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

Comparison of primary, secondary and tertiary structure of xylanase of Bacillus pumilus and cellulase of Aspergillus acleatus.

短小芽孢杆菌木聚糖酶和曲霉纤维素酶一级、二级和三级结构的比较。

基本信息

批准号：
03453129
负责人：
OKADA Hirosuke
金额：
$ 4.35万
依托单位：
The Kumamoto Institute of Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1993
项目状态：
已结题

项目摘要

The tertiary structure of F1-CM-cellulase of Aspergillus acleatus was found to be very similar to that of xylanase of Bacillus pumilus by X-ray crystallographic analysis, though no significant homology was observed in the amino acid sequence. It was considered that such a topology of both enzymes might be widely distributed among hydrolases of small molecular mass. We obtained many mutant xylanases by random mutagenesis to investigate the relationship between the function of xylanase and the substitution in the amino acid sequence.Four heat-resistant mutants of xylanase (N56, N102, N104 and F1) were obtained. The mutant xylanases had the following amino acid changes : N56, S26 to W, G38 to D, and Y126 to S ; N102, G38 to D ; N104, G38 to S and R48 to K ; F1, S12 to C.Kinetic studies showed that N104 is stabilized by an increase in the activation enthalpy, while the other mutants are stabilized by a decrease in the activation entropy.p-Nitrophenyl-beta-D-xylopyranobioside (pNPX2) was found to be a good model substrate of the xylanase to investigate the interaction between enzyme molecule and substrate. One mutant of xylanase having higher activity to pNPX2 was obtained. The amino acid change was S76 to G in the mutant. Kinetic studies showed that the higher activity was caused by an increase of Vmax value, not by a decrease of Km value.

X射线衍射分析表明，Acleatus曲霉F1-CM-纤维素酶的三级结构与短小芽孢杆菌木聚糖酶的三级结构非常相似，但氨基酸序列没有明显的同源性。据认为，这种拓扑结构的两种酶可能广泛分布在小分子量的水解酶。为了研究木聚糖酶的功能与其氨基酸序列的关系，我们采用随机诱变的方法获得了多株木聚糖酶突变株，得到了4株木聚糖酶耐热突变株（N56、N102、N104和F1）。突变体木聚糖酶具有以下氨基酸变化：N56，S26变为W，G38变为D，Y126变为S ; N102，G38变为D ; N104，G38变为S，R48变为K ;动力学研究表明，N104通过活化焓的增加而稳定，对硝基苯基-β-D-吡喃木糖苷（pNPX 2）是研究木聚糖酶分子与底物相互作用的良好模型底物。获得了一株对pNPX 2有较高酶活的木聚糖酶突变株。突变体的氨基酸由S76变为G。动力学研究表明，酶的高活性是由于Vmax值的增加而不是Km值的降低。