超声功率对胰蛋白酶的酶活性与结构变化的影响
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摘要
为研究超声功率引起胰蛋白酶的结构变化与酶活力之间的关系,研究了不同的超声功率对胰蛋白酶活力、热稳定性、酶解反应动力学和热力学参数的影响,并采用紫外光谱、傅立叶变换红外光谱和荧光光谱来分析超声功率对胰蛋白酶结构的影响。结果表明:除功率300,350 W外,胰蛋白酶活性与对照相比有显著提高。80℃处理时,在功率300,350 W时的胰蛋白酶热稳定性有显著性提升。20℃酶解时75 W处理的胰蛋白酶的反应速率最快。200 W处理时胰蛋白酶的表观活化能最小,150,200 W处理时胰蛋白酶的活化焓和活化熵与对照相比显著降低,75～250 W处理时胰蛋白酶的吉布斯自由能与对照相比无明显差异。光谱分析表明,超声功率不改变胰蛋白酶的肽键含量,然而显著改变其二级结构。超声功率诱导胰蛋白酶变性过程中,在200 W出现1个中间体,且中间体状态的胰蛋白酶的有序性高,具有高酶活和反应速率。
To study the relationship of changes induced by ultrasonic power in trypsin structure and activity.The effects of ultrasonic power on trypsin activity,thermal stability,kinetics and thermodynamic parameters were studied.The effects of ultrasonic power on the structure of trypsin were analyzed by UV,Fourier transform infrared spectroscopy and fluorescence spectroscopy.The results showed that,on the one hand,except 300 and 350 W,the enzyme activity was significantly higher than that of the control.At 80 °C,the thermal stability of trypsin at 300 W and 350 W was significant.When the trypsin was treated with ultrasonic power at 75 W and the enzyme hydrolysis temperature at 20 °C,the reaction rate was the fast.When the trypsin was treated with ultrasonic power at 200 W,the apparent energy of activation was the smallest.Enthalpy of activation and entropy of activation at 150 W and 200 W were significantly lower than the control.Gibbs free energy at 75-250 W was not significantly different from that of the control.On the other hand,spectral analysis showed that ultrasonic power did not alter the molecular configuration of trypsin,but significantly changed its secondary structure.In the process of ultrasonic power induced trypsin degeneration,one intermediate appeared at 200 W.And the intermediate state of the trypsin had high order,while high enzymatic activity and high response rate.
To study the relationship of changes induced by ultrasonic power in trypsin structure and activity.The effects of ultrasonic power on trypsin activity,thermal stability,kinetics and thermodynamic parameters were studied.The effects of ultrasonic power on the structure of trypsin were analyzed by UV,Fourier transform infrared spectroscopy and fluorescence spectroscopy.The results showed that,on the one hand,except 300 and 350 W,the enzyme activity was significantly higher than that of the control.At 80 °C,the thermal stability of trypsin at 300 W and 350 W was significant.When the trypsin was treated with ultrasonic power at 75 W and the enzyme hydrolysis temperature at 20 °C,the reaction rate was the fast.When the trypsin was treated with ultrasonic power at 200 W,the apparent energy of activation was the smallest.Enthalpy of activation and entropy of activation at 150 W and 200 W were significantly lower than the control.Gibbs free energy at 75-250 W was not significantly different from that of the control.On the other hand,spectral analysis showed that ultrasonic power did not alter the molecular configuration of trypsin,but significantly changed its secondary structure.In the process of ultrasonic power induced trypsin degeneration,one intermediate appeared at 200 W.And the intermediate state of the trypsin had high order,while high enzymatic activity and high response rate.
引文
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[24]贾俊强.超声对酶法制备小麦胚芽ACE抑制肽的影响及其作用机理[D].镇江:江苏大学,2010.
[25]QU P,WANG Y X,WU G H,et,al.Effect of polyethylene glycols on the alkaline-induced molten globule intermediate of bovine serum albumin[J].International Journal of Biological Macromolecules,2012,51(1/2):97-104.
[26]刘莉.猪胃蛋白酶变性复性过程及其部分折叠中间体的研究[D].杨凌:西北农林科技大学,2007.
[27]倪国华,宋焕禄.凝胶过滤分离鸡肉蛋白酶解物时紫外检测波长的选择[J].食品研究与开发,2002,23(5):73-75.
[28]MICHAEL J,HENRY H M.Protein secondary structure from FT-IR spectroscopy:Correlation with dihedral angles from three-dimensional Ramachandran plots[J].Canadian Journal of Chemistry,1991,69(11):1639-1642.
[29]唐传核,马正勇.采用FTIR技术研究高压处理对冻干大豆分离蛋白构象的影响[J].光谱学与光谱分析,2009,29(5):1237-1240.
[30]刘平,胡志和,吴子健,等.超高压引发胰蛋白构象变化与酶活性间的关系[J].光谱学与光谱分析,2015,35(5):1335-1339.
[2]WALSH K A.Trypsinogens and trypsins of various species[J].Methods enzymol,1970,19:41-63.
[3]曹玉华,王肇慈,俞斌.应用固定化胰蛋白酶制备大豆肽的研究[J].中国粮油学报,2002,17(3):18-21.
[4]温燕梅,邱彩虹.吸附-交联法固定胰蛋白酶及在澄清啤酒中的应用[J].广东海洋大学学报,2001,21(4):42-46.
[5]温燕梅,卢泽勤.磁性固定化胰蛋白酶的催化特性及应用的研究[J].生物技术,2005,15(4):56-58.
[6]王慧.以海藻酸钙为载体固定化胰蛋白酶[D].沈阳:沈阳药科大学,2011.
[7]MOMENI L,SHAREGHI B,SABOURY A,et,al A spectroscopic and thermal stability study on the interaction between putrescine and bovine trypsin[J].International Journal of Biological Macromolecules,2017,94:145-153.
[8]刘军平.聚乙二醇修饰对去折叠胰蛋白酶的影响[D]南昌:南昌大学,2012.
[9]GIZURARSON J G K,FILIPPUSSON H.Conjugation of D-glucosamine to bovine trypsin increases thermal stability and alters functional properties[J].Enzyme and Microbial Technology,2015,75:1-9.
[10]黄继红,张龙飞,王文.右旋糖苷修饰胰蛋白酶条件优化及性质研究[J].发酵科技通讯,2015,44(4):1-5.
[11]邹立强,刘伟,刘军平,等.m PEG-SS修饰对DH-PM去折叠胰蛋白酶相对活性、热稳定性和动力学参数的影响[J].食品工业科技,2011,32(12):103-105.
[12]MOMENI L,SHAREGHI B,SABOURY A A.Aspectroscopic and thermal stability study on the interaction between putrescine and bovine trypsin[J].International Journal of Biological Macromolecules,2017,94(Part A):145-153.
[13]TREETHARNMATHUROT B,OVARTLARNPORNC,WUNGSINTAWEEKUL J.Effect of PEG molecular weight and linking chemistry on the biological activity and thermal stability of PEGylated trypsin[J].International Journal of Pharmaceutics,2008,357(1/2):252-259.
[14]KHURANA R,FINK A.Do parallel beta-helix proteins have a unique fourier transform infrared spec trum[J].Biophysical Journal,2000,78(2):994-1000.
[15]张兆琴.动态高压微射流技术对胰蛋白酶和菠萝蛋白酶性质和构象变化的影响[D].南昌:南昌大学,2010.
[16]朱建华,杨晓泉,熊犍.超声波技术在食品工业中的最新应用进展[J].酿酒,2005,32(2):54-57.
[17]马亚琴,叶兴乾,吴厚玖,等.超声波辅助提取植物活性成分的研究进展[J].食品科学,2010,31(21):459-463.
[18]LI S Y,YANG X,ZHANG Y Y.Enzymolysis kinetics and structural characteristics of rice protein with energy-gathered ultrasound and ultrasound assisted alkali pretreatments[J].Ultrasonics Sonochemistry,2016,31:85-92.
[19]LI S Y,YANG X,ZHANG Y Y.Effects of ultrasound and ultrasound assisted alkaline pretreatments on the enzymolysis and structural characteristics of rice protein[J].Ultrasonics Sonochemistry,2016,31:20-28.
[20]JIN J,MA H L,WANG K.Effects of multi-frequency power ultrasound on the enzymolysis and structural characteristics of corn gluten meal[J].Ultrasonics Sonochemistry,2015,24:55-64.
[21]毕爽,齐宝坤,隋晓楠,等.超声处理对黑豆蛋白结构和功能性质的影响[J].中国食品学报,2016,16(6):153-160.
[22]蛋白酶制剂:GB/T 23527-2009[S].北京:中国标准出版社,2009.
[23]KADKHODAEE R,POVEY M J W.Ultrasonic inactivation of Bacillusα-amylase.I.effect of gas content and emitting face of probe[J].Ultrasonics Sonochemistry,2008,15(2):133-142.
[24]贾俊强.超声对酶法制备小麦胚芽ACE抑制肽的影响及其作用机理[D].镇江:江苏大学,2010.
[25]QU P,WANG Y X,WU G H,et,al.Effect of polyethylene glycols on the alkaline-induced molten globule intermediate of bovine serum albumin[J].International Journal of Biological Macromolecules,2012,51(1/2):97-104.
[26]刘莉.猪胃蛋白酶变性复性过程及其部分折叠中间体的研究[D].杨凌:西北农林科技大学,2007.
[27]倪国华,宋焕禄.凝胶过滤分离鸡肉蛋白酶解物时紫外检测波长的选择[J].食品研究与开发,2002,23(5):73-75.
[28]MICHAEL J,HENRY H M.Protein secondary structure from FT-IR spectroscopy:Correlation with dihedral angles from three-dimensional Ramachandran plots[J].Canadian Journal of Chemistry,1991,69(11):1639-1642.
[29]唐传核,马正勇.采用FTIR技术研究高压处理对冻干大豆分离蛋白构象的影响[J].光谱学与光谱分析,2009,29(5):1237-1240.
[30]刘平,胡志和,吴子健,等.超高压引发胰蛋白构象变化与酶活性间的关系[J].光谱学与光谱分析,2015,35(5):1335-1339.