酶法制备壳寡糖及其生物学功能

用正交试验方法考察温度、酶浓度、pH对蜗牛酶降解壳聚糖的影响,筛选蜗牛酶降解壳聚糖的最佳反应条件,采用SDS-PAGE方法分析降解产物,制备具有生物学功能的壳寡糖。用不同浓度的壳寡糖处理人肝癌HepG2细胞,观察细胞形态学变化,MTT法检测壳寡糖对其增殖的影响,琼脂糖凝胶电泳检测DNA变化,流式细胞术检测凋亡率(AR)。结果表明:蜗牛酶降解壳聚糖的产物主要是聚合度为4以上的寡糖,更多的接近壳六糖。最佳反应条件为pH 4.0、温度40℃、酶和底物质量比为4∶50;壳寡糖质量浓度在2~4 mg/mL时,对HepG2细胞增殖有抑制效应,细胞经壳寡糖处理48 h后,开始空泡化,DNA出现明显的凋亡条带,AR明显高于对照组。在最佳反应条件下蜗牛酶能较好地降解壳聚糖,制备的壳寡糖在一定浓度范围内能通过诱导HepG2细胞发生凋亡而抑制其增殖,其作用呈浓度依赖性。

Enzymatic production of chitooligosaccharide and its biological functions

The hydrolysis of chitosan by snail enzymes was investigated with orthogonal test in factors of temperature,enzyme concentration and pH.SDS-PAGE was used to analyze the hydrolysates for preparing chitooligosaccharides with biological functions.With different concentrations of chitooligosaccharide processing of human hepatoma HepG2 cells,morphological changes were observed,the proliferation activity by MTT assay and changes of DNA by agarose gel electrophoresis were detected,and following flow cytometry was used to detect the apoptotic rate（AR）.The results showed that polymerization degree of the major hydrolysis products was above 4.The optimal conditions for chitosan hydrolysis were as follows：pH 4.0,temperature 40 ℃,the ratio of enzyme to substrate 8∶100.When chitooligosaccharide concentration was 2～4 mg/mL,it affected HepG2 cell proliferation.After treating HepG2 cells with chitoosligoaccharide about 48 h,the cells began to vacuolization and DNA ladder strip appear distinctly,AR was higher than control.Chitosan was hydrolyzed by snail enzyme under the optimal condition on a certain concentration,and chitooligosaccharide inhibited the proliferation of HepG2 cells.