DETERMINATION OF CHITOSANASE STRUCTURE AND CREATION OF NEW CHITOSANASE.

1环状芽孢杆菌MH-K 1产壳聚糖酶的结构。以硫酸铵为沉淀剂，采用气相扩散法对壳聚糖酶进行结晶。棒状晶体属正交晶系，空间群P2_12_12，晶胞参数a=43.3，B=57.7，c=128.0。获得了3A分辨率的电子密度图。α-螺旋大多数是局部的。β折叠的定位正在进行中。2.定点突变选择MH-K1壳聚糖酶中与其它壳聚糖酶和溶菌酶具有相似性的保守氨基酸进行定点突变。测定了突变株的壳聚糖酶活力。37位的Glu突变体和55位的Asp突变体丧失了活性。表明这些氨基酸是重要的催化剂。Aspon 52也可能参与了催化反应。3其他几种壳聚糖酶的克隆：克隆了拟诺卡氏菌K-01的部分壳聚糖酶基因。利用蛋白质测序数据和克隆的序列确定壳聚糖酶的一级结构。

1 Structure of chitosanase from Bacillus circulans MH-K1.Chitosanase was crystallized by a vapor-diffusion procedure using ammonium sulfate as a precipitate. Rod-shaped crystals belong to the orthorhombic system and the space group P2_12_12 with unit-cell dimensions of a=43.3, b=57.7, c=128.0 A.MAD Diffraction pattern of the crystals containing Pt was analyzed. Electron density map of 3A resolution was obtained. Most of alpha-helices were localized. Localization of beta-sheets are on the way. Refinement of the map using the data of native crystals should be done.2 Site-directed mutagenesits.Amino acids of MH-K1 chitosanase, which were conserved and showed similarity to other chitosanases and lysozymes, were selected and changed by site-directed mutagenesis. The chitosanase activity of mutants were measured. Mutants of Glu on 37 or Asp on 55 were lost its activity. It indicated that these amino acids were catalytically importnat. Asp on 52 also suggested to participate in catalytic reaction.3 Cloning of other chitosanase.Part of chitosanase gene from Nocardioides sp K-01 was cloned. Primary structure of the chitosanase was determined using protein-sequencing data and the clone's sequence.