Chitinase is an important enzyme for many physiological processes. Mytichitin-1 is a chitinase-like protein in Mytilus coruscus， and its C-terminal 55-AA fragment (mytichitin-CB) is a novel antimicrobial peptide， suggesting a new immune process in which chitinase is involved; mytichtin-1 may have various forms in the different biological processes of M. coruscus. Thus， the study of mytichitin-1 will be helpful for understanding the mechanism of mussel immune biology and the functional diversity of chitinase. In this study， mytichitin-1 was recombinantly expressed with different lengths， full-length mytichtin-1 (rMchi-F) and the N-terminal region (rMchi-N) in Escherichia coli BL21 with codon optimizati... More
Chitinase is an important enzyme for many physiological processes. Mytichitin-1 is a chitinase-like protein in Mytilus coruscus， and its C-terminal 55-AA fragment (mytichitin-CB) is a novel antimicrobial peptide， suggesting a new immune process in which chitinase is involved; mytichtin-1 may have various forms in the different biological processes of M. coruscus. Thus， the study of mytichitin-1 will be helpful for understanding the mechanism of mussel immune biology and the functional diversity of chitinase. In this study， mytichitin-1 was recombinantly expressed with different lengths， full-length mytichtin-1 (rMchi-F) and the N-terminal region (rMchi-N) in Escherichia coli BL21 with codon optimization. The results of SDS-PAGE， Western blotting， and mass spectrometry confirmed that the two forms of mytichitin-1 had been successfully recombinant expressed with a yield of 40 mg purified enzyme per L culture. In addition， the 55-AA fragment of mytichitin-CB was chemically synthesized (sMchi-CB). After purification and oxidation， the functions of the three protein products were analysed， including chitin degradation， chitin binding， and antimicrobial activities. Both rMchi-F and rMchi-N displayed enzymatic activity with the optimum pH of 4.0 and optimum temperature of 40 °C， and rMchi-N showed a stronger activity than rMchi-F. Enzymatic activities of rMchi-F and rMchi-N were stimulated by the metal ions Fe， Ba， and Na and partially inhibited by Cu， Ni and Zn. rMchi-F， rMchi-N， and sMchi-CB had the ability to combine with colloid chitin. The antimicrobial activities of these proteins were tested against bacteria and fungi， and the results indicated the strongest activity for sMchi-CB and the weakest activity for rMchi-N. Using a prepared anti-rMchi-F polyclonal antibody， immunohistochemistry and immunoprecipitation were performed and the results revealed the location of mytichitin-1 in mantle， digestive gland and blood cells. In addition， two forms of mytichitin-1， mytichitin-CB (6 kD) and full-length mytichitin-1 (48 kD)， were detected， and a 35 kD protein was identified as the third form of mytichitin-1， existing in various tissues of M. coruscus. These findings suggest that mytichitin-1 may play different roles， with at least three forms， in different M. coruscus tissues.
