木瓜蛋白酶水解β-酪蛋白膜的體外實驗研究
發(fā)布時間：2019-04-17 18:41
【摘要】：目的:采用消散因子石英晶體微天平(QCM-D)體外原位、實時和動態(tài)監(jiān)測β-酪蛋白(β-CN)生物膜在Au石英晶體表面的組裝形成過程和木瓜蛋白酶水解β-CN生物膜的動力學(xué)過程,比較不同酶濃度的水解效率。采用接觸角測量儀(CAM)考察β-CN生物膜經(jīng)酶水解前后表面性狀的變化,通過原子力顯微鏡(AFM)觀察β-CN生物膜經(jīng)酶水解前后表面形貌和粗糙度的變化。方法:QCM-D監(jiān)測β-CN吸附于自組裝膜上造成的頻率(?F)、生物膜質(zhì)量(m)、和厚度(h)變化。根據(jù)Langmuir(L)和Freundlich(F)吸附等溫線的相關(guān)系數(shù)(R2)判斷β-CN在自組裝單層膜上的吸附行為。QCM-D上建立體外β-CN生物膜模型,采用不同濃度的木瓜蛋白酶水解蛋白膜,觀察?F以及m、h的改變,運用Boltzmann S形方程對實驗數(shù)據(jù)進行擬合,單因素方差分析和SNK-q檢驗比較水解參數(shù)B、Δm、Δh、V50、C和1/C。采用CAM和AFM分別考察純Au(a)、包被11-MUA(b)、激活羧基(c)、固定有β-CN(d)以及分別經(jīng)0.5μg/ml(e)和12.5μg/ml(f)的木瓜蛋白酶水解芯片表面的β-CN后接觸角的變化和芯片表面形貌和粗糙度變化。配對t檢驗比較F和L吸附等溫線的相關(guān)系數(shù),單因素方差分析和SNK-q檢驗比較成膜參數(shù)?F、Δm、Δh,水解參數(shù)B、Δm、Δh、V_(50)、C和1/C,接觸角測量值以及均方粗糙度之間的統(tǒng)計學(xué)差異。檢驗水平α= 0.05。結(jié)果:在臨界膠束濃度內(nèi),隨著β-CN濃度升高,膜的質(zhì)量和厚度增加(P 0.05),L吸附等溫線能夠更好的描述β-CN在SAM上的吸附行為。隨著酶濃度增大,膜的厚度Δh減薄,ΔF上升增多,水解參數(shù)B增大,C減小,V50縮短(P 0.05)。接觸角大小為c b d e f a(P 0.05)。芯片表面的β-CN顆粒經(jīng)酶水解后減少甚至消失,均方粗糙度大小為d e f c(P 0.05)。結(jié)論:在臨界膠束濃度內(nèi),β-CN在SAM上形成的膜為單層膜。在本實驗濃度范圍內(nèi)水解蛋白生物膜時,酶的水解效率隨濃度增大而提高。β-CN分子上的木瓜蛋白酶作用靶點位點位于肽鏈的疏水C端。高濃度的木瓜蛋白酶可均勻水解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 偽 = 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.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R346
本文編號：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 偽 = 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.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R346
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