碱性蛋白酶Alcalase酶解大米蛋白制备小分子肽的动力学研究

Enzymatic Kinetics of Small Peptides Prepared from Rice Proteins with Alcalase

摘要:

以大米蛋白为原料，用碱性蛋白酶Alcalase2.4 L酶解大米蛋白制备小分子多肽。采用单因素试验方法优化酶解条件，考察酶解过程中pH、加酶量、底物浓度和温度酶解初速度的影响，并建立了酶解动力学方程。研究了最优酶解条件下酶解过程中酶解产物的分子量分布状态。结果表明，Alcalase2.4 L酶解大米蛋白的最优pH 8.5、温度65 ℃、酶底比0.096 AU/g（底物），在酶解过程中存在产物抑制，在研究的底物浓度范围内（<90 g/kg）不存在底物抑制。主要动力学参数为：Km为5.76 (g·min)/mmol，Vmax为0.67 mmol/(kg·min)，k2为0.28 mmol/(AU·min)。酶解动力学方程为：1/V0=56.29/[S0][E0]+1/0.28[E0]。酶解至3 h时水解度达到16%，酶解产物的分子量在264~584 u之间的组分达到94 %，酶解3 h后酶解产物的分子量分布基本保持不变。本研究结果为制备大米蛋白小分子活性肽奠定基础。

Abstract:

Enzymatic conditions for preparing small peptides from rice proteins with alcalase 2.4L were optimized by single factor experiment and the enzymatic kinetics was established. The optimal enzymatic conditions were as follows: temperature 55 ℃ pH 8.5, enzyme-substrate ratio 0.096 AU/g (substrate). Product inhibition was observed during the enzymatic hydrolysis process, but no substrate inhibition was observed within tested substrate concentration (<90 g/kg). The enzymatic kinetic parameters were: k2 = 0.28 mmol/(AU·min), Km = 5.76 (g·min)/mmol, and Vmax = 0.67 mmol/(kg·min). The kinetic enzymatic equation was: 1/V0 = 56.29/[S0][E0] + 1/0.28[E0]. After enzymolysis for three hours under the optimal enzymatic condition, the degree of hydrolysis (DH) reached 16% and the hydrolysates content with molecular weight between 264~584 u reached 94%. The molecular weight distribution of hydrolysates was barely changed after 3 h enzymolysis.