毛栓菌原生质体制备和再生及单核菌株产漆酶特性

The protoplast preparation and regeneration of Trametes hirsuta and laccase production of monokaryons

毛栓菌Trametes hirsuta能有效地降解木质素,在生物燃料、制浆和饲料工业等方面具有很高应用价值。为了获得遗传性能稳定的T.hirsuta单核菌株,研究了其菌丝生长培养基的类型、菌丝生长时间(菌龄)、酶解时间、原生质体纯化离心速度和原生质体再生培养基类型对T.hirsuta YJ-9-1原生质体制备与再生的影响;采用DAPI染色和锁状联合缺失的观察,从再生株中筛选单核菌株并考察其产酶特性。结果表明:采用YGM菌丝生长培养基、88h菌龄、1h酶解时间、4,000r/min原生质体纯化离心速度以及YGMS再生培养基,最终可获得密度大约为5.0×106个/mL的原生质体悬浮液和9.1%的再生率;从200株再生菌株中筛选出了3株单核菌株,其中一株单核菌株D-2-1的漆酶产量比原菌T.hirsuta YJ-9-1明显提高,在第12天其漆酶酶活为771.67U/L,是原菌的1.51倍。

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 5.0×10 6/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.