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Construction and one-step purification of Bacillus kaustophilus leucine aminopeptidase fused to the starch-binding domain of Bacillus sp. strain TS-23 α-amylase

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Abstract

The starch-binding domain of Bacillus sp. strain TS-23 α-amylase was introduced into the C-terminal end of Bacillus kaustophilus leucine aminopeptidase (BkLAP) to generate a chimeric enzyme (BkLAPsbd) with raw-starch-binding activity. BkLAPsbd, with an apparent molecular mass of approximately 65 kDa, was overexpressed in Escherichia coli M15 cells and purified to homogeneity by nickel–chelate chromatography. Native PAGE and chromatographic analyses revealed that the purified fusion protein has a hexameric structure. The half-life for BkLAPsbd was 12 min at 70°C, while less than 20% of wild-type enzyme activity retained at the same heating condition. Compared with the wild-type enzyme, the 60% decrease in the catalytic efficiency of BkLAPsbd was due to a 91% increase in K m value. Starch-binding assays showed that the K d and B max values for the fusion enzyme were 2.3 μM and 0.35 μmol/g, respectively. The adsorption of the crude BkLAPsbd onto raw starch was affected by starch concentration, pH, and temperature. The adsorbed enzyme could be eluted from the adsorbent by 2% soluble starch in 20 mM Tris–HCl buffer (pH 8.0). About 49% of BkLAPsbd in the crude extract was recovered through one adsorption–elution cycle with a purification of 11.4-fold.

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Acknowledgements

This work was supported by a research grant (NSC 93-2313-B-415-004) from the National Science Council of the Republic of China.

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Authors and Affiliations

  1. Department of Life Sciences and Institute of Molecular Biology, National Chung Cheng University, Chiayi, 621, Taiwan

    Hsien-Bin Huang

  2. Department of Chemistry and Biochemistry, National Chung Cheng University, Chiayi, 621, Taiwan

    Meng-Chun Chi

  3. Institute of Molecular Biology, National Chung Hsing University, 402-27, Taichung, Taiwan

    Wen-Hwei Hsu

  4. Department of Applied Chemistry, National Chaiyi University, 300 University Road, Chiayi, 60083, Taiwan

    Wan-Chi Liang & Long-Liu Lin

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Correspondence to Long-Liu Lin.

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Huang, HB., Chi, MC., Hsu, WH. et al. Construction and one-step purification of Bacillus kaustophilus leucine aminopeptidase fused to the starch-binding domain of Bacillus sp. strain TS-23 α-amylase. Bioprocess Biosyst Eng 27, 389–398 (2005). https://doi.org/10.1007/s00449-005-0001-8

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