DETERMINATION OF CHITOSANASE STRUCTURE AND CREATION OF NEW CHITOSANASE.

壳聚糖酶结构的测定和新壳聚糖酶的产生。

• 批准号: 07660094
• 负责人: ANDO Akikazu
• 金额: $ 1.79万
• 依托单位: CHIBA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07660094/
• 关键词: CHITOSANASE; CRYSTALLIZATION; X-RAY CRYSTALLOGRAPHY; SITE-DIRECTED MUTAGENESIS; CATALYTIC AMINO ACIDS

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.

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。

项目成果

Jun-ichi Saito, Yoshiho Nagata, Akikazu Ando, Akiko Kita, Kunio Miki: "Crystalllization and preliminary X-ray crystallographic analysis of chitosanase from Bacillus circulans MH-K1" Acta Cryst.D51. 856-857 (1995)

Jun-ichi Saito、Yoshiho Nagata、Akikazu Ando、Akiko Kita、Kunio Miki：“环状芽孢杆菌 MH-K1 中壳聚糖酶的结晶和初步 X 射线晶体分析”Acta Cryst.D51。

斎藤純一: "キトサナーゼ研究の最前線" キチン・キトサン研究. 2. 187-196 (1996)

Junichi Saito：“壳聚糖酶研究的前沿”甲壳素和壳聚糖研究2. 187-196 (1996)。

Jun-ichi Saito,Yoshiho Nagata,Akikazu Ando,Akiko Kita,Kunio Miki: "Crystallization and preliminary X-ray crystallographic analysis of chitosanase from Bacillus circulans MH-K1." Acta Cryst.D51. 856-857 (1995)

Jun-ichi Saito、Yoshiho Nagata、Akikazu Ando、Akiko Kita、Kunio Miki：“环状芽孢杆菌 MH-K1 壳聚糖酶的结晶和初步 X 射线晶体分析。”

Jun-ichi Saito, Akikazu Ando: "The forefront of chitosanase research" Chitin & Chitosan Research.2. 187-196 (1996)

Jun-ichi Saito、Akzu Ando：“壳聚糖酶研究的最前沿”甲壳素

Shoji Okajima: "Purification and some properties of a chitosanase of Nocardioides sp." J. Gen. Appl. Microbiol.41. 351-357 (1995)

Shoji Okajima：“诺卡氏菌壳聚糖酶的纯化和一些特性。”