Structure and Function of Chitosanase from Streptomyces sp.N174

壳聚糖酶由lividans TK24产自Streptomyces sp.N174的壳聚糖酶基因，经S-Sepharose和Bio-Gel A柱层析纯化。由此得到的壳聚糖酶从-1,4-低聚糖底物中产生α -异头物，表明该酶是一种反相酶。以葡萄糖胺六糖为底物时，产物分布为三聚体>>二聚体>四聚体。低聚糖水解的时间过程表明底物从六聚体到五聚体再到四聚体的降解率降低。很有可能壳聚糖酶的底物结合间隙可以容纳至少6个氨基葡萄糖残基，并且裂解点位于结合间隙的中点。为了鉴定催化所需的氨基酸残基，对壳聚糖酶进行了定点诱变，并测定了每种突变壳聚糖酶的活性和稳定性。替换Glu22或Asp40显著降低了比活性和kappacat，而kappam仅略有变化。这些结果表明，Glu22和Asp40直接参与壳聚糖酶的催化中心。Asp37突变显著降低了酶活性，并显著降低了热展开转变的中点温度（Tm）。在晶体结构上，Asp37的羧基侧链远离结合间隙，但与His90的咪唑氮有密切的相互作用。可能是Asp37通过与His90的相互作用稳定了底物结合裂缝的结构，而其突变破坏了结构的稳定性，导致对底物的亲和力降低。

Chitosanase was produced by the strain of Streptomyces lividans TK24 bearing the chitosanase gene from Streptomyces sp.N174, and purified by S-Sepharose and Bio-Gel A column chromatography. The chitosanase thus obtained produced alpha-anomer from beta-1,4-linked oligosaccharide substrate, indicating that the enzyme is an inverter. When glucosamine hexasaccharide was used as the substrate, the product distribution was trimer>>dimer>tetramer. Time-courses of oligosaccharide hydrolysis showed a decrease in rate of substrate degradation from hexamer to pentamer to tetramer. It is most likely that the substrate binding cleft of the chitosanase can accommodate at least six glucosamine residues, and that the cleavage point is located at the midpoint of the binding cleft.For identification of the amino acid residues essential for catalysis, site-directed mutagenesis of the chitosanase was carried out, and the activity and stability were determined for each of the mutant chitosanases. Substitutions of Glu22 or Asp40 reduced drastically specific activity and kappacat, while KAPPAmwas only slightly changed. These results suggested that Glu22 and Asp40 are directly involved in the catalytic center of the chitosanase. The mutation of Asp37 considerably reduced the enzymatic activity, and significantly decreased the midpoint temperature of thermal unfolding transition (Tm). In the crystal structure, the carboxyl side chain of Asp37 points away from the binding cleft, but makes a close interaction with the imidazole nitrogen of His90. It is likely that Asp37 stabilize the structure of substrate binding cleft by interacting with His90 and its mutation destabilize the structure resulting in the lower affinity to the substrate.