制备条斑紫菜原生质体的工具酶研究

Study on the Tool Enzymes of Isolating Protoplasts of Porphyra yezoensis

通过比较自制的4种海螺酶(分别取自单齿螺、锈凹螺、短滨螺和石鳖)及国产的纤维素酶、果胶酶等对条斑紫菜细胞壁的降解效果,探索经济高效制备条斑紫菜原生质体的工具酶,并进一步研究了最佳酶解条件。结果表明,单独使用海螺酶及市售的纤维素酶、果胶酶制备紫菜原生质体的效果都不好,而用单齿螺酶与质量分数为1.5%的纤维素酶制成的混合酶液(体积比1∶1)效果最佳,在25℃,pH为6.5时酶解能力最强,酶解时间以1 h为宜,酶解率可达80.4%。温度和酸碱性过高、酶解时间过长都会造成紫菜细胞的死亡。加入2 mol/L的葡萄糖作为渗压剂可使制备的原生质体成活率从65.0%提高到80.7%。

The efficient and economical tool enzymes for isolating protoplasts of Porphyra yezoensis from the digestive glands of four shellfish （Monodonta labio, Chlorostoma rusticum, Littorina brevicula, Acanthochiton rubrolineatus）, cellulase and pectinase from internal market were studied. Results indicated that the mixture of shellfish enzyme and cellulase was more effective than any single enzyme in isolating protoplasts of Porphyra yezoensis. The mixture of digestive enzyme from Monodonta labio and cellulase was the best among them. The best conditions of enzymatic isolation were 25 ℃, pH6.5 and one hour was enough for isolation. The rate of enzymatic isolation could come up to 80.4%. A high temperature, unsuitable pH or a long time of isolation could raise the cell dead rate. The survival rate of protoplasts could raise from 65. 0% to 80. 7% with 2 mol/L of glucose as penetrant.