摘要
建立了监测葡萄糖异构酶在不同浓度变性剂中构象变化过程的高效液相色谱法。葡萄糖异构酶在天然条件下，以四聚体形式存在，在不同变性剂的不同浓度下，会解聚成二聚体或单体，或整体结构变松散。
Glucose isomerase (GI) can catalyze in vitro the isomerization of D glucose to D fructose. So it is an extremely important industrial enzyme in the commercial conversion of starch to high fructose syrups. In the previous papers, we have purified and characterized the enzyme from streptomyces diastaticus M1033 of China and obtained the crystal structures by X ray. In this paper, a method for measurement of the dynamic conformational change procedure of glucose isomerase in various concentrations of denaturants by HPLC has been established. At first the relative molecular mass of GI in solution is measured by HPLC on PROTEIN PAK 300SW (7.5 mm i.d.×30 cm) column The relative molecular mass of GI is about 150 000. So GI exists as tetramer in the solution without denaturants. In 0 5 mol/L guanidine hydrochloride, incubated at 30 ℃ for 30 min, GI is gradually dissociated into monomer, and at the same time its activity gradually disappears. In various concentrations of urea and incubation at 30 ℃ for 30 min (or 60℃ for 1 h), the results are different from that in guanidine, because the monomers peaks of GI is not found. Only in certain concentrations of urea, the small dimer peaks of GI is found, but the activity of GI significantly disappears. Moreover as the increase of the urea concentration, the retention time of tetramer peak is gradually decreases. From the fluorescence spectra, we found the conformation of GI changed in the solution of urea. So perhaps in urea, the conformation of GI become a little unfolded, and the active region is partly damaged, which makes GI partly inactive. Dissociation into inactive monomers and conformation partly unfolding are all the reason of GI inactivation in denaturants.
出处
《色谱》
CAS
CSCD
北大核心
1999年第5期462-465,共4页
Chinese Journal of Chromatography
基金
国家"八六三"资助
关键词
葡萄糖异构酶
变性剂
HPLC
构象变化
活性
high performance liquid chromatography, glucose isomerase, denaturant
