超高压处理菠萝蛋白酶引发构象变化与酶活力的关系

Relationship between Bromelain Activity and Conformational Changes Caused by Ultra High Static Pressure

采用超高压处理菠萝蛋白酶,研究菠萝蛋白酶结构变化与酶活力的关系。采用傅里叶红外光谱(Fourier transform infrared spectrometer,FTIR)检测二级结构变化,荧光光谱检测三级结构变化,采用福林酚法测定酶活力。结果显示,与0.1 MPa相比,在37℃,不同压力(100~500 MPa)处理20 min,对菠萝蛋白酶活力均有显著影响(P<0.05)。200 MPa处理时,酶活力达到最大值(3 524 U/g),提高了8.29%。200 MPa处理后的菠萝蛋白酶,经傅里叶红外光谱分析结果显示,β-折叠含量比常压下增加了1.76倍。内源性荧光光谱结果显示,280 nm波长处激发,荧光强度达到最高4 934。因此,菠萝蛋白酶的活性与β-折叠的含量以及亲水性Typ残基的暴露程度有关。

The relationship between structural changes and bromelain activity was investigated. After bromelain was treated at various pressure levels, its secondary structure changes were observed by Fourier transform infrared spectroscopy(FTIR), its tertiary structure changes were examined by fluorescence spectroscopy, and its activity was tested using Folin phenol method. The results showed that compared with the control group(0.1 MPa), the bromelain activity significantly changed after treatment at 37 ℃ for 20 min at 100-500 MPa(P < 0.05). The maximum level(3 524 U/g) of bromelain activity was observed after treatment of the enzyme at 200 MPa, representing a 8.29% increase over the control group. Moreover, the treated enzyme displayed a 2.76-fold increase in β-sheet content as analyzed by Fourier transform infrared spectroscopy. The maximum endogenous fluorescence spectral intensity of 4 934 was found at excitation wavelength of 280 nm. Therefore, bromelain activity is related to the β-sheet content and the exposure degree of Tyr residues.