22种海水鱼中ACEI的制备及其化学修饰

Preparation and modification of ACEI from 22 species of sea fishes

以取自中国黄海的22种鱼类为研究对象,测定其鱼肉组织上清液的酶解物对血管紧张素转化酶(ACE)的抑制作用。从6种商业用酶和1种新型海洋低温碱性蛋白酶(MLAP)中,筛选出MLAP作为酶解制备ACEI的理想水解酶。以该酶水解上述22种海水鱼鱼肉组织上清液,并将水解液经Sephadex G-25凝胶层析和DEAE Sepharose阴离子交换层析,得到ACEI纯品。测定各类ACEI体外抑制ACE的活性发现,这22种海水鱼中以鱼(Engraulis japonicus)所产A-CEI活性最高,其余鱼类无显著差异。以鱼制备血管紧张素转化酶抑制剂(ACEI-22),并对ACEI-22作进一步研究,结果发现,ACEI-22对自发性高血压大鼠(SHR)有降压效果,但不显著;ACEI-22经聚乙二醇化学修饰后,修饰率为68%,且给药SHR后,大鼠收缩压显著降低,与相同条件下同剂量的卡托普利的降压效果相当。故聚乙二醇修饰的A-CEI-22可有效地降低SHR的血压。[中国水产科学,2006,13(1):100-105]

Twenty-two species of fishes from the Yellow Sea in China were selected as the raw materials. The supernates of sea fishes protein homogenate from Coilia ectenes, Cynoglossus sernilaevis and Trichiu rus haumela were hydrolyzed respectively by single protease in 6 kinds of protease and a marine low-temperature alkaline protease （MLAP） for antihypertensive peptide, and the activity of angiotensin-converting enzyme inhibitor （ACEI） was measured. The MLAP digestion showed the highest inhibitory activity （40.28%, 48.03%, 48.20%）. Engraulis japonicus was described in detail as an example. The MLAP hydrolysate from Engraulis japonicus was purified by means of Sephadex G-25 and DEAE Sepharose anion-exchange chromatography by an elution with a linear gradient of 10 mmol/L Tris-HCl buffer （pH 7.5） containing 0.5 mol/L NaC1 and the inhibitory activity of the purified ACEI was determined. The result showed that the activity of hydrolysate from the supernate of Engraulis japonicus protein homogenate was the highest in vitro, but there was not yet notable difference in IC50 of any other MLAP hydrolysates except Engraulis japonicus. Engraulis japonicus was selected to prepare ACEI for further studies and the purified MLAP hydrolysate was named ACEI-22. ACEI-22 was composed of Asp, Ala, Val, Met, Leu, Phe and Trp according to the amino acid analysis. The stability of ACEI-22 was studied in vitro with artificial gastric juice and intestinal juice according to the method reported in China Pharmacopeia. The result showed that inhibitory activity of ACEI-22 was seriously affected in artificial gastric juice and intestinal iuice. In order to improve the stability of ACEI-22, the chemical modifications of ACEI-22 with polyethylene glycol （PEG）, dextran and heparin were conducted respectively. It was found that modification degrees of PEG, dextran and heparin were 68%, 55 % and 61% respectively. But residual activity of ACEI- 22 modified with PEG was higher than those of dextran and heparin in artificial gastric juice and intestinal juice. In vivo, ACEI-22 had slight effect on the blood pressure of spontaneously hypertensive rat （SHR） by oral administration, but the ACEI-22 modified with PEG decreased the blood pressure of SHR significantly. Though the inhibitory activity of ACEI-22 was much lower than that of Captopril （IC50 = 0.022 μmol/L） in vitro, there was equal effect on the blood pressure of SHR after the ACEI-22 was modified with PEG. The purpose of this study was to utilize enzyme engineering to convert low value biological protein resources from ocean into a series of active peptide with higher inhibitory activity of ACEI, which would be beneficial to the development of oceanic pharmaceutics.