摘要
分别用 PCMB、NEM、N- AI、NBS等对诺卡氏菌形放线菌β- D-甘露聚糖酶进行化学修饰 ,证明蛋白上的巯基、酪氨酸残基及色氨酸残基是维持酶活性的必需基团 .在加入少量底物后 ,β- D-甘露聚糖酶的最大荧光发射峰从天然状态下的 336nm处蓝移至 332 nm,且峰强度有所增大 .这表明其色氨酸残基隐藏在蛋白内部的疏水区域 .通过对该酶圆二色性扫描光谱的分析 ,表明蛋白内部有二硫键的存在 ;通过巯基乙醇化学修饰的研究 ,表明二硫键是影响该酶热稳定性的一个重要因素 .在蛋白的各种二级结构中 ,α-螺旋、β-折叠、β-转角、自由卷曲的比例分别为 1 6.6%、2 5.4%、2 0 .5%和 37.5% .
The modification of PCMB, NEM N AI, NBS resulted in complete and partial loss of the activity of β D mannanase (EC 3 2 1 78) from Nocardioform actinomycetes NA3 540 respectively. It was shown that sulfhydryl residues, tryptophan residues and tyrosine residues might be involved in the active site of the enzyme. In the presence of ligands binding, the λ max emission fluorescence spectrum of the enzyme changed from 336 nm to 332 nm. It appeared that tryptophan residues were involved in a widely hydrophobic micro environment near the active site of protein molecule. The states of disulfide bonds were analyzed by CD spectroscopy. That decreasing thermostability of the enzyme in the presence of α mercaptoethanol implied that disulfide bonds were essential for the enzyme stability in the high temperature. In addition, CD spectrum indicated that the secondary structure of the enzyme consisted of 16 6% α helix, 25 4% β sheet, 20 5% β turn and 37 5% random coil.
出处
《中国生物化学与分子生物学报》
CSCD
2000年第2期227-230,共4页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家"八五"科技攻关项目部分内容!( 85 -72 2 -14 -0 2 )
