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Highly thermostable and surfactant-activated chitinase from a subseafloor bacterium, Laceyella putida

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Abstract

A novel chitinase (LpChiA) was purified to homogeneity from a culture of Laceyella putida JAM FM3001. LpChiA hydrolyzed colloidal chitin optimally at a pH of 4 in an acetate buffer and temperature of 75 ºC. The enzyme was remarkably stable to incubation at 70 ºC up to 1 h at pH 5.2, and its activity half-life was 3 days. The molecular mass of the enzyme was around 38 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and around 75 kDa by gel filtration, suggesting it is a homodimer. The enzyme activity was enhanced about 60 % when pre-incubated with anionic, cationic, and nonionic surfactants. The gene for LpChiA was cloned by PCR and sequenced. The nucleotide sequence of the gene consisted of 1,683 bp encoding 560 amino acids. The N-terminal and internal amino acid sequences of the purified LpChiA from L. putida suggested that the mature enzyme was composed of 384 amino acids after cleaving its 176 N-terminal amino acids and dimerized to express its activity. The deduced amino acid sequence of the mature enzyme showed the highest similarity to chitinase of Laceyella sacchari with 79 % identity.

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Author notes

  1. Hiroki Shibasaki

    Present address: Present Address: Nippon Rika Co. Ltd., Tokyo, 108-0023, Japan

Authors and Affiliations

  1. Graduate School of Engineering, Toyo University, 2100 Kujirai, Kawagoe, Saitama, 350-8585, Japan

    Hiroki Shibasaki & Ron Usami

  2. Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima, Yokosuka, Kanagawa, 237-0061, Japan

    Kohsuke Uchimura, Tohru Kobayashi & Koki Horikoshi

  3. Graduate School of Life Sciences, Toyo University, 1-1-1 Izumino, Itakura, Oura-gun, Gunma, 374-0193, Japan

    Takeshi Miura

Authors
  1. Hiroki Shibasaki
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  2. Kohsuke Uchimura
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  3. Takeshi Miura
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  4. Tohru Kobayashi
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Correspondence to Tohru Kobayashi.

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Shibasaki, H., Uchimura, K., Miura, T. et al. Highly thermostable and surfactant-activated chitinase from a subseafloor bacterium, Laceyella putida . Appl Microbiol Biotechnol 98, 7845–7853 (2014). https://doi.org/10.1007/s00253-014-5692-9

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  • DOI: https://doi.org/10.1007/s00253-014-5692-9

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