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Properties of an ionic liquid-tolerant Bacillus amyloliquefaciens CMW1 and its extracellular protease

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Abstract

An ionic liquid-tolerant bacterium, Bacillus amyloliquefaciens CMW1, was isolated from a Japanese fermented soybean paste. Strain CMW1 grew in the presence of 10 % (v/v) 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), a commonly used ionic liquid. Additionally, strain CMW1 grew adequately in the presence of the hydrophilic ionic liquids 10 % (v/v) 1-ethyl-3-methylimidazolium trifluoromethanesulfonate ([EMIM]CF3SO3) or 2.5 % (v/v) 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([BMIM]CF3SO3). Strain CMW1 produced an extracellular protease (BapIL) in the culture medium. BapIL was stable in the presence of 80 % (v/v) ionic liquids, [EMIM]CF3SO3, [BMIM]Cl, [BMIM]CF3SO3, 1-butyl-3-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium hexafluorophosphate, and 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide, and functioned in 10 % (v/v) these ionic liquids. BapIL was stable at pH 4.0–12.6 or in 4004 mM NaCl solution, and exhibited activity in the presence of 50 % (v/v) hydrophilic or hydrophobic organic solvents. BapIL was completely inhibited by 1 mM PMSF and partially by 5 mM EDTA. BapIL belongs to the true subtilisins according to analysis of the deduced amino acid sequence. We showed that BapIL from the ionic liquid-tolerant B. amyloliquefaciens CMW1 exhibited tolerance to ionic liquid and halo, alkaline, and organic solvents.

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Acknowledgments

This work was supported by Grant-in-Aid for Young Scientists (B) (No. 26870731) from JSPS (to A.K.) and a Grant for Scientific Research from Faculty of Agriculture, Kindai University (to A.K.).

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    Authors and Affiliations

    1. Department of Applied Biological Chemistry, Faculty of Agriculture, Kinki University, 3327-204 Nakamachi, Nara, 631-8505, Japan

      Atsushi Kurata, Humiya Senoo, Yasuyuki Ikeda, Hideaki Kaida, Chiaki Matsuhara & Noriaki Kishimoto

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    1. Atsushi Kurata
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    2. Humiya Senoo
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    3. Yasuyuki Ikeda
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    4. Hideaki Kaida
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    5. Chiaki Matsuhara
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    6. Noriaki Kishimoto
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    Corresponding author

    Correspondence to Atsushi Kurata.

    Additional information

    Communicated by L. Huang.

    A. Kurata, H. Senoo, Y. Ikeda, H. Kaida, C. Matsuhara and N. Kishimoto contributed equally to this work.

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    792_2016_832_MOESM1_ESM.docx

    Supplemental Fig. 1. Effect of NaCl or pH on CMW1 growth. CMW1 was cultivated using the medium (pH 7.3) containing 1 % yeast extract, 0.25 % tryptone, 0.5 % KCl, 1 % MgSO4 7H2O and 0-16 % (w/v) NaCl (A). Using the medium containing 1 % yeast extract, 0.25 % tryptone, 0.5 % KCl, and 1 % MgSO4 7H2O at the pH values with 1.9-10.9, CMW1 was cultivated for 3 h (B). ●, without addition of NaCl; ○, 5.0 %(w/v); ■,10 %(w/v); □,16 %(w/v). Supplemental Fig. 2. Effects of various ionic liquids on CMW1 growth. CMW1 was cultivated in the presence or absence of (A) [EMIM]CF3SO3, (B) [BMIM]Cl, (C) [BMIM]CF3SO3, (D) [BMIM]BF4, (E) [BMIM]PF6, or (F) [BMIM]NTf2 as described in Materials and Methods. ●, without addition of ionic liquid; ■, 2.5 %(v/v); □, 5.0 %(v/v); ○, 10.0 %(v/v); △, 16.0 %(v/v) (DOCX 167 kb)

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    Kurata, A., Senoo, H., Ikeda, Y. et al. Properties of an ionic liquid-tolerant Bacillus amyloliquefaciens CMW1 and its extracellular protease. Extremophiles 20, 415–424 (2016). https://doi.org/10.1007/s00792-016-0832-z

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    • DOI: https://doi.org/10.1007/s00792-016-0832-z

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