改性琼脂糖微球固定化甘油脱氢酶的研究

Study of immobilized glycerol dehydrogenase on modified agarose microspheres

通过膜乳化法制备琼脂糖微球,利用环氧氯丙烷活化琼脂糖微球,并与聚乙烯亚胺(PEI)反应引入氨基,得到胺化琼脂糖微球。利用扫描电子显微镜(SEM)、X射线能谱(EDS)、X射线光电子能谱(XPS)、傅立叶变换红外光谱(FTIR)等手段对微球的形貌和结构进行表征。通过戊二醛(GA)共价固定甘油脱氢酶(GlyDH),并催化甘油生成1,3-二羟基丙酮(DHA)。结果表明,膜乳化过程可改善载体的球形度,PEI分子量对接枝微球的N元素含量有显著影响。固定化酶的储存稳定性和循环使用性均高于游离酶。

1,3-dihydroxyacetone(DHA)is one of the most valuable chemicals with a wide range of applications in cosmetics and pharmaceutical industry.Glycerol dehydrogenase(Gly DH)is able to selectively catalyze the transformation of glycerol to DHA,by which glycerol,the oversupply by-product of the biodiesel industry is highly valued.To make enzymatic production of DHA practically feasible,immobilizing GlyD H onto a suitable insoluble support is critical for facilitating biocatalyst recovery and improving stability of the enzyme.Agarose microspheres as carrier wereprepared by membrane emulsification method.Then the agarose microspheres were activated with epichlorohydrin and reacted with polyethyleneimine(PEI)to introduce the amino group into the agarose microspheres.The morphology and structure of the microspheres were characterized by scanning electron microscope(SEM),energy dispersion spectrum(EDS),X-ray photoelectron spectroscopy(XPS)and Fourier transform infrared spectroscopy(FT-IR).GlyD H was covalently fixed with glutaraldehyde and then catalyzed the formation of 1,3-dihydroxyacetone.The results showed that the spherical degree of microspheres was enhanced due to the membrane emulsification process.The N content in grafted products was obviously effected by the relative molecular weight of PEI.The N content of PEI graft was the highest when the relative molecular mass of PEI was 10000.The effect of p H and temperature dependence,thermal stability and the reusability of GlyD H were systematically investigated.The optimum p H value of free enzyme and immobilized enzyme activity was 10.0.The storage efficiency of immobilized GlyD H was higher than that of free Gly DH after storage at 4℃for 21 days.After 7 cycles,the activity of GlyD H was still partly remained.