1. Academic Validation
  2. Cloning and expression of the sucrose phosphorylase gene in Bacillus subtilis and synthesis of kojibiose using the recombinant enzyme

Cloning and expression of the sucrose phosphorylase gene in Bacillus subtilis and synthesis of kojibiose using the recombinant enzyme

  • Microb Cell Fact. 2018 Feb 15;17(1):23. doi: 10.1186/s12934-017-0842-2.
Miaomiao Wang 1 2 Jing Wu 1 2 3 Dan Wu 4 5 6
Affiliations

Affiliations

  • 1 State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China.
  • 2 School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China.
  • 3 International Joint Laboratory on Food Safety, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China.
  • 4 State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China. wudan@jiangnan.edu.cn.
  • 5 School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China. wudan@jiangnan.edu.cn.
  • 6 International Joint Laboratory on Food Safety, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China. wudan@jiangnan.edu.cn.
Abstract

Background: Kojibiose as a prebiotic and inhibitor of α-glucosidase exhibits potential for a wide range of applications in the food and medicine fields; however, large-scale separation and extraction of kojibiose from nature is difficult. Sucrose phosphorylase (SPase) can be used for the production of kojibiose, and currently, SPase is only heterologously expressed in E. coli, making it unsuitable for use in the food industry. However, Bacillus subtilis is generally considered to be a safe organism potentially useful for SPase expression.

Results: Here, for the first time, we heterologously expressed Bifidobacterium adolescentis SPase in a food-grade B. subtilis strain. The results showed that SPase was efficiently secreted into the extracellular medium in the absence of a signal peptide. After culturing the recombinant strain in a 3-L bioreactor, crude SPase yield and activity reached 7.5 g/L and 5.3 U/mL, respectively, the highest levels reported to date. The optimal reaction conditions for kojibiose synthesis catalyzed by recombinant SPase were as follows: 0.5 M sucrose, 0.5 M glucose, 0.02 UEnzyme/mgall_substrates, pH 7.0, 50 °C, and 30 h. Furthermore, the substrate-conversion rate reached 40.01%, with kojibiose accounting for 104.45 g/L and selectivity for kojibiose production at 97%.

Conclusions: Here, we successfully expressed SPase in B. subtilis in the absence of a signal peptide and demonstrated its secretion into the extracellular medium. Our results indicated high levels of recombinant Enzyme expression, with a substrate-conversion rate of 40.01%. These results provide a basis for large-scale preparation of kojibiose by the recombinant SPase.

Keywords

Bacillus subtilis; Enzymatic transformation; Heterologous expression; Kojibiose; Sucrose phosphorylase.

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