis中进行产酶研究,在3L发酵罐中高密度发酵最终酶活可达495U/ml,得到高表达量的DPEase酶液。通过硅藻土-海藻酸钠(吸附包埋法)对重组细胞进行固定化研究,结果表明,当海藻酸钠浓度为2%、细胞包埋量为50g/L、CaCl2浓度为2%、硅藻土浓度为1%时,固定化细胞酶活回收率可达64%,固定化细胞与游离细胞相比最适pH不变,最适温度提高5℃,热稳定性明显提高,连续反应7个批次后转化率仍然为28%,仍保持81%的残余酶活,具有很高的工业应用价值。
关键词： D-阿洛酮糖;  3-差向异构酶;  枯草芽孢杆菌;  高密度发酵;  固定化细胞    
Abstract:

The DPEase gene from Clostridium cellulolyticum H10 was studied on the enzyme production in the food grade expression system Bacillus subtilis. The final enzyme activity was 495U/ml by high-cell-density fermentation in the 3L fermentor. The recombinant cells were immobilized by diatomite-sodium alginate (adsorption-occlusion method); the optimized immobilized conditions were as follows: 2% sodium alginate, 50g/L cell concentration, 2% CaCl2 and 1% diatomite. Under the optimum condition, the recovery rate reached 64%. Compared with the free cells, the immobilized cells had the same optimal pH, the optimal temperature was increased by 5℃;and the thermal stability was significantly improved. The conversion rate was still 28% after 7 times repeated operations; it was also maintained 81% of the residual enzyme activity, which had a high industrial application value.
Key words: D-psicose    3-epimerase    Bacillus subtilis    High-cell-density fermentation    Immobilized cells
收稿日期: 2018-01-23 出版日期: 2018-08-13
ZTFLH:  Q814  
基金资助: 国家自然科学基金(31501419);国家杰出青年基金(31425020)
通讯作者: 吴敬     E-mail: jingwu@jiangnan.edu.cn
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孙帆
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引用本文:

孙帆,宿玲恰,张康,吴敬. D-阿洛酮糖 3-差向异构酶在枯草芽孢杆菌中的高效表达及固定化细胞研究 *[J]. 中国生物工程杂志, 2018, 38(7): 83-88.

Fan SUN,Ling-qia SU,Kang ZHANG,Jing WU. D-psicose 3-epimerase Gene Overexpression in Bacillus subtilis and Immobilization of Cells. China Biotechnology, 2018, 38(7): 83-88.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180711    或    https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I7/83

图1  重组菌在3L罐中的生长和产酶过程
图2  DPEase SDS-PAGE分析
图3  海藻酸钠浓度对固定化细胞酶活回收率的影响
图4  细胞包埋量对固定化细胞酶活回收率的影响
图5  CaCl2浓度对固定化细胞酶活回收率的影响
图6  硅藻土浓度对固定化细胞酶活回收率的影响
图7  固定化细胞与游离细胞的最适pH(a)和最适温度(b)
图8  固定化细胞的操作稳定性
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