采用超高压技术处理胰蛋白酶,改变其空间结构,研究酶空间结构变化与酶活力之间的关系.采用傅立叶红外光谱(FTIR)检测超高压处理后胰蛋白酶的二级结构变化;采用荧光光谱检测处理后胰蛋白酶的三级结构;酶活力的检测采用福林酚法.结果显示,与未处理的相比,在37 ℃,不同压力(100～600 MPa)条件处理20 min,对胰蛋白酶活力影响显著(p＜0.05).其中,300 MPa处理,胰蛋白酶活力达到最大,较未处理的酶活提高了0.386倍.FTIR检测分析显示,300 MPa处理的胰蛋白酶,α-螺旋与β-转角的峰面积比值达到最大(2.749);内源性荧光光谱检测结果显示,当激发波长为295 nm,其荧光强度达到最高值(1 353);激发波长为280 nm,其荧光强度达到最高(4 262);外源性荧光光谱结果显示,当激发波长为228 nm,疏水氨基酸残基的荧光强度达到最高(2 022);上述荧光强度的变化较0.1 MPa处理的胰蛋白酶均有显著差异(p＜0.05).结论:超高压处理影响胰蛋白酶的空间结构及酶活性.其中,胰蛋白酶活性与α-螺旋和β-转角的峰面积的比值、色氨酸等疏水氨基酸及酪氨酸残基暴露程度有关。

Trypsin was treated by high pressure technology,and its spatial structure was changed,the relationship between structural changes and trypsin activity was investigated.The secondary structure change of trypsin after pressure treatment was observed by Fourier transform infrared spectroscopy(FTIR).Moreover its tertiary structure change was observed by fluorescence spectroscopy;and its activity was tested using Folin phenol method.The results showed that,compared with the untreated(0.1 MPa),trypsin activity change was significant(p<0.05) under different pressure(100～600 MPa) treatment at 37 ℃ for 20 min.After treated with 300 MPa,its activity was 0.386 times higher than the untreated.Secondary structure of trypsin was analysed using FTIR,and the peak area ratio of α-helix and β-turn in secondary structure was the maximum(2.749);Endogenous fluorescence spectra intensity was the maximum(1 353) at excitation wavelength 295 nm,and was 4 262 at excitation wavelength 280 nm;exogenous fluorescent spectra intensity was 2 022 at excitation wavelength 228 nm,all these change was remarkable(p<0.05)comparing with the untreated.Therefore,ultrahigh pressure processing influence on the spatial structure of trypsin and induce enzyme activity change.Trypsin activity is relate to the peak area ratio of α-helix and β-turn and the exposure degree of Trp and other hydrophobic a mino acid residues and Tyr.