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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2018, Vol. 38 Issue (7): 83-88    DOI: 10.13523/j.cb.20180711
    
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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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     
Received: 23 January 2018      Published: 13 August 2018
ZTFLH:  Q814  
Corresponding Authors: Jing WU     E-mail: jingwu@jiangnan.edu.cn
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Fan SUN
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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.

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

Fig.1 OD600 and DPEase activity of the recombinant strain in the 3L fermentor
Fig.2 SDS-PAGE analysis of the DPEaseM:Protein marker; 1:Intracellular soluble fraction
Fig.3 Effects of sodium alginate amount on enzyme activity recovery of immobilized cells
Fig.4 Effects of cell concentration on enzyme activity recovery of immobilized cells
Fig.5 Effects of CaCl2 concentration on enzyme activity recovery of immobilized cells
Fig.6 Effects of diatomite concentration on enzyme activity recovery of immobilized cells
Fig.7 The optimum pH(a) and temperature(b) of the immobilized cells and free cells
Fig.8 The operational stability of immobilized cells
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