超声辅助酶解促进草鱼鳞胶原肽水解进程的内在机制解析

为探究超声辅助酶解促进草鱼磷胶原肽水解进程的内在原因,分别研究了超声对底物蛋白（草鱼鳞）的分子结构、表面疏水性、粒径等理化特性和蛋白酶酶解能力的影响机制,在此基础上,对超声酶解进程进行了动力学拟合,从酶解动力学角度进一步评估了促进草鱼鳞胶原肽水解进程的"超声-酶"耦合效应。结果表明,适当的超声强度（300 W、20 min）可以使底物蛋白的结构展开,此时其表面疏水性最大、粒径最小,使之更适合后续酶解,但当超声功率大于300 W时,底物蛋白会重新聚集,其中的部分疏水基团被掩埋,不利于酶解的进行。同时,当超声功率为300 W、时间为20 min时,单酶酶解组和分步酶解组的酶解能力从2.35×105 U/g、3.41×105 U/g分别提高至3.44×105 U/g、3.86×105 U/g,且表现出显著性差异（P<0.05）。通过动力学模型对超声辅助单酶酶解（r2=0.988 8）和分步酶解进程（r2=0.960 7）进行了动力学拟合,依据建立的反应动力学方程,证实了超声辅助分步酶解进程更快,研究结果为超声辅助酶解工艺制备胶原肽提供了一定理论基础。

Analysis of the internal mechanism for ultrasound-assisted enzymatic hydrolysis for promoting the hydrolysis of grass carp scale collagen peptide

Abstract: In order to explore the internal mechanism of the hydrolysis process of collagen peptide derived from grass carp scale (Ctenopharyngodon idellus), the effects of ultrasound treatment on the structure of substrate protein (grass carp scale) and the hydrolysis ability of protease were studied by Fourier transform infrared spectra, ultraviolet spectra, circular dichroism spectra, surface hydrophobicity, endogenous fluorescence spectra, particle size and enzyme activity. On this basis, the kinetics of ultrasound-assisted enzymatic hydrolysis was fitted. First of all, Fourier Transform Infrared Spectroscopy (FT-IR), Ultraviolet Spectroscopy (UV-vis), Circular Dichroism (CD), Surface Hydrophobicity (S0-ANS), endogenous fluorescence spectroscopy and particle size were all employed to investigate the effects of ultrasound on the structure of substrate protein (grass carp scale). The FT-IR results indicated that the absorption peak of the substrate protein in amide A band showed a trend of first blue-shift and then red-shift with the increase of ultrasound intensity. In the meanwhile, the intensity of negative absorption of the substrate protein at 198 nm in CD spectrum, the fluorescence intensity of the endogenous fluorescence peaks in endogenous fluorescence spectroscopy and absorbance values of absorption peaks in UV-vis were all showed a first increased and then decreased tendency with the increasing of ultrasound intensity. Moreover, with the increasing of ultrasound intensity, the surface hydrophobicity of the substrate protein was increased firstly and then decreased, and the particle size was reduced firstly and then increased. All of these results indicated that the appropriate ultrasound power (300 W, 20 min) led to the expansion of substrate protein, which made it more suitable for the subsequently enzymatic hydrolysis. However, when the ultrasound power was greater than 300 W, the substrate protein would be re-aggregated so that some of the hydrophobic groups were buried, which was not conducive to the enzymatic hydrolysis. At the same time, with the increasing of ultrasound intensity, enzymatic hydrolysis capacity of protease in single-enzyme hydrolysis group was increased firstly and then decreased. When the ultrasound treatment was conducted in 300 W for 20 min, the enzymatic hydrolysis capacity of alkaline protease was increased from 2.35×105 U/g to 3.44×105 U/g. Furthermore, when the ultrasound treatment of 300 W and 10 min was applied in each step of the step-by-step enzymatic hydrolysis group, the enzymatic hydrolysis capacity was increased from 3.41×105 U/g to 3.86×105 U/g. Finally, the kinetics of the ultrasound-assisted enzymatic hydrolysis process were fitted by the kinetic model, and the reaction kinetic equation was established, which further demonstrated that the ultrasound-assisted step-by-step enzymatic hydrolysis process was faster than single-enzyme hydrolysis. In summary, ultrasound treatment could speed up the enzymatic hydrolysis process by destroying the structure of the substrate protein and enhancing the enzymatic hydrolysis ability of protease, thereby improving the enzymatic hydrolysis efficiency.