van den Broek W J, Stunnenberg H G, Wennekes L M
Microbios. 1979;26(104):115-28.
A very effective lytic enzyme system for massive micro/macro-scale production of protoplasts from the filamentous fungus Aspergillus nidulans is described. A striking coincidence was observed between maximal lytic activity towards Aspergillus mycelium and the presece of both chitinase and alpha-(1 leads to 3)-glucanase activities. The release of protoplasts was greatly enhanced by preincubating the mycelium with 2-deoxy-D-glucose. Furthermore, protoplast formation was influenced by fungal age, culture conditions, pH of incubation and the osmotic stabilizer used. From 40 mg of fresh mycelium, grown for 14--16 h on 1% glucose in a low phosphate-citrate medium, preincubated with 2-deoxy-D-glucose for 45 min, and then incubated with the lytic enzyme mixture at pH 6.5 in the presence of 0.3--0.4 M (NH4) SO4, 2.5 x 10(8) stable protoplasts were produced within 3 h of incubation at 30 degrees C. Comparable results were obtained with 40--50 g of mycelium. At low osmotic stabilizer concentrations a peculiar type of regeneration was observed in the presence of the lytic enzyme system; within 12 h of incubation aberrant hyphal structure emerged from the large vacuolated protoplasts.
本文描述了一种非常有效的裂解酶系统，用于从丝状真菌构巢曲霉大规模地在微观/宏观尺度上生产原生质体。观察到对曲霉菌丝体的最大裂解活性与几丁质酶和α-(1→3)-葡聚糖酶活性的存在之间存在惊人的巧合。用2-脱氧-D-葡萄糖对菌丝体进行预孵育可大大增强原生质体的释放。此外，原生质体的形成受真菌年龄、培养条件、孵育pH值和所用渗透稳定剂的影响。在低磷酸盐-柠檬酸盐培养基中于1%葡萄糖上生长14-16小时的40毫克新鲜菌丝体，用2-脱氧-D-葡萄糖预孵育45分钟，然后在pH 6.5、0.3-0.4M硫酸铵存在的条件下与裂解酶混合物一起孵育，在30℃孵育3小时内可产生2.5×10⁸个稳定的原生质体。用40-50克菌丝体可获得类似结果。在低渗透稳定剂浓度下，在裂解酶系统存在的情况下观察到一种特殊类型的再生；在孵育12小时内，大的液泡化原生质体中出现异常的菌丝结构。
