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Function of aspartic acid residues in optimum pH control of l-arabinose isomerase from Lactobacillus fermentum

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Abstract

l-Arabinose isomerase (l-AI) catalyzes the isomerization of l-arabinose to l-ribulose and d-galactose to d-tagatose. Most reported l-AIs exhibit neutral or alkaline optimum pH, which is less beneficial than acidophilic ones in industrial d-tagatose production. Lactobacillus fermentum l-AI (LFAI) is a thermostable enzyme that can achieve a high conversion rate for d-galactose isomerization. However, its biocatalytic activity at acidic conditions can still be further improved. In this study, we report the single- and multiple-site mutagenesis on LFAI targeting three aspartic acid residues (D268, D269, and D299). Some of the lysine mutants, especially D268K/D269K/D299K, exhibited significant optimum pH shifts (from 6.5 to 5.0) and enhancement of pH stability (half-life time increased from 30 to 62 h at pH 6.0), which are more favorable for industrial applications. With the addition of borate, d-galactose was isomerized into d-tagatose by D268K/D269K/D299K at pH 5.0, resulting in a high conversion rate of 62 %. Based on the obtained 3.2-Å crystal structure of LFAI, the three aspartic acid residues were found to be distant from the active site and possibly did not participate in substrate catalysis. However, they were proven to possess similar optimum pH control ability in other l-AI, such as that derived from Escherichia coli. This study sheds light on the essential residues of l-AIs that can be modified for desired optimum pH and better pH stability, which are useful in d-tagatose bioproduction.

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Acknowledgements

This work was supported by National Basic Research Program of China (2013CB733600), the National High Technology Research and Development Program of China (2012AA021503), the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT) (IRT1066), Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP) (20113221130001), the National Natural Science Foundation of China (31371732).

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  1. State Key Laboratory of Materials-Oriented Chemical Engineering, College of Food Science and Light Industry, Nanjing University of Technology, No. 30 South Puzhu Road, Pukou District, Nanjing, 211816, China

    Zheng Xu, Sha Li, Xiaohai Feng, Yijing Zhan & Hong Xu

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  1. Zheng Xu
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  2. Sha Li
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  3. Xiaohai Feng
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  4. Yijing Zhan
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  5. Hong Xu
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Correspondence to Hong Xu.

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Xu, Z., Li, S., Feng, X. et al. Function of aspartic acid residues in optimum pH control of l-arabinose isomerase from Lactobacillus fermentum . Appl Microbiol Biotechnol 98, 3987–3996 (2014). https://doi.org/10.1007/s00253-013-5342-7

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