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Overproduction, purification, and property analysis of an extracellular recombinant fructosyltransferase

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Abstract

Fructooligosaccharides (FOS) have widely used for the manufacture of low-calorie and functional foods, because they can inhibit intestinal pathogenic microorganism growth and increase Ca2+ and Mg2+ absorption. In this study, the fructosyltransferase (FruSG) from Aspergillus niger strain SG610, which is used in the industrial production of FOS, was successfully overexpressed in Pichia pastoris strain GS115 and characterized. Recombinant FruSG had optimum temperature and pH of 50 °C and 5.5, respectively, and it was stable below 40 °C and at from pH 3.0 to 8.0. K m and k cat values of the recombinant FruSG were 424.5 mmol L−1 and 3.8 × 103 min−1, respectively. On addition of 50 and 100 g L−1 glucose, the K i was 130.6 and 170.0 mmol L−1, respectively. The recombinant FruSG had the highest activity (2294.7 U mL−1) after induction for 144 h in a 5-L fermenter, which was 956.1-fold higher than that of native FruSG from A. niger SG610, and produced a high yield of FOS (64.5 %, w/w). The recombinant FruSG was activated by 5 mmol L−1 Mg2+ and 1 mmol L−1 Fe2+ (relative activity of 125.2 and 120.9 %, respectively), indicating that the enzyme was Mg2+ and Fe2+ dependent. Moreover, toluene, n-butanol, trichloromethane, and ethyl acetate significantly activated FruSG. Together, these results indicate that the recombinant FruSG is an excellent candidate for the industrial production of FOS.

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Acknowledgments

This project was financially supported by the Open Project Program of the Key Laboratory of Industrial Biotechnology, Ministry of Education, China (KLIB-KF201301, KLIB-KF201404), the Open Project Program of the Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, China (KLCCB-KF201506), the Fundamental Research Funds for the Central Universities (JUSRP11429), the 111 Project (111-2-06), Natural Science Foundation of Jiangsu Province (BK20140152), National Natural Science Foundation of China (21406089), 863 Program (2014AA021304), the Priority Academic Program Development of Jiangsu Higher Education Institutions, and the Jiangsu Province “Collaborative Innovation Center for Advanced Industrial Fermentation” industry development program.

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    1. The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China

      Wenwen Guo, Haiquan Yang, Shumin Qiang, You Fan, Wei Shen & Xianzhong Chen

    2. The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China

      Wenwen Guo, Haiquan Yang & Xianzhong Chen

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    1. Wenwen Guo
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    Correspondence to Haiquan Yang, Wei Shen or Xianzhong Chen.

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    Wenwen Guo and Haiquan Yang have contributed equally to this work.

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    Guo, W., Yang, H., Qiang, S. et al. Overproduction, purification, and property analysis of an extracellular recombinant fructosyltransferase. Eur Food Res Technol 242, 1159–1168 (2016). https://doi.org/10.1007/s00217-015-2620-x

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