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Molecular analysis of the gene encoding a new chitinase from the marine psychrophilic bacterium Moritella marina and biochemical characterization of the recombinant enzyme

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Abstract

The marine psychrophilic bacterium Moritella marina, isolated from a sample raised from a depth of 1,200 m in the northern Pacific Ocean, secretes several chitinases in response to chitin induction. A gene coding for an extracellular chitinolytic enzyme was cloned and its nucleotide sequence was determined. The chitinase gene consists of an open reading frame of 1,650 nucleotides and encodes a protein of 550 amino acids with a calculated molecular weight of 60.788 kDa, named MmChi60. MmChi60 has a modular structure consisting of a glycosyl-hydrolase family 18 N-terminal catalytic region as well as a C-terminal chitin-binding domain (ChBD). The new chitinase was purified to homogeneity from the intracellular fraction of Escherichia coli. The optimum pH and temperature of the recombinant MmChi60 were 5.0 and 28°C, respectively. The mode of action of the new enzyme on N-acetylchitooligomers, chitin polymers, and other substrates was examined, and MmChi60 was classified as an endochitinase. Thermal unfolding of MmChi60 was studied using differential scanning microcalorimetry and revealed that the protein unfolds reversibly at 65°C. On the basis of the crystal structure of the chitinase C of Streptomyces griseus, a homology-based 3-D model of the ChBD of the MmChi60 was calculated.

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Acknowledgments

We thank Dr George Nounesis for helping in DSC measurements, Dr Nikos Papandreou for helping in producing the ChBD model, and Dr Dimitrios Hatzinikolaou for helping in HPLC measurements. This study was supported by the State Scholarships Foundation (IKY) grant scholarship of Greece to Eleni Stefanidi, by the GSRT PENED to Constantinos E. Vorgias and Eleni Stefanidi, and by the EMBO and Marie Curie fellowships of EU to Eleni Stefanidi.

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  1. Department of Biochemistry and Molecular Biology, Faculty of Biology, National and Kapodistrian University of Athens, Panepistimiopolis-Zographou, 15784, Athens, Greece

    Eleni Stefanidi & Constantinos E. Vorgias

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  1. Eleni Stefanidi
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  2. Constantinos E. Vorgias
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Correspondence to Constantinos E. Vorgias.

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Communicated by G. Antranikian.

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Stefanidi, E., Vorgias, C.E. Molecular analysis of the gene encoding a new chitinase from the marine psychrophilic bacterium Moritella marina and biochemical characterization of the recombinant enzyme. Extremophiles 12, 541–552 (2008). https://doi.org/10.1007/s00792-008-0155-9

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