Novel b-N-acetylglucosaminidases from Vibrio harveyi 650: Cloning, expression, enzymatic properties, and subsite identification

摘要

Background: Since chitin is a highly abundant natural biopolymer, many attempts have been made to convertthis insoluble polysaccharide into commercially valuable products using chitinases and b-N-acetylglucosaminidases(GlcNAcases). We have previously reported the structure and function of chitinase A from Vibrio harveyi 650. Thisstudy t reports the identification of two GlcNAcases from the same organism and their detailed functionalcharacterization.Results: The genes encoding two new members of family-20 GlcNAcases were isolated from the genome ofV. harveyi 650, cloned and expressed at a high level in E. coli. VhNag1 has a molecular mass of 89 kDa and anoptimum pH of 7.5, whereas VhNag2 has a molecular mass of 73 kDa and an optimum pH of 7.0. The recombinantGlcNAcases were found to hydrolyze all the natural substrates, VhNag2 being ten-fold more active than VhNag1.Product analysis by TLC and quantitative HPLC suggested that VhNag2 degraded chitooligosaccharides in asequential manner, its highest activity being with chitotetraose. Kinetic modeling of the enzymic reaction revealedthat binding at subsites (-2) and (+4) had unfavorable (positive) binding free energy changes and that the bindingpocket of VhNag2 contains four GlcNAc binding subsites, designated (-1),(+1),(+2), and (+3).Conclusions: Two novel GlcNAcases were identified as exolytic enzymes that degraded chitin oligosaccharides,releasing GlcNAc as the end product. In living cells, these intracellular enzymes may work after endolytic chitinasesto complete chitin degradation. The availability of the two GlcNAcases, together with the previously-reportedchitinase A from the same organism, suggests that a systematic development of the chitin-degrading enzymesmay provide a valuable tool in commercial chitin bioconversion.