Molecular analysis of the gene encoding a new chitinase from the marine psychrophilic bacterium Moritella marina and biochemical characterization of the recombinant enzyme.
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 degrees 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 degrees 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.
Stefanidi
Eleni
E
Department of Biochemistry and Molecular Biology, Faculty of Biology, National and Kapodistrian University of Athens, Panepistimiopolis-Zographou, 15784, Athens, Greece.
Vorgias
Constantinos E
CE
D000595:Amino Acid Sequence; D001483:Base Sequence; D002151:Calorimetry; D002686:Chitin / Q000737:chemistry; D002688:Chitinases / Q000235:genetics*; D003001:Cloning, Molecular; D003080:Cold Temperature; D008969:Molecular Sequence Data; D008970:Molecular Weight; D034221:Moritella / Q000201:enzymology* / Q000235:genetics; D016366:Open Reading Frames; D010940:Plant Proteins; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D010940:Plant Proteins; D011994:Recombinant Proteins; D002686:Chitin; D002688:Chitinases; C119825:chitinase C