软叶刺葵果实过氧化物酶的提取及其催化合成生物基印刷染料的研究

Purification and Characterization of Peroxidase from Fruit of Phenix roebelenii O.Brien for Catalytic Synthesis of Biobased-Dyes

本研究从软叶刺葵果实中提取出粗过氧化物酶,通过丙酮沉淀、离子交换层析和超滤等方法对其进行纯化,得到分子质量为39670的电泳纯级过氧化物酶(PRP),对愈创木酚的最适催化条件为pH值5、温度60℃。通过测试不同金属离子、有机溶剂以及表面活性剂对PRP酶活性的影响,结果表明,1 g/L的Span-80在常温、pH值5、添加Mn2+或乙醇条件下可提高PRP的酶活性。以愈创木酚、3,3′-二甲氧基联苯胺和邻苯二酚为底物,测得PRP的米氏常数分别为1.053、5.563和2.316 mmol/L。PRP催化邻苯二酚和儿茶素、愈创木酚、3,3′-二甲氧基联苯胺分别得到黑色、橙红色和棕红色的染料,用纤维素衍生物调制后可用于丝网印刷,其中黑色水性墨在A4纸上的印刷密度达到0.72。

Crude peroxidase was extracted from the fruit of Phenix roebelenii O.Brien,and purified by acetone precipitation,ion exchange chromatography and ultrafiltration.P.roebelenii Peroxidase(PRP)of electrophoretic purity was finally obtained with a molecular weight of 39670.The optimal catalytic conditions for guaiacol were pH value 5 and 60℃.By studying the effects of different metal ions,organic solvents and surfactants on PRP activity,it was found that Span-80 of 1 g/L could improve the activity of PRP at room temperature,pH value 5,Mn^(2+)or ethanol.Using guaiacol,3,3'-dimethoxybenzidine and catechol as substrates,the Michaelis constant of PRP were determined to be 1.053,5.563 and 2.316 mmol/L,respectively.Black,orange-red and brown-red dyes were obtained with PRP catalyzed catechol,catechin,guaiacol and 3,3'-dimethoxybenzidine,respectively.After being modulated with cellulose derivatives,it could be used for screen printing.The printing density of black water-based ink on A4 paper could reach 0.72.The peroxidase extracted from Phenix roebelenii O.Brien fruit is a green biocatalyst for synthesizing bio-based printing dyes.