桄榔蛋白酶的分离、纯化及性质的研究

Study on the Separation, Purification and Properties of the Arengain

在桄榔属的Arenga pinnata的果实中,发现了丰富的蛋白水解酶。我们称之为桄榔酶(Arengain)。用硫酸铵将果肉汁分级盐析获得粗酶制品,用DE52层析分出三个蛋白水解活性峰,其中层析成分5为主要活性组分,等电聚焦电泳证实该组分均一,等电点在pH3.99左右,用SDS-PAGE电泳法测算得分子量为44000。 10^(-3)mol/L的Hg^(2+)、PCMB、1AA、DTNB可计量地使酶失活.Hg^(2+)、PCMB抑制了的酶可为EDTA恢复活性。一定时间内巯基乙醇能令酶活性增加。二异丙基氯磷酸虽可抑制酶活性,但先加半胱氨酸,后加二异丙基氟磷酸,可使这种抑制不能实现,证明桄榔酶是巯基酶。酪蛋白做底物,桄榔酶反应的最适温度为60℃,最适pH为pH11。该酶的pH稳定性很好,pH6～12都表现了很高活性.酶液于pH6～12中24小时活性基本不变;该酶还表现了极高的乙醇耐受力,在50％～70％的乙醇中都显示出高活性。它还耐受3mol/L盐酸胍,50％曲拉通,0.1mol/L的二巯苏糖醇。

Rich protease, nemed arengain, is discovered from the fruits of Arenga pinnata. The crude powder of the arengain is fractionated by ammonium sulfate from the fruit flesh juice of Arenga pinnata. Three active compounds of proteolysis are separated out by DE52. The main active compound' is the fifth fraction of chromatography. It is proved that the compound is uniform on electrofocusing. Its isoelectric point is about pH3.99 and the molecular weight is 44000 by the method of SDS-PAGE electrophoresis. Hg^(2+), PCMB, IAA and DTNB can proportionally, inhibit the activity of the arengain. EDTA can reactivate the activity which is inhibited by Hg^(2+) and PCMB. The mercaptoethanol in certain time can increase the activity of the arengain. DFP can inhibit the activity, but the activity can be protected if adding cystine firstly than DEP. The facts prove that the aegain is one of the sulfydryl type. With casein as substrate, it can be used optimally at 60℃ and at pH11. The activity of the arengain in aqueous solution of pH6～12 could be kept for 24 hours. It has high activity in 50%～75% ethanol, and also has tolerance to 3mol/L guanidine hydrochloride, 50% Triton 100 and 10-1 mol/L DTT.