木瓜蛋白酶水解β-酪蛋白膜的体外实验研究
发布时间：2019-04-17 18:41
【摘要】：目的:采用消散因子石英晶体微天平(QCM-D)体外原位、实时和动态监测β-酪蛋白(β-CN)生物膜在Au石英晶体表面的组装形成过程和木瓜蛋白酶水解β-CN生物膜的动力学过程,比较不同酶浓度的水解效率。采用接触角测量仪(CAM)考察β-CN生物膜经酶水解前后表面性状的变化,通过原子力显微镜(AFM)观察β-CN生物膜经酶水解前后表面形貌和粗糙度的变化。方法:QCM-D监测β-CN吸附于自组装膜上造成的频率(?F)、生物膜质量(m)、和厚度(h)变化。根据Langmuir(L)和Freundlich(F)吸附等温线的相关系数(R2)判断β-CN在自组装单层膜上的吸附行为。QCM-D上建立体外β-CN生物膜模型,采用不同浓度的木瓜蛋白酶水解蛋白膜,观察?F以及m、h的改变,运用Boltzmann S形方程对实验数据进行拟合,单因素方差分析和SNK-q检验比较水解参数B、Δm、Δh、V50、C和1/C。采用CAM和AFM分别考察纯Au(a)、包被11-MUA(b)、激活羧基(c)、固定有β-CN(d)以及分别经0.5μg/ml(e)和12.5μg/ml(f)的木瓜蛋白酶水解芯片表面的β-CN后接触角的变化和芯片表面形貌和粗糙度变化。配对t检验比较F和L吸附等温线的相关系数,单因素方差分析和SNK-q检验比较成膜参数?F、Δm、Δh,水解参数B、Δm、Δh、V_(50)、C和1/C,接触角测量值以及均方粗糙度之间的统计学差异。检验水平α= 0.05。结果:在临界胶束浓度内,随着β-CN浓度升高,膜的质量和厚度增加(P 0.05),L吸附等温线能够更好的描述β-CN在SAM上的吸附行为。随着酶浓度增大,膜的厚度Δh减薄,ΔF上升增多,水解参数B增大,C减小,V50缩短(P 0.05)。接触角大小为c b d e f a(P 0.05)。芯片表面的β-CN颗粒经酶水解后减少甚至消失,均方粗糙度大小为d e f c(P 0.05)。结论:在临界胶束浓度内,β-CN在SAM上形成的膜为单层膜。在本实验浓度范围内水解蛋白生物膜时,酶的水解效率随浓度增大而提高。β-CN分子上的木瓜蛋白酶作用靶点位点位于肽链的疏水C端。高浓度的木瓜蛋白酶可均匀水解β-CN膜。
[Abstract]:Objective: to establish an in situ quartz crystal microbalance (QCM-D) with dissipation factor. The assembly process of 尾-casein (尾-CN) biomembrane on the surface of Au quartz crystal and the kinetics of papain hydrolysis of 尾-CN biofilm were monitored in real-time and dynamically. The hydrolysis efficiency of 尾-casein biofilm with different enzyme concentrations was compared. The surface characteristics of 尾-CN biofilm before and after enzymatic hydrolysis were investigated by contact angle meter (CAM), and the changes of surface morphology and roughness of 尾-CN biofilm before and after enzymatic hydrolysis were observed by atomic force microscopy (AFM). Methods: QCM-D was used to monitor the frequency of 尾-CN adsorbed on self-assembled membrane (? F), biofilm mass (m), and thickness (h). The adsorption behavior of 尾-CN on self-assembled monolayer membrane was evaluated by the correlation coefficient (R2) of adsorption isotherm between Langmuir (L) and Freundlich (F). The model of 尾-CN biofilm in vitro was established on QCM-D. The protein membrane was hydrolyzed by papain at different concentrations. The changes of? F and m, h were observed. The experimental data were fitted by Boltzmann S-form equation. The hydrolysis parameters B, 螖 m, 螖 h, V _ 50 were compared by one-way ANOVA and SNK-q test, and the hydrolytic parameters B, 螖 m, 螖 h, V _ (50) were compared with each other. C and 1 C The activation of carboxyl (c), by pure Au (a), coated with 11-MUA (b), was investigated by CAM and AFM, respectively. After 尾-CN (d) was immobilized and 尾-CN was hydrolyzed by 0.5 渭 g / ml (e) and 12.5 渭 g / ml (f) of papain, respectively, the changes of contact angle and surface morphology and roughness of the chip were observed. The correlation coefficients of adsorption isotherms of F and L were compared by paired t-test, and the parameters of membrane formation, F, 螖 m, 螖 h, B, 螖 m, 螖 h, V _ (50), C) and 1 渭 C, were compared by one-way ANOVA and SNK-q test. Statistical differences between contact angle measurements and mean square roughness. Test level 伪 = 0.05. Results: in critical micelle concentration, the mass and thickness of 尾-CN membrane increased with the increase of 尾-CN concentration (P 0.05), L adsorption isotherm could better describe the adsorption behavior of 尾-CN on SAM). With the increase of enzyme concentration, the membrane thickness 螖 h decreased, 螖 F increased, hydrolysis parameter B increased, C decreased and V50 shortened (P 0.05). The contact angle is c b d e f a (P 0.05). The 尾-CN particles on the chip surface decreased or even disappeared after enzymatic hydrolysis, and the mean square roughness was d e f c (P 0.05). Conclusion: in critical micelle concentration, 尾-CN formed on SAM as a monolayer. When the protein biofilm was hydrolyzed within the concentration range of the experiment, the hydrolysis efficiency of the enzyme increased with the increase of the concentration. The target of papain on 尾-CN molecule was located at the hydrophobic C-terminal of the peptide chain. High concentration of papain can hydrolyze 尾-CN membrane uniformly.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346
本文编号：2459692
[Abstract]:Objective: to establish an in situ quartz crystal microbalance (QCM-D) with dissipation factor. The assembly process of 尾-casein (尾-CN) biomembrane on the surface of Au quartz crystal and the kinetics of papain hydrolysis of 尾-CN biofilm were monitored in real-time and dynamically. The hydrolysis efficiency of 尾-casein biofilm with different enzyme concentrations was compared. The surface characteristics of 尾-CN biofilm before and after enzymatic hydrolysis were investigated by contact angle meter (CAM), and the changes of surface morphology and roughness of 尾-CN biofilm before and after enzymatic hydrolysis were observed by atomic force microscopy (AFM). Methods: QCM-D was used to monitor the frequency of 尾-CN adsorbed on self-assembled membrane (? F), biofilm mass (m), and thickness (h). The adsorption behavior of 尾-CN on self-assembled monolayer membrane was evaluated by the correlation coefficient (R2) of adsorption isotherm between Langmuir (L) and Freundlich (F). The model of 尾-CN biofilm in vitro was established on QCM-D. The protein membrane was hydrolyzed by papain at different concentrations. The changes of? F and m, h were observed. The experimental data were fitted by Boltzmann S-form equation. The hydrolysis parameters B, 螖 m, 螖 h, V _ 50 were compared by one-way ANOVA and SNK-q test, and the hydrolytic parameters B, 螖 m, 螖 h, V _ (50) were compared with each other. C and 1 C The activation of carboxyl (c), by pure Au (a), coated with 11-MUA (b), was investigated by CAM and AFM, respectively. After 尾-CN (d) was immobilized and 尾-CN was hydrolyzed by 0.5 渭 g / ml (e) and 12.5 渭 g / ml (f) of papain, respectively, the changes of contact angle and surface morphology and roughness of the chip were observed. The correlation coefficients of adsorption isotherms of F and L were compared by paired t-test, and the parameters of membrane formation, F, 螖 m, 螖 h, B, 螖 m, 螖 h, V _ (50), C) and 1 渭 C, were compared by one-way ANOVA and SNK-q test. Statistical differences between contact angle measurements and mean square roughness. Test level 伪 = 0.05. Results: in critical micelle concentration, the mass and thickness of 尾-CN membrane increased with the increase of 尾-CN concentration (P 0.05), L adsorption isotherm could better describe the adsorption behavior of 尾-CN on SAM). With the increase of enzyme concentration, the membrane thickness 螖 h decreased, 螖 F increased, hydrolysis parameter B increased, C decreased and V50 shortened (P 0.05). The contact angle is c b d e f a (P 0.05). The 尾-CN particles on the chip surface decreased or even disappeared after enzymatic hydrolysis, and the mean square roughness was d e f c (P 0.05). Conclusion: in critical micelle concentration, 尾-CN formed on SAM as a monolayer. When the protein biofilm was hydrolyzed within the concentration range of the experiment, the hydrolysis efficiency of the enzyme increased with the increase of the concentration. The target of papain on 尾-CN molecule was located at the hydrophobic C-terminal of the peptide chain. High concentration of papain can hydrolyze 尾-CN membrane uniformly.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346
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