Abstract
Chitinase, as one of the most important extracellular enzymes in the marine environment, has great ecological and applied values. In this study, two chitinases (Chi1557 and Chi4668) with 97.33% amino acid sequences identity were individually found in Vibrio rotiferianus and Vibrio harveyi. They both were encoding by 561 amino acids, but differed in 15 amino acids and showed different enzymatic properties. The optimal temperature and pH ranges were 45-50 °C and pH 5.0-7.0 for Chi1557, while ~50 °C and pH 3.0-6.0 for Chi4668. K+, Mg2+, and EDTA increased the enzymatic activity of Chi4668 significantly, yet these factors were inhibitory to Chi1557. Moreover, Chi1557 degraded colloidal chitin to produce (GlcNAc)2 and minor GlcNAc, whereas Chi4668 produce (GlcNAc)2 with minor (GlcNAc)3 and (GlcNAc)4. The Kcat/Km of Chi4668 was ~4.7 times higher than that of Chi1557, indicating that Chi4668 had stronger catalytic activity than Chi1557. Furthermore, site-directed mutagenesis was performed on Chi1557 focusing on seven conserved amino acid residues of family GH18 chitinases. Chi1557 was almost completely inactive after Glu154, Gln219, Tyr221, or Trp312 was individually mutated, retained ~50% activity after Tyr37 was mutated, and increased two times activity after Asp152 was mutated, indicating that these six amino acids were key sites for Chi1557.