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Tuning the catalytic performances of a sucrose isomerase for production of isomaltulose with high concentration

  • Biotechnologically Relevant Enzymes and Proteins
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Abstract

Obtaining a sucrose isomerase (SIase) with high catalytic performance is of great importance in industrial production of isomaltulose (a reducing sugar). In order to obtain such SIase mutant, a high-throughput screening system in microtiter plate format was developed based on a widely used 2,4-dinitrosalicylic acid (DNS) method for determination of reducing sugar. An SIase from Erwinia sp. Ejp617 (ErSIase) was selected to improve its catalytic efficiency. After screening of ~ 8000 mutants from a random mutagenesis library, Q209 and R456 were identified as beneficial positions. Saturation mutagenesis of the two positions resulted in a double-site mutant ErSIase_Q209S-R456H that showed the highest catalytic efficiency, and its specific activity reached 684 U/mg that is 17.5-fold higher than that of the wild-type ErSIase. By employing the lyophilized Escherichia coli (E. coli) cells harboring ErSIase_Q209S-R456H, a high space–time yield (STY = 3.9 kg/(L·d)) was achieved toward 600 g/L sucrose. Furthermore, the in silico analysis suggested that the hydrogen bond network was improved and steric hindrance was reduced due to the beneficial substitutions.

Key points

• A sucrose isomerase mutant with high catalytic efficiency was obtained.

• The highest space–time yield was achieved toward high-concentration sucrose.

• The optimized H-bond network contributed to the enhanced catalytic efficiency.

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Acknowledgements

This work was financially supported by the Leading Innovative and Entrepreneur Team Introduction Program of Zhejiang, P. R. China (2018R01014) and the Zhejiang Provincial Qianjiang Talent Project.

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Authors and Affiliations

  1. Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, 18 Chaowang Road, Hangzhou, 310014, People’s Republic of China

    Feng Zhang, Feng Cheng, Dong-Xu Jia, Qian Liu, Zhi-Qiang Liu & Yu-Guo Zheng

  2. The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou, 310014, People’s Republic of China

    Feng Zhang, Feng Cheng, Dong-Xu Jia, Qian Liu, Zhi-Qiang Liu & Yu-Guo Zheng

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  1. Feng Zhang
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  2. Feng Cheng
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  3. Dong-Xu Jia
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  4. Qian Liu
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  5. Zhi-Qiang Liu
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  6. Yu-Guo Zheng
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Contributions

FZ and FC conceived and designed research. ZL and YZ supervised the project. FZ conducted experiments. FZ, DJ and QL analyzed data. FZ, FC and ZL wrote the manuscript. All authors read and approved the manuscript.

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Correspondence to Zhi-Qiang Liu.

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Zhang, F., Cheng, F., Jia, DX. et al. Tuning the catalytic performances of a sucrose isomerase for production of isomaltulose with high concentration. Appl Microbiol Biotechnol 106, 2493–2501 (2022). https://doi.org/10.1007/s00253-022-11891-5

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  • DOI: https://doi.org/10.1007/s00253-022-11891-5

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