权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Structure and Function of Chitosanase from Streptomyces sp.N174

链霉菌 N174 壳聚糖酶的结构和功能

基本信息

批准号：
07660124
负责人：
FUKAMIZO Tamo
金额：
$ 1.41万
依托单位：
Kinki 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

项目摘要

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.

将链霉菌N174的壳聚糖酶基因导入变铅青链霉菌TK 24中，经S-Sepharose和Bio-Gel A柱层析纯化，得到一株产壳聚糖酶的菌株。由此获得的壳聚糖酶从β-1，4-连接的寡糖底物产生α-端基异构体，表明该酶是一种转化体。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.寡糖水解的时间过程显示，从六聚体到五聚体再到四聚体的底物降解速率降低。通过对该酶进行定点突变，并对突变体的酶活和稳定性进行了测定。Glu 22或Asp 40的取代显著降低了比活性和kappacat，而KAPPAm仅略有变化。这些结果表明Glu 22和Asp 40直接参与了壳聚糖酶的催化中心。Asp 37的突变显著降低了该酶的活性，并显著降低了热去折叠转变的中点温度（Tm）。在晶体结构中，Asp 37的羧基侧链远离结合裂缝，但与His 90的咪唑氮产生密切的相互作用。Asp 37可能通过与His 90相互作用来稳定底物结合裂缝的结构，并且其突变使结构不稳定，导致对底物的亲和力降低。