植物细胞脱壁酶Cellulase ZA-P的制备和应用效果

Preparation and Application of Cellulase ZA-P for the Isolation of Plant Protoplasts:

Cellulase ZA-P的产生菌为李氏木霉(Trichoderma reesei ZA-1)。研究出适于此菌产酶的液体发酵培养基配方和最佳发酵条件。其发酵液的滤纸酶活力达1.0～2.5IU/ml。发酵液经调节pH值,硫酸铵低浓度盐析和聚苯胺酚树脂脱色等处理后酶液得到纯化,最后经一种简便经济的脱盐干燥方法获得精制酶粉。Cellulase ZA-P可用于蚕豆、大小麦、番茄、烟草和青菜等多种科属植物的原生质体制备。在水稻和番茄上进行的测定试验表明,Cellulase ZA-P能分离产生大量的原生质体,其中番茄原生质体的成活率大于90％,经培养后能正常分裂形成细胞团,并进一步发育成植株。对细胞脱壁效果,原生质体成活率及原生质体分裂频率等方面与Onozuka R-10及RS的效果相当,而对原生质体无毒害。

Cellulase ZA-P was developed from Trichoderma reesei ZA-1.Optimum conditons for submerged culture for the strain were investigated. Its special activity of paperase in culture filtrate was assayed as 1.0-2.5 IU/ml. The enzyme was refined by adjusting pH of culture filtrate, precipitation with (NH_4)_2SO_4 sotution at low concentrations, then decolored and desalted. The whole process was simple and economical. Comparison tests between Cellulase ZA-P and Onozuka R—10 on protoplast preparation of rice and tomato were conducted, and they were well-matched in protoplast yield, recovery and regeneration of protoplasts. Cellulase ZA-P was not poisonous to plant protoplasts. Cellulase ZA-P has also been used to prepare other protoplasts such as that of tobacco, cabbage, broad bean, wheat, barley etc.