Mechanism involved in the expression of functional roles of chitinase domains in crystalline chitin hydrolysis

由于几丁质是几丁质酶的底物，是一种刚性的、不溶性的结晶性多糖，因此了解结晶性几丁质水解的机理是几丁质酶研究的关键问题。目前对晶体几丁质水解机理的研究大多集中在几丁质酶结构域氨基酸残基的作用上。在本研究中，除了氨基酸残基外，我们还关注几丁质酶分子的局部结构，以进一步了解结晶几丁质水解。1)过悬环结构在晶体水解中的重要性来自环孢杆菌（Bacillus circulans） WL-12的几丁质酶A1在催化裂口上具有过悬环结构，被认为对晶体几丁质水解具有重要意义。对该环结构进行了删除诱变，并分析了效果。突变体的几丁质酶对结晶几丁质的水解活性明显降低，因此，证明了环结构对结晶几丁质水解的重要作用。B.circulans几丁质酶A1中的ChBD是一个独特的几丁质结合结构域，它对结晶几丁质具有特异性。在筛选该ChBD中参与几丁质结合活性的氨基酸残基时，发现除了W687.3外，Gln679是一个对几丁质结合具有重要作用的新残基。为了明确与催化裂口对齐的4个芳香氨基酸残基（Y240、W252、W479和Y481）的作用，我们进行了位点定向诱变，将Y替换为W，将W替换为Y。Y - W突变增加了结合活性，W - Y突变降低了结合活性，但所有突变均显著降低了水解活性。综上所述，酶的迁移率和结合活性之间的平衡对晶体甲壳素的水解至关重要。少

Since chitin, the substrate for chitinases, is a rigid, insoluble and crystalline polysaccharide, understanding of mechanism for crystalline chitin hydrolysis is a critical issue for chitinase study. Most studies aimed at understanding mechanism for crystalline chitin hydrolysis carried out so far are focused on the role of amino acid residues in the chitinase domains. In this study, we newly focused on the local structure of the chitinase molecules in addition to the amino acid residues, to get further insight into crystalline chitin hydrolysis.1)Importance of over hung-loop structure in crystalline hydrolysisChitinase A1 from Bacillus circulans WL-12 has an overhung-loop structure on the catalytic cleft and it has been suggested to be important for crystalline chitin hydrolysis. Deletion mutagenesis of this loop structure was carried out and the effect was analyzed. The mutant chitinase decreased the hydrolytic activity against crystalline chitin significantly and, thus, the loop str … More ucture was proved to be important for crystalline chitin hydrolysis.2)The mechanism for chitin binding of ChBD specific for crytalline chitinChBD in B.circulans chitinase A1 is a unique chitin-binding domain, since it is specific for crystalline chitin. Site-directed mutagenesis for screening amino acid residues involved in chitin binding activity of this ChBD revealed Gln679 as a new residue important for chitin binding, in addition to W687.3)The role of aromatic amino acid residues exposed on the surface of Serratia chitinase BTo clarify the roles of the four aromatic amino acid residues (Y240,W252,W479 and Y481) aligned to the catalytic cleft, site-directed mutagenesis to replace Y with W and W with Y was carried out. Y to W mutation increased and W to Y mutation decreased binding activity, while all mutations decreased hydrolytic activity significantly. From these results, it was concluded that proper balance between mobility of enzyme and binding activity is important for crystalline chitin hydrolysis. Less