菠萝茎蛋白酶的纯化及酶活力受超声处理影响的机理

Purification of Stem Bromelain and Mechanism of Effect of Ultrasound on Its Catalysis Activity

DOI：10.3969/j.issn.1673-1689.2012.05.005

中文关键词: 菠萝茎蛋白酶 纯化 酶活力 超声波 构象

英文关键词: stem bromelain purification enzyme activity ultrasound conformation

基金项目:国家自然科学基金项目(10074016和10174021)

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中文摘要:

对菠萝茎蛋白酶进行纯化和鉴定,并研究酶活力受超声处理影响的机理。采用羧甲基纤维素柱层析纯化、PAGE鉴定纯度,测定纯化的酶经超声波处理后的酶活性、动力学参数以及紫外和荧光光谱。柱层析分离出3个蛋白峰区域,其中第二蛋白峰经电泳鉴定纯度较高。酶经超声处理后Km变小,Vm也减小;紫外吸收光谱不变,而差示光谱出现了明显的正峰和负峰,荧光发射光谱基本不变。羧甲基纤维素柱层析对菠萝茎蛋白酶纯化效果较好,超声处理可能通过改变菠萝茎蛋白酶的构象从而改变酶活力。

英文摘要:

To purify and identify stem bromelain for further study on its activity and mechanism after treated with ultrasound.Stem bromelain was purified and identified with carboxymethyl cellulose chromatography and polyacrylamide gel electrophoresis(PAGE) respectively.Enzyme activity,kinetic parameter,ultraviolet spectrum and fluorescence spectrum were studied on purified enzyme treated with ultrasound.Three single protein apex regions were separated with gradient elution.The ingredient with the highest activity in the second protein apex region was identified with PAGE,showing considerably high purity.After treatment with ultrasound,both Km and Vm decreased.No clear changes were observed in the ultraviolet absorption spectrum and the fluorescence emission spectrum while positive and negative peaks occurred in the differential ultraviolet spectrum.Conclusion: Considerably pure component of stem bromelain can be obtained after purified with carboxymethyl cellulose chromatography.It’s probably conformation change that results activity change after treatment of the ultrasound on stem bromelain.

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