The protoplast preparation and regeneration of Trametes hirsuta and laccase production of monokaryons
毛栓菌原生质体制备和再生及单核菌株产漆酶特性

Trametes hirsuta is very effective on degradation of lignin, and is proved to be of high value in biofuel, pulping, feed and other industries. To get the monokaryons of T. hirsuta YJ-9-1, we investigated the effect of several factors on the protoplast preparation and regeneration, such as growth medium type, fungal age, hydrolysis time, centrifugal speed for purification of protoplasts and regeneration medium type. The monokaryons were identified by using DAPI staining and clamp connection deficiency method, and ligninolytic enzyme production characteristics of the monokaryons were investigated. The protoplast forming number and regeneration rate reached 5.0×106/mL and 9.1% respectively under the optimal conditions, viz. yeast glucose malt agar (YGM) medium for fungus growth, fungal age of 88h, hydrolisis duration of 1h, centrifugal speed of 4,000r/min, and yeast glucose malt sucrose agar (YGMS) medium for protoplasts regeneration. Three strains of monokaryons were obtained and the strain named D-2-1 had a higher laccase production level than that of YJ-9-1, and the laccase activity of D-2-1 reached 771.67U/L on day 12, which is 1.5 times as high as that of YJ-9-1.