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Cloning, Expression, Characterization, and Mutagenesis of a Thermostable Exoinulinase From Kluyveromyces cicerisporus

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Abstract

Inulinase is an enzyme that belongs to glycoside hydrolase family 32. It converts inulin into high-fructose syrups and fructoligosaccharides, both of which are widely used in pharmaceutical and food industries. In this study, the kcINU1 gene (GenBank accession number AF178979) encoding an exoinulinase was cloned from Kluyveromyces cicerisporus CBS4857 and expressed in Pichia pastoris X-33, yielding a maximum of 45.2 ± 0.6 U mL−1 of inulinase activity of culture supernatant. The expressed inulinase was purified and characterized. The enzyme had an optimum temperature of 55 °C and an optimum pH of 4.5. It had a K m of 0.322 mM and a V max of 4317 μM min−1 mg−1 protein when inulin was used as a substrate. It retained nearly 90 % of the maximal activity after pre-incubation at 50 °C for 1 h or at pH ranging from 3.0 to 6.0 at 4 °C for 24 h, demonstrating that KcINU1 was stable at high temperature and low pH. Moreover, we constructed two KcINU1 mutants, Asp30Ala and Glu215Ala, by site-directed mutagenesis and confirmed via zymogram analysis that Asp-30 and Glu-215 of the enzyme were the catalytic active center. The present study has provided important information for understanding the catalytic mechanism of exoinulinase.

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Acknowledgments

We are grateful to Professor Hong Lv for the plasmid Y179U/pUKDS-INU presented to us and Dr. Qun Zhao for the MALDI-TOF analysis of molecular weight of the protein. This study was supported by grants from the Chinese High-tech Research and Development program (2011AA10A205, 2014AA093511) and the Open Funding of National Key Laboratory of Biochemical Engineering (2012KF-06). Heng Yin was supported by Youth Innovation Promotion Association of Chinese Academy of Sciences.

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Authors and Affiliations

  1. Natural Products and Glyco-Biotechnology Research Group, Liaoning Provincial Key Laboratory of Carbohydrates, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, CAS, Dalian, 116023, People’s Republic of China

    Jun-Yan Ma, Hai-Long Cao, Hai-Dong Tan, Wu-Jun Liu, Yu-Guang Du & Heng Yin

  2. Medical College of Dalian University, Dalian, 116022, People’s Republic of China

    Jun-Yan Ma & Xue-Jun Hu

  3. University of Chinese Academy of Sciences, Beijing, 100049, People’s Republic of China

    Jun-Yan Ma

  4. Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, People’s Republic of China

    Yu-Guang Du

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Correspondence to Yu-Guang Du or Heng Yin.

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Fig. S1

Lineweaver-Burk plot showing the activity of KcINU1 against different concentrations of inulin. V represents the initial reaction rates, which were calculated from the amount of reducing sugars produced per minute per milligram protein, and [S] represents the concentration of inulin. The initial reaction rates were determined at pH 4.5 and 55 °C for 10 min with the concentrations of inulin ranging from 4.2 to 83.3 mM. The kinetic parameters K m and V max were calculated according to the Lineweaver-Burk plot. (GIF 31 kb)

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Ma, JY., Cao, HL., Tan, HD. et al. Cloning, Expression, Characterization, and Mutagenesis of a Thermostable Exoinulinase From Kluyveromyces cicerisporus . Appl Biochem Biotechnol 178, 144–158 (2016). https://doi.org/10.1007/s12010-015-1864-z

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