利用坛紫菜藻红蛋白制备脯氨酰内肽酶抑制肽

以坛紫菜(Porphyra haitanensis)为原料,通过硫酸铵盐析和DEAE-Sepharose阴离子交换柱层析等方法分离纯化得到一种高纯度藻红蛋白(R-phycoerythrin,R-PE)(A565 nm/A280 nm=5.4)。利用胃、肠液消化酶对R-PE进行酶解制备脯氨酰内肽酶(prolyl endopeptidase,PEP)抑制肽。酶解条件为:1)胃蛋白酶与R-PE比例0.5%(m/m),酶解时间30 min,pH 1.2,温度37℃;2)胰蛋白酶和胰凝乳蛋白酶质量比为1∶1的混合酶,其与R-PE比例0.25%(m/m),酶解时间30 min,pH 7.5,温度37℃。Tricine-十二烷基硫酸钠-聚丙烯酰氨凝胶电泳分析发现,经两步酶解,R-PE被完全降解。采用凝胶过滤色谱法测得R-PE水解液中分子质量在3 kDa以下的小肽含量为86.06%。R-PE水解液对PEP抑制水平IC50为136.35μg/mL。酶抑制动力学研究发现,R-PE水解液对PEP表现为可逆的非竞争性抑制作用,抑制常数Ki为14μg/mL。本研究利用坛紫菜R-PE制备活性高的PEP抑制肽,将为坛紫菜深加工及高值化利用提供一定的理论参考。

Preparation of Prolyl Endopeptidase Inhibitory Peptides from R-phycoerythrin Hydrolysate of Porphyra haitanensis

In this paper, R-phycoerythrin with relatively high purity (A565 nm/A280 nm = 5.4) was obtained from Porphyra haitanensis by ammonium sulfate fractionation and DEAE-Sepharose anion exchange chromatography. Prolyl endopeptidase (PEP) inhibitory peptides from R-phycoerythrin were prepared by two-step enzymatic digestion with gastrointestinal fluid proteases. Optimal hydrolysis conditions were determined as sequential hydrolysis with 0.5% pepsin at an enzyme-tosubstrate ratio of for 30 min at an initial pH of 1.2 and 37 ℃ followed by a 1:1 mixture of trypsin and chymotrypsin at an enzyme-to-substrate ratio of 0.25% for 30 min at pH 7.5 and 37 ℃. Tricine-SDS-PAGE showed that R-phycoerythrin was completely degraded by two-step enzymatic hydrolysis. Gel filtration chromatography analysis revealed that fractions with molecular mass lower than 3 kDa accounted for 86.06% of the total peptides. The half-maximum inhibitory concentration (IC50) of R-phycoerythrin hydrolysate toward PEP was 136.35 μg/mL. Inhibition kinetic study revealed that the hydrolysate acted as a noncompetitive inhibitor and the constant of inhibition (Ki) was 14 μg/mL. Successful preparation of R-phycoerythrin peptides with potential PEP inhibitory activity provides a theoretical rationale for the processing and utilization of P. haitanensis.